elife01832.xml

<?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 article-type="research-article" dtd-version="1.1d1" xmlns:xlink="http://www.w3.org/1999/xlink"><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">01832</article-id><article-id pub-id-type="doi">10.7554/eLife.01832</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>ErbB expressing Schwann cells control lateral line progenitor cells via non-cell-autonomous regulation of Wnt/β-catenin</article-title></title-group><contrib-group><contrib contrib-type="author" id="author-9029"><name><surname>Lush</surname><given-names>Mark E</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="other" rid="par-1"/><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-8858"><name><surname>Piotrowski</surname><given-names>Tatjana</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="other" rid="par-2"/><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf1"/></contrib><aff id="aff1"><institution>Stowers Institute for Medical Research</institution>, <addr-line><named-content content-type="city">Kansas City</named-content></addr-line>, <country>United States</country></aff><aff id="aff2"><institution content-type="dept">Department of Neurobiology and Anatomy</institution>, <institution>University of Utah School of Medicine</institution>, <addr-line><named-content content-type="city">Salt Lake City</named-content></addr-line>, <country>United States</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Whitfield</surname><given-names>Tanya T</given-names></name><role>Reviewing editor</role><aff><institution>University of Sheffield</institution>, <country>United Kingdom</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>pio@stowers.org</email></corresp></author-notes><pub-date date-type="pub" publication-format="electronic"><day>18</day><month>03</month><year>2014</year></pub-date><pub-date pub-type="collection"><year>2014</year></pub-date><volume>3</volume><elocation-id>e01832</elocation-id><history><date date-type="received"><day>06</day><month>11</month><year>2013</year></date><date date-type="accepted"><day>13</day><month>02</month><year>2014</year></date></history><permissions><copyright-statement>© 2014, Lush and Piotrowski</copyright-statement><copyright-year>2014</copyright-year><copyright-holder>Lush and Piotrowski</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/3.0/"><license-p>This article is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/3.0/">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="elife01832.pdf"/><abstract><object-id pub-id-type="doi">10.7554/eLife.01832.001</object-id><p>Proper orchestration of quiescence and activation of progenitor cells is crucial during embryonic development and adult homeostasis. We took advantage of the zebrafish sensory lateral line to define niche-progenitor interactions to understand how integration of diverse signaling pathways spatially and temporally regulates the coordination of these processes. Our previous studies demonstrated that Schwann cells play a crucial role in negatively regulating lateral line progenitor proliferation. Here we demonstrate that ErbB/Neuregulin signaling is not only required for Schwann cell migration but that it plays a continued role in postmigratory Schwann cells. ErbB expressing Schwann cells inhibit lateral line progenitor proliferation and differentiation through non-cell-autonomous inhibition of Wnt/β-catenin signaling. Subsequent activation of Fgf signaling controls sensory organ differentiation, but not progenitor proliferation. In addition to the lateral line, these findings have important implications for understanding how niche-progenitor cells segregate interactions during development, and how they may go wrong in disease states.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01832.001">http://dx.doi.org/10.7554/eLife.01832.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.01832.002</object-id><title>eLife digest</title><p>All the different types of cells that make up the body of an animal are descended from a single fertilized egg. As this egg develops into an embryo, the cells divide and specialize to become a specific type of cell, such as: a liver cell, a muscle cell or a nerve cell. The cells in the embryo that are destined to become specific cell types are called progenitor cells. However, these cells are also found within adult tissues, where they wait until they are needed to replace old or damaged cells.</p><p>Zebrafish are commonly used in scientific research and, like other fish, they have a ‘lateral line’ that runs along both sides of the body and contains cells that detect movements in the surrounding water. During its development, the lateral line contains many progenitors that are primed to form more of these sense organs. The lateral line is also connected to nerve cells that relay information about water movements to the central nervous system, while other cells called Schwann cells support the nerve cells. The local environment or ‘niche’ created by the Schwann cells is known to prevent the progenitor cells within the lateral line from becoming their specific cell type too early. However, the molecules that cause progenitor cells to stop dividing, and later restart dividing and change in to their predestined cell type is not well understood.</p><p>Now Lush and Piotrowski have discovered that signaling through a protein called ErbB causes the Schwann cells to multiply, but has the opposite effect on nearby progenitor cells in the lateral line. ErbB signaling in the Schwann cells inhibited various signaling pathways in the progenitor cells; and whilst some of these pathways normally encourage the progenitors to multiply, others cause them to change into their specific cell type.</p><p>The findings of Lush and Piotrowski have important implications for understanding how the interactions between progenitor cells and the cells around them affect their development. These findings may be useful for understanding diseases caused when the control of cell multiplication or cell-type changes goes awry—such as developmental abnormalities or cancer.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01832.002">http://dx.doi.org/10.7554/eLife.01832.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>neuromast</kwd><kwd>glia</kwd><kwd>stem cells</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd>zebrafish</kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution>Huntsman Cancer Institute Multidisciplinary Cancer Research Training Program Grant, University of Utah</institution></institution-wrap></funding-source><award-id>5T32CA093247</award-id><principal-award-recipient><name><surname>Lush</surname><given-names>Mark E</given-names></name></principal-award-recipient></award-group><award-group id="par-2"><funding-source><institution-wrap><institution>Institutional Support from the Stowers Institute for Medical Research</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Piotrowski</surname><given-names>Tatjana</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</meta-value></custom-meta><custom-meta specific-use="meta-only"><meta-name>Author impact statement</meta-name><meta-value>Schwann cell induced quiescence of mechanosensory progenitor cells is mediated by inhibition of Wnt/β-catenin signaling.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="s1" sec-type="intro"><title>Introduction</title><p>The cell signaling events that govern progenitor quiescence, activation and differentiation are incompletely understood, but emerging data in many tissues indicate that dynamic interactions between progenitors and specialized niche environments play key roles in regulating the properties of progenitor pools. Hence, understanding niche-progenitor interactions at the cellular level is crucial for building a general understanding of this process. The development of the zebrafish lateral line is an excellent model system to study progenitor cell regulation, as it consists of relatively few cells that are easily accessible and amenable to experimental manipulations.</p><p>The sensory organs of the lateral line are called neuromasts. Neuromasts contain support and mechanosensory hair cells that detect water motion. The first set of neuromasts is laid down by a migrating primordium (primI) that develops from a placode just posterior to the otic vesicle. As the primordium migrates posteriorly along the trunk of the embryo it deposits five to six primary neuromasts and a chain of interneuromast cells that connects each neuromast (<xref ref-type="bibr" rid="bib19">Ghysen and Dambly-Chaudiere, 2007</xref>). Before the placode becomes migratory, its anterior portion splits off and forms the posterior lateral line ganglion (<xref ref-type="bibr" rid="bib53">Northcutt and Brandle, 1995</xref>). Lateral line axons closely follow the migrating primordium and eventually innervate deposited neuromasts (<xref ref-type="bibr" rid="bib20">Gilmour et al., 2004</xref>). In turn, neural crest-derived Schwann cells migrate along the axons which they eventually myelinate (<xref ref-type="bibr" rid="bib21">Gilmour et al., 2002</xref>; <xref ref-type="bibr" rid="bib41">Lyons et al., 2005</xref>). Thus, interneuromast cells, axons and Schwann cells are in close contact during the early stages of lateral line development (see diagram in <xref ref-type="fig" rid="fig1">Figure 1A</xref>; <xref ref-type="bibr" rid="bib75">Whitfield, 2005</xref>).<fig-group><fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.01832.003</object-id><label>Figure 1.</label><caption><title>Illustration of cell types in the migrating lateral line.</title><p>(<bold>A</bold>) As the primordium migrates it deposits neuromasts and a chain of interneuromast cells (green cells). Pioneer axons (yellow line) of the posterior lateral line ganglion grow out with the primordium. Schwann cells (red cells) migrate and proliferate along axons. <italic>nrg1-3</italic><sup><italic>z26</italic></sup> mutants and pharmacological inhibition of ErbB signaling mimics the <italic>erbb</italic> phenotype. (<bold>B</bold>–<bold>E</bold>) Double in situ hybridization was performed to label Schwann cells with <italic>myelin basic protein</italic> (<italic>mbp)</italic> and neuromasts with <italic>klf4</italic> at 5 dpf. (<bold>B</bold>) Control siblings with Schwann cells (arrows) along the lateral line nerve and normal neuromast number. <italic>nrg1-3</italic><sup><italic>z26</italic></sup> mutants mimic <italic>erbb2</italic> and <italic>erbb3b</italic> mutants in that they lack Schwann cells along the lateral line and have increased neuromast number (<bold>C</bold>). The brown cells along the midline in both sibling and <italic>nrg1-3</italic><sup><italic>z26</italic></sup> are pigment cells. (<bold>D</bold> and <bold>E</bold>) Double in situ hybridization for <italic>mbp</italic> and <italic>klf4</italic> in DMSO or AG1478 treated larvae from 50 hpf. Compared to DMSO treatment (<bold>D</bold>), increased neuromasts are seen in AG1478 treated larvae (<bold>E</bold>). <italic>mbp</italic> expression along the midline shows that Schwann cells (arrows) are still present at 5 dpf when AG1478 was given at 50 hpf (<bold>E</bold>), compare to DMSO treated (<bold>D</bold>).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01832.003">http://dx.doi.org/10.7554/eLife.01832.003</ext-link></p></caption><graphic xlink:href="elife01832f001"/></fig><fig id="fig1s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01832.004</object-id><label>Figure 1—figure supplement 1.</label><caption><title>Mutations in the <italic>erbb</italic> signaling pathway show precocious neuromast formation by 5 dpf.</title><p>Alkaline phosphatase staining of control (<bold>A</bold>), <italic>erbb2</italic> (<bold>B</bold>), <italic>erbb3b</italic> (<bold>C</bold>) and <italic>nrg1-3</italic><sup><italic>z26</italic></sup> (<bold>D</bold>) zebrafish at 5 dpf. Quantification of alkaline phosphatase stained larvae shows significant increase in neuromast number in all mutants compared to control siblings (<bold>E</bold>, Student's <italic>t</italic>-test, p≤1.0E<sup>−44</sup>). (<bold>F</bold>) AG1478 induces extra neuromasts even if given after Schwann cell migration is complete. AG1478 was added at 24, 50, 59, 72, or 80 hpf and neuromasts were counted at 5 dpf. For the negative control DMSO was added at 24 hpf only. AG1478 induces a significant increase in neuromasts if given up to 59 hpf (<bold>F</bold>, one-way ANOVA with Tukey pairwise comparison, p≤4.0 E<sup>−6</sup>).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01832.004">http://dx.doi.org/10.7554/eLife.01832.004</ext-link></p></caption><graphic xlink:href="elife01832fs001"/></fig><fig id="fig1s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01832.005</object-id><label>Figure 1—figure supplement 2.</label><caption><title><italic>nrg1-3</italic><sup><italic>z26</italic></sup> mutants have defects in adult pigment pattern.</title><p>Control siblings at one month of age show typical stripe pattern of melanophores (<bold>A</bold>–<bold>A′</bold>). <italic>nrg1-3</italic><sup><italic>z26</italic></sup> at 1-month-old show patchy placement of melanophores in the anterior trunk with a more adult like pattern in the posterior region reminiscent of <italic>erbb3b</italic> mutants (<bold>B</bold>–<bold>B′</bold>).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01832.005">http://dx.doi.org/10.7554/eLife.01832.005</ext-link></p></caption><graphic xlink:href="elife01832fs002"/></fig><fig id="fig1s3" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01832.006</object-id><label>Figure 1—figure supplement 3.</label><caption><title><italic>nrg1-3</italic><sup><italic>z26</italic></sup> mutants lose neuromasts as they age.</title><p>Control sibling <italic>Tg(SqET20:gfp)</italic> (<bold>A</bold>) or <italic>nrg1-3</italic><sup><italic>z26</italic></sup><italic>/Tg(SqET20:gfp)</italic> (<bold>B</bold>), were imaged at 1 month of age. Neuromasts that stay along the midline can be seen in control siblings (<bold>A</bold>, arrowhead). These neuromasts are lost from the more posterior region in <italic>nrg1-3</italic><sup><italic>z26</italic></sup> adult zebrafish (<bold>B</bold>, arrowhead). Similarly neuromasts are also lost from the more ventral lateral line (arrows), which are mostly derived from primI, in <italic>nrg1-3</italic><sup><italic>z26</italic></sup> (<bold>B</bold>)<italic>.</italic> (<bold>C</bold>–<bold>D′</bold>) At 4 months of age the degeneration of neuromasts is even more severe. In controls at four months multiple stitches of neuromasts can be seen after DASPEI staining along the ventral line (<bold>C</bold>) and tail fin (<bold>C′</bold>). <italic>nrg1-3</italic><sup><italic>z26</italic></sup> have no ventral lateral line (<bold>D</bold>) or tail fin (<bold>D′</bold>) neuromasts remaining at 4 months.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01832.006">http://dx.doi.org/10.7554/eLife.01832.006</ext-link></p></caption><graphic xlink:href="elife01832fs003"/></fig><fig id="fig1s4" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01832.007</object-id><label>Figure 1—figure supplement 4.</label><caption><title>ErbB inhibition after lateral line migration is complete causes a decrease in proliferation and number of lateral line Schwann cells.</title><p>BrdU plus DMSO or AG1478 was given to <italic>Tg(foxd3:gfp)</italic> fish at 48 hpf then fixed at 6, 14, or 24 hr post treatment. BrdU index is decreased (<bold>A</bold>, Student's <italic>t</italic>-test, p=0.0006), but Schwann cell number is normal 6 hr post AG1478 treatment (<bold>B</bold>, Student's t-test, p=0.16). There is an average count of 12 or 13 Schwann cells in control or AG1478 treatments respectively. At 14 hr post AG1478 treatment both BrdU index (<bold>C</bold>, Student's <italic>t</italic>-test, p=5.8 E<sup>−6</sup>), and Schwann cell numbers are decreased (<bold>D</bold>, Student's <italic>t</italic>-test, p=0.0008). There is an average count of 20 or 14 Schwann cells in control or AG1478 treatments respectively. By 24 hr post treatment, BrdU index (<bold>E</bold>, Student's <italic>t</italic>-test, p=2.6E<sup>−19</sup>), and Schwann cell numbers (<bold>F</bold>, Student's <italic>t</italic>-test, p=2.4E<sup>−28</sup>), are even more decreased by AG1478 compared to DMSO. There is an average count of 14 or 6 Schwann cells in control or AG1478 treatments respectively.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01832.007">http://dx.doi.org/10.7554/eLife.01832.007</ext-link></p></caption><graphic xlink:href="elife01832fs004"/></fig></fig-group></p><p>The adult posterior lateral line contains many more neuromasts than the 7–8 neuromasts initially laid down by primI. These additional ‘secondary’ neuromasts originate from several sources. (A) A second primordium, primII, develops at 40 hr post fertilization (hpf) and deposits neuromasts in between the previously deposited sensory organs (<xref ref-type="bibr" rid="bib63">Sapede et al., 2002</xref>; <xref ref-type="bibr" rid="bib54">Nunez et al., 2009</xref>). (B) Intercalary neuromasts arise during the first 2 weeks of development by proliferation and differentiation of primI deposited interneuromast cells (<xref ref-type="bibr" rid="bib63">Sapede et al., 2002</xref>; <xref ref-type="bibr" rid="bib22">Grant et al., 2005</xref>; <xref ref-type="bibr" rid="bib40">Lopez-Schier and Hudspeth, 2005</xref>; <xref ref-type="bibr" rid="bib54">Nunez et al., 2009</xref>). and (C) During juvenile stages neuromast stitches arise through budding from primary neuromasts (<xref ref-type="bibr" rid="bib37">Ledent, 2002</xref>; <xref ref-type="bibr" rid="bib72">Wada et al., 2013a</xref>).</p><p>We and others have previously shown that Schwann cells play a crucial role in negatively regulating the timing of differentiation of interneuromast cells into intercalary neuromasts (<xref ref-type="bibr" rid="bib22">Grant et al., 2005</xref>; <xref ref-type="bibr" rid="bib40">Lopez-Schier and Hudspeth, 2005</xref>). In zebrafish that lack Schwann cells along the lateral line, such as in mutants for <italic>sox10</italic> and the ErbB pathway members <italic>erbb2</italic>, <italic>erbb3b and nrg1-3,</italic> intercalary neuromasts form precociously (<xref ref-type="bibr" rid="bib22">Grant et al., 2005</xref>; <xref ref-type="bibr" rid="bib62">Rojas-Munoz et al., 2009</xref>; <xref ref-type="bibr" rid="bib58">Perlin et al., 2011</xref>). As Schwann cells require axons for migration along the lateral line, <italic>neurogenin</italic> mutants that lack a posterior lateral line ganglion, also show extra neuromasts (<xref ref-type="bibr" rid="bib40">Lopez-Schier and Hudspeth, 2005</xref>). Likewise, extra neuromasts form after posterior lateral line ganglion extirpation or Schwann cell ablation (<xref ref-type="bibr" rid="bib22">Grant et al., 2005</xref>; <xref ref-type="bibr" rid="bib40">Lopez-Schier and Hudspeth, 2005</xref>). These experiments suggest that Schwann cells contribute to an inhibitory niche that keeps lateral line progenitor cells from undergoing precocious proliferation and differentiation.</p><p>The signaling pathways that orchestrate intercalary neuromast formation are currently unknown. In contrast, the early development of the migrating lateral line has been extensively studied. Complex cell signaling interactions between Wnt/β-catenin, Fgf, Notch and chemokine pathways regulate proliferation, neuromast formation and migration (<xref ref-type="bibr" rid="bib4">Aman and Piotrowski, 2009</xref>; <xref ref-type="bibr" rid="bib42">Ma and Raible, 2009</xref>; <xref ref-type="bibr" rid="bib12">Chitnis et al., 2012</xref>). Wnt/β-catenin signaling in the leading region of the primordium initiates and restricts Fgf signaling to the trailing region. In turn, Fgf signaling upregulates <italic>dkk1b</italic>, a secreted Wnt/β-catenin inhibitor, that restricts Wnt/β-catenin signaling to the leading region (<xref ref-type="bibr" rid="bib3">Aman and Piotrowski, 2008</xref>). Fgf signaling induces apical constriction in clusters of cells resulting in the morphogenesis of rosette shaped protoneuromasts (<xref ref-type="bibr" rid="bib36">Lecaudey et al., 2008</xref>; <xref ref-type="bibr" rid="bib52">Nechiporuk and Raible, 2008</xref>). Fgf signaling is also required for hair cell differentiation (<xref ref-type="bibr" rid="bib47">Millimaki et al., 2007</xref>; <xref ref-type="bibr" rid="bib52">Nechiporuk and Raible, 2008</xref>), and both Wnt/β-catenin and Fgf signaling are required for proliferation within the migrating primordium (<xref ref-type="bibr" rid="bib2">Aman et al., 2011</xref>).</p><p>This study focuses on the development of intercalary neuromasts to elucidate the molecules that regulate progenitor cell proliferation and development. We characterize the signaling pathways required for precocious intercalary neuromast formation downstream of ErbB signaling. In the absence of Schwann cells, or ErbB/Neuregulin signaling, Wnt/β-catenin and Fgf signaling are increased. Wnt/β-catenin signaling is required for interneuromast proliferation while Fgf signaling is required for subsequent rosette formation and cellular differentiation. Schwann cells maintain interneuromast cells as quiescent progenitors by expressing a, as yet unidentified Wnt/β-catenin inhibitor. These findings illustrate the intricate manner in which diverse signaling pathways coordinate distinct aspects of the niche-progenitor interaction needed to maintain the proper balance and timing of this dynamic cell population.</p></sec><sec id="s2" sec-type="results"><title>Results</title><sec id="s2-1"><title>Mutations in ErbB/Neuregulin pathway members cause precocious differentiation of intercalary neuromasts</title><p>Intercalary neuromasts arise during a 2-week period from interneuromast cells, which are initially deposited by primI as a chain of single cells in between primary neuromasts (<xref ref-type="bibr" rid="bib22">Grant et al., 2005</xref>). The cellular relationships within the migrating lateral line are outlined in <xref ref-type="fig" rid="fig1">Figure 1A</xref>. Deposited interneuromast cells are initially in close contact with Schwann cells (<xref ref-type="fig" rid="fig1">Figure 1A</xref>, green and red cells respectively). A variety of lines of genetic evidence, including our own, demonstrates that ErbB signaling plays a fundamental role in the migration of Schwann cells that control the proliferation of interneuromast cells. Mutations in ErbB receptors (<italic>row/erbb2</italic>, <italic>hps/erbb3b</italic>) cause a loss of Schwann cells along the lateral line nerve leading to precocious interneuromast proliferation and intercalary neuromast differentiation (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1A–E</xref>; <xref ref-type="bibr" rid="bib22">Grant et al., 2005</xref>; <xref ref-type="bibr" rid="bib41">Lyons et al., 2005</xref>; <xref ref-type="bibr" rid="bib62">Rojas-Munoz et al., 2009</xref>). We recently identified a mutation in the ErbB ligand <italic>neuregulin 1-3 (nrg1-3</italic><sup><italic>z26</italic></sup><italic>)</italic> that also lacks Schwann cell migration along lateral line axons (<xref ref-type="bibr" rid="bib58">Perlin et al., 2011</xref>), and forms supernumerary neuromasts (<xref ref-type="fig" rid="fig1">Figure 1B–C</xref>). <italic>nrg1-3</italic><sup><italic>z26</italic></sup> mutants survive to adulthood but exhibit an adult pigment pattern and neuromast degeneration phenotype (<xref ref-type="fig" rid="fig1s2 fig1s3">Figure 1—figure supplement 2,3</xref>), similarly to <italic>erbb3b</italic> adult mutant fish (<xref ref-type="bibr" rid="bib7">Budi et al., 2008</xref>; <xref ref-type="bibr" rid="bib27">Honjo et al., 2011</xref>). Below we identified in which cell types different members of the ErbB/Neuregulin pathway are functioning to control Schwann cell migration and lateral line progenitor proliferation and differentiation.</p></sec><sec id="s2-2"><title>Pharmacological inhibition of ErbB signaling mimics the <italic>erbb2/3b</italic> mutant phenotype</title><p>During development, signaling pathways are repeatedly employed. We therefore wanted to test if the extra neuromast phenotype is due solely to loss of Schwann cells along the lateral line, or if ErbB signaling plays an additional role in inhibiting proliferation of interneuromast cells. Therefore, ErbB signaling was inhibited with the ErbB tyrosine kinase inhibitor AG1478 (<xref ref-type="bibr" rid="bib55">Osherov and Levitzki, 1994</xref>), before (24 hpf) and after (48 hpf) completion of Schwann cell migration, and neuromast number was assessed at 5 days post fertilization (dpf). As expected, inhibition of ErbB signaling at 24 hpf, when Schwann cells migrate, leads to a loss of Schwann cells and the formation of extra neuromasts (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1F</xref>; <xref ref-type="bibr" rid="bib62">Rojas-Munoz et al., 2009</xref>). Interestingly, ErbB inhibition is able to increase neuromast numbers even in the presence of Schwann cells, if supplied between 50–59 hpf (<xref ref-type="fig" rid="fig1">Figure 1D–E</xref>, <xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1F</xref>). The presence of Schwann cells is based on detection of <italic>myelin basic protein</italic> (<italic>mbp)</italic> expression (<xref ref-type="fig" rid="fig1">Figure 1D–E</xref>, arrows). These data suggest that ErbB signaling not only regulates Schwann cell migration but also plays a continued role in post-migratory Schwann cells in inhibiting interneuromast cell proliferation.</p><p>A potential caveat for that interpretation is that ErbB signaling is also required for Schwann cell proliferation (<xref ref-type="bibr" rid="bib41">Lyons et al., 2005</xref>; <xref ref-type="bibr" rid="bib59">Raphael et al., 2011</xref>), and pharmacologically lowering the number of Schwann cells could secondarily affect interneuromast proliferation. To test when Schwann cell numbers are reduced upon ErbB inhibition at 48 hpf we used the <italic>Tg(foxd3:gfp)</italic> zebrafish line that expresses EGFP in neural crest derived tissues including Schwann cells (<xref ref-type="bibr" rid="bib21">Gilmour et al., 2002</xref>). Using BrdU labeling in control and AG1478 treated <italic>Tg(foxd3:gfp)</italic> fish, we counted BrdU positive <italic>Tg(foxd3:gfp)</italic> Schwann cells at 6, 14 or 24 hr post treatment. ErbB inhibition induces a decrease in BrdU incorporation in Schwann cells at 6 hr post treatment, however the total Schwann cell number remains unchanged (<xref ref-type="fig" rid="fig1s4">Figure 1—figure supplement 4A–B</xref>). A reduction in Schwann cell proliferation continues at 14 and 24 hr post treatment, at which point it is accompanied by a decrease in total Schwann cell numbers (<xref ref-type="fig" rid="fig1s4">Figure 1—figure supplement 4C–F</xref>). The finding that Schwann cell numbers are not affected at 6 hr post treatment is important, as the first molecular changes in interneuromast cells are already observed at this stage (see below, 'Wntβ-catenin signaling activation occurs prior to Notch and Fgf activation within interneuromast cells after ErbB inhibition'). This suggests that ErbB signaling affects lateral line proliferation directly, rather than indirectly via the regulation of Schwann cell number. Thus, ErbB signaling has independent functions in Schwann cell migration and lateral line progenitor proliferation and differentiation. To elucidate if ErbB signaling controls progenitor proliferation cell-autonomously or non-cell-autonomously, we performed transplantation experiments between mutant and wild type embryos.</p></sec><sec id="s2-3"><title>ErbB2 is required in Schwann cells and Nrg1-3 is required in lateral line axons to inhibit precocious formation of intercalary neuromasts</title><p>Transplantation experiments revealed that <italic>erbb3b</italic> and <italic>sox10</italic> act cell-autonomously in Schwann cells to regulate their migration and inhibit precocious interneuromast proliferation (<xref ref-type="bibr" rid="bib22">Grant et al., 2005</xref>). As <italic>erbb2</italic> is ubiquitously expressed and AG1478 blocks ErbB signaling globally, we wanted to clarify in which cell type ErbB2 signaling is required. We transplanted dextran-Alexa568 labeled cells from <italic>erbb2</italic> mutant blastomere stage donor embryos into wild type <italic>Tg(foxd3:gfp)</italic> host embryos and analyzed clones that gave rise to interneuromast cells. <italic>erbb2</italic> mutant interneuromast cell clones failed to produce extra neuromasts by 4 dpf, suggesting that ErbB2 signaling is not required in interneuromast cells to prevent intercalary neuromast formation (<xref ref-type="fig" rid="fig2">Figure 2A–A′</xref>, n = 0/4). On the other hand, when transplanted <italic>Tg(foxd3:gfp)</italic> wild type cells gave rise to Schwann cell clones in an <italic>erbb2</italic> mutant host embryo, the extra neuromasts phenotype at 4 dpf was rescued (<xref ref-type="fig" rid="fig2">Figure 2B–B“’</xref>, n = 9/9). Rescue was only achieved when Schwann cell clones extended all the way along the trunk by 48 hpf. Transplanted cells that only gave rise to interneuromast cells, or a few Schwann cells that did not reach the tail tip, failed to rescue the <italic>erbb2</italic> mutant phenotype (n = 0/12). These transplant experiments illustrate that, similar to ErbB3b, ErbB2 is required in Schwann cells to inhibit intercalary neuromast formation.<fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.01832.008</object-id><label>Figure 2.</label><caption><title>Transplantation and transgenic analysis demonstrates that ErbB2 is required within Schwann cells and Nrg1-3 within lateral line neurons to inhibit extra neuromast formation.</title><p>(<bold>A</bold> and <bold>A’</bold>) Alexa-568 dextran (red) labeled <italic>erbb2</italic> mutant cells were transplanted into <italic>Tg(foxd3:gfp)</italic> (green) wild type fish. (<bold>A</bold>) High magnification view shows <italic>erbb2</italic> interneuromast and mantle cells along the lateral line and around neuromasts (arrows). These <italic>erbb2</italic> interneuromast cells fail to induce extra neuromasts by 4 dpf (<bold>A’</bold>). (<bold>B</bold>–<bold>B’’’</bold>) Alexa-568 dextran (red)/<italic>Tg(foxd3:gfp)</italic> (green) wild type cells were transplanted into <italic>erbb2</italic> mutant host. At 4 dpf neuromast were labeled by DASPEI staining (green). (<bold>B</bold> and <bold>B’</bold>) On the untransplanted side there are no Schwann cells and eighteen neuromasts. (<bold>B’’</bold> and <bold>B’’’</bold>) On the transplanted side you can see complete migration of wild type Schwann cells in an otherwise <italic>erbb2</italic> mutant fish and rescue of neuromast number (arrows). (<bold>C</bold> and <bold>D</bold>) Dominant negative ErbB receptor expression in neural crest derived cells mimics <italic>erbb</italic> mutant phenotype. (<bold>C</bold>) Control <italic>Tg(SqET20:gfp)</italic> siblings at 4 dpf. (<bold>D</bold>) <italic>Tg(SqET20:gfp)/Tg(sox10:DNerbb4)</italic> showing extra neuromasts. (<bold>E</bold>–<bold>E’’’</bold>) Alexa-568 dextran (red)/<italic>Tg(clndB:lyngfp)</italic>(green) wild type cells were transplanted into <italic>nrg1-3</italic><sup><italic>z26</italic></sup> mutant host. (<bold>E</bold>–<bold>E’</bold>) At 4 dpf the untransplanted side has nineteen neuromasts. (<bold>E’’</bold> and <bold>E’’’</bold>) On the transplanted side there are GFP labeled axons (arrowhead) along the entire length of the lateral line with rescue of neuromast number. All axons cannot be seen because some are obscured underneath pigment cells.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01832.008">http://dx.doi.org/10.7554/eLife.01832.008</ext-link></p></caption><graphic xlink:href="elife01832f002"/></fig></p><p>We confirmed these results genetically by generating a transgenic zebrafish line that drives human dominant-negative ErbB4 (DNErbB4) in neural crest-derived tissues, including Schwann cells, using the <italic>sox10</italic> promoter. DNErbB4 blocks Neuregulin-induced signaling in cell culture and in vivo (<xref ref-type="bibr" rid="bib61">Rio et al., 1997</xref>; <xref ref-type="bibr" rid="bib11">Chen et al., 2006</xref>). We isolated a stable transgenic line for analysis designated <italic>Tg(sox10:DNhsaerbb4-rfp),</italic> from now on called <italic>Tg(sox10:DNerbb4)</italic>. As expected, in transgenic embryos Schwann cells fail to migrate along the lateral line and they develop extra neuromasts (data not shown and <xref ref-type="fig" rid="fig2">Figure 2C–D</xref>). Transgenic fish survive to adulthood, are fertile and exhibit no other obvious phenotypes. Along with the transplantation experiments these results demonstrate that ErbB2/3b signaling is required in Schwann cells in order to non-cell-autonomously regulate neuromast number.</p><p><italic>nrg1-3</italic> is expressed in lateral line ganglia suggesting that it is required in lateral line axons (<xref ref-type="bibr" rid="bib58">Perlin et al., 2011</xref>). To functionally test in which cell type Nrg1-3 is required we performed transplantation experiments between wild type and <italic>nrg1-3</italic><sup><italic>z26</italic></sup> embryos. As donors we used <italic>Tg(cldnb:lyngfp)</italic> embryos that express EGFP in all lateral line cells including the ganglion (<xref ref-type="bibr" rid="bib24">Haas and Gilmour, 2006</xref>)<italic>.</italic> We rescued the extra neuromast phenotype in <italic>nrg1-3</italic><sup><italic>z26</italic></sup> mutants with transplanted wild type cells that gave rise to large posterior lateral line ganglion clones (<xref ref-type="fig" rid="fig2">Figure 2E–E</xref>’’’, n = 13/13). Transplanted cells that only contributed to interneuromast cells or to few lateral line ganglion neurons failed to rescue <italic>nrg1-3</italic><sup><italic>z26</italic></sup> (n = 0/19). This is consistent with prior findings that wild type posterior lateral line ganglion clones rescue Schwann cell migration in <italic>nrg1-3</italic><sup><italic>z26</italic></sup> mutant embryos (<xref ref-type="bibr" rid="bib58">Perlin et al., 2011</xref>). Thus, the ligand Nrg1-3 is required in axons to induce migration and proliferation of ErbB expressing Schwann cells and inhibit precocious formation of intercalary neuromasts.</p><p>Combined, these experiments revealed that the quiescent niche consists of axonal, membrane bound, Nrg1-3 that signals to ErbB receptors within Schwann cells. In response, Schwann cells send a signal to interneuromast cells that inhibits their precocious differentiation into neuromasts. The following experiments were designed to identify signaling pathways that are regulated in interneuromast cells in response to Schwann cell-derived signals.</p></sec><sec id="s2-4"><title>Proliferation is the first cellular response in interneuromast cells after abrogation of the ErbB pathway</title><p>The identification of interneuromast cell behaviors that are inhibited by ErbB signaling provides clues to which signaling pathways might be regulated by ErbB signaling. To identify the earliest changes in lateral line cell behavior in response to the loss of ErbB signaling we performed time-lapse analyses. We imaged interneuromast cells in Schwann cell-depleted larvae derived from crosses between <italic>Tg(sox10:DNerbb4)</italic> and <italic>Tg(SqET20:gfp). Tg(SqET20:gfp)</italic> larvae express EGFP in neuromast mantle cells and interneuromast cells (<xref ref-type="bibr" rid="bib56">Parinov et al., 2004</xref>). In a 40-hr time-lapse four intercalary neuromasts are formed from interneuromasts cells (<xref ref-type="other" rid="video1">Video 1</xref>). The time-lapse analyses revealed that interneuromast cell proliferation precedes clustering of interneuromast cells. In addition, interneuromast cells are highly motile and migrate into and out of the forming neuromasts. The clusters of interneuromast cells continue to proliferate and differentiate into neuromasts as evident by the mature pattern of a ring of <italic>Tg(SqET20:gfp)</italic> positive mantle cells that surrounds GFP-negative sensory hair cells. In contrast, control <italic>Tg(SqET20:gfp)</italic> larvae show no proliferation and little migration of interneuromast cells during the same time period (<xref ref-type="other" rid="video2">Video 2</xref>). In conclusion, the absence of Schwann cells, leads first to interneuromast cell proliferation, followed by an increase in migration and clustering of interneuromast cells that eventually differentiate into sensory hair and support cells.<media content-type="glencoe play-in-place height-250 width-310" id="video1" mime-subtype="avi" mimetype="video" xlink:href="elife01832v001.avi"><object-id pub-id-type="doi">10.7554/eLife.01832.012</object-id><label>Video 1.</label><caption><title>Time-lapse recording of <italic>Tg(sox10:DNerbb4)/Tg(SqET20:gfp)/Tg(clndB:H2B-mcherry)</italic> during intercalary neuromast formation.</title><p>The time-lapse runs from 32 to 72 hpf. One frame was taken every 7 min. Four intercalary neuromasts form during this time.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01832.012">http://dx.doi.org/10.7554/eLife.01832.012</ext-link></p></caption></media><media content-type="glencoe play-in-place height-250 width-310" id="video2" mime-subtype="avi" mimetype="video" xlink:href="elife01832v002.avi"><object-id pub-id-type="doi">10.7554/eLife.01832.013</object-id><label>Video 2.</label><caption><title>Time-lapse recording of control <italic>Tg(SqET20:gfp)/(Tg(clndB:H2A-mcherry)</italic> from approximately 48–72 hpf.</title><p>One frame was taken every 7 min. No interneuromast cell proliferation is seen during this time.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01832.013">http://dx.doi.org/10.7554/eLife.01832.013</ext-link></p></caption></media></p><p>To be able to correlate cell behavior with gene expression changes (see below), we sought to determine how many hours after ErbB inhibition interneuromast proliferation begins. We added BrdU plus DMSO or AG1478 to <italic>Tg(SqET20:gfp)</italic> larvae at 48 hpf, after Schwann cell migration is completed. After 14 hr of ErbB signaling inhibition there is no significant increase in BrdU incorporation in GFP-positive interneuromast cells (<xref ref-type="fig" rid="fig3">Figure 3A–B,E</xref>). After 24 hr of treatment we detected a significant increase in BrdU labeling in interneuromast cells (<xref ref-type="fig" rid="fig3">Figure 3C–E</xref>). Concurrent with the increase in BrdU incorporation, an increase in interneuromast cells is observed after 24 hr of AG1478 treatment (<xref ref-type="fig" rid="fig3">Figure 3F</xref>). The increase in proliferation begins sometime between 14 and 24 hr post ErbB inhibition.<fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.01832.009</object-id><label>Figure 3.</label><caption><title>ErbB inhibition, after Schwann cell lateral line migration is completed, induces proliferation of interneuromast cells.</title><p>BrdU plus DMSO or AG1478 was given to <italic>Tg(SqET20:gfp)</italic> fish at 48 hpf then fixed at 14 or 24 hr post treatment. Immunohistochemistry for BrdU (red) and GFP (green) reveals no difference in BrdU incorporation within interneuromast cells between DMSO (<bold>A</bold>) or AG1478 (<bold>B</bold>) 14 hr post treatment. At 24 hr post treatment DMSO (<bold>C</bold>) treated fish show little BrdU incorporation while AG1478 (<bold>D</bold>) treated fish show increased BrdU incorporation and interneuromast cell number. Quantification of both BrdU index (<bold>E</bold>, Student’s <italic>t</italic>-test, p=0.18 for 14 hr and p=4.5E<sup>−18</sup> for 24 hr time point) and interneuromast cell number (<bold>F</bold>, Student’s <italic>t</italic>-test, p=0.69 for 14 hr and p=0.003 for 24 hr time point) shows a significant increase with AG1478 only after 24 hr.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01832.009">http://dx.doi.org/10.7554/eLife.01832.009</ext-link></p></caption><graphic xlink:href="elife01832f003"/></fig></p><p>As ErbB signaling acts cell-autonomously in Schwann cells but proliferation occurs in interneuromast cells, we aimed to identify the signaling pathways that are activated in interneuromast cells when ErbB signaling is inhibited.</p></sec><sec id="s2-5"><title>Wnt/β-catenin, Fgf and Notch signaling pathways are upregulated in ErbB/Neuregulin pathway mutants</title><p>The Wnt/β-catenin, Fgf and Notch signaling pathways are excellent candidates for being involved in intercalary neuromast formation as they regulate progenitor cell proliferation in several other organs, such as the CNS (<xref ref-type="bibr" rid="bib39">Logan and Nusse, 2004</xref>; <xref ref-type="bibr" rid="bib23">Guillemot and Zimmer, 2011</xref>; <xref ref-type="bibr" rid="bib33">Koch et al., 2013</xref>). In addition, these three pathways play multiple, crucial roles in the primordium of the lateral line (reviewed in <xref ref-type="bibr" rid="bib4">Aman and Piotrowski, 2009</xref>; <xref ref-type="bibr" rid="bib42">Ma and Raible, 2009</xref>; <xref ref-type="bibr" rid="bib12">Chitnis et al., 2012</xref>). Briefly, Wnt/β-catenin and Fgf signaling regulate cell proliferation, while Fgf is also required for neuromast rosette formation and hair cell differentiation (<xref ref-type="bibr" rid="bib3">Aman and Piotrowski, 2008</xref>; <xref ref-type="bibr" rid="bib36">Lecaudey et al., 2008</xref>; <xref ref-type="bibr" rid="bib52">Nechiporuk and Raible, 2008</xref>; <xref ref-type="bibr" rid="bib2">Aman et al., 2011</xref>). Notch signaling regulates sensory hair cell production and primordium cohesion (<xref ref-type="bibr" rid="bib29">Itoh and Chitnis, 2001</xref>; <xref ref-type="bibr" rid="bib43">Matsuda and Chitnis, 2010</xref>). To test if the Wnt/β-catenin, Fgf and Notch pathways are also involved in the extra neuromast phenotype we performed an in situ expression screen on 48 hpf <italic>nrg1-3</italic><sup><italic>z26</italic></sup> mutant larvae (<xref ref-type="fig" rid="fig4">Figure 4</xref>). The Wnt/β-catenin pathway members <italic>wnt10a</italic>, <italic>lef1</italic>, <italic>myca,</italic> and <italic>β-catenin-2</italic> (<italic>ctnnb2)</italic> are upregulated in interneuromast cells in <italic>nrg1-3</italic><sup><italic>z26</italic></sup> larvae (<xref ref-type="fig" rid="fig4">Figure 4A–H</xref>). <italic>lef1</italic> and <italic>ctnnb2</italic> are also expressed at lower levels in interneuromast cells in control animals while <italic>wnt10a</italic> and <italic>myca</italic> show no expression. The expression of <italic>wnt10a</italic> correlates with the differentiation status of intercalary neuromasts. <italic>wnt10a</italic> is expressed in proliferating interneuromast cells but is down regulated in differentiating neuromasts (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1A–D</xref>). The Notch receptor <italic>notch3</italic> and the Notch target gene <italic>her4.1</italic> are expressed in primary neuromasts in control animals but not in interneuromast cells (<xref ref-type="fig" rid="fig4">Figure 4I,K</xref>, arrows). <italic>notch3</italic> is broadly induced in <italic>nrg1-3</italic><sup><italic>z26</italic></sup> interneuromast cells (<xref ref-type="fig" rid="fig4">Figure 4J</xref>). <italic>her4.1</italic> is also induced in <italic>nrg1-3</italic><sup><italic>z26</italic></sup> but in a more discrete cluster of cells (<xref ref-type="fig" rid="fig4">Figure 4L</xref>, arrowhead). <italic>her4.1</italic> is not expressed in proliferating interneuromast cells but is induced as intercalary neuromasts mature (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1E–H</xref>). This suggests that Notch signaling is only active in differentiating neuromasts. Fgf signaling pathway components are also upregulated in interneuromast cells in <italic>nrg1-3</italic><sup><italic>z26</italic></sup> mutant larvae (<xref ref-type="fig" rid="fig4">Figure 4N,P,R,T</xref>). In control embryos, <italic>fgfr1a</italic>, <italic>fgf3</italic>, <italic>fgf10</italic> and the Fgf target <italic>pea3</italic> only show strong expression in primary neuromasts but not interneuromast cells (<xref ref-type="fig" rid="fig4">Figure 4M,O,Q,S</xref>, arrows), suggesting that, similar to Notch signaling, Fgf signaling might be involved in neuromast differentiation.<fig-group><fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.01832.010</object-id><label>Figure 4.</label><caption><title>Increase in Wnt/β-catenin, Notch and Fgf signaling pathway gene expression in <italic>nrg1-3</italic><sup><italic>z26</italic></sup> mutant interneuromast cells.</title><p>Control siblings and <italic>nrg1-3</italic><sup><italic>z26</italic></sup> mutants were processed for in situ hybridization at 48 hpf. A Wnt ligand, <italic>wnt10a</italic>, is not expressed in control interneuromast cells (<bold>A</bold>) but is increased in <italic>nrg1-3</italic><sup><italic>z26</italic></sup> (<bold>B</bold>). The Wnt/β-catenin target gene <italic>lef1</italic> is expressed in interneuromast cells in control siblings (<bold>C</bold>) but is greatly increased in <italic>nrg1-3</italic><sup><italic>z26</italic></sup> (<bold>D</bold>). An additional Wnt/β-catenin target <italic>myca</italic> shows no expression in interneuromast cells (<bold>E</bold>) but strong expression in clumps of interneuromast cells in <italic>nrg1-3</italic><sup><italic>z26</italic></sup> (<bold>F</bold>). <italic>ctnnb2</italic> shows weak expression in control interneuromast cells (<bold>G</bold>) which is upregulated in <italic>nrg1-3</italic><sup><italic>z26</italic></sup> (<bold>H</bold>). (<bold>I</bold>) In controls, <italic>notch3</italic> is expressed in primary neuromasts (arrow) but not interneuromast cells. (<bold>J</bold>) <italic>notch3</italic> is upregulated in mutant interneuromast cells. (<bold>K</bold>) In controls, the Notch target <italic>her4.1</italic> is expressed in primary neuromasts (arrow) but not interneuromast cells. (<bold>L</bold>) In mutants, <italic>her4.1</italic> is expressed in primary neuromasts (arrow) but is also increased in discrete clusters of cells (arrowhead). In controls the Fgf pathway genes including the receptor <italic>fgfr1a</italic> (<bold>M</bold>), the two ligands <italic>fgf3</italic> (<bold>O</bold>) and <italic>fgf10</italic> (<bold>Q</bold>) and the Fgf target gene <italic>pea3</italic> (<bold>S</bold>) are all expressed in primary neuromasts (arrow) but not in interneuromast cells. All Fgf pathway genes, <italic>fgfr1a</italic> (<bold>N</bold>), <italic>fgf3</italic> (<bold>P</bold>) <italic>fgf10</italic> (<bold>R</bold>) and <italic>pea3</italic> (<bold>T</bold>), retain expression in primary neuromasts (arrow) but are upregulated in interneuromast cells of <italic>nrg1-3</italic><sup><italic>z26</italic></sup>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01832.010">http://dx.doi.org/10.7554/eLife.01832.010</ext-link></p></caption><graphic xlink:href="elife01832f004"/></fig><fig id="fig4s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01832.011</object-id><label>Figure 4—figure supplement 1.</label><caption><title><italic>wnt10a</italic> is expressed in proliferating interneuromast cells while <italic>her4.1</italic> is expressed in differentiating and mature neuromasts.</title><p>Lateral line cells from <italic>Tg(sox10:DNerbb4)/Tg(SqET20:gfp)</italic> were imaged at 48 hpf. Larvae were then fixed at processed for <italic>wnt10a</italic> or <italic>her4.1</italic> in situ hybridization and photographed at the same level. (<bold>A</bold>–<bold>D</bold>) Proliferating interneuromast cells (arrowhead) express higher <italic>wnt10a</italic> than more mature intercalary neuromasts (square). (<bold>E</bold>–<bold>H</bold>) <italic>her4.1</italic> is expressed in more mature intercalary neuromasts (squares), but is absent from more immature proliferating interneuromast cells (arrowhead).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01832.011">http://dx.doi.org/10.7554/eLife.01832.011</ext-link></p></caption><graphic xlink:href="elife01832fs005"/></fig><fig id="fig4s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01832.017</object-id><label>Figure 4—figure supplement 2.</label><caption><title><italic>wnt10a, fgf3</italic> and <italic>fgf10</italic> expression depends on Wnt/β-catenin signaling.</title><p>Control or <italic>Tg(sox10:DNerbb4</italic>) siblings were treated with DMSO, IWR-1, PD173074 or LY411575 at 32 hpf then fixed at 48 hpf for in situ hybridizations. In control siblings, inhibitor treatment has no effect on <italic>wnt10a</italic> (<bold>A</bold>–<bold>D</bold>), <italic>fgf3</italic> (<bold>I</bold>–<bold>L</bold>) or <italic>fgf10</italic> (<bold>Q</bold>–<bold>T</bold>) expression in interneuromast cells. <italic>wnt10a</italic> is strongly induced in <italic>Tg(sox10:DNerbb4</italic>) DMSO (<bold>E</bold>), PD173074 (<bold>G</bold>) or LY411575 (<bold>H</bold>) treated embryos but is greatly inhibited in IWR-1 treated embryos (<bold>F</bold>). Likewise, IWR-1 inhibits <italic>fgf3</italic> (<bold>N</bold>) and <italic>fgf10</italic> (<bold>V</bold>) expression in <italic>Tg(sox10:DNerbb4</italic>). Neither PD173074 or LY411575 effect <italic>fgf3</italic> (<bold>O</bold>–<bold>P</bold>) or <italic>fgf10</italic> expression (<bold>W</bold>–<bold>X</bold>) in <italic>Tg(sox10:DNerbb4</italic>).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01832.017">http://dx.doi.org/10.7554/eLife.01832.017</ext-link></p></caption><graphic xlink:href="elife01832fs006"/></fig><fig id="fig4s3" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01832.018</object-id><label>Figure 4—figure supplement 3.</label><caption><title><italic>wnt10a, fgf3</italic> and <italic>fgf10</italic> expression are induced within interneuromast cells after Wnt/β-catenin activation.</title><p>Wild type zebrafish were treated with DMSO or BIO at 48 hpf then fixed at 6, 12 or 24 hr post treatment for in situ hybridizations. The known Wnt/β-catenin target gene <italic>lef1</italic> is expressed in DMSO treated interneuromast cells (<bold>A</bold>, <bold>C</bold>, <bold>E</bold>) and is greatly increased by BIO treatment (<bold>B</bold>, <bold>D</bold>, <bold>F</bold>). <italic>wnt10a</italic> is not expressed in control interneuromast cells from 6 to 24 hr post treatment (<bold>G</bold>, <bold>I</bold>, <bold>K</bold>). <italic>wnt10a</italic> is induced by BIO within 6 hr post (<bold>H</bold>) and this increase is maintained from 12 to 24 hr post (<bold>J</bold>, <bold>L</bold>). <italic>fgf3</italic> (<bold>M</bold>, <bold>O</bold>, <bold>Q</bold>) and <italic>fgf10</italic> (<bold>S</bold>, <bold>U</bold>, <bold>W</bold>) are not expressed within DMSO treated interneuromast cells. <italic>fgf3</italic> is induced by 24 hr post BIO treatment (<bold>N</bold>, <bold>P</bold>, <bold>R</bold>), while <italic>fgf10</italic> is induced within 12 hr of treatment (<bold>T</bold>, <bold>V</bold>, <bold>X</bold>).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01832.018">http://dx.doi.org/10.7554/eLife.01832.018</ext-link></p></caption><graphic xlink:href="elife01832fs007"/></fig></fig-group></p></sec><sec id="s2-6"><title>Wnt/β-catenin and Fgf pathway members are activated during intercalary neuromast development in wild type larvae</title><p>If increased Wnt/β-catenin and Fgf signaling are important for intercalary neuromast formation in Schwann cell-deficient larvae, these pathways should also be upregulated during post-embryonic intercalary neuromast formation in wild type larvae.</p><p>To enable us to compare gene expression changes with neuromast formation we quantified neuromasts formed between 2–6 dpf. In wild type larvae two primordia form the majority of the posterior lateral line system (<xref ref-type="bibr" rid="bib19">Ghysen and Dambly-Chaudiere, 2007</xref>). PrimI deposits five to six primary neuromasts between 20–40 hpf. PrimII begins migration at around 40 hpf along the same path as primI (<xref ref-type="bibr" rid="bib63">Sapede et al., 2002</xref>; <xref ref-type="bibr" rid="bib54">Nunez et al., 2009</xref>). We counted the number of neuromasts after alkaline phosphatase staining (<xref ref-type="fig" rid="fig5">Figure 5D–G</xref>). The total number of neuromasts increases steadily from six to seven neuromasts at 2 dpf to 12 at 6 dpf (<xref ref-type="fig" rid="fig5">Figure 5A</xref>). Of these 12 neuromasts at 6 dpf, three have been deposited by primII (<xref ref-type="fig" rid="fig5">Figure 5B,E–G</xref>, asterisks). PrimII-derived neuromasts are always located dorsally to the primI-derived chain of interneuromast cells. The first clusters of interneuromast cells that will differentiate into intercalary neuromasts appear by 3 dpf (<xref ref-type="fig" rid="fig5">Figure 5E</xref>, arrowhead). Typically, at least one intercalary neuromast has formed by 4 dpf, with a second formed by 6 dpf (<xref ref-type="fig" rid="fig5">Figure 5C,F–G</xref>, squares). Therefore, any genes crucial for interneuromast proliferation and differentiation should commence expression between 2–3 dpf.<fig-group><fig id="fig5" position="float"><object-id pub-id-type="doi">10.7554/eLife.01832.014</object-id><label>Figure 5.</label><caption><title>Wnt/β-catenin and Fgf signaling target genes are expressed in interneuromast cells during wild type intercalary neuromast formation.</title><p>To see when intercalary neuromasts first arise we alkaline phosphatase stained wild type zebrafish at 2, 3, 4, 5 and 6 dpf. Quantification of total neuromast number shows a steady increase from 2 to 6 dpf (<bold>A</bold>). Most of the increase comes from primII deposited neuromasts (<bold>B</bold>). There is one intercalary neuromast by 4 dpf and two by 6 dpf (<bold>C</bold>). (<bold>D</bold>–<bold>G</bold>) Alkaline phosphatase staining from 2–5 dpf. The images were taken so that the first primI deposited neuromast is always at the left (without a label). Asterisk labels primII-derived neuromasts, the arrowhead labels interneuromast cells and squares label intercalary neuromasts. (<bold>H</bold>–<bold>K</bold>) <italic>lef1</italic> in situ hybridization from 2–5 dpf. <italic>lef1</italic> is expressed in interneuromast cells at 2 dpf (<bold>H</bold>, arrowhead) and is maintained in clumps of interneuromast cells from 3–5 dpf (<bold>I</bold>–<bold>K</bold>, arrowheads) that will likely give rise to intercalary neuromasts. The strong cluster of <italic>lef1</italic> expression at 3 dpf is the leading edge of primII (<bold>I</bold>, arrow). <italic>lef1</italic> is not expressed in mature neuromasts. (<bold>L</bold>–<bold>O</bold>) <italic>beta-catenin2</italic> (<italic>ctnnb2</italic>) expression from 2–5 dpf. Similar to <italic>lef1, ctnnb2</italic> is expressed in interneuromast cells at 2 dpf (L, arrowhead) and is maintained in clumps of interneuromast cells from 3–5 dpf (<bold>M</bold>–<bold>O</bold>, arrowhead). Unlike <italic>lef1</italic>, <italic>ctnnb2</italic> is expressed in primary neuromasts (<bold>M</bold>–<bold>O</bold>, asterisk). (<bold>P</bold>–<bold>S</bold>) <italic>pea3</italic> expression from 2–5 dpf. (<bold>P</bold>) At 2 dpf <italic>pea3</italic> shows strong expression in primary neuromasts but not in interneuromast cells. (<bold>Q</bold>–<bold>R</bold>) At 3 and 4 dpf <italic>pea3</italic> shows expression in primII (arrow) and primII derived neuromasts (asterisk) but still no interneuromast cell expression. At 5 dpf <italic>pea3</italic> can be seen in a few cells near somite boundaries (<bold>S</bold>, arrowhead).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01832.014">http://dx.doi.org/10.7554/eLife.01832.014</ext-link></p></caption><graphic xlink:href="elife01832f005"/></fig><fig id="fig5s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01832.015</object-id><label>Figure 5—figure supplement 1.</label><caption><title><italic>lef1</italic> is expressed in clusters of interneuromast cells before they become intercalary neuromasts.</title><p>Lateral line cells expressing <italic>Tg(SqET20:gfp)</italic> were imaged at 4 or 6 dpf. Larvae were then fixed at processed for <italic>lef1</italic> in situ hybridization and photographed at the same level. (<bold>A</bold>) At 4 dpf a small group of <italic>Tg(SqET20:gfp)</italic> positive interneuromast cells (arrowhead) can be seen between two primII deposited primary neuromasts (asterisk). This small cluster of interneuromast cells is positive for <italic>lef1</italic> (<bold>B</bold>, arrowhead). (<bold>C</bold>) At 6 dpf a larger cluster of interneuromast cells is seen forming. Again this cluster of cells is <italic>lef1</italic> positive (<bold>D</bold>, arrowhead). Interestingly, at 6 dpf one intercalary neuromast has formed (<bold>C</bold>, square), but it is not positive for <italic>lef1</italic> (<bold>D</bold>). Suggesting <italic>lef1</italic> is quickly decreased as neuromasts mature.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01832.015">http://dx.doi.org/10.7554/eLife.01832.015</ext-link></p></caption><graphic xlink:href="elife01832fs008"/></fig></fig-group></p><p>In situ hybridization experiments for the Wnt/β-catenin targets <italic>lef1</italic> and <italic>ctnnb2</italic> revealed that <italic>lef1</italic> and <italic>ctnnb2</italic> are expressed in deposited interneuromast cells at 2 dpf (<xref ref-type="fig" rid="fig5">Figure 5H,L</xref>, arrowheads). Unlike <italic>lef1</italic>, <italic>ctnnb2</italic> is also expressed in differentiating neuromasts (<xref ref-type="fig" rid="fig5">Figure 5M–O</xref>, asterisk). <italic>lef1</italic> and <italic>ctnnb2</italic> are downregulated in interneuromast cells between 3–5 dpf, with the exception of forming clusters of interneuromast cells that will differentiate into intercalary neuromasts (<xref ref-type="fig" rid="fig5">Figure 5I–K,M–O</xref>, arrowheads). To study <italic>lef1</italic> expression at single cell resolution we photographed the lateral line in <italic>Tg(SqEt20:gfp)</italic> larvae at 4 or 6 dpf and then performed <italic>lef1</italic> in situ hybridization on the same larvae (<xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1</xref>). These experiments illustrate that <italic>lef1</italic> is expressed only in the few interneuromast cells that begin to proliferate to form clusters (<xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1A–D</xref>, arrowheads). As <italic>lef1</italic> expression is shut off in mature neuromasts, Wnt/β-catenin signaling is likely involved in the initiation of intercalary neuromast proliferation and not differentiation.</p><p>In contrast, the Fgf target <italic>pea3</italic> is never expressed in interneuromast cells but is strongly expressed in differentiated neuromasts from 2–5 dpf (<xref ref-type="fig" rid="fig5">Figure 5P–S</xref>, asterisks). Discrete clusters of <italic>pea3</italic> expressing interneuromast cells are observed at 5 dpf (<xref ref-type="fig" rid="fig5">Figure 5S</xref>, arrowhead). The expression analyses shows that Wnt/β-catenin pathway activation coincides with interneuromast cell proliferation and cluster formation, whereas Fgf signaling is initiated later, during the differentiation phase of intercalary neuromast formation.</p></sec><sec id="s2-7"><title>Wnt/β-catenin activation occurs prior to Notch and Fgf activation within interneuromast cells after ErbB inhibition</title><p>The expression analyses during wild type intercalary neuromast formation suggest that the onset of Wnt/β-catenin expression precedes Fgf and Notch signaling. To determine the temporal dynamics of pathway activations after ErbB inhibition we blocked ErbB signaling with AG1478 at 48 hpf and fixed larvae at 6, 12, 24 and 36 hr post treatment followed by in situ hybridization. We focused our analysis on interneuromast cells between the first and second deposited primary neuromasts. We performed in situ hybridization with the Wnt/β-catenin target <italic>lef1</italic> and a <italic>dgfp</italic> probe for a transgenic Wnt/β-catenin reporter line, <italic>Tg(Tcf/Lef-miniP:dGFP)</italic> that contains 6 copies of a consensus TCF/Lef binding site followed by destabilized EGFP (<xref ref-type="bibr" rid="bib66">Shimizu et al., 2012</xref>). At 6 and 12 hr post treatment, DMSO treated fish show strong expression of the Wnt/β-catenin reporter in primII (<xref ref-type="fig" rid="fig6">Figure 6A–B</xref>, arrow), and in some interneuromast cells (arrowhead). At 24 hr post DMSO the Wnt/β-catenin reporter is expressed in few interneuromast cells (<xref ref-type="fig" rid="fig6">Figure 6C</xref>, arrowhead). By 36 hr post DMSO the Wnt/β-catenin reporter is only expressed in clusters of newly forming intercalary neuromasts and is downregulated in other interneuromast cells (<xref ref-type="fig" rid="fig6">Figure 6D</xref>, arrowhead). This correlates with the clusters of <italic>lef1</italic> expression seen in wild type fish at 3–5 dpf (<xref ref-type="fig" rid="fig5">Figure 5I–K</xref>). ErbB inhibition induces a large increase of the Wnt/β-catenin reporter expression in interneuromast cells at 6 and 12 hr post treatment (<xref ref-type="fig" rid="fig6">Figure 6E–F</xref>, arrowheads). By 24 and 36 hr post ErbB inhibition the Wnt/β-catenin reporter expression level has decreased and is only maintained in a few clumps of interneuromast cells (<xref ref-type="fig" rid="fig6">Figure 6G–H</xref>, arrowhead). The Wnt/β-catenin reporter is not seen in primary neuromasts (<xref ref-type="bibr" rid="bib73">Wada et al., 2013b</xref>), and is turned off in interneuromast cell clusters as they differentiate into intercalary neuromasts (data not shown). The pattern and timing of <italic>lef1</italic> expression mirrors the Wnt/β-catenin reporter expression, with ErbB inhibition inducing high expression at early stages followed by a gradual decrease (<xref ref-type="fig" rid="fig6">Figure 6I–P</xref>).<fig id="fig6" position="float"><object-id pub-id-type="doi">10.7554/eLife.01832.016</object-id><label>Figure 6.</label><caption><title>Wnt/β-catenin expression precedes Fgf and Notch expression after ErbB inhibition.</title><p>To determine which signaling pathways are induced first we treated wild type zebrafish starting at 48 hpf with DMSO or AG1478 then fixed at 6, 12, 24 and 36 hr post treatment. All images were taken between the first two primary neuromasts. In DMSO treated Wnt reporter Tg(<italic>Tcf/Lef-miniP:dGFP)</italic> strong expression is seen in primII (arrow) at 6 and 12 hr post treatment but also in some interneuromast cells (<bold>A</bold>–<bold>B</bold>, arrowhead). By 12 and 36 hr there are only clumps of interneuromast cells expressing the reporter (<bold>C</bold>–<bold>D</bold>, arrowhead). 6 hr post AG1478 treatment there is a large increase in Wnt reporter expression specifically within interneuromast cells (<bold>E</bold>, arrowhead). This AG1478 induced increased expression is maintained after 12 hr (<bold>F</bold>) but has started to go down by 24 hr (<bold>G</bold>) and is only seen in a few interneuromast cells by 36 hr (<bold>H</bold>). (<bold>I</bold>–<bold>P</bold>) <italic>lef1</italic> mirrors Wnt reporter expression. In DMSO, <italic>lef1</italic> is expressed in primII (arrow) and few interneuromast cells at 6 (<bold>I</bold>) and 12 hr (<bold>J</bold>). At 24 hr post DMSO <italic>lef1</italic> is maintained in a few interneuromast cells (<bold>K</bold>). At 36 hr post DMSO, <italic>lef1</italic> is seen in clumps of cells likely corresponding to normally developing intercalary neuromasts (<bold>L</bold>, arrowhead). AG1478 induces <italic>lef1</italic> within interneuromast cells at 6 (<bold>M</bold>) and 12 hr (<bold>N</bold>). By 24 (<bold>O</bold>) and 36 hr (<bold>P</bold>) post AG1478 treatment <italic>lef1</italic> is decreased in interneuromast cells compared to the 6 and 12 hr time points, and is maintained in a few clumps of interneuromast cells (arrowhead). (<bold>Q</bold>–<bold>X</bold>) <italic>pea3</italic> shows a later induction than Wnt target genes. In controls <italic>pea3</italic> is only seen in primII (arrow) or primII deposited neuromasts (asterisk) at all time points tested (<bold>Q</bold>–<bold>T</bold>). (<bold>U</bold>–<bold>X</bold>) After AG1478 treatment, <italic>pea3</italic> is maintained in deposited neuromasts (asterisk) and begins to be expressed in interneuromast cells only at 36 hr post treatment (<bold>X</bold>, arrowhead). (<bold>Y</bold>–<bold>FF</bold>) <italic>her4.1</italic> expression is seen before <italic>pea3</italic> expression. (<bold>Y</bold>–<bold>BB</bold>) In controls, <italic>her4.1</italic> is seen in primII (arrow) and deposited neuromasts (asterisk). In AG1478 treated larvae <italic>her4.1</italic> is seen in large clusters of cells starting at 24 hr post treatment and can still be seen at 36 hr (<bold>EE</bold>–<bold>FF</bold>, arrowhead).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01832.016">http://dx.doi.org/10.7554/eLife.01832.016</ext-link></p></caption><graphic xlink:href="elife01832f006"/></fig></p><p>The temporal expression analysis of the Fgf target <italic>pea3</italic> shows that the Fgf pathway is also induced after ErbB inhibition but that the induction happens at a much later time point compared to the Wnt/β-catenin pathway. From 6 to 24 hr post treatment with DMSO or AG1478 <italic>pea3</italic> shows no expression in interneuromast cells, even though it is strongly expressed in primII and in deposited neuromasts (<xref ref-type="fig" rid="fig6">Figure 6Q–S,U–W</xref>). At 36 hr post ErbB inhibition <italic>pea3</italic> appears in small clusters of interneuromast cells (<xref ref-type="fig" rid="fig6">Figure 6X</xref>, arrowhead). No <italic>pea3</italic> expression is seen in interneuromast cells of control treated fish at 36 hr (<xref ref-type="fig" rid="fig6">Figure 6T</xref>). This later induction of <italic>pea3</italic> expression after AG1478 correlates with the later induction seen during wild type intercalary neuromast formation (<xref ref-type="fig" rid="fig5">Figure 5</xref>).</p><p>To test when the Notch pathway becomes active after ErbB inhibition we examined the expression of its target gene <italic>her4.1</italic>. Similar to <italic>pea3</italic>, <italic>her4.1</italic> is only expressed in primII and deposited neuromasts but not in interneuromast cells in control embryos (<xref ref-type="fig" rid="fig6">Figure 6Y–BB</xref>). After ErbB inhibition <italic>her4.1</italic> is upregulated in large clusters of cells at 24 hr post treatment. The Notch pathway is induced many hours later than the Wnt/β-catenin pathway but before the activation of Fgf signaling (<xref ref-type="fig" rid="fig6">Figure 6EE–FF</xref>, arrowhead).</p></sec><sec id="s2-8"><title>Wnt and Fgf ligands are dependent on Wnt/β-catenin signaling</title><p>In the migrating primordium the Fgf ligands <italic>fgf3</italic> and <italic>fgf10</italic> are Wnt/β-catenin targets, whereas it is not known what induces <italic>wnt10a</italic> (<xref ref-type="bibr" rid="bib3">Aman and Piotrowski, 2008</xref>). To test if <italic>wnt10a</italic>, <italic>fgf3</italic> and <italic>fgf10</italic> are Wnt/β-catenin targets during intercalary neuromast formation we treated control or <italic>Tg(sox10:DNerbb4)</italic> larvae with pharmacological inhibitors of Wnt/β-catenin, Fgfr or Notch signaling at 32 hpf then fixed for in situ hybridization at 48 hpf. To block Wnt/β-catenin signaling we used the Axin2 stabilizing drug IWR-1 (<xref ref-type="bibr" rid="bib10">Chen et al., 2009</xref>). We blocked Fgfr or Notch signaling with PD173074 and the γ-secretase inhibitor LY411575, respectively (<xref ref-type="bibr" rid="bib49">Mohammadi et al., 1998</xref>; <xref ref-type="bibr" rid="bib76">Wong et al., 2004</xref>). <italic>wnt10a, fgf3 and fgf10</italic> expression were only inhibited by blocking Wnt/β-catenin, but not Fgfr or Notch signaling (<xref ref-type="fig" rid="fig4s2">Figure 4—figure supplement 2</xref>). To test if Wnt/β-catenin signaling induces Wnt or Fgf ligand expression we treated larvae with BIO, a pharmacological inhibitor of the Wnt/β-catenin inhibitor GSK-3 (<xref ref-type="bibr" rid="bib46">Meijer et al., 2003</xref>). <italic>wnt10a</italic> is induced within 6 hr of treatment and expression is maintained up to 24 hr post-treatment (<xref ref-type="fig" rid="fig4s3">Figure 4—figure supplement 3G-L</xref>). <italic>fgf3</italic> and <italic>fgf10</italic> are also induced by BIO treatment, but induction takes longer (<xref ref-type="fig" rid="fig4s3">Figure 4—figure supplement 3M–X</xref>). These experiments show that Wnt/β-catenin signaling is both necessary and sufficient for expression of <italic>wnt10a</italic>, <italic>fgf3</italic> and <italic>fgf10</italic> within interneuromast cells of Schwann cell deficient larvae.</p><p>The expression time course analysis of Wnt/β-catenin, Fgf and Notch target genes demonstrates that the loss of ErbB signaling leads to a fast activation of the Wnt/β-catenin pathway followed by Notch and Fgf pathway activation several hours later. As Wnt/β-catenin signaling precedes and coincides with interneuromast cell proliferation, Wnt/β-catenin signaling might be required for proliferation. At the same time Wnt/β-catenin signaling is required for <italic>fgf</italic> ligand induction. Notch and Fgf signaling are only upregulated in clusters of cells suggesting that they might be playing a later role in hair cell differentiation and rosettogenesis as during early development.</p></sec><sec id="s2-9"><title>Wnt/β-catenin signaling is required for extra neuromast formation</title><p>To test if Wnt/β-catenin signaling is necessary for extra neuromast formation we employed several methods to block Wnt/β-catenin signaling. First we analyzed larvae mutant for the Wnt/β-catenin signal transducer <italic>lef1</italic>, as they show a decrease in primordium cell proliferation (<xref ref-type="bibr" rid="bib18">Gamba et al., 2010</xref>; <xref ref-type="bibr" rid="bib45">McGraw et al., 2011</xref>; <xref ref-type="bibr" rid="bib69">Valdivia et al., 2011</xref>). During development <italic>lef1</italic> mutant larvae generate intercalary neuromasts, but not as many as control larvae (<xref ref-type="fig" rid="fig7s1">Figure 7—figure supplement 1A</xref>). Lack of <italic>lef1</italic> also partially rescued the extra neuromast phenotype induced by ErbB inhibition (<xref ref-type="fig" rid="fig7s1">Figure 7—figure supplement 1B–F</xref>). It is likely that intercalary neuromast formation in <italic>lef1</italic> mutant larvae was only incompletely inhibited due to redundancy with three additional TCF family members that are also expressed in the developing lateral line (<xref ref-type="bibr" rid="bib45">McGraw et al., 2011</xref>; <xref ref-type="bibr" rid="bib69">Valdivia et al., 2011</xref>).</p><p>To pharmacologically block Wnt/β-catenin signaling we soaked the larvae in Tankyrase inhibitors. We treated 32 hpf wild type and <italic>Tg(sox10:DNerbb4)</italic> larvae with IWR-1 or XAV939 (<xref ref-type="bibr" rid="bib28">Huang et al., 2009</xref>). Larvae were soaked in the inhibitors for 24 hr and then transferred to embryo medium. At 3 dpf we counted the number of alkaline phosphatase stained neuromasts (<xref ref-type="fig" rid="fig7">Figure 7C–H</xref>), up to somite 14 because both inhibitors induced stalling of primI migration (data not shown and <xref ref-type="bibr" rid="bib44">Matsuda et al., 2013</xref>). Wnt/β-catenin inhibition induced a decrease in neuromast number in wild type siblings due to an effect on primII deposition (<xref ref-type="fig" rid="fig7">Figure 7A</xref>, blue bars). Importantly, both Wnt/β-catenin pathway inhibitors significantly decreased extra neuromast formation compared to DMSO in <italic>Tg(sox10:DNerbb4)</italic> larvae (<xref ref-type="fig" rid="fig7">Figure 7A</xref>, red bars).<fig-group><fig id="fig7" position="float"><object-id pub-id-type="doi">10.7554/eLife.01832.019</object-id><label>Figure 7.</label><caption><title>Wnt/β-catenin signaling is required for extra neuromast formation in the absence of ErbB signaling.</title><p>To block Wnt/β-catenin signaling wild type or <italic>Tg(sox10:DNerbb4</italic>) fish were treated with two different inhibitors IWR-1 or XAV939 for 24 hr starting at 32 hpf. Neuromast number up to somite 14 was counted at 3 dpf (<bold>A</bold>). Compared to DMSO, both IWR-1 and XAV939 significantly inhibited neuromast formation in <italic>Tg(sox10:DNerbb4</italic>) (<bold>A</bold>, red bars, One-way ANOVA with Tukey pairwise comparison, p≤0.05). Representative images of alkaline phosphatase stained control siblings treated with DMSO (<bold>C</bold>), IWR-1 (<bold>E</bold>) or XAV939 (<bold>G</bold>) or <italic>Tg(sox10:DNerbb4</italic>) treated with DMSO (<bold>D</bold>), IWR-1 (<bold>F</bold>) or XAV939 (<bold>H</bold>). (<bold>B</bold>) Neuromast counts at 3 dpf of control, <italic>Tg(sox10:DNerbb4</italic>), <italic>Tg(hsp70l:dkk1b)</italic> or <italic>Tg(sox10:DNerbb4</italic>)/<italic>Tg(hsp70l:dkk1b)</italic> after heat shock at 32 hpf. <italic>Tg(sox10:DNerbb4</italic>)/<italic>Tg(hsp70l:dkk1b)</italic> double transgenics show a complete loss of extra neuromast formation seen in <italic>Tg(sox10:DNerbb4</italic>) (<bold>B</bold>, Student’s <italic>t</italic>-test, p=2.4E<sup>−16</sup>). Representative images of alkaline phosphatase stained sibling (<bold>I</bold>), <italic>Tg(sox10:DNerbb4</italic>) (<bold>J</bold>), <italic>Tg(hsp70l:dkk1b)</italic> (<bold>K</bold>) or <italic>Tg(sox10:DNerbb4</italic>)/<italic>Tg(hsp70l:dkk1b)</italic> (<bold>L</bold>) at 3 dpf. The first deposited neuromast is to the left for all alkaline phosphatase images.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01832.019">http://dx.doi.org/10.7554/eLife.01832.019</ext-link></p></caption><graphic xlink:href="elife01832f007"/></fig><fig id="fig7s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01832.020</object-id><label>Figure 7—figure supplement 1.</label><caption><title><italic>lef1</italic> mutants have decreased intercalary neuromast formation in the absence of ErbB signaling.</title><p>Control siblings or <italic>lef1</italic> mutants were fixed at 2, 3, 5, 7, 10 and 14 dpf and processed for alkaline phosphatase staining. Because <italic>lef1</italic> mutants have a decrease in neuromast deposition, we counted neuromasts just up to somite 14. At 2 and 3 dpf <italic>lef1</italic> mutants have the same number of neuromasts as controls (<bold>A</bold>). From 5 dpf onward there is a significant decrease in neuromast formation in <italic>lef1</italic> (<bold>A</bold>, Student's <italic>t</italic>-test, p≤0.001). Challenging <italic>lef1</italic> mutants with AG1478 at 48 hpf shows only a 19.5% increase in neuromast formation compared to DMSO treatment by 5 dpf, vs 35% increase seen in AG1478 treated control siblings (<bold>B</bold>, Student's <italic>t</italic>-test, p=1.7 E<sup>−8</sup>). Representative images of alkaline phosphatase stained DMSO treated sibling (<bold>C</bold>) or <italic>lef1</italic> mutant (<bold>E</bold>) or AG1478 treated sibling (<bold>D</bold>) or <italic>lef1</italic> (<bold>F</bold>).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01832.020">http://dx.doi.org/10.7554/eLife.01832.020</ext-link></p></caption><graphic xlink:href="elife01832fs009"/></fig></fig-group></p><p>As a third method to inhibit Wnt/β-catenin signaling we heat shock induced Dkk1b (<xref ref-type="bibr" rid="bib67">Stoick-Cooper et al., 2007</xref>), a secreted antagonist of Wnt/β-catenin signaling that blocks Wnt/β-catenin dependent gene expression and cell proliferation in the lateral line primordium (<xref ref-type="bibr" rid="bib3">Aman and Piotrowski, 2008</xref>; <xref ref-type="bibr" rid="bib2">Aman et al., 2011</xref>). <italic>Tg(hsp70l:dkk1b)</italic> zebrafish were crossed to <italic>Tg(sox10:DNerbb4)</italic> and embryos were heat shocked at 32 hpf. Alkaline phosphatase staining at 3 dpf shows a clear reduction in intercalary neuromasts in <italic>Tg(hsp70l:dkk1b</italic>)/<italic>Tg(sox10:DNerbb4)</italic> compared to <italic>Tg(sox10:DNerbb4)</italic> (<xref ref-type="fig" rid="fig7">Figure 7I–L</xref>)<italic>.</italic> Quantification of neuromast number up to somite fourteen verified a complete absence of extra neuromast formation induced by dominant-negative ErbB when Wnt/β-catenin signaling is inhibited by Dkk1b overexpression (<xref ref-type="fig" rid="fig7">Figure 7B</xref>). These different methods of blocking Wnt/β-catenin signaling all show that this pathway is necessary for intercalary neuromast formation in the absence of ErbB signaling.</p></sec><sec id="s2-10"><title>Wnt/β-catenin signaling is required for interneuromast cell proliferation</title><p>The lack of extra neuromast formation after Wnt/β-catenin inhibition could be due to a decrease in cell proliferation or a lack of differentiation. Because ErbB inhibition induces an increase in Wnt/β-catenin pathway expression prior to the increase in interneuromast proliferation (<xref ref-type="fig" rid="fig3 fig6">Figures 3 and 6</xref>), we hypothesized that the inability to induce extra neuromasts after Wnt/β-catenin inhibition is due to a lack of proliferation. We performed BrdU incorporation analyses on <italic>Tg(hsp70l:dkk1b</italic>)/<italic>Tg(sox10:DNerbb4)/Tg(SqET20:gfp)</italic> larvae<italic>.</italic> Larvae were heat shocked at 32 hpf, then raised in BrdU solution until 48 hpf and fixed. Immunostaining for BrdU and GFP shows a significant increase in double labeling in <italic>Tg(sox10:DNerbb4)</italic> compared to control siblings (<xref ref-type="fig" rid="fig8">Figure 8A–B,E</xref>). Such increase in BrdU incorporation is not observed in <italic>Tg(hsp70l:dkk1b</italic>)/<italic>Tg(sox10:DNerbb4)</italic> double transgenic larvae (<xref ref-type="fig" rid="fig8">Figure 8D–E</xref>). <italic>Tg(hsp70l:dkk1b</italic>) larvae show no significant difference in the BrdU index compared to control larvae (<xref ref-type="fig" rid="fig8">Figure 8C,E</xref>). Quantification of the number of <italic>Tg(SqET20:gfp)-</italic>positive interneuromast cells shows a significant decrease in <italic>Tg(hsp70l:dkk1b</italic>)/<italic>Tg(sox10:DNerbb4)</italic> larvae compared to <italic>Tg(sox10:DNerbb4)</italic> larvae (<xref ref-type="fig" rid="fig8">Figure 8F</xref>). These experiments demonstrate that inhibition of Wnt/β-catenin prevents the formation of intercalary neuromasts in ErbB signaling deficient larvae by inhibiting proliferation of progenitor cells.<fig id="fig8" position="float"><object-id pub-id-type="doi">10.7554/eLife.01832.021</object-id><label>Figure 8.</label><caption><title>Wnt/β-catenin signaling is required for proliferation of interneuromast cells in the absence of ErbB signaling.</title><p>Control sibling, <italic>Tg(sox10:DNerbb4</italic>), <italic>Tg(hsp70l:dkk1b)</italic> or <italic>Tg(sox10:DNerbb4</italic>)/<italic>Tg(hsp70l:dkk1b)</italic>, all with <italic>Tg(SqET20:gfp)</italic> in the background, were heat shocked at 32 hpf then placed in BrdU solution. Fish were fixed at 48 hpf and processed for BrdU and GFP immunohistochemistry (<bold>A</bold>–<bold>D</bold>). Compared to control siblings (<bold>A</bold>), <italic>Tg(sox10:DNerbb4</italic>) (<bold>B</bold>) show a strong increase in BrdU incorporation into <italic>Tg(SqET20:gfp)</italic> positive interneuromast cells. BrdU incorporation in <italic>Tg(sox10:DNerbb4</italic>) is blocked by over expression of Dkk1b (<bold>D</bold>). (<bold>E</bold>) Quantification of BrdU index shows no difference between control and <italic>Tg(hsp70l:dkk1b)</italic> (Student’s <italic>t</italic>-test, p=0.6) but a large decrease between <italic>Tg(sox10:DNerbb4</italic>) and <italic>Tg(sox10:DNerbb4</italic>)/<italic>Tg(hsp70l:dkk1b)</italic> (Student’s <italic>t</italic>-test, p=3.4 E<sup>−8</sup>). (<bold>F</bold>) Quantification of <italic>Tg(SqET20:gfp)</italic> cell number again shows no difference between control and <italic>Tg(hsp70l:dkk1b)</italic> (Student’s <italic>t</italic>-test, p=0.14) but a large decrease between <italic>Tg(sox10:DNerbb4</italic>) and <italic>Tg(sox10:DNerbb4</italic>)/<italic>Tg(hsp70l:dkk1b)</italic> (Student’s <italic>t</italic>-test, p=1 E<sup>−4</sup>).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01832.021">http://dx.doi.org/10.7554/eLife.01832.021</ext-link></p></caption><graphic xlink:href="elife01832f008"/></fig></p></sec><sec id="s2-11"><title>Increased Wnt/β-catenin signaling is sufficient to induce interneuromast cell proliferation</title><p>To examine if Wnt/β-catenin signaling is sufficient to induce proliferation we treated 48 hpf larvae with BIO. In DMSO treated larvae <italic>lef1</italic> is expressed in primII and weakly in a few interneuromast cells (<xref ref-type="fig" rid="fig9">Figure 9A</xref>, arrowhead). BIO treatment induced expression of <italic>lef1</italic> in neuromasts and interneuromast cells (<xref ref-type="fig" rid="fig9">Figure 9B</xref>, <xref ref-type="fig" rid="fig4s3">Figure 4—figure supplement 3</xref>). To study proliferation in interneuromast cells <italic>Tg(SqET20:gfp)</italic> larvae were treated with DMSO or BIO in the presence of BrdU. Similarly to ErbB inhibition, BIO induces an increase in proliferation and the number of interneuromast cells 24 hr post treatment (<xref ref-type="fig" rid="fig9">Figure 9C–J</xref>). BIO treatment does not result in a reduction of Schwann cells along the lateral line (<xref ref-type="fig" rid="fig9s1">Figure 9—figure supplement 1</xref>). The BIO-induced increase in interneuromast cell proliferation did not result in extra neuromasts, due to a strong increase in cell death in interneuromast cells after prolonged BIO treatment (data not shown). We also transplanted <italic>apc</italic><sup><italic>mcr</italic></sup> mutant lateral line cells into wild type hosts and similarly observed that these clones did not survive more than 48 hpf (data not shown). This increase in cell death was only seen in interneuromast cells and not in primary neuromasts, suggesting that interneuromast cells are particularly sensitive to the levels of Wnt/β-catenin signaling. Combined, our experiments demonstrate that Wnt/β-catenin signaling is sufficient and necessary for inducing interneuromast cell proliferation and is absolutely required for the extra neuromast formation that occurs in the absence of Schwann cells.<fig-group><fig id="fig9" position="float"><object-id pub-id-type="doi">10.7554/eLife.01832.022</object-id><label>Figure 9.</label><caption><title>Pharmacological activation of Wnt/β-catenin signaling is sufficient to induce interneuromast cell proliferation.</title><p>To verify BIO induces Wnt/β-catenin signaling we treated 48 hpf zebrafish with DMSO or BIO for 6 hr and then fixed and stained for <italic>lef1</italic> expression. Compared to DMSO treated fish, BIO induces expression of the Wnt/β-catenin target <italic>lef1</italic> in neuromasts and interneuromast cells (<bold>A</bold>–<bold>B</bold>). The large cluster of cells that are labeled in both (<bold>A</bold>) and (<bold>B</bold>) is primII (arrowhead). To measure proliferation <italic>Tg(SqET20:gfp)</italic> fish were treated with BrdU plus DMSO or BIO at 48 hpf and fixed at 72 hpf. (<bold>C</bold>–<bold>E</bold>) DMSO treated interneuromast cells (arrow) show single chain morphology with rare BrdU incorporation. BrdU incorporation is strong in primary neuromasts. (<bold>F</bold>–<bold>H</bold>) BIO treated fish show increased interneuromast cells with BrdU incorporation (arrows). Quantification of BrdU index (<bold>I</bold>, Student’s t-test, p=0.0003) and <italic>Tg(SqET20:gfp)</italic> positive cell number (<bold>J</bold>, Student’s <italic>t</italic>-test, p=2.75 E<sup>−5</sup>) shows a significant increase in both after BIO treatment.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01832.022">http://dx.doi.org/10.7554/eLife.01832.022</ext-link></p></caption><graphic xlink:href="elife01832f009"/></fig><fig id="fig9s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01832.023</object-id><label>Figure 9—figure supplement 1.</label><caption><title>Schwann cells are still present after BIO treatment.</title><p><italic>Tg(foxd3:gfp)</italic> larvae were treated with DMSO or BIO at 32 hpf then imaged at 48 hpf. (<bold>A</bold>) DMSO treated larvae show a line of <italic>Tg(foxd3:gfp)</italic> positive Schwann cells along the midline. (<bold>B</bold>) BIO treated larvae also show Schwann cells along the midline. Schwann cell migration is not complete due to stalling of the primordium.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01832.023">http://dx.doi.org/10.7554/eLife.01832.023</ext-link></p></caption><graphic xlink:href="elife01832fs010"/></fig></fig-group></p></sec><sec id="s2-12"><title>Fgf signaling is required for intercalary neuromast formation</title><p>Fgf signaling is upregulated in interneuromast cells in <italic>nrg1-3</italic><sup><italic>z26</italic></sup> mutant larvae (<xref ref-type="fig" rid="fig4">Figure 4</xref>). To test if Fgf signaling is also required for intercalary neuromast formation we used both genetic and pharmacological methods to block Fgfr signaling. We added the Fgfr inhibitors SU5402 or PD173074 to 32 hpf <italic>Tg(SqET20:gfp)</italic> or <italic>Tg(SqET20:gfp)/Tg(sox10:DNerbb4)</italic> larvae and observed neuromasts at 3 dpf. DMSO treated <italic>Tg(sox10:DNerbb4)</italic> larvae show formation of intercalary neuromasts (<xref ref-type="fig" rid="fig10">Figure 10B</xref>, arrowheads). In contrast, intercalary neuromast formation is blocked after Fgfr inhibition in <italic>Tg(sox10:DNerbb4)</italic> (<xref ref-type="fig" rid="fig10">Figure 10D,F</xref>). In Fgfr inhibitor treated <italic>Tg(sox10:DNerbb4)</italic> larvae a thickening of the chain of interneuromast cells occurs that is not observed in Fgfr inhibited siblings (<xref ref-type="fig" rid="fig10">Figure 10C–F</xref>, arrows). However, this increase in interneuromast cells does not lead to the formation of rosettes or differentiated neuromasts. Quantification of neuromast numbers revealed a significant reduction in Fgfr inhibitor treated <italic>Tg(sox10:DNerbb4)</italic> larvae (<xref ref-type="fig" rid="fig10">Figure 10G</xref>, red bars). Fgfr is required for maintenance of rosettes in the primordium (<xref ref-type="bibr" rid="bib52">Nechiporuk and Raible, 2008</xref>). SU5402 and to a lesser extent PD173074, also induced loss of the rosette shape of primary neuromasts, decreasing the number of neuromasts in treated control siblings (<xref ref-type="fig" rid="fig10">Figure 10G</xref>, blue bars). As another means to inhibit Fgf signaling, we heat shock induced dominant negative Fgfr1 expression in 32 hpf <italic>Tg(sox10:DNerbb4)</italic> and sibling larvae. Dominant negative Fgfr1 also inhibited extra neuromast formation in <italic>Tg(sox10:DNerbb4)</italic> larvae by 3 dpf (<xref ref-type="fig" rid="fig10">Figure 10H</xref>). The observation that Fgfr inhibited <italic>Tg(sox10:DNerbb4)</italic> larvae still form large clusters of interneuromast cells that fail to differentiate into neuromasts, shows that Fgf signaling is crucial for interneuromast differentiation and rosette formation, but not proliferation.<fig-group><fig id="fig10" position="float"><object-id pub-id-type="doi">10.7554/eLife.01832.024</object-id><label>Figure 10.</label><caption><title>Fgf signaling is required for neuromast formation but not interneuromast proliferation in the absence of ErbB signaling.</title><p>Control <italic>Tg(SqET20:gfp)</italic> or <italic>Tg(sox10:DNerbb4</italic>)/<italic>Tg(SqET20:gfp)</italic> larvae were treated for 24 hr starting at 32 hpf with DMSO, SU5402 or PD173074 then allowed to develop until 3 dpf and imaged (<bold>A</bold>–<bold>F</bold>). The first primary neuromast is to the left in all images. Control siblings (<bold>A</bold>) show no intercalary neuromasts, while <italic>Tg(sox10:DNerbb4</italic>) have several (<bold>B</bold>, arrowhead). <italic>Tg(sox10:DNerbb4</italic>) treated with SU5402 (<bold>D</bold>) or PD173074 (<bold>F</bold>) show clumps of interneuromast cells (arrows) compared to sibling SU5402 (<bold>C</bold>) or PD173074 (<bold>E</bold>) treated but no intercalary neuromasts. (<bold>G</bold>) Quantification of neuromast number up to somite 14 shows a decrease when control siblings are treated with SU5402 or PD173074 (blue bars, One-way ANOVA with Tukey pairwise comparison, p≤0.05). (<bold>G</bold>) Extra neuromast formation is inhibited when <italic>Tg(sox10:DNerbb4</italic>) fish are treated with SU5402 or PD173074 (red bars, one-way ANOVA with Tukey pairwise comparison, p≤0.05). (<bold>H</bold>) <italic>Tg(sox10:DNerbb4</italic>) were crossed to <italic>Tg(hsp70l:dnfgfr1-EGFP</italic>) and larvae were heat shocked at 32 hpf then allowed to grow to 3 dpf. DNFgfr1 reduces neuromasts slightly in control siblings (blue bars, Student’s <italic>t</italic>-test, p=7.1 E<sup>−9</sup>) and completely blocks extra neuromast formation in <italic>Tg(sox10:DNerbb4</italic>) (red bars, Student’s <italic>t</italic>-test, p=1.5 E<sup>−7</sup>). To measure proliferation, control <italic>Tg(SqET20:gfp)</italic> or <italic>Tg(sox10:DNerbb4</italic>)/<italic>Tg(SqET20:gfp)</italic> larvae were treated with BrdU plus DMSO, SU5402 or PD173074 at 32 hpf then fixed at 48 hpf. Fish were then processed for BrdU and GFP immunohistochemistry (<bold>I</bold>–<bold>N</bold>). All <italic>Tg(sox10:DNerbb4</italic>)/<italic>Tg(SqET20:gfp)</italic> treated fish (<bold>J</bold>, <bold>L</bold> and <bold>N</bold>) show higher BrdU incorporation when compared to control <italic>Tg(SqET20:gfp)</italic> siblings (<bold>I</bold>, <bold>K</bold> and <bold>M</bold>). (<bold>O</bold>) Quantification of BrdU index shows no significant difference between DMSO, SU5402 or PD173074 treated <italic>Tg(sox10:DNerbb4</italic>) larvae (red bars, one-way ANOVA with Tukey pairwise comparison, p≥0.1). (<bold>P</bold>) Quantification of <italic>Tg(SqET20:gfp)</italic> cell number shows no difference in <italic>Tg(sox10:DNerbb4</italic>) larvae treated with DMSO, SU5402 or PD173074 (red bars, one-way ANOVA with Tukey pairwise comparison, p≥0.9).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01832.024">http://dx.doi.org/10.7554/eLife.01832.024</ext-link></p></caption><graphic xlink:href="elife01832f010"/></fig><fig id="fig10s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01832.025</object-id><label>Figure 10—figure supplement 1.</label><caption><title>Posterior lateral line ganglion neurons are affected differently by SU5042 or PD173074 treatment.</title><p><italic>Tg(HGN39D:gfp)</italic> transgenics were treated with indicated drugs at 48 hpf, allowed to develop for 24 hr then imaged. DMSO (<bold>A</bold>) and AG1478 (<bold>B</bold>) have similar ganglion size. SU5402 reduces ganglion size (<bold>C</bold>), while PD173074 increases ganglion size (<bold>D</bold>). (<bold>E</bold>) Quantification of GFP positive cells per ganglion. There is no significant difference between DMSO and AG1478 (one-way ANOVA with Tukey pairwise comparison, p=0.9). SU5402 induces a reduction in cell number (one-way ANOVA with Tukey pairwise comparison, p≤0.05). PD173074 induces a mild increase in cell number (one-way ANOVA with Tukey pairwise comparison, p≤0.05).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01832.025">http://dx.doi.org/10.7554/eLife.01832.025</ext-link></p></caption><graphic xlink:href="elife01832fs011"/></fig></fig-group></p></sec><sec id="s2-13"><title>Fgf signaling is not required for interneuromast cell proliferation but for differentiation</title><p>To test if Fgf signaling is required for proliferation we performed BrdU analyses of Fgfr inhibitor treated <italic>Tg(SqET20:gfp)</italic> and <italic>Tg(sox10:DNerbb4)/Tg(SqET20:gfp)</italic> larvae. BrdU plus DMSO, SU5402 or PD173074 were added at 32 hpf, and the fish were fixed and stained at 48 hpf. Non-transgenic siblings treated with DMSO or Fgfr inhibitors display a single chain of interneuromast cells with rare BrdU positive cells (<xref ref-type="fig" rid="fig10">Figure 10I,K,M</xref>). SU5402 treated wild type larvae show an increase in BrdU incorporation compared to DMSO treated larvae. The increase is significant by a Student’s <italic>t</italic>-test but not by an ANOVA analysis that compares all groups. PD173074 treated wild type fish show no significant change in BrdU incorporation compared to DMSO (<xref ref-type="fig" rid="fig10">Figure 10O</xref>, blue bars). When characterizing SU5402 and PD173074 treated fish we noticed an effect on the posterior lateral ganglion (pllg) size. Quantification of the number of cells in the pllg showed a decrease induced by SU5402 but an increase induced by PD173074 (<xref ref-type="fig" rid="fig10s1">Figure 10—figure supplement 1</xref>). The increase in the posterior lateral line ganglion size after PD173074 treatment is similar to recent results that showed that Fgf inhibition resulted in a larger statoacoustic ganglion (<xref ref-type="bibr" rid="bib70">Vemaraju et al., 2012</xref>). Possibly, the two inhibitors affect a different set of Fgf receptors or pathways. Irrespective of their opposing effect on ganglia size<bold>,</bold> both SU5402 and PD173074 treated <italic>Tg(sox10:DNerbb4)</italic> transgenic larvae show BrdU positive clusters of interneuromast cells (<xref ref-type="fig" rid="fig10">Figure 10J,L,N</xref>). Likewise, quantification of BrdU indices and interneuromast cell number indicates that neither inhibitor significantly decreases the proportion of proliferating cells or cell number in <italic>Tg(sox10:DNerbb4)</italic> larvae (<xref ref-type="fig" rid="fig10">Figure 10O–P</xref>, red bars). These results confirm that Fgf signaling is not required for the increase in interneuromast cell proliferation in Schwann cell deficient zebrafish.</p><p>In addition to rosette formation, Fgf signaling also regulates <italic>atoh1a</italic>, which is crucial for sensory hair cell differentiation in the mouse and zebrafish ear and lateral line (<xref ref-type="bibr" rid="bib5">Bermingham et al., 1999</xref>; <xref ref-type="bibr" rid="bib64">Sarrazin et al., 2006</xref>; <xref ref-type="bibr" rid="bib47">Millimaki et al., 2007</xref>; <xref ref-type="bibr" rid="bib36">Lecaudey et al., 2008</xref>; <xref ref-type="bibr" rid="bib52">Nechiporuk and Raible, 2008</xref>). We tested if Fgf signaling plays similar roles in postembryonic and precocious intercalary neuromast formation in wild type and Schwann cell deficient larvae, respectively. We examined the expression of the Fgf targets <italic>pea3</italic> and <italic>atoh1a</italic> after DMSO or PD173074 treatment from 32 to 48 hpf in wild type and <italic>Tg(sox10:DNerbb4)</italic> larvae (<xref ref-type="fig" rid="fig11">Figure 11</xref>)<italic>.</italic> In DMSO treated larvae <italic>pea3</italic> and <italic>atoh1a</italic> are only expressed in primary neuromasts but not in interneuromast cells (<xref ref-type="fig" rid="fig11">Figure 11A,E</xref>, inset). After Fgfr inhibition the expression of <italic>pea3</italic> and <italic>atoh1a</italic> are downregulated in primary neuromasts (<xref ref-type="fig" rid="fig11">Figure 11B,F</xref>, inset). <italic>pea3</italic> and <italic>atoh1a</italic> are strongly upregulated in precociously differentiating interneuromast cells of DMSO treated <italic>Tg(sox10:DNerbb4)</italic> larvae (<xref ref-type="fig" rid="fig11">Figure 11C,G</xref>). However, this induction of <italic>pea3</italic> and <italic>atoh1a</italic> in <italic>Tg(sox10:DNerbb4)</italic> larvae is blocked by Fgfr inhibition (<xref ref-type="fig" rid="fig11">Figure 11D,H</xref>). Therefore, Fgf signaling has the same function in rosette formation and hair cell differentiation in postembryonic intercalary neuromast formation, as in primary neuromast formation during primordium migration.<fig id="fig11" position="float"><object-id pub-id-type="doi">10.7554/eLife.01832.026</object-id><label>Figure 11.</label><caption><title>Fgf signaling is required for neuromast differentiation.</title><p>Control or <italic>Tg(sox10:DNerbb4</italic>) siblings were treated with DMSO or PD173074 at 32 hpf then fixed at 48 hpf. To verify that Fgf signaling was blocked, we performed in situ hybridazation for the Fgf target <italic>pea3</italic> (<bold>A</bold>–<bold>D</bold>). (<bold>A</bold>) In controls treated with DMSO, <italic>pea3</italic> is not expressed in interneuromast cells but is expressed in neuromasts (inset). (<bold>B</bold>) Control siblings treated with PD173074 show downregulation of <italic>pea3</italic> in neuromasts (inset). (<bold>C</bold>) As shown for <italic>nrg1-3</italic><sup><italic>z26</italic></sup>, <italic>Tg(sox10:DNerbb4</italic>) have an upregulation of <italic>pea3</italic> within interneuromast cells. (<bold>D</bold>) This upregulation of <italic>pea3</italic> is blocked by PD173074, illustrating that Fgfr signaling is inhibited. (<bold>E</bold>) In controls, <italic>atoh1a</italic> is only expressed in neuromasts (inset). (<bold>F</bold>) PD173074 decreases <italic>atoh1a</italic> in primary neuromasts (inset). (<bold>G</bold>) <italic>atoh1a</italic> is upregulated in differentiating interneuromast cells in <italic>Tg(sox10:DNerbb4</italic>). (<bold>H</bold>) This upregulation of <italic>atoh1a</italic> in interneuromast cells is completely blocked by PD173074, while some expression is still retained in primary neuromasts (inset).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01832.026">http://dx.doi.org/10.7554/eLife.01832.026</ext-link></p></caption><graphic xlink:href="elife01832f011"/></fig></p></sec></sec><sec id="s3" sec-type="discussion"><title>Discussion</title><sec id="s3-1"><title>ErbB signaling regulates interneuromast cell proliferation by activating a Wnt/β-catenin inhibitor</title><p>Interneuromast cells are a latent lateral line progenitor cell population capable of giving rise to all cell types of the neuromast. We uncovered a novel mechanism whereby Schwann cells constitute part of an inhibitory niche, regulating interneuromast cell proliferation and differentiation through non-cell-autonomous regulation of Wnt/β-catenin and Fgf signaling downstream of ErbB signaling (see model <xref ref-type="fig" rid="fig12">Figure 12</xref>).<fig id="fig12" position="float"><object-id pub-id-type="doi">10.7554/eLife.01832.027</object-id><label>Figure 12.</label><caption><title>Model of Schwann cell inhibition of intercalary neuromast formation.</title><p>(<bold>A</bold>) Schwann cells (red) co-migrate with lateral line axons (yellow) and interneuromast cells (green). Nrg1-3 present on the axon induces Schwann cell migration and proliferation through activation of ErbB2/3B. As interneuromast cells are deposited they remain in close proximity to Schwann cells. This interaction induces inhibition of Wnt/β-catenin signaling by an unknown mechanism. (<bold>B</bold>) As interneuromast cells migrate ventrally away from Schwann cells, this Wnt/β-catenin inhibition is released. (<bold>C</bold>) Release of inhibition leads to increased Wnt/β-catenin signaling and proliferation followed by Fgf signaling and differentiation.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01832.027">http://dx.doi.org/10.7554/eLife.01832.027</ext-link></p></caption><graphic xlink:href="elife01832f012"/></fig></p><p>ErbB receptor expressing Schwann cells co-migrate with the primordium due to the presence of Nrg1-3 ligand in lateral line axons. As interneuromast cells are deposited by the primordium, they are in close contact with Schwann cells. Schwann cells keep interneuromast cells in a quiescent state via the action of an unknown inhibitor (<xref ref-type="fig" rid="fig12">Figure 12A</xref>). After deposition, interneuromast cells migrate ventrally away from the midline, which coincides with the commencement of proliferation in interneuromast cells (<xref ref-type="fig" rid="fig12">Figure 12B</xref>). Simultaneously, Schwann cells migrate medially, crossing the basement membrane (<xref ref-type="bibr" rid="bib60">Raphael et al., 2010</xref>). Both of these steps likely contribute to the release of the Schwann cell inhibitory signal during normal development. The release of this inhibitory signal causes the upregulation of Wnt/β-catenin signaling leading to interneuromast proliferation (<xref ref-type="fig" rid="fig12">Figure 12C</xref>). Eventually, Notch and Fgf signaling is initiated inducing rosette formation and differentiation.</p><p>In zebrafish, ErbB signaling serves several functions during the lifetime of a Schwann cell (<xref ref-type="bibr" rid="bib41">Lyons et al., 2005</xref>; <xref ref-type="bibr" rid="bib59">Raphael et al., 2011</xref>). Our ErbB inhibitor treatment, after Schwann cell migration is complete, suggests that ErbB signaling plays an additional role in Schwann cells by regulating an inhibitor of Wnt/β-catenin signaling in interneuromast cells. As pharmacological inhibition of ErbB signaling also causes a reduction in Schwann cell proliferation, it is possible that this secondarily affects interneuromast proliferation via reduction of the number of Schwann cells. However, pharmacological inhibition of ErbB signaling induces an increase in Wnt/β-catenin signaling before we observe a decrease in Schwann cell number (<xref ref-type="fig" rid="fig6">Figure 6</xref> and <xref ref-type="fig" rid="fig1s4">Figure 1—figure supplement 4</xref>), suggesting that ErbB signaling directly regulates the expression or activity of the Wnt/β-catenin inhibitor. The inhibitor could either be expressed by Schwann or interneuromast cells.</p><p>The list of potential candidates for the Wnt/β-catenin pathway inhibitor is growing (<xref ref-type="bibr" rid="bib14">Cruciat and Niehrs, 2013</xref>). The Wnt/β-catenin inhibitor controlling interneuromast progenitor proliferation should be expressed in wild type Schwann cells until at least 48 hpf, when intercalary neuromasts begin to form. In embryos in which ErbB signaling is abrogated and interneuromast cells proliferate, the Wnt/β-catenin inhibitor should be downregulated. However, the known Wnt/β-catenin inhibitors <italic>sfrp1a, wif1</italic>, <italic>dkk1b</italic> and <italic>dkk2</italic> are upregulated in <italic>nrg1-3</italic><sup><italic>z26</italic></sup> mutants or <italic>Tg(sox10:DNerbb4)</italic> larvae and <italic>wif1, dkk1b and dkk2</italic> are not expressed in 48 hpf wild type interneuromast or Schwann cells (<xref ref-type="fig" rid="fig13">Figure 13</xref>). Therefore, the expression patterns of these inhibitors do not correlate with a function in inhibiting Wnt/β-catenin signaling in 48 hpf wild type interneuromast cells or the release of inhibition in <italic>nrg1-3</italic><sup><italic>z26</italic></sup> mutant larvae. Our results are consistent with several recent reports that have shown the importance for Wnt/β-catenin signaling in regulating proliferation in mammalian inner ear support cells and zebrafish neuromasts (<xref ref-type="bibr" rid="bib9">Chai et al., 2012</xref>; <xref ref-type="bibr" rid="bib30">Jacques et al., 2012</xref>; <xref ref-type="bibr" rid="bib31">Jan et al., 2013</xref>; <xref ref-type="bibr" rid="bib65">Shi et al., 2012</xref>; <xref ref-type="bibr" rid="bib26">Head et al., 2013</xref>; <xref ref-type="bibr" rid="bib73">Wada et al., 2013b</xref>). We identified <italic>wnt10a</italic> as the potential ligand required for intercalary neuromast formation, which should be either inhibited by Schwann cells or induced in their absence. <italic>wnt10a</italic> is expressed in interneuromast cells of Schwann cell deficient mutants (<xref ref-type="fig" rid="fig4">Figure 4A–B</xref>, <xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1</xref>) and is induced 6 hr post AG1478 treatment (data not shown). However, even though <italic>lef1</italic> and the Wnt/β-catenin reporter are expressed in wild type interneuromast cells at 48 hpf (<xref ref-type="fig" rid="fig6">Figure 6</xref>), we did not detect <italic>wnt10a</italic> expression, suggesting that additional Wnt ligands upregulate Wnt/β-catenin signaling at this time.<fig id="fig13" position="float"><object-id pub-id-type="doi">10.7554/eLife.01832.028</object-id><label>Figure 13.</label><caption><title>Expression of several Wnt/β-catenin inhibitors are increased in <italic>nrg1-3</italic><sup><italic>z26</italic></sup>.</title><p>Control sibling and <italic>nrg1-3</italic><sup><italic>z26</italic></sup> mutants were fixed at 48 hpf and processed for in situ hybridization. Control larvae show <italic>sfrp1a</italic> expression in interneuromast and mantle cells (<bold>A</bold>). Expression of <italic>sfrp1a</italic> is increased in <italic>nrg1-3</italic><sup><italic>z26</italic></sup> (<bold>B</bold>). Control larvae show no expression of <italic>wif1</italic> (<bold>C</bold>) or <italic>dkk1b</italic> (<bold>F</bold>) in interneuromast or Schwann cells. Both <italic>wif1</italic> (<bold>D</bold>) and <italic>dkk1b</italic> (<bold>E</bold>) are induced in interneuromast cells of <italic>nrg1-3</italic><sup><italic>z26</italic></sup>. (<bold>G</bold>) Control larvae show expression of <italic>dkk2</italic> in primary neuromasts (inset) but not in interneuromast or Schwann cells. (<bold>H</bold>) In <italic>Tg(sox10:DNerbb4) dkk2</italic> is expressed in neuromasts (inset), and is upregulated in interneuromast cells.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01832.028">http://dx.doi.org/10.7554/eLife.01832.028</ext-link></p></caption><graphic xlink:href="elife01832f013"/></fig></p></sec><sec id="s3-2"><title>ErbB receptor tyrosine kinases regulate multiple stem cell niches</title><p>Formation of the adult zebrafish pigment pattern depends on ErbB3b, in part non-cell-autonomously, by regulating the formation of dorsal root ganglion neurons, which act as a niche for adult melanophore precursors (<xref ref-type="bibr" rid="bib8">Budi et al., 2011</xref>; <xref ref-type="bibr" rid="bib16">Dooley et al., 2013</xref>). We found that <italic>nrg1-3</italic><sup><italic>z26</italic></sup> mutants mimic the <italic>erbb3b</italic> pigment pattern phenotype (<xref ref-type="fig" rid="fig1s2">Figure 1—figure supplement 2</xref>). Whether Nrg1-3 is required for melanophore progenitor niche formation or maintenance is currently unknown. The signaling pathways regulating the niche downstream of ErbB3b have not been identified, but it is interesting to speculate that Wnt/β-catenin signaling may also be involved.</p><p>Another ErbB family member, ErbB1, inhibits neural stem cell proliferation non-cell-autonomously through inhibition of Notch activity (<xref ref-type="bibr" rid="bib1">Aguirre et al., 2010</xref>). Notch signaling serves several functions in the development of the lateral line and therefore, is another likely candidate to regulate intercalary neuromast formation. Notch signaling is not active in wild type interneuromast cells but is activated in <italic>nrg1-3</italic><sup><italic>z26</italic></sup> mutants (<xref ref-type="fig" rid="fig4">Figure 4</xref>). However, the time course expression analysis after ErbB inhibition uncovered that Notch signaling is not upregulated until 24 hr post treatment, well after the initiation of interneuromast cell proliferation (<xref ref-type="fig" rid="fig3 fig6">Figures 3 and 6</xref>, <xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1</xref>). In addition, pharmacological inhibition of Notch activity, using the γ-secretase inhibitors DAPT or LY411575, did not induce Wnt or Fgf ligand expression (<xref ref-type="fig" rid="fig4s2">Figure 4—figure supplement 2</xref>) or intercalary neuromasts in wild type larvae (data not shown). Therefore we conclude that, similar to Fgf, Notch signaling is only playing a later role in intercalary neuromast development, likely regulating the choice between support or hair cell specification as in primary neuromasts (<xref ref-type="bibr" rid="bib29">Itoh and Chitnis, 2001</xref>).</p><p>Recently, it was shown that non-myelinating Schwann cells constitute part of a niche, non-cell-autonomously controlling the quiescence of hematopoietic stem cells (HSC) (<xref ref-type="bibr" rid="bib77">Yamazaki et al., 2011</xref>). Removing Schwann cells in mice by axotomy lead to loss of active TGF-β and an increase in HSC proliferation (<xref ref-type="bibr" rid="bib77">Yamazaki et al., 2011</xref>). The TGF-β pathway has been implicated to interact with ErbB signaling (<xref ref-type="bibr" rid="bib13">Chow et al., 2011</xref>). <italic>tgfb1a</italic> is also robustly expressed in the migrating lateral line primordium and we therefore examined if this pathway acts as an intermediary between ErbB and Wnt/β-catenin signaling (ZFIN, <ext-link ext-link-type="uri" xlink:href="http://zfin.org">http://zfin.org</ext-link>). We treated wild type larvae with two pharmacological inhibitors of Tgf-β receptors, SB505124 or SB431542 (<xref ref-type="bibr" rid="bib25">Hagos and Dougan, 2007</xref>). Neither drug induced an increase in interneuromast number or clusters of interneuromast cells, suggesting that Tgf-β signaling does not inhibit interneuromast cell proliferation and is not involved in their regulation (data not shown).</p><p>In <italic>Drosophila</italic>, glial cells control quiescence and proliferation of neural progenitors depending on the developmental stage (<xref ref-type="bibr" rid="bib15">Doe, 2008</xref>). This non-cell-autonomous interaction is regulated by ErbB (<italic>spitz/TGF-α),</italic> HSPGs (<italic>trol/perlecan</italic>), and a secreted glycoprotein, <italic>anachronism</italic> (<xref ref-type="bibr" rid="bib17">Ebens et al., 1993</xref>; <xref ref-type="bibr" rid="bib71">Voigt et al., 2002</xref>; <xref ref-type="bibr" rid="bib50">Morante et al., 2013</xref>). Based on our recent results it would be interesting to examine if these molecules or pathways interact with the Wnt/β-catenin pathway. For example, Perlecan interacts with Wnt/β-catenin and Fgf signaling, however the other pathways have not been examined yet (<xref ref-type="bibr" rid="bib57">Park et al., 2003</xref>; <xref ref-type="bibr" rid="bib32">Kamimura et al., 2013</xref>).</p></sec><sec id="s3-3"><title>Developmental and postembryonic intercalary neuromast formation relies on similar but not identical mechanisms</title><p>We find that a set of the same signaling pathways are involved in neuromast differentiation from interneuromast cells as in neuromast formation in the primordium. However, primary neuromasts form within the migrating primordium and are deposited, whereas interneuromast cells proliferate to form a cluster of progenitor cells that eventually differentiate (<xref ref-type="other" rid="video1">Video 1</xref>). Therefore some differences exist. Both Fgf and Wnt/β-catenin are required for proliferation in the primordium (<xref ref-type="bibr" rid="bib2">Aman et al., 2011</xref>), whereas interneuromast proliferation depends on Wnt/β-catenin signaling only. Nevertheless, both neuromast types require Fgf signaling for rosette formation and hair cell specification (<xref ref-type="fig" rid="fig10 fig11">Figure 10A–F; 11E–H</xref> and <xref ref-type="bibr" rid="bib36">Lecaudey et al., 2008</xref>; <xref ref-type="bibr" rid="bib52">Nechiporuk and Raible, 2008</xref>). Fgf signaling is induced by Wnt/β-catenin signaling in the primordium, and likely also in interneuromast cells (<xref ref-type="fig" rid="fig4s2 fig4s3">Figure 4—figure supplement 2 and 3</xref>). However, it is not upregulated for many hours after the Wnt/β-catenin pathway is activated post ErbB inhibition (<xref ref-type="fig" rid="fig5 fig6">Figures 5 and 6</xref>) or after BIO treatment (<xref ref-type="fig" rid="fig4s3">Figure 4—figure supplement 3</xref>). Also, activation of the Wnt/β-catenin pathway by GSK-3 inhibition does not induce <italic>pea3</italic> in interneuromast cells during the first 24 hr post treatment (data not shown). Therefore, in contrast to the primordium, Fgf may not be a direct target of Wnt/β-catenin signaling in interneuromast cells. Neuromast formation in the primordium occurs in an existing and migrating tissue whereas intercalary neuromasts form from a string of cells. Therefore, it is not surprising that some of the signaling interactions between these two modes of neuromast formations are different.</p></sec><sec id="s3-4"><title>ErbB signaling can exert oncogenic or tumor suppressive functions</title><p>A detailed analysis of ErbB receptor functions is important as upregulation of ErbB family receptor tyrosine kinases is characteristic of many human cancers, notably breast cancer, resulting in enhanced tumorigenesis. Consequently, ErbB receptors are important therapeutic targets (<xref ref-type="bibr" rid="bib48">Moasser, 2007</xref>). Although cancers typically originate from unique cell types, tumors can be heterogeneous, containing multiple cell types. As the repertoire of pharmacological kinase inhibitors that are being developed to treat cancer increases, it becomes crucial to take into consideration that different ErbB receptor/ligand combinations can result in counterproductive cellular responses. Our results from the zebrafish lateral line stress the importance that ErbB signaling also has non-cell-autonomous, anti-proliferative functions, and that these anti-proliferative functions play a role during the regulation of neuronal progenitor regulation during development.</p><p>In conclusion, we identified that ErbB signaling in Schwann cells non-cell-autonomously regulates a molecular signaling network consisting of Wnt/β-catenin, Fgf and Notch pathways within neural progenitors, regulating their quiescence and activation. Given how conserved signaling interactions are during development and across species, it is tempting to speculate that these four pathways also interact in other neural progenitor cell populations.</p></sec></sec><sec id="s4" sec-type="materials|methods"><title>Materials and methods</title><sec id="s4-1"><title>Fish maintenance and fish strains</title><p>We used the following fish strains; <italic>rowgain/erbb2</italic> and <italic>hypersensitive/erbb3b</italic> (<xref ref-type="bibr" rid="bib22">Grant et al., 2005</xref>), <italic>nrg1-3</italic><sup><italic>z26</italic></sup> (<xref ref-type="bibr" rid="bib58">Perlin et al., 2011</xref>), <italic>erbb2</italic><sup><italic>st61</italic></sup> (<xref ref-type="bibr" rid="bib41">Lyons et al., 2005</xref>), <italic>Tg(foxd3:gfp)</italic><sup><italic>zf15</italic></sup> (<xref ref-type="bibr" rid="bib21">Gilmour et al., 2002</xref>), <italic>Tg(clndB:lyngfp)</italic><sup><italic>zf106</italic></sup> (<xref ref-type="bibr" rid="bib24">Haas and Gilmour, 2006</xref>), <italic>Et(krt4:EGFP)</italic><sup><italic>sqet20</italic></sup> (<xref ref-type="bibr" rid="bib56">Parinov et al., 2004</xref>), <italic>Tg(hsp70l:dkk1b-GFP)</italic><sup><italic>w32</italic></sup> (<xref ref-type="bibr" rid="bib67">Stoick-Cooper et al., 2007</xref>), <italic>Tg(hsp70l:dnfgfr1-EGFP)</italic><sup><italic>pd1</italic></sup> (<xref ref-type="bibr" rid="bib38">Lee et al., 2005</xref>), <italic>Tg(OTM:d2EGFP)</italic><sup><italic>kyu1</italic></sup> referred to as <italic>Tg(Tcf/Lef-miniP:dGFP)</italic> (<xref ref-type="bibr" rid="bib66">Shimizu et al., 2012</xref>), <italic>cntnap2a</italic><sup><italic>nkhgn39dET</italic></sup> referred to as HGN39D (<xref ref-type="bibr" rid="bib51">Nagayoshi et al., 2008</xref>) and <italic>lef1</italic><sup><italic>zd11</italic></sup> (<xref ref-type="bibr" rid="bib74">Wang et al., 2012</xref>). To generate the <italic>Tg(sox10:DNhsaerbb4-RFP)</italic>, we used the zebrafish Tol2 kit (<xref ref-type="bibr" rid="bib35">Kwan et al., 2007</xref>). We cloned the zebrafish 7.2 kb <italic>sox10</italic> promoter, obtained from Thomas Carney, into the 5′ entry vector. Human dominant-negative ErbB4 (hsaDNerbb4) construct was a gift of Gabriel Corfas (<xref ref-type="bibr" rid="bib61">Rio et al., 1997</xref>). The flag tag on hsaDNerbb4 was replaced with mRFP and cloned into the middle entry vector. To generate the final vector the 5′ entry, middle entry and 3′ polyA entry vector were recombined with the destination vector containing the <italic>cmcl2:gfp</italic> expression cassette, in order to identify transgenics based on GFP expression in the heart (<xref ref-type="bibr" rid="bib35">Kwan et al., 2007</xref>). The recombined vector was then injected into one cell stage embryos from the Tubingen strain along with transposase mRNA. Founder GFP positive heart carriers were raised and identified by crossing to wild type fish. The positive carriers have been maintained over three generations.</p></sec><sec id="s4-2"><title>In situ hybridization</title><p>In situ hybridization was performed as previously described (<xref ref-type="bibr" rid="bib34">Kopinke et al., 2006</xref>). The following probes were used <italic>lef1, fgf3, fgf10, fgfr1a</italic>, <italic>pea3, atoh1a, dkk1b, klf4</italic> (<xref ref-type="bibr" rid="bib3">Aman and Piotrowski, 2008</xref>), <italic>sfrp1a</italic> (<xref ref-type="bibr" rid="bib68">Tendeng and Houart, 2006</xref>), <italic>mbp</italic> (<xref ref-type="bibr" rid="bib6">Brosamle and Halpern, 2002</xref>), <italic>dkk2</italic> (<xref ref-type="bibr" rid="bib73">Wada et al., 2013b</xref>). To clone additional probes the following primers were used, for <italic>myca;</italic> forward 5′-ggtcctggacactccaccta-3′ and reverse 5′-atgcactctgtcgccttctt-3′, <italic>beta-catenin-2 (ctnnb2);</italic> forward 5’-cgactctgctcatccaacaa-3’ and reverse 5′-aggatctgcaggcagtctgt-3′, <italic>wnt10a</italic>; forward 5′-cttcagcaggggtttcagag-3′ and reverse 5′-tccctggctggtcttgttac-3′, <italic>wif1</italic>; forward 5′-aaccaaaggatggxtttcagg-3′ and reverse 5′- aggtttaaaccacatagttggtttcag-3′. For low magnification images, individual images were stitch together using ImageJ.</p></sec><sec id="s4-3"><title>Alkaline phosphatase and DASPEI staining</title><p>For alkaline phosphatase staining larvae were fixed overnight at room temperature in 4% paraformaldehyde. Larvae were then washed three times 5 min each in PBS/0.3% Tween-20 followed by three 5-min washes in staining buffer (50 mM MgCl<sub>2</sub>, 100 mM NaCl, 100 mM Tris pH 9.5 and 0.1% Tween-20). Larvae were then placed in staining buffer plus NBT/BCIP (Roche, USA) and stained at room temperature in the dark. The staining reaction was stopped with 4% paraformaldehyde. To label hair cells embryos were placed in a 0.06 mg/ml solution of DASPEI (2-(4-(dimethylamino)styryl)-N-ethylpyridinium iodide, [Invitrogen, USA]) diluted in embryo media for 10 min. Embryos were then briefly washed and anesthetized with Tricane for imaging under a dissecting fluorescent microscope. For low magnification images, individual images were stitch together using ImageJ.</p></sec><sec id="s4-4"><title>Transplantation assay</title><p>Transplantation assays were performed as previously described (<xref ref-type="bibr" rid="bib3">Aman and Piotrowski, 2008</xref>). Donor embryos were injected with 5% Alexa-568 and 3% lysine-fixable biotinylated-dextran (Invitrogen, USA) at the one cell stage. <italic>Tg(foxd3:gfp)</italic> or <italic>Tg(clndB:lyngfp)</italic> wild type donors were used to test rescue of <italic>erbb2</italic> mutant hosts. <italic>Tg(clndB:lyngfp)</italic> donors were used to test rescue of <italic>nrg1-3</italic><sup><italic>z26</italic></sup> mutant hosts. Host embryos were screened for lateral line or Schwann cell clones at 24 hr post fertilization (hpf). Embryos were imaged at 48 hpf to record the extent of the transplanted clone. To identify mutant hosts or donors, neuromasts were counted after DASPEI staining at 4 days post fertilization (dpf). Both transplanted and untransplanted sides of the hosts were imaged with a Zeiss LSM 510 or 780 confocal microscope at 20X. For low magnification images, individual images were stitch together using ImageJ.</p></sec><sec id="s4-5"><title>Pharmacological inhibitors</title><p>All chemical inhibitors were added to embryo media with a final concentration of 1% DMSO. Negative controls consisted of 1% DMSO only. The ErbB inhibitor AG1478 was used at 3 μM. The Fgf receptor inhibitors PD173074 or SU5402 were added at 100 μM or 10 μM respectively. The Wnt/β-catenin inhibitors IWR-1 or XAV939 were used at 40 μM and 20 μM respectively. The GSK-3 inhibitor BIO was used at 2 μM. The γ-secretase inhibitor LY411575 was used at 100 μM. LY411575 was obtained from Santa Cruz (USA) and all other inhibitors were purchased from Tocris (USA).</p></sec><sec id="s4-6"><title>BrdU assay</title><p>BrdU incorporation was performed in <italic>Tg(foxd3:gfp)</italic> or <italic>Tg(SqET20:gfp)</italic> transgenics by addition of 10 mM BrdU (Sigma, USA) with 1% DMSO or chemical inhibitors for various lengths of times as indicated in the text. Embryos were fixed in 4% paraformaldehyde overnight. BrdU immunostaining was performed as described except embryos were treated for 15 min with proteinase K (<xref ref-type="bibr" rid="bib2">Aman et al., 2011</xref>). To visualize GFP, larvae were also immunostained with rabbit anti-GFP (Invitrogen, USA) at 1/400 dilution. All embryos were counterstained with DAPI (Invitrogen, USA). To quantify BrdU index we counted BrdU and GFP double positive cells between the first and second deposited neuromasts. Immunostained embryos were imaged with a Zeiss LSM 510 or 780 confocal microscopes at 40X.</p></sec><sec id="s4-7"><title>Heat shock induction of gene expression</title><p>Heat shock induction was done at various developmental ages as indicated in the text. Embryos were placed at 39°C for 20 min, room temperature for 20 min and then another 20 min at 39°C. Embryos were then allowed to develop at 28.5°C.</p></sec><sec id="s4-8"><title>Time-lapse imaging</title><p>Time-lapse imaging was performed similar as described (<xref ref-type="bibr" rid="bib3">Aman and Piotrowski, 2008</xref>). <italic>Tg(SqET20:gfp)</italic> or <italic>Tg(SqET20:gfp)/Tg(sox10:DNhsaerbb4-RFP)</italic> were anesthetized with Tricaine and mounted in 0.8% low melting agarose on glass bottom dishes (MatTek, USA). Embryos were imaged with a Zeiss 710 or 780 confocal microscopes using a 40X water objective in a climate-controlled chamber set to 28°C.</p></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>We are grateful to Dr Gabriel Corfas for the human dominant negative ErbB4 plasmid, Dr Thomas Carney for the Sox10 promoter and Drs Chi-Bin Chen and Kristen Kwan for the Tol2-kit. We thank the zebrafish cores of both the University of Utah and the Stowers Institute for Medical Research for excellent zebrafish husbandry. We thank Drs Rich Dorsky, William Talbot, Darren Gilmour, Vladimir Korzh, Randall Moon, Kenneth Poss and Tohru Ishitani for providing zebrafish strains and Dr Robb Krumlauf and Marina Venero-Galanternik for valuable comments on the manuscript and Hua Li for help with statistics. We thank Dr Andy Aman and Robert Duncan for cloning of in situ probes, MinhTu Nguyen and Megan Smith for technical help, Joshua Sasine for help with pilot experiments and members of the Piotrowski laboratory for helpful discussions. We would also like to thank the reviewers for valuable suggestions and Mark Miller for help with graphic design.</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>MEL, 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>TP, 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: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. 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pub-id-type="doi">10.1016/j.cell.2011.09.053</pub-id></element-citation></ref></ref-list></back><sub-article article-type="article-commentary" id="SA1"><front-stub><article-id pub-id-type="doi">10.7554/eLife.01832.029</article-id><title-group><article-title>Decision letter</article-title></title-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Whitfield</surname><given-names>Tanya T</given-names></name><role>Reviewing editor</role><aff><institution>University of Sheffield</institution>, <country>United Kingdom</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://elife.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 “ErbB expressing Schwann cells control lateral line progenitor cells via non-cell-autonomous regulation of Wnt/β-catenin” for consideration at <italic>eLife</italic>. Your article has been favorably evaluated by a Senior editor and 2 reviewers, one of whom is a member of our Board of Reviewing Editors.</p><p>The Reviewing editor and the other reviewer 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>This work adds to our understanding of the mechanism of formation of intercalary neuromasts in the zebrafish lateral line system. The authors show that Wnt signaling-mediated proliferation in interneuromast cells is an early step in intercalary neuromast formation from these cells, which serve as a quiescent progenitor population. The study is a nice follow up of previous work from this group and others, which demonstrated a role for ErbB signaling in preventing premature intercalary neuromast formation. The figures are clear and easy to follow, and in general, the study is careful and thorough.</p><p>The following points should be addressed before publication:</p><p>1) Wnt signalling in the interneuromast cells. It is very interesting that the authors describe the expression of <italic>wnt10a</italic> in the context of intercalary neuromast formation, especially since no Wnt homologue has yet been shown to play a clear role primary neuromast formation. However, the role of <italic>wnt10a</italic> has not been tested rigorously. Which cells express it? Which signaling pathway determines its expression? Though obviously its expression contributes to Wnt signaling, is its transcription dependent on Wnt, FGF or Notch signaling?</p><p>2) Is the pattern of induced expression of <italic>fgf3</italic> and <italic>fgf10</italic> consistent with its dependence on Wnt signaling as in the leading end of the primordium or is it determined by <italic>atoh1a</italic> expression?</p><p>3) Which cells express <italic>her4.1</italic>?</p><p>4) Why does Wnt signaling reduce prematurely following AG1478? Does the down regulation of Wnt activity correlate with an upregulation of some other signaling system at this time?</p><p>5) Continued role of ErbB signalling in Schwann cells. The data supporting this claim (<xref ref-type="fig" rid="fig1s4">Figure 1–figure supplement 4</xref>) are not entirely clear. A graph of cell counts indicates a significant difference between 0.035 and 0.025 cells/µm (<xref ref-type="fig" rid="fig1s4">Figure 1–figure supplement 4</xref>), and the methods indicate that counts of cells were taken between the first and second neuromasts. In the first place, the distance between first and second neuromasts will be very different between wild-type and inhibitor-treated embryos (and can be variable even in wild-type). The authors should state the actual distance and the numbers of cells counted, which must be quite small. Later experiments merely seem to confirm the requirement for Schwann cells, rather than the requirement for continued ErbB signalling in Schwann cells, and so this conclusion should be toned down. In particular, the authors should remove the words 'ErbB signaling in' from the sentence “In conclusion, the absence of ErbB signaling in Schwann cells, leads first to interneuromast cell proliferation…”, as this particular experiment uses a transgenic line in which Schwann cells are depleted.</p><p>6) Neuregulin mutant. The authors should change the wording of the sentence:</p><p>“Here we show that a mutation in <italic>neuregulin 1-3 (nrg1-3)</italic> also exhibits precocious neuromast formation”. The authors have previously reported that this mutant was isolated through a screen for supernumerary neuromasts, so this finding was effectively reported in their previous publication (Perlin et al., 2011), which is not cited on this page.</p><p>7) Statistical tests. Student's t-tests are the only statistical tests mentioned in the manuscript, but there are a number of instances throughout (e.g., control vs multiple experimental treatments) where t-tests would not be appropriate. All the statistical tests should be re-evaluated carefully to make sure that appropriate tests are chosen for each experimental situation.</p><p>8) A reference for the PD173074 Fgf inhibitor should be given. This is not widely used, and it is surprising that it affects the lateral line ganglion so differently to SU5402 (<xref ref-type="fig" rid="fig10s1">Figure 10–figure supplement 1</xref>). The authors state that “likely, the two inhibitors affect a different set of Fgf receptors or pathways”, but this is not backed up with a citation or other precedent.</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.01832.030</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p><italic>1) Wnt signalling in the interneuromast cells. It is very interesting that the authors describe the expression of</italic> wnt10a <italic>in the context of intercalary neuromast formation, especially since no Wnt homologue has yet been shown to play a clear role primary neuromast formation. However, the role of</italic> wnt10a <italic>has not been tested rigorously. Which cells express it? Which signaling pathway determines its expression? Though obviously its expression contributes to Wnt signaling, is its transcription dependent on Wnt, FGF or Notch signaling</italic>?</p><p><italic>Which cells express</italic> wnt10a?</p><p>We have now included data showing that <italic>wnt10a</italic> is expressed in proliferating interneuromast cells only (<xref ref-type="fig" rid="fig4s1">Figure 4–figure supplement 1</xref>, described within the paragraph ‘Wnt/β-catenin, Fgf and Notch signaling pathways are upregulated in ErbB/Neuregulin pathway mutants’). To determine which cells express <italic>wnt10a</italic> we imaged <italic>Tg(ET20:gfp)</italic> siblings and <italic>Tg(ET20:gfp)</italic>/<italic>Tg(sox10:DNerbb4)</italic> larvae at 48hpf, then fixed and performed in situ hybridization for <italic>wnt10a</italic> on the same animals<italic>.</italic> We only show the data for <italic>Tg(ET20:gfp)</italic>/<italic>Tg(sox10:DNerbb4)</italic> larvae because, as shown in <xref ref-type="fig" rid="fig4">Figure 4</xref>, <italic>wnt10a</italic> is not expressed in control interneuromast cells. <italic>wnt10a</italic> is expressed within interneuromast cells of <italic>Tg(sox10:DNerbb4)</italic> but is downregulated in differentiating neuromasts.</p><p><italic>Which signaling pathways activate</italic> wnt10a?</p><p>We have now determined that <italic>wnt10a</italic> transcription is dependent on Wnt/β-catenin activity but is not dependent on Fgf or Notch signaling (Figure 6–figure supplement 1A-H, described within the paragraph ‘Wnt and Fgf ligands are dependent on Wnt/β-catenin signaling’). To test which signaling pathways regulate <italic>wnt10a</italic> expression we treated control sibling or <italic>Tg(sox10:DNerbb4)</italic> embryos at 32hpf (hours post fertilization) with Wnt/ β-catenin, Fgf or Notch signaling inhibitors, and subsequently fixed them at 48hpf and performed in situ hybridization. <italic>wnt10a</italic> is downregulated in <italic>Tg(sox10:DNerbb4)</italic> embryos by inhibition of Wnt/β-catenin signaling but not by inhibition of Fgf or Notch signaling (Figure 6–figure supplement 1, E-H).</p><p>We also tested if activation of the Wnt/β-catenin pathway with BIO leads to upregulation of <italic>wnt10a</italic> transcription in interneuromast cells. Wild type embryos were treated with DMSO or BIO at 48hpf then fixed at 6, 12 or 24 hours post-treatment. The known Wnt/β-catenin target gene <italic>lef1</italic> was induced at all time points (Figure 6–figure supplement 2, A-F, described within the paragraph ‘Wnt and Fgf ligands are dependent on Wnt/β-catenin signaling’). Likewise <italic>wnt10a</italic> was induced from 6-24 hours post treatment (Figure 6–figure supplement 2, G-L, described within the paragraph ‘Wnt and Fgf ligands are dependent on Wnt/β-catenin signaling’). These results support the conclusion of the loss of function studies that Wnt/β-catenin signaling acts upstream of <italic>wnt10a</italic>, whereas Fgf and Notch signaling do not affect <italic>wnt10a</italic> expression.</p><p><italic>2) Is the pattern of induced expression of</italic> fgf3 <italic>and</italic> fgf10 <italic>consistent with its dependence on Wnt signaling as in the leading end of the primordium or is it determined by</italic> atoh1a <italic>expression?</italic></p><p>We have now performed experiments that reveal that in interneuromast cells Fgf signaling depends on Wnt/β-catenin signaling as in the leading region of the primordium. However, Fgf ligand activation takes 12-24h suggesting that Fgf signaling might not be a direct target. To determine which signaling pathways induce <italic>fgf3</italic> and <italic>fgf10</italic>, we treated 32hpf control siblings or <italic>Tg(sox10:DNerbb4)</italic> embryos with Wnt/β-catenin, Fgf or Notch signaling inhibitors. We subsequently fixed the embryos at 48hpf and performed in situ hybridization with <italic>fgf3</italic> and <italic>fgf10</italic>. Both <italic>fgf3</italic> and <italic>fgf10</italic> expression are reduced in <italic>Tg(sox10:DNerbb4)</italic> embryos after blocking Wnt/β-catenin signaling but not after inhibition of Fgf or Notch signaling (Figure 6–figure supplement 1, M-P and U-X, described within the paragraph ‘Wnt and Fgf ligands are dependent on Wnt/β-catenin signaling’).</p><p>Our new experiments also demonstrate that Wnt/β-catenin activation induces transcription of <italic>fgf3</italic> and <italic>fgf10</italic> in interneuromast cells (Figure 6–figure supplement 2M-X, described within the paragraph ‘Wnt and Fgf ligands are dependent on Wnt/β-catenin signaling’). We treated wild type siblings with DMSO or BIO at 48hpf and fixed the larvae at 6, 12 or 24 hours post-treatment. <italic>fgf10</italic> is induced by 12 hours of BIO treatment while <italic>fgf3</italic> is only induced at 24 hours post treatment. This relatively long time span between Wnt/β-catenin activation and induction of <italic>fgf</italic> ligands suggests that Fgf signaling might not be a direct target of Wnt/β-catenin signaling in interneuromast cells.</p><p><italic>3) Which cells express her4.1</italic>?</p><p>We imaged 48pf <italic>Tg(ET20:gfp)</italic> siblings and <italic>Tg(ET20:gfp)</italic>/<italic>Tg(sox10:DNerbb4)</italic> larvae and fixed and performed in situ for <italic>her4.1.</italic> We only show the data for <italic>Tg(ET20:gfp)</italic>/<italic>Tg(sox10:DNerbb4)</italic> larvae because, as shown in <xref ref-type="fig" rid="fig4">Figure 4</xref>, <italic>her4.1</italic> is not expressed in control interneuromast cells. In <italic>Tg(ET20:gfp)</italic>/<italic>Tg(sox10:DNerbb4)</italic> larvae <italic>her4.1</italic> is expressed in deposited neuromasts (<xref ref-type="fig" rid="fig4s1">Figure 4–figure supplement 1 E-H</xref>, described within the paragraph ‘Wnt/β-catenin, Fgf and Notch signaling pathways are upregulated in ErbB/Neuregulin pathway mutants’) and also in some cells within newly differentiating neuromasts (<xref ref-type="fig" rid="fig4s1">Figure 4–figure supplement 1 E-H</xref>, yellow squares). However, <italic>her4.1</italic> is not expressed in proliferating interneuromast cells (<xref ref-type="fig" rid="fig4s1">Figure 4–figure supplement 1 E-H</xref>, yellow arrowheads).</p><p><italic>4) Why does Wnt signaling reduce prematurely following AG1478? Does the down regulation of Wnt activity correlate with an upregulation of some other signaling system at this time</italic>?</p><p>As noted by the reviewer Wnt/β-catenin signaling is upregulated at 6 and 12 hours post treatment but returns close to wild type levels at 24 hours post AG1478 treatment (<xref ref-type="fig" rid="fig6">Figure 6</xref>). It is possible that other signaling pathways are induced at this later stage and are responsible for inducing a Wnt/β-catenin inhibitor in interneuromast cells. One Wnt/β-catenin inhibitor, <italic>dkk1b,</italic> is an Fgf target in the primordium (Aman and Piotrowski, 2008). However, Fgf signaling, as judged by <italic>pea3</italic> expression, is not induced until after 24 hours of AG1478 treatment. We also performed an expression analysis with <italic>dkk1b</italic> and <italic>dkk2</italic> after AG1478 addition and neither gene was induced by 36 hours post treatment (data not shown), illustrating that <italic>dkk1b</italic> and <italic>dkk2</italic> are likely not the responsible Wnt/β-catenin signaling inhibitors at the AG1478 24 hour time point. This suggest that if Fgf is also regulating <italic>dkk</italic> expression during intercalary neuromast formation, as it is in the primordium, it is acting at a time point later than 24 hours post AG1478 treatment. Therefore, Fgf signaling is not a likely candidate to cause the decrease in Wnt/β-catenin signaling (<xref ref-type="fig" rid="fig6">Figure 6X</xref>).</p><p>Based on our experiments with the Notch inhibitor LY411575 (Figure 6–figure supplement 1D,L,T), and the absence of Notch signaling in wild type interneuromast cells, we concluded that Notch signaling is not required for keeping interneuromast cells quiescent by inhibiting Wnt/β-catenin signaling in wild type embryos. However, Notch signaling, as judged by <italic>her4.1</italic> expression, is induced at 24 hours after AG1478 and could therefore be responsible for the upregulation of a Wnt/β-catenin inhibitor at this stage (<xref ref-type="fig" rid="fig6">Figure 6EE</xref>). To investigate if Wnt/β-catenin signaling persists if Notch signaling is inhibited at 24 hour post AG1478 treatment we soaked embryos simultaneously in AG1478 and LY411575. Unfortunately, the combination of these two drugs had toxic effects on the embryos. We think this down regulation of Wnt/β-catenin signaling is interesting; however, it would require more elaborate experiments that are beyond the focus of this study.</p><p><italic>5) Continued role of ErbB signalling in Schwann cells. The data supporting this claim (</italic><xref ref-type="fig" rid="fig1s4"><italic>Figure 1–figure supplement 4</italic></xref><italic>) are not entirely clear. A graph of cell counts indicates a significant difference between 0.035 and 0.025 cells/µm (</italic><xref ref-type="fig" rid="fig1s4"><italic>Figure 1–figure supplement 4</italic></xref><italic>), and the methods indicate that counts of cells were taken between the first and second neuromasts. In the first place, the distance between first and second neuromasts will be very different between wild-type and inhibitor-treated embryos (and can be variable even in wild-type). The authors should state the actual distance and the numbers of cells counted, which must be quite small. Later experiments merely seem to confirm the requirement for Schwann cells, rather than the requirement for continued ErbB signalling in Schwann cells, and so this conclusion should be toned down. In particular, the authors should remove the words 'ErbB signaling in' from the sentence “In conclusion, the absence of ErbB signaling in Schwann cells, leads first to interneuromast cell proliferation…”, as this particular experiment uses a transgenic line in which Schwann cells are depleted</italic>.</p><p>We counted Schwann cells between the first and second deposited neuromasts, L1 and L2. We re-worded the methods section to make this clearer. We agree that the distance between neuromasts L1 and L2 varies and therefore we chose to calculate the number of Schwann cells per μm to control for the variation in distance between neuromasts. We have added within the figure legend the average number of Schwann cells counted for each group (<xref ref-type="fig" rid="fig1s4">Figure 1–figure supplement 4</xref>). At 6 hours post DMSO treatment we counted an average of 12 Schwann cells and the average distance between L1 and L2 was 531μm. In AG1478 treated larvae we counted on average 13 Schwann cells over 546 μm. At 14 hours post DMSO treatment we counted an average of 20 Schwann cells over 576 μm and 14 Schwann cells over 536 μm after AG1478 treatment. At 24 hours post DMSO treatment we counted an average of 14 Schwann cells over 511 μm and 6 Schwann cells over 508 μm after AG1478 treatment.</p><p>We have removed 'ErbB signaling in' from the sentence indicated, as we agree the experiment described is examining Schwann cell deficient zebrafish.</p><p><italic>6) Neuregulin mutant. The authors should change the wording of the sentence</italic>:</p><p><italic>“Here we show that a mutation in neuregulin 1-3 (nrg1-3) also exhibits precocious neuromast formation”. The authors have previously reported that this mutant was isolated through a screen for supernumerary neuromasts, so this finding was effectively reported in their previous publication (Perlin et al., 2011), which is not cited on this page</italic>.</p><p>We have deleted this sentence from the manuscript and added <italic>nrg1-3</italic> to the preceding sentence of the manuscript.</p><p><italic>7) Statistical tests. Student's t-tests are the only statistical tests mentioned in the manuscript, but there are a number of instances throughout (e.g., control vs multiple experimental treatments) where t-tests would not be appropriate. All the statistical tests should be re-evaluated carefully to make sure that appropriate tests are chosen for each experimental situation</italic>.</p><p>We have re-evaluated the statistical analysis and have performed a one-way ANOVA with Tukey pairwise comparison for experiments that have multiple experimental treatments. This new analysis was performed on data graphed in <xref ref-type="fig" rid="fig7 fig10">Figures 7A, 10G, 10O-P</xref>, <xref ref-type="fig" rid="fig1s1">1–figure supplement 1F</xref> and <xref ref-type="fig" rid="fig10s1">10–figure supplement 1</xref>. For two of these experiments the results are now no longer significant, as the p-value is greater than 0.05. One of these experiments is the test of neuromast formation after AG1478 is added at different developmental time points (<xref ref-type="fig" rid="fig1s1">Figure1–figure supplement 1F</xref>). The 72 hour time point is no longer significantly different from the DMSO treated group. We have updated the text and figure to reflect this change. This does not change the main conclusion of this experiment which is AG1478 induces an increase in neuromast number, even if given after Schwann cell migration is completed at 50 or 59 hpf.</p><p>The second experiment is described in <xref ref-type="fig" rid="fig10">Figure 10O</xref>. The difference in BrdU incorporation within <italic>Tg(SqET20:gfp)</italic> cells between DMSO and SU5402 treated control siblings is not significant. We have updated the text to reflect this change. Again this does not change the conclusion of the experiment that Fgf pathway inhibition, either by SU5402 or PD173074, does not block interneuromast cell proliferation in <italic>Tg(sox10:DNerbb4)</italic> fish.</p><p>For the remaining experiments we continue to use a Student’s t-test<bold>,</bold> as we are comparing only two groups<bold>.</bold> We now state within the figure legends which statistical analysis was used for each graph.</p><p><italic>8) A reference for the PD173074 Fgf inhibitor should be given. This is not widely used, and it is surprising that it affects the lateral line ganglion so differently to SU5402 (</italic><xref ref-type="fig" rid="fig10s1"><italic>Figure 10–figure supplement 1</italic></xref><italic>). The authors state that “likely, the two inhibitors affect a different set of Fgf receptors or pathways”, but this is not backed up with a citation or other precedent</italic>.</p><p>We have not found a reference that directly compares the two different FGFR inhibitors with different FGF receptors and we therefore only reference the original paper that describes PD173074 (<xref ref-type="bibr" rid="bib49">Mohammadi et al., 1998</xref>). As mentioned in the text, the fact that PD173074 causes an increase in posterior lateral line ganglion cell number matches the results the Riley laboratory acquired using a heat-shock dominant negative FGFR transgenic in the zebrafish statoacoustic ganglion (<xref ref-type="bibr" rid="bib70">Vemaraju et al., 2012</xref>). As dominant negative Fgfr is likely a more specific inhibitor than pharmacological inhibitors we suspect that PD173074 is more specific in blocking Fgf signaling within the posterior lateral line ganglion than SU5402 that is possibly affecting other receptor tyrosine kinases. We would like to emphasize that this difference in drug effect was only seen in the posterior lateral line ganglion. SU5402 and PD173074 induced the same phenotypes during primordium migration (data not shown) as well as intercalary neuromast formation described in this paper.</p></body></sub-article></article>