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| <?xml version="1.0" encoding="UTF-8"?><!DOCTYPE article PUBLIC "-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD v1.1d1 20130915//EN" "JATS-archivearticle1.dtd"><article xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink" article-type="research-article" dtd-version="1.1d1"><front><journal-meta><journal-id journal-id-type="nlm-ta">elife</journal-id><journal-id journal-id-type="hwp">eLife</journal-id><journal-id journal-id-type="publisher-id">eLife</journal-id><journal-title-group><journal-title>eLife</journal-title></journal-title-group><issn publication-format="electronic">2050-084X</issn><publisher><publisher-name>eLife Sciences Publications, Ltd</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="publisher-id">04000</article-id><article-id pub-id-type="doi">10.7554/eLife.04000</article-id><article-categories><subj-group subj-group-type="display-channel"><subject>Research article</subject></subj-group><subj-group subj-group-type="heading"><subject>Developmental biology and stem cells</subject></subj-group></article-categories><title-group><article-title>Integrated β-catenin, BMP, PTEN, and Notch signalling patterns the nephron</article-title></title-group><contrib-group><contrib contrib-type="author" corresp="yes" id="author-16627"><name><surname>Lindström</surname><given-names>Nils O</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="aff" rid="aff2">2</xref><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-2"/><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-16628"><name><surname>Lawrence</surname><given-names>Melanie L</given-names></name><xref ref-type="aff" rid="aff3">3</xref><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-16629" equal-contrib="yes"><name><surname>Burn</surname><given-names>Sally F</given-names></name><xref ref-type="aff" rid="aff4">4</xref><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="fn" rid="con4"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-16633" equal-contrib="yes"><name><surname>Johansson</surname><given-names>Jeanette A</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="other" rid="par-2"/><xref ref-type="fn" rid="con5"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-16630" equal-contrib="yes"><name><surname>Bakker</surname><given-names>Elvira RM</given-names></name><xref ref-type="aff" rid="aff5">5</xref><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="fn" rid="con7"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-16631" equal-contrib="yes"><name><surname>Ridgway</surname><given-names>Rachel A</given-names></name><xref ref-type="aff" rid="aff6">6</xref><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="fn" rid="con9"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-16632"><name><surname>Chang</surname><given-names>C-Hong</given-names></name><xref ref-type="aff" rid="aff3">3</xref><xref ref-type="fn" rid="con3"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-16634"><name><surname>Karolak</surname><given-names>Michele J</given-names></name><xref ref-type="aff" rid="aff7">7</xref><xref ref-type="fn" rid="con11"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-16635"><name><surname>Oxburgh</surname><given-names>Leif</given-names></name><xref ref-type="aff" rid="aff7">7</xref><xref ref-type="fn" rid="con12"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-16636"><name><surname>Headon</surname><given-names>Denis J</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="other" rid="par-2"/><xref ref-type="fn" rid="con6"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-8520"><name><surname>Sansom</surname><given-names>Owen J</given-names></name><xref ref-type="aff" rid="aff8">8</xref><xref ref-type="fn" rid="con10"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-16637"><name><surname>Smits</surname><given-names>Ron</given-names></name><xref ref-type="aff" rid="aff5">5</xref><xref ref-type="fn" rid="con8"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-16638"><name><surname>Davies</surname><given-names>Jamie A</given-names></name><xref ref-type="aff" rid="aff3">3</xref><xref ref-type="fn" rid="con13"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-15921"><name><surname>Hohenstein</surname><given-names>Peter</given-names></name><contrib-id contrib-id-type="orcid">http://orcid.org/0000-0001-8548-4734</contrib-id><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="aff" rid="aff2">2</xref><xref ref-type="corresp" rid="cor2">*</xref><xref ref-type="other" rid="par-2"/><xref ref-type="fn" rid="con14"/><xref ref-type="fn" rid="conf1"/></contrib><aff id="aff1"><label>1</label><institution content-type="dept">Division of Developmental Biology</institution>, <institution>The Roslin Institute, University of Edinburgh</institution>, <addr-line><named-content content-type="city">Easter Bush</named-content></addr-line>, <country>United Kingdom</country></aff><aff id="aff2"><label>2</label><institution content-type="dept">MRC Human Genetics Unit</institution>, <institution>MRC Institute of Genetics and Molecular Medicine</institution>, <addr-line><named-content content-type="city">Edinburgh</named-content></addr-line>, <country>United Kingdom</country></aff><aff id="aff3"><label>3</label><institution content-type="dept">Centre for Integrated Physiology</institution>, <institution>University of Edinburgh</institution>, <addr-line><named-content content-type="city">Edinburgh</named-content></addr-line>, <country>United Kingdom</country></aff><aff id="aff4"><label>4</label><institution content-type="dept">Department of Genetics and Development</institution>, <institution>Columbia University</institution>, <addr-line><named-content content-type="city">New York</named-content></addr-line>, <country>United States</country></aff><aff id="aff5"><label>5</label><institution content-type="dept">Laboratory of Gastroenterology and Hepatology</institution>, <institution>Erasmus MC, University Medical Centre</institution>, <addr-line><named-content content-type="city">Rotterdam</named-content></addr-line>, <country>Netherlands</country></aff><aff id="aff6"><label>6</label><institution content-type="dept">Department of Invasion and Metastasis</institution>, <institution>Cancer Research UK Beatson Institute</institution>, <addr-line><named-content content-type="city">Glasgow</named-content></addr-line>, <country>United Kingdom</country></aff><aff id="aff7"><label>7</label><institution content-type="dept">Center for Molecular Medicine</institution>, <institution>Maine Medical Center Research Institute</institution>, <addr-line><named-content content-type="city">Scarborough</named-content></addr-line>, <country>United States</country></aff><aff id="aff8"><label>8</label><institution>Beatston Institute for Cancer Research</institution>, <addr-line><named-content content-type="city">Glasgow</named-content></addr-line>, <country>United Kingdom</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Fuchs</surname><given-names>Elaine</given-names></name><role>Reviewing editor</role><aff><institution>Rockefeller University</institution>, <country>United States</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>nils.lindstrom@roslin.ed.ac.uk</email> (NOL);</corresp><corresp id="cor2"><email>peter.hohenstein@roslin.ed.ac.uk</email> (PH)</corresp><fn fn-type="con" id="equal-contrib"><label>†</label><p>These authors contributed equally to this work</p></fn></author-notes><pub-date publication-format="electronic" date-type="pub"><day>03</day><month>02</month><year>2015</year></pub-date><pub-date pub-type="collection"><year>2015</year></pub-date><volume>4</volume><elocation-id>e04000</elocation-id><history><date date-type="received"><day>13</day><month>07</month><year>2014</year></date><date date-type="accepted"><day>28</day><month>12</month><year>2014</year></date></history><permissions><copyright-statement>© 2014, Lindström et al</copyright-statement><copyright-year>2014</copyright-year><copyright-holder>Lindström et al</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This article is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="elife04000.pdf"/><abstract><object-id pub-id-type="doi">10.7554/eLife.04000.001</object-id><p>The different segments of the nephron and glomerulus in the kidney balance the processes of water homeostasis, solute recovery, blood filtration, and metabolite excretion. When segment function is disrupted, a range of pathological features are presented. Little is known about nephron patterning during embryogenesis. In this study, we demonstrate that the early nephron is patterned by a gradient in β-catenin activity along the axis of the nephron tubule. By modifying β-catenin activity, we force cells within nephrons to differentiate according to the imposed β-catenin activity level, thereby causing spatial shifts in nephron segments. The β-catenin signalling gradient interacts with the BMP pathway which, through PTEN/PI3K/AKT signalling, antagonises β-catenin activity and promotes segment identities associated with low β-catenin activity. β-catenin activity and PI3K signalling also integrate with Notch signalling to control segmentation: modulating β-catenin activity or PI3K rescues segment identities normally lost by inhibition of Notch. Our data therefore identifies a molecular network for nephron patterning.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04000.001">http://dx.doi.org/10.7554/eLife.04000.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.04000.002</object-id><title>eLife digest</title><p>The main function of the kidney is to filter blood to remove waste and regulate the amount of water and salt in the body. Structures in the kidney—called nephrons—do much of this work and blood is filtered in a part of each nephron called the glomerulus. The substances filtered out of the blood move into a series of ‘tubules’, another part of the nephrons, from where water and soluble substances are reabsorbed or excreted as the body requires. If the nephrons do not work correctly, it can lead to a wide range of health problems—from abnormal water and salt loss to dangerously high blood pressure.</p><p>For organs and tissues to develop in an embryo, signalling pathways help cells to communicate with each other. These pathways control what type of cells the embryonic cells become and also help neighbouring cells work together to form specialised structures with particular functions. Much is unknown about how the nephron develops, including how its different structures coordinate their development with each other so that they form in the right position in the nephron.</p><p>A protein called beta-catenin was already known to play an important role in the signalling pathways that trigger the earliest stages of nephron formation. Lindström et al. further investigated how this protein helps the nephron to develop by using a wide range of techniques, including growing genetically altered mouse kidneys in culture and capturing images of the developing nephrons with time-lapse microscopy. The combined results reveal that the levels of beta-catenin activity coordinate the development of the different structures in the nephron. The beta-catenin protein is not equally active in all parts of the nephron; instead, it forms a gradient of different activity levels. The highest levels of beta-catenin activity occur in the tubules at the furthest end of the developing nephron; this activity gradually decreases along the length of the nephron, and the glomerulus itself lacks beta-catenin activity altogether. Experimentally manipulating the levels of beta-catenin at different points along the nephron caused those cells to take on the wrong identity, causing parts of the nephron to form in the wrong place.</p><p>Lindström et al. were also able to establish that the signalling pathway controlled by beta-catenin activity interacts with three other well-known signalling pathways as part of a network that controls nephron development. More research is required to find out which signal activates beta-catenin in the first place and from where in the kidney this signal comes. It also remains to be discovered how a particular cell in the tubule interprets the exact activities of the different signals to give the cell its specific identity for that place in the nephron. A better understanding of these sorts of processes will eventually help build new kidneys for people with kidney failure.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04000.002">http://dx.doi.org/10.7554/eLife.04000.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>kidney development</kwd><kwd>patterning</kwd><kwd>beta-catenin</kwd><kwd>PI3K</kwd><kwd>Notch</kwd><kwd>BMP</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd>mouse</kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100000849</institution-id><institution>National Centre for the Replacement, Refinement and Reduction of Animals in Research</institution></institution-wrap></funding-source><award-id>94808</award-id><principal-award-recipient><name><surname>Lindström</surname><given-names>Nils O</given-names></name></principal-award-recipient></award-group><award-group id="par-2"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100000268</institution-id><institution>Biotechnology and Biological Sciences Research Council</institution></institution-wrap></funding-source><award-id>BB/J003416/1</award-id><principal-award-recipient><name><surname>Lindström</surname><given-names>Nils O</given-names></name><name><surname>Johansson</surname><given-names>Jeanette A</given-names></name><name><surname>Headon</surname><given-names>Denis J</given-names></name><name><surname>Hohenstein</surname><given-names>Peter</given-names></name></principal-award-recipient></award-group><funding-statement>The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.</funding-statement></funding-group><custom-meta-group><custom-meta><meta-name>elife-xml-version</meta-name><meta-value>2.0</meta-value></custom-meta><custom-meta specific-use="meta-only"><meta-name>Author impact statement</meta-name><meta-value>The patterning of the nephron is driven by a gradient in the activity of β-catenin and further defined by a network of BMP, PTEN and NOTCH signalling.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec sec-type="intro" id="s1"><title>Introduction</title><p>All adult vertebrates depend on renal nephrons accurately performing a diverse set of roles that protect and maintain blood homeostasis. The diversity of roles that the nephron performs is reflected in the range of symptoms of abnormal nephron function and the consequent diseases range from, for example, Bartter syndrome (abnormal water and salt loss), acidosis, and rickets to essential hypertension (<xref ref-type="bibr" rid="bib70">Simon et al., 1996</xref>; <xref ref-type="bibr" rid="bib69">Schedl, 2007</xref>). In spite of the importance of proper nephron function and segmentation, little is known regarding how the nephron patterns and how different specialised segments form during nephrogenesis. Thus far, only a handful of genes have been shown to control the development of specific nephron segments of which none have been explicitly connected to an explanation for how patterning is regulated along the whole length of the nephron.</p><p>Nephrons form during embryonic development, when Wnt9b, secreted by the ureteric bud, activates a canonical β-catenin-mediated pathway in a population of overlying Six2<sup>+</sup> mesenchymal nephron progenitor-cells (<xref ref-type="bibr" rid="bib51">Kobayashi et al., 2008</xref>; <xref ref-type="bibr" rid="bib47">Karner et al., 2011</xref>; <xref ref-type="bibr" rid="bib63">Park et al., 2012</xref>; <xref ref-type="bibr" rid="bib23">Das et al., 2013</xref>). In the canonical WNT pathway, WNT signalling results in the destabilisation of the GSK-3β/CK1α/AXIN2/APC complex and prevents the normal tagging of cytosolic β-catenin for degradation. Stabilised β-catenin translocates to the nucleus and together with members of the TCF family of transcription factors controls the expression of a wide range of target genes (<xref ref-type="bibr" rid="bib22">Clevers and Nusse, 2012</xref>). One of these β-catenin target genes, <italic>Wnt4</italic>, triggers a mesenchymal-to-epithelial transition (MET) (<xref ref-type="bibr" rid="bib72">Stark et al., 1994</xref>; <xref ref-type="bibr" rid="bib50">Kispert et al., 1998</xref>) in the nephron progenitor cells and these reconfigure into an epithelial renal vesicle (RV). Following the MET, the RV becomes polarised and connects to the ureteric bud at its distal end (<xref ref-type="bibr" rid="bib38">Georgas et al., 2009</xref>), and during subsequent developmental steps, the nephron starts to display additional signs of pattern-formation along its proximal–distal axis. Several distinct cell-populations form and these produce the different segments of the adult nephron (<xref ref-type="bibr" rid="bib68">Saxen, 1987</xref>); a Wt1<sup><italic>+</italic></sup> cell population gives rise to proximal structures, a Jag1<sup>+</sup> population to the medial part and Lgr5<sup><italic>+</italic></sup> cells generate the distal nephron segments (<xref ref-type="bibr" rid="bib4">Armstrong et al., 1993</xref>; <xref ref-type="bibr" rid="bib20">Cheng et al., 2003</xref>; <xref ref-type="bibr" rid="bib18">Chen and Al-Awqati, 2005</xref>; <xref ref-type="bibr" rid="bib19">Cheng et al., 2007</xref>; <xref ref-type="bibr" rid="bib52">Kreidberg, 2010</xref>; <xref ref-type="bibr" rid="bib7">Barker et al., 2012</xref>). These segments are in turn further subdivided into functionally specialised portions, which express specific combinations of transmembrane transporters/channels for salts, glucose, and metals (<xref ref-type="bibr" rid="bib65">Raciti et al., 2008</xref>). How the differentiation of these segments is regulated remains unknown. The initiation of the nephron MET is driven by β-catenin signalling (<xref ref-type="bibr" rid="bib51">Kobayashi et al., 2008</xref>; <xref ref-type="bibr" rid="bib47">Karner et al., 2011</xref>; <xref ref-type="bibr" rid="bib63">Park et al., 2012</xref>), but the Wnt4 driven MET is most likely mediated by the non-canonical Ca<sup>2+</sup>–NFAT pathway (<xref ref-type="bibr" rid="bib15">Burn et al., 2011</xref>; <xref ref-type="bibr" rid="bib73">Tanigawa et al., 2011</xref>). It remains uncertain by what mechanism and at what precise stage the Six2<sup>+</sup> cells or the RV develop distinct nephron segment lineages (<xref ref-type="bibr" rid="bib55">Lindstrom et al., 2013</xref>). Post-MET, Wnt9b acts via the planar cell polarity pathway and controls the orientation of cell division and the elongation of collecting tubules (<xref ref-type="bibr" rid="bib46">Karner et al., 2009</xref>). Wnt7b also has a role as it controls the development of the medulla and papilla of the kidney (<xref ref-type="bibr" rid="bib76">Yu et al., 2009</xref>). Notch signalling has previously been identified as being important for the formation of the proximal tubule (<xref ref-type="bibr" rid="bib20">Cheng et al., 2003</xref>, <xref ref-type="bibr" rid="bib19">2007</xref>). <italic>Notch2</italic><sup><italic>−/−</italic></sup> nephrons form no proximal tubules or glomeruli (<xref ref-type="bibr" rid="bib19">Cheng et al., 2007</xref>). However, ectopic expression of the intracellular and active Notch1-domain (N1ICD) in nephrons blocks glomerular development (<xref ref-type="bibr" rid="bib20">Cheng et al., 2003</xref>, <xref ref-type="bibr" rid="bib19">2007</xref>; <xref ref-type="bibr" rid="bib11">Boyle et al., 2011</xref>). N1ICD expression in Six2<sup>+</sup> cells can actually substitute for Wnt9b and trigger nephron induction and MET (<xref ref-type="bibr" rid="bib11">Boyle et al., 2011</xref>). Whether Notch or Wnt is important for the initial patterning of the nephron immediately post-MET remains to be determined.</p><p>Using in vivo and ex vivo techniques we demonstrate that a gradient of β-catenin activity, along the proximal–distal nephron axis, controls the differentiation of segment-specific cell fates. We further investigate how β-catenin activity is prevented in the proximal and medial segments and show that BMP/PTEN/PI3K signalling in the medial nephron actively promotes the medial segment identity whilst blocking β-catenin activity. In addition, we show that modulating β-catenin or PI3K activity partially rescues the nephron segment defect phenotypes associated with the loss of Notch function. Our findings provide a model where multiple signalling pathways are integrated to control nephron segment-identity specification.</p></sec><sec sec-type="results" id="s2"><title>Results</title><sec id="s2-1"><title>A β-catenin activity gradient is generated along the nephron axis</title><p>Regulation of β-catenin activity is essential for nephron induction and MET (<xref ref-type="bibr" rid="bib27">Davies and Garrod, 1995</xref>; <xref ref-type="bibr" rid="bib53">Kuure et al., 2007</xref>; <xref ref-type="bibr" rid="bib64">Park et al., 2007</xref>). To determine whether β-catenin is involved in post-MET stages of nephron development, we tracked its activity in embryonic kidney organ cultures using a β-catenin signalling reporter mouse strain (<italic>TCF/Lef::H2B-GFP</italic>; <xref ref-type="bibr" rid="bib32">Ferrer-Vaquer et al., 2010</xref>). Confocal and time-lapse microscopy indicated strong activity of the reporter in the ureteric bud as described before (<xref ref-type="bibr" rid="bib32">Ferrer-Vaquer et al., 2010</xref>; <xref ref-type="bibr" rid="bib15">Burn et al., 2011</xref>). Importantly, we also detected different GFP intensities, reporting β-catenin activity, along the proximal–distal axis of the nephron. The nomenclature we use to describe the domains of the proximal–distal axis is as defined by the GenitoUrinary Development Molecular Anatomy Project (<ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://gudmap.org">gudmap.org</ext-link>) for S-shaped body nephrons. Confocal z-projections (<xref ref-type="fig" rid="fig1">Figures 1A</xref>, 3D rendering <xref ref-type="other" rid="video1">Video 1</xref>) of whole nephrons at an early stage of development show the signal being highest at the distal end of the nephron, where it connects to the ureteric bud, and gradually decreasing towards the proximal end. Time-lapse imaging of developing <italic>TCF/Lef::H2B-GFP</italic> expressing nephrons showed that the different GFP signal intensities propagated in a distal-to-proximal direction over time alongside the normal nephron growth and segmentation (<xref ref-type="fig" rid="fig1s1">Figure 1<bold>—</bold>figure supplement 1A</xref> and <xref ref-type="other" rid="video2">Video 2</xref>). Confocal imaging confirmed different GFP intensities in nephrons at later stages: S-shaped bodies (<xref ref-type="fig" rid="fig1">Figure 1B</xref> and <xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1B</xref>) and more mature nephrons (data not shown), and we consistently found that the podocytes and their precursors at the extreme proximal end of the nephrons were almost completely devoid of β-catenin activity (<xref ref-type="fig" rid="fig1">Figure 1A,B</xref>, <xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1B</xref>; <xref ref-type="other" rid="video1">Video 1</xref>). We quantified the <italic>TCF/Lef::H2B-GFP</italic> signal in cells located in the distal, medial, and proximal segments of nephrons and plotted their intensities against their position. The segments were defined with antibodies for Jag1 (medial segment; <xref ref-type="bibr" rid="bib18">Chen and Al-Awqati, 2005</xref>; <xref ref-type="bibr" rid="bib38">Georgas et al., 2009</xref>), Cdh1 (distal segment; <xref ref-type="bibr" rid="bib21">Cho et al., 1998</xref>), and by morphology. The <italic>TCF/Lef::H2B-GFP</italic> signal intensities showed an exponentially decreasing gradient (R2 = 0.999; n = 11 nephrons) suggestive of a single source and first-order decay of the activating signal (<xref ref-type="fig" rid="fig1">Figure 1C</xref>). Jag1 immunofluorescent intensity data were used to indicate positions within nephrons. The GFP intensities measured in the distal segments differed from those in proximal segments by a minimum of a 15-fold difference to a maximum of a 72-fold difference (mean = 39-fold difference; n = 10 nephrons; <xref ref-type="fig" rid="fig1">Figure 1C</xref>).<fig-group><fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.04000.003</object-id><label>Figure 1.</label><caption><title>β-catenin activity levels form a reversed gradient along the axis of the nephron.</title><p>(<bold>A</bold>–<bold>B</bold>) <italic>TCF/Lef::H2B-GFP</italic> expression in nephrons: (<bold>A</bold>) late renal vesicle/early comma-shaped body nephron, (<bold>B</bold>) S-shaped body nephron, lines: white–nephron axis, purple–ureteric bud, green–distal nephron, red–medial nephron, blue–proximal nephron/glomerular precursors. Heat-maps display signal intensity in different nephron segments. (<bold>C</bold>) Quantification of nuclear H2B-GFP and cell-membrane Jag1 antibody stain signal-intensity along the proximal–distal axis. Error bars represent SEM of pixels representative of 10 µm segments. Right-hand side graph shows mean values for segments, as identified by H2B-GFP and Jag1 profiles (n = 11 nephrons), error bars indicate SEM. p-values derived from t-tests. (<bold>D</bold>) Antibody stains against total β-catenin and phosphorylated β-catenin in S-shaped body nephron—Jag1 marking the medial segment. White dashed line indicating nephron axis.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04000.003">http://dx.doi.org/10.7554/eLife.04000.003</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife04000f001"/></fig><fig id="fig1s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.04000.004</object-id><label>Figure 1—figure supplement 1.</label><caption><title>β-catenin reporter and antibody data show different β-catenin activity levels along the axis of the nephron.</title><p>(<bold>A</bold>) <italic>TCF/Lef:H2B-GFP</italic> time-lapse data of a nephron developing through post-MET Pretubular Aggregate (PTA), Renal Vesicle (RV), Comma-shaped (CB), and S-shaped (SB) stages. These data are shown as time-lapse in <xref ref-type="other" rid="video2">Video 2</xref>. (<bold>B</bold>) S-shaped body nephron from <xref ref-type="fig" rid="fig1">Figure 1B</xref> shown at four different brightness settings that represent a doubling in brightness for each field going left to right. This clearly shows that all segments of the nephron are positive for the <italic>TCF/Lef:H2B-GFP</italic> reporter but follow a visual gradient. Green arrows point to positive cells in the dimmest portion of the nephron and an area of the kidney that even in at the brightest settings show up as negative. (<bold>C</bold>) Quantification of antibody stain for β-catenin phosphorylated at Ser33/Ser33/Thr41 in the distal, medial, and proximal segments (nine measurements per nephron in five nephrons). Error bars represent SEM, p-values derived from t-tests. (<bold>D</bold>–<bold>F</bold>) Lef1 and Ccnd1 (CycD1) antibody stains in S-shaped body nephrons; Pax2 is used as a structural marker against all nuclei within the nephron, Jag1 is used to detect the medial segment, Wt1 is used to detect the developing podocytes in the proximal segment.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04000.004">http://dx.doi.org/10.7554/eLife.04000.004</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife04000fs001"/></fig></fig-group><media id="video1" content-type="glencoe play-in-place height-250 width-310" xlink:href="elife04000v001.avi" mimetype="video" mime-subtype="avi"><object-id pub-id-type="doi">10.7554/eLife.04000.007</object-id><label>Video 1.</label><caption><title>3D reconstructions of nephrons.</title><p>3D reconstruction of renal vesicle (top), S-shaped body (middle), and more mature nephron (bottom). Nephrons are positive for <italic>TCF/Lef::H2B-GFP</italic>, Jag1-red, Cdh1-blue (left panel) and the <italic>TCF/Lef::H2B-GFP</italic> reporter is shown in a heat-map overlay (right panel).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04000.007">http://dx.doi.org/10.7554/eLife.04000.007</ext-link></p></caption></media><media id="video2" content-type="glencoe play-in-place height-250 width-310" xlink:href="elife04000v002.avi" mimetype="video" mime-subtype="avi"><object-id pub-id-type="doi">10.7554/eLife.04000.008</object-id><label>Video 2.</label><caption><title>Time-lapse capture of nephron.</title><p>The nephron is the same as shown in <xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1A</xref> (from a <italic>TCF/Lef::H2B-GFP</italic> reporter kidney) is shown during the earliest stages of nephron development. Segments and stages are annotated based on the brightfield channel (not shown).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04000.008">http://dx.doi.org/10.7554/eLife.04000.008</ext-link></p></caption></media></p><p>To further assess the observed gradient in β-catenin activity, we analysed the expression and activity of the β-catenin protein directly. Using pan-β-catenin antibodies, we found β-catenin to be expressed throughout the developing nephrons (<xref ref-type="fig" rid="fig1">Figure 1D</xref>). Antibodies for β-catenin that was already tagged for degradation (phosphorylated at Ser33/Ser37/Thr41), most strongly labelled the proximal domain (twofold higher compared to medial and distal, p = 6.2 × 10<sup>−5</sup> and 1.2 × 10<sup>−4</sup>) (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1C</xref>). Target genes of β-catenin (<italic>Lef1</italic> and <italic>Ccnd1</italic>) were also expressed along the gradient (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1D–F</xref>). The distribution of β-catenin protein and the expression of direct β-catenin target genes support the existence of the activity gradient.</p></sec><sec id="s2-2"><title>Proximal and distal positional identities are controlled by different β-catenin activity levels</title><p>To investigate whether the β-catenin activity gradient has functional implications for the development of the nephron proximal–distal axis, we used inhibitors of GSK-3β (CHIR99021) and Tankyrase (IWR1) to positively and negatively modify β-catenin signalling ex vivo in whole organ cultures. We extensively characterised these small molecule inhibitors to ensure that they had their expected effect on the β-catenin signalling pathway; these data can be found in <xref ref-type="fig" rid="fig2s1 fig2s2 fig2s3 fig2s4">Figure 2—figure supplements 1–4</xref> and <xref ref-type="other" rid="video3 video4">Videos 3–4</xref> and are briefly described here. First, we used the β-catenin reporter <italic>TCF/Lef::H2B-GFP</italic> and qRT-PCR analyses and confirmed that the inhibitors acted as expected (<xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1</xref>). Second, because maximal activation of β-catenin has previously been suggested to be incompatible with MET (<xref ref-type="bibr" rid="bib53">Kuure et al., 2007</xref>; <xref ref-type="bibr" rid="bib64">Park et al., 2007</xref>, <xref ref-type="bibr" rid="bib63">2012</xref>), we identified CHIR concentrations that induced nephron differentiation (PAX2 and PAX8 expression) but still permitted epithelialisation (CDH1 expression; <xref ref-type="fig" rid="fig2s2">Figure 2—figure supplement 2</xref>). Increased β-catenin signalling induced ectopic nephrons to form at the periphery of the kidneys and large nephron structures developed within the ureteric tree (<xref ref-type="fig" rid="fig2s1 fig2s2">Figure 2—figure supplements 1,2</xref>). Third, we demonstrated that these ectopic structures, just like the tree-bound structures, were derived from Six2<sup>+</sup> nephron progenitors by fate-mapping these using a <italic>Six2-CreGFP</italic> (<xref ref-type="bibr" rid="bib29">Dolt et al., 2013</xref>) and a <italic>Rosa26</italic><sup><italic>tdRFP</italic></sup> Cre reporter mouse model (<xref ref-type="bibr" rid="bib58">Luche et al., 2007</xref>) (<xref ref-type="fig" rid="fig2s3">Figure 2—figure supplement 3</xref> and <xref ref-type="other" rid="video3">Video 3</xref>). Fourth, since pharmacological inhibitors can have multiple targets, we confirmed their specificity by combining activators and inhibitors of the WNT-pathway. <italic>Pax8</italic>-Cre lineage tracing and immunofluorescent analyses allowed us to show that by blocking β-catenin signalling downstream of GSK-3β (by administering ICG001 which binds CBP and prevents β-catenin/CBP interaction), but not upstream (IWR1), CHIR-induced ectopic nephron formation was inhibited (<xref ref-type="fig" rid="fig2s4">Figure 2—figure supplement 4A</xref> and <xref ref-type="other" rid="video4">Video 4</xref>). Fifth, additional inhibitors against components of the β-catenin/Canonical WNT-pathway also confirmed our findings: (BIO (GSK3β inhibitor) induced effects similar to CHIR; salinomycin (LRP6 inhibitor) induced effects similar to IWR1 (<xref ref-type="bibr" rid="bib36">Gandhirajan et al., 2010</xref>; <xref ref-type="bibr" rid="bib57">Lu et al., 2011</xref>); <xref ref-type="fig" rid="fig2s4">Figure 2—figure supplement 4B</xref>).<media id="video3" content-type="glencoe play-in-place height-250 width-310" xlink:href="elife04000v003.avi" mimetype="video" mime-subtype="avi"><object-id pub-id-type="doi">10.7554/eLife.04000.009</object-id><label>Video 3.</label><caption><title>Time-lapse capture of <italic>Six2</italic><sup>GFPCre</sup>/Rosa26<sup>tdRFP</sup> kidneys.</title><p>Kidneys cultured in control medium and CHIR medium. Timing and conditions as shown in videos.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04000.009">http://dx.doi.org/10.7554/eLife.04000.009</ext-link></p></caption></media><media id="video4" content-type="glencoe play-in-place height-250 width-310" xlink:href="elife04000v004.avi" mimetype="video" mime-subtype="avi"><object-id pub-id-type="doi">10.7554/eLife.04000.010</object-id><label>Video 4.</label><caption><title>Time-lapse capture of Pax8<sup>Cre</sup> YFP<sup>lox-stop</sup> kidneys.</title><p>Kidneys cultured in control medium, IWR1 and CHIR medium, or ICG001 and CHIR medium. Timing and conditions as shown in videos.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04000.010">http://dx.doi.org/10.7554/eLife.04000.010</ext-link></p></caption></media></p><p>Using these inhibitors we modulated β-catenin signalling in <italic>Wt1</italic><sup>+/GFP</sup> nephrons, where GFP labels the proximal cell population, allowing maturing podocytes within glomeruli to be recognized by their bright concentrated GFP signal (<xref ref-type="fig" rid="fig2">Figure 2A</xref>, <xref ref-type="other" rid="video5">Video 5</xref>). The <italic>Wt1</italic><sup>+/GFP</sup> also labels the nephron progenitor cells and the whole of the pre-tubular aggregate during the MET, but at lower intensities, in accordance with the known expression pattern of <italic>Wt1</italic> (<xref ref-type="bibr" rid="bib52">Kreidberg, 2010</xref>). Time-lapse analysis showed that, by decreasing β-catenin activity, we favoured the differentiation of the proximal cell identity as was seen by an increase in maturation rate of glomeruli; in contrast, increased β-catenin activity blocked proximal identity formation and the formation of glomeruli (<xref ref-type="fig" rid="fig2">Figure 2A–C</xref>). Podxl antibody staining (<xref ref-type="fig" rid="fig2">Figure 2D</xref>), qRT-PCR analysis of additional markers for proximal differentiation (<italic>Nphs2</italic>, <italic>Synpo</italic>, <italic>Nphs1</italic>, <italic>Podxl</italic>), and <italic>Wt1</italic><sup>+/GFP</sup> reporter kidneys (<xref ref-type="fig" rid="fig2s5">Figure 2—figure supplement 5A,B</xref>) confirmed that these markers were expressed earlier when β-catenin activity was decreased and that they were repressed when β-catenin activity was increased. We consistently noticed a small number of individual cells in CHIR conditions that remained positive for Wt1 or Podxl (<xref ref-type="fig" rid="fig2">Figure 2A,D</xref>). Releasing these cells from increased β-catenin activity, by removing CHIR, allowed them to resume their development (<xref ref-type="fig" rid="fig2">Figure 2E</xref>). The proximal identity almost fully recovered and glomerular structures formed at almost the same size as those found in controls, show that the ectopically increased β-catenin activity had been actively suppressing the proximal identity (<xref ref-type="fig" rid="fig2s5">Figure 2—figure supplement 5C</xref>). In contrast, removing IWR1 did not result in the reversal of the phenotype (<xref ref-type="fig" rid="fig2s5">Figure 2—figure supplement 5D</xref>).<fig-group><fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.04000.011</object-id><label>Figure 2.</label><caption><title>Pharmacological modulation of β-catenin signalling alters proximal segment development.</title><p>(<bold>A</bold>) Time-lapse analysis of treated <italic>Wt1</italic><sup>+/GFP</sup> kidneys—same as shown in <xref ref-type="other" rid="video5">Video 5</xref>. (<bold>B</bold>) Quantification of mean time taken for first glomeruli to mature to crescent-shaped stage where glomeruli are tightly packed and exhibit a bright signal. (<bold>C</bold>) Mean number of mature glomeruli after 3800 min of culture. (<bold>D</bold>) Kidneys stained for podocyte marker Podxl, β-catenin target Lef1, and epithelial marker Cdh1. Arrowheads indicating structures positive for Podxl. (<bold>E</bold>) The proximal identity resumed its formation when CHIR was removed after 48 hr—white arrowheads indicate larger Podxl positive structures, blue arrowheads indicate very small Podxl positive structures, dashed line separates ectopic nephron zone (e.n.z.) nephrons from those inside the ureteric tree (UB).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04000.011">http://dx.doi.org/10.7554/eLife.04000.011</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife04000f002"/></fig><fig id="fig2s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.04000.012</object-id><label>Figure 2—figure supplement 1.</label><caption><title>β-catenin signalling is altered in response to pharmacological inhibitors.</title><p>(<bold>A</bold>–<bold>B</bold>) <italic>TCF/Lef:H2B-GFP</italic> kidneys displaying low levels of β-catenin signalling in IWR1 conditions whereas strong activation of β-catenin signalling is detected in CHIR conditions. Typical nephrons within respective conditions are indicated with yellow arrowheads and nephrons captured at 60× and stained Jag1 and Cdh1 are shown in (<bold>B</bold>). (<bold>C</bold>) Signal intensity of <italic>TCF/Lef:H2B-GFP</italic> β-catenin reporter is increased and decreased in the tips of the ureteric bud in response to CHIR and IWR1, respectively. (<bold>D</bold>) qRT-PCR of known β-catenin target genes in response to IWR1 and CHIR treatment. Some β-catenin target genes responded as predicted (<italic>Axin2</italic>, <italic>Lef1</italic>), others did not (<italic>c-Myc</italic> and <italic>Ccnd1</italic>) as is expected from cell type-specific β-catenin targets (<xref ref-type="bibr" rid="bib67">Sansom et al., 2005</xref>; <xref ref-type="bibr" rid="bib66">Sansom et al., 2007</xref>)—<italic>Axin2</italic> and <italic>Lef1</italic> data is also shown in <xref ref-type="fig" rid="fig4">Figure 4D</xref> to put them into the context of other expression changes. (<bold>E</bold>) Effects of different β-catenin activity levels on kidneys morphogenesis and nephron-formation.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04000.012">http://dx.doi.org/10.7554/eLife.04000.012</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife04000fs002"/></fig><fig id="fig2s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.04000.013</object-id><label>Figure 2—figure supplement 2.</label><caption><title>‘Just-right’ β-catenin signalling levels drive MET.</title><p>(<bold>A</bold>) Response of β-catenin target gene (Lef1), induction markers (Pax2 and Pax8), and epithelialisation marker (Cdh1) to different CHIR concentrations. Inserts showing all channels for kidneys stained for Jag1, Cdh1, and indicated marker. As in previous studies, strong activation of β-catenin disrupted the normal MET. Arrowheads indicate ectopic nephrons. All error bars indicate SEM.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04000.013">http://dx.doi.org/10.7554/eLife.04000.013</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife04000fs003"/></fig><fig id="fig2s3" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.04000.014</object-id><label>Figure 2—figure supplement 3.</label><caption><title>Ectopic nephrons form from Six2 expressing progenitors.</title><p><italic>Six2</italic><sup>GFPCre</sup> with conditional RFP lineage tracing highlighting all nephron progenitors cell (GFP<sup>+</sup>) and all nephron lineages (RFP<sup>+</sup>). Kidneys cultured in CHIR show ectopic RFP<sup>+</sup> structures forming from previous GFP<sup>+</sup> nephron progenitors. Dashed line indicates separation of ectopic nephrons from the ureteric bud tree and endogenous ‘tree-bound’ nephrons. Data also presented in <xref ref-type="other" rid="video3">Video 3</xref>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04000.014">http://dx.doi.org/10.7554/eLife.04000.014</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife04000fs004"/></fig><fig id="fig2s4" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.04000.015</object-id><label>Figure 2—figure supplement 4.</label><caption><title>Co-inhibition experiments confirm specificity of pharmacological inhibitors.</title><p>(<bold>A</bold>) Co-inhibition of GSK-3β (CHIR) and Tankyrase (IWR1) or CBP (ICG001). ICG001 blocks TCF interacting with the CBP co-activator (<xref ref-type="bibr" rid="bib30">Emami et al., 2004</xref>) downstream of (CHIR). CHIR effects are not blocked by IWR1 inhibiting Tankyrase upstream of GSK-3β but are blocked by ICG001. Dashed line separates ectopic nephrons from those adjacent to the ureteric bud. Error bars indicate SEM. Arrowheads indicate ectopic nephrons. (<bold>B</bold>) 7.5 µM BIO (GSK-3β inhibitor) mimics 1.5 µM CHIR and ectopic nephrons form and the UB (ureteric tree) branches is as in (<bold>A</bold>). Arrowheads indicate ectopic nephrons, dashed line separates ectopic nephron zone (e.n.z.) nephrons from those inside the ureteric tree (UB). (<bold>B</bold>) 100 nM salinomycin (LRP6 inhibitor) treated samples show UB branching similar to that caused by IWR1. The effect on nephrons is also similar. Antibody stains and scale bars as indicated.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04000.015">http://dx.doi.org/10.7554/eLife.04000.015</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife04000fs005"/></fig><fig id="fig2s5" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.04000.016</object-id><label>Figure 2—figure supplement 5.</label><caption><title>The proximal cell-identity is promoted by decreased β-catenin signalling.</title><p>(<bold>A</bold>) qRT-PCR data for genes indicative of terminally differentiated glomerular cells. (<bold>B</bold>) <italic>Wt1</italic><sup>+/GFP</sup> kidneys stained for Pax8 and Cdh1–arrowheads indicating structures positive for GFP. (<bold>C</bold>) Size of glomerular structures in rescued nephrons as shown in <xref ref-type="fig" rid="fig2">Figure 2E</xref>. All error bars indicate SEM. (<bold>D</bold>) Kidney development in kidneys cultured for a full 96 hr in IWR1 or 48 hr in IWR1 followed by 48 hr in control medium. Antibody stains as indicated.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04000.016">http://dx.doi.org/10.7554/eLife.04000.016</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife04000fs006"/></fig></fig-group><media id="video5" content-type="glencoe play-in-place height-250 width-310" xlink:href="elife04000v005.avi" mimetype="video" mime-subtype="avi"><object-id pub-id-type="doi">10.7554/eLife.04000.017</object-id><label>Video 5.</label><caption><title>Time-lapse capture of Wt1<sup>+/GFP</sup> kidneys.</title><p>Kidneys cultured in control conditions or treated with IWR1 or CHIR. Timing, conditions, and scale as specified.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04000.017">http://dx.doi.org/10.7554/eLife.04000.017</ext-link></p></caption></media></p><p>Distal cells express the epithelial stem-cell marker and β-catenin target <italic>Lgr5</italic> (<xref ref-type="bibr" rid="bib7">Barker et al., 2012</xref>). To test whether the β-catenin gradient also controls the formation of the distal identity, we monitored <italic>Lgr5-EGFP</italic> expression in kidneys at different β-catenin activity levels using the <italic>Lgr5</italic><sup>+/EGFP-IRES-CreERT2</sup> mouse model (<xref ref-type="bibr" rid="bib7">Barker et al., 2012</xref>). <italic>Lgr5</italic> was expressed as previously described (<xref ref-type="bibr" rid="bib7">Barker et al., 2012</xref>), although we primarily detected strong <italic>Lgr5-EGFP</italic> expression in a subsection of the distal domain adjacent to the medial segment (<xref ref-type="fig" rid="fig3">Figure 3A</xref>). <italic>Lgr5</italic> expression levels were increased at high levels of β-catenin activity. When β-catenin activity was decreased, the expression domain extended longer although the actual GFP signal was at lower levels compared to controls and samples where β-catenin activity was increased (<xref ref-type="fig" rid="fig3">Figure 3A</xref>). We analysed the dynamics of <italic>Lgr5</italic> expression under different β-catenin activity levels using time-lapse imaging of <italic>Lgr5</italic><sup>+/EGFP-IRES-CreERT2</sup> kidneys. The number of <italic>Lgr5</italic> positive nephrons increased significantly at higher β-catenin activity levels (3.0×; p = 0.05) compared to controls or kidneys with decreased β-catenin activity (<xref ref-type="fig" rid="fig3">Figure 3B,C</xref>). However, in samples with decreased β-catenin activity, <italic>Lgr5</italic> positive nephrons emerged at an earlier time-point and these nephrons again appeared to be more elongated, but the GFP signal was lower compared to controls (<xref ref-type="fig" rid="fig3">Figure 3B</xref> and <xref ref-type="other" rid="video6">Video 6</xref>). Compared to controls, the actual number of <italic>Lgr5</italic> positive nephrons remained unchanged in IWR1 treated samples (<xref ref-type="fig" rid="fig3">Figure 3C</xref>). <italic>Lgr5</italic> is also an R-spondin receptor and mediates β-catenin signalling (<xref ref-type="bibr" rid="bib28">de Lau et al., 2011</xref>). To test whether <italic>Lgr5</italic> was functionally modulating the β-catenin signalling gradient, and thereby controlling nephron patterning, we intercrossed the <italic>Lgr5</italic><sup>+/EGFP-IRES-CreERT2</sup> knockin mice to homozygosity and analysed these for segmentation defects. Homozygous mutants displayed no obvious phenotype in the developing kidney (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1</xref> and <xref ref-type="other" rid="video7">Video 7</xref>).<fig-group><fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.04000.005</object-id><label>Figure 3.</label><caption><title>Pharmacological modulation of β-catenin signalling alters distal segment development.</title><p>(<bold>A</bold>) <italic>Lgr5-EGFP</italic> expression in treated nephrons with segmentation markers. (<bold>B</bold>) Time-lapse analysis of treated <italic>Lgr5</italic><sup><italic>+/</italic>EGFP-IRES-CreERT2</sup> kidneys–arrowheads indicate developing nephrons, red-dashed line indicates ureteric bud (UB). (<bold>C</bold>) Mean number of <italic>Lgr5-EGFP</italic> positive nephrons per kidney.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04000.005">http://dx.doi.org/10.7554/eLife.04000.005</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife04000f003"/></fig><fig id="fig3s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.04000.006</object-id><label>Figure 3—figure supplement 1.</label><caption><title>Lgr5 expression domain in heterozygous and homozygous Lgr5<sup>+/EGFP-IRES-CreERT2</sup> kidneys.</title><p>Cultured Lgr5<sup>+/EGFP-IRES-CreERT2</sup> kidneys stained for segmentation marker Jag1 and epithelial marker Cdh1. (<bold>A</bold> and <bold>C</bold>) Lgr5<sup>+/EGFP-IRES-CreERT2</sup> heterozygous kidneys and kidneys and (<bold>B</bold> and <bold>D</bold>) Lgr5 <sup>EGFP-IRES-CreERT2/EGFP-IRES-CreERT2</sup> homozygous kidneys and nephrons display normal segmentation. Homozygous tissue displays stronger EGFP expression as expected but nephrons appear morphologically normal.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04000.006">http://dx.doi.org/10.7554/eLife.04000.006</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife04000fs007"/></fig></fig-group><media id="video6" content-type="glencoe play-in-place height-250 width-310" xlink:href="elife04000v006.avi" mimetype="video" mime-subtype="avi"><object-id pub-id-type="doi">10.7554/eLife.04000.018</object-id><label>Video 6.</label><caption><title>Time-lapse capture of <italic>Lgr5</italic><sup><italic>+/EGFP-IRES-CreERT2</italic></sup> kidneys.</title><p>Kidneys cultured in control conditions or treated with IWR1 or CHIR. Timing, conditions, and scale as specified.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04000.018">http://dx.doi.org/10.7554/eLife.04000.018</ext-link></p></caption></media><media id="video7" content-type="glencoe play-in-place height-250 width-310" xlink:href="elife04000v007.avi" mimetype="video" mime-subtype="avi"><object-id pub-id-type="doi">10.7554/eLife.04000.019</object-id><label>Video 7.</label><caption><title>Time-lapse capture of <italic>Lgr5</italic><sup><italic>+/EGFP-IRES-CreERT2</italic></sup> and <italic>Lgr5</italic> <sup><italic>EGFP-IRES-CreERT2/EGFP-IRES-CreERT2</italic></sup> kidneys.</title><p>Kidneys cultured in control conditions over 6 days. Timing and scale as specified.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04000.019">http://dx.doi.org/10.7554/eLife.04000.019</ext-link></p></caption></media></p><p>Collectively, these data confirm that the cell populations along the proximal–distal axis of the nephrons are responsive to changes in β-catenin signalling and respond positively and negatively as would be predicted from the proposed β-catenin activity gradient.</p></sec><sec id="s2-3"><title>Modulating β-catenin activity shifts positional identities along the nephron</title><p>Having shown that the proximal and distal cell populations are both affected by increased β-catenin activity and the proximal is affected by decreased β-catenin activity, we hypothesized that the different levels of β-catenin signalling within the gradient specify the positions of identities in the nephron. If so, modulating β-catenin signalling might impose abnormal distal or proximal identities along the axis of the developing nephron (<xref ref-type="fig" rid="fig4">Figure 4A</xref>). We immunostained inhibitor treated nephrons with markers for proximal, medial, and distal identities (Wt1, Jag1, Cdh1) and measured the size of the domains in which each was expressed (<xref ref-type="fig" rid="fig4">Figure 4B</xref>). Increasing β-catenin did mildly increase total nephron lengths (1.17×, p = 0.017) compared to controls (<xref ref-type="fig" rid="fig4">Figure 4C</xref>), but the distal segment was significantly longer (1.8×, p = 0.0003) and the proximal segment was severely reduced (0.2×, p = 1.5 × 10<sup>−6</sup>). The medial segment showed no significant change in length (1.3×, p = 0.067). These data confirm that both the distal and proximal nephron segments respond to increased β-catenin signalling according to our hypothesis, but the medial remained unchanged in size. Decreasing β-catenin signalling, on the other hand, resulted in much elongated nephrons (<xref ref-type="fig" rid="fig4">Figure 4B,C</xref>: 1.7×, p = 1.8 × 10<sup>−14</sup>), as we had previously noticed (<xref ref-type="fig" rid="fig3">Figure 3A</xref>). Here, the length of the proximal segment was unchanged compared to controls (1.0×, p = 0.93), but as we showed above, this segment was more mature in appearance and gene expression (<xref ref-type="fig" rid="fig2">Figure 2</xref>). The elongation of the nephron was primarily due to the increases in both the distal (2.5×, p = 0.0002) and the medial segments (1.7×, p = 0.0007) and the nephrons appeared thinner. Although nephron identities responded to increased β-catenin as our model predicted, the large morphological changes obscured any subtle changes in segmentation in those nephrons with decreased β-catenin activity. To address this, we tested whether a gradual increase in β-catenin activity, which only mildly shifted activity levels away from the normal, would give a gradual decrease in proximal identity as would be expected if identities are β-catenin dosage-dependent. This was observed in nephrons that we exposed to different incremental concentrations of CHIR (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1</xref>). qRT-PCR analysis of RNA from whole treated kidneys confirmed that a mild increase in β-catenin activity promoted expression of most distal segment genes (<italic>Wnt4</italic>, <italic>Pax8</italic>, <italic>Fgf8</italic>, <italic>Lhx1, Lgr5</italic>) (<xref ref-type="fig" rid="fig4">Figure 4D</xref>) thus mirroring the effect of decreasing β-catenin activity; surprisingly, <italic>Pax2</italic> did not respond similarly to <italic>Pax8</italic>. Medial segment control genes (<italic>Jag1, Dll1, Heyl,</italic> and <italic>Irx2</italic>) did not increase in response to CHIR as distal genes did. At the highest β-catenin activity levels used [6 µM], nephron formation was reduced (<xref ref-type="fig" rid="fig2s1 fig2s2">Figure 2—figure supplements 1–2</xref>) and the expression profile thus dropped as would be expected (<xref ref-type="fig" rid="fig4">Figure 4D</xref>). To test if the observed phenotypes were due to a direct effect on the nephrogenic lineage or an indirect effect via the ureteric bud, we isolated metanephric mesenchyme away from the ureteric bud, induced it to form nephrons with spinal cord (<xref ref-type="bibr" rid="bib41">Grobstein, 1953</xref>, <xref ref-type="bibr" rid="bib40">1955</xref>), and modulated β-catenin activity therein. Comparable to the phenotype observed in whole kidney rudiments, we found that inhibition of β-catenin activity favoured patterning towards the proximal fate, whereas increasing its activity had the opposite effect (<xref ref-type="fig" rid="fig4">Figure 4E</xref>).<fig-group><fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.04000.020</object-id><label>Figure 4.</label><caption><title>Shifts in positional identity by altered β-catenin activity.</title><p>(<bold>A</bold>) Model of predicted changes in segmentation if the gradient of β-catenin activity specifies positional identities in the nephron. Nephrons depicted as spheres representing renal vesicle stage. Dashed line indicates nephron segments. Gradient bar indicates β-catenin activity. (<bold>B</bold>) Antibody stains against segment specific markers in nephrons with different β-catenin signalling conditions. (<bold>C</bold>) Proximal, medial, and distal nephron domain-sizes in Control, CHIR, and IWR1 treated kidneys. Mean values and SEMs indicated within bars on graph. (<bold>D</bold>) qRT-PCR analysis of markers for nephron induction displayed as a heat-map with information displayed in figure. The RNA was isolated after 48 hr of culture from whole kidneys. (<bold>E</bold>) Antibody stains on nephrons developed in isolated mesenchyme. (<bold>F</bold> and <bold>G</bold>) <italic>Apc</italic><sup>+/1638N</sup> <italic>Ctnnb1</italic><sup>Y654/E654</sup> kidneys where <italic>Ctnnb1</italic><sup>Y654</sup> is the wild-type allele. Kidneys characterised using anti-Wt1, Jag1, Podxl, Lam, and Cdh1. Arrowheads and boxed area indicate ectopic nephrons in <bold>E</bold>–<bold>F</bold>. Dashed lines separate metanephric and mesonephric regions in <bold>E</bold>–<bold>F</bold>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04000.020">http://dx.doi.org/10.7554/eLife.04000.020</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife04000f004"/></fig><fig id="fig4s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.04000.021</object-id><label>Figure 4—figure supplement 1.</label><caption><title>Gradual shifts in positional identity by gentle changes in β-catenin activity.</title><p>(<bold>A</bold>) Typical nephrons for their conditions as cultured at incremental CHIR dosages (0 µM, 0.50 µM, 0.75 µM, 1 µM, 1.25 µM, and 1.5 µM). Dashed lines indicate the axis and lengths of nephron segments. Kidneys stained for Wt1, Jag1, and Cdh1. UB–ureteric bud.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04000.021">http://dx.doi.org/10.7554/eLife.04000.021</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife04000fs008"/></fig><fig id="fig4s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.04000.022</object-id><label>Figure 4—figure supplement 2.</label><caption><title>β-catenin activity dosage-dependent phenotypes in series of <italic>Apc</italic> and <italic>Ctnnb1</italic> models.</title><p>Kidneys characterised using anti-Wt1, Jag1, Podxl, Lam, and Cdh1. (<bold>A</bold>) <italic>Ctnnb1</italic><sup>Y654/E654</sup> and <italic>Ctnnb1</italic><sup>E654/E654</sup>, (<bold>B</bold>) <italic>Apc</italic><sup>+/1572T</sup> <italic>Ctnnb1</italic><sup>Y654/Y654</sup> and <italic>Apc</italic> <sup>+/1572T</sup> <italic>Ctnnb1</italic><sup>Y654/E654</sup>. <italic>Ctnnb1</italic><sup>Y654</sup> is the wild-type allele. Dashed lines separate metanephric and mesonephric regions.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04000.022">http://dx.doi.org/10.7554/eLife.04000.022</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife04000fs009"/></fig></fig-group></p><p>Previous genetic approaches to investigate the role of β-catenin during nephron formation have been hampered by the problem that when β-catenin is maximally activated in nephron progenitors MET is concurrently blocked (<xref ref-type="bibr" rid="bib53">Kuure et al., 2007</xref>; <xref ref-type="bibr" rid="bib64">Park et al., 2007</xref>). Our data, however, suggest that MET is only incompatible with very high levels of β-catenin activity (<xref ref-type="fig" rid="fig4">Figure 4D</xref> and <xref ref-type="fig" rid="fig2s1 fig2s2">Figure 2—figure supplements 1–2</xref>), therefore identifying a dose–response correlation between β-catenin activity and renal MET. To confirm this, and to test if nephron segmentation defects could also be generated by genetic means, we made use of <italic>Apc</italic> and <italic>Ctnnb1</italic> (the gene encoding β-catenin) mutants that only result in mild increases in β-catenin activity. The <italic>Ctnnb1</italic><sup>E654</sup> model (<xref ref-type="bibr" rid="bib74">van Veelen et al., 2011</xref>) expresses a Y654E mutation in the endogenous β-catenin gene (<italic>Ctnnb1</italic>) leading to an increased propensity to signal, whereas the <italic>Apc</italic><sup>1572T</sup> and <italic>Apc</italic><sup>1638N</sup> models (<xref ref-type="bibr" rid="bib34">Fodde et al., 1994</xref>; <xref ref-type="bibr" rid="bib37">Gaspar et al., 2009</xref>) carry endogenous <italic>Apc</italic>-truncations associated with low or moderate activation of β-catenin signalling, respectively. Such hypomorphic alleles by themselves or in combinations can provide a series of increasing β-catenin activity levels (<xref ref-type="bibr" rid="bib49">Kielman et al., 2002</xref>; <xref ref-type="bibr" rid="bib14">Buchert et al., 2010</xref>). We generated different combinations of these alleles that display increasing levels of β-catenin activity in order of increasing β-catenin activity: <italic>Ctnnb1</italic><sup>+/E654</sup>, <italic>Ctnnb1</italic><sup>E654/E654</sup>, <italic>Apc</italic><sup>+/1572T</sup> <italic>Ctnnb1</italic><sup>+/E654</sup>, and <italic>Apc</italic><sup>+/1638N</sup> <italic>Ctnnb1</italic><sup>+/E654</sup> (<xref ref-type="bibr" rid="bib37">Gaspar et al., 2009</xref>; <xref ref-type="bibr" rid="bib74">van Veelen et al., 2011</xref>). We cultured E12.5 kidney rudiments for 5 days and stained them for different compartments of the kidneys with antibodies against Wt1, Jag1, Podxl, Lam, and Cdh1. <italic>Ctnnb1</italic><sup>+/E654</sup> and <italic>Ctnnb1</italic><sup>E654/E654</sup> kidneys (<xref ref-type="fig" rid="fig4s2">Figure 4—figure supplement 2</xref>) were indistinguishable from wild-type (<italic>Ctnnb1</italic><sup>+/+</sup>) kidneys (data not shown). Kidneys from <italic>Apc</italic><sup>+/1572T</sup> <italic>Ctnnb1</italic><sup>+/+</sup> and <italic>Apc</italic><sup>+/1572</sup> <italic>Ctnnb1</italic><sup>+/E654</sup> embryos had normal nephrons (<xref ref-type="fig" rid="fig4s2">Figure 4—figure supplement 2</xref>), though some kidneys developed supernumerary ureteric buds (data not shown). The mutants with the highest level of β-catenin activity, <italic>Apc</italic><sup>+/1638N</sup> <italic>Ctnnb1</italic><sup>+/E654</sup> displayed the most severe phenotype. The kidneys from such embryos showed effects ranging from those with severely reduced and morphologically abnormal branching and growth (<xref ref-type="fig" rid="fig4">Figure 4F,G</xref>) to those with more normal ureteric bud trees. Significantly, a small number of nephrons formed ectopically away from the ureteric bud in several kidneys. The ectopic nephrons were found to be positive for Jag1, Cdh1, and laminin but lacked podocytes, as shown by the absence of Wt1 and Podxl signal (<xref ref-type="fig" rid="fig4">Figure 4F,G</xref> right panels, respectively), in accordance with our pharmacological data presented above. A few endogenous nephrons within the ureteric bud tree managed to produce proximal domains (<xref ref-type="fig" rid="fig4">Figure 4F,G</xref>). The ectopic nephrons lacking proximal domains together with the absence of β-catenin signalling in this end of the nephrons (<xref ref-type="fig" rid="fig1">Figure 1</xref>) and our inhibitor experiments, strongly suggest that increased β-catenin activity disrupted normal patterning of the developing nephron and blocked the development of the most proximal end.</p></sec><sec id="s2-4"><title>Modulating β-catenin activity shifts positional identities along the nephron without altering proliferation or apoptosis</title><p>The canonical Wnt signalling pathway is known to have a direct effect on cell proliferation (<xref ref-type="bibr" rid="bib24">Davidson and Niehrs, 2010</xref>). We tested if this was part of the mechanism via which β-catenin controls nephron patterning. Neither increasing nor decreasing β-catenin signalling levels changed the number of mitotic nuclei per nephron (<xref ref-type="fig" rid="fig5">Figure 5A,B</xref>) nor did blocking cell proliferation with methotrexate (MTX; <xref ref-type="bibr" rid="bib16">Chabner and Young, 1973</xref>) affect the patterning of the nephron. Nephrons that formed in MTX were much smaller compared to control nephrons but expressed Cdh1, Wt1, and Jag1 correctly (<xref ref-type="fig" rid="fig5">Figure 5C</xref>), and the ratio of the size of the medial and proximal segment was unchanged (<xref ref-type="fig" rid="fig5">Figure 5D–F</xref>). Moreover, nephrons treated simultaneously with MTX and either CHIR or IWR produced nephrons with the same segmentation changes as caused by IWR1 or CHIR on their own (<xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1A</xref>), in spite of the nephrons now being smaller as a result of the MTX treatment. Finally, MTX treatment did not affect the existence of the β-catenin activity gradient as observed with the <italic>TCF/Lef::H2B-GFP</italic> reporter. Moreover, co-inhibition with MTX and either IWR1 or CHIR still decreased and increased the reporter signal, respectively (<xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1B–D</xref>).<fig-group><fig id="fig5" position="float"><object-id pub-id-type="doi">10.7554/eLife.04000.023</object-id><label>Figure 5.</label><caption><title>Modulating β-catenin activity shifts positional identities along the nephron without altering proliferation.</title><p>(<bold>A</bold>) Proliferation in <italic>TCF/Lef::H2B-GFP</italic> expressing nephrons treated with CHIR and IWR1. Nephron axis–dashed white line. Phosphorylated Histone 3 used as a marker for mitotic cells. (<bold>B</bold>) Quantification of mitotic nuclei per nephron. (<bold>C</bold>) Effect of Methotrexate (MTX) on nephron development and patterning. Nephrons outlined with white dashed line. (<bold>D</bold>–<bold>F</bold>) Measurement of Jag1<sup>+</sup> and Wt1<sup>+</sup> segment sizes of nephrons in control, 250 nM Methotrexate (MTX), and 500 nM MTX conditions. All error bars indicate SEM. p-values generated using Student's <italic>t</italic> test. Scale bars and antibodies as indicated on fields. UB–ureteric bud.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04000.023">http://dx.doi.org/10.7554/eLife.04000.023</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife04000f005"/></fig><fig id="fig5s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.04000.024</object-id><label>Figure 5—figure supplement 1.</label><caption><title>Modulating β-catenin activity shifts positional identities along the nephron regardless of proliferation levels.</title><p>(<bold>A</bold>) CHIR and IWR1 alter segmentation similarly with or without MTX. Dashed-white line indicates nephron axis. (<bold>B</bold>–<bold>C</bold>) Inhibiting proliferation does not block the formation of a GFP-gradient in <italic>TCF/Lef:H2B-GFP</italic> expressing nephrons. Nephrons outlined with white dashed line. Segment labelled on images. Red dots indicate examples of nuclei that were quantified for graph (<bold>C</bold>). Error bars indicate SEM. p-values were calculated using t-tests. (<bold>D</bold>) Signal intensity of <italic>TCF/Lef:H2B-GFP</italic> β-catenin reporter is decreased and increased with IWR1 and CHIR also, when MTX is added. Nephrons outlined with white dashed line. Scale bars and antibodies as indicated on fields. UB–ureteric bud.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04000.024">http://dx.doi.org/10.7554/eLife.04000.024</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife04000fs010"/></fig><fig id="fig5s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.04000.025</object-id><label>Figure 5—figure supplement 2.</label><caption><title>Modulating β-catenin activity shifts positional identities along the nephron regardless of apoptosis levels.</title><p>(<bold>A</bold>) Treated kidneys stained with TUNEL-assay to detect apoptotic cells. (<bold>B</bold>) Treated kidneys (live) stained with Annexin V assay to detect apoptotic cells. 6-CF marks proximal tubules and PT and as specified. Scale bars and antibodies as indicated on fields. UB–ureteric bud, N–Nephron.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04000.025">http://dx.doi.org/10.7554/eLife.04000.025</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife04000fs011"/></fig></fig-group></p><p>Treatment with neither IWR1 nor CHIR altered apoptosis in the nephrons (<xref ref-type="fig" rid="fig5s2">Figure 5—figure supplement 2</xref>). Only very low levels of apoptosis were detected, primarily at the periphery of the kidneys, in line with previous data (<xref ref-type="bibr" rid="bib35">Foley and Bard, 2002</xref>). CHIR treated kidneys did display some apoptotic nuclei, but these were located in the mesenchymal progenitor population surrounding ureteric bud tips. Collectively, these data indicate that changes in proliferation and apoptosis are not the driving force behind the β-catenin signalling gradient or the patterning of the nephron.</p></sec><sec id="s2-5"><title>β-catenin activity is negatively modulated in the nephron by BMP signalling which positively regulates medial segment development</title><p>In the intestine BMP negatively regulates β-catenin signalling by inhibiting the PTEN/PI3K/AKT pathway to maintain high levels of active GSK3β (<xref ref-type="bibr" rid="bib44">He et al., 2004</xref>). BMP2/4/7 are expressed in the nephron (<xref ref-type="bibr" rid="bib61">Oxburgh et al., 2011</xref>) and the BMP/SMAD pathway is active (<xref ref-type="bibr" rid="bib8">Blank et al., 2008</xref>; <xref ref-type="bibr" rid="bib13">Brown et al., 2013</xref>); albeit with an unknown function. We confirmed BMP pathway activity in the medial segment of the nephron using pSMAD1/5/8 reporter <italic>BRE-LacZ</italic> (<xref ref-type="bibr" rid="bib8">Blank et al., 2008</xref>) (<xref ref-type="fig" rid="fig6">Figure 6A</xref>). This closely correlated with the Jag1<sup>+</sup> domain (<xref ref-type="fig" rid="fig6">Figure 6A</xref>). BMP activity was further confirmed using antibodies specific for pSMAD1/5/8 (<xref ref-type="fig" rid="fig6">Figure 6B</xref>).<fig-group><fig id="fig6" position="float"><object-id pub-id-type="doi">10.7554/eLife.04000.026</object-id><label>Figure 6.</label><caption><title>The β-catenin activity gradient is modified by changes to BMP and PI3K signalling.</title><p>(<bold>A</bold>) BRE-LacZ pSMAD reporter shows strong labelling in medial segment; co-stained for Wt1, Jag1, Cdh1. (<bold>B</bold>) pSMAD1/5/8 specific antibody stain. Lines and labelling in <bold>A</bold>–<bold>B</bold> indicate different segments. (<bold>C</bold>) pPTEN and pAKT levels in the nephron after inhibition of BMPR with 4 µM LDN-193189 or PI3K with 20 µM Ly294002. White arrowheads and arrows indicate staining in ureteric bud and nephrons, respectively. (<bold>D</bold>) Time-lapse of single <italic>TCF/Lef::H2B-GFP</italic> positive nephrons developing from induction stage through S-shaped body stage. Nephron axis in red. Nephrons treated as specified. CM -cap mesenchyme, UB–ureteric bud. (<bold>E</bold>) Quantification of <italic>TCF/Lef::H2B-GFP</italic> intensities at different positions along the proximal–distal axis at S-shaped body stage/22 hr. Multiple nephrons used as indicated on graph. Error bars indicate SEM. Average lengths of nephrons at 22 hr for each condition are indicated on the graph. (<bold>F</bold>) Inhibition of BMPR or PI3K alters the medial segment negatively and positively, respectively. (<bold>G</bold>) qRT-PCR data of segment-specific markers and β-catenin target genes displayed as a heat-map with gene information displayed in figure. (<bold>H</bold>) p-β-catenin Ser552 localisation in S-shaped nephron. Antibody stains and scale bars as indicated.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04000.026">http://dx.doi.org/10.7554/eLife.04000.026</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife04000f006"/></fig><fig id="fig6s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.04000.027</object-id><label>Figure 6—figure supplement 1.</label><caption><title>Rescue/reversals experiments for kidneys treated with 20 µM Ly294002 or 4 µM LDN193189.</title><p>Kidneys were cultured for 96 hr in the inhibitors or for 48 hr in inhibitor followed by another 48 hr in control medium. Kidneys were stained for Wt1, Jag1, and Cdh1 to display nephron formation and overall morphology.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04000.027">http://dx.doi.org/10.7554/eLife.04000.027</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife04000fs012"/></fig><fig id="fig6s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.04000.028</object-id><label>Figure 6—figure supplement 2.</label><caption><title>Changes to BMP and PI3K signalling alters nephron segmentation and β-catenin signalling.</title><p>(<bold>A</bold>) Data from time-lapse captured <italic>TCF/Lef:H2B-GFP</italic> nephrons treated with LDN-193189 or Ly294002 or as controls. The percentage frequency is plotted against pixel intensity values. The GFP intensity was measured for all pixels throughout the nephron at 22 hr of culture. Ly294002 treated nephrons had fewer bright nuclei whilst LDN-193189 and control nephrons were not distinguishable at this stage with this method. Error bars indicate SEM. (<bold>B</bold>) Whole <italic>TCF/Lef:H2B-GFP</italic> kidneys treated with LDN-193189, Ly294002, CHIR, and combinations thereof. Yellow and red dashed circles indicate ureteric bud tree-bound endogenous and ectopic nephrons, respectively. (<bold>C</bold>) Inhibition of BMPR, PI3K, and activation of β-catenin predictably alters the medial and proximal segments. Dashed line indicates separation between ectopic and endogenous ureteric bud tree-bound nephron structures.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04000.028">http://dx.doi.org/10.7554/eLife.04000.028</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife04000fs013"/></fig></fig-group></p><p>To test if BMP signalling could inhibit β-catenin signalling via the PTEN pathway as it does in the intestine (<xref ref-type="bibr" rid="bib44">He et al., 2004</xref>) we first investigated if BMP can signal via PTEN in the kidney. To do this we blocked BMP signalling at the BMP receptor (BMPR) level with inhibitor LDN-193189 (<xref ref-type="bibr" rid="bib9">Boergermann et al., 2010</xref>) and PI3K with inhibitor Ly294002 (<xref ref-type="bibr" rid="bib75">Vlahos et al., 1994</xref>; <xref ref-type="bibr" rid="bib39">Gharbi et al., 2007</xref>). Inhibiting PI3K is equivalent to further activating any existing BMP/PTEN signalling (<xref ref-type="bibr" rid="bib44">He et al., 2004</xref>). If BMP signals via PTEN, the expected outcome of BMPR inhibition would be a rise in the levels of phosphorylated PTEN (inactive) and phosphorylated AKT (active) (<xref ref-type="bibr" rid="bib44">He et al., 2004</xref>), which was indeed found in nephrons and the ureteric bud (<xref ref-type="fig" rid="fig6">Figure 6C</xref>). We tested whether the inhibitors had reversible effects by releasing the kidneys from inhibition after 48 hr of treatment. A moderate recovery towards the control phenotype was observed after removal of the inhibitors (<xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1</xref>). To test if BMP and PI3K signalling in the nephron affects β-catenin activity, we treated <italic>TCF/Lef::H2B-GFP</italic> kidneys with both inhibitors. If the system operates as in the intestine, BMPR inhibition should prevent BMP/PTEN signalling from antagonising β-catenin activity thus resulting in increased β-catenin reporter activity (<xref ref-type="bibr" rid="bib44">He et al., 2004</xref>). Inhibition of PI3K should do the opposite. The total β-catenin reporter activity as measured throughout nephrons, 22 hr after nephron formation, was indistinguishable between controls and BMPR inhibited nephrons, but PI3K inhibition reduced β-catenin reporter activity levels as expected (<xref ref-type="fig" rid="fig6s2">Figure 6—figure supplement 2A</xref>). The time-lapse data did however visually show higher and lower <italic>TCF/Lef::H2B-GFP</italic> activity in the BMPR and PI3K inhibited nephrons, respectively, as we had anticipated (<xref ref-type="other" rid="video8 video9">Videos 8, 9</xref> top part). Thus, we monitored the β-catenin reporter of each nephron over the whole 22 hr time-span and measured the activity along the whole distal-to-proximal axis (<xref ref-type="fig" rid="fig6">Figure 6D,E</xref>). Nephrons were randomly picked during the whole time-span of the kidney being monitored (96 hr) and the averages were determined for each treatment. This showed that the high to medium β-catenin reporter activity extended ectopically into the middle portion of the nephron when BMPR was inhibited and low levels were detected when PI3K was blocked (<xref ref-type="fig" rid="fig6">Figure 6D,E</xref> and <xref ref-type="other" rid="video8">Video 8</xref>, top part).<media id="video8" content-type="glencoe play-in-place height-250 width-310" xlink:href="elife04000v008.avi" mimetype="video" mime-subtype="avi"><object-id pub-id-type="doi">10.7554/eLife.04000.029</object-id><label>Video 8.</label><caption><title>Time-lapse capture <italic>TCF/Lef::H2B-GFP</italic> kidneys.</title><p>Kidneys cultured in control medium, 4 µM LDN-193189, 20 µM Ly294003, 1.5 µM CHIR, 4 µM LDN-193189, and 1.5 µM CHIR, or 20 µM Ly294003 and 1.5 µM CHIR. Timing and scales are as specified.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04000.029">http://dx.doi.org/10.7554/eLife.04000.029</ext-link></p></caption></media><media id="video9" content-type="glencoe play-in-place height-250 width-310" xlink:href="elife04000v009.avi" mimetype="video" mime-subtype="avi"><object-id pub-id-type="doi">10.7554/eLife.04000.030</object-id><label>Video 9.</label><caption><title>Time-lapse capture <italic>TCF/Lef::H2B-GFP</italic> kidneys.</title><p>Kidneys cultured in control medium, 4 µM LDN-193189, 20 µM Ly294003, 1.5 µM CHIR, 4 µM LDN-193189, and 1.5 µM CHIR, or 20 µM Ly294003 and 1.5 µM CHIR. Timing and scales are as specified.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04000.030">http://dx.doi.org/10.7554/eLife.04000.030</ext-link></p></caption></media></p><p>Moreover, when BMPR or PI3K were inhibited, nephrons elongated at different rates compared to controls; 11.2 µm/hr (BMPR), 10.6 µm/hr (PI3K), and 8.4 µm/hr (control) (data not shown).</p><p>Given the observed increase of the β-catenin reporter in response to blocking BMPR, by inhibiting BMPR and simultaneously activating β-catenin with CHIR, this should synergistically expand β-catenin signalling in the medial nephron segment where BMP activity was detected. The ectopic and tree-bound nephrons that formed in these dual-inhibitor conditions displayed very high β-catenin activity throughout the nephron structures (<xref ref-type="fig" rid="fig6s2">Figure 6—figure supplement 2B</xref> and <xref ref-type="other" rid="video8 video9">Videos 8, 9</xref>, lower part). Interestingly, co-inhibition of PI3K and activation of β-catenin also positively affected β-catenin reporter activity; however, the dynamics of this was clearly different from that caused by inhibition of BMPR and activation of β-catenin with CHIR (<xref ref-type="other" rid="video8 video9">Videos 8, 9</xref>, lower part). To test if segmentation was altered by inhibition of BMPR or PI3K, we stained kidneys for Wt1, Jag1, and Cdh1. Activating β-catenin and inhibiting BMPR at the same time completely removed the Wt1<sup>+</sup> cells, strongly reduced the Jag1<sup>+</sup> cells, and formed nephrons made of distal regions (<xref ref-type="fig" rid="fig6s2">Figure 6—figure supplement 2C</xref>). Activating β-catenin and inhibiting PI3K at the same time made nephrons form large medial/Jag1<sup>+</sup> segments with few WT1<sup>+</sup> proximal cells present (<xref ref-type="fig" rid="fig6s2">Figure 6—figure supplement 2C</xref>). These data suggest that PI3K inhibition positively promotes a medial fate. Inhibiting PI3K on its own strongly promoted the development of the Jag1<sup>+</sup> segment and inhibiting BMPR had a slight negative effect on the Jag1<sup>+</sup> segment as expected, the latter perhaps partially hidden by the overall increase in nephron length (<xref ref-type="fig" rid="fig6">Figure 6F</xref> and <xref ref-type="fig" rid="fig6s2">Figure 6—figure supplement 2C</xref>). qRT-PCR analyses on segment-specific genes confirmed that inhibiting PI3K had a positive effect on the medial domain; medial markers <italic>Dll1</italic>, <italic>Jag1</italic>, <italic>Irx2</italic>, and <italic>Fgf8</italic> increased, as did distal marker <italic>Lhx1</italic> (<xref ref-type="fig" rid="fig6">Figure 6G</xref>). Inhibition of BMPR positively affected distal markers <italic>Bmp2</italic> and <italic>Lgr5</italic> expression and also some medial markers <italic>Heyl</italic> and <italic>Irx2</italic> (<xref ref-type="fig" rid="fig6">Figure 6G</xref>). <italic>Pax2</italic> and <italic>Wnt4</italic> were both down-regulated. Co-inhibiting BMPR and PI3K resulted in nephrons highly similar to those in just PI3K-inhibitor conditions confirming that PI3K is downstream of BMPR and that PI3K can positively affect the medial segment independently of BMPR/SMAD (data not shown).</p><p>Since both β-catenin signalling and nephron patterning was modulated via the PI3K pathway, we tested whether AKT positively regulates β-catenin signalling via phosphorylation of β-catenin at Ser552, as demonstrated elsewhere (<xref ref-type="bibr" rid="bib31">Fang et al., 2007</xref>). β-catenin phosphorylated at Ser552 was localised primarily to the apical surfaces of the nephrons in the distal and medial segments (<xref ref-type="fig" rid="fig6">Figure 6H</xref>). In a subset of cells, the phosphorylated Ser552 β-catenin was detected at lateral and basal surfaces. 80% of such cells were found in the distal and medial segments (45/56 positive cells counted in five nephrons) and the remaining 20% in proximal cells. Unlike phosphorylated PTEN and phosphorylated AKT, which responded to PI3K and BMPR inhibition, phosphorylated Ser552 β-catenin did not change on inhibition of either (data not shown).</p><p>These data indicate that the positional identities in the nephron can be modified through BMP mediated PI3K/AKT signalling and that simulated increased BMP signalling (via inhibition of PI3K) leads to reduced β-catenin signalling, whereas simulated decreased BMP signalling (via inhibition of BMPR) results in the opposite effect.</p></sec><sec id="s2-6"><title>Low β-catenin and PI3K activity partially rescue defective Notch-signalling</title><p>To date, the only other major pathway known to control nephron patterning is the Notch-signalling pathway (<xref ref-type="bibr" rid="bib19">Cheng et al., 2007</xref>). Notch-signalling specifies the proximal and medial identity, but it can also block the adjacent glomerular identity when over-activated in these cells (<xref ref-type="bibr" rid="bib19">Cheng et al., 2007</xref>; <xref ref-type="bibr" rid="bib11">Boyle et al., 2011</xref>). In many tissues, Notch and Wnt pathways act both in synergy and in opposition (<xref ref-type="bibr" rid="bib3">Andersson et al., 2011</xref>), and Notch has also been reported to antagonise the PTEN/PI3K/AKT pathway in clear-cell renal-cell carcinoma (<xref ref-type="bibr" rid="bib56">Liu et al., 2013</xref>). Our data already pointed to a negative correlation between PI3K activity and the expression of Notch ligands <italic>Dll1</italic> and <italic>Jag1</italic>. Furthermore, we showed activity of the BMP pathway in the segment affected when Notch is absent. Because we have shown that decreased β-catenin activity enhances the formation of the glomerular segment and PI3K inhibition promotes the medial segment, we tested if inhibition of β-catenin or PI3K could rescue these respective identities when Notch activity is lost. We first inhibited Notch using the γ-secretase DAPT as previously published (<xref ref-type="bibr" rid="bib20">Cheng et al., 2003</xref>). This resulted in a loss of proximal and medial structures and glomerular precursors (positive for Podxl and <italic>Lotus tetragonolobus</italic> lectin (LTL), <xref ref-type="fig" rid="fig7">Figure 7A</xref>) and these effects mimic those phenotypes seen in <italic>Notch2</italic><sup><italic>−/−</italic></sup> animals (<xref ref-type="bibr" rid="bib19">Cheng et al., 2007</xref>). Removal of the inhibitor led to a partial recovery of proximal segments (<xref ref-type="fig" rid="fig7s1">Figure 7—figure supplement 1</xref>). Co-inhibition of β-catenin and Notch-signalling resulted in the reappearance of nephrons positive for LTL and Podxl that displayed glomerular structures and medial and proximal development (<xref ref-type="fig" rid="fig7">Figure 7A</xref>). The Wt1<sup>+/GFP</sup> reporter kidneys in time-lapse (<xref ref-type="fig" rid="fig7">Figure 7B</xref>) and antibody stains that identify these segments (<xref ref-type="fig" rid="fig7">Figure 7C</xref>) confirmed that by decreasing β-catenin activity in Notch-inhibited kidneys we partially rescued the absence of normal Notch-signalling. The inhibition of β-catenin did not restore the expression of key Notch target genes (<italic>Jag1</italic>, <italic>Dll1</italic>, <italic>Heyl</italic>, <italic>Hey1</italic>) (<xref ref-type="fig" rid="fig7">Figure 7D</xref>). Co-inhibition of PI3K and Notch also resulted in a partial rescue (<xref ref-type="fig" rid="fig7s2">Figure 7—figure supplement 2A</xref> and <xref ref-type="other" rid="video10 video11">Videos 10, 11</xref>). Wt1<sup>+</sup> cells were not evident but Jag1 was again expressed, indicating that PI3K exerts an effect specific to the medial segment (<xref ref-type="fig" rid="fig7s2">Figure 7—figure supplement 2A</xref>). We assessed how inhibition of Notch, with or without inhibition of β-catenin and PI3K signalling, affected β-catenin activity using the β-catenin reporter <italic>TCF/Lef::H2B-GFP.</italic> Inhibition of Notch resulted in stunted nephrons without Wt1<sup>+</sup> or Jag1<sup>+</sup> cells with medium to high level of β-catenin activity throughout (<xref ref-type="fig" rid="fig7s2">Figure 7—figure supplement 2B–C</xref>; <xref ref-type="other" rid="video11">Video 11</xref>). Co-inhibition of Notch and β-catenin signalling strongly decreased the β-catenin activity and Wt1<sup>+</sup> proximal cells were again present. Co-inhibition of Notch and PI3K increased the size of the nephrons and Jag1<sup>+</sup> cells were again present and the β-catenin reporter was slightly decreased. Triple inhibition of Notch, PI3K, and β-catenin signalling did not result in an improved rescue (data not shown). Together, these data show that proximal and medial nephron cells require Notch signalling and simultaneously need β-catenin and PI3K signalling to be kept at low levels, respectively.<fig-group><fig id="fig7" position="float"><object-id pub-id-type="doi">10.7554/eLife.04000.031</object-id><label>Figure 7.</label><caption><title>Altered β-catenin activity rescues the loss of Notch.</title><p>(<bold>A</bold>) Kidneys treated with DAPT and DAPT/IWR1 and stained for LTL, β-laminin, Cdh1, and Podxl–arrowheads indicate LTL-positive nephrons, inserts show magnified nephrons with Podxl staining for podocytes, yellow line outlines nephron tubules. (<bold>B</bold>) Time-lapse analysis of Wt1<sup>+/GFP</sup> kidneys treated with DAPT and DAPT + IWR1–arrowheads show GFP<sup>HIGH</sup> structures in developing proximal segments, red dashed line indicates ureteric bud positions (UB). (<bold>C</bold>) Structures positive for Jag1 and Wt1 in treated kidneys–arrowheads indicating double-positive structures. (<bold>D</bold>) qRT-PCR data for Notch target genes (<italic>Jag1</italic>, <italic>Dll1</italic>, <italic>Heyl</italic>, <italic>Hey1</italic>). All error bars indicate SEM.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04000.031">http://dx.doi.org/10.7554/eLife.04000.031</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife04000f007"/></fig><fig id="fig7s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.04000.032</object-id><label>Figure 7—figure supplement 1.</label><caption><title>Rescue/reversals experiments for kidneys treated with 2 µM DAPT.</title><p>Kidneys were cultured for 96 hr in DAPT or for 48 hr in DAPT followed by another 48 hr in control medium. Kidneys were stained for Wt1, Jag1, and Cdh1 to display nephron formation and overall morphology. Antibody stains indicated.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04000.032">http://dx.doi.org/10.7554/eLife.04000.032</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife04000fs014"/></fig><fig id="fig7s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.04000.033</object-id><label>Figure 7—figure supplement 2.</label><caption><title>Altered β-catenin activity or PI3K signalling rescues the loss of Notch.</title><p>Data from kidneys treated with 2 µM DAPT and in combination with 20 µM Ly294002 or 2 µM IWR1. (<bold>A</bold>–<bold>B</bold>) <italic>TCF/Lef:H2B-GFP</italic> nephrons stained for WT1, JAG1, and CDH1. WT1 and JAG1 stains are not optimally compatible with the PFA fixation required to preserve the GFP signal. Thus, wild-type kidneys were also treated and stained for WT1 JAG1 and CDH1—shown in (<bold>C</bold>). Jag1<sup>+</sup> structures indicated with arrowheads. Stains, scales, and treatments as specified.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04000.033">http://dx.doi.org/10.7554/eLife.04000.033</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife04000fs015"/></fig></fig-group><media id="video10" content-type="glencoe play-in-place height-250 width-310" xlink:href="elife04000v010.avi" mimetype="video" mime-subtype="avi"><object-id pub-id-type="doi">10.7554/eLife.04000.034</object-id><label>Video 10.</label><caption><title>Time-lapse capture of <italic>TCF/Lef::H2B-GFP</italic> kidneys.</title><p>Kidneys in this video were cultured in control medium or 20 µM Ly294003. Timing and scales are as specified.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04000.034">http://dx.doi.org/10.7554/eLife.04000.034</ext-link></p></caption></media><media id="video11" content-type="glencoe play-in-place height-250 width-310" xlink:href="elife04000v011.avi" mimetype="video" mime-subtype="avi"><object-id pub-id-type="doi">10.7554/eLife.04000.035</object-id><label>Video 11.</label><caption><title>Time-lapse capture of <italic>TCF/Lef::H2B-GFP</italic> kidneys.</title><p>Kidneys in this video were cultured in 2 µM DAPT or 20 µM Ly294003 with 2 µM DAPT. Timing and scales are as specified.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04000.035">http://dx.doi.org/10.7554/eLife.04000.035</ext-link></p></caption></media></p></sec></sec><sec sec-type="discussion" id="s3"><title>Discussion</title><sec id="s3-1"><title>An integrated model for β-catenin controlled nephron patterning</title><p>A major outstanding question in kidney development has been to understand how the nephrons are patterned. It has been shown that Wnt via canonical β-catenin signalling induces nephrons to form and through non-canonical pathways regulate the nephron mesenchymal-to-epithelial transition as well as subsequent tubule elongation (<xref ref-type="bibr" rid="bib27">Davies and Garrod, 1995</xref>; <xref ref-type="bibr" rid="bib53">Kuure et al., 2007</xref>; <xref ref-type="bibr" rid="bib64">Park et al., 2007</xref>; <xref ref-type="bibr" rid="bib46">Karner et al., 2009</xref>; <xref ref-type="bibr" rid="bib15">Burn et al., 2011</xref>; <xref ref-type="bibr" rid="bib73">Tanigawa et al., 2011</xref>). Our data now show that β-catenin activity is essential throughout nephron development by regulating the patterning of the nephron through interactions with the BMP, PTEN/PI3K, and Notch pathways (<xref ref-type="fig" rid="fig8">Figure 8</xref>).<fig id="fig8" position="float"><object-id pub-id-type="doi">10.7554/eLife.04000.036</object-id><label>Figure 8.</label><caption><title>Model for molecular pathways interacting to control the patterning of the nephron.</title><p>β-catenin activity is necessary to determine a distal cell identity but must be excluded from the proximal nephron. BMP/PTEN/PI3K antagonises β-catenin activity in the medial segment and positively promotes a medial fate and Notch ligand expression. Notch is essential for medial and proximal development. Glomerular progenitor cells strongly inhibit β-catenin function, possibly via a WT1-dependent mechanism as in Sertoli cells (<xref ref-type="bibr" rid="bib17">Chang et al., 2008</xref>).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04000.036">http://dx.doi.org/10.7554/eLife.04000.036</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife04000f008"/></fig></p><p>Our data combined suggest a model where a gradient of β-catenin activity specifies positional identities along the nephron axis. Additional signal transduction pathways modulate local β-catenin activity levels. Its action has a differential effect on nephron segments by controlling differentiation, maturation, and their size. While cell proliferation and apoptosis are likely to be essential in other aspects of nephrogenesis, our data exclude a direct role in the initial patterning mechanism. With the current data, it is difficult to identify key downstream targets and that way describe the next level of mechanism of this process, as many targets are also widely used as segmentation markers.</p></sec><sec id="s3-2"><title>Nephrons are patterned by a gradient of β-catenin activity</title><p>Using a variety of methods, our combined data strongly suggest the existence of a gradient in β-catenin activity along the proximal–distal axis of the developing nephron. First, the <italic>TCF/Lef::H2B-GFP</italic> activity reporter showed a clearly recognisable visual gradient which we confirmed by quantifying the reporter signal along the nephron axis. Reporter activity is high at the distal end of the nephron, where it connects to the ureteric bud, and gradually reduces in the proximal direction, with the developing glomerulus at the extreme proximal end being devoid of β-catenin activity (<xref ref-type="fig" rid="fig1">Figure 1A–C</xref>). Second, while antibody staining for pan β-catenin showed equal expression of the protein throughout the post-MET nephron, staining with phospho-specific antibodies (indicative of protein targeted for break-down) was greatly enhanced in the proximal end of nephrons, the same segment where the reporter is least active. This also indicates that the gradient is an activity gradient, not an expression gradient. Third, the pharmacological and genetic modulation of the β-catenin signal result in a phenotype that can be predicted from the reporter and antibody data. In isolation, none of these analyses would prove the existence of a β-catenin activity gradient. Reporters of the type used here can sometimes give unreliable data, however, the model we used was found to give a very reproducible activity patterns that overlaps with many other β-catenin activity reporters (<xref ref-type="bibr" rid="bib32">Ferrer-Vaquer et al., 2010</xref>). Phospho-specific antibodies are powerful tools to monitor activity of signal transduction pathways, but their use can be difficult to optimize, especially in the kidney organ culture system. Maybe surprisingly, our antibody data do not show nuclear β-catenin as would be expected for active signalling. It should be realized, however, that this is the same for the signal in the ureteric bud which is generally accepted to have active β-catenin signalling (<xref ref-type="bibr" rid="bib12">Bridgewater et al., 2008</xref>; <xref ref-type="bibr" rid="bib59">Marose et al., 2008</xref>). This could indicate technical limitations of the whole-mount staining, but it should also be noted that in many other developing systems it can be difficult to detect nuclear β-catenin. Even in colorectal cancer it was shown that in cells from the same tumours, all assumed to have the same initiating loss of <italic>APC</italic> or oncogenic activation of β-catenin, dynamic localisation changes between nucleus and cytoplasm can be found (<xref ref-type="bibr" rid="bib33">Fodde and Brabletz, 2007</xref>). Similarly, expression of a constitutively activated β-catenin mutant in mice throughout the intestine results in different responses in different parts of the intestine, including different localization (varying between nuclear, cytoplasmic, or membranous; <xref ref-type="bibr" rid="bib54">Leedham et al., 2013</xref>). Finally, a functional role for β-catenin in the patterning process as shown by our pharmacological and genetic data does not prove that this is dependent on an activity gradient. However, combined, these data strongly suggests that this gradient is real and functional in the patterning of the nephron.</p><p>The reporter signal showed an exponential decrease, suggesting first-order decay of a single source from the distal end. The proximal domain (Wt1<sup>+</sup>), which is normally characterized by low or absent β-catenin activity, does not form when β-catenin activity is increased, whereas it forms quicker when the activity of the pathway is decreased (<xref ref-type="fig" rid="fig2">Figure 2A–D</xref>). In the other domains of the developing nephron (Lgr5<sup>+</sup> and Jag1<sup>+</sup>), cells also respond to the level of β-catenin activity that is forced on them (<xref ref-type="fig" rid="fig3 fig4">Figure 3A,B, 4C,D</xref>). Increasing β-catenin activity had a positive effect on the distal domain (<xref ref-type="fig" rid="fig3 fig4">Figures 3B, 4C</xref>) as expected. Whilst the effect of decreasing β-catenin signalling was clear on the proximal segment it led to a more complex effect in the other segments. Lgr5 became expressed at earlier time-points, albeit at lower levels (both in terms of GFP fluorescence in the <italic>Lgr5</italic><sup>+/EGFP-IRES-CreERT2</sup> model and also mRNA levels analysed by qRT-PCR) compared to controls and when β-catenin signalling was increased (<xref ref-type="fig" rid="fig3">Figure 3B</xref>). The tubules also elongated excessively (<xref ref-type="fig" rid="fig4">Figure 4C</xref>). We speculate that the reason for this is that decreasing β-catenin signalling might have resulted in the whole of the nephron maturing faster and therefore elongating more, however, decreasing β-catenin signalling particularly favoured the proximal domain. Since proliferation was not affected by either increasing or decreasing β-catenin signalling, the changes in tubular morphology and length must have been caused by another mechanism. The tubules appeared thinner, suggesting cellular rearrangements as a likely cause.</p><p>In addition to using genetic models we also utilised pharmacological inhibitors to enable us to fine-tune the β-catenin activity dosage through titration of the concentration of the compounds (<xref ref-type="bibr" rid="bib26">Davies, 2009</xref>). This made it possible to inhibit as well as activate the pathway and, crucially, to achieve temporal control in the experimental system. Not only did this approach exclude the possibility that the observed phenotypes were due to cellular toxicity, but by washing away the β-catenin activator we showed that proximal nephron structures could recover and reform if the sustained β-catenin signalling, imposed on them by CHIR, was removed (<xref ref-type="fig" rid="fig2">Figure 2E</xref>). Genetic models for activation of β-catenin are available, but inhibiting the pathway is not possible with current mouse models. The series of different <italic>Apc</italic> and <italic>Ctnnb1</italic> mouse models that we employed have previously been used to show that β-catenin activity levels are selected for by different types of cancer, and these ectopic increases in signalling activity can also lead to a range of developmental defects (<xref ref-type="bibr" rid="bib49">Kielman et al., 2002</xref>; <xref ref-type="bibr" rid="bib37">Gaspar et al., 2009</xref>; <xref ref-type="bibr" rid="bib14">Buchert et al., 2010</xref>; <xref ref-type="bibr" rid="bib6">Bakker et al., 2013</xref>). Our data indicate that increasing levels of β-catenin activity through <italic>Apc</italic> and <italic>Ctnnb1</italic> mutations lead to successively more severe kidney phenotypes, but only at the highest activity–dose achieved in this study did we detect ectopic nephron development (<xref ref-type="fig" rid="fig4">Figure 4F,G</xref>). It has been known for a long time that LiCl, which is an inhibitor of GSK3β, can induce nephron formation but it also blocks MET at high concentrations (<xref ref-type="bibr" rid="bib27">Davies and Garrod, 1995</xref>). This has since been confirmed by other, more specific, GSK3β inhibitors as well as genetic activation of β-catenin (<xref ref-type="bibr" rid="bib53">Kuure et al., 2007</xref>; <xref ref-type="bibr" rid="bib64">Park et al., 2007</xref>). Therefore, the appearance of the epithelialized, though abnormal, nephrons in the <italic>Apc</italic><sup>+/1638N</sup> <italic>Ctnnb1</italic><sup>+/E654</sup> mutants and the ectopic nephrons in the inhibitor studies indicate that β-catenin activation has dosage-dependent effects on the developing kidney. The absence of proximal markers in these ectopic nephrons correlated with the lack of reporter activity as well as the phosphorylation state of β-catenin in that part of the nephron. It is important to note that some nephrons within the ureteric bud trees of <italic>Apc</italic><sup>+/1638N</sup> <italic>Ctnnb1</italic><sup>+/E654</sup> mutant animals still managed to produce proximal domains. Whether this indicates redundancy or an ability to overcome increased β-catenin levels through ureteric bud-derived signals remains to be determined. Using cultures of spinal cord-induced metanephric mesenchymes, we also show that the observed phenotypes are not an indirect effect of changes in the ureteric bud but are a direct effect in the mesenchyme.</p></sec><sec id="s3-3"><title>Nephron patterning and the β-catenin activity gradient are independent of proliferation</title><p>Cell proliferation could be a mechanism through which β-catenin controls the patterning of the nephron. However, we found that proliferation along the nephron axis does not change in response to stimulating or inhibiting β-catenin activity, nor does blocking cell proliferation affect the patterning (<xref ref-type="fig" rid="fig5">Figure 5C</xref>), suggesting that proliferation does not play a mechanistic role in this patterning process. Even in nephrons which were treated with MTX and which were significantly smaller compared to controls did quantification of the <italic>TCF/Lef::H2B-GFP</italic> activity reporter show that there was a gradient in β-catenin signalling as also seen in normal nephrons. Similarly, when nephrons were treated with MTX it was still possible to induce segmentation changes by adding either IWR1 or CHIR. This implies that the underlying mechanisms behind nephron segmentation can adequately adapt to reduced cell numbers and an overall reduction in nephron size. We also found that apoptosis in nephrons was not altered by either CHIR or IWR1. Together, these findings would therefore suggest that increasing and decreasing β-catenin signalling changes segmentation by forcing cells to change their identity and that this is the most likely mechanism underlying the alterations to segmentation.</p></sec><sec id="s3-4"><title>The β-catenin gradient is spatially limited by BMP/PTEN/PI3K signalling</title><p>We also investigated how β-catenin signalling interacts with other pathways in the nephron and how the β-catenin activity gradient is limited from expanding proximally (<xref ref-type="fig" rid="fig6">Figure 6</xref>). Our data indicate that, similar to the case in intestinal stem cells (<xref ref-type="bibr" rid="bib44">He et al., 2004</xref>), β-catenin activity is negatively regulated by BMP/PTEN/PI3K signalling in the medial domain of the nephron. This domain is normally positive for a BMP/pSMAD reporter and pSMAD staining. This link between BMP and β-catenin is likely to reflect a normal function of BMP in this nephron segment since inhibition of BMPR and PI3K provoked opposite effects on the medial segment and BMPR inhibition increased levels of both phosphorylated PTEN and AKT. Furthermore, inhibition of PI3K on its own made the medial segment visually grow much larger and Jag1 and other medial markers were expressed more strongly. The significance of PI3K signalling in connection with Wnt/β-catenin pathway is strongly debated (<xref ref-type="bibr" rid="bib60">Ng et al., 2009</xref>), but the PI3K pathway has not only been shown to alter Wnt signalling in the gut but also in embryonic stem cells (<xref ref-type="bibr" rid="bib62">Paling et al., 2004</xref>). We tested whether β-catenin was phosphorylated at Ser552 as it has been reported to be by AKT (<xref ref-type="bibr" rid="bib31">Fang et al., 2007</xref>). Based on our data, we expect low levels of PI3K/AKT signalling in the medial segment, and consequently, we would therefore anticipate that β-catenin would only be phosphorylated at Ser552 at very low levels if at all. We did detect phosphorylated β-catenin at the apical surfaces and lateral surfaces mainly within the distal and medial domain but never within the nucleus (<xref ref-type="fig" rid="fig6">Figure 6H</xref>). A number of cells displayed stronger β-catenin staining which also extended to the lateral and basal surfaces. We currently do not know the significance of this and the staining pattern did not change in response to either BMPR inhibition of PI3K inhibition. It will be important to explore precisely how PI3K and BMP/pSMAD signalling regulate the Wnt/β-catenin pathway, in the nephron and elsewhere. This should be the subject of future studies.</p></sec><sec id="s3-5"><title>The medial and proximal nephron segments are dependent on Notch and low levels of PI3K and β-catenin</title><p>To date, one of the best characterised signalling pathways that have been implicated in nephron patterning is the Notch pathway (<xref ref-type="bibr" rid="bib19">Cheng et al., 2007</xref>; <xref ref-type="bibr" rid="bib11">Boyle et al., 2011</xref>). As Wnt and Notch signalling are tightly linked during development (<xref ref-type="bibr" rid="bib43">Hayward et al., 2008</xref>), we examined if the role of Notch signalling in nephron pattern formation is linked to the β-catenin activity gradient. We found that inhibition of Notch resulted in nephrons with medium to high levels of β-catenin activity throughout. By co-inhibiting β-catenin signalling in Notch-inhibited kidneys we partially reversed the latter's patterning phenotype. This was also true of co-inhibition of PI3K and Notch. In common to inhibition of β-catenin and PI3K was that they both decreased β-catenin signalling in nephrons and positively affected the development of the proximal-most and medial segments, respectively. Inhibition of β-catenin signalling in Notch-inhibited nephrons reduced the level of β-catenin signalling and rescued the proximal-most cells whilst co-inhibition of Notch and PI3K partially rescued the medial segment. It is therefore tempting to speculate that the mechanism for these rescues is by reduction of the level of β-catenin signalling. The mechanism of the rescue needs to be further studied, particularly in light of that the expression of known Notch target genes was not rescued by the β-catenin inhibitor (<xref ref-type="fig" rid="fig7">Figure 7D</xref>).</p></sec><sec id="s3-6"><title>Implications for morphogen signalling</title><p>Wnt ligands have long been known to act as one of the classic morphogens that drive patterning during development (<xref ref-type="bibr" rid="bib77">Zecca et al., 1996</xref>). To be effective as a gradient, the cells within the gradient should be able to respond to the different concentrations of extracellular Wnt ligand. In previous studies, a gradient of recombinant XWnt8 has been shown to be able to drive anterior–posterior neural patterning in dissociated <italic>Xenopus</italic> embryos with a corresponding gradient of β-catenin activity (<xref ref-type="bibr" rid="bib48">Kiecker and Niehrs, 2001</xref>). Similarly, a gradient in nuclear β-catenin was described in the patterning of the segmentation clock that controls somitogenesis (<xref ref-type="bibr" rid="bib5">Aulehla et al., 2008</xref>). Although this study analysed different β-catenin levels, in contrast to our approach, the authors focused on the extreme ends of the gradient (‘off’ vs ‘maximal’), and it was concluded that an unidentified co-factor was essential for interpreting the Wnt gradient. We show that in the nephron variations in the levels of β-catenin activity within the observed β-catenin gradient are directly responsible for correct patterning. Whilst the phenotypic effect of the β-catenin activity gradient in patterning the nephron is obvious, the mechanistic rationale for this is unclear. It is difficult to see how the present molecular/biochemical model for β-catenin signalling would allow the existence of β-catenin dosage-dependent differential gene expression. The model predicts that once β-catenin is stabilized, it will translocate to the nucleus and activate its target genes (<xref ref-type="bibr" rid="bib22">Clevers and Nusse, 2012</xref>). The data presented here, together with the ‘just-right’ signalling model for the role of <italic>APC</italic> mutations and β-catenin activity in cancer (<xref ref-type="bibr" rid="bib2">Albuquerque et al., 2002</xref>; <xref ref-type="bibr" rid="bib37">Gaspar et al., 2009</xref>) clearly show that β-catenin signalling is not a binary process and suggest additional levels of control of the β-catenin transcriptional output. Further, biochemical analysis of β-catenin function will be needed to elucidate this mechanism.</p><p>At present we do not know which Wnt (assuming it is a Wnt) it is that drives the gradient or how β-catenin activity is antagonised in the proximal cells. For the Wnts, none of the published <italic>Wnt</italic> knockout mouse models display a phenotype that would suggest a clear role in patterning of the nephron, although both <italic>Wnt9b</italic> and <italic>Wnt7b</italic>, when deleted, alter nephron morphogenesis. The <italic>Wnt7b</italic> knockout throughout the embryo proper or in the ureteric bud was described to lack a medulla due to disturbances of the plane of cell division, resulting in a failure of the Loop of Henle to elongate (<xref ref-type="bibr" rid="bib76">Yu et al., 2009</xref>). As this phenotype was mainly analysed at later stages of kidney development, it would require the inclusion of the <italic>TCF/Lef::H2B-GFP</italic> reporter and analysis of appropriate markers to determine if this phenotype is linked to the patterning defects we describe here. A conditional <italic>Wnt9b</italic> knockout in post-MET nephrons resulted in disturbed planar cell polarity as well but resulted in expansion of tubule diameter instead of the Loop of Henle defect (<xref ref-type="bibr" rid="bib46">Karner et al., 2009</xref>). Wnt9b is known to regulate planar cell polarity in the nephron, and the canonical pathway and non-canonical pathways are widely believed to be mutually competitive and inhibitory (<xref ref-type="bibr" rid="bib42">Grumolato et al., 2010</xref>). Although competition is known to occur mainly at the receptor level, it could be imagined that competition for intracellular downstream targets have the consequence that when decreasing the β-catenin activity using IWR1, there is shift from the canonical pathway towards the planar cell polarity pathway resulting in the observed nephron elongation. Again, extensive further analysis of the <italic>Wnt9b</italic> model is required to demonstrate involvement in the processes we describe here.</p><p>We analysed <italic>Lgr5</italic> as a potential modifier of Wnt signalling establishing the β-catenin gradient. The nephrons forming in <italic>Lgr5</italic><sup>+/EGFP-IRES-CreERT2</sup> homozygotes were morphologically indistinguishable to heterozygotes or control animals (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1</xref>). This is maybe not surprising as <italic>Lgr5</italic> knockouts have previously been shown to be without phenotype in the intestine where it is an important marker of intestinal stem cells (<xref ref-type="bibr" rid="bib28">de Lau et al., 2011</xref>).</p><p>A second question is how a single source of Wnt could establish a β-catenin signalling gradient within a morphologically convoluted tissue. It is plausible that the responsible Wnt forms an intraluminal gradient which would therefore potentially form a gradient irrespective of the actual nephron shape. It is also possible that all the nephron cells are exposed to a homogenous level of Wnt ligand and that they by cell-specific means modulate this to specific levels via, for example, Notch or BMP signalling. In the proximal domain, the β-catenin activity levels are even lower than in the medial domain, but no BMP activity is detected there. A plausible candidate for preventing β-catenin activity in this segment would be Wt1, as in in Sertoli cells Wt1 was shown to limit β-catenin activation (<xref ref-type="bibr" rid="bib17">Chang et al., 2008</xref>). Whilst additional <italic>Wnt</italic> and other knockout studies, either conventional or conditional, might provide new clues to extend our data into a yet more complete genetic pathway, the rate of nephron formation makes this a particularly challenging process. Since patterned S-shaped body nephrons form from mesenchymal progenitors within a 24 hr time-span, to conditionally knock out <italic>Wnts</italic>, it would be necessary to identify potential Cre-driving genes that are expressed at the very earliest stages of nephrogenesis but which are not active prior to nephron formation, since deletion at that time-point is likely to block the process. At present we do not know of any Cre driver that would be able to do this, so this might be a long-term goal.</p></sec></sec><sec sec-type="materials|methods" id="s4"><title>Materials and methods</title><sec id="s4-1"><title>Ethics statement for experimental animals</title><p>Animals were kept at facilities at the MRC Human Genetics Unit and the University of Edinburgh (UK); Columbia University, New York (USA); Maine Medical Center, Maine (USA); the Beatson Institute, Glasgow (UK); and the Erasmus Medical Centre, Rotterdam (The Netherlands). All animal experiments and animal use were carried out according to regulations specified by the Home Office (MRC/UoE), approved by the Animal Ethics Committee and carried out in accordance with Dutch and international legislation (Erasmus) and conducted under PHS guidelines and approved by the relevant Institutional Animal Care and Use Committees (Columbia/Maine). All animal experiments were performed under Project Licenses 60/3788 and 60/4473.</p></sec><sec id="s4-2"><title>Experimental animals</title><p>Outbred CD1 animals were obtained from Charles Rivers (UK). Embryos were generated through timed mating, with noon of the day a vaginal plug was found considered E0.5. <italic>TCF/Lef:H2B-EGFP</italic> (Tg(TCF/Lef1-HIST1H2BB/EGFP)61Hadj) (<xref ref-type="bibr" rid="bib32">Ferrer-Vaquer et al., 2010</xref>) were crossed with 129/SvEV and <italic>Wt1</italic><sup>+/GFP</sup> (Wt1<sup>tm1Nhsn</sup>) (<xref ref-type="bibr" rid="bib45">Hosen et al., 2007</xref>) mice were crossed with CD1. <italic>Pax8</italic><sup>+/Cre</sup> (Pax8<sup>tm1(cre)Mbu</sup>) mice (<xref ref-type="bibr" rid="bib10">Bouchard et al., 2004</xref>) were crossed to <italic>Rosa26</italic><sup>eYFP/eYFP</sup> (Gt(ROSA)26<sup>Sortm1(EYFP)Cos</sup>) animals (<xref ref-type="bibr" rid="bib71">Srinivas et al., 2001</xref>). <italic>Six2</italic><sup>+/GCiP</sup> (<xref ref-type="bibr" rid="bib29">Dolt et al., 2013</xref>) mice were crossed with Rosa26<sup>tdRFP</sup> (Gt(ROSA)26Sor<sup>tm1Hjf</sup>) (<xref ref-type="bibr" rid="bib58">Luche et al., 2007</xref>). <italic>Ctnnb1</italic><sup>E654</sup> (Ctnnb1<sup>tm1.2Wvv</sup>) (<xref ref-type="bibr" rid="bib74">van Veelen et al., 2011</xref>), <italic>Apc</italic><sup>+/1572T</sup> (Apc<sup>tm2Rfo</sup>) (<xref ref-type="bibr" rid="bib37">Gaspar et al., 2009</xref>), and <italic>Apc</italic><sup>+/1638N</sup> (Apc<sup>tm1Rak</sup>) (<xref ref-type="bibr" rid="bib34">Fodde et al., 1994</xref>) were intercrossed as required. <italic>Lgr5</italic><sup>+/EGFP-IRES-CreERT2</sup> (B6.129P2-Lgr5<sup>tm1(cre/ERT2)Cle</sup>/J) (<xref ref-type="bibr" rid="bib7">Barker et al., 2012</xref>) were crossed with C57BL/6 (Harlan) or intercrossed. <italic>BRE-Hspa1a-LacZ</italic> were described before (<xref ref-type="bibr" rid="bib8">Blank et al., 2008</xref>).</p></sec><sec id="s4-3"><title>Organ culture and pharmaceutical inhibitors</title><p>Kidneys were dissected from E12.5 embryos and cultured at 37°C with 5% CO<sub>2</sub> on 0.4 µm PET Transwell membranes (Corning, New York, NY). The culture medium consisted of MEM (SIGMA, UK M5650), 10% FCS, and 1% Pen/Strep. Isolated mesenchyme was induced with spinal cord as previously described (<xref ref-type="bibr" rid="bib25">Davies, 1994</xref>; <xref ref-type="bibr" rid="bib27">Davies and Garrod, 1995</xref>). Media was changed to contain IWR1 or CHIR after 48 hr and cultures kept for an additional 48 hr. Isolated Six2<sup>+</sup> cells were pelleted for 5 min at 800×<italic>g</italic> and induced with spinal cord for 24 hr. The cultures were treated with CHIR or IWR1. CHIR99021 (University of Dundee, UK), ICG001 (Enzo LifeSciences, UK), IWR1 (TOCRIS), salinomycin (SIGMA), BIO (TOCRIS, UK), Ly294002 (TOCRIS), LDN-193189 (STEMGENT, Cambridge, MA), methotrexate (SIGMA), were used as specified in the text.</p></sec><sec id="s4-4"><title>Immunofluorescent staining, image capture, and image analysis</title><sec id="s4-4-1"><title>Primary antibodies</title><p>anti-jagged1 (R&D Systems, Minneapolis, MN), anti-E-cadherin (BD Transduction Laboratories, UK), anti-wt1 (Santa Cruz, Dallas, TX), anti-LEF1 (Cell Signaling, The Netherlands), anti-Pax8 (Proteintech Europe, UK), anti-Pax2 (Covance, Princeton, NJ), anti-phospho-β-cat (S33, S37, T41) (Cell Signaling), anti-phospho-β-cat (S45) (Cell Signaling), pan β-cat (SIGMA), anti-dephospho-β-cat (AG Scientific, San Diego, CA), anti-podocalyxin, anti-laminin (SIGMA), LTL (Vector Labs, Burlingame, CA), anti-ZO1 (DSHB, Iowa City, IA), anti-pSMAD (Cell Signaling), anti-pAKT (Cell Signaling), 6-CF (SIGMA), PNA (Vector Labs), Annexin V (Biovision, San Francisco, CA), TUNEL (ROCHE, Switzerland), anti-phospho-β-cat (S552) (Cell Signaling).</p></sec><sec id="s4-4-2"><title>Fluorescent secondary antibodies</title><p>Fluorescent secondary antibodies were purchased from Invitrogen Molecular Probes: anti-mouse IgG 488, anti-rabbit IgG 594, anti-goat IgG 488, anti-goat IgG 594, anti-mouse IgG 647, anti-rabbit IgG 488, anti-goat IgG 350.</p></sec><sec id="s4-4-3"><title>Immunfluorescent staining</title><p>Organ cultures were fixed in −20°C methanol or 4% PFA in 1× PBS. 4% PFA was used for all tissues with fluorescently labelled proteins. Fixed tissues were washed thoroughly in 1× PBS prior to addition of primary antibodies and incubation at 4°C O/N. Samples were again washed thoroughly in 1× PBS before incubation with secondary antibodies at 4°C O/N. Excess secondary antibodies were washed off thoroughly with 1× PBS before mounting in Vectashield (Vectorlabs).</p></sec><sec id="s4-4-4"><title>Microscopy and image processing</title><p>Live imaging microscopy was performed on a Nikon TiE (Perfect Focus System) with NIS-Elements 4.0. Imaging was performed using a 4× objective and a CoolSnap HQ2 CCD camera (Photometrics, Tucson, AZ). Confocal microscopy was carried out on a Nikon A1R and an N-STORM/A1 Confocal and 10×–60× objectives were used and image stitching was carried out at 10× to capture whole kidneys. Nikon NIS-Elements 4.0, Fiji (<ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://fiji.sc/">http://fiji.sc/</ext-link>), Volocity (PerkinElmer, Waltham, MA), and ImageJ (<ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://rsb.info.nih.gov/ij/">http://rsb.info.nih.gov/ij/</ext-link>) were used for image analysis and presentation. 3D videos were made in Fiji. Time-lapse videos were presented using ImageJ, Fiji, and IP-Lab. Brightness and contrast levels were adjusted in Adobe Photoshop CS5 and IP-Lab after quantification and for presentational purposes only.</p></sec><sec id="s4-4-5"><title>Quantification of H2B-GFP signal along the nephron axis</title><p>Kidneys were fixed to preserve H2B-GFP fluorescence using 4% PFA in 1× PBS. Antibody stains were performed as described above. Whole nephrons were scanned at 40× at 0.5 µm z-intervals. Nephrons were visualised on NIS-Elements 4.0. Using the line intensity-profile tool, 10 µm wide lines were constructed centrally through each nephron segment and the maximum pixel value was determined along the line at each z-plane. The mean of the maximum values was calculated for 10 µm intervals along the whole nephron axis. The percentage of the mean maximum value was plotted against the corresponding position along the nephron axis. A total of 11 nephrons were analysed that encompassed: renal vesicle, comma-shaped body, S-shaped body, and capillary-loop stage nephrons. For the multi-nephron analysis, the mean value of each segment (distal, medial, proximal) was calculated and combined for all the nephrons analysed. Two-tailed Student's <italic>t</italic> tests were used to compare the medial with distal and medial with proximal segment.</p></sec><sec id="s4-4-6"><title>Quantification of phospho-β-cat (S33, S37, T41) signal along the nephron axis</title><p>Kidneys were stained for phospho-β-cat (S33/S37/T41), Jag1, and Cdh1. Scans were made every 1 µm with a 63× objective through whole nephrons with an N-STORM/A1 confocal microscope. 3D volume reconstructions were generated in IMARIS. Very bright pixels from background noise were made into a volume and masked onto the original channel and removed. Volume measurements were made in 75 × 75 × 10 µm cubes. Three cubes were fitted per nephron segment, thus nine measurements were made per nephron. The Image intensity sum was calculated and the average found per segment and across five different nephrons. Two-tailed Student's <italic>t</italic> tests were used to compare the medial with distal and medial with proximal segment.</p></sec><sec id="s4-4-7"><title>Measuring image intensities in live nephrons</title><p>Whole E12.5 kidneys were cultured in control, LDN-193189, or Ly294002 containing media. Time-lapse imaging was performed to capture GFP and brightfield. The GFP signal was captured far below saturation levels (saturated pixels in figures and videos are present due to brightness and contrast changes performed for publication purposes) as a 16-bit image every 20 min. To measure the GFP signal intensity, individual nephrons were selected from their point of formation and monitored for 22 hr. The point of formation was determined based on the brightfield view and the GFP signal, which was detectable at low levels in the pre-epithelial cells. The earliest signs of nephron formation were when cells aggregated prior to epithelialisation (pretubular aggregates). That was chosen as ‘time 0’. The 22 hr time-frame was chosen as this was the time taken for nephrons to develop to an S-shaped body stage in controls. To measure the intensity of nuclei along the nephron distal-to-proximal axis a 5-µm wide line was drawn down the length of the axis. The mean value across the line was measured along the whole length of the nephron. This gave readouts of the intensities of nuclei along the nephron and also provided the nephron lengths. Several nuclei were thus picked up within each nephron segment. The nephron axis was visible in the GFP and the brightfield channels and both were used to accurately reconstruct the nephron axis. The intensities were plotted against the axis length in 8-bit values vs µm position. Several nephrons from separate kidneys were measured in this fashion for each condition: 11 control nephrons, 12 LDN-193189 nephrons, 11 Ly294002 nephrons. The nephron lengths were used to calculate the changes in nephron lengths between each time-point and over the total 22 hr time-period. This allowed us to calculate the rate of nephron growth. To measure the ‘total intensity’ at the 22 hr time-point, the intensity of all pixels within the whole nephron at that time-point was measured. For this, 17 control nephrons, 18 LDN-193189 nephrons, and 18 Ly294002 nephrons were measured. The pixel values were plotted against their percentage frequency to obtain curves displaying the frequency of GFP signal intensities.</p></sec><sec id="s4-4-8"><title>Identification of CHIR99021 concentrations stimulating epithelialisation and nephron induction</title><p>Kidneys were treated with a range of CHIR concentrations (1.5 µM, 3 µM, or 6 µM shown) and immunostained for Lef1, Pax2, Pax8, Chd1. Using Fiji the mean secondary antibody signal-intensity was measured within 50-µm diameter circular sections in all ectopic nephrons using two kidneys per treatment. All comparisons between treatments were made using samples that had been simultaneously stained and imaged.</p></sec><sec id="s4-4-9"><title>Quantification of TCF/Lef::H2B-GFP fluorescence to determine inhibitor effects</title><p>H2B-GFP fluorescence was measured in all ureteric bud tips of four kidneys per treatment. Images for measurements were captured using the same settings throughout. Statistical analysis was performed using single factor ANOVAs (α 0.05) for single time-points (0 hr, 24 hr, 48 hr).</p></sec><sec id="s4-4-10"><title>Glomerular maturation rates</title><p>Kidneys were cultured and imaged as described above. Kidneys were scored for the development of crescent-shaped glomeruli and the time was recorded. Statistical analysis of timing of crescent-shaped glomerular formation was carried out using two-tailed Student's <italic>t</italic> tests of each group compared to controls. 5 <italic>Wt1</italic><sup>+/GFP</sup> (Wt1<sup>tm1Nhsn</sup>) kidneys were tested per group.</p></sec><sec id="s4-4-11"><title>Analysis of segment transformations and sizes</title><p>Confocal scans were carried out on kidneys cultured in specified conditions. Wt1, Jag1, Cdh1 immunofluorescent stains were used to measure the size of each nephron domain. Fiji software was used to measure the length of each domain. Where overlap of markers was seen, the mid-point of overlaps was taken as end of each domain. The mean domain-size was calculated and separate two-tailed Student's <italic>t</italic> tests were used to analyse statistical significance compared to controls.</p></sec><sec id="s4-4-12"><title>Quantification of H3(S10<sub>P</sub>) nuclei</title><p>Confocal scans were performed on <italic>TCF/Lef:H2B-EGFP</italic> kidneys cultured in the specified conditions. Kidneys were stained for H3(S10<sub>P</sub>), Cdh1, and Hoechst. Positive nuclei were counted within nephrons. 21–22 nephrons were counted per condition.</p></sec></sec><sec id="s4-5"><title>TaqMan qRT-PCR</title><p>For TaqMan experiments, samples were placed in RNALater (Ambion). A minimum of three kidneys were used per replicate of each condition and all experiments were performed in triplicates. RNA was isolated from cultured kidneys using RNAeasy micro kits (QIAGEN, The Netherlands). cDNA was made using SuperScript II (Invitrogen, Carlsbad, CA) and random primers (Promega, Madison, WI). Assays were done with the Universal Probe Library (Roche) and were designed using the Universal Probe Library Assay Design Center (<ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://www.roche-applied-science.com/sis/rtpcr/upl/index.jsp?id=UP030000">http://www.roche-applied-science.com/sis/rtpcr/upl/index.jsp?id=UP030000</ext-link>); primer and probe details can be found in <xref ref-type="supplementary-material" rid="SD1-data">Supplementary file 1</xref>. All TaqMan assays used a mouse GAPDH internal control (Roche). Heat-maps were generated using the matrix2png interface (<ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://www.chibi.ubc.ca/matrix2png/bin/matrix2png.cgi">http://www.chibi.ubc.ca/matrix2png/bin/matrix2png.cgi</ext-link>).</p></sec><sec id="s4-6"><title>Abbreviations</title><p>GSK-3β, glycogen synthase kinase 3 beta; WNT, wingless-related MMTV integration site; Cited1, Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 1; Wt1, Wilms' tumour 1 homologue; Six2, sine oculis-related homeobox 2; Jag1, jagged 1; Ctnnb1, catenin (cadherin associated protein) beta 1 (β-catenin); PTEN, phosphatase and tensin homolog; PI3K, phosphatidylinositide 3-kinase; AKT, thymoma viral proto-oncogene 1; BMP, bone morphogenetic protein; Apc, adenomatosis polyposis coli; Lgr5, leucine rich repeat containing G protein coupled receptor 5; Lhx1, LIM homeobox protein 1; Fgf8, fibroblast growth factor 8; Pax8, paired box gene 8; UB, ureteric bud; Pod., podocyte; Pt., parietal epithelium.</p></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>We thank the members of the Hohenstein lab and Nick Hastie for valuable discussions. We thank Shahida Sheraz, Anna Thornburn, and Rachel Berry for help with animals, Paul Perry, Matthew Pearson, and Ann Wheeler for help with confocal and time-lapse imaging, Kat Norrby for providing reagents, and Elizabeth Freyer for FACS sorting. NOL was supported by the National Centre for Replacement, Refinement, and Reduction of Animals in Research (Grant 94808). The Roslin Institute receives strategic funding from the BBSRC.</p></ack><sec sec-type="additional-information"><title>Additional information</title><fn-group content-type="competing-interest"><title>Competing interests</title><fn fn-type="conflict" id="conf1"><p>The authors declare that no competing interests exist.</p></fn></fn-group><fn-group content-type="author-contribution"><title>Author contributions</title><fn fn-type="con" id="con1"><p>NOL, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con2"><p>MLL, Performed the Annexin V assays</p></fn><fn fn-type="con" id="con3"><p>C-HC, Performed the Annexin V assays</p></fn><fn fn-type="con" id="con4"><p>SFB, Provided <italic>TCF/Lef:H2B-EGFP</italic> animals</p></fn><fn fn-type="con" id="con5"><p>JAJ, Helped with the analysis of BMP signalling</p></fn><fn fn-type="con" id="con6"><p>DJH, Helped with the analysis of BMP signalling</p></fn><fn fn-type="con" id="con7"><p>ERMB, Provided <italic>Apc</italic> and <italic>Ctnnb1</italic> transgenic animals</p></fn><fn fn-type="con" id="con8"><p>RS, Provided <italic>Apc</italic> and <italic>Ctnnb1</italic> transgenic animals</p></fn><fn fn-type="con" id="con9"><p>RAR, Provided <italic>Lgr5</italic><sup>+/EGFP-IRES-CreERT2</sup> animals</p></fn><fn fn-type="con" id="con10"><p>OJS, Provided <italic>Lgr5</italic><sup>+/EGFP-IRES-CreERT2</sup> animals</p></fn><fn fn-type="con" id="con11"><p>MJK, Provided BRE-LacZ animals</p></fn><fn fn-type="con" id="con12"><p>LO, Provided BRE-LacZ animals</p></fn><fn fn-type="con" id="con13"><p>JAD, Conception and design, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con14"><p>PH, Conception and design, Analysis and interpretation of data, Drafting or revising the article</p></fn></fn-group><fn-group content-type="ethics-information"><title>Ethics</title><fn fn-type="other"><p>Animal experimentation: Animals were kept at facilities at the MRC Human Genetics Unit, The Roslin Institute and the University of Edinburgh (UK), Columbia University, New York (USA), Maine Medical Center, Maine (USA), the Beatson Institute, Glasgow (UK), and the Erasmus Medical Centre, Rotterdam (The Netherlands). All animal experiments and animal use was carried out according to regulations specified by the Home Office (MRC/UoE), approved by the Animal Ethics Committee and carried out in accordance with Dutch and international legislation (Erasmus) and conducted under PHS guidelines and approved by the relevant Institutional Animal Care and Use Committees (Columbia/Maine). All animal experiments were performed under Project Licenses 60/3788 and 60/4473.</p></fn></fn-group></sec><sec sec-type="supplementary-material"><title>Additional files</title><supplementary-material id="SD1-data"><object-id pub-id-type="doi">10.7554/eLife.04000.037</object-id><label>Supplementary file 1.</label><caption><p>Primers and UPL probes are used in qRT-PCR analysis.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04000.037">http://dx.doi.org/10.7554/eLife.04000.037</ext-link></p></caption><media xlink:href="elife04000s001.pdf" mimetype="application" mime-subtype="pdf"/></supplementary-material></sec><ref-list><title>References</title><ref id="bib2"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Albuquerque</surname><given-names>C</given-names></name><name><surname>Breukel</surname><given-names>C</given-names></name><name><surname>van der 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letter</article-title></title-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Fuchs</surname><given-names>Elaine</given-names></name><role>Reviewing editor</role><aff><institution>Rockefeller University</institution>, <country>United States</country></aff></contrib></contrib-group></front-stub><body><boxed-text><p>eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see <ext-link ext-link-type="uri" xlink:href="http://elifesciences.org/review-process">review process</ext-link>). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.</p></boxed-text><p>Thank you for sending your work entitled “Integrated β-catenin, BMP, PTEN, and Notch signalling patterns the nephron” for consideration at <italic>eLife</italic>. Your article has been favorably evaluated by Janet Rossant (Senior editor), a Reviewing editor, and 4 reviewers.</p><p>The Reviewing editor and the reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.</p><p>All four reviewers found your work to be comprehensive in scope and for the most part found the data to justify the conclusions drawn, and the manuscript well-written. In fact, one of the strengths cited by multiple reviewers was the comprehensiveness of the study relative to other existing work on the signaling pathways controlling kidney morphogenesis. That said, although your work is in principle suitable for publication in <italic>eLife</italic>, there are a few issues raised by the reviewers that merit your attention prior to further consideration.</p><p>Below, I delineate the issues raised that if satisfactorily addressed would allow us to move forward. In the reviewers' view, these should not be insurmountable hurdles for you, and we'd expect that you should be able to address the issues expeditiously:</p><p>Experimental issues:</p><p>Nephron patterning is assessed using several parameters: maturation, segment length, protein expression and gene expression. The results of these distinct assessments are not explicitly compared and integrated into one conclusion.</p><p>A changing set of segment specific markers is used for nephron segment identification (e.g. <xref ref-type="fig" rid="fig4 fig6">Figure 4D and 6G</xref>).</p><p>Although some of the pathway modulations are performed intermittently (canonical Wnt activation by CHIR, followed by CHIR withdrawal, <xref ref-type="fig" rid="fig2">Figure 2F</xref>), other pathway modulations are performed only continuously (e.g. BMP).</p><p>Reviewer 2 points out that β-catenin staining in <xref ref-type="fig" rid="fig1">Figure 1F</xref> and Figure 1–figure supplement 2B, D only shows membrane bound β-catenin and not the nuclear β-catenin necessary for the transcription of Wnt target genes. In addition, the presence of phosphorylated β-catenin in the developing nephron in Figure F appears to be limited to one cell, which is insufficient to support the claim of a gradient of β-catenin phosphorylation (422). In addition, the supposed gradient of phosphorylated β-catenin along the tubule is insufficiently demonstrated by Figure 1–figure supplement 2B. These figures do not sufficiently explain the β-catenin activity gradient. Reviewer 1 points out an issue with the reliability of WNT/beta catenin reporters in detecting WNT signaling and that they should not be considered definitive (<xref ref-type="fig" rid="fig1">Figure 1</xref>). Moreover, the images don't completely agree with the quantifications shown. On this point, multiple reviewers have raised questions as to how a single WNT source might generate such a gradient and whether a gradient has been definitively shown. A final issue relating to this point is what (where) is the source of the Wnt?</p><p>Continuing along this line of ambiguity is the issue of why does both decrease and increase of WNT give increased maturation rate? (<xref ref-type="fig" rid="fig2">Figure 2B</xref>). And why is the CTRL showing no crescent-shaped glomeruli until 3500 minutes (<xref ref-type="fig" rid="fig2">Figure 2B</xref>)?</p><p>Several of your reviewers also felt that your section on the link to Notch signalling is somewhat confusing and is not well-integrated with the first two major findings. This section needs bolstering with additional data to make a more compelling link.</p><p>Reviewer 3 points out that it would be straightforward to relate to other systems your finding that the PI3K pathway regulates beta-catenin. One way to explore these parallels is to evaluate whether Akt activates beta-catenin by phosphorylation at Ser552 in the nephron.</p><p>Writing/Text issues:</p><p>The number and organization of the figures, movies and supplementary figures interferes with the readability of the manuscript. Reducing the number of main and supplementary movies, figures and panels will enhance the legibility of the manuscript.</p><p>The presentation of the data and conclusions drawn needs to be more structured, and some of the statements, e.g. those of reviewer 2, need to be more explicitly stated upfront (issues with interpreting the Lgr5 data, fact that Wnt KO's have no consequences thus far to nephron development, etc.). Reviewers 1-3 provided some specific recommendations for recrafting the text to more coherently flow for the reader.</p><p>Please pay attention to reviewer 3's comment that more is known regarding nephron development than the authors sometimes claim, as this is important to correct. Among them, the example cited is a good one, point out your claim that “The nephron β-catenin activity gradient is to our knowledge the first instance of a β-catenin activity gradient being described in any system”, when in fact this has been shown for multiple systems, including the hair follicle, hematopoietic and intestinal stem cell lineages.</p><p>The review comments below should put this summary into context:</p><p><italic>Reviewer #1:</italic></p><p>This is a very interesting manuscript that defines an important role for canonical WNT signaling in the patterning of the nephron. Some evidence is provided suggesting that there is normally a WNT gradient going from distal (strong) to proximal (weak). Increasing WNT, chemically or genetically, results in increased distal and decreased proximal regions of the nephron. Decreasing WNT gives more complicated results, although it is argued that there is a more rapid maturation of the proximal nephron.</p><p>The work is quite thorough in many respects, for example in using a multitude of chemical modifiers of WNT signaling, as well as genetic approaches. The characterization of resulting phenotypes is also generally excellent, with many outstanding images provided and a reasonable set of markers employed.</p><p>There are some significant unanswered questions that are raised by the results and the proposed model. As mentioned in the paper, which WNT is responsible for the proposed gradient? And why don't any Wnt knockouts show the predicted nephron patterning defect? Further, why does knockdown of WNT in the proximal region of the nephron, where WNT is already proposed to be extremely low, partially rescue the Notch mutant loss of proximal structures?</p><p>Despite these questions there is a wealth of interesting data presented. There is a real lack of understanding of the forces that pattern the nephron and this paper begins to fill that important gap.</p><p><italic>Reviewer #2</italic>:</p><p>The authors have investigated the role of canonical Wnt signaling in nephron patterning during murine renal development in ex vivo culture experiments. In addition, the effect of BMP and PTEN/PI3K/AKT signaling on nephron patterning as well as the interaction between these pathways was investigated.</p><p>Extensive intervention studies have been undertaken with both pharmacological compounds as well as mutant mouse models, and the effect on patterning is assessed morphologically, immunohistochemically and by gene expression analysis. The results provide relevant and new insight into mechanisms involved in nephron patterning.</p><p>However, I do have serious concerns regarding the way the data is presented and some concerns about the coherence of the experiments.</p><p>Major comments:</p><p>The number and organization of the figures and movies interferes with the readability of the manuscript. Reducing the number of movies, figures and panels will enhance the legibility of the manuscript.</p><p>The presentation of the data and conclusion should be more structured:</p><p>Conclusions are presented inconsistently in the manuscript body, the figure legends (<xref ref-type="fig" rid="fig2s1">Figure 2–figure supplement 1</xref>) and the figure itself (<xref ref-type="fig" rid="fig6s1">Figure 6–figure supplement 1 D</xref>).</p><p>Paragraphs in the Results section sometimes end with a conclusion and sometimes with just research results. The manuscript contains redundant information and conclusions: e.g.: “Since proliferation was not affected by either increasing or decreasing β-catenin signaling” and “we found that proliferation along the nephron axis does not change in response to stimulating or inhibiting β-catenin”, both in the Discussion, and “phospho- and dephospho-specific antibodies (indicative of protein targeted for break-down and active 400 β-catenin respectively)” and “antibodies for β-catenin that was already tagged for degradation”, in the Discussion and in the Results respectively. Improving the cohesion (in the presentation) of the different experiments would strengthen the manuscript.</p><p>Nephron patterning is assessed using several parameters: maturation, segment length, protein expression and gene expression. The results of these distinct assessments are not explicitly compared and integrated into one conclusion.</p><p>A changing set of segment specific markers is used for nephron segment identification (e.g. <xref ref-type="fig" rid="fig4 fig6">Figure 4D and 6G</xref>).</p><p>Although some of the pathway modulations are performed intermittently (canonical Wnt activation by CHIR, followed by CHIR withdrawal, <xref ref-type="fig" rid="fig2">Figure 2F</xref>), other pathway modulations are performed only continuously (e.g. BMP). Vague remarks should be replaced by more clearly defined statements.</p><p>Different terms are used interchangeably in the Discussion section, but remain devoid of proper definition. Please clarify how these terms relate to the parameters measured (see above): “more efficient formation of the proximal domain”, “a strong positive effect on the development of the medial segment”, “increased rate of maturation”, “change in cell identity”.</p><p>In the Discussion section, the sentence “Exploring the roles… of future studies”: In the Results, the authors mention that Lgr-5 is a β-catenin target, however Lgr5 is also a Wnt receptor component that can enhance canonical Wnt signaling (<xref ref-type="bibr" rid="bib28">de Lau, Nature, 2011</xref>). The Lgr-5 expression (gradient) might therefore not be the result but the cause of the β-catenin gradient. The latter hypothesis is rejected by the experiments with Lgr-5 KO kidneys. This consideration should be mentioned more explicitly in the manuscript.</p><p>The authors state that <xref ref-type="fig" rid="fig1">Figure 1E</xref> suggests that β-catenin activity is induced by (factors secreted by (?)) a single source with a first order decay. This concept is difficult to visualize due to the spiral morphology of the immature nephron. Please clarify. (One might however hypothesize that the β-catenin activity-inducing signal is propagated intraluminally.)</p><p>β-catenin staining in <xref ref-type="fig" rid="fig1">Figure 1F</xref> and Figure 1–figure supplement 2B, D only shows membrane bound β-catenin and not the nuclear β-catenin necessary for the transcription of Wnt target genes. In addition, the presence of phosphorylated β-catenin in the developing nephron in Figure F appears to be limited to one cell, which is insufficient to support the claim of a gradient of β-catenin phosphorylation made in the Discussion section. In addition, the supposed gradient of phosphorylated β-catenin along the tubule is insufficiently demonstrated by Figure 1–figure supplement 2B. These figures do not sufficiently explain the β-catenin activity gradient.</p><p><italic>Reviewer #3</italic>:</p><p>In this manuscript, the authors investigate pattern formation of the nephron. Although nephron development is a relatively neglected area of research, more is known regarding its development than the authors sometimes claim. Nonetheless, this manuscript provides a far more detailed and integrated view of nephron pattern formation than any other study to date. First, the authors conduct a detailed series of experiments showing that a beta-catenin activity gradient determines positional identity in a morphogenetic manner. This is shown primarily by using pharmacological inhibitors/activators of Wnt signaling along with a reporter of beta-catenin activity. The authors do a nice job verifying the specificity and fidelity of their system. Importantly, this pharmacological study is further supported genetically using various hypomorphic alleles of beta-catenin. The authors then show that, similar to the intestine, nephron patterning is further regulated through a BMP/PI3K network, which acts in modulating the more central-acting beta-catenin activity. Given the structural and functional similarities between the nephron and intestine, this is an attractive model. The authors also attempt to link their data with Notch signaling, which is also known to regulate nephron patterning. This section is somewhat confusing and is not well-integrated with the first two major findings. Overall, the authors have provided a more comprehensive study of nephron patterning than any previous study, and their study sheds some light on its subject that could not be gleaned from the current literature. However, some questions/concerns should be addressed prior to publication.</p><p>Minor comments:</p><p>The results showing no changes in proliferation or apoptosis should be better explained and integrated into the manuscript. Do the authors suggest this implies that differentiation is mainly responsible for the patterning effects? Actually, changes in proliferation and apoptosis are well-known mechanisms for regulating morphogenesis (along with differentiation). At any rate, the authors should better clarify the purpose of these experiments and what they conclude from the results.</p><p>The finding that the PI3K pathway is regulating beta-catenin is interesting. These pathways are known to interact, especially in the intestinal and hematopoietic systems, where Akt can activate beta-catenin by phosphorylation at Ser552. It would be interesting to see whether and where pS552-bcat positive cells were present during nephron patterning. Such data could further support the author's model.</p><p>The authors sometimes discount previous findings. For instance, they claim that “The nephron β-catenin activity gradient is to our knowledge the first instance of a β-catenin activity gradient being described in any system”. This is surprising since such gradients have been described in multiple systems, including mammals.</p><p>The authors should consider either better integrating and explaining the Notch data or removing this section from the manuscript. It would be appropriate to discuss what's known about Notch and how it might relate to their new findings in the Discussion section; however, the current presentation tends to leave the reader more confused than enlightened.</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.04000.039</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p><italic>Experimental issues</italic>:</p><p><italic>Nephron patterning is assessed using several parameters: maturation, segment length, protein expression and gene expression. The results of these distinct assessments are not explicitly compared and integrated into one conclusion</italic>.</p><p>We have added a combining/concluding paragraph to the beginning of the Discussion. We have limited this to what are in our opinion the clearly proven facts. Other parts we consider more speculative are discussed elsewhere in the Discussion.</p><p><italic>A changing set of segment specific markers is used for nephron segment identification (e.g.</italic> <xref ref-type="fig" rid="fig4 fig6"><italic>Figure 4D and 6G</italic></xref><italic>)</italic>.</p><p>In the original manuscript we chose markers that best illustrated the phenotype described in the specific part of the manuscript. In particular, in cases where markers can be used in different ways, for instance genes that are known to be downstream targets of the pathways, we study as well as markers for spatial segments of the nephron, we thought this would help to illustrate our points. However, we appreciate that this has led to confusion for the reader. We now provide a consistent set of markers in the different experiments.</p><p>In the original manuscript, we presented these data as overlapping line diagrams in different colours. However, the expression patterns of the additional genes we now show made these very confusing figures, even if different colours were combined with different line styles. Eventually we decided to present these data in the revised manuscripts in the form of heat maps. Although these have the disadvantage of that we cannot include statistical information in the form of error bars, this presentation is the clearest in showing the biological message coming from the experiments.</p><p><italic>Although some of the pathway modulations are performed intermittently (canonical Wnt activation by CHIR, followed by CHIR withdrawal,</italic> <xref ref-type="fig" rid="fig2"><italic>Figure 2F</italic></xref><italic>), other pathway modulations are performed only continuously (e.g. BMP)</italic>.</p><p>We now provide the reversal experiments for the important inhibitors we use in the manuscript. In all but one of the inhibitors (the exception being IWR1) this resulted in at least partial reversal of the phenotype. Please note that a failure to reverse the phenotype does not necessarily imply the experiment is flawed. For instance, in some case inhibitors can bind irreversibly to their target making phenotypic rescue impossible. In other instances, the change that is made might be of such a nature that the patterning would simply not be able to reverse. IWR1, for example, causes the nephrons to mature quicker. Once segments have fully matured, they might no longer have the capacity to generate those segments that didn’t form due to the inhibitor’s effect. Therefore, withdrawal of the inhibitor would simply not allow for a reversal of the effects.</p><p><italic>Reviewer 2 points out that</italic> β-<italic>catenin staining in</italic> <xref ref-type="fig" rid="fig1"><italic>Figure 1F</italic></xref> <italic>and Figure 1–figure supplement 2B, D only shows membrane bound</italic> β-<italic>catenin and not the nuclear</italic> β-<italic>catenin necessary for the transcription of Wnt target genes. In addition, the presence of phosphorylated</italic> β-<italic>catenin in the developing nephron in Figure F appears to be limited to one cell</italic>, <italic>which is insufficient to support the claim of a gradient of</italic> β-<italic>catenin phosphorylation. In addition, the supposed gradient of phosphorylated B catenin along the tubule is insufficiently demonstrated by Figure 1–figure supplement 2B. These figures do not sufficiently explain the</italic> β-<italic>catenin activity gradient.</italic></p><p>We appreciate the original data using the phospho- and dephospho-specific antibodies on its own was insufficient to prove the existence of a β-catenin activity gradient, and for this reason we interpreted it in the context of all other data (reporter and pathway modulation). Unfortunately, these antibodies are very difficult to use in the whole mount kidney organ culture system (we are not aware of other publications that show their use in this) and optimization options are limited. We did succeed in improving the data with the Ser33/37/Thr41 phospho-specific antibody and these data are now provided. To further support the activity gradient this signal is now quantified in different parts of the nephron which confirms the different signals in different parts of the nephron.</p><p>We did not succeed in further improving the pSer45 and dephospho-specific data. As we do not want to present data that has, for technical reasons, limited relevance these have now been removed from the manuscript to reduce the complexity of the presented data. We stress the pSer33/37/Thr41 data should still be considered in the context of the complete manuscript, not as stand-alone data. This is now more clearly discussed in the Discussion section.</p><p>As for the lack of nuclear β-catenin, although we cannot exclude technical complications with the whole organ staining, the model that active signalling automatically leads to and requires nuclear localization is likely an oversimplification. For instance, in colorectal cancer (the archetypal example of a tumour caused by activation of this pathway through either loss of APC or activating mutation in β-catenin) it has been shown that within the tumour there is a dynamic change in localization in different parts of the same tumour although all cells will carry the same cancer-inducing mutation (PMID 17306971). Likewise, in mice expressing the same activating <italic>Ctnnb1</italic><sup>ex 3</sup> mutation throughout the intestine, the biological effect <italic>as well as the intracellular localization of the mutant β-catenin</italic> varies in different parts of the gut (PMID 22287596). This is now also discussed in the manuscript. Therefore, although we agree this is an important observation, in our opinion it does not necessarily disprove the existence of the gradient.</p><p><italic>Reviewer 1 points out an issue with the reliability of WNT/beta catenin reporters in detecting WNT signaling and that they should not be considered definitive (</italic><xref ref-type="fig" rid="fig1"><italic>Figure 1</italic></xref><italic>)</italic>.</p><p>We fully agree with this reviewer that activity reporters need to be used with common sense. Whereas we have chosen to use an artificial mini-construct reporter, other labs prefer to use the <italic>Axin2-lacZ</italic> knock-in line (<italic>Axin2</italic> being an endogenous β-catenin target). Our preference for the mini-construct is based on the following: i) the relevance of endogenous target genes can differ in a cell type-dependent manner. For instance, c-Myc is a key β-catenin target in colorectal cancer (PMID 17377531) but Ccnd1 is not (PMID 15946945). If endogenous transcriptional responses are not uniform, reporters based on them will not be reliable in their responses; ii) endogenous reporters will only be fully reliable if β-catenin is the only pathway that controls its expression. This is unlikely to be the case. A reporter based on an endogenous β-catenin target gene can in that case truthfully report increased activity of the pathway, but reduction is not necessarily detected as other pathways could maintain its expression. In this respect, the simplicity of mini constructs as we used is a clear advantage, and we show that IWR1 treatment results in the expected decrease in signal intensity; iii) the use of <italic>lacZ</italic> in the <italic>Axin2</italic> model gives an additional layer of arbitrary experimental conditions. Staining time can be chosen at will and the <italic>lacZ</italic> signal rapidly reaches a maximum. For subtle differences, as we show here, a fluorescent reporter and quantification methods as we employ them here is far superior.</p><p>We do emphasize that none of the experiments on the gradient should be seen in isolation (see the previous point), and this is now discussed in more detail.</p><p><italic>Moreover, the images don't completely agree with the quantifications shown</italic>.</p><p>The detectors of the imaging systems, and therefore quantification of the signal, have a far better dynamic range than computer screens and printers. RGB images have a value range 0-255, with 255 being a fully saturated pixel. In the example of, for instance, the 39x difference between the proximal and distal end of the nephron (in the Results section), this would mean that an almost-but-just-not saturated pixel at the high end (value 254) would give a pixel with value 6.5 at the low end. On every printer and all but the best calibrated screens this would simply be black. For this reason we have chosen to quantify as much of our data as possible, it is the only way to see the nuances in the actual data. Visual recognition is due to the technical limitations of screens and paper simply not good enough. The fact that many structures are in close proximity of the ureteric bud (with very high reporter activity) makes it even more difficult to present proper imaging data that shows all the nuances that exist in the data. However, because this was an issue raised by several reviewers we chose to include additional data to more clearly display the different intensities. <xref ref-type="fig" rid="fig1s1">Figure 1–figure supplement 1B</xref> now shows the S-Shaped body nephron in <xref ref-type="fig" rid="fig1">Figure 1B</xref> but at four differently enhanced brightness levels. In the brightest field, we now indicate that the podocytes are very weakly positive whilst other areas of the kidney are negative indicating that this is a real difference.</p><p><italic>On this point, multiple reviewers have raised questions as to how a single WNT source might generate such a gradient and whether a gradient has been definitively shown</italic>.</p><p>The details of the mode of Wnt secretion and spreading are hotly debated in the literature. We can only speculate that it is unlikely the (hypothetical) Wnt molecule would be traveling on the outside of the nephron, as this would imply movement through the basement membrane in the responding cell. This would indeed also be complicated, as implicated by the reviewers, that the convoluted 3D structure of the nephron would be difficult to imagine in a gradient, especially in the context of the growing organ. Intraluminal transport however would, by definition, follow the shape of the nephron irrespective of the shape and organ as a whole. It would allow the Wnt signal to be taken up by receiving cell via the apical side (i.e. no basement membrane), and would fit with the observation that cilia are present on that side of the cell. We could have included this speculation in the Discussion, but as at the moment our manuscript is written from the β-catenin angle and not the Wnt angle we have not yet done so. The second issue on the gradient is discussed above.</p><p><italic>A final issue relating to this point is what (where) is the source of the Wnt?</italic></p><p>We have extended the Discussion with some speculation of a potential role of <italic>Wnt7b</italic> and <italic>Wnt9b</italic> in the patterning of the nephron as we describe, and indicate that extensive additional analyses beyond the scope of this work are required to prove or disprove this. We also like to stress that because of this we refer consistently to a β-catenin activity gradient, not a Wnt gradient. We even mention that at present we cannot exclude the possibility this is a hypothetical Wnt-independent β-catenin function (though we think this is highly unlikely).</p><p><italic>Continuing along this line of ambiguity is the issue of why does both decrease and increase of WNT give increased maturation rate? (</italic><xref ref-type="fig" rid="fig2"><italic>Figure 2B</italic></xref><italic>)</italic>. <italic>And why is the CTRL showing no crescent-shaped glomeruli until 3500 minutes (</italic><xref ref-type="fig" rid="fig2"><italic>Figure 2B</italic></xref><italic>)?</italic></p><p>We think there is a misunderstanding here. Only the decreased β-catenin signalling results in increased maturation rate (<xref ref-type="fig" rid="fig2">Figure 2</xref>). We agree that the labelling on the figure and perhaps also the terminology used was confusing. In the controls, there are nephrons continuously forming during the experiment. However, only those that appear mature in their morphology are counted. In IWR1 treated kidneys the glomeruli reached this mature stage faster compared to controls. The timing in this experiment is consistent with that in other experiments we show, e.g. <xref ref-type="fig" rid="fig2">Figure 2D</xref>. There we show kidneys after 2880 min (48 hrs) of culture. Whilst there are several Podxl positive structures in the control, <xref ref-type="fig" rid="fig2">Figure 2D</xref> illustrates that these are not yet at the stage of 'crescent-shaped' glomeruli. We have now attempted to make the figure more accessible and the figure legend clearer.</p><p><italic>Several of your reviewers also felt that your section on the link to Notch signalling is somewhat confusing and is not well-integrated with the first two major findings. This section needs bolstering with additional data to make a more compelling link</italic>.</p><p>We thank the reviewers for this criticism. We have added data on the β-catenin activity reporter under Notch/β-catenin and Notch/PI3K modulated conditions. These data have made the link between Notch and the other pathways considerably stronger.</p><p><italic>Reviewer 3 points out that it would be straightforward to relate to other systems your finding that the PI3K pathway regulates beta-catenin. One way to explore these parallels is to evaluate whether Akt activates beta-catenin by phosphorylation at Ser552 in the nephron</italic>.</p><p>We have added this experiment but our current data cannot support the reviewers’ hypothesis; we find staining for this antibody in the medial and distal segment (<xref ref-type="fig" rid="fig6">Figure 6H</xref>) but this doesn’t change upon inhibition of BMPR or PI3K.</p><p><italic>Writing/Text issues</italic>:</p><p><italic>The number and organization of the figures, movies and supplementary figures interferes with the readability of the manuscript. Reducing the number of main and supplementary movies, figures and panels will enhance the legibility of the manuscript</italic>.</p><p>We have tried to find a balance between reducing the figures and maintaining essential data. For instance, given the wide scepticism of specificity of pharmacological inhibitors we decided to keep all control experiments for this in figure supplements. Unfortunately, where in a biochemical paper this might be combined in a single western blot experiment, in our organ culture system this requires a lot more figures. On the other hand, in the old manuscript we showed the whole kidneys in figure supplements that our close-up data in the main figures was taken from to confirm we showed representative parts of the kidneys. To reduce complexity the majority of fields showing whole kidneys have now been removed and we have left the close-ups of nephrons.</p><p><italic>The presentation of the data and conclusions drawn needs to be more structured, and some of the statements, e.g. those of Reviewer 2, need to be more explicitly stated upfront (issues with interpreting the Lgr5 data, fact that Wnt KO's have no consequences thus far to nephron development, etc.). Reviewers 1-3 provided some specific recommendations for recrafting the text to more coherently flow for the reader</italic>.</p><p>We have extended this; more reviewer-specific comments are addressed below.</p><p><italic>Please pay attention to Reviewer 3's comment that more is known regarding nephron development than the authors sometimes claim, as this is important to correct. Among them, the example cited is a good one, point out your claim that “The nephron β-catenin activity gradient is to our knowledge the first instance of a β-catenin activity gradient being described in any system”, when in fact this has been shown for multiple systems, including the hair follicle, hematopoietic and intestinal stem cell lineages</italic>.</p><p>We thank the editor and reviewers’ for this comment and we have now toned down such statements or deleted them altogether where appropriate as this is not central to our work described here.</p><p><italic>The review comments below should put this summary into context</italic>:</p><p>Reviewer #1:</p><p><italic>This is a very interesting manuscript that defines an important role for canonical WNT signaling in the patterning of the nephron. Some evidence is provided suggesting that there is normally a WNT gradient going from distal (strong) to proximal (weak). Increasing WNT, chemically or genetically, results in increased distal and decreased proximal regions of the nephron. Decreasing WNT gives more complicated results, although it is argued that there is a more rapid maturation of the proximal nephron</italic>.</p><p><italic>The work is quite thorough in many respects, for example in using a multitude of chemical modifiers of WNT signaling, as well as genetic approaches. The characterization of resulting phenotypes is also generally excellent, with many outstanding images provided and a reasonable set of markers employed</italic>.</p><p>We thank this reviewer for these kind and positive remarks.</p><p><italic>There are some significant unanswered questions that are raised by the results and the proposed model. As mentioned in the paper</italic>, <italic>which WNT is responsible for the proposed gradient? And why don't any Wnt knockouts show the predicted nephron patterning defect?</italic></p><p>We address this point in the response to the joint review.</p><p><italic>Further, why does knockdown of WNT in the proximal region of the nephron, where WNT is already proposed to be extremely low</italic>, <italic>partially rescue the Notch mutant loss of proximal structures?</italic></p><p>We hypothesize that under normal conditions proximal cells try to maintain a sufficiently low β-catenin activity level. By inhibiting this further using IWR1 we would simply be giving these cells a helping hand, and reaching the proximal fate becomes easier and is reached quicker. Our new Notch inhibition data shows more homogenous medium to high β-catenin activity levels throughout the nephron. By combining this with IWR1 or PI3K inhibition, which on their own decreases β-catenin activity throughout the nephron or in the medial segment (<xref ref-type="fig" rid="fig6">Figure 6</xref>), we would reduce β-catenin activities again to compensate for the Notch inhibition effects. However, we emphasize that we do not claim that inhibiting β-catenin is the only role of PI3K or Notch, making the above merely speculative although we added it to the extended discussion on the role of Notch.</p><p><italic>Reviewer #2</italic>:</p><p><italic>The authors have investigated the role of canonical Wnt signaling in nephron patterning during murine renal development in ex vivo culture experiments. In addition, the effect of BMP and PTEN/PI3K/AKT signaling on nephron patterning as well as the interaction between these pathways was investigated</italic>.</p><p><italic>Extensive intervention studies have been undertaken with both pharmacological compounds as well as mutant mouse models, and the effect on patterning is assessed morphologically, immunohistochemically and by gene expression analysis. The results provide relevant and new insight into mechanisms involved in nephron patterning</italic>.</p><p>We thank the reviewer for these kind words.</p><p><italic>Major comments</italic>:</p><p><italic>The number and organization of the figures and movies interferes with the readability of the manuscript. Reducing the number of movies, figures and panels will enhance the legibility of the manuscript</italic>.</p><p>As discussed above and described in more detail below, we have addressed this in the best possible way, at least in our opinion.</p><p><italic>The presentation of the data and conclusion should be more structured. Conclusions are presented inconsistently in the manuscript body, the figure legends (</italic><xref ref-type="fig" rid="fig2s1"><italic>Figure 2–figure supplement 1</italic></xref><italic>) and the figure itself (</italic><xref ref-type="fig" rid="fig6s1"><italic>Figure 6–figure supplement 1 D</italic></xref><italic>). Paragraphs in the Results section sometimes end with a conclusion and sometimes with just research results. The manuscript contains redundant information and conclusions: e.g.: “Since proliferation was not affected by either increasing or decreasing β-catenin signaling” and “we found that proliferation along the nephron axis does not change in response to stimulating or inhibiting β-catenin”, both in the Discussion, and “phospho- and dephospho-specific antibodies (indicative of protein targeted for break-down and active 400 β-catenin respectively)” and “antibodies for β-catenin that was already tagged for degradation”, in the Discussion and in the Results respectively. Improving the cohesion (in the presentation) of the different experiments would strengthen the manuscript</italic>.</p><p>We have, with the help of the reviewers’ comments, hopefully addressed some of the inconsistencies within the manuscript. We believe that most of these came from not explaining clearly enough our conclusions. We have now added short conclusions to the different paragraph which should be helpful to the reader. Some of the examples that the reviewer considered being redundant information and conclusions have been modified, but others we chose to keep as they have been touched open by other reviewers as important.</p><p>We have also taken into account all the reviewers’ and editor’s comments to improve the flow and cohesion of the manuscript.</p><p>We have had a critical look at which figure panels are essential, and as discussed above, we have extended the range of markers to be consistent throughout the manuscript in parts where there was some inconsistent use.</p><p><italic>Nephron patterning is assessed using several parameters: maturation, segment length, protein expression and gene expression. The results of these distinct assessments are not explicitly compared and integrated into one conclusion</italic>.</p><p>We have added a section to the start of the Discussion combining these different types of data in a single model.</p><p><italic>A changing set of segment specific markers is used for nephron segment identification (e.g.</italic> <xref ref-type="fig" rid="fig4 fig6"><italic>Figure 4D and 6G</italic></xref><italic>)</italic>.</p><p><italic>Although some of the pathway modulations are performed intermittently (canonical Wnt activation by CHIR, followed by CHIR withdrawal,</italic> <xref ref-type="fig" rid="fig2"><italic>Figure 2F</italic></xref><italic>), other pathway modulations are performed only continuously (e.g. BMP)</italic>.</p><p>As discussed above, we now use a consistent set of markers and we have included a more general interpretation of the data. We have also added the reversal experiments for the other pathways as discussed above.</p><p><italic>Vague remarks should be replaced by more clearly defined statements. Different terms are used interchangeably in the Discussion section, but remain devoid of proper definition. Please clarify how these terms relate to the parameters measured (see above): “more efficient formation of the proximal domain”, “a strong positive effect on the development of the medial segment”, “increased rate of maturation”, “change in cell identity”</italic>.</p><p>We now specifically state that we use the nomenclature used by the GenitorUrinary Development Molecular Anatomy Project (<ext-link ext-link-type="uri" xlink:href="http://gudmap.org">gudmap.org</ext-link>) for S-Shaped body nephrons to refer to distal, medial, and proximal segments. This is also mentioned in the text.</p><p><italic>In the Discussion section, the sentence “Exploring the roles… of future studies”: In the Results, the authors mention that Lgr-5 is a β-catenin target, however Lgr5 is also a Wnt receptor component that can enhance canonical Wnt signaling (</italic><xref ref-type="bibr" rid="bib28"><italic>de Lau, Nature, 2011</italic></xref><italic>). The Lgr-5 expression (gradient) might therefore not be the result but the cause of the β-catenin gradient. The latter hypothesis is rejected by the experiments with Lgr-5 KO kidneys. This consideration should be mentioned more explicitly in the manuscript</italic>.</p><p>This is a very helpful comment and it has been added to the manuscript.</p><p><italic>The authors state that</italic> <xref ref-type="fig" rid="fig1"><italic>Figure 1E</italic></xref> <italic>suggest that</italic> β-<italic>catenin activity is induced by (factors secreted by (?)) a single source with a first order decay</italic>. <italic>This concept is difficult to visualize due to the spiral morphology of the immature nephron. Please clarify. (One might however hypothesize that the</italic> β-<italic>catenin activity-inducing signal is propagated intraluminally.)</italic></p><p>As discussed in our response to reviewer 1, this is indeed something we cannot address within the scope of this manuscript. Moreover, tools for tracking Wnt molecules in the 3D context of a developing context would be as useful as they would be technically challenging (for multiple reasons). Transport within the lumen is indeed a possibility as we now discuss.</p><p><italic>β-catenin staining in</italic> <xref ref-type="fig" rid="fig1"><italic>Figure 1F</italic></xref> <italic>and Figure 1–figure supplement 2B, D only shows membrane bound β-catenin and not the nuclear β-catenin necessary for the transcription of Wnt target genes. In addition, the presence of phosphorylated β-catenin in the developing nephron in Figure F appears to be limited to one cell, which is insufficient to support the claim of a gradient of β-catenin phosphorylation made in the Discussion section. In addition, the supposed gradient of phosphorylated β-catenin along the tubule is insufficiently demonstrated by Figure 1–figure supplement 2B. These figures do not sufficiently explain the β-catenin activity gradient</italic>.</p><p>This is addressed in the response to the joint review.</p><p><italic>Reviewer #3</italic>:</p><p><italic>In this manuscript, the authors investigate pattern formation of the nephron. Although nephron development is a relatively neglected area of research, more is known regarding its development than the authors sometimes claim. Nonetheless, this manuscript provides a far more detailed and integrated view of nephron pattern formation than any other study to date. First, the authors conduct a detailed series of experiments showing that a beta-catenin activity gradient determines positional identity in a morphogenetic manner. This is shown primarily by using pharmacological inhibitors/activators of Wnt signaling along with a reporter of beta-catenin activity. The authors do a nice job verifying the specificity and fidelity of their system. Importantly, this pharmacological study is further supported genetically using various hypomorphic alleles of beta-catenin. The authors then show that, similar to the intestine, nephron patterning is further regulated through a BMP/PI3K network, which acts in modulating the more central-acting beta-catenin activity. Given the structural and functional similarities between the nephron and intestine, this is an attractive model. The authors also attempt to link their data with Notch signaling, which is also known to regulate nephron patterning. This section is somewhat confusing and is not well-integrated with the first two major findings. Overall, the authors have provided a more comprehensive study of nephron patterning than any previous study, and their study sheds some light on its subject that could not be gleaned from the current literature</italic>.</p><p>We also thank this reviewer for the kind words.</p><p><italic>The results showing no changes in proliferation or apoptosis should be better explained and integrated into the manuscript. Do the authors suggest this implies that differentiation is mainly responsible for the patterning effects? Actually, changes in proliferation and apoptosis are well-known mechanisms for regulating morphogenesis (along with differentiation). At any rate, the authors should better clarify the purpose of these experiments and what they conclude from the results</italic>.</p><p>We now explain this in more detail in the Discussion.</p><p><italic>The finding that the PI3K pathway is regulating beta-catenin is interesting. These pathways are known to interact, especially in the intestinal and hematopoietic systems, where Akt can activate beta-catenin by phosphorylation at Ser552. It would be interesting to see whether and where pS552-bcat positive cells were present during nephron patterning. Such data could further support the author's model</italic>.</p><p>As discussed above, this experiment has been added in the new manuscript and has indeed strengthened our model. We thank the reviewer for this suggestion.</p><p><italic>The authors sometimes discount previous findings. For instance, they claim that “The nephron β-catenin activity gradient is to our knowledge the first instance of a β-catenin activity gradient being described in any system”. This is surprising since such gradients have been described in multiple systems, including mammals</italic>.</p><p>This is addressed in the response to the joint review.</p><p><italic>The authors should consider either better integrating and explaining the Notch data or removing this section from the manuscript. It would be appropriate to discuss what's known about Notch and how it might relate to their new findings in the Discussion section; however, the current presentation tends to leave the reader more confused than enlightened</italic>.</p><p>As discussed in the response to the joint review, we have added data to strengthen the links between Notch, PI3K and β-catenin and this has greatly improved the robustness of our proposed model.</p><p>We have also introduced new data showing 60x 1 µm scans through TCF/LEF nephrons treated with DAPT and in combination with Ly294002 and IWR1. These new data show how in DAPT treated nephrons the whole nephron is positive of GFP. It is not clear whether this is a cause or effect since DAPT treatment stops the proximal segments from forming. IWR1 treatment on top of DAPT treatment effectively reduces the signaling further and this might explain why some Wt1+ cells are now rescued. DAPT+ Ly29 does not clearly reduce GFP expression but it does rescue growth and expression of Jag1.</p></body></sub-article></article> |