elife02663.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">02663</article-id><article-id pub-id-type="doi">10.7554/eLife.02663</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><subj-group subj-group-type="heading"><subject>Neuroscience</subject></subj-group></article-categories><title-group><article-title>GSK-3 signaling in developing cortical neurons is essential for radial migration and dendritic orientation</article-title></title-group><contrib-group><contrib contrib-type="author" corresp="yes" id="author-12025"><name><surname>Morgan-Smith</surname><given-names>Meghan</given-names></name><contrib-id contrib-id-type="orcid">http://orcid.org/0000-0001-5122-2330</contrib-id><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="con1"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-12026"><name><surname>Wu</surname><given-names>Yaohong</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-12027"><name><surname>Zhu</surname><given-names>Xiaoqin</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con3"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-12028"><name><surname>Pringle</surname><given-names>Julia</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con4"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-11678"><name><surname>Snider</surname><given-names>William D</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="corresp" rid="cor2">*</xref><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-3"/><xref ref-type="fn" rid="con5"/><xref ref-type="fn" rid="conf1"/></contrib><aff id="aff1"><institution content-type="dept">UNC Neuroscience Center</institution>, <institution>University of North Carolina</institution>, <addr-line><named-content content-type="city">Chapel Hill</named-content></addr-line>, <country>United States</country></aff><aff id="aff2"><institution content-type="dept">Neurobiology Curriculum</institution>, <institution>University of North Carolina</institution>, <addr-line><named-content content-type="city">Chapel Hill</named-content></addr-line>, <country>United States</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Miller</surname><given-names>Freda</given-names></name><role>Reviewing editor</role><aff><institution>The Hospital for Sick Children Research Institute, University of Toronto</institution>, <country>Canada</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>meghan_morgan@med.unc.edu</email> (MM);</corresp><corresp id="cor2"><label>*</label>For correspondence: <email>wsnider@med.unc.edu</email> (WDS)</corresp></author-notes><pub-date date-type="pub" publication-format="electronic"><day>29</day><month>07</month><year>2014</year></pub-date><pub-date pub-type="collection"><year>2014</year></pub-date><volume>3</volume><elocation-id>e02663</elocation-id><history><date date-type="received"><day>27</day><month>02</month><year>2014</year></date><date date-type="accepted"><day>03</day><month>07</month><year>2014</year></date></history><permissions><copyright-statement>© 2014, Morgan-Smith et al</copyright-statement><copyright-year>2014</copyright-year><copyright-holder>Morgan-Smith et al</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This article is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="elife02663.pdf"/><abstract><object-id pub-id-type="doi">10.7554/eLife.02663.001</object-id><p>GSK-3 is an essential mediator of several signaling pathways that regulate cortical development. We therefore created conditional mouse mutants lacking both GSK-3α and GSK-3β in newly born cortical excitatory neurons. <italic>Gsk3</italic>-deleted neurons expressing upper layer markers exhibited striking migration failure in all areas of the cortex. Radial migration in hippocampus was similarly affected. In contrast, tangential migration was not grossly impaired after <italic>Gsk3</italic> deletion in interneuron precursors. <italic>Gsk3</italic>-deleted neurons extended axons and developed dendritic arbors. However, the apical dendrite was frequently branched while basal dendrites exhibited abnormal orientation. GSK-3 regulation of migration in neurons was independent of Wnt/β-catenin signaling. Importantly, phosphorylation of the migration mediator, DCX, at ser327, and phosphorylation of the semaphorin signaling mediator, CRMP-2, at Thr514 were markedly decreased. Our data demonstrate that GSK-3 signaling is essential for radial migration and dendritic orientation and suggest that GSK-3 mediates these effects by phosphorylating key microtubule regulatory proteins.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02663.001">http://dx.doi.org/10.7554/eLife.02663.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.02663.002</object-id><title>eLife digest</title><p>In the brain, one of the most striking features of the cerebral cortex is that its neurons are organized into different layers that are specifically connected to one another and to other regions of the brain. How newly generated neurons find their appropriate layer during the development of the brain is an important question; and, in humans, when this process goes awry, it can often result in seizures and mental retardation.</p><p>An enzyme called GSK-3 regulates several major signaling pathways important to brain development. The GSK-3 enzyme switches other proteins on or off by adding phosphate groups to them.</p><p>Morgan-Smith et al. set out to better understand the role of GSK-3 in brain development by deleting the genes for this enzyme specifically in the cerebral cortex of mice. Mice have two genes that encode slightly different forms of the GSK-3 enzyme. Deleting both of these in different groups of neurons during brain development revealed that a major group of neurons need GSK-3 in order to migrate to the correct layer. Specifically, the movement of neurons from where they arise in the central region of the brain to the outermost layer (a process called radial migration) was disrupted when the GSK-3 genes were deleted.</p><p>Morgan-Smith et al. further found that cortical neurons without GSK-3 were unable to develop the shape needed to undertake radial migration because they failed to switch from having many branches to having just two main branches. Additional experiments revealed that these abnormalities did not depend on certain signaling pathways, such as the Wnt-signaling pathway or the PI3K signaling pathway that can control GSK-3 activity.</p><p>Instead, Morgan-Smith et al. found that two proteins that are normally targeted by the GSK-3 enzyme have fewer phosphate groups than normal in the cortical neurons that did not contain the enzyme: both of these proteins regulate the shape of neurons by interacting with the molecular ‘scaffolding’ within the cell. The GSK-3 enzyme was already known to modify the activities of many other proteins that affect the migration of cells. Thus, the findings of Morgan-Smith et al. suggest that this enzyme may coordinate many of the mechanisms thought to underlie this process during brain development.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02663.002">http://dx.doi.org/10.7554/eLife.02663.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>neuronal migration</kwd><kwd>neuronal morphology</kwd><kwd>cortical development</kwd><kwd>GSK-3</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd>mouse</kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000002</institution-id><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>NS050968</award-id><principal-award-recipient><name><surname>Snider</surname><given-names>William D</given-names></name></principal-award-recipient></award-group><award-group id="par-2"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000002</institution-id><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>5F31NS067838</award-id><principal-award-recipient><name><surname>Morgan-Smith</surname><given-names>Meghan</given-names></name></principal-award-recipient></award-group><award-group id="par-3"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000002</institution-id><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>NS045892</award-id><principal-award-recipient><name><surname>Snider</surname><given-names>William D</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>The kinase GSK-3 regulates cortical neuronal migration and dendritic orientation by phosphorylating key cytoskeletal proteins.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="s1" sec-type="intro"><title>Introduction</title><p>Glycogen synthase kinase (GSK-3) α and β are serine/threonine kinases that act as key downstream regulators in multiple signaling pathways, including Wnt/β-catenin, receptor tyrosine kinase (RTK)/PI3K, and Sonic hedgehog (Shh) (<xref ref-type="bibr" rid="bib44">Kaidanovich-Beilin et al., 2012</xref>). GSK-3s act via mechanisms that include regulation of transcription factors, control of multiple aspects of cellular metabolism, and phosphorylation of cytoskeletal proteins (<xref ref-type="bibr" rid="bib39">Hur and Zhou, 2010</xref>; <xref ref-type="bibr" rid="bib45">Kaidanovich-Beilin and Woodgett, 2011</xref>). Most often, although not invariably, GSK-3s function as negatively acting kinases by inhibiting the functions of substrates at baseline. Inhibition is then relieved via signaling pathways that engage GSK-3 (<xref ref-type="bibr" rid="bib21">Doble and Woodgett, 2003</xref>; <xref ref-type="bibr" rid="bib45">Kaidanovich-Beilin and Woodgett, 2011</xref>). For most GSK-3 substrates, phosphorylation by another kinase near the GSK-3 site (‘priming’) is required for, or enhances, GSK-3 substrate phosphorylation (<xref ref-type="bibr" rid="bib18">Cohen and Frame, 2001</xref>; <xref ref-type="bibr" rid="bib21">Doble and Woodgett, 2003</xref>; <xref ref-type="bibr" rid="bib45">Kaidanovich-Beilin and Woodgett, 2011</xref>). Priming kinases for GSK-3 substrates include cyclin dependent kinase-5 (cdk5), a kinase that is known to regulate important neurodevelopmental events like radial migration (<xref ref-type="bibr" rid="bib68">Tanaka et al., 2004</xref>; <xref ref-type="bibr" rid="bib19">Cole et al., 2006</xref>; <xref ref-type="bibr" rid="bib49">Li et al., 2006</xref>; <xref ref-type="bibr" rid="bib73">Xie et al., 2006</xref>).</p><p>In the nervous system, GSK-3β has long been thought to be a target of lithium used in treatment of bipolar disorder (<xref ref-type="bibr" rid="bib47">Klein and Melton, 1996</xref>; <xref ref-type="bibr" rid="bib56">O'Brien et al., 2004</xref>). Some of the GSK-3β effects related to lithium actions are due to regulation of signaling downstream of dopamine receptors (<xref ref-type="bibr" rid="bib6">Beaulieu et al., 2004</xref>, <xref ref-type="bibr" rid="bib5">2008</xref>; <xref ref-type="bibr" rid="bib72">Urs et al., 2012</xref>). More recently GSK-3 signaling has been implicated in the pathogenesis of schizophrenia (<xref ref-type="bibr" rid="bib26">Emamian et al., 2004</xref>; <xref ref-type="bibr" rid="bib51">Mao et al., 2009</xref>; <xref ref-type="bibr" rid="bib25">Emamian, 2012</xref>). Disrupted in Schizophrenia-1 (DISC1), mutated in some familial cases of schizophrenia, is thought to function in part by modulating GSK-3β effects on progenitor proliferation (<xref ref-type="bibr" rid="bib51">Mao et al., 2009</xref>; <xref ref-type="bibr" rid="bib65">Singh et al., 2011</xref>).</p><p>Despite the obvious importance of GSK-3 signaling in pathogenesis and treatment of psychiatric disorders, there are important gaps in information on the role of GSK-3 in the developing brain. It has clearly been established that GSK-3 signaling is a strong regulator of radial progenitor proliferation in the developing cerebral cortex and that these effects are at least partly meditated through β-catenin (<xref ref-type="bibr" rid="bib15">Chenn and Walsh, 2002</xref>; <xref ref-type="bibr" rid="bib46">Kim et al., 2009</xref>). Additionally, a recent study demonstrated an important role for GSK-3 in regulating INP amplification, an effect associated with GSK-3β binding to the scaffolding protein Axin (<xref ref-type="bibr" rid="bib28">Fang et al., 2013</xref>). Thus, critical roles for GSK-3 signaling in processes that control neuron number in the developing telencephalon have been established.</p><p>In contrast, functions of GSK-3 in regulating developing cortical neurons are much less clear. Multiple in vitro studies have suggested roles for GSK-3 in regulating neuronal polarity and axon growth and branching (see <xref ref-type="bibr" rid="bib39">Hur and Zhou, 2010</xref> for review). These functions are thought to be mediated via GSK-3 phosphorylation of microtubule-associated proteins (MAPs) including Collapsin response mediator protein-2 (CRMP-2) (<xref ref-type="bibr" rid="bib76">Yoshimura et al., 2005</xref>), Adenomatous polypsis coli (APC) (<xref ref-type="bibr" rid="bib64">Shi et al., 2004</xref>; <xref ref-type="bibr" rid="bib77">Zhou et al., 2004</xref>), Tau (<xref ref-type="bibr" rid="bib67">Stoothoff and Johnson, 2005</xref>), microtubule-associated protein 1B (MAP1B) (<xref ref-type="bibr" rid="bib69">Trivedi et al., 2005</xref>), Doublecortin (DCX) (<xref ref-type="bibr" rid="bib10">Bilimoria et al., 2010</xref>), and subsequent regulation of cytoskeletal dynamics. In general, inhibition of GSK-3β via serine 9 (ser9) and GSK-3α via serine 21 (ser21) phosphorylation and subsequent relief of phosphorylation of downstream targets is thought to be required for the formation of the axon and subsequent axonal growth (<xref ref-type="bibr" rid="bib43">Jiang et al., 2005</xref>; <xref ref-type="bibr" rid="bib39">Hur and Zhou, 2010</xref>). In a similar vein, a study employing in utero electroporation of an activating construct suggested that GSK-3 inhibition was essential for radial-guided cortical neuronal migration downstream of STK11 (LKB1) via a mechanism involving APC (<xref ref-type="bibr" rid="bib2">Asada and Sanada, 2010</xref>). A prediction of this work might be that GSK-3 deletion would enhance axon growth and radial migration. However, to date these effects of GSK-3 on neuronal polarity and migration have not been confirmed with mouse genetic studies. Further, mice with point mutation knockins that prevent ser9/21 phosphorylation are viable and have not been reported to show defects in neuronal morphology or migration (<xref ref-type="bibr" rid="bib52">McManus et al., 2005</xref>; <xref ref-type="bibr" rid="bib30">Gartner et al., 2006</xref>). Finally, GSK-3 may regulate migration and morphology of cortical neurons via entirely different pathways for example via mediating effects of semaphorin signaling (<xref ref-type="bibr" rid="bib13">Chen et al., 2008</xref>; <xref ref-type="bibr" rid="bib62">Renaud et al., 2008</xref>; <xref ref-type="bibr" rid="bib54">Nakamura et al., 2009</xref>).</p><p>We have now assessed GSK-3 functions in newly born cortical neurons using <italic>Neurod6</italic> (<italic>Nex</italic>)-Cre (<xref ref-type="bibr" rid="bib32">Goebbels et al., 2006</xref>) to mediate recombination of <italic>Gsk3</italic> floxed alleles in INPs and newly born excitatory neurons. We demonstrate, surprisingly, that GSK-3 activity is essential for radial neuron migration in all areas of the cortex and in the hippocampus. In contrast, tangential migration is not affected after <italic>Gsk3</italic> deletion in cortical interneuron precursors. Remarkably, the migration effects appear to be independent of Wnt/β-catenin signaling that mediates GSK-3 functions in neuronal progenitors. The few upper layer neurons that reached their normal location exhibited strikingly abnormal orientation of basal dendrites. GSK-3 control of migration and morphology is correlated with the regulation of phosphorylation of DCX on ser327 and CRMP-2 on Thr514. We conclude that GSK-3 is a critical regulator of neuronal migration and morphogenesis and that GSK-3 regulation is mediated by phosphorylation of key cytoskeletal proteins.</p></sec><sec id="s2" sec-type="results"><title>Results</title><sec id="s2-1"><title>GSK-3 signaling regulates migration of cortical excitatory neurons</title><p>To investigate the function of GSK-3 in developing cortical neurons, we generated <italic>Gsk3a</italic><sup><italic>−/−</italic></sup><italic>Gsk3b</italic><sup><italic>loxp/loxp</italic></sup><italic>:Neurod6</italic>-<italic>Cre</italic> mice (<italic>Gsk3:Neurod6</italic>). The <italic>Neurod6</italic>-<italic>Cre</italic> line induces recombination in intermediate progenitors of the dorsal telencephalon and early postmitotic neurons beginning at approximately embryonic day 11 (E11) (<xref ref-type="bibr" rid="bib32">Goebbels et al., 2006</xref>). Prior studies using crosses with reporter lines indicate that recombination occurs in virtually all excitatory pyramidal neurons in the dorsal telencephalon (<xref ref-type="bibr" rid="bib32">Goebbels et al., 2006</xref>; <xref ref-type="bibr" rid="bib53">Monory et al., 2006</xref>). Western blot analysis of lysates from the whole cortex, shows a 60% decrease in GSK-3β protein at E19.5 as compared with heterozygous litter mates (<xref ref-type="fig" rid="fig1">Figure 1C</xref>). Remaining GSK-3β protein in the mutants is likely due to lack of recombination in interneurons and developing glia. At E19, <italic>Gsk3:Neurod6</italic> brains are roughly the same size as littermate controls. However, <italic>Gsk3:Neurod6</italic> mice die shortly after birth (P0–P3) for reasons that have not yet been determined.<fig-group><fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.02663.003</object-id><label>Figure 1.</label><caption><title>GSK-3 signaling is essential for proper lamination of the developing cortex.</title><p>(<bold>A–A'</bold>) Cux-1 staining (red) in coronal sections from control and <italic>Gsk3:Neurod6</italic> mice at E19.5. Cux-1 neurons are strikingly mislocalized in <italic>Gsk3:Neurod6</italic> mutants (orange arrows) including a small population of neurons that remain in the ventricular zone (yellow arrowhead). Nuclei were counterstained with DRAQ5. Scale bar = 500 μm. (n = 4). (<bold>B–B'</bold>) Cux-1 staining in parasagittal vibratome sections from control and <italic>Gsk3:Neurod6</italic> mutants at E18.5. Cux-1 expressing neurons (arrows) are mislocalized in <italic>Gsk3:Neurod6</italic> mutants and populate the deeper layers of the cortex along the entire rostrol/caudal axis. Scale bar = 200 μm. (<bold>C</bold>) Representative Western blot confirms strongly reduced GSK-3β protein levels in the E19.5 <italic>Gsk3:Neurod6</italic> cortex compared to heterozygous control (n = 3 het control, n = 3 CKO). Relative Density *p&lt;0.05, unpaired t-test.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02663.003">http://dx.doi.org/10.7554/eLife.02663.003</ext-link></p></caption><graphic xlink:href="elife02663f001"/></fig><fig id="fig1s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02663.004</object-id><label>Figure 1—figure supplement 1.</label><caption><title>Migration defect apparent by E16 after <italic>Gsk3</italic> deletion in cortical excitatory neurons.</title><p>Coronal cryostat sections at E16. Layer 6 TBR1 neurons populate appropriate layers in control and <italic>Gsk3:Neurod6</italic> mutants. Cux-1 (red) expressing neurons, marker for upper layer 2/3 neurons, are mislocalized in <italic>Gsk3:Neurod6</italic> mutant coronal sections. L1 staining labels axon tracts.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02663.004">http://dx.doi.org/10.7554/eLife.02663.004</ext-link></p></caption><graphic xlink:href="elife02663fs001"/></fig></fig-group></p><p>To explore neuronal functions of GSK-3, we first assessed cortical lamination at E16, a time of rapid neuronal migration along radial glial processes. Deep layer neurons appeared to be normally positioned (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>). Thus staining with Tbr1, a layer 6 marker, revealed a distinctive band in the deeper layers of the cortex in both controls and <italic>Gsk3:Neurod6</italic> mutants. In contrast, we noted clear abnormalities in the localization of Cux1 expressing neurons that normally populate Layers 2/3 (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>). A clear band of Cux1 positive neurons has formed in controls by E16. In contrast <italic>Gsk3:Neurod6</italic> mice exhibit a dispersion of Cux1 cells with fewer neurons reaching the outermost layer, even at this early developmental stage.</p><p>Dramatic mislocalization of layer 2/3 neurons was apparent by E19.5. Coronal sections through developing somatosensory cortex showed a large population of Cux1 expressing neurons essentially 'stuck' in the intermediate zone and throughout the deeper cortical layers (orange arrows) (<xref ref-type="fig" rid="fig1">Figure 1A–A’</xref>). Indeed some Cux1-expressing neurons in mutants were observed in the ventricular zone (yellow arrowhead). The migration defect in <italic>Gsk3:Neurod6</italic> mutants was striking along the entire rostrol/caudal axis at E18.5, as observed in parasagittal sections (arrows) (<xref ref-type="fig" rid="fig1">Figure 1B–B'</xref>). The migration defect was particularly prominent anteriorly, a developmental profile corresponding to the neurogenic gradient of the developing cortex (<xref ref-type="bibr" rid="bib12">Caviness et al., 2009</xref>).</p><p>We also generated <italic>Gsk3a</italic><sup><italic>loxp/loxp</italic></sup><italic>, Gsk3b</italic><sup><italic>loxp/loxp</italic></sup><italic>: Neurod6-Cre</italic> mice (<italic>Gsk3</italic><sup><italic>loxp</italic></sup><italic>:Neurod6</italic>) using a <italic>Gsk3a</italic><sup><italic>loxp/loxp</italic></sup> mouse line, which harbors loxp sites flanking exon 2 of <italic>Gsk3a</italic>. Western blot analysis of lysates from the whole cortex at P0, verified an 85% decrease in GSK-3α and a 76% decrease in GSK-3β protein when compared with wild-type littermate controls (<xref ref-type="fig" rid="fig2">Figure 2B</xref>). At P0 as expected, <italic>Gsk3</italic><sup><italic>loxp</italic></sup><italic>:Neurod6</italic> mice showed the same migration failure of upper layer neurons as <italic>Gsk3:Neurod6</italic> (<xref ref-type="fig" rid="fig2">Figure 2A</xref>). Inspection and quantification, (<xref ref-type="fig" rid="fig2">Figure 2A,C</xref>) showed most Cux1+ neurons stuck in deeper layers in mutants, whereas in controls a heavy majority of neurons had already migrated to the most superficial layers. An increase in neurogenesis could in theory account for some of the Cux1+ neurons found in deeper layers, but counts of Cux1+ cells revealed no major difference in numbers between <italic>Gsk3</italic> mutants and controls (control Cux1/total = 32.45% ± 3.74, mutant = 34.31% ± 0.649, p=0.561, unpaired t-test).<fig-group><fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.02663.005</object-id><label>Figure 2.</label><caption><title>Migration defects in <italic>Gsk3</italic>-deleted mice are persistent.</title><p>(<bold>A</bold>) P0:Cux 1 staining (red) in coronal sections of <italic>Gsk3</italic><sup><italic>loxp</italic></sup><italic>:Neurod6</italic> mutants and littermate heterozygote controls. Cux1-expressing neurons are localized to layer2/3 in controls (denoted by yellow dashed lines) while Cux1-expressing neurons are localized throughout the cortical plate in the mutants. (n = 5, scale bar = 100 μm). (<bold>B</bold>) Representative Western blot of P0 cortical lysates confirms strongly reduced GSK-3α and GSK-3β protein levels in <italic>Gsk3</italic><sup><italic>loxp</italic></sup><italic>:Neurod6</italic> mutants when compared to <italic>Gsk3a</italic><sup><italic>loxp/loxp</italic></sup><italic>Gsk3b</italic><sup><italic>loxp/loxp</italic></sup> controls. GAPDH was probed as a loading control (n = 3 control, n = 3 CKO). (<bold>C</bold>) P0 quantification of control and <italic>Gsk3</italic><sup><italic>loxp</italic></sup><italic>:Neurod6</italic> Cux1 neurons using 8 bin analysis spanning white matter (WM) to the pial surface (PS), (n = 2 het control, n = 2 CKO). (<bold>D–D'</bold>) P7: <italic>Gsk3</italic><sup><italic>loxp</italic></sup><italic>:Neurod6</italic> mutants stained with Cux1 (red) show persistent altered lamination with Cux1-expressing neurons spread throughout all layers of the cortex. Littermate controls show normal Cux1 distribution in layer 2/3. Scale bar = 200 μm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02663.005">http://dx.doi.org/10.7554/eLife.02663.005</ext-link></p></caption><graphic xlink:href="elife02663f002"/></fig><fig id="fig2s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02663.006</object-id><label>Figure 2—figure supplement 1.</label><caption><title>Gsk3 overexpression enhances radial migration.</title><p>(<bold>A</bold>) E19.5 coronal sections showing normal migration after in utero electroporation of <italic>Neurod1-Cre</italic> and <italic>lox-STOP-lox-Ai9</italic> (red neurons) and (<bold>B</bold>) Coronal sections showing abnormal neuronal migration after electroporation of <italic>Neurod1-Gsk3b</italic> and <italic>Neurod1</italic>-GFP (green neurons). Enhanced migration is apparent in <bold>B</bold>. Scale bar = 200 μm. (<bold>C</bold>) E19.5 quantification of migration in control and <italic>Gsk3</italic> over-expressing neurons using 8 bin analysis as previously described. Significantly more <italic>Gsk3</italic> over-expressing neurons were found in the outermost bin 8 and fewer <italic>Gsk3</italic> over-expressing neurons populated bin 6. p-values shown in figure, unpaired t-test. (control n = 2 mice from two individual litters, 1276 neurons vs <italic>Gsk3</italic> overexpression n = 3 mice from two individual litters, 1959 neurons).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02663.006">http://dx.doi.org/10.7554/eLife.02663.006</ext-link></p></caption><graphic xlink:href="elife02663fs002"/></fig></fig-group></p><p>A few Cux1 neurons migrated successfully to layer 2/3 (area denoted by yellow lines) in <italic>Gsk3</italic><sup><italic>loxp</italic></sup><italic>:Neurod6</italic> mutants (<xref ref-type="fig" rid="fig2">Figure 2A</xref>). Whether this subset of normally placed neurons did not undergo recombination at an early enough stage for migration to be regulated could not be determined. However, these properly positioned neurons exhibited abnormalities of dendritic orientation (see <xref ref-type="fig" rid="fig5">Figure 5</xref>).</p><p>Interestingly, mice with floxed alpha rather than null alpha alleles survived somewhat longer than the <italic>Gsk3:Neurod6</italic> mice but died after the second postnatal week (P15–P17). At P7, <italic>Gsk3</italic><sup><italic>loxp</italic></sup><italic>:Neurod6</italic> mutants exhibited a striking lamination phenotype involving Cux1 neurons (<xref ref-type="fig" rid="fig2">Figure 2D–D'</xref>). This persistent lamination defect demonstrates that <italic>Gsk3</italic> deletion results in permanent migration failure and does not simply result in a delay.</p><p>To further assess functions of GSK-3 in radial migration, we over-expressed <italic>Gsk3b</italic> in newly born cortical neurons using a p<italic>Neurod1</italic> (<italic>Neurod1</italic>) vector (<xref ref-type="bibr" rid="bib35">Guerrier et al., 2009</xref>). Co-electroporation of <italic>Neurod1-Cre</italic> and lox-STOP-lox Ai9 in control embryos at E15 (<xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1A</xref>) was compared to co-electroporation of <italic>Neurod1</italic>-<italic>Gsk3b</italic> and <italic>Neurod1</italic>-Egfp in experimental embryos (<xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1B</xref>). Importantly, over-expression of <italic>Gsk3b</italic> did not inhibit migration as might have been expected from prior studies (<xref ref-type="bibr" rid="bib2">Asada and Sanada, 2010</xref>). In fact, <italic>Gsk3b</italic> over-expression enhanced neuronal migration and resulted in greater than normal numbers of neurons populating the outermost layers of the cortex by E19.5 (<xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1C</xref>).</p></sec><sec id="s2-2"><title>Specificity of GSK-3 signaling for radial migration</title><p>Importantly, the migration defect in the developing cortex is specific to excitatory pyramidal neurons. In order to assess interneurons, we used the <italic>Dlx5/6</italic>-<italic>Cre</italic> (<xref ref-type="bibr" rid="bib66">Stenman et al., 2003</xref>) line to generate conditional mice lacking <italic>Gsk3</italic> in GABAergic interneurons. A robust decrease of GSK-3β protein (84%) was observed in E18 MGE lysates from <italic>Gsk3:DLX5/6-Cre</italic> mice when compared to littermate controls (<xref ref-type="fig" rid="fig3">Figure 3D,E</xref>). Interneuron migration was monitored using the AI3 reporter line (<italic>Gsk3-Ai3:Dlx</italic>). Surprisingly, in both controls and <italic>Gsk3</italic> mutants, interneurons exhibited robust migration along the two migratory streams (yellow arrows) from the medial ganglionic eminence (MGE) (<xref ref-type="fig" rid="fig3">Figure 3A–A'</xref>). In <italic>Gsk3</italic> mutants, as in controls, interneurons entered all areas of the cortical plate by E19.5. Quantification is shown in (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1</xref>). These results are not meant to imply that migration of interneurons was normal in every respect as we did not assess migration of specific interneuron subsets.<fig-group><fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.02663.007</object-id><label>Figure 3.</label><caption><title>GSK-3 signaling is dispensable for tangential migration, but required for radial hippocampal migration.</title><p>(<bold>A–A'</bold>) E19.5 coronal sections showing EYFP-expressing interneurons in heterozygous control and <italic>Gsk3:Dlx5/6</italic> mutants crossed with the Ai3 reporter line. <italic>Gsk3</italic>-deleted interneurons (green) enter the cortex in two streams in both controls and mutants (arrowheads). Mutants showed no overt migration defect. Nuclei were counterstained with Hoechst. (n = 3). (<bold>B–B'</bold>) E19 coronal sections of control and <italic>Gsk3:Neurod6</italic> mutants showing CTIP2 (green) expressing neurons in the hippocampus. In the <italic>Gsk3:Neurod6</italic> mutants, the pyramidal cell layer (green) does not extend laterally into a compact CA1 region and remains dispersed (yellow arrowheads). Fimbrial axonal projections appear normal in <italic>Gsk3:Neurod6</italic> mutants (orange arrow). Nuclei were counterstained with DRAQ5. Scale bar = 500 μm. (n = 3). (<bold>C–C'</bold>) Higher magnification of hippocampal area shown in (<bold>B</bold>). The <italic>Gsk3:Neurod6</italic> mutants show disrupted cytoarchitecture. In the mutants, DRAQ5-labeled cells are mislocalized and diffuse (arrowheads) and fail to form clearly defined CA1/CA3 regions of the hippocampus. The <italic>Gsk3:Neurod6</italic> mutant mice also lack a clearly defined hippocampal sulcus (green bars) and dentate gyrus (DG). (<bold>D</bold>) Representative Western blot of E18 MGE lysates confirm strongly reduced GSK-3β protein after recombination with <italic>Dlx5/6-Cre</italic>. (<bold>E</bold>) Quantification of protein knockdown in D (n = 3 WT, n = 3 CKO, unpaired t-test).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02663.007">http://dx.doi.org/10.7554/eLife.02663.007</ext-link></p></caption><graphic xlink:href="elife02663f003"/></fig><fig id="fig3s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02663.008</object-id><label>Figure 3—figure supplement 1.</label><caption><title>No apparent migration defect in <italic>Gsk3:Dlx5/6</italic> mice.</title><p>P0 quantification of Ai3-positive neurons in control and <italic>Gsk3</italic>-deleted interneurons using 8 bin analysis spanning white matter to the dorsal stream. p-values reaching significance are shown in figure, unpaired t-test. (n = 2 controls, 3894 total cux1 neurons, n = 2 cko, 3681 total cux1 neurons).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02663.008">http://dx.doi.org/10.7554/eLife.02663.008</ext-link></p></caption><graphic xlink:href="elife02663fs003"/></fig></fig-group></p><p>In order to assess the generality of GSK-3 regulation of radial migration, we assessed migration in developing hippocampus. The hippocampus, like the cortex, is an area where developing neurons migrate along radial glial-like processes (<xref ref-type="bibr" rid="bib55">Nowakowski and Rakic, 1979</xref>; <xref ref-type="bibr" rid="bib23">Eckenhoff and Rakic, 1984</xref>). <italic>Neurod6</italic>-<italic>Cre</italic> expression is evident in developing hippocampal neurons as early as E14 (<xref ref-type="bibr" rid="bib32">Goebbels et al., 2006</xref>), allowing us to delete <italic>Gsk3</italic> in those cells. Pyramidal neurons generated from the hippocampal primordia undergo migration along radial processes to form CA1/CA3 and the dentate gyrus (DG) (<xref ref-type="bibr" rid="bib1">Altman and Bayer, 1990</xref>). The transcription factor CTIP2 marks neurons in the developing CA1 region (<xref ref-type="fig" rid="fig3">Figure 3B</xref>). <italic>Gsk3:Neurod6</italic> mice exhibited a striking hippocampal migration defect. CTIP2 expressing neurons did not migrate properly (yellow arrows) and as a consequence CA1 did not fully develop (<xref ref-type="fig" rid="fig3">Figure 3B–B'</xref>). As a result, CA1-3 and the DG (arrowheads) were disorganized, and the hippocampal sulcus was not well defined (<xref ref-type="fig" rid="fig3">Figure 3C–C'</xref>). These defects were striking in rostral areas, as shown, although somewhat less pronounced in caudal sections (data not shown). Interestingly, fimbrial axonal projections formed in <italic>Gsk3:Neurod6</italic> mice (<xref ref-type="fig" rid="fig3">Figure 3B–B'</xref>, orange arrows) demonstrating that even though migration fails, hippocampal neurons were able to polarize and extend appropriately directed axons.</p></sec><sec id="s2-3"><title><italic>Gsk3</italic> deletion delays the multipolar to bipolar transition</title><p>To verify that the migration defect was cell autonomous and to visualize morphology of <italic>Gsk3</italic>-deleted neurons, we introduced Cre and EGFP into a subpopulation of developing neurons in <italic>Gsk3a</italic><sup><italic>−/−</italic></sup><italic>Gsk3b</italic><sup><italic>loxp/loxp</italic></sup> mice. <italic>Neurod1</italic>-Cre and lox-STOP-lox <italic>lac<underline>Z</underline>/<underline>Egfp</underline></italic> (Z/EG) plasmids were injected into the ventricles and co-electroporated at E14–15.5. Electroporation at this age targets radial progenitors that generate mainly upper layer neurons. This co-electroporation technique allowed us to visualize individual <italic>Gsk3</italic>-deleted neurons in an otherwise control background. Labeled neurons were imaged at late embryonic and postnatal stages.</p><p>Deletion of <italic>Gsk3</italic> in individual neurons phenocopies the migration delay seen in the <italic>Gsk3:Neurod6</italic> mutants. At E19 most control neurons were located in the upper cortical layers as expected (<xref ref-type="fig" rid="fig4">Figure 4A</xref>). In contrast, most <italic>Gsk3</italic>-deleted neurons had cell somas that were localized to the deeper layers of the cortex and very few were found in upper layers (<xref ref-type="fig" rid="fig4">Figure 4A'</xref>). Importantly, <italic>Gsk3</italic>-deleted neurons were clearly able to project axons (orange arrows) suggesting that initial polarization had proceeded in the absence of <italic>Gsk3</italic>. Further, most <italic>Gsk3</italic> null neurons in the cortical plate elaborated a long leading process directed toward the pial surface (yellow arrowheads) (<xref ref-type="fig" rid="fig4">Figure 4A</xref>'). Thus at least the initial stages of dendritic arborization also appeared to proceed in the absence of <italic>Gsk3</italic>.<fig-group><fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.02663.009</object-id><label>Figure 4.</label><caption><title>GSK-3 deletion delays the multipolar to bipolar transition.</title><p>(<bold>A–A'</bold>) Representative E19 coronal sections after in utero electroporation at E14.5 with <italic>Neurod1-Cre</italic> and Z/EG plasmids. Electroporated cells were visualized with anti-EGFP (green), and nuclei were stained with DAPI (blue). <italic>Gsk3</italic>-deleted neurons remain in the deeper layers of the cortex but elaborate a long pial-directed process (yellow arrowheads). <italic>Gsk3</italic>-deleted neurons elaborate axons projecting towards the corpus callosum (orange arrows). Scale bar = 200 μm (n = 5, two independent litters). (<bold>B–B'</bold>) Coronal sections at P10 after E14.5 electroporation, as in <bold>A</bold>. <italic>Gsk3</italic>-deleted neurons remain in the deeper layers of the cortical plate and fail to reach layer 2/3 (denoted with yellow bars). Scale bar = 200 μm (n = 3, 2 independent litters). (<bold>C</bold>) Higher magnification of <italic>Gsk3</italic>-deleted neurons in <bold>B'</bold> (box). <italic>Gsk3</italic>-deleted neurons (green) in deeper layers co-label with Cux (red) (orange arrows). Nuclei were stained with Dapi. (<bold>D</bold>) Quantification of control and <italic>Gsk3</italic>-deleted neurons in upper (layer 2–3) vs deeper layers of the cortex at P10. (n = 3, 4209 total neurons counted, 2234 control vs 1975 <italic>Gsk3</italic> deleted) **p=0.003, unpaired t-test. (<bold>E</bold>) <italic>Gsk3</italic> deletion delays the multipolar to bipolar transition. Still images from time-lapse imaging of slice cultures at 3DIV. pCAG-dsRED or <italic>Neurod1-Cre</italic>;Z/EG was injected into the ventricles of <italic>Gsk3a</italic><sup><italic>−/−</italic></sup><italic>Gsk3b</italic><sup><italic>loxp/loxp</italic></sup> embryos and electroporated at E15. Representative images were taken at time 0, 6, and 12 hr. Control dsRed neurons migrate through the cortical plate (yellow, red, and blue arrows show individual neurons at the different time points). (n = 2 controls). <italic>Gsk3</italic>-deleted neurons fail to migrate through the cortical plate and exhibit persistent multi-polar morphology (yellow arrowheads). (n = 4 mutants).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02663.009">http://dx.doi.org/10.7554/eLife.02663.009</ext-link></p></caption><graphic xlink:href="elife02663f004"/></fig><fig id="fig4s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02663.010</object-id><label>Figure 4—figure supplement 1.</label><caption><title><italic>Gsk3-deleted</italic> neurons polarize and are highly dynamic.</title><p>(<bold>A</bold>) Stage progression analysis of dissociated control and <italic>Gsk3</italic>-deleted neurons. Stage 1 immature neurons display lamellapodial and filopodial protrusions. Stage 2 neurons have transitioned to form multiple short neurites (multipolar morphology), and stage 3 neurons exhibit a single neurite extending to become an axon (see <xref ref-type="bibr" rid="bib22">Dotti et al., 1988</xref>). There is no significant delay in polarization (n = 3, 1341 neurons). (<bold>B</bold>) Live imaging of dissociated <italic>Gsk3</italic>-deleted neurons reveal dynamic neurites. Images were taken every 11 min. Representative images at time 0 (red), 220 min (blue), and 440 min (green). Merged image is pseudo colored.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02663.010">http://dx.doi.org/10.7554/eLife.02663.010</ext-link></p></caption><graphic xlink:href="elife02663fs004"/></fig></fig-group></p><p>The GSK-3 migration defect was strikingly persistent into the postnatal period. In mice electroporated at E14–15.5 and analyzed at P10, <italic>Gsk3</italic>-deleted neurons remained in the deeper layers and subcortical white matter (<xref ref-type="fig" rid="fig4">Figure 4B–B'</xref>). Egfp-expressing neurons co-labeled with Cux1 (red) demonstrating that they had acquired the proper laminar markers but were unable to attain the proper position (<xref ref-type="fig" rid="fig4">Figure 4C</xref>, arrows). Again, long apical processes that reached the pial surface were elaborated by <italic>Gsk3</italic>-deleted neurons, basal dendrites formed, and axons projecting towards the corpus callosum were evident. Thus, <italic>Gsk3</italic> regulates some critical aspect of migration but not the early stages of axon and dendrite formation.</p><p>In quantifying our results, we found that in control animals the majority of electroporated neurons (73.7%) were found in the upper layers of the cortex (yellow dashed lines) by P10 (<xref ref-type="fig" rid="fig4">Figure 4B–B',D</xref>). In contrast, <italic>Gsk3</italic>-deleted neurons remained in the deeper layers of the cortex and only 23% had reached the upper layer 2/3 by P10 (p=&lt;0.005).</p><p>A number of in vitro studies have suggested that <italic>Gsk3</italic> regulates neuronal polarization. It is plausible that some defect or delay in the polarization might account for migration failure. To test this idea, we electroporated <italic>Neurod1-Cre</italic> and Z/EG into the <italic>Gsk3a</italic><sup><italic>−/−</italic></sup><italic>Gsk3b</italic><sup><italic>loxp/loxp</italic></sup> cortex, plated cortical cells in dissociated culture and assessed stage progression at 3 days in vitro (3DIV). We observed no statistically significant difference in the stage progression between control and <italic>Gsk3</italic>-deleted neurons (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1A</xref>). Further, <italic>Gsk3</italic>-deleted neurons that successfully extended an axon remained highly dynamic (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1B</xref>) and extended and retracted neurites. Thus at least some processes that require complex cytoskeletal regulation proceed normally in the <italic>Gsk3</italic>-deleted neurons.</p><p>To assess the cell biological mechanisms of GSK-3 regulation of neuronal migration, we co-electroporated <italic>Neurod1-Cre</italic>;Z/EG or a control pCAG-dsRED construct into the lateral ventricles of control and <italic>Gsk3a</italic><sup><italic>−/−</italic></sup><italic>Gsk3b</italic><sup><italic>loxp/loxp</italic></sup> mice. This was followed by live imaging of migration ex vivo in a cortical slice preparation (<xref ref-type="bibr" rid="bib37">Hand et al., 2005</xref>) at 3DIV. In controls electroporated with pCAG-dsRED, labeled neurons transitioned from multipolar to bipolar morphology and migrated through the cortical plate over a period of 12 hr, as expected (<xref ref-type="fig" rid="fig4">Figure 4E</xref>). The progress of individual neurons could readily be tracked and is indicated for three examples by the progress of the colored arrowheads in the three panels. In contrast, most <italic>Gsk3</italic>-deleted neurons failed to translocate through the intermediate zone and remained in a multipolar state in the outer subventricular zone (arrowheads) (<xref ref-type="fig" rid="fig4">Figure 4E</xref> and <xref ref-type="other" rid="video1 video2">Videos 1, 2</xref>). Further, during the 12 hr of observation most <italic>Gsk3</italic>-deleted neurons did not transition to a bipolar morphology, a step thought to be required for radial-guided migration.<media content-type="glencoe play-in-place height-250 width-310" id="video1" mime-subtype="mov" mimetype="video" xlink:href="elife02663v001.mov"><object-id pub-id-type="doi">10.7554/eLife.02663.011</object-id><label>Video 1.</label><caption><title>Live cell imaging of radially migrating neurons in a cortical slice preparation.</title><p>Control neurons electroporated at E15.5 migrate towards the pial surface after 3 days ex vivo. Neurons are imaged using time-lapse microscopy with images taken every 45 min for a 20-hr session.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02663.011">http://dx.doi.org/10.7554/eLife.02663.011</ext-link></p></caption></media><media content-type="glencoe play-in-place height-250 width-310" id="video2" mime-subtype="mov" mimetype="video" xlink:href="elife02663v002.mov"><object-id pub-id-type="doi">10.7554/eLife.02663.012</object-id><label>Video 2.</label><caption><title>Gsk3-deleted neurons fail to migrate in cortical slice preparation.</title><p><italic>Gsk3a</italic><sup>−/−</sup><italic>Gsk3b</italic><sup>loxp/loxp</sup> mice electroporated at E15.5 with <italic>Neurod1-cre</italic> and Z/EG have an elongated multipolar stage and do not migrate towards the pial surface. Images were taken every 45 min over a 20-hr imaging session.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02663.012">http://dx.doi.org/10.7554/eLife.02663.012</ext-link></p></caption></media></p></sec><sec id="s2-4"><title>Abnormal dendritic orientation in <italic>Gsk3</italic> mutants</title><p>That fact that multiple cytoskeletal proteins are GSK-3 substrates might suggest that <italic>Gsk3</italic> deletion would have profound effects on dendrite and axonal arborization. To address GSK-3 regulation of cortical neuronal morphology, we deleted <italic>Gsk3</italic> using in utero electroporation of <italic>Neurod1</italic>-<italic>Cre</italic> as outlined above, and analyzed dendritic morphology at P15.</p><p>As demonstrated above, most <italic>Gsk3</italic>-deleted neurons failed to migrate and populated the deeper layers of the cortex at P15. Many of these migration arrested neurons exhibited abnormal dendritic arbors (data not shown). Because improper laminar position might affect dendritic arborization, we focused analysis on a small subset of <italic>Gsk3</italic>-deleted neurons that reached layer 2/3. Images at E16 (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>) and E19 (<xref ref-type="fig" rid="fig4">Figure 4A'</xref>) show that a few <italic>Gsk3</italic>-deleted neurons are normally positioned and suggest that these normally positioned neurons did not undergo a substantial delay in migration. All of the neurons elaborated dendritic arbors and extended an axon into the corpus callosum. However, many of these normally positioned <italic>Gsk3</italic>-deleted neurons exhibited markedly abnormally oriented basal dendrites (<xref ref-type="fig" rid="fig5">Figure 5A</xref>, arrows). In many cases, basal dendrites were oriented towards the pial surface rather than towards the deeper cortical layers (<xref ref-type="fig" rid="fig5">Figure 5B–D</xref>). Additionally, <italic>Gsk3</italic>-deleted basal processes grew longer and were more branched than those of control neurons (<xref ref-type="fig" rid="fig5">Figure 5D</xref> and <xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1C,D</xref>). Many <italic>Gsk3</italic>-deleted neurons also exhibited striking defects in the apical dendrite (<xref ref-type="fig" rid="fig5">Figure 5C,C'</xref>,E orange arrows and <xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1B</xref>). Thus, apical dendrites of the <italic>Gsk3</italic>-deleted neurons, although properly oriented towards the pial surface often extended branches close to the soma that extended apically rather than laterally (<xref ref-type="fig" rid="fig5">Figure 5E</xref>, orange arrows and <xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1B</xref>).<fig-group><fig id="fig5" position="float"><object-id pub-id-type="doi">10.7554/eLife.02663.013</object-id><label>Figure 5.</label><caption><title>GSK-3 signaling is required for proper dendrite orientation.</title><p>(<bold>A</bold>) Gsk3 deleted neurons at P15 shown after in utero electroporation at E14.5 with <italic>Neurod1-Cre</italic> and Z/EG plasmids. Multiple neurons with obvious abnormalities in dendritic orientation were observed in the upper layers of the cortex (orange arrows). Scale bar = 200 μm. (<bold>B–B'</bold>) Control and <italic>Gsk3</italic>-deleted neurons in the upper layers at P15, immunostained with antibodies against eGFP (black) using same methods as <xref ref-type="fig" rid="fig4">Figure 4</xref>. Gsk3-deleted neurons have abnormally polarized arbors indicated by orange arrows. Scale bar = 50 μm. (<bold>C–C'</bold>) Neurolucida reconstructions of control and <italic>Gsk3</italic>-deleted neurons in the upper layers of the cortex. The axon (red) projects towards the ventricle in control and <italic>Gsk3</italic>-deleted neurons. Both apical dendrites (orange) and basal dendrites (blue) are more branched (orange arrows) and basal dendrites (blue) are mispolarized (blue arrowheads) in <italic>Gsk3</italic>-deleted neurons. Scale bar = 100 μm. (<bold>D</bold>) Basal dendrite quantification. Dendrogram shows that basal dendrites more frequently project towards the pial surface in <italic>Gsk3</italic>-deleted neurons when compared to control basal dendrite orientation. (n = 3, n = 3 CKO; 15 control and 15 <italic>Gsk3</italic>-deleted neurons quantified). (<bold>E</bold>) Apical dendrite dendrogram indicates polarization and length of processes. Control apical dendrites project pially (90°). Numerous small apical branches form near the soma and project laterally (orange arrows). <italic>Gsk3</italic>-deleted neurons also project pially-directed apical dendrites. However, Apical branches have a pially-directed orientation, resulting in abnormal morphology (orange arrows, also see <bold>C'</bold>).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02663.013">http://dx.doi.org/10.7554/eLife.02663.013</ext-link></p></caption><graphic xlink:href="elife02663f005"/></fig><fig id="fig5s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02663.014</object-id><label>Figure 5—figure supplement 1.</label><caption><title>Quantification of dendritic branching at P15 in control and <italic>Gsk3-deleted</italic> neurons.</title><p>(<bold>A</bold>) Representitive images of control and <italic>Gsk3</italic>-deleted neurons used for Sholl analysis with specific processes pseudocolored for identification. (<bold>B</bold>) Apical dendrite Sholl analysis of <italic>Gsk3</italic>-deleted neurons shows significantly increased branching close to the soma. p=0.034. Additionally, <italic>Gsk3</italic>-deleted neurons display a trend toward increased branching in areas further from the soma. (<bold>C</bold>) Basal dendrite Sholl analysis of <italic>Gsk3</italic>-deleted neurons shows significantly increased branching in basal dendrites in areas furthest away from the soma (p=0.015). (<bold>D</bold>) Basal dendrite sholl analysis of dendritic lengths reveals altered morphology of <italic>Gsk3</italic>-deleted neurons. <italic>Gsk3</italic>-deleted neurons have increased lengths of basal dendrites in areas beginning 75 μm away from the soma. p=0.023, p=0.0118, and p=0.0202.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02663.014">http://dx.doi.org/10.7554/eLife.02663.014</ext-link></p></caption><graphic xlink:href="elife02663fs005"/></fig></fig-group></p><p>Perhaps surprisingly <italic>Gsk3</italic>-deleted neurons extended axons into the callosum towards the contralateral cortex (<xref ref-type="fig" rid="fig4">Figure 4A,A’</xref> orange arrows). Axonal arborization in the contralateral cortex had reduced density, an abnormality that is under further investigation (data not shown).</p></sec><sec id="s2-5"><title>GSK-3 regulation of migration is independent of Wnt/β-catenin signaling</title><p>Signaling via β-catenin is an obvious candidate to mediate GSK-3 regulation of migration. In the canonical Wnt cascade, Wnt signaling through frizzled receptors leads to dishevelled and GSK-3 sequestration, β-catenin accumulation and enhanced β-catenin/TCF-mediated transcription (<xref ref-type="bibr" rid="bib45">Kaidanovich-Beilin and Woodgett, 2011</xref>). In radial progenitors, β-catenin signaling is clearly an important mediator of the effects of GSK-3 deletion on proliferation (<xref ref-type="bibr" rid="bib15">Chenn and Walsh, 2002</xref>; <xref ref-type="bibr" rid="bib46">Kim et al., 2009</xref>).</p><p>To determine the role of GSK-3 regulation of β-catenin in developing cortical excitatory neurons, we utilized a β-catenin (<italic>Ctnnb1</italic>) mouse that harbors loxP sites flanking exon 3 (<italic>Ctnnb1</italic><sup><italic>Ex3</italic></sup><italic>:Neurod6</italic>) (<xref ref-type="bibr" rid="bib38">Harada et al., 1999</xref>). Exon 3 encodes the residues that GSK-3 phosphorylates to signal β-catenin degradation; thus deleting exon 3 stabilizes β-catenin. Using a β-catenin antibody directed at the residues encoded by exon3 verified a reduction in this protein fragment at P0 as expected after <italic>Neurod6</italic>-mediated recombination (<xref ref-type="fig" rid="fig6">Figure 6C,H</xref>). Migration of Cux1 neurons was entirely normal in these animals (<xref ref-type="fig" rid="fig6">Figure 6A</xref>, <xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1A</xref>). Further, in contrast to <italic>Gsk3:Neurod6</italic>, <italic>Ctnnb1</italic><sup><italic>Ex3</italic></sup><italic>:Neurod6</italic> mice survive, breed, and have no overt behavioral phenotype. They display a rostral midline defect resulting from lack of the hippocampal commissure (data not shown), as seen in other models using stabilized β-catenin (<xref ref-type="bibr" rid="bib16">Chenn and Walsh, 2003</xref>).<fig-group><fig id="fig6" position="float"><object-id pub-id-type="doi">10.7554/eLife.02663.015</object-id><label>Figure 6.</label><caption><title>Lamination in other signaling mutants.</title><p>(<bold>A</bold>) P0 representative coronal sections of control (heterozygous for floxed allele) and <italic>Ctnnb1</italic><sup><italic>Ex3</italic></sup><italic>:Neurod6</italic> mutants stained for Cux1 (red) and Hoechst (blue). (n = 5) Scale bar = 100 μm. (<bold>B</bold>) E18 Coronal sections of control and <italic>Dvl123:Neurod6</italic> showing Cux-1 (red) and DRAQ5 (blue) staining. Cux-1 neurons reach layer 2/3 in both controls and <italic>Dvl123:Neurod6</italic> triple mutants. (n = 3). (<bold>C</bold>) Western blot verification of protein deletion after recombination of floxed alleles with <italic>Neurod6</italic>-<italic>Cre. Ctnnb1</italic><sup><italic>Ex3</italic></sup><italic>:NeuroD6</italic> (n = 3 WT, n = 3 CKO), <italic>Dvl123:NeuroD6</italic> (n = 3 WT, n = 3 CKO). GAPDH was probed as a loading control. (<bold>D–F</bold>) P0 representative coronal sections of control (heterozygous for floxed allele) and indicated mutant lines stained for Cux1 and Hoechst. Scale bars are 100 μm, at least n = 5 per line. (<bold>G</bold>) Western blot verification of protein deletion in mutant lines. GAPDH was probed as a loading control. N's refer to numbers of mutants and paired controls. <italic>Stk11:Neurod6</italic> (n = 2), <italic>Cdc42:Neurod6</italic> (n = 2), <italic>Pten:Neurod6</italic> (n = 3). (<bold>H</bold>) P0 Western Blot quantification of <italic>Ctnnb1</italic><sup><italic>Ex3</italic></sup><italic>:Neurod6</italic>, <italic>Dvl123:Neurod6</italic>, <italic>Stk11:Neurod6</italic>, <italic>Pten:Neurod6</italic> and <italic>Cdc42:Neurod6</italic> lines.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02663.015">http://dx.doi.org/10.7554/eLife.02663.015</ext-link></p></caption><graphic xlink:href="elife02663f006"/></fig><fig id="fig6s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02663.016</object-id><label>Figure 6—figure supplement 1.</label><caption><title>Quantification of lamination in other signaling mutants.</title><p>(<bold>A–D</bold>) P0 quantification of Cux1 expressing neurons using 8 Bin quantification in conditional mutants and control shown in <xref ref-type="fig" rid="fig6">Figure 6</xref>. (<bold>A</bold>) <italic>Ctnnb1</italic><sup><italic>Ex3</italic></sup><italic>:Neurod6,</italic> (<bold>B</bold>) <italic>Stk11:Neurod6,</italic> (<bold>C</bold>) <italic>Cdc42:Neurod6,</italic> (<bold>D</bold>) <italic>Pten:Neurod6</italic>. (n = 2 het control mice, n = 2 CKO mice per line). Between 2000 and 3000 Cux1 neurons were scored in each of the control pairs and each of the mutant pairs.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02663.016">http://dx.doi.org/10.7554/eLife.02663.016</ext-link></p></caption><graphic xlink:href="elife02663fs006"/></fig></fig-group></p><p>For additional assessment of the role of β-catenin signaling, we performed global analysis of GSK-3 transcriptional targets at E18 in control and <italic>Gsk3:Neurod6</italic> cortical lysates using Affymetrix microarray analysis. Consistent with loss of <italic>Gsk3</italic> in our conditional mutants, probe level information specific to exon 2 of <italic>Gsk3b</italic> showed that exon 2 was decreased by an average 2.15-fold. However, classic Wnt pathway target genes downstream of β-catenin/(TCF)/LEF-1 transcription factors, including CyclinD1, Brachyury, Wisp1, Cdx1, and Engrailed2 were unchanged (data made available in GEO, GSE58727). Finally mice in which β-catenin signaling is abrogated after <italic>Neurod6-Cre</italic> mediated recombination (<italic>Ctnnb1</italic><sup><italic>loxp/loxp</italic></sup><italic>:Neurod6)</italic> also show no change in migration of Cux1 neurons (ES Anton, personal communication, June 2014).</p><p>To further assess a potential role of Wnt/β-catenin signaling in cortical lamination, we created a conditional dishevelled 2<sup>loxp/loxp</sup> (<italic>Dvl2</italic>) mouse (<xref ref-type="bibr" rid="bib57">Ohata et al., 2014</xref>). We then generated a triple mutant by crossing our floxed <italic>Dvl2</italic><sup><italic>loxp/loxp</italic></sup> with existing <italic>Dvl1</italic> and <italic>Dvl3</italic> nulls and the <italic>Neurod6-Cre</italic> line (<italic>Dvl123:Neurod6</italic>). Deletion of all three <italic>Dvls</italic> presumably completely abrogates Wnt signaling via the canonical pathway. A robust decrease of DVL2 protein (91%) was observed in P0 cortical lysates from <italic>Dvl123:Neurod6</italic> mutants when compared to Cre<sup>−</sup> <italic>Dvl2</italic><sup><italic>loxp/loxp</italic></sup> controls (<xref ref-type="fig" rid="fig6">Figure 6C,H</xref>). These mice are born but die immediately at P0. Remarkably, lamination in the triple allele mutant <italic>Dvl123:Neurod6</italic> appears relatively normal at E18 (<xref ref-type="fig" rid="fig6">Figure 6B</xref>). This result further supports the idea that GSK-3 regulation of migration is not mediated by the WNT/β-catenin cascade.</p></sec><sec id="s2-6"><title>Surprising lack regulation by STK11 (LKB1), CDC42, and PTEN</title><p>Recent work utilized RNAi and GSK-3 S9A mutant constructs to conclude that STK11 (LKB1) inactivation of GSK-3 via ser9 phosphorylation alters neuronal migration (<xref ref-type="bibr" rid="bib2">Asada and Sanada, 2010</xref>). However, <italic>Gsk3</italic> knock-in S9A/S21A <italic>Gsk3</italic> ‘constitutively active’ mice develop normally and migration defects have not been reported (<xref ref-type="bibr" rid="bib43">Jiang et al., 2005</xref>; <xref ref-type="bibr" rid="bib30">Gartner et al., 2006</xref>). To further address the issue of GSK-3 inhibition downstream of STK11, we genetically deleted <italic>Stk11</italic> from developing excitatory neurons <italic>(Stk11:Neurod6</italic>). <italic>Stk11:Neurod6</italic> mutant mice die around P20. Western blot analysis verified a 66% decrease in STK11 (LKB1) protein in <italic>Stk11:Neurod6</italic> mutant mice when compared to heterozygous littermate controls (<xref ref-type="fig" rid="fig6">Figure 6G,H</xref>). Perhaps surprisingly, no gross lamination abnormalities were observed (<xref ref-type="fig" rid="fig6">Figure 6D</xref>, <xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1B</xref>). Thus in our hands, STK11 (LKB1) regulation of GSK-3 activity is not essential for radial-guided neuronal migration in vivo.</p><p>We also deleted a key regulator of Par6-aPKC, <italic>Cdc42</italic>, using <italic>Neurod6-Cre</italic>. aPKCs are also known to phosphorylate Ser9/Ser21 of GSK-3 (<xref ref-type="bibr" rid="bib27">Etienne-Manneville and Hall, 2003</xref>). <italic>Cdc42:Neurod6</italic> mutants also die shortly after birth, but again we observed no gross lamination defect in these mutant mice (<xref ref-type="fig" rid="fig6">Figure 6E</xref>, <xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1C</xref>). Western blot analysis verified an 87% decrease in CDC42 protein levels in mutants when compared to littermate wild type controls at P0 (<xref ref-type="fig" rid="fig6">Figure 6G,H</xref>).</p><p>In receptor tyrosine kinase (RTK) cascades, GSK-3 lies downstream of PI3K/ AKT signaling. Signals transduced through this cascade inhibit GSK-3 via phosphorylation on Ser9/Ser21 (<xref ref-type="bibr" rid="bib39">Hur and Zhou, 2010</xref>). The phosphatase PTEN suppresses PI3K signaling. To determine if PI3K signaling affects migration, we genetically deleted <italic>Pten</italic> in neurons using <italic>Neurod6-Cre</italic>. Western blot analysis at P0 revealed an 88% decrease in PTEN protein in our conditional knockouts when compared to littermate wild-type controls (<xref ref-type="fig" rid="fig6">Figure 6G,H</xref>). Though <italic>Pten:Neurod6</italic> mutants die around birth, no overt lamination defects were observed (<xref ref-type="fig" rid="fig6">Figure 6F</xref>, <xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1D</xref>).</p><p>These findings taken together suggest that regulation of GSK-3 phosphorylation at ser9/ser21 is not important in the control of radial migration.</p></sec><sec id="s2-7"><title>Reduced phosphorylation of DCX and CRMP-2 in GSK-3 mutants</title><p>To determine the status of relevant GSK-3 targets, we conducted western blot analysis of GSK-3 substrates that have been implicated in migration regulation. Recently, the key migration regulator doublecortin (DCX) was shown to be a GSK-3 substrate (<xref ref-type="bibr" rid="bib10">Bilimoria et al., 2010</xref>). In P0 cortical lysates of <italic>Gsk3</italic><sup><italic>loxp</italic></sup><italic>:Neurod6</italic> mutants, we observed a 61% decrease in phosphorylated DCX on Ser327 (<xref ref-type="fig" rid="fig7">Figure 7A–A'</xref>). These results demonstrate that DCX is importantly regulated by GSK-3 in developing cortical neurons.<fig id="fig7" position="float"><object-id pub-id-type="doi">10.7554/eLife.02663.017</object-id><label>Figure 7.</label><caption><title>Phosphorylation status of GSK-3 substrates.</title><p>(<bold>A–A'</bold>) Western blots of P0 cortical lysates from <italic>Gsk3</italic><sup><italic>loxp</italic></sup><italic>:Neurod6</italic> mutants and wild-type controls performed in triplicate. Levels of GSK-3 proteins and phospho-target proteins are shown. Strong reductions in phosphorylation of doublecortin on ser327/Thr321 and CRMP-2 on Thr514 are evident. (<bold>A'</bold>) Quantification of relative densities from <bold>A</bold>. p values shown in figure (n = 3, unpaired t-test) (<bold>B–B'</bold>) Western blots of cortical lysates at P0 showing levels of other GSK-3 targets. No changes were observed in phosphorylation of dynamin, pCREB, or pFAK. No change was observed in cleaved caspase-3. GAPDH was used as a loading control. (<bold>B'</bold>) Quantification of relative protein densities (n = 3, unpaired t-test).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02663.017">http://dx.doi.org/10.7554/eLife.02663.017</ext-link></p></caption><graphic xlink:href="elife02663f007"/></fig></p><p>CRMP-2 family proteins have also recently been implicated in control of radial migration (<xref ref-type="bibr" rid="bib40">Ip et al., 2014</xref>). We found an 80% decrease in phosphorylated CRMP-2 on Thr514 in P0 cortical lysates from <italic>Gsk3</italic><sup><italic>loxp</italic></sup><italic>:Neurod6</italic> mutants (<xref ref-type="fig" rid="fig7">Figure 7A–A'</xref>). Changes in the phosphorylation status of DCX and CRMP-2 have been implicated in migration control, raising the possibility that reduced functions of these proteins toward microtubules are responsible for the effects of <italic>Gsk3</italic> deletion.</p><p>GSK-3 regulation appeared to be surprisingly specific. Thus, we did not detect changes in pSer722 FAK (<xref ref-type="bibr" rid="bib9">Bianchi et al., 2005</xref>), pSer744 dynamin-1 (<xref ref-type="bibr" rid="bib17">Clayton et al., 2010</xref>), and pSer129 CREB (<xref ref-type="bibr" rid="bib29">Fiol et al., 1994</xref>; <xref ref-type="bibr" rid="bib11">Bullock and Habener, 1998</xref>) (<xref ref-type="fig" rid="fig7">Figure 7B–B'</xref>). Presumably other kinases contribute to phosphorylation of the putative GSK-3 sites in vivo. However, we cannot rule out changes that would be masked by the dilution effect of non-recombined cells, changes confined to specific cellular compartments, or changes that might be more apparent later in development. There was no increase in cleaved caspase-3 staining or other evidence of apoptosis in the mutants at P0 (<xref ref-type="fig" rid="fig7">Figure 7B–B'</xref>). We did see increases in cleaved caspase-3 staining in cortex in <italic>Gsk3</italic><sup><italic>loxp</italic></sup><italic>:Neurod6</italic> mice starting at later postnatal stages and these changes currently under investigation (data not shown).</p></sec></sec><sec id="s3" sec-type="discussion"><title>Discussion</title><sec id="s3-1"><title>Overview</title><p>In this work, we have documented a cell autonomous requirement for GSK-3 signaling in migration and dendritic orientation of cortical excitatory neurons. GSK-3 activity is critical for the radial migration of later born, Cux1-expressing neurons in all regions of cortex, and for radial migration in the hippocampus. The GSK-3 requirement is specific for radial migration as tangential migration proceeded despite <italic>Gsk3</italic> deletion. GSK-3 regulation of migration appears to be independent of Wnt/β-catenin and PI3K signaling. Rather, <italic>Gsk3</italic> deletion is associated with striking reductions in phosphorylation of key microtubule regulatory proteins, DCX and CRMP-2.</p></sec><sec id="s3-2"><title>GSK-3 regulation of cortical neuronal development</title><p>Our work builds on a growing body of evidence establishing critical requirements for GSK-3 in cortical neuronal development, with GSK-3 signaling having different functions at different developmental stages. In radial progenitors, GSK-3 is a critical mediator of proliferation via regulation of β-catenin (<xref ref-type="bibr" rid="bib46">Kim et al., 2009</xref>). At later stages, recent work has demonstrated that GSK-3 is a key mediator of the amplification of the INP pool via interactions with the scaffolding protein Axin (<xref ref-type="bibr" rid="bib28">Fang et al., 2013</xref>). Axin-GSK-3 binding in the cytoplasm is clearly required for expansion of the INP pool, although the mechanism of GSK-3 action was not specified in this study. Interestingly, β-catenin regulation of transcription in the nucleus was required for differentiation of INPs into neurons. These effects of GSK-3 signaling on radial progenitors and INPs are presumably a strong determinant of final cortical neuronal number in mature animals.</p><p>We now find that excitatory neuron development is also under important GSK-3 regulation. Thus, Cux1-expressing neurons require GSK-3 signaling for timely multipolar to bipolar transition and migration along radial processes. Deeper layer neurons expressing Tbr1 were not strongly influenced, but we cannot be certain that GSK-3 protein was fully depleted in these earlier born neurons at the time migration was occurring. The behavioral consequences of migration failure and dendritic arbor abnormalities could not be assessed due to early death of the <italic>Gsk3</italic>-mutant animals. Whether death was due to cortical abnormalities or defects in other cells that underwent recombination with <italic>Neurod6-Cre</italic> could not be determined. However, even a very mild form of this type of migration defect would presumably be catastrophic for human brain development.</p><p>In contrast to regulation of progenitor proliferation and neural differentiation, GSK-3 regulation of radial migration appears to be independent of Wnt/β-catenin signaling. Thus, neither the stabilization of β-catenin nor deletion of all <italic>Dvl</italic>s in developing cortical neurons using the <italic>Neurod6-Cre</italic> driver produced major defects in cortical layering. Interestingly, the migration defect also appears to be independent from a migration defect associated with mutations of the schizophrenia-associated protein, DISC1. DISC1 regulates progenitor proliferation via interactions with GSK-3, decreased GSK-3 kinase activity and, ultimately, increased β-catenin signaling (<xref ref-type="bibr" rid="bib51">Mao et al., 2009</xref>). Interestingly, a recent study demonstrated that DISC1 regulates migration independently of GSK-3 via effects on the centrosome (<xref ref-type="bibr" rid="bib42">Ishizuka et al., 2011</xref>). However, our work reported here clearly demonstrates that GSK-3 also has a critical role in the regulation of cortical neuronal migration.</p><p>Radial migration is an enormously complex process requiring timely progenitor differentiation, dramatic morphological change, intricate mechanisms for cell and nuclear movements, and dynamic sensing of multiple cues that start and stop the process. Not surprisingly, GSK-3 joins dozens of other molecules implicated in the control of radial migration (see <xref ref-type="bibr" rid="bib3">Ayala et al., 2007</xref> for review). Comparisons with the literature suggest that effects of GSK-3 deletion are among the most severe that have yet been observed.</p><p>Interestingly, GSK-3 importantly regulates multiple proteins implicated in migration and is an important mediator in several of the signaling pathways involved. Thus GSK-3 is known to phosphorylate DCX (<xref ref-type="bibr" rid="bib10">Bilimoria et al., 2010</xref>), FAK (<xref ref-type="bibr" rid="bib9">Bianchi et al., 2005</xref>), dynamin (<xref ref-type="bibr" rid="bib17">Clayton et al., 2010</xref>), neurogenin (<xref ref-type="bibr" rid="bib48">Li et al., 2012</xref>), CRMP-2 (<xref ref-type="bibr" rid="bib71">Uchida et al., 2005</xref>; <xref ref-type="bibr" rid="bib76">Yoshimura et al., 2005</xref>), and MAP1B (<xref ref-type="bibr" rid="bib69">Trivedi et al., 2005</xref>). Further, GSK-3 mediates Reelin signaling (<xref ref-type="bibr" rid="bib7">Beffert et al., 2002</xref>), LKB1 effects (<xref ref-type="bibr" rid="bib2">Asada and Sanada, 2010</xref>), Cdc42 effects (<xref ref-type="bibr" rid="bib27">Etienne-Manneville and Hall, 2003</xref>), integrin signaling (<xref ref-type="bibr" rid="bib36">Guo et al., 2007</xref>), semaphorin signaling (<xref ref-type="bibr" rid="bib24">Eickholt et al., 2002</xref>; <xref ref-type="bibr" rid="bib71">Uchida et al., 2005</xref>), and other pathways that have been implicated in control of radial migration. Finally, GSK-3 shares multiple substrates with cdk5, a kinase that is situated among the most important regulators of neuronal migration and acts as a ‘priming’ kinase for GSK-3 signaling (<xref ref-type="bibr" rid="bib73">Xie et al., 2006</xref>).</p><p>Clearly GSK-3 effects on neuronal development extend well beyond the phase of migration. For example, although dendritic arbors form, abnormalities in dendritic orientation were striking in <italic>Gsk3</italic>-deleted neurons. Importantly, interference with semaphorin signaling mediators also produces abnormalities of apical process development and orientation of basal dendrites (see below).</p></sec><sec id="s3-3"><title>Mechanisms of GSK-3 regulation</title><p>In general, GSK-3 acts via two classes of mechanisms: one where GSK-3 activity inhibits substrate function or availability and another where GSK-3 activity is required for substrate function. Therefore, we might expect <italic>Gsk3</italic> deletion to enhance processes normally inhibited by GSK-3 activity and to inhibit processes that require GSK-3 activity.</p><p>Multiple studies have suggested that inhibition of GSK-3 kinase activity is important for morphological functions such as establishment of neuronal polarity and cellular migration. The effects of GSK-3 inhibition are thought to be mediated by dephosphorylation of CRMP-2, APC and other cytoskeletal mediators with resulting stabilization of microtubules at the tips of axons (<xref ref-type="bibr" rid="bib27">Etienne-Manneville and Hall, 2003</xref>; <xref ref-type="bibr" rid="bib43">Jiang et al., 2005</xref>; <xref ref-type="bibr" rid="bib76">Yoshimura et al., 2005</xref>; <xref ref-type="bibr" rid="bib39">Hur and Zhou, 2010</xref>). Another recent study employing in utero electroporation suggested that an STK11(LKB1)/GSK-3 pathway resulting in GSK-3β ser9 phosphorylation and APC localization at the leading edge was important in cortical neuronal migration (<xref ref-type="bibr" rid="bib2">Asada and Sanada, 2010</xref>).</p><p>In the studies outlined above, inhibition of GSK-3β kinase activity is indicated by phosphorylation on Ser9 and inhibition of GSK-3α by phosphorylation on Ser21. However, a major unresolved paradox is that mice <italic>with Gsk3a and Gsk3b</italic> point mutation knock-ins that prevent ser9 and ser21 phosphorylation respectively do not exhibit widespread morphological abnormalities (<xref ref-type="bibr" rid="bib30">Gartner et al., 2006</xref>). Similarly, truncation mutants of the <italic>Drosophila</italic> GSK-3 homologue, <italic>Shaggy</italic>, that lack inhibitory phosphorylation sites are not associated with morphological abnormalities (<xref ref-type="bibr" rid="bib58">Papadopoulou et al., 2004</xref>). In the context of neuronal migration, although GSK-3 ser9/21 phosphorylation is enhanced downstream of Reelin signaling (<xref ref-type="bibr" rid="bib7">Beffert et al., 2002</xref>; <xref ref-type="bibr" rid="bib33">Gonzalez-Billault et al., 2005</xref>), GSK-3β kinase activity towards the important substrate MAP1B is actually increased rather than diminished (<xref ref-type="bibr" rid="bib33">Gonzalez-Billault et al., 2005</xref>). Finally, our findings reported here do not support critical roles in radial migration for STK11, CDC42, or PTEN, all of which are known to regulate GSK-3 ser9/21 phosphorylation. These findings, taken together, raise questions about the importance of negative regulation of GSK-3 via Ser9/21 phosphorylation on functions of microtubule binding proteins related to migration in vivo.</p><p>Our results demonstrate that <italic>Gsk3</italic> deletion blocks radial migration and that <italic>Gsk3</italic> over-expression may enhance it. Thus our results are more in line with a mechanism in which GSK-3 activity is required for normal substrate function. Several proteins have been shown to require GSK-3 kinase activity for normal function. These include DCX where GSK-3 mediated phosphorylation is thought to be required for DCX's actions in regulating axon branching and possibly in migration (<xref ref-type="bibr" rid="bib10">Bilimoria et al., 2010</xref>). Additional examples possibly relevant to migration are GSK-3 regulation of FAK phosphorylation at ser722 (<xref ref-type="bibr" rid="bib9">Bianchi et al., 2005</xref>) and dynamin-1 phosphorylation of ser774 (<xref ref-type="bibr" rid="bib17">Clayton et al., 2010</xref>). GSK-3 phosphorylation regulates functions of these proteins in complex ways, but possibly enhances functions that may be required for neuronal migration.</p><p>A well-studied example of a pathway that is associated with upregulation of GSK-3 activity is semaphorin signaling. Semaphorin family members have multiple roles as chemorepellants and chemoattractants for many classes of axons and dendrites (for review see <xref ref-type="bibr" rid="bib59">Pasterkamp, 2012</xref>). Semaphorin signaling strongly activates GSK-3 (<xref ref-type="bibr" rid="bib24">Eickholt et al., 2002</xref>) resulting in phosphorylation of a key MAP, CRMP-2 (<xref ref-type="bibr" rid="bib71">Uchida et al., 2005</xref>), as well as other CRMP family members (<xref ref-type="bibr" rid="bib19">Cole et al., 2006</xref>; <xref ref-type="bibr" rid="bib74">Yamashita and Goshima, 2012</xref>). Intriguingly, during neuronal polarization semaphorin signaling is associated with suppression of axons, and formation of dendrites (<xref ref-type="bibr" rid="bib63">Shelly et al., 2011</xref>), consistent with the idea that dephosphorylated CRMP-2 may favor axon formation, whereas phosphorylated CRMP-2 may be critical to proper formation of dendritic arbors.</p><p>Importantly, migration and dendritic abnormalities that we have described here are consistent with cortical neuron phenotypes reported due to manipulation of semaphorin and CRMP signaling. Interfering with semaphorin, NP1 or Plexin via silencing RNAs and gene knockouts has been reported to interfere with radial migration (<xref ref-type="bibr" rid="bib13">Chen et al., 2008</xref>; <xref ref-type="bibr" rid="bib62">Renaud et al., 2008</xref>). Application of exogenous semaphorin supports apical process formation (<xref ref-type="bibr" rid="bib61">Polleux et al., 2000</xref>). Further, reduced semaphorin signaling causes bifurcation of CA1 pyramidal dendrites in the developing hippocampus (<xref ref-type="bibr" rid="bib54">Nakamura et al., 2009</xref>). Downstream of semaphorins, interfering with CRMP functions has been reported to affect migration and result in branched apical dendrites and abnormal dendrite orientation (<xref ref-type="bibr" rid="bib75">Yamashita et al., 2012</xref>; <xref ref-type="bibr" rid="bib74">Yamashita and Goshima, 2012</xref>; <xref ref-type="bibr" rid="bib41">Ip et al., 2012</xref>; for a review see, <xref ref-type="bibr" rid="bib40">Ip et al., 2014</xref>). Finally, in the context of cortical neuronal migration, phosphorylation of CRMP-2 promotes migration. Thus a GSK-3 phosphomimetic CRMP-2 can rescue migration defects associated with knockdown of the semaphorin signaling mediator, α2-chimaerin (<xref ref-type="bibr" rid="bib41">Ip et al., 2012</xref>). Taken together, these findings suggest that inhibition of CRMP2 phosphorylation at Thr514 in <italic>Gsk3</italic>-deleted neurons may partially explain the migration and dendritic orientation abnormalities we have observed.</p></sec><sec id="s3-4"><title>Conclusion</title><p>In sum, we have demonstrated a key cell autonomous role for GSK-3 signaling in regulating radial migration and dendritic orientation of cortical excitatory neurons. <italic>Our Gsk3</italic> deletion results are not in line with what might have been predicted from prior studies that have correlated ser9/21 phosphorylation with relief of negative GSK-3 phosphorylation and negative regulation of cytoskeletal-associated proteins. In particular, we do not find evidence that an STK11(LKB1)/GSK-3 inhibitory pathway is a key migration regulator. In contrast, our results are consistent with the idea GSK-3 phosphorylation of DCX and CRMP-2 is required for appropriate cytoskeletal regulation and migration. Finally, our data are consistent with the idea that GSK-3 activity is essential for the effects of semaphorin and CRMP-2 signaling on cortical neuronal development in vivo.</p></sec></sec><sec id="s4" sec-type="materials|methods"><title>Materials and methods</title><sec id="s4-1"><title>Mice</title><p>Mice were cared for according to animal protocols approved by the Institutional Animal Care and Use Committees of the University of North Carolina at Chapel Hill. GSK-3 lines were generously provided by Jim Woodgett. <italic>Gsk3a</italic> mice possessing exon 2 deletions and <italic>Gsk3a</italic> and <italic>Gsk3b</italic> loxp flanked exon 2 mice have been previously described (<xref ref-type="bibr" rid="bib20">Doble et al., 2007</xref>; <xref ref-type="bibr" rid="bib50">MacAulay et al., 2007</xref>; <xref ref-type="bibr" rid="bib60">Patel et al., 2008</xref>). <italic>Gsk3:Neurod6</italic> mice were generated by mating <italic>Gsk3a</italic><sup><italic>−/−</italic></sup><italic>, Gsk3b</italic><sup><italic>loxp/loxp</italic></sup> with <italic>Neurod6:Cre</italic> mice generously provided by Dr KA Nave (<xref ref-type="bibr" rid="bib32">Goebbels et al., 2006</xref>). <italic>Gsk3</italic> mutant mice and <italic>Neurod6-Cre</italic> mice were maintained on mixed genetic backgrounds. For all experiments, four allele mutants were compared with littermate controls. Triple allelic male mutants (<italic>Gsk3a</italic><sup><italic>+/-</italic></sup><italic>b</italic><sup><italic>f/f</italic></sup><italic>:Neurod6</italic>) fail to survive into adulthood with death occurring around P25 for reasons not yet determined.</p><p><italic>Ctnnb1</italic> exon 3 (<italic>Ctnnb1</italic><sup><italic>Ex3</italic></sup>) floxed mice were previously described (<xref ref-type="bibr" rid="bib38">Harada et al., 1999</xref>) and maintained on a mixed C57/Bl6 129 background. <italic>Stk11</italic>-floxed mice have been previously described (<xref ref-type="bibr" rid="bib4">Bardeesy et al., 2002</xref>) and were maintained on a mixed C57/Bl6 129, CF1 background. <italic>Pten</italic>-floxed mice (<xref ref-type="bibr" rid="bib34">Groszer et al., 2001</xref>), <italic>Cdc42</italic>-floxed mice were purchased from Jackson lab and have been previously described (<xref ref-type="bibr" rid="bib14">Chen et al., 2006</xref>).</p><p><italic>Dvl2</italic> floxed allele was generated in the UNC Neuroscience Center Molecular Neuroscience Core using conventional methodology (<xref ref-type="bibr" rid="bib57">Ohata et al., 2014</xref>). <italic>Dvl1</italic> (<xref ref-type="bibr" rid="bib8">Beier et al., 1992</xref>) and <italic>Dvl3</italic> (<xref ref-type="bibr" rid="bib70">Tsang et al., 1996</xref>) mutant mice were purchased from The Jackson Laboratory (Bar Harbor, Maine) have been previously described. All <italic>Dvl</italic> mutant mice were maintained on a mixed background.</p><p>Results from mutants and control littermates shown in all of the figure panels were based on at least three experiments from independent litters, unless otherwise noted.</p></sec><sec id="s4-2"><title>In utero electroporation</title><p>Mice were anesthetized using 2,2,2-Tribromoethanol (4 mg/10 g mouse) or isofluorine, and embryos were exposed at E14.5 or E15.5. Plasmids were mixed with fast green and microinjected into the lateral ventricle of embryos using a picospritzer. Embryos were electroporated with five 50 ms pulses at 30–35V with a 950 ms interval and returned to the abdominal cavity. Plasmids used were: <italic>Neurod1-Cre</italic>, <italic>lac<underline>Z</underline>/<underline>Egfp</underline></italic>, lox-STOP-lox Ai9, <italic>Neurod1-Gsk3b</italic>, and <italic>Neurod1</italic>-EGFP. A CF1 foster dam was used to aid postnatal survival studies. Depending on the experiment, mice were perfused at E19.5, P0, P10, or P15 with 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO) in phosphate buffered saline (PBS) and post fixed overnight.</p></sec><sec id="s4-3"><title>Ex vivo electroporation and organotypic cortical slice culture</title><p>Cortical progenitor cells were electroporated ex vivo at E15.5 as described previously (<xref ref-type="bibr" rid="bib37">Hand et al., 2005</xref>). Briefly, E15.5 embryos were decapitated, plasmids were injected into lateral ventricle followed by electroporation with four 30V pulses that were 30–40 ms in duration and separated by a 100-ms interval. Following electroporation, brains were dissected and vibratome sectioned at 250 μm. Slices were transferred to Poly-D-Lysine and laminin coated culture insert (Millipore, Billerica, MA) in a FluoroDish (World Precision Instruments, Sarasota, FL), then 2 ml Basal Medium Eagle with FBS, N2 (Gemini Bio-Poducts, West Sacramento, CA), B27 (Life Technologies, Grand Island, NY), penicillin-streptomycin (Life Technologies), and L-glutamine (Life Technologies) supplements were added. Slices were cultured for 3 days at 37°C and live imaged using an Olympus FV1000 Confocal microscope with stage incubator.</p></sec><sec id="s4-4"><title>Cortical progenitor cultures</title><p>E14.5–15.5 dorsal cortices were electroporated with <italic>Neurod1-Cre</italic>; Z/EG, dissected in 4°C Hank's Balanced Salt Solution (HBSS) (Life Technologies) and dissociated into single cells using Trypsin (Life Technologies) according to previously described methods (<xref ref-type="bibr" rid="bib37">Hand et al., 2005</xref>). Neurons were plated on glass bottom dishes (MatTek) coated with 0.1 mg/ml Poly-D-Lysine (Sigma) and 5 μg/ml Laminin (Sigma). Cells were cultured in Neuralbasal-A Medium (Life Technologies), supplemented with 1X B-27 (Gibco, 17,054-044), L-glutamine (Life Technologies), penicillin-streptomycin (Life Technologies), N2 (Life Technologies), and FBS. Neurons were fixed with 4% PFA and stained for stage progression analysis.</p></sec><sec id="s4-5"><title>Western Blotting</title><p>Mouse cortices were dissected from three control (<italic>Gsk3a</italic><sup><italic>−/−</italic></sup><italic>Gsk3b</italic><sup><italic>loxp/+</italic></sup><italic>:Neurod6</italic>) and mutant (<italic>Gsk3:NeuroD6</italic>) or three control (<italic>Gsk3a</italic><sup><italic>loxp/loxp</italic></sup><italic>Gsk3b</italic><sup><italic>loxp/loxp</italic></sup>) and three mutant (<italic>Gsk3a</italic><sup><italic>loxp/loxp</italic></sup><italic>Gsk3b</italic><sup><italic>loxp/loxp</italic></sup><italic>:NeuroD6</italic>) mice from independent litters, collected in RIPA lysis buffer supplemented with protease and phosphatase inhibitors and cleared by centrifugation. Proteins were separated on SDS-PAGE gradient gels, transferred to a PVDF membrane and probed for proteins of interests and secondary HRP-conjugated antibodies were used for detection. Blots were washed and detection was performed with a commercially available ECL kit. ImageJ software (NIH) was used for quantification of band intensities, and intensities were normalized with total protein or the load control. Statistical analyses were conductive using Prism (GraphPad Software Inc., La Jolla, CA). Differences were considered statistically significant at p&lt;0.05 and are included in each figure. The following antibodies were used: GSK-3 (Invitrogen, Carlsbad, CA), Actin (Cell Signaling Technology Inc., Danvers, MA), pFAK ser722 (Santa Cruz Biotechnology, Inc., Dallas, TX), FAK (Cell Signaling Technology Inc), pCRMP-2 Thr514 (Cell Signaling Technology Inc), CRMP-2 (Cell Signaling Technology Inc), pCREB ser129 (Santa Cruz Biotechnology, Inc.), CREB (Santa Cruz Biotechnology, Inc.), pDCX ser327, DCX (Cell Signaling Technology Inc.), pDynamin-1 ser744 (Santa Cruz Biotechnology, Inc.), Dynamin-1 (Santa Cruz Biotechnology, Inc.), PTEN (Cell Signaling Technology Inc.), LKB1 (Upstate), β-catenin (Cell Signaling Technology Inc), DVL2 (Cell Signaling Technology Inc), GAPDH (Cell Signaling Technology Inc), Cleaved Caspase-3 (Cell Signaling Technology Inc).</p></sec><sec id="s4-6"><title>Immunohistochemistry</title><p>Briefly, 100–150 μm free-floating vibratome sections of brains were collected in PBS, blocked with 5% normal serum in PBS with 0.1% Triton X-100 and incubated with primary antibodies in blocking solution overnight at 4°C. Cryosectioned tissue samples were immunostained as previously described (see <xref ref-type="bibr" rid="bib78">Newbern et al., 2011</xref>). P15 in utero vibratome sections were blocked with 5% normal serum in PBS with 0/1% Triton X-100 and 2% DMSO, and incubated with primary antibodies for 3 days. Slices were rinsed with PBST for 24 hr and incubated with secondary antibodies for 3 days in PBS with 0/1% Triton X-100.</p><p>The following primary antibodies were used in our study: L1 (Millipore), Cux1 (Santa Cruz Biotechnology, Inc.), Neuronal Nuclei (Millipore), GFP (Aves Labs, Inc., Tigard, OR), CTIP2 (Abcam, Cambridge, MA) DRAQ5 (Fisher), TBR1 (Abcam, Cambridge, MA), HOECHST (Sigma), RFP (Rockland, Gilbertscille, PA). After rinsing, sections were then incubated with Alexa-conjugated secondary antibodies (Invitrogen) overnight at 4°C, rinsed three times in PBS, and mounted with gel/mount (Sigma).</p></sec><sec id="s4-7"><title>Quantification of cortical layering</title><p><italic>Gsk3</italic> overexpression analysis was conducted with <italic>Neurod1-Cre</italic> and lox-STOP-lox-Ai9 plasmids as control and <italic>Neurod1-Gsk3b</italic> and <italic>Neurod1</italic>-eGFP plasmids for overexpression. Electroporations were performed at E14.5 and analyzed at E19.5. For controls and <italic>Gsk3</italic> over-expression, multiple comparable cortical sections were analyzed. For analysis of layering, the cortical plate was equally divided into eight bins, and cells in each bin were counted using ImageJ software. Bin 7–8 included layers 1–3. The percentage of GFP+ neurons in each bin were determined from multiple comparable sections.</p><p>Postnatal day 10 analysis after in utero electroporation was conducted with <italic>Gsk3a</italic><sup><italic>+/−</italic></sup><italic>Gsk3b</italic><sup><italic>loxp/loxp</italic></sup> control <italic>Gsk3</italic><sup><italic>−/−</italic></sup><italic>Gsk3b</italic><sup><italic>loxp/loxp</italic></sup> embryos. EGFP-filled neurons were counted and localized to either upper or deeper layers of cortex. Differences were considered statistically significant at p≤0.05 using an unpaired t-test.</p><p>Lamination quantification of mutant mice at P0 (<italic>Gsk3</italic><sup><italic>loxp</italic></sup><italic>:Neurod6</italic>, <italic>Gsk3:Dlx5/6</italic>, <italic>Ctnnb1</italic><sup><italic>Ex3</italic></sup><italic>:Neurod6</italic>, <italic>Dvl123:Neurod6</italic>, <italic>Stk11:Neurod6</italic>, <italic>Cdc42:Neurod6</italic>, <italic>Pten:Neurod6</italic>) was conducted using heterozygous controls (Cre+, loxP/+) compared to conditional knockouts. Vibratome sections (3–5 per mouse) were imaged at 40X with a 2.4-μm optical slice and Cux-1 cells were counted in an 8 bin analysis. Data were quantified as average percentage of Cux-1 neurons per bin with differences considered statistically significant at p≤0.05 using an unpaired t-test. Note that the histograms for controls <xref ref-type="fig" rid="fig2">Figure 2</xref> and <xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1</xref> (based on a total of 10 control animals) all look very similar reflecting the fact that almost all Cux1 neurons have reached Layer 2–3 by P0 in control mice. The histogram for the <italic>Gsk3</italic> mutants in <xref ref-type="fig" rid="fig2">Figure 2</xref> is drastically different and reflects the distribution of Cux 1 neurons observed by inspection in many additional <italic>Gsk3:Neurod6</italic> and <italic>Gsk3</italic><sup><italic>loxp</italic></sup><italic>:Neurod6</italic> mice. In contrast, the histograms showing Cux1 neuron distribution for the other mutants were indistinguishable from controls.</p><p>Determination of number of Cux1 neurons at P0 in <italic>Gsk3</italic><sup><italic>loxp</italic></sup><italic>:Neurod6</italic> mice with <italic>Gsk3a</italic><sup><italic>loxp/loxp</italic></sup><italic>Gsk3b</italic><sup><italic>loxp/WT</italic></sup><italic>:Neurod6</italic> heterozygous as controls was performed as follows: Three 100-μm thick vibratome sections were imaged and one to two 100-μm cortical columns were obtained per slice for a total of five columns per mouse. Total Cux1 cells and total cell numbers were counted using an 8 bin analysis with ImageJ software. Data were quantified as average percentage of Cux1/total per bin and differences were analyzed using an unpaired t-test.</p></sec><sec id="s4-8"><title>Image Acquisition and analysis</title><p>Images were collected on Zeiss LSM 710 and Zeiss LSM 780 confocal microscopes. Z-stack images were collected with 10X or 20X objectives and tiled together to generate high-resolution images of whole brain sections. Binned quantification images were taken using a 20× objective and a 2-μm optical slice. Randomly identified sections of the electroporated area in presumptive somatosensory cortex were imaged for quantification. Live imaging of migrating neurons was acquired on an Olympus FV1000 Confocal microscope with stage incubator. On average, 40 μm Z-stack images were acquired and merged every 30–60 min for 8–20 hr. A 40X objective at a 0.8-μm optical slice was used for GSK-3<sup>loxp</sup>:Nex cux-1 cell counting.</p></sec><sec id="s4-9"><title>Dendritic arborization</title><p>P15 in utero experiments used <italic>Gsk3a</italic><sup><italic>+/+</italic></sup><italic>Gsk3b</italic><sup><italic>loxp/loxp</italic></sup> control <italic>Gsk3a</italic><sup><italic>−/−</italic></sup> <italic>Gsk3b</italic><sup><italic>loxp/loxp</italic></sup> or <italic>Gsk3a</italic><sup><italic>loxp/loxp</italic></sup> <italic>Gsk3b</italic><sup><italic>loxp/loxp</italic></sup> embryos. Images for the dendritic arbor reconstruction were acquired on a Zeiss LSM 7 MP multiphoton system using a W-PlanApochromat 20x/1.0 IR-corrected water immersion objective. On average, Z-stacks were acquired from 90 to 100 μm range with 0.65 μm steps. Dendritic reconstructions were performed using Neurolucida. A total of 15 control and 15 <italic>Gsk3</italic>-deleted cells from three mice each were reconstructed for dendritic analysis. Apical and basal dendrite polarity was quantified by generating dendrograms (Neurolucida) from reconstructed images. Sholl analysis calculated the number of dendrite intersections and dendritic lengths, Sholl quantification was conducted with an initial 15-μm somal radius and 20 μm concentric radial steps.</p></sec><sec id="s4-10"><title>Microarray</title><p>Dorsal cortices were dissected from three E18 control (<italic>Gsk3a</italic><sup><italic>−/−</italic></sup><italic>Gsk3b</italic><sup><italic>loxp/loxp</italic></sup>) and mutant (<italic>Gsk3;Neurod6</italic>) embryos derived from two independent litters. RNA was prepared using the MiRNAeasy kit (Qiagen, Valencia, CA) and analyzed using the Affymetrix Mouse Gene 2.0 St array (Affymetrix, Santa Clara, CA). Following scanning of the array, basic data analysis was carried out using the Partek Genomics Suite Version 6.12.0712 (Partek, Inc., St. Louis, MO) Transcripts up or down-regulated by 1.5-fold were considered interesting candidates. Microarray data have been made available through Gene Expression Omnibus, accession number GSE58727 (GEO, NCBI).</p></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>The authors would like to thank all members of the Snider Lab for thoughtful discussion. Dr Franck Polleux (Columbia University) provided the <italic>Neurod1-Cre</italic>, Lox-STOP-lox <italic>lac<underline>Z</underline>/<underline>Egfp</underline></italic> (Z/EG) plasmids, and <italic>Stk11</italic> mutant mice. The phospho-DCX ser327/Thr321 antibody was provided by Dr Orley Reiner (Weizmann Institute of Science, Israel) and has been previously described (<xref ref-type="bibr" rid="bib31">Gdalyahu et al., 2004</xref>).</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>MM-S, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con2"><p>YW, Acquisition of data, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con3"><p>XZ, Conception and design, Acquisition of data</p></fn><fn fn-type="con" id="con4"><p>JP, Acquisition of data, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con5"><p>WDS, 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 to animal protocol (#14-006)approved by the Institutional Animal Care and Use Committees (IACUC) of the University of North Carolina at Chapel Hill.</p></fn></fn-group></sec><ref-list><title>References</title><ref id="bib1"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Altman</surname><given-names>J</given-names></name><name><surname>Bayer</surname><given-names>SA</given-names></name></person-group><year>1990</year><article-title>Mosaic organization of the hippocampal neuroepithelium and the multiple 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contrib-type="editor"><name><surname>Miller</surname><given-names>Freda</given-names></name><role>Reviewing editor</role><aff><institution>The Hospital for Sick Children Research Institute, University of Toronto</institution>, <country>Canada</country></aff></contrib></contrib-group></front-stub><body><boxed-text><p>eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see <ext-link ext-link-type="uri" xlink:href="http://elifesciences.org/review-process">review process</ext-link>). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.</p></boxed-text><p>Thank you for choosing to send your work entitled “GSK-3 signaling in developing cortical neurons regulates migration and dendritic polarization” for consideration at <italic>eLife</italic>. Your full submission has been evaluated by a Senior editor and 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the decision was reached after discussions between the reviewers.</p><p>While all three of the reviewers appreciated the general high quality of the data that were presented in this manuscript, after a lengthy discussion our concern was that, as is, the data presented in the manuscript are not sufficiently novel for publication in <italic>eLife.</italic> However, given the high quality of the manuscript, and potential interest of the findings, we would be happy to consider a revised version of the paper in which you have added perhaps another piece of data that adds a conceptual advance as well as data addressing the reviewers concerns. In that regard, if you were to validate all of the different conditional mouse knockout models that you have used with specific regard to their efficacy, then all of the reviewers agreed that this would considerably increase the novelty of the conclusions that you could draw.</p><p><italic>Reviewer #1:</italic> In this manuscript by Morgan-Smith et al., the authors have defined an important role for GSK-3 signaling in the migration of excitatory cortical and hippocampal neurons during embryonic murine development. In general, the studies are very nicely performed, and the amount of work is extensive. Having said that, some of their conclusions do not seem to be justified on the basis of the data that are presented, and the manuscript could be significantly improved by consideration of a number of issues, as follows.</p><p>1) The authors demonstrate a robust and clearly-defined deficit in the migration of newborn Cux1-positive cortical neurons as soon as they are born. In contrast, they see that the deeper-layer Tbr1-positive neurons are unaffected. Since these neurons are born quite a bit earlier, are the authors confident that their driver line Nex-Cre actually causes recombination early enough to affect these earlier-born neurons? Also, most Tbr1-positive neurons are generated directly by radial glia, while the Cux1-positive neurons are largely generated via intermediate progenitors. Since intermediate progenitors are targeted by their Nex-Cre construct, might this provide an explanation for these differences?</p><p>While it is not essential for the authors to address these issues experimentally here, they should be more cautious in their interpretation of these differences.</p><p>2) In <xref ref-type="fig" rid="fig2">Figure 2A,B</xref>, using mice floxed for both GSK3A and B, it looks like there are many more Cux1-positive neurons in addition to their deficit in migration. Is it possible that inducible deletion of GSK3 in intermediate progenitors causes an expansion of cortical neurons which in turn affects their migration indirectly? The authors need to address this possibility by quantifying the relative proportions of Cux1-positive neurons in these floxed mice relative to the controls.</p><p>3) In <xref ref-type="fig" rid="fig2">Figure 2D</xref>, the authors show statistical significance, but they only have an n = 2 for controls. How can this be the case? I realize that some authors count each section as an n, but this isn't really valid for these types of analyses.</p><p>4) One of the main conclusions of this manuscript is that GSK3 is essential for radial but not tangential migration, based upon their analysis of cortical excitatory vs MGE-generated inhibitory interneurons. This conclusion isn't really warranted by the data they show. In particular, while <xref ref-type="fig" rid="fig3">Figure 3A–A'</xref> does show that inhibitory interneurons make it into the cortex, these data need to be quantified and it needs to be shown that GSK3 has actually been deleted in these neurons to draw this conclusion.</p><p>5) Similarly, the authors conclusions about direct effects of GSK3 ablation on dendritic orientation are also difficult to draw based upon their experimental design. In particular, while I agree that their data convincingly show inappropriate orientation of the dendrites of Cux1-positive neurons in layers 2-3, the authors cannot rule out the possibility that these neurons were delayed or perturbed in their migration to this location, and that the dendritic deficits are a secondary consequence of any such perturbations. The authors need to deemphasize their conclusions that these are direct effects unless they have data dissociating migration from later events such as dendritic development.</p><p>6) Data in <xref ref-type="fig" rid="fig6 fig7">Figures 6 and 7</xref> showing negative results vis a vis migration with multiple signaling proteins are a nice addition to the manuscript. However, to really substantiate these negative data, it would be important to (i) show that the Cre-mediated recombination really did occur as predicted, and (ii) show some kind of quantification to rule out the possibility that there are indeed neuronal deficits, but that they are just more subtle than with GSK3 knockout.</p><p>7) I'm not sure the small amount of microarray data shown in <xref ref-type="fig" rid="fig6">Figure 6B</xref> really adds anything to the manuscript. I would either add all of the microarray data or remove this.</p><p>8) The data on Crmp-2 are important and interesting. The Dcx data are potentially also very interesting, but the authors don't show that there is no change in total Dcx levels. In the absence of such data, it is difficult to know whether relative phosphorylation of Dcx is actually decreased.</p><p><italic>Reviewer #2:</italic> In the manuscript 26-02-2014-RA-eLife-02663 entitled “GSK-3 signaling in developing cortical neurons regulates migration and dendritic polarization”, Morgan-Smith and colleagues address the question of molecular mechanisms of neuronal migration, a timely and very interesting problem with many significant knowledge gaps. Since many of the molecules implicated in this process are major players in important intracellular signalling events, this question is of considerable interest to a general audience. Specifically, the manuscript focuses on the regulation of cytoskeleton dynamics in migrating posmitotic neurons in the vertebrate cortex, and the role of LKB1, GSK-3 and APC signalling cascade. These proteins have been previously implicated in neuronal migration in a study using non-genetic and acute manipulations (Asada, N. &amp; Sanada, 2010).</p><p>Taking advantage of the Nex:cre driver, the authors first analyse two different post-mitiotic neuron knockouts of GSK-3 alpha and beta and show that they are required for the positioning of Cux1+ neurons in cortical layers 2/3. Overexpression of GSK-3 through in utero electroporation results in more neurons occupying superficial cortical layers. Tangential neuronal migration in GSK-3 mutants is not affected suggesting that it GSK-3 is specifically involved in radial migration. Cell-autonomous deletion of GSK-3 reproduced the migration defect and revealed a neuronal morphology defect (the authors argue for dendrite orientation defects), as well as the persistence of the migration defects in postnatal animals. Next, the authors used Nex:cre to delete beta catenin, disheveled, LKB1, CDC42 and Pten, and show no effect on cortical neuron migration. However, the authors note, that in such knockouts, there was no change of Wnt target gene expression, or in the phosphorylation state of some of the GSK-3 substrates. However, the authors do finally show that GSK-3 loss of function does lead to a consistent decrease of pDCX and pCRMP-2 levels.</p><p>The data are mostly well-documented and convincing. Indeed, as authors claim, to my knowledge, this is the first genetic demonstration that GSK-3's are required for specific aspects of neuronal migration. My main concern is with the novelty of the findings, relative to the J Nsci study by Asada and Sanada which also showed a requirement for GSK-3 in migration, albeit through an acute, non-genetic manipulation. Other studies, including Shi et al. Curr Biol 14, 2025-2032 (2004) have also implicated GSK-3 in dendrite vs axon polarity.</p><p>Additionally, while I appreciate the lack of migration phenotypes in other KOs examined here, contrasted with Asada's manipulations of LKB1, I am concerned about the extent of the loss of function induced. The fact that in the b-cat KO, the authors fail to detect any effect on Wnt signalling targets makes me wary of their strong conclusions. Especially, since the effect of KO on b-catenin expression is barely more than a two-fold change. How do we know that the induction of those KOs is efficient enough to warrant the strong conclusion that LKB1, Pten, b-catenin etc, are not required for migration?</p><p><italic>Reviewer #3:</italic> The study by Morgan-Smith and colleagues investigates the role of GSK3 in the developing mouse telencephalon. Using a line allowing efficient deletion of GSK3 in newly-born excitatory neurons, the study documents migration failures both in the developing cerebral cortex and the hippocampus. These defects are not observed with medial eminence-derived interneurons. The conclusion that GSK play a critical role in the migration of cortical excitatory neurons is supported by data based on a GSK-overexpression approach causing enhanced migration. A thorough analysis of the canonical Wnt pathway suggests that it is not involved in the phenotype described here. By contrast, based on results illustrating a dramatic reduction of phosphorylated CRMP-2 it would appear that GSK may be critically involved in a semaphorin pathway. These results fit well with recent observations by Ip and colleagues as well as with a report by Chen and colleague on the role of Semaphorin 3A in the radial migration of cortical neurons. This is a very well executed, thorough and interesting study. In addition to the main results summarised in the above, another virtue of this study is that it reports on a range of negative, but surprising and useful results. Beyond the lack of involvement of the Wnt pathway, the lack of effects of the deletions of PTEN and especially of Cdc42 are quite surprising. Clearly then, this important study should be published in a scholarly, high profile journal as it is likely to be of high interest to developmental neurobiologists as well as to human neurogeneticists.</p><p>My main recommendation would be to revise the Abstract to better highlight the interest of the study. In particular, the first sentence is difficult to understand (“strongly regulates progenitors”?) and a bit discouraging as well since one of the virtue of the deleter line used here is to leave aside these previously investigated aspects of GSK3. They were also somewhat less interesting from a developmental neurobiology viewpoint since they dealt in essence with proliferation as opposed to migration. In a similar vein, it is unfortunate that the Abstract fails to mention that GSK does not seem to be involved in the migration of cortical interneurons. Finally, the role of GSK in the Semaphorin pathway would be worth emphasizing in the Abstract and perhaps better explained in the Introduction. The important <xref ref-type="bibr" rid="bib13">Chen et al., 2008</xref> study is briefly mentioned there as well, but it would be useful if it would be better emphasize at the expense of some of the Wnt pathway discussion that could be kept for the Discussion.</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.02663.019</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p>We have thoroughly revised our manuscript based on helpful comments of the reviewers. In particular, a weakness of the prior version that diminished novelty was inadequate verification of several of the lines of mutant mice on which we reported negative findings. As instructed by the editors, we have carefully verified protein reduction after <italic>Neurod6-Cre</italic> mediated recombination in each of the six lines and provided quantification of cortical neuronal migration. Most of these results are shown in a new <xref ref-type="fig" rid="fig6">Figure 6</xref> where new confocal images of Cux1 expressing neurons in mutants and controls are shown together with Western blots documenting strongly reduced levels of proteins in the mutants.</p><p>Quantification is shown in supplemental material linked to this new data in <xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement figure 1</xref>.</p><p>We have also carefully commented on all of the points raised by each reviewer and revised wording in the text in accordance with reviewer suggestions.</p><p>Reviewer #1: <italic>[…] 1) The authors demonstrate a robust and clearly-defined deficit in the migration of newborn Cux1-positive cortical neurons as soon as they are born. In contrast, they see that the deeper-layer Tbr1-positive neurons are unaffected. Since these neurons are born quite a bit earlier, are the authors confident that their driver line Nex-Cre actually causes recombination early enough to affect these earlier-born neurons? Also, most Tbr1-positive neurons are generated directly by radial glia, while the Cux1-positive neurons are largely generated</italic> via <italic>intermediate progenitors. Since intermediate progenitors are targeted by their Nex-Cre construct, might this provide an explanation for these differences?</italic></p><p><italic>While it is not essential for the authors to address these issues experimentally here, they should be more cautious in their interpretation of these differences</italic>.</p><p>We are in complete agreement with the reviewer on this point. In fact we did not perform extensive analysis of the Tbr1-positive cells precisely because we did not think we could draw a strong conclusion about GSK-3 regulation of this population.</p><p>In order to make it clear that the strong GSK-3 regulation of migration may not apply to early born Tbr1-positive neurons, we modified the text in the Discussion as follows: “Deeper layer neurons expressing Tbr1 were not strongly influenced, but we cannot be certain that GSK-3 protein was fully depleted in these earlier born neurons at the time migration was occurring.”</p><p><italic>2) In</italic> <xref ref-type="fig" rid="fig2"><italic>Figure 2A,B</italic></xref><italic>, using mice floxed for both GSK3A and B, it looks like there are many more Cux1-positive neurons in addition to their deficit in migration. Is it possible that inducible deletion of GSK3 in intermediate progenitors causes an expansion of cortical neurons which in turn affects their migration indirectly? The authors need to address this possibility by quantifying the relative proportions of Cux1-positive neurons in these floxed mice relative to the controls</italic>.</p><p>We think this unlikely because our cell autonomous recombination studies (<xref ref-type="fig" rid="fig4">Figure 4A’,B’</xref>) where recombination is targeted to postmitotic neurons with <italic>Neurod1-Cre</italic> show that a heavy majority of cells did not migrate to the upper layers. Also time lapse imaging of cells that are clearly neurons show that migration is strikingly delayed for almost all neurons imaged (<xref ref-type="fig" rid="fig4">Figure 4E</xref>).</p><p>In addition, as suggested by the reviewer, we quantified numbers of Cux1 positive cells in relation to total cell number in both <italic>Gsk3</italic> mutants and controls. The number of Cux1 neurons in mutants was very similar to the number in controls.</p><p><italic>3) In</italic> <xref ref-type="fig" rid="fig2"><italic>Figure 2</italic>D</xref><italic>, the authors show statistical significance, but they only have an n=2 for controls. How</italic> can <italic>this be the case? I realize that some authors count each section as an n, but this isn't really valid for these types of analyses</italic>.</p><p>N = 2 for controls and n = 3 for the experimental refers to number of animals studied. These were littermate controls obtained from 2 separate pregnant females, multiple separate coronal sections of the electroporated cortex was studied in each animal (on average, 300 neurons per section). We do not necessarily get appropriate genotypes with matched controls from every litter electroporated. Since the n is low and the effects are relatively modest, we moved the overexpression image to <xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1</xref> and emphasize the point that overexpression of <italic>Gsk3</italic> does not delay migration (based on n = 3 animals) as would have been predicted from prior studies.</p><p><italic>4) One of the main conclusions of this manuscript is that GSK3 is essential for radial but not tangential migration, based upon their analysis of cortical excitatory versus MGE-generated inhibitory interneurons. This conclusion isn't really warranted by the data they show. In particular, while</italic> <xref ref-type="fig" rid="fig3"><italic>Figure 3A–A'</italic></xref> <italic>does show that inhibitory interneurons make it into the cortex, these data need to be quantified and it needs to be shown that GSK3 has actually been deleted in these neurons to draw this conclusion</italic>.</p><p>We have addressed both of these important points. A Western blot of MGE lystates has been added to supplementary material, <xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1</xref>, which demonstrates strong reduction GSK-3 protein in precursors and newly generated inhibitory neurons after recombination with <italic>Dlx5/6-Cre</italic>. Quantification showing only modest if any effects on migration is included in this supplement. We have also added the caveat to the text that we assessed only gross measures of tangential migration as we did not asses migration of interneuron subtypes in this study: “These results are not meant to imply that migration of interneurons was normal in every respect as we did not assess migration of specific interneuron subsets.”</p><p><italic>5) Similarly, the authors conclusions about direct effects of GSK3 ablation on dendritic orientation are also difficult to draw based upon their experimental design. In particular, while I agree that their data convincingly show inappropriate orientation of the dendrites of Cux1-positive neurons in layers 2-3, the authors cannot rule out the possibility that these neurons were delayed or perturbed in their migration to this location, and that the dendritic deficits are a secondary consequence of any such perturbations. The authors need to deemphasize their conclusions that these are direct effects unless they have data dissociating migration from later events such as dendritic development</italic>.</p><p>The reviewer makes an interesting point. However, we are not in complete agreement. We specifically chose the few neurons in normal locations in Layer 2-3 for analysis. <xref ref-type="fig" rid="fig4">Figure 4A,A’</xref> shows that some <italic>Gsk3</italic> deleted neurons are normally positioned by E19 and <xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1B</xref> shows a few Cux 1 neurons normally positioned as early as E16. It seems fair to assume that these neurons did not undergo a prolonged migration delay, presumably because GSK-3 levels were not drastically reduced in these few neurons at these early stages. As the reviewer acknowledges, the abnormal orientation of the dendrites is striking. We think the most plausible explanation is that dendrites of <italic>Gsk3</italic> deleted neurons do not respond normally to directional cues that are present.</p><p>We agree that we can’t absolutely prove that dendritic polarization is directly related to GSK-3 abrogating signaling as opposed to an abnormality acquired during migration. We have therefore modified the title to: <italic>“</italic>GSK-3 signaling in developing cortical neurons is essential for radial migration and dendritic orientation” which is certainly true and avoids the terms “regulate” and “polarization”. We have also modified our text to reflect reviewer concerns on this point as follows: A) We changed the section heading from: “GSK-3 regulates dendritic orientation” to “Abnormal dendritic orientation in <italic>Gsk3</italic> mutants”</p><p>B) We added text: “Images at E16 (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1B</xref>) and E19 (<xref ref-type="fig" rid="fig4">Figure 4A</xref>’) show that a few <italic>Gsk3</italic> deleted neurons are normally positioned and suggest that these normally positioned neurons did not undergo a substantial delay in migration.”</p><p><italic>6) Data in</italic> <xref ref-type="fig" rid="fig6 fig7"><italic>Figures 6 and 7</italic></xref> <italic>showing negative results vis a vis migration with multiple signaling proteins are a nice addition to the manuscript. However, to really substantiate these negative data, it would be important to (i) show that the Cre-mediated recombination really did occur as predicted, and (ii) show some kind of quantification to rule out the possibility that there are indeed neuronal deficits, but that they are just more subtle than with GSK3 knockout</italic>.</p><p>Again, a very helpful point. As noted above, we have carefully verified protein reduction and provided quantification of migration in five of the six lines where we report negative results, <xref ref-type="fig" rid="fig6">Figure 6</xref> and <xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement figure 1</xref>. For comparison we show quantification of the strikingly abnormal quantification in <italic>Gsk3</italic> mutants in <xref ref-type="fig" rid="fig2">Figure 2</xref>, new panels A and C. For <italic>Dvl123:Neurod6</italic> we did not obtain enough 6 allele mutant mice for both Westerns and detailed quantification. We do show a representative image based on inspection of multiple sections from n = 3 of the 6 allele mutant mice. We hope we can keep the image in the paper as it will be of substantial interest to researchers in this field.</p><p><italic>7) I'm not sure the small amount of microarray data shown in</italic> <xref ref-type="fig" rid="fig6"><italic>Figure 6</italic>B</xref> <italic>really adds anything to the manuscript. I would either add all of the microarray data or remove this</italic>.</p><p>We have now removed the Table shown in former <xref ref-type="fig" rid="fig6">Figure 6B</xref> and simply mention results in the text. The full array has been deposited in GEO, NCBI for reference (GEO accession number GSE58727) and will be released to the public shortly.</p><p><italic>8) The data on Crmp-2 are important and interesting. The Dcx data are potentially also very interesting, but the authors don't show that there is no change in total Dcx levels. In the absence of such data, it is difficult to know whether relative phosphorylation of Dcx is actually decreased</italic>.</p><p>We probed for total DCX in these blots as directed. The reduction in pDCX remains very striking. The new results are shown in a revised <xref ref-type="fig" rid="fig7">Figure 7A</xref>.</p><p>Reviewer #2: <italic>[…] The data are mostly well-documented and convincing. Indeed, as authors claim, to my knowledge, this is the first genetic demonstration that GSK-3's are required for specific aspects of neuronal migration. My main concern is with the novelty of the findings, relative to the J Nsci study by Asada and Sanada which also showed a requirement for GSK-3 in migration, albeit through an acute, non-genetic manipulation. Other studies, including Shi et al. Curr Biol 14, 2025-2032 (2004) have also implicated GSK-3 in dendrite vs. axon polarity</italic>.</p><p>We addressed this point in an exchange with the editors: our findings are in direct conflict with the Asada study. We did <italic>not</italic> verify that <italic>inhibition</italic> of GSK-3 via an LKB1→GSK-3→APC (dephosphorylation) signaling cascade is required for neuronal migration as was published in that paper. Analysis of GSK-3 in the Asada paper relied exclusively on activation of GSK-3 via the GSK-3β S9A construct and the demonstrated effects on migration were transient. We understand that GSK-3 inhibition due to ser9/ser21 phosphorylation and subsequent dephosphorylation of key cytoskeletal regulators has previously been implicated in vitro in elaboration of axons and migration of non-neuronal cells. However, we and others (Courchet et al. Cell 2013) do not find evidence that LKB1 signaling regulates migration in vivo<italic>.</italic> Finally, published work that we cite on GSK-3 mutant knock-ins that are viable and exhibit grossly normal brain development, suggest it is unlikely that ser9/ser21 phosphorylation of GSK-3 strongly regulates cortical neuron migration in vivo<italic>.</italic> Rather, our study shows that GSK-3 activity is required for cortical neuronal migration in vivo. Our evidence suggests that GSK-3 phosphorylation of key downstream cytoskeletal regulators (rather than dephosphorylation) is crucial for migration.</p><p><italic>Additionally, while I appreciate the lack of migration phenotypes in other KOs examined here, contrasted with Asada's manipulations of LKB1, I am concerned about the extent of the loss of function induced. The fact that in the b-cat KO, the authors fail to detect any effect on Wnt signalling targets makes me wary of their strong conclusions. Especially, since the effect of KO on b-catenin expression is barely more than a two-fold change. How do we know that the induction of those KOs is efficient enough to warrant the strong conclusion that LKB1, Pten, b-catenin etc, are not required for migration?</italic></p><p>To clarify, we did not assess Wnt target genes in <italic>Ctnnb1</italic><sup><italic>Ex3</italic></sup><italic>:Neurod6</italic> mice. Rather, we assessed Wnt target genes in <italic>Gsk3:Neurod6</italic> mice. Reviewer 1 felt that this aspect should be given less emphasis and we have changed the presentation of the data as directed (see response to point 7 above)</p><p>We now show strikingly reduced protein levels after <italic>Neurod6-Cre</italic> mediated recombination for all of signaling mediators studied (new <xref ref-type="fig" rid="fig6s1">Figure 6 and—figure supplement 1</xref>) as noted in point 6 above. These strong protein reductions demonstrate that <italic>Neurod6-Cre</italic> was highly effective in generating recombination of all of the floxed alleles used in this study.</p><p>Reviewer 3: <italic>[…] My main recommendation would be to revise the Abstract to better highlight the interest of the study. In particular, the first sentence is difficult to understand (“strongly regulates progenitors”?) and a bit discouraging as well since one of the virtue of the deleter line used here is to leave aside these previously investigated aspects of GSK3. They were also somewhat less interesting from a developmental neurobiology viewpoint since they dealt in essence with proliferation as opposed to migration. We have revised the Abstract and removed the first sentence as recommended</italic>.</p><p><italic>In a similar vein, it is unfortunate that the Abstract fails to mention that GSK does not seem to be involved in the migration of cortical interneurons</italic>.</p><p>We have added a new sentence to the Abstract: “In contrast, tangential migration is not grossly impaired after <italic>Gsk3</italic> deletion in interneuron precursors”. This point is also now mentioned in the Introduction.</p><p><italic>Finally, the role of GSK in the Semaphorin pathway would be worth emphasizing in the Abstract and perhaps better explained in the Introduction. The important</italic> <xref ref-type="bibr" rid="bib13"><italic>Chen et al. 2008</italic></xref> <italic>study is briefly mentioned there as well, but it would be useful if it would be better emphasize at the expense of some of the Wnt pathway discussion that could be kept for the Discussion</italic>.</p><p>Because of the word limitations of the Abstract, we cannot emphasize the Sema pathway here. However, we have revised the Introduction to include information on the Sema pathway as instructed.</p></body></sub-article></article>