elife03596.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">03596</article-id><article-id pub-id-type="doi">10.7554/eLife.03596</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>The Drosophila Sp8 transcription factor Buttonhead prevents premature differentiation of intermediate neural progenitors</article-title></title-group><contrib-group><contrib contrib-type="author" id="author-15035"><name><surname>Xie</surname><given-names>Yonggang</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-15037"><name><surname>Li</surname><given-names>Xiaosu</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-15036"><name><surname>Zhang</surname><given-names>Xian</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" id="author-15038"><name><surname>Mei</surname><given-names>Shaolin</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con5"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-15039"><name><surname>Li</surname><given-names>Hongyu</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con6"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-15040"><name><surname>Urso</surname><given-names>Andreacarola</given-names></name><xref ref-type="aff" rid="aff2"/><xref ref-type="fn" rid="con7"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-14606"><name><surname>Zhu</surname><given-names>Sijun</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-2"/><xref ref-type="other" rid="par-3"/><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf1"/></contrib><aff id="aff1"><institution content-type="dept">Department of Neuroscience and Physiology</institution>, <institution>State University of New York Upstate Medical University</institution>, <addr-line><named-content content-type="city">Syracuse</named-content></addr-line>, <country>United States</country></aff><aff id="aff2"><institution content-type="dept">Department of Biology</institution>, <institution>Syracuse University</institution>, <addr-line><named-content content-type="city">Syracuse</named-content></addr-line>, <country>United States</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Bronner</surname><given-names>Marianne E</given-names></name><role>Reviewing editor</role><aff><institution>California Institute of Technology</institution>, <country>United States</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>zhus@upstate.edu</email></corresp></author-notes><pub-date date-type="pub" publication-format="electronic"><day>06</day><month>10</month><year>2014</year></pub-date><pub-date pub-type="collection"><year>2014</year></pub-date><volume>3</volume><elocation-id>e03596</elocation-id><history><date date-type="received"><day>07</day><month>06</month><year>2014</year></date><date date-type="accepted"><day>28</day><month>09</month><year>2014</year></date></history><permissions><copyright-statement>© 2014, Xie et al</copyright-statement><copyright-year>2014</copyright-year><copyright-holder>Xie 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="elife03596.pdf"/><related-article ext-link-type="doi" id="ra1" related-article-type="article-reference" xlink:href="10.7554/eLife.03502"/><abstract><object-id pub-id-type="doi">10.7554/eLife.03596.001</object-id><p>Intermediate neural progenitor cells (INPs) need to avoid differentiation and cell cycle exit while maintaining restricted developmental potential, but mechanisms preventing differentiation and cell cycle exit of INPs are not well understood. In this study, we report that the <italic>Drosophila</italic> homolog of mammalian Sp8 transcription factor Buttonhead (Btd) prevents premature differentiation and cell cycle exit of INPs in <italic>Drosophila</italic> larval type II neuroblast (NB) lineages. We show that the loss of Btd leads to elimination of mature INPs due to premature differentiation of INPs into terminally dividing ganglion mother cells. We provide evidence to demonstrate that Btd prevents the premature differentiation by suppressing the expression of the homeodomain protein Prospero in immature INPs. We further show that Btd functions cooperatively with the Ets transcription factor Pointed P1 to promote the generation of INPs. Thus, our work reveals a critical mechanism that prevents premature differentiation and cell cycle exit of <italic>Drosophila</italic> INPs.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03596.001">http://dx.doi.org/10.7554/eLife.03596.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.03596.002</object-id><title>eLife digest</title><p>Whereas the majority of cells in the brain are unable to divide to produce new cells, neural stem cells can divide numerous times and have the potential to become many different types of brain cells. However, in between these two extremes there is another group of cells called neural progenitors. These cells can give rise to multiple types of neurons but, in contrast to stem cells, they can undergo only a limited number of divisions.</p><p>Many of the molecular mechanisms by which stem cells give rise to progenitors are similar in mammals and in the fruit fly <italic>Drosophila</italic>. In the brains of fruit fly larvae, neural stem cells called neuroblasts give rise to ‘intermediate neural progenitors', each of which can divide between four and six times. Every division generates a replacement intermediate progenitor and a cell called a GMC, which divides one last time to produce two brain cells.</p><p>Intermediate progenitors must be tightly regulated to ensure that they undergo an appropriate number of divisions: too few divisions will result in a shortage of cells, disrupting brain development, whereas too many divisions will result in the formation of tumors. Now, using <italic>Drosophila</italic> brains in the laboratory, Xie et al.—and, independently, Komori et al.—have shown that a protein called ‘Buttonhead’ is responsible for maintaining this balance.</p><p>Xie et al. show that deletion of the gene for Buttonhead gene caused the progenitor cells to become GMCs before they had undergone the correct number of divisions. Further experiments revealed that Buttonhead prevents this problem by suppressing a protein called Prospero.</p><p>The mammalian equivalent of Buttonhead—a protein called Sp8—can substitute for Buttonhead in <italic>Drosophila</italic> neural progenitors, suggesting that the observed mechanisms may also apply to mammals. Further work is required to test this possibility directly and to examine the involvement of Sp8 in brain development and tumor formation.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03596.002">http://dx.doi.org/10.7554/eLife.03596.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>Sp8</kwd><kwd>Buttonhead</kwd><kwd>intermediate neural progenitor</kwd><kwd>neuroblast</kwd><kwd>pointed</kwd><kwd>Prospero</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd><italic>D. melanogaster</italic></kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100007258</institution-id><institution>State University of New York</institution></institution-wrap></funding-source><award-id>Upstate Medical School</award-id><principal-award-recipient><name><surname>Zhu</surname><given-names>Sijun</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/10.13039/100000065</institution-id><institution>National Institute of Neurological Disorders and Stroke</institution></institution-wrap></funding-source><award-id>R01NS085232</award-id><principal-award-recipient><name><surname>Zhu</surname><given-names>Sijun</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/10.13039/100000912</institution-id><institution>March of Dimes Foundation</institution></institution-wrap></funding-source><award-id>#5-FY14-59</award-id><principal-award-recipient><name><surname>Zhu</surname><given-names>Sijun</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 Drosophila equivalent of the human transcription factor Sp8 acts to ensure that neural progenitor cells undergo an appropriate number of cell divisions, thereby helping to regulate brain development and guard against tumor formation.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="s1" sec-type="intro"><title>Introduction</title><p>Intermediate neural progenitor cells (INPs) play a critical role in increasing the brain size and complexity. Transient amplification of INPs dramatically boosts the neural output from neural stem cells (NSCs) (<xref ref-type="bibr" rid="bib24">Kriegstein and Alvarez-Buylla, 2009</xref>; <xref ref-type="bibr" rid="bib13">Florio and Huttner, 2014</xref>). Recent studies in developing human brains as well as other mammalian brains suggest that an expansion of the number of transiently amplifying INPs, the outer sub-ventricular zone radial glia-like cells (oRGs), likely contributes to the increased cortical size and complexity in humans and other gyrencephalic animals (<xref ref-type="bibr" rid="bib12">Fietz et al., 2010</xref>; <xref ref-type="bibr" rid="bib16">Hansen et al., 2010</xref>; <xref ref-type="bibr" rid="bib28">Lui et al., 2011</xref>; <xref ref-type="bibr" rid="bib43">Wang et al., 2011</xref>). On the other hand, accumulating body of evidence suggests that brain tumors could originate from dedifferentiation and unrestricted proliferation of INPs (<xref ref-type="bibr" rid="bib17">Holland et al., 2000</xref>; <xref ref-type="bibr" rid="bib8">Dai et al., 2001</xref>; <xref ref-type="bibr" rid="bib42">Walton et al., 2009</xref>; <xref ref-type="bibr" rid="bib32">Persson et al., 2010</xref>; <xref ref-type="bibr" rid="bib55">Zong et al., 2012</xref>). Therefore, it is fundamentally important to understand how the generation and proliferation of INPs are regulated.</p><p>The recently discovered type II neuroblasts (NBs, the <italic>Drosophila</italic> NSCs) in developing <italic>Drosophila</italic> larval brains provide an excellent model system for studying mechanisms regulating the generation and proliferation of INPs (<xref ref-type="bibr" rid="bib3">Bello et al., 2008</xref>; <xref ref-type="bibr" rid="bib4">Boone and Doe, 2008</xref>; <xref ref-type="bibr" rid="bib5">Bowman et al., 2008</xref>). There are 8 type II NBs in each brain lobe. Like mammalian NSCs, <italic>Drosophila</italic> type II NBs produce neurons and glia indirectly by generating INPs. Individual INPs undergo 4–6 rounds of asymmetric divisions to produce a new INP to self-renew and a ganglion mother cell (GMC), which divides terminally to produce neurons and/or glia (<xref ref-type="bibr" rid="bib1">Bayraktar et al., 2010</xref>; <xref ref-type="bibr" rid="bib40">Viktorin et al., 2011</xref>; <xref ref-type="bibr" rid="bib48">Yang et al., 2013</xref>). Meanwhile, individual INPs produce distinct types of neurons by sequentially expressing a set of distinct transcription factors to specify the identity of their progeny (<xref ref-type="bibr" rid="bib2">Bayraktar and Doe, 2013</xref>; <xref ref-type="bibr" rid="bib44">Wang et al., 2014</xref>). Through self-renewing divisions, INPs not only amplify the number but also increase the diversity of neural progeny generated from type II NBs. Therefore, the neurogenesis pattern in type II NB lineages is remarkably similar to that in mammalian brains and the <italic>Drosophila</italic> INPs are functionally analogous to mammalian INPs, particularly oRGs.</p><p>The generation of INPs in type II NB lineages involves multiple steps (<xref ref-type="bibr" rid="bib3">Bello et al., 2008</xref>; <xref ref-type="bibr" rid="bib4">Boone and Doe, 2008</xref>; <xref ref-type="bibr" rid="bib5">Bowman et al., 2008</xref>). Newly generated INPs are immature and do not express any NB markers, such as the proneural protein Asense (Ase) or the bHLH protein Deadpan (Dpn), except for Miranda (Mira). The Ase<sup>−</sup> immature INPs first turn on the expression of Ase to become Ase<sup>+</sup> immature INPs. Ase<sup>+</sup> immature INPs then further differentiate to become mature INPs, which express both Ase and Dpn. INPs do not divide until they are fully mature. The maturation of INPs requires Numb, the NHL family protein Brain tumor (Brat), the transcription factor Earmuff (Erm), as well as the BAP and Histone deacetylase 3 (HDAC3) chromatin remodeling complexes (<xref ref-type="bibr" rid="bib5">Bowman et al., 2008</xref>; <xref ref-type="bibr" rid="bib45">Weng et al., 2010</xref>; <xref ref-type="bibr" rid="bib9">Eroglu et al., 2014</xref>; <xref ref-type="bibr" rid="bib22">Koe et al., 2014</xref>). Both Numb and Brat are segregated into Ase<sup>−</sup> immature INPs during the division of type II NBs to prevent them from dedifferentiating into NB fate, but they function through independent pathways. Numb inhibits Notch activity in Ase<sup>−</sup> immature INPs, whereas Brat likely antagonizes the activity of the EGR family transcription factor Klumpfuss (Klu) and Armadillo/β-Catenin in Ase<sup>−</sup> immature INPs (<xref ref-type="bibr" rid="bib5">Bowman et al., 2008</xref>; <xref ref-type="bibr" rid="bib23">Komori et al., 2014</xref>). Erm functions together with BAP and HDAC3 chromatin remodeling complexes after Brat and Numb to further restrict the developmental potential of INPs by attenuating the response of INPs to self-renewal factors such as Klu and Dpn (<xref ref-type="bibr" rid="bib20">Janssens et al., 2014</xref>; <xref ref-type="bibr" rid="bib22">Koe et al., 2014</xref>). In addition, the BAP chromatin remodeling complex limits the self-renewal of INPs by activating the expression of Prdm protein Hamlet (<xref ref-type="bibr" rid="bib9">Eroglu et al., 2014</xref>). In the absence of Numb, Brat, Erm, or chromatin remodeling complexes, INPs dedifferentiate into type II NBs and initiate tumorigenic overproliferation (<xref ref-type="bibr" rid="bib5">Bowman et al., 2008</xref>; <xref ref-type="bibr" rid="bib45">Weng et al., 2010</xref>; <xref ref-type="bibr" rid="bib9">Eroglu et al., 2014</xref>; <xref ref-type="bibr" rid="bib22">Koe et al., 2014</xref>). Therefore, these proteins are critical to prevent dedifferentiation of INPs.</p><p>However, despite the significant progress on elucidating mechanisms that promote maturation and prevent dedifferentiation of INPs in the past few years, much less is known about why only type II NBs produce self-renewing INPs but not the type I NBs, which produce neurons by generating terminally dividing GMCs. One major difference between INPs and GMCs is that INPs divide to self-renew whereas GMCs divide terminally (<xref ref-type="bibr" rid="bib3">Bello et al., 2008</xref>; <xref ref-type="bibr" rid="bib4">Boone and Doe, 2008</xref>; <xref ref-type="bibr" rid="bib5">Bowman et al., 2008</xref>; <xref ref-type="bibr" rid="bib48">Yang et al., 2013</xref>). Therefore, in addition to avoiding dedifferentiation and unrestricted tumorigenic overproliferation, INPs need to overcome another challenge–to avoid over-differentiation and cell cycle exit–in order to maintain their progenitor state and self-renewal while they differentiate to mature and undergo self-renewing divisions. Type II NBs and newly born Ase<sup>−</sup> immature INPs differ from type I NBs and GMCs by the lack of the expression of Ase and the homeodomain protein Prospero (Pros) (<xref ref-type="bibr" rid="bib3">Bello et al., 2008</xref>; <xref ref-type="bibr" rid="bib4">Boone and Doe, 2008</xref>; <xref ref-type="bibr" rid="bib5">Bowman et al., 2008</xref>). In type I NB lineages, Pros is expressed in the cytoplasm in the NBs and translocates to the nucleus in GMCs to promote differentiation and cell cycle exit by inhibiting NB self-renewing genes and activating neural differentiation genes (<xref ref-type="bibr" rid="bib26">Li and Vaessin, 2000</xref>; <xref ref-type="bibr" rid="bib7">Choksi et al., 2006</xref>). In type II NB lineages, Pros is not expressed in the NB or Ase<sup>−</sup> immature INPs. In Ase<sup>+</sup> immature INPs and mature INPs, Pros is expressed at low levels in the cytoplasm (<xref ref-type="bibr" rid="bib3">Bello et al., 2008</xref>; <xref ref-type="bibr" rid="bib4">Boone and Doe, 2008</xref>; <xref ref-type="bibr" rid="bib5">Bowman et al., 2008</xref>). It has been demonstrated that the lack of Ase and Pros in type II NBs and Ase<sup>−</sup> immature INPs is essential for the generation of self-renewing INPs in type II NB lineages. Forced expression of Ase or Pros in type II NBs and their progeny is sufficient to eliminate INPs although removing Ase or Pros in type I NBs does not change the identity of type I NBs or induce the generation of INPs (<xref ref-type="bibr" rid="bib5">Bowman et al., 2008</xref>; <xref ref-type="bibr" rid="bib1">Bayraktar et al., 2010</xref>; <xref ref-type="bibr" rid="bib54">Zhu et al., 2012</xref>). Our recent studies demonstrated that the Ets family transcription factor Pointed P1 (PntP1) suppresses Ase in type II NBs and is required for the generation of INPs (<xref ref-type="bibr" rid="bib53">Zhu et al., 2011</xref>). However, mechanisms that prevent premature differentiation of INPs and/or inhibit Pros expression in type II NBs and immature INPs are not known.</p><p>In this study, we investigate the role of the Sp family transcription factor Buttonhead (Btd) in type II NB lineage development. Btd is a homolog of mammalian Sp8 (<xref ref-type="bibr" rid="bib39">Treichel et al., 2003</xref>; <xref ref-type="bibr" rid="bib10">Estella and Mann, 2010</xref>). In developing mammalian brains, Sp8 is expressed in neural progenitor cells to regulate forebrain patterning and interneuron development (<xref ref-type="bibr" rid="bib14">Griesel et al., 2006</xref>; <xref ref-type="bibr" rid="bib41">Waclaw et al., 2006</xref>; <xref ref-type="bibr" rid="bib35">Sahara et al., 2007</xref>; <xref ref-type="bibr" rid="bib27">Li et al., 2011</xref>). In <italic>Drosophila</italic> embryos, Btd is required for the formation of specific head segments and NB formation (<xref ref-type="bibr" rid="bib46">Wimmer et al., 1993</xref>; <xref ref-type="bibr" rid="bib51">Younossi-Hartenstein et al., 1997</xref>). In addition, Sp8/Btd also promotes the growth of limbs and other appendages (<xref ref-type="bibr" rid="bib11">Estella et al., 2003</xref>; <xref ref-type="bibr" rid="bib39">Treichel et al., 2003</xref>; <xref ref-type="bibr" rid="bib21">Kawakami et al., 2004</xref>; <xref ref-type="bibr" rid="bib10">Estella and Mann, 2010</xref>). In this study, we report that Btd is expressed in type II NB lineages to prevent premature differentiation of INPs into GMCs by suppressing Pros expression in immature INPs. We also demonstrate that PntP1 and Btd function cooperatively to specify type II NB lineages and promote the generation of INPs.</p></sec><sec id="s2" sec-type="results"><title>Results</title><sec id="s2-1"><title>Loss of Btd results in a complete elimination of mature INPs in type II NB lineages</title><p>Our recent studies demonstrated that the Ets family transcription factor PntP1 is specifically expressed in type II NB lineages to promote the generation of INPs. However, although forced expression of PntP1 suppress Ase expression in nearly all type I NB lineages, it induces the generation of INP-like cells only in a subset of type I NBs (<xref ref-type="bibr" rid="bib53">Zhu et al., 2011</xref>). Therefore, it is likely that other protein(s) may function together with PntP1 to specify type II NB lineages and promote the generation of INPs. A recent functional genomic study showed that in addition to PntP1, there are other nine genes that are highly expressed in brain tumors derived from type II NB lineages (<xref ref-type="bibr" rid="bib6">Carney et al., 2012</xref>). We wondered whether any of these genes could function together with PntP1 to promote INP generation. To test this idea, we first examined how knockdown of these genes would affect INP generation in type II NB lineages. A normal type II NB lineage contains 2–3 Ase<sup>−</sup> immature INPs, 2–3 Ase<sup>+</sup> immature INP, and about 20–30 (26.9 ± 4.1, mean ± SD) Ase<sup>+</sup> Dpn<sup>+</sup> mature INPs (<xref ref-type="fig" rid="fig1">Figure 1A–A′,C–C′,G</xref>). Interestingly, RNAi knockdown of Btd using the type II NB lineage-specific <italic>pntP1-GAL4</italic> (named as <italic>GAL4</italic><sup><italic>14−94</italic></sup> previously) (<xref ref-type="bibr" rid="bib53">Zhu et al., 2011</xref>) as a driver led to a complete elimination of mature INPs in about 50% of type II NB lineages (<xref ref-type="fig" rid="fig1">Figure 1B–B′,G–H</xref>). Instead, only a few (3.7 ± 1.2) Ase<sup>+</sup> Dpn<sup>−</sup> cells were observed next to the Ase<sup>−</sup> immature INPs (<xref ref-type="fig" rid="fig1">Figure 1B–B′</xref>). However, type II NBs remain Ase<sup>−</sup> as normal type II NBs (<xref ref-type="fig" rid="fig1">Figure 1B–B′</xref>), suggesting that the identity of the type II NBs was not affected by Btd RNAi knockdown.<fig-group><fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.03596.003</object-id><label>Figure 1.</label><caption><title>Loss of Btd eliminates mature INPs in type II NB lineages.</title><p>(<bold>A</bold>–<bold>A′</bold>, <bold>C</bold>–<bold>C′</bold>) Wild-type type II NB lineages in a third instar larval brain. mCD8-GFP driven by pntP1-GAL4 labels all type II NB lineages (<bold>A</bold>–<bold>A′</bold>) or a single type II NB clone (<bold>C</bold>–<bold>C′</bold>). Ase<sup>−</sup> immature INPs, Ase<sup>+</sup> immature INPs, and mature INPs are indicated by open arrows, solid arrows, and arrowheads, respectively. (<bold>B</bold>–<bold>B′</bold>, <bold>D</bold>–<bold>F′</bold>) Btd RNAi knockdown type II NB lineages (<bold>B</bold>–<bold>B′</bold>) or type II NB clones homozygous mutant for <italic>btd</italic><sup><italic>XG81</italic></sup> (<bold>D</bold>–<bold>E′</bold>) or <italic>btd</italic><sup><italic>XA</italic></sup> (<bold>F</bold>–<bold>F′</bold>) in 3rd instar larval brains produce Ase<sup>−</sup> immature INPs (open arrows) and a few Ase<sup>+</sup> daughter cells (arrows) but no mature INPs. Only 3 out of total 8 type II NB lineages are shown in (<bold>A</bold>–<bold>A′</bold>) and (<bold>B</bold>–<bold>B′</bold>). In this and all other figures, asterisks indicate type II NBs and scale bars equal to 20 µm. Dpn staining alone shows the NB and mature INPs. (<bold>G</bold>–<bold>H</bold>) Quantifications of the number of mature INPs (<bold>G</bold>) and the percentage of type II NB lineages with mature INPs (<bold>H</bold>) in the wild type, Btd RNAi knockdown, and btd mutant type II NB lineages. The numbers on top of each bar are the numbers of type II NB lineages analyzed except for the numbers for the wt and btd RNAi in (<bold>H</bold>), which are the number of brain lobes examined. The mean and stdev for <italic>btd</italic><sup><italic>XG81</italic></sup> and <italic>btd</italic><sup><italic>XA</italic></sup> in (<bold>H</bold>) are calculated by bootstrapping. **p &lt; 0.01, *p &lt; 0.05 (Student <italic>t</italic> test).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03596.003">http://dx.doi.org/10.7554/eLife.03596.003</ext-link></p></caption><graphic xlink:href="elife03596f001"/></fig><fig id="fig1s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03596.004</object-id><label>Figure 1—figure supplement 1.</label><caption><title>Expression of mouse Sp8 (mSp8).</title><p>(<bold>A</bold>–<bold>A′′</bold>) and <italic>Drosophila</italic> Btd (<bold>B</bold>–<bold>B′′</bold>) rescues the loss of mature INPs in <italic>btd</italic> mutant type II NB clones. Multiple mature INPs are observed in <italic>btd</italic> mutant type II NB clone that expresses <italic>UAS-mSp8</italic> (<bold>A</bold>–<bold>A′′</bold>) or <italic>UAS-btd</italic> (<bold>B</bold>–<bold>B′′</bold>). Type II NBs, Ase<sup>−</sup> immature INPs, Ase<sup>+</sup> immature INPs, and mature INPs are indicated by asterisks, open arrows, solid arrows, and arrowheads, respectively.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03596.004">http://dx.doi.org/10.7554/eLife.03596.004</ext-link></p></caption><graphic xlink:href="elife03596fs001"/></fig></fig-group></p><p>To confirm that the loss of INPs indeed results from the knockdown of Btd rather than off-target effects of <italic>UAS-Btd RNAi</italic>, we generated <italic>btd</italic> mutant type II NB clones using two loss-of-function alleles, <italic>btd</italic><sup><italic>XA</italic></sup> and <italic>btd</italic><sup><italic>XG81</italic></sup> (<xref ref-type="bibr" rid="bib46">Wimmer et al., 1993</xref>; <xref ref-type="bibr" rid="bib10">Estella and Mann, 2010</xref>). Consistent with the Btd RNAi knockdown, all <italic>btd</italic><sup><italic>XG81</italic></sup> mutant and 90% of <italic>btd</italic><sup><italic>XA</italic></sup> mutant type II NB clones failed to generate any mature INPs except for 4–6 Ase<sup>+</sup> Dpn<sup>−</sup> cells (<xref ref-type="fig" rid="fig1">Figure 1D–F′,G–H</xref>). Moreover, about 40% of <italic>btd</italic> mutant type II NBs ectopically express Ase, making them appear as type I NB lineages (<xref ref-type="fig" rid="fig1">Figure 1E</xref>). The loss of INPs resulting from the Btd RNAi knockdown and <italic>btd</italic> loss-of-function mutations suggests that Btd is required for the generation of INPs. Remarkably, the loss of INPs in <italic>btd</italic> mutant clones can be similarly rescued by the expression of mouse Sp8 or <italic>Drosophila</italic> Btd (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>), suggesting that mammalian Sp8 could have a conserved role in promoting the generation of transient amplifying INPs.</p><p>Since the loss of mature INPs occurred even when Ase was not ectopically expressed in <italic>btd</italic> mutant type II NBs, the loss of INPs is not primarily due to the ectopic Ase expression or transformation of type II NBs into type I NBs. Therefore, we first focused our phenotypic analyses on lineages without the ectopic Ase expression in the NB. We also used the <italic>btd</italic><sup><italic>XG81</italic></sup> allele for further mutant phenotypic analyses below, given that <italic>btd</italic><sup><italic>XG81</italic></sup> shows slightly stronger phenotypes than <italic>btd</italic><sup><italic>XA</italic></sup>.</p></sec><sec id="s2-2"><title>Ase<sup>−</sup> immature INPs differentiate into Ase<sup>+</sup> immature INPs normally in the absence of Btd</title><p>Why does the loss of Btd lead to the elimination of mature INPs? When mature INPs are eliminated in the absence of Btd, the type II NBs without the ectopic Ase expression still produce Ase<sup>−</sup> immature INPs and a few Ase<sup>+</sup> Dpn<sup>−</sup> daughter cells. In normal type II NB lineages, Ase<sup>+</sup> Dpn<sup>−</sup> cells can be either Ase<sup>+</sup> immature INPs or GMCs. Therefore, three possible scenarios could happen when mature INPs are eliminated in the absence of Btd: 1) Ase<sup>−</sup> immature INPs differentiate into GMCs instead of Ase<sup>+</sup> immature INPs; 2) Ase<sup>−</sup> immature INPs differentiate into Ase<sup>+</sup> immature INPs, which then directly differentiate into neurons/glia without further dividing; 3) Ase<sup>−</sup> immature INPs differentiate into Ase<sup>+</sup> immature INPs, which in turn differentiate into terminally dividing GMCs. To distinguish these possibilities, we first wanted to determine if Ase<sup>−</sup> immature INPs still differentiate into Ase<sup>+</sup> immature INP in the absence of Btd by examining the expression of INP specific marker R9D11<italic>-</italic>CD4<italic>-tdTomato</italic> and progenitor marker Miranda (Mira) in the Ase<sup>+</sup> cells next to the Ase<sup>−</sup> immature INPs<italic>.</italic> R9D11<italic>-</italic>CD4<italic>-tdTomato</italic> utilizes a DNA fragment R9D11 from the <italic>erm</italic> promoter to drive the expression of CD4-tdTomato (<xref ref-type="bibr" rid="bib15">Han et al., 2011</xref>). In normal type II NB lineages, R9D11<italic>-</italic>CD4<italic>-tdTomato</italic> is first turned on in Ase<sup>+</sup> immature INPs and becomes stronger as INPs mature (<xref ref-type="fig" rid="fig2">Figure 2A–A′</xref>), which is similar to R9D11<italic>-mCD8-GFP</italic> (<xref ref-type="bibr" rid="bib53">Zhu et al., 2011</xref>). Mira is expressed in all NBs as well as INPs but not (or very weakly) in GMCs (<xref ref-type="fig" rid="fig2s1">Figure 2—–figure supplement 1</xref>). In Btd RNAi knockdown type II NB lineages without mature INPs, we found that R9D11<italic>-</italic>CD4<italic>-tdTomato</italic> was expressed in Ase<sup>+</sup> daughter cells next to the Ase<sup>−</sup> immature INPs but its overall expression was much weaker than that in normal type II NB lineages (<xref ref-type="fig" rid="fig2">Figure 2B–B′,I</xref>). Consistently, Mira is also expressed in those Ase<sup>+</sup> cells next to the Ase<sup>−</sup> immature INPs in the Btd RNAi knockdown type II NB lineages (<xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1</xref>). The expression of R9D11<italic>-</italic>CD4<italic>-tdTomato</italic> and Mira suggests that Ase<sup>−</sup> immature INPs still differentiate into Ase<sup>+</sup> immature INP in the absence of Btd as in wild-type type II NB lineages (<xref ref-type="fig" rid="fig2">Figure 2J</xref>).<fig-group><fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.03596.005</object-id><label>Figure 2.</label><caption><title>Loss of Btd results in ectopic nuclear Pros in immature INPs and premature differentiation of Ase<sup>+</sup> immature INPs into GMCs.</title><p>(<bold>A</bold>–<bold>A′</bold>) R9D11-CD4-tdTomato is expressed in Ase<sup>+</sup> immature INPs (solid arrows) and mature INPs (arrowheads) but not in Ase<sup>−</sup> immature INPs (open arrows) in a wild-type type II NB lineage. (<bold>B</bold>–<bold>B′</bold>) R9D11-CD4-tdTomato remains expressed in Ase<sup>+</sup> daughter cells (solide arrows) next to the Ase<sup>−</sup> immature INPs (open arrows) when mature INPs are eliminated by Btd RNAi knockdown. (<bold>C</bold>–<bold>C′</bold>) pH3 is not detected in Ase<sup>+</sup> immature INPs (solid arrows) in a wild-type type II NB lineage. (<bold>D</bold>–<bold>E′</bold>) pH3 is expressed in Ase<sup>+</sup> daughter cells (solid arrows) that are the furthest from the Ase<sup>−</sup> immature INPs (open arrows) in a Btd RNAi knockdown type II NB lineage without mature INPs (<bold>D</bold>–<bold>D′</bold>) or <italic>btd</italic> mutant type II NB lineages (<bold>E</bold>–<bold>E′</bold>). (<bold>F</bold>–<bold>F′</bold>) Nuclear Pros is not expressed in Ase<sup>−</sup> immature INPs (open arrows) in a wild-type type II NB lineage. (<bold>G</bold>-<bold>H′</bold>) Nuclear Pros is ectopically expressed in both Ase<sup>−</sup> immature INPs (open arrows) and Ase<sup>+</sup> cells (solid arrows) in Btd RNAi knockdown (<bold>G</bold>–<bold>G′</bold>) or btd mutant (<bold>H</bold>–<bold>H′</bold>) type II NB lineages. (<bold>I</bold>) Quantifications of relative overall expression levels of R9D11-CD4-tdTomato in wild-type (<bold>A</bold>–<bold>A′</bold>) and Btd RNAi knockdown (<bold>B</bold>–<bold>B′</bold>) type II NB lineages. (<bold>J</bold>) A diagram of neurogenesis patterns in type II NB lineages in the presence or absence of Btd.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03596.005">http://dx.doi.org/10.7554/eLife.03596.005</ext-link></p></caption><graphic xlink:href="elife03596f002"/></fig><fig id="fig2s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03596.006</object-id><label>Figure 2—figure supplement 1.</label><caption><title>Mira expression in Btd RNAi knockdown type II NB clone.</title><p>(<bold>A</bold>–<bold>A′</bold>) Mira is expressed in INPs (yellow arrows) in a wild-type type II NB lineage and forms a basal crescent (white arrows) at metaphase. (<bold>B</bold>–<bold>B′</bold>) In a Btd RNAi knockdown type II NB lineage without mature INPs, Mira is expressed in Ase<sup>+</sup> daughter cells (yellow arrows) next to the Ase<sup>−</sup> immature INP (open arrows) but does not form a basal crescent at metaphase in the dividing Ase<sup>+</sup> cell (white arrows).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03596.006">http://dx.doi.org/10.7554/eLife.03596.006</ext-link></p></caption><graphic xlink:href="elife03596fs002"/></fig></fig-group></p></sec><sec id="s2-3"><title>Loss of Btd leads to ectopic expression of Pros in immature INPs and premature differentiation of Ase<sup>+</sup> immature INPs into GMCs</title><p>Next we asked if Ase<sup>+</sup> immature INPs differentiate into neurons/glia directly or GMCs in the absence of Btd. GMCs express both Ase and nuclear Pros and divide terminally but do not form a Mira crescent while dividing. If Ase<sup>+</sup> immature INPs directly differentiate into neurons/glia, then all the Ase<sup>+</sup> daughter cells should be Ase<sup>+</sup> immature INPs and none of them should be dividing. In contrast, if Ase<sup>+</sup> immature INPs differentiate into GMCs, then some Ase<sup>+</sup> daughter cells will become mitotically active and express nuclear Pros but will not form a Mira crescent at the metaphase or telophase. Immunostaining with the mitotic marker phospho-histone 3 (pH3) showed that unlike Ase<sup>+</sup> immature INPs, which never become pH3-positive (<xref ref-type="fig" rid="fig2">Figure 2C–C′</xref>), some Ase<sup>+</sup> daughter cells generated in both Btd RNAi knockdown type II NB lineages without mature INPs (<xref ref-type="fig" rid="fig2">Figure 2D–D′</xref>) and <italic>btd</italic> mutant type II NB lineages became mitotically active (<xref ref-type="fig" rid="fig2">Figure 2E–E′</xref>). However, the pH3 positive cells were always the furthest from the Ase<sup>−</sup> immature INPs among the Ase<sup>+</sup> daughter cells (<xref ref-type="fig" rid="fig2">Figure 2D–D′</xref>), suggesting that the Ase<sup>+</sup> daughter cells divide terminally like GMCs. Consistently, unlike in mature INPs, which form a Mira crescent at metaphase (<xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1</xref>), we did not observe any Mira crescents in the Ase<sup>+</sup> daughter cells at metaphase (<xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1</xref>). The terminal division and the lack of the Mira crescent strongly argue that Ase<sup>+</sup> immature INPs differentiate into GMCs in the absence of Btd.</p><p>To further confirm that late immature INPs differentiate into GMCs in the absence of Btd, we then examined the expression of nuclear Pros in the Ase<sup>+</sup> daughter cells. Nuclear Pros is a cell fate determinant of GMCs. In normal type II NBs lineages, Pros is expressed in the cytoplasm of Ase<sup>+</sup> immature INPs and mature INPs and in the nucleus of GMCs and post-mitotic neurons, but not in type II NBs or Ase<sup>−</sup> immature INPs (<xref ref-type="fig" rid="fig2">Figure 2F–F′</xref>). If Ase<sup>+</sup> immature INPs differentiate into GMCs in the absence of Btd, we expected that some of the Ase<sup>+</sup> daughter cells express nuclear Pros. Interestingly, immunostaining of Pros showed that nuclear Pros was expressed not only in all Ase<sup>+</sup> daughter cells but also in Ase<sup>−</sup> immature INPs generated in Btd RNAi knockdown or <italic>btd</italic> mutant type II NB lineages (<xref ref-type="fig" rid="fig2">Figure 2G–H′</xref>). Given that Pros promotes cell cycle exit and GMC differentiation and that forced expression of Pros is sufficient to eliminate INPs in type II NB lineages (<xref ref-type="bibr" rid="bib26">Li and Vaessin, 2000</xref>; <xref ref-type="bibr" rid="bib7">Choksi et al., 2006</xref>; <xref ref-type="bibr" rid="bib1">Bayraktar et al., 2010</xref>), the ectopic expression of nuclear Pros in immature INPs resulting from the loss of Btd very likely promotes the premature differentiation of Ase<sup>+</sup> immature INPs into GMCs and cell cycle exit, leading to the loss of mature INPs (<xref ref-type="fig" rid="fig2">Figure 2J</xref>). These results also reveal that it is Btd that is responsible for the suppression of Pros in immature INPs.</p></sec><sec id="s2-4"><title>Reducing Pros expression rescues the elimination of mature INPs resulting from the loss of Btd</title><p>To determine if the ectopic expression of nuclear Pros in immature INPs is indeed responsible for the elimination of mature INPs in the absence of Btd, we next examined if reducing Pros expression was able to rescue the elimination of INPs in Btd RNAi knockdown or <italic>btd</italic> mutant type II NB lineages. To reduce Pros expression, we either removed one wild-type copy of <italic>pros</italic> or knocked down Pros by RNAi in type II NB lineages. Remarkably, the elimination of mature INPs resulting from the Btd RNAi knockdown was nearly fully rescued even just by removing one wild-type copy of <italic>pros</italic> (<xref ref-type="fig" rid="fig3">Figure 3A–D′,I–J</xref>). Unlike Btd RNAi knockdown in wild-type background, which resulted in a completely elimination of mature INPs in about 50% of type II NB lineages (<xref ref-type="fig" rid="fig1">Figure 1B–B′,G–H</xref>, <xref ref-type="fig" rid="fig3">Figure 3B–B′,I–J</xref>), knockdown of Btd in <italic>pros</italic><sup><italic>17</italic></sup> or <italic>pros</italic><sup><italic>10419</italic></sup> heterozygous mutant animals no longer led to an obvious loss of mature INPs (<xref ref-type="fig" rid="fig3">Figure 3D–D′,I–J</xref>, and data not shown), although type II NB lineages develop normally in <italic>pros</italic><sup><italic>17</italic></sup> or <italic>pros</italic><sup><italic>10419</italic></sup> heterozygous mutant animals (<xref ref-type="fig" rid="fig3">Figure 3C–C′,I–J</xref>, and data not shown). Similarly, the loss of INPs in <italic>btd</italic> mutant clones was also largely rescued when <italic>btd</italic> mutant type II NB clones were generated in <italic>pros</italic><sup><italic>17</italic></sup> heterozygous mutant background (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1</xref>).<fig-group><fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.03596.007</object-id><label>Figure 3.</label><caption><title>Reducing Pros rescues the elimination of INPs resulting from the loss of Btd.</title><p>(<bold>A</bold>–<bold>D′</bold>) The loss of INPs resulting from Btd RNAi knockdown is rescued in <italic>pros</italic><sup><italic>17</italic></sup> heterozygous mutant background. Only two lineages are shown in each brain. (<bold>A</bold>–<bold>A′</bold>) Wild-type type II NB lineages have multiple mature INPs. (<bold>B</bold>–<bold>B′</bold>) Btd RNAi knockdown causes a loss of mature INPs. (<bold>C</bold>–<bold>C′</bold>) Type II NB lineages in <italic>pros</italic><sup><italic>17</italic></sup> heterozygous mutant larvae produce a similar number of mature INPs as in wild-type larvae. (<bold>D</bold>–<bold>D′</bold>) Btd RNAi knockdown no long leads to the loss of mature INPs in <italic>pros</italic><sup><italic>17</italic></sup> heterozygous mutant type II NB lineages. (<bold>E</bold>–<bold>H′</bold>) Pros RNAi knockdown rescues the loss of INPs in btd mutant type II NB clones. (<bold>E</bold>–<bold>E′</bold>) A wild-type type II NB clone has multiple mature INPs. (<bold>F</bold>–<bold>F′</bold>) A btd mutant type II NB clone contains no mature INPs. (<bold>G</bold>–<bold>G′</bold>) Pros RNAi knockdown causes overproliferation of mature INPs in a type II NB clone. (<bold>H</bold>–<bold>H′</bold>) Pros RNAi knockdown rescues the loss of mature INPs in a <italic>btd</italic> mutant type II clone. Arrowheads point to mature INPs in all images. (<bold>I</bold>–<bold>L</bold>) Quantifications of the number of mature INPs (<bold>I</bold>–<bold>K</bold>) and the percentage of lineages with mature INPs (<bold>J</bold>–<bold>L</bold>) for the rescue of Btd RNAi knockdown phenotypes in <italic>pros</italic><sup><italic>17</italic></sup>/+ larvae (<bold>I</bold>–<bold>J</bold>) or the rescue of <italic>btd</italic> mutant phenotypes by Pros RNAi knockdown (<bold>K</bold>–<bold>L</bold>). The samples sizes on top of each bar represent the number of type II NB lineages (<bold>I</bold>, <bold>K</bold>, <bold>L</bold>) or the number of brain lobes (<bold>J</bold>). The mean and stdev in (<bold>L</bold>) are calculated by bootstrapping. **p &lt; 0.01, *p &lt; 0.05 (Student <italic>t</italic> test). NS: not significant.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03596.007">http://dx.doi.org/10.7554/eLife.03596.007</ext-link></p></caption><graphic xlink:href="elife03596f003"/></fig><fig id="fig3s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03596.008</object-id><label>Figure 3—figure supplement 1.</label><caption><title>Reducing Pros rescues the elimination of INPs resulting from the loss of Btd.</title><p>(<bold>A</bold>–<bold>D′</bold>) The loss of INPs resulting from Btd RNAi knockdown is rescued by Pros RNAi knockdown. (<bold>A</bold>–<bold>A′</bold>) Wide-type type II lineages labeled with mCD8-GFP driven by <italic>insc-GAL4</italic> have multiple mature INPs. (<bold>B</bold>–<bold>B′</bold>) Btd RNAi knockdown driven by <italic>insc-GAL4</italic> eliminates mature INPs in a subset of type II NB lineages. (<bold>C</bold>–<bold>C′</bold>) RNAi knockdown of Pros results in overproliferation of mature INPs. (<bold>D</bold>–<bold>D′</bold>) Simultaneous knockdown of Btd and Pros leads to a similar overproliferation of mature INPs in type II NB lineages as Pros RNAi knockdown alone. (<bold>E</bold>–<bold>H′</bold>) The loss of mature INPs in <italic>btd</italic> mutant clones is rescued in <italic>pros</italic><sup><italic>17</italic></sup> heterozygous mutant larvae. (<bold>E</bold>–<bold>E′</bold>) A wild-type type II NB clone has multiple mature INPs. (<bold>F</bold>–<bold>F′</bold>) A <italic>btd</italic> mutant type II NB clone contains no mature INPs. (<bold>G</bold>–<bold>G′</bold>) <italic>pros</italic><sup><italic>17</italic></sup> heterozygous mutant type II NB clone contains multiple mature INPs as the wild-type type II NB clone (<bold>A</bold>–<bold>A′</bold>). (<bold>H</bold>–<bold>H′</bold>) A <italic>btd</italic> mutant type II NB clone generated in <italic>pros</italic><sup><italic>17</italic></sup> heterozygous mutant larvae has multiple mature INPs. Asterisks indicate NBs and arrowheads point to mature INPs in all images. (<bold>I</bold>–<bold>L</bold>) Quantifications of the number of mature INPs (<bold>I</bold>–<bold>K</bold>) or the percentage of type II NB lineages with mature INPs (<bold>J</bold>–<bold>L</bold>) for the rescue of Btd RNAi knockdown phenotypes by Pros RNAi knockdown (<bold>I</bold>–<bold>J</bold>) or the rescue of <italic>btd</italic> mutant phenotypes in <italic>pros</italic><sup><italic>17</italic></sup> heterozygous mutant larvae (<bold>K</bold>–<bold>L</bold>). The sample size on top of each bar represents the number of lineages (<bold>I</bold>, <bold>K</bold>, <bold>L</bold>) or the number of brain lobes (<bold>J</bold>). The mean and stdev in (<bold>L</bold>) are calculated by bootstrapping. **, p &lt; 0.01; NS: not significant.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03596.008">http://dx.doi.org/10.7554/eLife.03596.008</ext-link></p></caption><graphic xlink:href="elife03596fs003"/></fig></fig-group></p><p>Consistent with the rescue in <italic>pros</italic> heterozygous mutant animals, Pros RNAi knockdown also rescued the loss of INPs resulting from the loss of Btd (<xref ref-type="fig" rid="fig3">Figure 3E–H′, K–L</xref>, <xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1</xref>). Pros RNAi knockdown led to overproliferation of mature INPs as observed in <italic>pros</italic> mutant type II NB clones (<xref ref-type="fig" rid="fig3">Figure 3G–G′,K</xref>, <xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1</xref>) (<xref ref-type="bibr" rid="bib5">Bowman et al., 2008</xref>). When Pros was knocked down in <italic>btd</italic> mutant type II NB clones, mature INPs were rescued in all <italic>btd</italic> mutant clones (<xref ref-type="fig" rid="fig3">Figure 3H–H′,L</xref>). In about 70% of <italic>btd</italic> mutant type II NB clones, Pros RNAi knockdown led to a similar mature INP overproliferation as in wild-type clones. In other 30% of <italic>btd</italic> mutant clones, Pros RNAi knockdown partially or fully rescued mature INPs without causing the overproliferation of mature INPs. Similarly, Pros RNAi knockdown also rescued the loss of mature INPs resulting from Btd RNAi knockdown (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1</xref>). Results from these rescue experiments demonstrate that the ectopic nuclear Pros in immature INPs is indeed responsible for the loss of mature INPs.</p><p>Interestingly, removing one wild-type copy of <italic>pros</italic> or knocking down Pros not only rescued the loss of mature INPs but also suppressed the ectopic Ase expression in all <italic>btd</italic> mutant type II NBs (<xref ref-type="fig" rid="fig3">Figure 3H</xref>, <xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1</xref>), suggesting that the ectopic Ase expression in <italic>btd</italic> mutant type II NBs also results from the ectopic expression of nuclear Pros in immature INPs.</p></sec><sec id="s2-5"><title>Btd likely functions only in newly born immature INPs</title><p>Our results showed that Btd is required to suppress Pros in Ase<sup>−</sup> immature INPs. We next asked if Btd is also required to partially suppress Pros at later stages of INP development. In normal type II NB lineages, Pros is absent in Ase<sup>−</sup> immature INPs but is expressed at low levels in the cytoplasm of Ase<sup>+</sup> immature INPs and mature INPs (<xref ref-type="bibr" rid="bib3">Bello et al., 2008</xref>; <xref ref-type="bibr" rid="bib4">Boone and Doe, 2008</xref>; <xref ref-type="bibr" rid="bib5">Bowman et al., 2008</xref>). Maintaining the expression of Pros at low levels is essential for the self-renewal of INPs (<xref ref-type="bibr" rid="bib1">Bayraktar et al., 2010</xref>). However, the complete elimination of mature INPs makes it difficult to assess the role of Btd in mature INPs. Therefore, we used <italic>erm-GAL4 (III)</italic> and <italic>erm-GAL4 (II)</italic> to knock down Btd. Both <italic>erm-GAL4 (III)</italic> and <italic>erm-GAL4 (II)</italic> are expressed in Ase<sup>+</sup> immature INPs and mature INPs, whereas <italic>erm-GAL4 (II)</italic> is also expressed in Ase<sup>−</sup> immature INPs except for the newly born Ase<sup>−</sup> immature INPs (<xref ref-type="bibr" rid="bib47">Xiao et al., 2012</xref>). However, knockdown of Btd using either <italic>erm-GAL4 (III)</italic> (<xref ref-type="fig" rid="fig4">Figure 4A–B′′,E</xref>) or <italic>erm-GAL4 (II)</italic> (<xref ref-type="fig" rid="fig4">Figure 4C–D′′,F</xref>) did not result in any obvious loss of mature INPs in type II NB lineages. In line with these RNAi knockdown results, we were able to recover multicellular <italic>btd</italic> mutant INP clones that were comparable to wild-type INP clones (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1</xref>) while we generated <italic>btd</italic> mutant type II NB clones, indicating that <italic>btd</italic> mutant INPs were still able to divide multiple rounds like wild-type INPs and did not prematurely differentiate into GMCs. These data suggest that Btd likely suppresses Pros expression only in newly born Ase<sup>−</sup> immature INPs but not in immature INPs at later developmental stages or mature INPs.<fig-group><fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.03596.009</object-id><label>Figure 4.</label><caption><title>Knockdown of Btd in immature or mature INPs by <italic>erm-GAL4</italic> lines does not lead to the loss of mature INPs.</title><p>(<bold>A</bold>–<bold>A′′</bold>, <bold>C</bold>–<bold>C′′</bold>) Wild-type type II NB lineages are labeled with mCD8-GFP driven by <italic>erm-GAL4</italic> (III) (<bold>A</bold>–<bold>A′′</bold>) or <italic>erm-GAL4</italic> (II) (<bold>C</bold>–<bold>C′′</bold>). (<bold>B</bold>–<bold>B′′</bold>, <bold>D</bold>–<bold>D′′</bold>) Knockdown of Btd in Ase<sup>+</sup> immature INPs and mature INPs by <italic>erm-GAL4</italic> (III) (<bold>B</bold>–<bold>B′′</bold>) or in Ase<sup>−</sup> immature INPs as well as Ase<sup>+</sup> immature INP and mature INPs by <italic>erm-GAL4</italic> (II) (<bold>D</bold>–<bold>D′′</bold>) does not cause a reduction of the number of mature INPs. Only two lineages are shown in each brain. (<bold>E</bold>–<bold>F</bold>) Quantifications of the number of mature INPs in type II NB lineages in which Btd is knocked down by <italic>erm-GAL4</italic> (III) (<bold>E</bold>) or <italic>erm-GAL4</italic> (II) (<bold>F</bold>). NS: not significant.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03596.009">http://dx.doi.org/10.7554/eLife.03596.009</ext-link></p></caption><graphic xlink:href="elife03596f004"/></fig><fig id="fig4s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03596.010</object-id><label>Figure 4—figure supplement 1.</label><caption><title>Btd likely does not function in mature INPs.</title><p>(<bold>A</bold>) A wild-type INP clone with 4 post-mitotic cells. (<bold>B</bold>) A <italic>btd</italic> mutant INP clone with 6 post-mitotic cells. INP clones labeled with mCD8-GFP are outlined by dashed circles. INP clones are identified as clones that have more than two cells but no NBs.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03596.010">http://dx.doi.org/10.7554/eLife.03596.010</ext-link></p></caption><graphic xlink:href="elife03596fs004"/></fig></fig-group></p></sec><sec id="s2-6"><title>Reduction of PntP1 expression is responsible for the ectopic Ase expression but not the loss of mature INPs in btd mutant type II NB lineages</title><p>Our <italic>btd</italic> mutant MARCM analyses showed that in addition to the loss of INPs, Ase was ectopically expressed in about 40% of <italic>btd</italic> mutant type II NBs. We showed previously that PntP1 is expressed in type II NBs as well as Ase<sup>−</sup> and Ase<sup>+</sup> immature INPs (<xref ref-type="fig" rid="fig5">Figure 5A–A′′</xref>) (<xref ref-type="bibr" rid="bib53">Zhu et al., 2011</xref>). Inhibiting PntP1 activity results in ectopic Ase expression in type II NBs and elimination of INPs (<xref ref-type="bibr" rid="bib53">Zhu et al., 2011</xref>). Therefore, we wondered if PntP1 expression was reduced or even lost in <italic>btd</italic> mutant type II NBs. Immunostaining of PntP1 showed that PntP1 was expressed at reduced levels in most <italic>btd</italic> mutant type II NB lineages without the ectopic Ase expression (<xref ref-type="fig" rid="fig5">Figure 5B–B′</xref>). The reduction is about 10% in the NBs and 50% in the immature INPs (<xref ref-type="fig" rid="fig5">Figure 5E–F</xref>). In those <italic>btd</italic> mutant clones, PntP1 was also detected in the Ase<sup>+</sup> daughter cells next to the Ase<sup>−</sup> immature INPs (<xref ref-type="fig" rid="fig5">Figure 5B–B′</xref>), providing additional evidence to support that Ase<sup>−</sup> immature INPs still differentiate into Ase<sup>+</sup> immature INPs in the absence of Btd. However, in <italic>btd</italic> mutant type II NB lineages with the ectopic Ase expression in the NB, PntP1 was largely abolished in both the NBs and their progeny (<xref ref-type="fig" rid="fig5">Figure 5C–C′,E–F</xref>). The correlation of the ectopic Ase expression in the NB and the severe reduction or loss of PntP1 suggesting that the ectopic Ase expression in <italic>btd</italic> mutant type II NBs could result from the severe reduction or loss of PntP1 expression.<fig-group><fig id="fig5" position="float"><object-id pub-id-type="doi">10.7554/eLife.03596.011</object-id><label>Figure 5.</label><caption><title>PntP1 expression is reduced in btd mutant type II NB clones.</title><p>(<bold>A</bold>–<bold>A′′</bold>) PntP1 is expressed in the NB (*), Ase<sup>−</sup> immature INPs (open arrows), as well as Ase<sup>+</sup> immature INPs (solid arrows) in a wild-type type II NB clone. (<bold>B</bold>–<bold>B′′</bold>) PntP1 expression is much lower in a <italic>btd</italic> mutant type II NB clone without the ectopic Ase expression in the NB than that in a neighboring <italic>btd</italic> heterozygous type II NB lineage. The reduction is particularly obvious in the Ase<sup>−</sup> immature INPs (open arrows). Note that PntP1 remains expressed in Ase<sup>+</sup> daughter cells (arrows) next to the Ase<sup>−</sup> immature INPs. (<bold>C</bold>–<bold>C′′</bold>) PntP1 expression is largely abolished in a <italic>btd</italic> mutant type II NB clone with the ectopic Ase expression in the NB. In a neighboring <italic>btd</italic> heterozygous type II NB lineages, PntP1 is still detected in the NBs (*), Ase<sup>-</sup> immature INPs (open arrows) and Ase<sup>+</sup> immature INPs (arrows). (<bold>D</bold>–<bold>D′′</bold>) Knocking down Pros restores the expression of PntP1 in the NB (*), Ase<sup>−</sup> immature INPs (open arrows), and Ase<sup>+</sup> immature INPs (arrows) in a <italic>btd</italic> mutant clone to levels comparable to those in a neighboring btd heterozygous type II NB lineage. Wild-type (<bold>A</bold>–<bold>A′′</bold>) or <italic>btd</italic> mutant type II NB clones (<bold>B</bold>–<bold>D′′</bold>) are outlined by dashed lines and neighboring <italic>btd</italic> heterozygous type II NB lineages (<bold>B</bold>–<bold>D′′</bold>) are marked with dotted lines. (<bold>E</bold>–<bold>F</bold>) Quantifications of PntP1 expression levels in type II NBs (<bold>E</bold>) and Ase<sup>−</sup> immature INPs (<bold>F</bold>) in <italic>btd</italic> mutant type II NB clones relative to neighboring type II NB lineages in the same brains. **p &lt; 0.01; NS: not significant.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03596.011">http://dx.doi.org/10.7554/eLife.03596.011</ext-link></p></caption><graphic xlink:href="elife03596f005"/></fig><fig id="fig5s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03596.012</object-id><label>Figure 5—figure supplement 1.</label><caption><title>The reduction of PntP1 is unlikely responsible for the loss of INPs in btd mutant type II NB clones.</title><p>(<bold>A</bold>–<bold>A′′</bold>) A <italic>btd</italic> mutant type II NB clone has similar expression levels of PntP1 as a neighboring btd heterozygous mutant type II NB lineage but fails to produce mature INPs. (<bold>B</bold>–<bold>B′′</bold>) Expressing <italic>UAS-PntP1</italic> does not rescue the loss of mature INPs in a <italic>btd</italic> mutant clone, even though the PntP1 expression in the <italic>btd</italic> mutant clone is much higher than that in a neighboring <italic>btd</italic> heterozygous mutant type II NB lineage. (<bold>C</bold>–<bold>C′′</bold>) Nuclear Pros remains ectopically expressed in Ase<sup>−</sup> immature INPs and Ase<sup>+</sup> daughter cells in a btd mutant type II NB clone expressing <italic>UAS-PntP1</italic>. (<bold>D</bold>–<bold>D′′</bold>) Mature INPs are partially rescued by <italic>UAS-PntP1</italic> in a <italic>btd</italic> mutant type II NB clone. In all images, <italic>btd</italic> mutant clones are outlined by dashed lines and their neighboring <italic>btd</italic> heterozygous mutant type II NB lineages are marked by dotted lines. Open arrows: Ase<sup>−</sup> immature INPs; solid arrows: Ase<sup>+</sup> immature INPs; arrowheads: mature INPs.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03596.012">http://dx.doi.org/10.7554/eLife.03596.012</ext-link></p></caption><graphic xlink:href="elife03596fs005"/></fig></fig-group></p><p>To determine if the reduction/loss of PntP1 is responsible for the ectopic Ase expression and/or the loss of INPs in <italic>btd</italic> mutant type II NB lineages, we examined if restoring PntP1 expression was sufficient to suppress ectopic Ase expression and/or rescue the loss of INPs resulting from the loss of Btd by expressing <italic>UAS-pntP1</italic> in <italic>btd</italic> mutant type II NB clones. Our results showed that expressing <italic>UAS-pntP1</italic> resulted in higher expression of PntP1 in <italic>btd</italic> mutant type II NB clones than that in neighboring <italic>btd</italic> heterozygous mutant type II NB lineages and suppressed the ectopic Ase expression in all <italic>btd</italic> mutant type II NBs (n = 12) (<xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1</xref>). However, unlike reducing Pros expression, which rescued mature INPs in nearly all <italic>btd</italic> mutant type II NB clones (<xref ref-type="fig" rid="fig3">Figure 3</xref>, <xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1</xref>), expressing <italic>UAS-pntP1</italic> failed to rescue mature INPs or suppress the ectopic nuclear Pros in Ase<sup>−</sup> immature INPs or Ase<sup>+</sup> daughter cells in the majority of <italic>btd</italic> mutant clones (<xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1</xref>). Only in 3 out of total 10 <italic>btd</italic> mutant clones expressing <italic>UAS-PntP1</italic>, we observed that mature INPs were partially rescued to 9.3 ± 3.2 per lineages (<xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1</xref>), which is still much fewer than the number of mature INPs (20–30 per lineages) in normal type II NB lineages. Consistent with the inability of <italic>UAS-pntP1</italic> to fully rescue the loss of mature INPs, we found occasionally that <italic>btd</italic> mutant clones that did not show an obvious reduction of PntP1 in either the NBs or early immature INPs still failed to generate any mature INPs (<xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1</xref>). Therefore, these results demonstrate that the severe reduction/loss of PntP1 accounts for the ectopic Ase expression in <italic>btd</italic> mutant type II NBs but is not the primary reason for the loss of mature INPs.</p><p>Given that reducing the expression of Pros suppressed the ectopic Ase expression in <italic>btd</italic> mutant type II NBs, we then asked if reducing Pros expression could also rescue the reduction/loss of PntP1 in <italic>btd</italic> mutant type II NB clones. Indeed, consistent with the suppression of the ectopic Ase expression by Pros RNAi knockdown, PntP1 expression in both the NBs and immature INPs returned to normal levels when the loss of INPs was rescued by Pros RNAi knockdown in <italic>btd</italic> mutant type II NB clones (<xref ref-type="fig" rid="fig5">Figure 5D–F</xref>). These results suggest that the reduction/loss of PntP1 in <italic>btd</italic> mutant type II NB clones is due to the ectopic Pros expression in immature INPs. However, given that ectopic nuclear Pros is only observed in immature INPs but not in the NBs in <italic>btd</italic> mutant type II NB clones, the reduction/loss of PntP1 and the subsequent ectopic Ase expression in the NB is most likely a secondary effect of the ectopic nuclear Pros expression in immature INPs.</p></sec><sec id="s2-7"><title>Btd is expressed in type II NB lineages and a subset of type I NB lineages</title><p>Our Btd loss of function analyses demonstrated that Btd is critical for the generation of INPs in type II NBs lineages. We next examined if Btd is only expressed type II NB lineages. Since Btd antibodies are not available and our in situ hybridization signals of btd mRNAs in the central brain were barely detectable (data now shown), we used the <italic>btd-GAL4</italic> as a reporter for <italic>btd</italic> expression. <italic>btd-GAL4</italic> is a <italic>GAL4</italic> enhancer trap line, in which the <italic>GAL4</italic> transgene is inserted at 753bp upstream of the transcription start site of <italic>btd</italic> (<xref ref-type="bibr" rid="bib11">Estella et al., 2003</xref>). <italic>btd-GAL4</italic> shows similar expression patterns as endogenous Btd in ventral imaginal discs (<xref ref-type="bibr" rid="bib11">Estella et al., 2003</xref>). We found that mCD8-GFP driven by the <italic>btd-GAL4</italic> is expressed in all type II NB lineages but not type I NB lineages on the dorsal side of larval brains (<xref ref-type="fig" rid="fig6">Figure 6A–A′</xref>). In type II NB lineages, the expression of mCD8-GFP driven by <italic>btd-GAL4</italic> is detected in the NB but becomes much stronger in immature INPs next to the NBs (<xref ref-type="fig" rid="fig6">Figure 6A</xref>). The expression of mCD8-GFP then becomes progressively weak in cells away from the NBs and is barely detectable in some mature INPs distal from the NB (<xref ref-type="fig" rid="fig6">Figure 6A–A′</xref>). The expression pattern of <italic>btd-GAL4</italic> in type II NB lineages is similar to that of <italic>pntP1-GAL4</italic> (e.g. <xref ref-type="fig" rid="fig1">Figure 1A–A′</xref>) and is consistent with our results that Btd mainly functions in immature INPs.<fig-group><fig id="fig6" position="float"><object-id pub-id-type="doi">10.7554/eLife.03596.013</object-id><label>Figure 6.</label><caption><title>Btd is expressed in type II NB lineages and a subset of type I NB lineages.</title><p>(<bold>A</bold>–<bold>A′</bold>) mCD8-GFP driven by <italic>btd-Gal4</italic> is expressed in all type II NB lineages (outlined by dashed lines) but not type I NB lineages (e.g. arrows) on the dorsal side of a 3rd instar larval brain. The expression of mCD8-GFP becomes progressively weak in cell away from the NB. Some mature INPs (e.g. arrowheads) distant from the NB have no obvious expression of mCD8-GFP. Only seven out of total eight type II NB lineages are shown in this particular focal plane. (<bold>B</bold>–<bold>B′</bold>) Two type I NB lineages are labeled by mCD8-GFP driven by <italic>btd-GAL4</italic> on the ventral side of a 3rd instar larval brain. (<bold>C</bold>–<bold>C′</bold>) mCD8-GFP driven by <italic>btd-Gal4</italic> labels a subset of type I NB lineages (e.g. arrows) in the ventral nerve cord (VNC).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03596.013">http://dx.doi.org/10.7554/eLife.03596.013</ext-link></p></caption><graphic xlink:href="elife03596f006"/></fig><fig id="fig6s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03596.014</object-id><label>Figure 6—figure supplement 1.</label><caption><title>The GAL4 insertion in the btd-GAL4 line does not affect type II NB lineage development.</title><p>(<bold>A</bold>–<bold>A′′</bold>) A wild-type type II NB clone contains multiple mature INP. (<bold>B</bold>–<bold>B′′</bold>) A <italic>btd-GAL4</italic> mutant type II NB clone has a similar number of mature INPs as wild-type type II NB clone. (<bold>C</bold>) Quantifications of the number of mature INPs in wild-type and <italic>btd-GAL4</italic> mutant type II NB clones. Arrowheads: mature INPs; NS: not significant.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03596.014">http://dx.doi.org/10.7554/eLife.03596.014</ext-link></p></caption><graphic xlink:href="elife03596fs006"/></fig><fig id="fig6s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03596.015</object-id><label>Figure 6—figure supplement 2.</label><caption><title>The loss of INP phenotype resulting from Btd RNAi knockdown is rescued by the expression of mouse Sp8 (mSp8) or Drosophila Btd.</title><p>(<bold>A</bold>–<bold>A′′</bold>) Btd RNAi knockdown driven by <italic>btd-GAL4</italic> completely eliminates mature INPs in all type II NB lineages in a 3rd instar larval brain. (<bold>B</bold>–<bold>B′′</bold>) The expression of <italic>UAS-mSp8</italic> driven by <italic>btd-GAL4</italic> fully rescues the loss of mature INPs in all type II NB lineages resulting from Btd RNAi knockdown. (<bold>C</bold>–<bold>C′′</bold>) The expression of <italic>UAS-btd</italic> driven by <italic>btd-GAL4</italic> partially rescues the loss of mature INPs in type II NB lineages resulting from Btd RNAi knockdown. Arrowheads point to mature INPs in all images. (<bold>D</bold>–<bold>E</bold>) Quantifications of the percentage of type II NB lineages with mature INPs (<bold>D</bold>) and the number of mature INPs (<bold>E</bold>) in 3rd instar larvae with indicated genotypes. Sample sizes represent the number of brain lobes (<bold>D</bold>) or the number of type II NB lineages (<bold>E</bold>). *, p &lt; 0.05; **, p &lt; 0.01; NS: not significant.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03596.015">http://dx.doi.org/10.7554/eLife.03596.015</ext-link></p></caption><graphic xlink:href="elife03596fs007"/></fig></fig-group></p><p>In addition to type II NB lineages, mCD8-GFP driven by <italic>btd-GAL4</italic> is also expressed in two type I NB lineages on the ventral side of larval brains as well as about 31 type I NB lineages in the ventral nerve cord (VNC) (<xref ref-type="fig" rid="fig6">Figure 6B–C′</xref>). In those type I NB lineages, mCD8-GFP driven by <italic>btd-GAL4</italic> is expressed in the NBs, GMCs, and newly born neurons. The expression pattern of <italic>btd-GAL4</italic> in single type I NB lineages is similar to those of other NB GAL4 lines such as <italic>insc-GAL4</italic> (e.g. <xref ref-type="fig" rid="fig7">Figure 7D</xref>), suggesting that Btd likely functions in the NB in these type I NB lineages.<fig-group><fig id="fig7" position="float"><object-id pub-id-type="doi">10.7554/eLife.03596.016</object-id><label>Figure 7.</label><caption><title>Btd and PntP1 function cooperatively to promote the generation of INPs.</title><p>(<bold>A</bold>–<bold>C′</bold>) Ectopic expression of PntP1 consistently promotes the generation of INP-like cells in Btd-positive type I NB lineages in larval brains. (<bold>A</bold>–<bold>A′</bold>) A wild-type type I NB lineage labeled by <italic>btd-GAL4</italic> has no INP-like cells. (<bold>B</bold>–<bold>C′</bold>) The ectopic expression of PntP1 driven by <italic>btd-GAL4</italic> suppresses Ase in the NB (*) and induces the generation of Ase<sup>+</sup> Dpn<sup>+</sup> INP-like cells (arrowheads) (<bold>B</bold>–<bold>B′</bold>), which also express INP-specific marker R9D11-CD4-tdTomato (<bold>C</bold>–<bold>C′</bold>). (<bold>D</bold>–<bold>E′</bold>) The expression of PntP1 driven by <italic>tub-GAL4</italic> suppresses Ase in both wild-type (<bold>D</bold>) and <italic>btd</italic> mutant (<bold>E</bold>) type I NBs (*) but only induced the generation of INP-like cells in the wild-type type I NB clone (<bold>D</bold>–<bold>D′</bold>) but not in the <italic>btd</italic> mutant clone (<bold>E</bold>–<bold>E′</bold>). (<bold>F</bold>–<bold>I′</bold>) <italic>insc-GAL4</italic> drives the expression of <italic>UAS-mCD8-GFP</italic> alone (<bold>F</bold>–<bold>F′</bold>) or together with <italic>UAS-btd</italic> (<bold>G</bold>–<bold>G′</bold>), <italic>UAS-pntP1</italic> (<bold>H</bold>–<bold>H′</bold>), or <italic>UAS-btd</italic> plus <italic>UAS-pntP1</italic> (<bold>I</bold>–<bold>I′</bold>) in type I NB lineages. Images are from the ventral side of larval brains, where only type I NB lineages are observed in wild-type animals (<bold>F</bold>–<bold>F′</bold>). The expression of <italic>UAS-btd</italic> (<bold>G</bold>–<bold>G′</bold>) or <italic>UAS-pntP1</italic> (<bold>H</bold>–<bold>H′</bold>) alone promotes the generation of INP-like cells only in small subset of type I NB lineages (dashed circles). The expression of Btd only suppresses/reduces Ase expression in type I NBs (arrows) that produce INP-like cells (<bold>G</bold>–<bold>G′</bold>) but not in other type I NBs (arrowheads), where PntP1 suppresses Ase in nearly all type I NBs (e.g. arrows) regardless of the generation of INP-like cells (<bold>H</bold>–<bold>H′</bold>). Co-expression of Btd and PntP1 promotes the generation of INP-like cells nearly in all type I NB lineages (dashed circles) (<bold>I</bold>–<bold>I′</bold>). (<bold>J</bold>–<bold>J′</bold>) Quantifications of the percentage of type I NB lineages that produce INP-like cells in larvae with indicated genotypes. The number on top of each bar represents the number of brain lobes except for numbers for the expression of <italic>UAS-pntP1</italic> driven by <italic>tub-GAL4</italic>, which are the number of clones. The mean and stdev for the percentage of wild-type or <italic>btd</italic> mutant clones expressing <italic>UAS-pntP1</italic> driven by <italic>tub-GAL4</italic> are calculated by bootstrapping. **p &lt; 0.01.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03596.016">http://dx.doi.org/10.7554/eLife.03596.016</ext-link></p></caption><graphic xlink:href="elife03596f007"/></fig><fig id="fig7s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03596.017</object-id><label>Figure 7—figure supplement 1.</label><caption><title>Btd and PntP1 function cooperatively to induce the generation of INP-like cells in type I NB lineages in the VNC.</title><p>(<bold>A</bold>–<bold>A′</bold>) Type I NB lineages labeled by mCD8-GFP driven by <italic>btd-GAL4</italic> in the VNCs have no INPs (e.g. dashed circles). (<bold>B</bold>–<bold>B′</bold>) Expression of PntP1 driven by <italic>btd-GAL4</italic> induces the generation of INP-like cells (small Dpn<sup>+</sup> cells) in nearly all Btd-positive lineages (dashed circles). (<bold>C</bold>–<bold>C′</bold>) Type I NB lineages labeled by mCD8-GFP driven by <italic>insc-GAL4</italic> in the VNC have no INPs. (<bold>D</bold>–<bold>D′</bold>) Expression of <italic>UAS-btd</italic> driven by <italic>insc-GAL4</italic> neither suppresses Ase in the NB nor induces the generation of INP-like cells in type I NB lineages (e.g. dashed circles) in the VNC. (<bold>E</bold>–<bold>E′</bold>) Expression of <italic>UAS-pntP1</italic> driven by <italic>insc-GAL4</italic> suppresses Ase in nearly all NBs (*) and induces the generation of INP-like cells in a subset of type I NB lineages (dashed circles) in the VNC. (<bold>F</bold>–<bold>F′</bold>) Co-expression of <italic>UAS-btd</italic> and <italic>UAS-pntP1</italic> driven by <italic>insc-GAL4</italic> induces INP-like cells in almost all type I NB lineages (dashed circles) in the VNC. (<bold>G</bold>) Quantifications of the percentage of type I NB lineages with INP-like cells in brain lobes with indicated genotypes. **p &lt; 0.01.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03596.017">http://dx.doi.org/10.7554/eLife.03596.017</ext-link></p></caption><graphic xlink:href="elife03596fs008"/></fig><fig id="fig7s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03596.018</object-id><label>Figure 7—figure supplement 2.</label><caption><title>Overexpression of Btd promotes the generation of INP-like cells.</title><p>(<bold>A</bold>–<bold>A′′</bold>) INP specific marker R9D11-CD4-tdTomato is not expressed in any type I NB lineages (e.g. arrows) on the ventral side of a larval brain. (<bold>B</bold>–<bold>B′′</bold>) Overexpression of Btd induces the generation of INP-like cells in a subset of type I NB lineages on the ventral side of a larval brain as indicated by the expression of R9D11-CD4-tdTomato (e.g. arrows).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03596.018">http://dx.doi.org/10.7554/eLife.03596.018</ext-link></p></caption><graphic xlink:href="elife03596fs009"/></fig></fig-group></p><p>In order to determine if the <italic>btd-GAL4</italic> reflects the endogenous Btd expression pattern in the central brain, we tried to rescue the <italic>btd</italic> loss-of-function phenotypes by expressing <italic>UAS-mSp8</italic> or <italic>UAS-btd</italic> driven by <italic>btd-GAL4</italic>. However, the <italic>GAL4</italic> insertion in the <italic>btd-GAL4</italic> line does not affect the type II NB lineage development although it causes a lethal mutation of <italic>btd</italic> (<xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1</xref>). Therefore, we tried to rescue Btd RNAi knockdown phenotypes by the expression of <italic>UAS-mSp8</italic> or <italic>UAS-btd</italic> driven by <italic>btd-GAL4</italic> instead. The expression of <italic>UAS-btd RNAi</italic> driven by <italic>btd-GAL4</italic> completely eliminated mature INPs in nearly all type II NB lineages (<xref ref-type="fig" rid="fig6s2">Figure 6—figure supplement 2</xref>), which was much stronger than the phenotype of Btd RNAi knockdown driven by <italic>pntP1-GAL4</italic>. As expected, the expression of <italic>UAS-mSp8</italic> driven by <italic>btd-GAL4</italic> fully rescued the loss of INPs resulting from Btd RNAi knockdown in all type II NB lineages (<xref ref-type="fig" rid="fig6s2">Figure 6—figure supplement 2</xref>). Similarly, the expression of <italic>UAS-btd</italic> driven by <italic>btd-GAL4</italic> partially rescued the loss of INPs in about 67% of lineages (<xref ref-type="fig" rid="fig6s2">Figure 6—figure supplement 2</xref>). The incomplete rescue by <italic>UAS-btd</italic> is likely because <italic>UAS-btd</italic> contains the sequence targeted by <italic>UAS-btd RNAi</italic>. The rescue of Btd RNAi knockdown phenotypes by the expression of <italic>UAS-mSp8</italic> and <italic>UAS-btd</italic> driven by <italic>btd-GAL4</italic> together with the strong loss of INP phenotypes in all <italic>btd</italic> mutant type II NB clones strongly argue that <italic>btd-GAL4</italic> expression is likely consistent with the endogenous Btd expression pattern in the central brain.</p></sec><sec id="s2-8"><title>PntP1 and Btd function cooperatively to promote the generation of INPs</title><p>Our previous studies showed that ectopic expression of PntP1 could induce the generation of INP-like cells in more type I NB lineages in VNCs than in larval brains (<xref ref-type="fig" rid="fig7">Figure 7H–H′,J</xref>, <xref ref-type="fig" rid="fig7s1">Figure 7—figure supplement 1</xref>) (<xref ref-type="bibr" rid="bib53">Zhu et al., 2011</xref>). Since Btd is expressed in much more type I NB lineages in VNCs than in larval brains and Btd is required to prevent the premature differentiation of INPs, we wondered if ectopic PntP1-induced generation of INP-like cells requires Btd activity and occurs mostly in Btd-positive type I NB lineages. To test this idea, we examined if co-expression of PntP1 and Btd in type I NB lineages was sufficient to induce the generation of INPs and if the ectopic PntP1-induced generation of INP-like cells would be impaired in the absence of Btd.</p><p>To coexpress PntP1 and Btd in type I NB lineages, we used either <italic>btd-GAL4</italic> to drive the expression of <italic>UAS-pntP1</italic> in Btd-positive type I NB lineages or <italic>insc-GAL4</italic> to drive the expression of <italic>UAS-pntP1</italic> and <italic>UAS-btd</italic> simultaneously in all type I NB lineages. INP-like cells were identified by their expression of Ase and Dpn as well as INP-specific marker R9D11<italic>-</italic>CD4<italic>-tdTomato</italic>. Since the <italic>GAL4</italic> insertion in the <italic>btd-GAL4</italic> line causes a lethal mutation in <italic>btd</italic> (<xref ref-type="bibr" rid="bib11">Estella et al., 2003</xref>), we examined the phenotype of the expression of <italic>UAS-pntP1</italic> driven by <italic>btd-GAL4</italic> only in <italic>btd-GAL4</italic> heterozygous female larvae. As shown in <xref ref-type="fig" rid="fig6">Figure 6A–A′</xref>, type II NB lineages in <italic>btd-GAL4</italic> heterozygous mutant larvae are indistinguishable from those in wild-type animals (e.g. <xref ref-type="fig" rid="fig1">Figure 1A–A′</xref>). Furthermore, as mentioned above, <italic>btd-GAL4</italic> homozygous mutant type II NB clones develop normally (<xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1</xref>). Therefore, the generation of INPs is not affected in the <italic>btd-GAL4</italic> line. Interestingly, ectopic expression of PntP1 using <italic>btd-GAL4</italic> as a driver induced the generation of INP-like cells in about 90% Btd-positive type I NB lineages in both larval brains (<xref ref-type="fig" rid="fig7">Figure 7A–C′,J</xref>) and VNCs (<xref ref-type="fig" rid="fig7s1">Figure 7—figure supplement 1</xref>). Consistently, the co-expression of <italic>UAS-pntP1</italic> and <italic>UAS-btd</italic> driven by <italic>insc-GAL4</italic> induced INP-like cells in about 95% of type I NB lineages in both larval brains and VNCs (<xref ref-type="fig" rid="fig7">Figure 7F–F′,I–I′,J</xref>, <xref ref-type="fig" rid="fig7s1">Figure 7—figure supplement 1</xref>). In contrast, the expression of <italic>UAS-pntP1</italic> alone driven by <italic>insc-GAL4</italic> only induced INP-like cells in about 10% and 46% of type I NB lineages in larval brains (<xref ref-type="fig" rid="fig7">Figure 7H–H′,J</xref>) and VNCs (<xref ref-type="fig" rid="fig7s1">Figure 7—figure supplement 1</xref>), respectively, although ectopic PntP1 expression suppressed Ase in nearly all type I NBs (<xref ref-type="fig" rid="fig7">Figure 7H</xref>, <xref ref-type="fig" rid="fig7s1">Figure 7—figure supplement 1</xref>). The expression of <italic>UAS-btd</italic> alone neither suppressed Ase nor induced the generation of INP-like cells in VNCs (<xref ref-type="fig" rid="fig7s1">Figure 7—figure supplement 1</xref>), whereas in larval brains, the expression of <italic>UAS-btd</italic> driven by <italic>insc-GAL4</italic> suppressed/reduced the expression of Ase in the NB and promoted the generation of INP-like cells in about 20% of type I NB lineages (<xref ref-type="fig" rid="fig7">Figure 7G–G′,J</xref>). These ectopic INP-like cells induced by the expression of <italic>UAS-btd</italic> also expressed INP-specific marker R9D11<italic>-</italic>CD4<italic>-tdTomato</italic> (<xref ref-type="fig" rid="fig7s2">Figure 7—figure supplement 2</xref>). These results indicate that the generation of INP-like cells induced by the ectopic PntP1 expression requires Btd activity, whereas the expression of either PntP1 or Btd alone has limited ability to induce the generation of INP-like cells in type I NB lineages.</p><p>To further confirm if Btd is required for ectopic PntP1 to induce the generation of INP-like cells, we then examined if the generation of INP-like cells induced by ectopic PntP1 expression would be impaired in the absence of Btd. To this end, we expressed <italic>UAS-pntP1</italic> in wild-type or <italic>btd</italic> mutant type I NB clones in VNCs in that there are more Btd-positive type I NB lineages and the expression of <italic>UAS-pntP1</italic> can induce INP-like cells in much more type I NB lineages in VNCs than in larval brains. Consistent with the induction of INP-like cells in nearly all type I NB lineages when PntP1 and Btd were coexpressed, the efficiency of PntP1 to induce the generation of INP-like cell was drastically reduced in the absence of Btd. Our results showed that the expression of <italic>UAS-pntP1</italic> driven by <italic>tub-GAL4</italic> could have induced the generation of INP-like cells in about 50% of wild-type type I NB clones but not in <italic>btd</italic> mutant type I NB clones, although the expression of PntP1 equally suppressed the expression of Ase in both wild-type and <italic>btd</italic> mutant type I NBs (<xref ref-type="fig" rid="fig7">Figure 7D–E′,J</xref>). These results provide additional evidence to support that only in the presence of Btd could PntP1 induce the generation of INP-like cells in type I NB lineages.</p><p>Taken together, these results suggest that the generation of INPs requires the cooperative action of PntP1 and Btd. Thus this study together with our previous work (<xref ref-type="bibr" rid="bib53">Zhu et al., 2011</xref>) identified two key factors, PntP1 and Btd, a combination of which is sufficient to specify type II NB lineages and promote INP generation.</p></sec></sec><sec id="s3" sec-type="discussion"><title>Discussion</title><p>In this study, we show that the Sp family transcription factor Btd is required to prevent the premature differentiation of INPs by suppressing the expression of Pros in immature INPs. Furthermore, we provide evidence to demonstrate that the combination of Btd and PntP1 is sufficient to specify type II NB lineages and promote the generation of INPs. Thus, our work reveals a critical mechanism that regulates INP generation.</p><sec id="s3-1"><title>Btd prevents premature differentiation of INPs into GMCs</title><p>The most striking phenotype resulting from the loss of Btd is the elimination of mature INPs. In addition, about 40% of <italic>btd</italic> mutant type II NB lineages ectopically express Ase in the NB and become type I-like NB lineages. However, although forced expression of Ase in type II NBs is sufficient to eliminate INPs in type II NB lineages (<xref ref-type="bibr" rid="bib5">Bowman et al., 2008</xref>; <xref ref-type="bibr" rid="bib54">Zhu et al., 2012</xref>), the loss of INPs is obviously not primarily due to the ectopic Ase expression or the transformation of type II NB lineages into type I-like NB lineage in that the loss of mature INPs occurs independently of the ectopic Ase expression in most <italic>btd</italic> mutant or Btd RNAi knockdown type II NB lineages. Instead, we demonstrate that the loss of mature INPs in the absence of Btd is due to the premature differentiation of Ase<sup>+</sup> immature INPs into GMCs. We show that in Btd RNAi knockdown or <italic>btd</italic> mutant type II NB lineages without the ectopic Ase expression, Ase<sup>−</sup> immature INPs differentiate into Ase<sup>+</sup> immature INPs normally as indicated by the expression of R9D11-mCD8-GFP, Mira, as well as PntP1 in Ase<sup>+</sup> daughter cells next to the Ase<sup>−</sup> immature INPs. However, instead of differentiating into mature INPs, we argue that Ase<sup>+</sup> immature INPs prematurely differentiate into GMCs based on the following two pieces of evidence. First, Ase<sup>+</sup> daughter cells eventually undergo terminal divisions as indicated by the positive pH3 staining and the position of the pH3-positive cells. Second, unlike mature INPs, the dividing Ase<sup>+</sup> daughter cells do not form basal Mira crescent at metaphase. The terminal division and the lack of Mira crescent during the division are two unique features that distinguish GMCs from INPs in addition to the expression of nuclear Pros (<xref ref-type="bibr" rid="bib19">Ikeshima-Kataoka et al., 1997</xref>; <xref ref-type="bibr" rid="bib31">Matsuzaki et al., 1998</xref>; <xref ref-type="bibr" rid="bib37">Schuldt et al., 1998</xref>). Therefore, the elimination of mature INPs resulting from the loss of Btd is due to the premature differentiation of Ase<sup>+</sup> immature INPs into GMCs.</p><p>Why does the loss of Btd lead to premature differentiation of INPs? Our results show that the loss of Btd results in a reduction or loss of PntP1 in type II NBs and immature INPs as well as ectopic expression of Pros in early immature INPs. Our previous studies show that PntP1 suppresses Ase in type II NBs and that inhibiting PntP1 activity leads to ectopic expression of Ase in type II NBs and elimination of INPs (<xref ref-type="bibr" rid="bib53">Zhu et al., 2011</xref>). Given that the ectopic Ase expression in <italic>btd</italic> mutant type II NBs is closely associated with the severe reduction or complete loss of PntP1 and that expression of <italic>UAS-pntP1</italic> largely suppresses the ectopic Ase expression in <italic>btd</italic> mutant type II NBs, the severe reduction or loss of PntP1 most likely accounts for the ectopic Ase expression in <italic>btd</italic> mutant type II NBs. However, although the loss of PntP1 could lead to the loss of INPs, we provide several lines of evidence to demonstrate that the elimination of INPs in <italic>btd</italic> mutant or Btd RNAi knockdown type II NB lineages is primarily due to the ectopic activation of Pros in immature INPs rather than the reduction or loss of PntP1. First, ectopic nuclear Pros is consistently expressed in Ase<sup>−</sup> immature INPs when mature INPs are eliminated. Second, the loss of mature INPs can be fully rescued by Pros RNAi knockdown or even just by removing one wild-type copy of <italic>pros</italic>. Third, Pros RNAi knockdown also rescues the reduction of PntP1 and suppresses the ectopic Ase expression in <italic>btd</italic> mutant type II NBs. In contrast, the expression of <italic>UAS-pntP1</italic> fails to rescue mature INPs in most <italic>btd</italic> mutant type II NB lineages although it largely suppresses the ectopic Ase expression in the NBs. Furthermore, the complete elimination of mature INPs is also observed occasionally in <italic>btd</italic> mutant type II NB lineages without the reduction of PntP1. Therefore, the elimination of mature INPs resulting from the loss of Btd is primarily due to the ectopic Pros expression, which likely promotes premature differentiation of INPs into GMCs and cell cycle exit. The severe reduction or loss of PntP1 is responsible for the ectopic Ase expression in <italic>btd</italic> mutant type II NBs and is more likely a secondary effect due to the ectopic Pros expression and/or the loss of INPs. INPs and/or other progeny may provide feedback signals to the NBs as has been demonstrated in other systems (<xref ref-type="bibr" rid="bib50">Yoon et al., 2008</xref>; <xref ref-type="bibr" rid="bib18">Hsu et al., 2014</xref>).</p><p>The ectopic expression of Pros in Ase<sup>−</sup> immature INPs resulting from the loss of Btd suggests that Btd is critical for suppressing Pros expression in Ase<sup>−</sup> immature INPs. Btd was known as a head gap gene. It has been suggested that gap factors act largely as transcriptional repressors (<xref ref-type="bibr" rid="bib36">Schroeder et al., 2004</xref>). Btd could directly suppress Pros by binding to the <italic>pros</italic> promoter as a transcriptional repressor. Alternatively, Btd could suppress Pros indirectly by regulating the expression or antagonizing the activity of factor(s) that activate(s) <italic>pros</italic> expression. Our results show that ectopic/overly expression of Btd in type I NB lineages or mature INPs does not lead to overproliferation of type I NBs as observed in <italic>pros</italic> mutant type I NB lineages. Instead, ectopic expression of Btd promotes the generation of INP-like cells from type I NBs and transforms some type I NB lineages into type II-like NB lineages. Therefore, it is more likely that Btd suppresses Pros indirectly by regulating the expression or antagonizing the activity of <italic>pros</italic> activator(s). Previous studies have suggested that Ase, Daughterless, Numb, and Erm could activate <italic>pros</italic> expression (<xref ref-type="bibr" rid="bib34">Reddy and Rodrigues, 1999</xref>; <xref ref-type="bibr" rid="bib38">Southall and Brand, 2009</xref>; <xref ref-type="bibr" rid="bib45">Weng et al., 2010</xref>; <xref ref-type="bibr" rid="bib49">Yasugi et al., 2014</xref>). Since Ase and R9D11-Cd4-tdTomato, which are under the control of <italic>erm</italic> promoter, are not expressed in Ase<sup>−</sup> immature INPs in the absence of Btd, it is unlikely that they are involved in the activation of <italic>pros</italic> in immature INPs. It would be interesting to investigate in the future if Numb or Daughterless could activate <italic>pros</italic> in immature INPs in the absence of Btd.</p></sec><sec id="s3-2"><title>Btd and PntP1 function cooperatively to promote the generation of INPs</title><p>In this study, we provided several lines of evidence to demonstrate that Btd and PntP1 function cooperatively to specify type II NB lineages and promote the generation of INPs. Results from this study as well as our previous study (<xref ref-type="bibr" rid="bib53">Zhu et al., 2011</xref>) show that ectopic expression of <italic>UAS-pntP1</italic> or <italic>UAS-btd</italic> alone can only promote the generation of INP-like cells in a subset of type I NB lineage, whereas ectopic expression of <italic>UAS-pntP1</italic> in Btd-positive type I NB lineages or co-expression of <italic>UAS-btd</italic> and <italic>UAS-pntP1</italic> can promote the generation of INP-like cells in nearly all type I NB lineages and transforms all these lineages into type II-like NB lineages. Consistently, the ability of PntP1 to promote the generation of INP-like cells in <italic>btd</italic> mutant type I NB lineages is largely impaired. These results suggest that the specification of type II NB lineages and the generation of INPs requires both PntP1 and Btd and that the combinatorial PntP1 and Btd is sufficient to promote the generation of INPs.</p><p>We propose that PntP1 and Btd function cooperatively but through different mechanisms to promote INP generation. PntP1 is responsible for the suppression of Ase in type II NBs. Meanwhile, PntP1 must be regulating the expression of other unknown target gene(s) that are/is essential for the generation of INPs, such as specification of immature INPs, because loss of Ase is not sufficient to promote the generation of INP-like cells in any type I NB lineages. Btd likely acts after PntP1 to mainly prevent premature differentiation of INPs into GMCs by indirectly suppressing <italic>pros</italic> in immature INPs. The role of Btd in suppressing Ase in type II NBs is minimal if there is any because unlike PntP1, which suppresses <italic>ase</italic> in nearly all type I NBs when it is ectopically expressed, overexpression of Btd only suppresses Ase in a small subset of type I NBs that produce INP-like cells in larval brains. Furthermore, Ase is expressed in Btd<sup>+</sup> type I NBs, indicating that Btd does not suppress Ase in type I NBs when it is expressed at normal levels. Studies in mammals as well as in <italic>Drosophila</italic> suggest that the Btd/Sp8 could function downstream of Wnt signaling to regulate the expression of Fgf8 as well as Distal-less (Dll) and Headcase (Hdc) during the forebrain patterning as well as limb development (<xref ref-type="bibr" rid="bib11">Estella et al., 2003</xref>; <xref ref-type="bibr" rid="bib39">Treichel et al., 2003</xref>; <xref ref-type="bibr" rid="bib21">Kawakami et al., 2004</xref>; <xref ref-type="bibr" rid="bib35">Sahara et al., 2007</xref>; <xref ref-type="bibr" rid="bib10">Estella and Mann, 2010</xref>). However, inhibiting Wnt signaling alone in type II NB lineages does not have any obvious phenotypes (<xref ref-type="bibr" rid="bib23">Komori et al., 2014</xref>), indicating that Btd unlikely functions downstream of Wnt signaling in type II NB lineages. Whether Fgf8, Dll, or Hdc could function downstream of Btd to regulate INP generation remains to be investigated in the future.</p><p>In mammals, the Btd homolog Sp8 plays important roles in brain development. In the developing mouse forebrain, Sp8 is expressed in cortical progenitors in a mediolateral gradient across the ventricular zone as well as in the lateral ganglionic eminence (LGE) and medial ganglionic eminence (MGE) (<xref ref-type="bibr" rid="bib35">Sahara et al., 2007</xref>; <xref ref-type="bibr" rid="bib52">Zembrzycki et al., 2007</xref>). In developing human brains, Sp8 is abundantly expressed in the ventricular zone and the outer sub-ventricular zone where RGs and oRGs reside (<xref ref-type="bibr" rid="bib30">Ma et al., 2013</xref>). In addition to its roles in interneuron development and the patterning of developing mammalian brains and spinal cords (<xref ref-type="bibr" rid="bib14">Griesel et al., 2006</xref>; <xref ref-type="bibr" rid="bib41">Waclaw et al., 2006</xref>; <xref ref-type="bibr" rid="bib35">Sahara et al., 2007</xref>; <xref ref-type="bibr" rid="bib27">Li et al., 2011</xref>), it was also shown that the loss of Sp8 led to the reduction of the progenitor pool (<xref ref-type="bibr" rid="bib52">Zembrzycki et al., 2007</xref>). Our results show that mammalian Sp8 can rescue the loss of mature INPs resulting from the loss of Btd in <italic>Drosophila</italic>, suggesting that Btd/Sp8 could have conserved functions across different species. It would be interesting to investigate if Sp8 has similar roles in promoting the generation of transient amplifying INPs, such as oRGs, in developing mammalian brains.</p></sec></sec><sec id="s4" sec-type="materials|methods"><title>Materials and methods</title><sec id="s4-1"><title>Fly stocks</title><p>For <italic>btd</italic> loss-of-function analyses, <italic>yw btd</italic><sup><italic>XA</italic></sup> <italic>FRT19A/FM7c</italic> and <italic>yw btd</italic><sup><italic>XG81</italic></sup> <italic>FRT19A/FM7c</italic> (<xref ref-type="bibr" rid="bib46">Wimmer et al., 1993</xref>; <xref ref-type="bibr" rid="bib10">Estella and Mann, 2010</xref>) were used for generating <italic>btd</italic> mutant clones and <italic>UAS-btd RNAi</italic> (#29453; Bloomington <italic>Drosophila</italic> stock Center, Bloomington, Indiana) for Btd RNAi knockdown<italic>.</italic> Type II NB lineage-specific <italic>pntP1-GAL4</italic> (also named as <italic>GAL4</italic><sup><italic>14−94</italic></sup>) (<xref ref-type="bibr" rid="bib53">Zhu et al., 2011</xref>) and <italic>erm-GAL4</italic> (II) or (III) (<xref ref-type="bibr" rid="bib33">Pfeiffer et al., 2008</xref>; <xref ref-type="bibr" rid="bib47">Xiao et al., 2012</xref>) were used to drive the expression of <italic>UAS-transgenes</italic> in type II NB lineages or in immature as well as mature INPs, respectively. <italic>insc-Gal4</italic> (<italic>Gal4</italic><sup><italic>1407</italic></sup> inserted in <italic>inscuteable</italic> promoter) (<xref ref-type="bibr" rid="bib29">Luo et al., 1994</xref>) was used to drive <italic>UAS-transgenes</italic> in all NB lineages. <italic>UAS-mCD8-GFP</italic> driven by <italic>Btd-GAL4</italic> was utilized as reporter for <italic>btd</italic> expression<italic>.</italic> The R9D11<italic>-</italic>CD4<italic>-tdTomato</italic> transgenic line (<xref ref-type="bibr" rid="bib15">Han et al., 2011</xref>) was used for labeling Ase<sup>+</sup> immature INPs and mature INPs<italic>.</italic> Other fly stocks include: <italic>hs-Flpase tub-GAL80 FRT19A; UAS-mCD8-GFP; pntP1-GAL4</italic> for generating type II NB clones; <italic>pros</italic><sup><italic>17</italic></sup><italic>/TM6 Tb, pros</italic><sup><italic>10419</italic></sup><italic>/</italic>Tm3 <italic>Sb,</italic> and <italic>UAS-pros RNAi</italic> (#26745; Bloomington stock) for rescuing Btd loss-of-function phenotypes.</p></sec><sec id="s4-2"><title>RNAi knockdown and clonal analyses</title><p>For RNAi knockdown analyses of Btd and Pros, larvae were raised at 29°C to increase the expression of <italic>UAS-RNAi</italic> transgenes after hatching. Furthermore, <italic>UAS-Dcr2</italic> was expressed together with <italic>UAS-RNAi</italic> transgenes to enhance the efficiency of RNAi knockdown. For clonal analyses, MARCM (<xref ref-type="bibr" rid="bib25">Lee and Luo, 1999</xref>) clones were induced by 1 hr heat shock at 38°C for 1 day after larval hatching. Larval brains were dissected at third instar larval stages for the examination of phenotypes.</p></sec><sec id="s4-3"><title>Immunostaining and confocal microscopy</title><p>Larval brains were dissected, fixed, and stained as described before (<xref ref-type="bibr" rid="bib25">Lee and Luo, 1999</xref>). Primary antibodies used in this study include: rabbit anti-Mira (1:500), guinea pig anti-Ase (1:5000), rabbit anti-Dpn (1:500) (a gift from Y.N. Jan), rat anti-mCD8 (Life Technologies, Grand Island, New York, 1:100), mouse anti-Pros (Developmental Studies Hybridoma Bank, Iowa City, Iowa, 1:20), mouse monoclonal anti-α-tubulin (Sigma, St. Louis, Missouri, 1:1000), rabbit anti-dsRed (Clontech, Mountain View, California, 1:500), rabbit anti-PntP1 (1:500, a gift from JS Skeath). Secondary antibodies conjugated to Cy2, Cy3, Cy5, or DyLight 647 (Jackson ImmunoResearch, West Grove, Pennsylvania) were used at 1:100, 1:500, or 1:500, respectively. Images were taken with a Zeiss LSM510 confocal microscopy and processed with Adobe Photoshop. For quantifications of the number of mature INPs or the percentage of type II NB lineages with mature INPs, we focus on the medial group of type II NB lineages (lineages DM1–DM6). Two-tailed t-tests were used for statistics analyses.</p></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>We thank RS Mann, YN Jan, JS Skeath, CY Lee, C Han, the Bloomington stock center, and the Developmental Studies Hybridoma Bank for fly stocks and antibodies; F Pignoni, ME Zuber, AS Viczian for thoughtful discussion and comments; F Pignoni for sharing research facilities. This work was supported by March of Dimes Basil O'Connor Starter Scholar Research Award (#5-FY14-59, S.Z), the National Institute of Neurological Disorders and Stroke of the National Institutes of Health under Award Number R01NS085232 (S.Z), and SUNY Upstate Medical University startup fund (S.Z).</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>YX, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con2"><p>SZ, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con3"><p>XL, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con4"><p>XZ, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con5"><p>SM, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con6"><p>HL, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con7"><p>AU, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</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>Bayraktar</surname><given-names>OA</given-names></name><name><surname>Boone</surname><given-names>JQ</given-names></name><name><surname>Drummond</surname><given-names>ML</given-names></name><name><surname>Doe</surname><given-names>CQ</given-names></name></person-group><year>2010</year><article-title>Drosophila type II neuroblast lineages keep Prospero levels low to generate large clones that contribute to the adult brain central complex</article-title><source>Neural Development</source><volume>5</volume><fpage>26</fpage><pub-id pub-id-type="doi">10.1186/1749-8104-5-26</pub-id></element-citation></ref><ref id="bib2"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bayraktar</surname><given-names>OA</given-names></name><name><surname>Doe</surname><given-names>CQ</given-names></name></person-group><year>2013</year><article-title>Combinatorial temporal patterning in progenitors expands neural diversity</article-title><source>Nature</source><volume>498</volume><fpage>449</fpage><lpage>455</lpage><pub-id pub-id-type="doi">10.1038/nature12266</pub-id></element-citation></ref><ref id="bib3"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bello</surname><given-names>BC</given-names></name><name><surname>Izergina</surname><given-names>N</given-names></name><name><surname>Caussinus</surname><given-names>E</given-names></name><name><surname>Reichert</surname><given-names>H</given-names></name></person-group><year>2008</year><article-title>Amplification of neural stem cell proliferation by intermediate progenitor cells in Drosophila brain development</article-title><source>Neural Development</source><volume>3</volume><fpage>5</fpage><pub-id pub-id-type="doi">10.1186/1749-8104-3-5</pub-id></element-citation></ref><ref id="bib4"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Boone</surname><given-names>JQ</given-names></name><name><surname>Doe</surname><given-names>CQ</given-names></name></person-group><year>2008</year><article-title>Identification of Drosophila type II neuroblast lineages containing transit amplifying ganglion mother cells</article-title><source>Developmental Neurobiology</source><volume>68</volume><fpage>1185</fpage><lpage>1195</lpage><pub-id pub-id-type="doi">10.1002/dneu.20648</pub-id></element-citation></ref><ref id="bib5"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bowman</surname><given-names>SK</given-names></name><name><surname>Rolland</surname><given-names>V</given-names></name><name><surname>Betschinger</surname><given-names>J</given-names></name><name><surname>Kinsey</surname><given-names>KA</given-names></name><name><surname>Emery</surname><given-names>G</given-names></name><name><surname>Knoblich</surname><given-names>JA</given-names></name></person-group><year>2008</year><article-title>The tumor suppressors Brat and Numb regulate transit-amplifying neuroblast lineages in Drosophila</article-title><source>Developmental Cell</source><volume>14</volume><fpage>535</fpage><lpage>546</lpage><pub-id pub-id-type="doi">10.1016/j.devcel.2008.03.004</pub-id></element-citation></ref><ref id="bib6"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Carney</surname><given-names>TD</given-names></name><name><surname>Miller</surname><given-names>MR</given-names></name><name><surname>Robinson</surname><given-names>KJ</given-names></name><name><surname>Bayraktar</surname><given-names>OA</given-names></name><name><surname>Osterhout</surname><given-names>JA</given-names></name><name><surname>Doe</surname><given-names>CQ</given-names></name></person-group><year>2012</year><article-title>Functional genomics identifies neural stem cell sub-type expression profiles and genes regulating neuroblast homeostasis</article-title><source>Developmental Biology</source><volume>361</volume><fpage>137</fpage><lpage>146</lpage><pub-id pub-id-type="doi">10.1016/j.ydbio.2011.10.020</pub-id></element-citation></ref><ref id="bib7"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Choksi</surname><given-names>SP</given-names></name><name><surname>Southall</surname><given-names>TD</given-names></name><name><surname>Bossing</surname><given-names>T</given-names></name><name><surname>Edoff</surname><given-names>K</given-names></name><name><surname>de Wit</surname><given-names>E</given-names></name><name><surname>Fischer</surname><given-names>BE</given-names></name><name><surname>van Steensel</surname><given-names>B</given-names></name><name><surname>Micklem</surname><given-names>G</given-names></name><name><surname>Brand</surname><given-names>AH</given-names></name></person-group><year>2006</year><article-title>Prospero acts as a binary switch between self-renewal and differentiation in Drosophila neural stem cells</article-title><source>Developmental Cell</source><volume>11</volume><fpage>775</fpage><lpage>789</lpage><pub-id pub-id-type="doi">10.1016/j.devcel.2006.09.015</pub-id></element-citation></ref><ref id="bib8"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Dai</surname><given-names>C</given-names></name><name><surname>Celestino</surname><given-names>JC</given-names></name><name><surname>Okada</surname><given-names>Y</given-names></name><name><surname>Louis</surname><given-names>DN</given-names></name><name><surname>Fuller</surname><given-names>GN</given-names></name><name><surname>Holland</surname><given-names>EC</given-names></name></person-group><year>2001</year><article-title>PDGF autocrine stimulation dedifferentiates cultured astrocytes and induces oligodendrogliomas and oligoastrocytomas from neural progenitors and astrocytes in vivo</article-title><source>Genes &amp; Development</source><volume>15</volume><fpage>1913</fpage><lpage>1925</lpage><pub-id pub-id-type="doi">10.1101/gad.903001</pub-id></element-citation></ref><ref id="bib9"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Eroglu</surname><given-names>E</given-names></name><name><surname>Burkard</surname><given-names>TR</given-names></name><name><surname>Jiang</surname><given-names>Y</given-names></name><name><surname>Saini</surname><given-names>N</given-names></name><name><surname>Homem</surname><given-names>CC</given-names></name><name><surname>Reichert</surname><given-names>H</given-names></name><name><surname>Knoblich</surname><given-names>JA</given-names></name></person-group><year>2014</year><article-title>SWI/SNF complex prevents lineage reversion and induces temporal patterning in neural stem cells</article-title><source>Cell</source><volume>156</volume><fpage>1259</fpage><lpage>1273</lpage><pub-id pub-id-type="doi">10.1016/j.cell.2014.01.053</pub-id></element-citation></ref><ref id="bib10"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Estella</surname><given-names>C</given-names></name><name><surname>Mann</surname><given-names>RS</given-names></name></person-group><year>2010</year><article-title>Non-redundant selector and growth-promoting functions of two sister genes, buttonhead and Sp1, in Drosophila leg development</article-title><source>PLOS Genetics</source><volume>6</volume><fpage>e1001001</fpage><pub-id pub-id-type="doi">10.1371/journal.pgen.1001001</pub-id></element-citation></ref><ref id="bib11"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Estella</surname><given-names>C</given-names></name><name><surname>Rieckhof</surname><given-names>G</given-names></name><name><surname>Calleja</surname><given-names>M</given-names></name><name><surname>Morata</surname><given-names>G</given-names></name></person-group><year>2003</year><article-title>The role of buttonhead and Sp1 in the development of the ventral imaginal discs of Drosophila</article-title><source>Development</source><volume>130</volume><fpage>5929</fpage><lpage>5941</lpage><pub-id pub-id-type="doi">10.1242/dev.00832</pub-id></element-citation></ref><ref id="bib12"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Fietz</surname><given-names>SA</given-names></name><name><surname>Kelava</surname><given-names>I</given-names></name><name><surname>Vogt</surname><given-names>J</given-names></name><name><surname>Wilsch-Brauninger</surname><given-names>M</given-names></name><name><surname>Stenzel</surname><given-names>D</given-names></name><name><surname>Fish</surname><given-names>JL</given-names></name><name><surname>Corbeil</surname><given-names>D</given-names></name><name><surname>Riehn</surname><given-names>A</given-names></name><name><surname>Distler</surname><given-names>W</given-names></name><name><surname>Nitsch</surname><given-names>R</given-names></name><name><surname>Huttner</surname><given-names>WB</given-names></name></person-group><year>2010</year><article-title>OSVZ progenitors of human and ferret neocortex are epithelial-like and expand by integrin signaling</article-title><source>Nature Neuroscience</source><volume>13</volume><fpage>690</fpage><lpage>699</lpage><pub-id pub-id-type="doi">10.1038/nn.2553</pub-id></element-citation></ref><ref id="bib13"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Florio</surname><given-names>M</given-names></name><name><surname>Huttner</surname><given-names>WB</given-names></name></person-group><year>2014</year><article-title>Neural progenitors, neurogenesis and the evolution of the neocortex</article-title><source>Development</source><volume>141</volume><fpage>2182</fpage><lpage>2194</lpage><pub-id pub-id-type="doi">10.1242/dev.090571</pub-id></element-citation></ref><ref id="bib14"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Griesel</surname><given-names>G</given-names></name><name><surname>Treichel</surname><given-names>D</given-names></name><name><surname>Collombat</surname><given-names>P</given-names></name><name><surname>Krull</surname><given-names>J</given-names></name><name><surname>Zembrzycki</surname><given-names>A</given-names></name><name><surname>van den Akker</surname><given-names>WM</given-names></name><name><surname>Gruss</surname><given-names>P</given-names></name><name><surname>Simeone</surname><given-names>A</given-names></name><name><surname>Mansouri</surname><given-names>A</given-names></name></person-group><year>2006</year><article-title>Sp8 controls the anteroposterior patterning at the midbrain-hindbrain border</article-title><source>Development</source><volume>133</volume><fpage>1779</fpage><lpage>1787</lpage><pub-id pub-id-type="doi">10.1242/dev.02326</pub-id></element-citation></ref><ref id="bib15"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Han</surname><given-names>C</given-names></name><name><surname>Jan</surname><given-names>LY</given-names></name><name><surname>Jan</surname><given-names>YN</given-names></name></person-group><year>2011</year><article-title>Enhancer-driven membrane markers for analysis of nonautonomous mechanisms reveal neuron-glia interactions in Drosophila</article-title><source>Proceedings of the National Academy of Sciences of USA</source><volume>108</volume><fpage>9673</fpage><lpage>9678</lpage><pub-id pub-id-type="doi">10.1073/pnas.1106386108</pub-id></element-citation></ref><ref id="bib16"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hansen</surname><given-names>DV</given-names></name><name><surname>Lui</surname><given-names>JH</given-names></name><name><surname>Parker</surname><given-names>PR</given-names></name><name><surname>Kriegstein</surname><given-names>AR</given-names></name></person-group><year>2010</year><article-title>Neurogenic radial glia in the outer subventricular zone of human neocortex</article-title><source>Nature</source><volume>464</volume><fpage>554</fpage><lpage>561</lpage><pub-id pub-id-type="doi">10.1038/nature08845</pub-id></element-citation></ref><ref id="bib17"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Holland</surname><given-names>EC</given-names></name><name><surname>Celestino</surname><given-names>J</given-names></name><name><surname>Dai</surname><given-names>C</given-names></name><name><surname>Schaefer</surname><given-names>L</given-names></name><name><surname>Sawaya</surname><given-names>RE</given-names></name><name><surname>Fuller</surname><given-names>GN</given-names></name></person-group><year>2000</year><article-title>Combined activation of Ras and Akt in neural progenitors induces glioblastoma formation in mice</article-title><source>Nature Genetics</source><volume>25</volume><fpage>55</fpage><lpage>57</lpage><pub-id pub-id-type="doi">10.1038/75596</pub-id></element-citation></ref><ref id="bib18"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hsu</surname><given-names>YC</given-names></name><name><surname>Li</surname><given-names>L</given-names></name><name><surname>Fuchs</surname><given-names>E</given-names></name></person-group><year>2014</year><article-title>Transit-amplifying cells orchestrate stem cell activity and tissue regeneration</article-title><source>Cell</source><volume>157</volume><fpage>935</fpage><lpage>949</lpage><pub-id pub-id-type="doi">10.1016/j.cell.2014.02.057</pub-id></element-citation></ref><ref id="bib19"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ikeshima-Kataoka</surname><given-names>H</given-names></name><name><surname>Skeath</surname><given-names>JB</given-names></name><name><surname>Nabeshima</surname><given-names>Y</given-names></name><name><surname>Doe</surname><given-names>CQ</given-names></name><name><surname>Matsuzaki</surname><given-names>F</given-names></name></person-group><year>1997</year><article-title>Miranda directs Prospero to a daughter cell during Drosophila asymmetric divisions</article-title><source>Nature</source><volume>390</volume><fpage>625</fpage><lpage>629</lpage><pub-id pub-id-type="doi">10.1038/37641</pub-id></element-citation></ref><ref id="bib20"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Janssens</surname><given-names>DH</given-names></name><name><surname>Komori</surname><given-names>H</given-names></name><name><surname>Grbac</surname><given-names>D</given-names></name><name><surname>Chen</surname><given-names>K</given-names></name><name><surname>Koe</surname><given-names>CT</given-names></name><name><surname>Wang</surname><given-names>H</given-names></name><name><surname>Lee</surname><given-names>CY</given-names></name></person-group><year>2014</year><article-title>Earmuff restricts progenitor cell potential by attenuating the competence to respond to self-renewal factors</article-title><source>Development</source><volume>141</volume><fpage>1036</fpage><lpage>1046</lpage><pub-id pub-id-type="doi">10.1242/dev.106534</pub-id></element-citation></ref><ref id="bib21"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kawakami</surname><given-names>Y</given-names></name><name><surname>Esteban</surname><given-names>CR</given-names></name><name><surname>Matsui</surname><given-names>T</given-names></name><name><surname>Rodriguez-Leon</surname><given-names>J</given-names></name><name><surname>Kato</surname><given-names>S</given-names></name><name><surname>Izpisua Belmonte</surname><given-names>JC</given-names></name></person-group><year>2004</year><article-title>Sp8 and Sp9, two closely related buttonhead-like transcription factors, regulate Fgf8 expression and limb outgrowth in vertebrate embryos</article-title><source>Development</source><volume>131</volume><fpage>4763</fpage><lpage>4774</lpage><pub-id pub-id-type="doi">10.1242/dev.01331</pub-id></element-citation></ref><ref id="bib22"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Koe</surname><given-names>CT</given-names></name><name><surname>Li</surname><given-names>S</given-names></name><name><surname>Rossi</surname><given-names>F</given-names></name><name><surname>Wong</surname><given-names>JJ</given-names></name><name><surname>Wang</surname><given-names>Y</given-names></name><name><surname>Zhang</surname><given-names>Z</given-names></name><name><surname>Chen</surname><given-names>K</given-names></name><name><surname>Aw</surname><given-names>SS</given-names></name><name><surname>Richardson</surname><given-names>HE</given-names></name><name><surname>Robson</surname><given-names>P</given-names></name><name><surname>Sung</surname><given-names>WK</given-names></name><name><surname>Yu</surname><given-names>F</given-names></name><name><surname>Gonzalez</surname><given-names>C</given-names></name><name><surname>Wang</surname><given-names>H</given-names></name></person-group><year>2014</year><article-title>The Brm-HDAC3-Erm repressor complex suppresses dedifferentiation in Drosophila type II neuroblast lineages</article-title><source>eLife</source><volume>3</volume><fpage>e01906</fpage><pub-id pub-id-type="doi">10.7554/eLife.01906</pub-id></element-citation></ref><ref id="bib23"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Komori</surname><given-names>H</given-names></name><name><surname>Xiao</surname><given-names>Q</given-names></name><name><surname>McCartney</surname><given-names>BM</given-names></name><name><surname>Lee</surname><given-names>CY</given-names></name></person-group><year>2014</year><article-title>Brain tumor specifies intermediate progenitor cell identity by attenuating beta-catenin/Armadillo activity</article-title><source>Development</source><volume>141</volume><fpage>51</fpage><lpage>62</lpage><pub-id pub-id-type="doi">10.1242/dev.099382</pub-id></element-citation></ref><ref id="bib24"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kriegstein</surname><given-names>A</given-names></name><name><surname>Alvarez-Buylla</surname><given-names>A</given-names></name></person-group><year>2009</year><article-title>The glial nature of embryonic and adult neural stem cells</article-title><source>Annual Review of Neuroscience</source><volume>32</volume><fpage>149</fpage><lpage>184</lpage><pub-id pub-id-type="doi">10.1146/annurev.neuro.051508.135600</pub-id></element-citation></ref><ref id="bib25"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lee</surname><given-names>T</given-names></name><name><surname>Luo</surname><given-names>L</given-names></name></person-group><year>1999</year><article-title>Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis</article-title><source>Neuron</source><volume>22</volume><fpage>451</fpage><lpage>461</lpage><pub-id pub-id-type="doi">10.1016/S0896-6273(00)80701-1</pub-id></element-citation></ref><ref id="bib26"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Li</surname><given-names>L</given-names></name><name><surname>Vaessin</surname><given-names>H</given-names></name></person-group><year>2000</year><article-title>Pan-neural Prospero terminates cell proliferation during Drosophila neurogenesis</article-title><source>Genes &amp; Development</source><volume>14</volume><fpage>147</fpage><lpage>151</lpage></element-citation></ref><ref id="bib27"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Li</surname><given-names>X</given-names></name><name><surname>Sun</surname><given-names>C</given-names></name><name><surname>Lin</surname><given-names>C</given-names></name><name><surname>Ma</surname><given-names>T</given-names></name><name><surname>Madhavan</surname><given-names>MC</given-names></name><name><surname>Campbell</surname><given-names>K</given-names></name><name><surname>Yang</surname><given-names>Z</given-names></name></person-group><year>2011</year><article-title>The transcription factor Sp8 is required for the production of parvalbumin-expressing interneurons in the olfactory bulb</article-title><source>The Journal of Neuroscience</source><volume>31</volume><fpage>8450</fpage><lpage>8455</lpage><pub-id pub-id-type="doi">10.1523/JNEUROSCI.0939-11.2011</pub-id></element-citation></ref><ref id="bib28"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lui</surname><given-names>JH</given-names></name><name><surname>Hansen</surname><given-names>DV</given-names></name><name><surname>Kriegstein</surname><given-names>AR</given-names></name></person-group><year>2011</year><article-title>Development and evolution of the human neocortex</article-title><source>Cell</source><volume>146</volume><fpage>18</fpage><lpage>36</lpage><pub-id pub-id-type="doi">10.1016/j.cell.2011.06.030</pub-id></element-citation></ref><ref id="bib29"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Luo</surname><given-names>L</given-names></name><name><surname>Liao</surname><given-names>YJ</given-names></name><name><surname>Jan</surname><given-names>LY</given-names></name><name><surname>Jan</surname><given-names>YN</given-names></name></person-group><year>1994</year><article-title>Distinct morphogenetic functions of similar small GTPases: Drosophila Drac1 is involved in axonal outgrowth and myoblast fusion</article-title><source>Genes &amp; Development</source><volume>8</volume><fpage>1787</fpage><lpage>1802</lpage><pub-id pub-id-type="doi">10.1101/gad.8.15.1787</pub-id></element-citation></ref><ref id="bib30"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ma</surname><given-names>T</given-names></name><name><surname>Wang</surname><given-names>C</given-names></name><name><surname>Wang</surname><given-names>L</given-names></name><name><surname>Zhou</surname><given-names>X</given-names></name><name><surname>Tian</surname><given-names>M</given-names></name><name><surname>Zhang</surname><given-names>Q</given-names></name><name><surname>Zhang</surname><given-names>Y</given-names></name><name><surname>Li</surname><given-names>J</given-names></name><name><surname>Liu</surname><given-names>Z</given-names></name><name><surname>Cai</surname><given-names>Y</given-names></name><name><surname>Liu</surname><given-names>F</given-names></name><name><surname>You</surname><given-names>Y</given-names></name><name><surname>Chen</surname><given-names>C</given-names></name><name><surname>Campbell</surname><given-names>K</given-names></name><name><surname>Song</surname><given-names>H</given-names></name><name><surname>Ma</surname><given-names>L</given-names></name><name><surname>Rubenstein</surname><given-names>JL</given-names></name><name><surname>Yang</surname><given-names>Z</given-names></name></person-group><year>2013</year><article-title>Subcortical origins of human and monkey neocortical interneurons</article-title><source>Nature Neuroscience</source><volume>16</volume><fpage>1588</fpage><lpage>1597</lpage><pub-id pub-id-type="doi">10.1038/nn.3536</pub-id></element-citation></ref><ref id="bib31"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Matsuzaki</surname><given-names>F</given-names></name><name><surname>Ohshiro</surname><given-names>T</given-names></name><name><surname>Ikeshima-Kataoka</surname><given-names>H</given-names></name><name><surname>Izumi</surname><given-names>H</given-names></name></person-group><year>1998</year><article-title>miranda localizes staufen and prospero asymmetrically in mitotic neuroblasts and epithelial cells in early Drosophila embryogenesis</article-title><source>Development</source><volume>125</volume><fpage>4089</fpage><lpage>4098</lpage></element-citation></ref><ref id="bib32"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Persson</surname><given-names>AI</given-names></name><name><surname>Petritsch</surname><given-names>C</given-names></name><name><surname>Swartling</surname><given-names>FJ</given-names></name><name><surname>Itsara</surname><given-names>M</given-names></name><name><surname>Sim</surname><given-names>FJ</given-names></name><name><surname>Auvergne</surname><given-names>R</given-names></name><name><surname>Goldenberg</surname><given-names>DD</given-names></name><name><surname>Vandenberg</surname><given-names>SR</given-names></name><name><surname>Nguyen</surname><given-names>KN</given-names></name><name><surname>Yakovenko</surname><given-names>S</given-names></name><name><surname>Ayers-Ringler</surname><given-names>J</given-names></name><name><surname>Nishiyama</surname><given-names>A</given-names></name><name><surname>Stallcup</surname><given-names>WB</given-names></name><name><surname>Berger</surname><given-names>MS</given-names></name><name><surname>Bergers</surname><given-names>G</given-names></name><name><surname>McKnight</surname><given-names>TR</given-names></name><name><surname>Goldman</surname><given-names>SA</given-names></name><name><surname>Weiss</surname><given-names>WA</given-names></name></person-group><year>2010</year><article-title>Non-stem cell origin for oligodendroglioma</article-title><source>Cancer Cell</source><volume>18</volume><fpage>669</fpage><lpage>682</lpage><pub-id pub-id-type="doi">10.1016/j.ccr.2010.10.033</pub-id></element-citation></ref><ref id="bib33"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Pfeiffer</surname><given-names>BD</given-names></name><name><surname>Jenett</surname><given-names>A</given-names></name><name><surname>Hammonds</surname><given-names>AS</given-names></name><name><surname>Ngo</surname><given-names>TT</given-names></name><name><surname>Misra</surname><given-names>S</given-names></name><name><surname>Murphy</surname><given-names>C</given-names></name><name><surname>Scully</surname><given-names>A</given-names></name><name><surname>Carlson</surname><given-names>JW</given-names></name><name><surname>Wan</surname><given-names>KH</given-names></name><name><surname>Laverty</surname><given-names>TR</given-names></name><name><surname>Mungall</surname><given-names>C</given-names></name><name><surname>Svirskas</surname><given-names>R</given-names></name><name><surname>Kadonaga</surname><given-names>JT</given-names></name><name><surname>Doe</surname><given-names>CQ</given-names></name><name><surname>Eisen</surname><given-names>MB</given-names></name><name><surname>Celniker</surname><given-names>SE</given-names></name><name><surname>Rubin</surname><given-names>GM</given-names></name></person-group><year>2008</year><article-title>Tools for neuroanatomy and neurogenetics in Drosophila</article-title><source>Proceedings of the National Academy of Sciences of USA</source><volume>105</volume><fpage>9715</fpage><lpage>9720</lpage><pub-id pub-id-type="doi">10.1073/pnas.0803697105</pub-id></element-citation></ref><ref id="bib34"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Reddy</surname><given-names>GV</given-names></name><name><surname>Rodrigues</surname><given-names>V</given-names></name></person-group><year>1999</year><article-title>Sibling cell fate in the Drosophila adult external sense organ lineage is specified by prospero function, which is regulated by Numb and Notch</article-title><source>Development</source><volume>126</volume><fpage>2083</fpage><lpage>2092</lpage></element-citation></ref><ref id="bib35"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Sahara</surname><given-names>S</given-names></name><name><surname>Kawakami</surname><given-names>Y</given-names></name><name><surname>Izpisua Belmonte</surname><given-names>JC</given-names></name><name><surname>O'Leary</surname><given-names>DD</given-names></name></person-group><year>2007</year><article-title>Sp8 exhibits reciprocal induction with Fgf8 but has an opposing effect on anterior-posterior cortical area patterning</article-title><source>Neural Development</source><volume>2</volume><fpage>10</fpage><pub-id pub-id-type="doi">10.1186/1749-8104-2-10</pub-id></element-citation></ref><ref id="bib36"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Schroeder</surname><given-names>MD</given-names></name><name><surname>Pearce</surname><given-names>M</given-names></name><name><surname>Fak</surname><given-names>J</given-names></name><name><surname>Fan</surname><given-names>H</given-names></name><name><surname>Unnerstall</surname><given-names>U</given-names></name><name><surname>Emberly</surname><given-names>E</given-names></name><name><surname>Rajewsky</surname><given-names>N</given-names></name><name><surname>Siggia</surname><given-names>ED</given-names></name><name><surname>Gaul</surname><given-names>U</given-names></name></person-group><year>2004</year><article-title>Transcriptional control in the segmentation gene network of Drosophila</article-title><source>PLOS Biology</source><volume>2</volume><fpage>E271</fpage><pub-id pub-id-type="doi">10.1371/journal.pbio.0020271</pub-id></element-citation></ref><ref id="bib37"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Schuldt</surname><given-names>AJ</given-names></name><name><surname>Adams</surname><given-names>JH</given-names></name><name><surname>Davidson</surname><given-names>CM</given-names></name><name><surname>Micklem</surname><given-names>DR</given-names></name><name><surname>Haseloff</surname><given-names>J</given-names></name><name><surname>St Johnston</surname><given-names>D</given-names></name><name><surname>Brand</surname><given-names>AH</given-names></name></person-group><year>1998</year><article-title>Miranda mediates asymmetric protein and RNA localization in the developing nervous system</article-title><source>Genes &amp; Development</source><volume>12</volume><fpage>1847</fpage><lpage>1857</lpage><pub-id pub-id-type="doi">10.1101/gad.12.12.1847</pub-id></element-citation></ref><ref id="bib38"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Southall</surname><given-names>TD</given-names></name><name><surname>Brand</surname><given-names>AH</given-names></name></person-group><year>2009</year><article-title>Neural stem cell transcriptional networks highlight genes essential for nervous system development</article-title><source>The EMBO Journal</source><volume>28</volume><fpage>3799</fpage><lpage>3807</lpage><pub-id pub-id-type="doi">10.1038/emboj.2009.309</pub-id></element-citation></ref><ref id="bib39"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Treichel</surname><given-names>D</given-names></name><name><surname>Schock</surname><given-names>F</given-names></name><name><surname>Jackle</surname><given-names>H</given-names></name><name><surname>Gruss</surname><given-names>P</given-names></name><name><surname>Mansouri</surname><given-names>A</given-names></name></person-group><year>2003</year><article-title>mBtd is required to maintain signaling during murine limb development</article-title><source>Genes &amp; Development</source><volume>17</volume><fpage>2630</fpage><lpage>2635</lpage><pub-id pub-id-type="doi">10.1101/gad.274103</pub-id></element-citation></ref><ref id="bib40"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Viktorin</surname><given-names>G</given-names></name><name><surname>Riebli</surname><given-names>N</given-names></name><name><surname>Popkova</surname><given-names>A</given-names></name><name><surname>Giangrande</surname><given-names>A</given-names></name><name><surname>Reichert</surname><given-names>H</given-names></name></person-group><year>2011</year><article-title>Multipotent neural stem cells generate glial cells of the central complex through transit amplifying intermediate progenitors in Drosophila brain development</article-title><source>Developmental Biology</source><volume>356</volume><fpage>553</fpage><lpage>565</lpage><pub-id pub-id-type="doi">10.1016/j.ydbio.2011.06.013</pub-id></element-citation></ref><ref id="bib41"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Waclaw</surname><given-names>RR</given-names></name><name><surname>Allen</surname><given-names>ZJ</given-names><suffix>II</suffix></name><name><surname>Bell</surname><given-names>SM</given-names></name><name><surname>Erdelyi</surname><given-names>F</given-names></name><name><surname>Szabo</surname><given-names>G</given-names></name><name><surname>Potter</surname><given-names>SS</given-names></name><name><surname>Campbell</surname><given-names>K</given-names></name></person-group><year>2006</year><article-title>The zinc finger transcription factor Sp8 regulates the generation and diversity of olfactory bulb interneurons</article-title><source>Neuron</source><volume>49</volume><fpage>503</fpage><lpage>516</lpage><pub-id pub-id-type="doi">10.1016/j.neuron.2006.01.018</pub-id></element-citation></ref><ref id="bib42"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Walton</surname><given-names>NM</given-names></name><name><surname>Snyder</surname><given-names>GE</given-names></name><name><surname>Park</surname><given-names>D</given-names></name><name><surname>Kobeissy</surname><given-names>F</given-names></name><name><surname>Scheffler</surname><given-names>B</given-names></name><name><surname>Steindler</surname><given-names>DA</given-names></name></person-group><year>2009</year><article-title>Gliotypic neural stem cells transiently adopt tumorigenic properties during normal differentiation</article-title><source>Stem Cells</source><volume>27</volume><fpage>280</fpage><lpage>289</lpage><pub-id pub-id-type="doi">10.1634/stemcells.2008-0842</pub-id></element-citation></ref><ref id="bib43"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wang</surname><given-names>X</given-names></name><name><surname>Tsai</surname><given-names>JW</given-names></name><name><surname>LaMonica</surname><given-names>B</given-names></name><name><surname>Kriegstein</surname><given-names>AR</given-names></name></person-group><year>2011</year><article-title>A new subtype of progenitor cell in the mouse embryonic neocortex</article-title><source>Nature Neuroscience</source><volume>14</volume><fpage>555</fpage><lpage>561</lpage><pub-id pub-id-type="doi">10.1038/nn.2807</pub-id></element-citation></ref><ref id="bib44"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wang</surname><given-names>YC</given-names></name><name><surname>Yang</surname><given-names>JS</given-names></name><name><surname>Johnston</surname><given-names>R</given-names></name><name><surname>Ren</surname><given-names>Q</given-names></name><name><surname>Luan</surname><given-names>H</given-names></name><name><surname>Brody</surname><given-names>T</given-names></name><name><surname>Odenwald</surname><given-names>WF</given-names></name><name><surname>Lee</surname><given-names>T</given-names></name></person-group><year>2014</year><article-title>Drosophila intermediate neural progenitors produce lineage-dependent related series of diverse neurons</article-title><source>Development</source><volume>141</volume><fpage>253</fpage><lpage>258</lpage><pub-id pub-id-type="doi">10.1242/dev.103069</pub-id></element-citation></ref><ref id="bib45"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Weng</surname><given-names>M</given-names></name><name><surname>Golden</surname><given-names>KL</given-names></name><name><surname>Lee</surname><given-names>CY</given-names></name></person-group><year>2010</year><article-title>dFezf/Earmuff maintains the restricted developmental potential of intermediate neural progenitors in Drosophila</article-title><source>Developmental Cell</source><volume>18</volume><fpage>126</fpage><lpage>135</lpage><pub-id pub-id-type="doi">10.1016/j.devcel.2009.12.007</pub-id></element-citation></ref><ref id="bib46"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wimmer</surname><given-names>EA</given-names></name><name><surname>Jackle</surname><given-names>H</given-names></name><name><surname>Pfeifle</surname><given-names>C</given-names></name><name><surname>Cohen</surname><given-names>SM</given-names></name></person-group><year>1993</year><article-title>A Drosophila homologue of human Sp1 is a head-specific segmentation gene</article-title><source>Nature</source><volume>366</volume><fpage>690</fpage><lpage>694</lpage><pub-id pub-id-type="doi">10.1038/366690a0</pub-id></element-citation></ref><ref id="bib47"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Xiao</surname><given-names>Q</given-names></name><name><surname>Komori</surname><given-names>H</given-names></name><name><surname>Lee</surname><given-names>CY</given-names></name></person-group><year>2012</year><article-title>klumpfuss distinguishes stem cells from progenitor cells during asymmetric neuroblast division</article-title><source>Development</source><volume>139</volume><fpage>2670</fpage><lpage>2680</lpage><pub-id pub-id-type="doi">10.1242/dev.081687</pub-id></element-citation></ref><ref id="bib48"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Yang</surname><given-names>JS</given-names></name><name><surname>Awasaki</surname><given-names>T</given-names></name><name><surname>Yu</surname><given-names>HH</given-names></name><name><surname>He</surname><given-names>Y</given-names></name><name><surname>Ding</surname><given-names>P</given-names></name><name><surname>Kao</surname><given-names>JC</given-names></name><name><surname>Lee</surname><given-names>T</given-names></name></person-group><year>2013</year><article-title>Diverse neuronal lineages make stereotyped contributions to the Drosophila locomotor control center, the central complex</article-title><source>The Journal of Comparative Neurology</source><volume>521</volume><fpage>2645</fpage><lpage>2662</lpage><pub-id pub-id-type="doi">10.1002/cne.23339</pub-id></element-citation></ref><ref id="bib49"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Yasugi</surname><given-names>T</given-names></name><name><surname>Fischer</surname><given-names>A</given-names></name><name><surname>Jiang</surname><given-names>Y</given-names></name><name><surname>Reichert</surname><given-names>H</given-names></name><name><surname>Knoblich</surname><given-names>JA</given-names></name></person-group><year>2014</year><article-title>A regulatory transcriptional loop controls proliferation and differentiation in Drosophila neural stem cells</article-title><source>PLOS ONE</source><volume>9</volume><fpage>e97034</fpage><pub-id pub-id-type="doi">10.1371/journal.pone.0097034</pub-id></element-citation></ref><ref id="bib50"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Yoon</surname><given-names>KJ</given-names></name><name><surname>Koo</surname><given-names>BK</given-names></name><name><surname>Im</surname><given-names>SK</given-names></name><name><surname>Jeong</surname><given-names>HW</given-names></name><name><surname>Ghim</surname><given-names>J</given-names></name><name><surname>Kwon</surname><given-names>MC</given-names></name><name><surname>Moon</surname><given-names>JS</given-names></name><name><surname>Miyata</surname><given-names>T</given-names></name><name><surname>Kong</surname><given-names>YY</given-names></name></person-group><year>2008</year><article-title>Mind bomb 1-expressing intermediate progenitors generate notch signaling to maintain radial glial cells</article-title><source>Neuron</source><volume>58</volume><fpage>519</fpage><lpage>531</lpage><pub-id pub-id-type="doi">10.1016/j.neuron.2008.03.018</pub-id></element-citation></ref><ref id="bib51"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Younossi-Hartenstein</surname><given-names>A</given-names></name><name><surname>Green</surname><given-names>P</given-names></name><name><surname>Liaw</surname><given-names>GJ</given-names></name><name><surname>Rudolph</surname><given-names>K</given-names></name><name><surname>Lengyel</surname><given-names>J</given-names></name><name><surname>Hartenstein</surname><given-names>V</given-names></name></person-group><year>1997</year><article-title>Control of early neurogenesis of the Drosophila brain by the head gap genes tll, otd, ems, and btd</article-title><source>Developmental Biology</source><volume>182</volume><fpage>270</fpage><lpage>283</lpage><pub-id pub-id-type="doi">10.1006/dbio.1996.8475</pub-id></element-citation></ref><ref id="bib52"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Zembrzycki</surname><given-names>A</given-names></name><name><surname>Griesel</surname><given-names>G</given-names></name><name><surname>Stoykova</surname><given-names>A</given-names></name><name><surname>Mansouri</surname><given-names>A</given-names></name></person-group><year>2007</year><article-title>Genetic interplay between the transcription factors Sp8 and Emx2 in the patterning of the forebrain</article-title><source>Neural Development</source><volume>2</volume><fpage>8</fpage><pub-id pub-id-type="doi">10.1186/1749-8104-2-8</pub-id></element-citation></ref><ref id="bib53"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Zhu</surname><given-names>S</given-names></name><name><surname>Barshow</surname><given-names>S</given-names></name><name><surname>Wildonger</surname><given-names>J</given-names></name><name><surname>Jan</surname><given-names>LY</given-names></name><name><surname>Jan</surname><given-names>YN</given-names></name></person-group><year>2011</year><article-title>Ets transcription factor Pointed promotes the generation of intermediate neural progenitors in Drosophila larval brains</article-title><source>Proceedings of the National Academy of Sciences of USA</source><volume>108</volume><fpage>20615</fpage><lpage>20620</lpage><pub-id pub-id-type="doi">10.1073/pnas.1118595109</pub-id></element-citation></ref><ref id="bib54"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Zhu</surname><given-names>S</given-names></name><name><surname>Wildonger</surname><given-names>J</given-names></name><name><surname>Barshow</surname><given-names>S</given-names></name><name><surname>Younger</surname><given-names>S</given-names></name><name><surname>Huang</surname><given-names>Y</given-names></name><name><surname>Lee</surname><given-names>T</given-names></name></person-group><year>2012</year><article-title>The bHLH repressor Deadpan regulates the self-renewal and specification of Drosophila larval neural stem cells independently of Notch</article-title><source>PLOS ONE</source><volume>7</volume><fpage>e46724</fpage><pub-id pub-id-type="doi">10.1371/journal.pone.0046724</pub-id></element-citation></ref><ref id="bib55"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Zong</surname><given-names>H</given-names></name><name><surname>Verhaak</surname><given-names>RG</given-names></name><name><surname>Canolk</surname><given-names>P</given-names></name></person-group><year>2012</year><article-title>The cellular origin for malignant glioma and prospects for clinical advancements</article-title><source>Expert Review of Molecular Diagnostics</source><volume>12</volume><fpage>383</fpage><lpage>394</lpage><pub-id pub-id-type="doi">10.1586/erm.12.30</pub-id></element-citation></ref></ref-list></back><sub-article article-type="article-commentary" id="SA1"><front-stub><article-id pub-id-type="doi">10.7554/eLife.03596.019</article-id><title-group><article-title>Decision letter</article-title></title-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Bronner</surname><given-names>Marianne E</given-names></name><role>Reviewing editor</role><aff><institution>California Institute of Technology</institution>, <country>United States</country></aff></contrib></contrib-group></front-stub><body><boxed-text><p>eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see <ext-link ext-link-type="uri" xlink:href="http://elifesciences.org/review-process">review process</ext-link>). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.</p></boxed-text><p>Thank you for sending your work entitled “The <italic>Drosophila</italic> Sp8 Transcription Factor Buttonhead Prevents Premature Differentiation of Intermediate Neural Progenitors” for consideration at <italic>eLife</italic>. Your article has been favorably evaluated by Janet Rossant (Senior editor), Marianne Bronner (Reviewing editor), and 2 reviewers.</p><p>The Reviewing editor and the reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.</p><p><italic>Drosophila</italic> type-II neuroblast (NB) lineages produce neural cells indirectly, by generating transiently amplifying Intermediate Neural Progenitors (INPs). INPs play a crucial role in mammalian brain development and understanding how they form and proliferate is therefore a central question in neurobiology. In a previous study, the authors have shown that the Ets transcription factor Pointed P1 (PntP1) is required for the specification of Type-II NB lineages in <italic>Drosophila</italic> larval brains. PntP1 suppresses Asense (As) expression in NBs, which promotes the formation of INPs. Here, Xie et al. show that the product of the button head gene (btd) works cooperatively with PntP1 to generate functional INPs.</p><p>They first show that btd is required for the maturation of INPs: In its absence, INPs are still generated but they undergo premature cell cycle exit and differentiate into Ganglion Mother Cells (GMCs). Xie et al. elegantly demonstrate that this phenotype is due to the ectopic expression of Prospero (Pros) in the nuclei of newly generated INPs. Thus, btd prevents premature differentiation of INPs by repressing early Pros expression. The authors then test whether the btd phenotype might be due to the loss of PntP1. They show that although PntP1 expression decreases in the NBs of btd mutant type-II lineages (leading to ectopic Ase expression in a subset of these NBs), this defect is not at the origin of the loss of mature INPs. It is instead a secondary effect of the ectopic expression of Pros. These results nicely confirm the key role played by btd in the formation of mature INPs. Finally, Xie et al. show that co-expressing btd with PntP1 in type-I NB lineages is sufficient to convert most of them into type-II NB lineages. The major finding reported is that the transcription factor encoding btd gene is required in INPs to prevent their premature differentiation, and that this occurs because btd suppresses the (nuclear) expression of the homeodomain protein Prospero in INP sublineages. These interesting data add to our knowledge of the molecular mechanisms involved in the control of the (limited) proliferative potential of INPs in the <italic>Drosophila</italic> neural stem cell model. This paper is interesting, well written, and the data are convincing.</p><p>Major concerns that need to be addressed:</p><p>1) Some of the data presented has been published previously and are not declared as such. The data in <xref ref-type="fig" rid="fig1">Figure 1A, A'</xref> in this paper correspond to that <xref ref-type="fig" rid="fig1">Figure 1B, B'</xref> in previously published paper by the same senior author (<xref ref-type="bibr" rid="bib53">Zhu et al., 2011</xref>), with only artwork and magnification changed. The authors should check all figures carefully to ensure that this does not happen and not recycle the same data.</p><p>2) The authors should mention clearly that the use of btd-Gal4 to study Btd expression and drive transgenes might not faithfully reproduce the endogenous expression pattern of Btd. There are two reasons for this. First, enhancer-reporter constructs do not necessarily reflect endogenous protein expression. Moreover, if enhancer-reporter constructs do mimic endogenous expression in one tissue, this does not prove that the constructs mimic endogenous expression in other tissues. Clearly, immunolabelling or <italic>in situ</italic> hybridization would be preferable and should be added to the paper if an antibody is available. If this is not available, the authors should make sure that they explain that they have made all the controls to test whether the Gal4 line reproduces the expression pattern. In particular, the ability to rescue the loss of function phenotype with the human construct is a good argument that the Gal4 line is expressed in the right tissue. Rescue with the fly UAS construct should be added to confirm this.</p><p>3) Second, the btd-Gal4 construct used by the authors is an insertion in the <italic>btd</italic> gene that actually causes a mutation in the gene (Estella et al., 5929). This is not a serious issue since the authors always use it as heterozygous (the authors should confirm this in the text), but this fact should be explained and the authors should explain the controls that explain why this is not an issue.</p><p>4) In some quantifications, the authors have a sample size of n=3. They should quantify at least 6 or 7 samples for each experiment to further support their conclusions.</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.03596.020</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p><italic>1) Some of the data presented has been published previously and are not declared as such. The data in <xref ref-type="fig" rid="fig1">Figure 1A, A'</xref> in this paper correspond to that <xref ref-type="fig" rid="fig1">Figure 1B, B'</xref> in previously published paper by the same senior author (<xref ref-type="bibr" rid="bib53">Zhu et al., 2011</xref>), with only artwork and magnification changed. The authors should check all figures carefully to ensure that this does not happen and not recycle the same data.</italic></p><p>We apologize that we reused an image that has been published before without realizing it. We did not intentionally recycle the data, but we really appreciate that the reviewers noticed this problem. We should have looked more carefully to make sure the same image was not used again. We have replaced the reused image in <xref ref-type="fig" rid="fig1">Figure 1A, A’</xref> with new ones with similar quality in the revision. We will also make sure that similar things do not happen again in the future.</p><p><italic>2) The authors should mention clearly that the use of btd-Gal4 to study Btd expression and drive transgenes might not faithfully reproduce the endogenous expression pattern of Btd. There are two reasons for this. First, enhancer-reporter constructs do not necessarily reflect endogenous protein expression. Moreover, if enhancer-reporter constructs do mimic endogenous expression in one tissue, this does not prove that the constructs mimic endogenous expression in other tissues. Clearly, immunolabelling or in situ hybridization would be preferable and should be added to the paper if an antibody is available. If this is not available, the authors should make sure that they explain that they have made all the controls to test whether the Gal4 line reproduces the expression pattern. In particular, the ability to rescue the loss of function phenotype with the human construct is a good argument that the Gal4 line is expressed in the right tissue. Rescue with the fly UAS construct should be added to confirm this.</italic></p><p>We agree with the reviewers that a GAL4 enhancer trap line like <italic>btd-GAL4</italic> does not always reflect endogenous expression patterns of the affected gene and it is essential to verify the <italic>btd-GAL4</italic> expression by immunostaining or <italic>in situ</italic> hybridization. However, we tried to generate btd antibodies using two different approaches (using a synthesized peptide and GST-btd fusion proteins), but had no luck. We also tried very hard to detect <italic>btd</italic> mRNAs by <italic>in situ</italic> hybridization. We got very nice in situ hybridization signals in the larval optic lobe (which is consistent with the expression of <italic>btd-GAL4</italic> in the optic lobe) and ventral imaginal discs, but we were not able to detect strong signals in the central brain or in the ventral nerve cord. Therefore, in order to test whether the <italic>btd-GAL4</italic> line reproduces the endogenous expression pattern, we tried to rescue the loss of function phenotype of Btd with the expression of mouse Sp8 construct (sorry we did not have the human Sp8 construct) or fly btd construct driven by <italic>btd-GAL4</italic> as reviewers suggested. Ideally this rescue should be done in <italic>btd</italic> mutant clones. However, we were not able to do such rescue experiments for the following two reasons:</p><p>(A) Although the <italic>GAL4</italic> insertion in the <italic>btd-GAL4</italic> line causes a lethal mutation of <italic>btd</italic>, we did not observe any obvious phenotypes in <italic>btd-GAL4</italic> homozygous mutant type II NB clones, indicating that the <italic>GAL4</italic> insertion does not affect the expression of <italic>btd</italic> in type II NB lineages (please see the new figure: <xref ref-type="fig" rid="fig6s1">Figure 6–figure supplement 1</xref>), which is possible given that the <italic>GAL4</italic> is inserted at 753bp upstream of the transcription start site of <italic>btd</italic> and the insertion of <italic>GAL4</italic> may not disrupt the enhancer elements that drive the expression of <italic>btd</italic> in type II NB lineages. The embryonic lethality of the <italic>btd-GAL4</italic> line could be due to the loss of Btd in other tissue or cells. Therefore, we could not do the rescue by using <italic>btd-GAL4</italic> to drive the expression of <italic>UAS-mSp8</italic> or <italic>UAS-btd</italic> in <italic>btd-GAL4</italic> homozygous mutant type II NB clones. In any event, we were able to rescue the lethality of <italic>btd-GAL4</italic> line by driving the expression of <italic>UAS-btd</italic> and type II NB lineages developed normally in those rescued <italic>btd-GAL4</italic> mutant larvae (data not shown, but we would be happy to provide images if needed).</p><p>(B) Since <italic>btd-GAL4</italic> is inserted in the promoter of <italic>btd</italic>, we could not do the rescue by using <italic>btd-GAL4</italic> to drive the expression of <italic>UAS-mSp8</italic> or <italic>UAS-btd</italic> in type II NB clones homozygous mutant for other <italic>btd</italic> mutant alleles, such as <italic>btd</italic><sup><italic>XG81</italic></sup>.</p><p>Therefore, in order to rescue the loss of function phenotype of Btd with the expression of mouse Sp8 or fly Btd driven by <italic>btd-GAL4</italic>, we tried to rescue the Btd RNAi knockdown phenotypes in type II NB lineages by the expression of <italic>UAS-mSp8</italic> or <italic>UAS-btd</italic> driven by <italic>btd-GAL4</italic>. In a new supplemental figure (<xref ref-type="fig" rid="fig6s2">Figure 6–figure supplement 2</xref>), we showed that Btd RNAi knockdown driven by <italic>btd-GAL4</italic> completely eliminated mature INPs in nearly all type II NB lineages, which is much stronger than that of Btd RNAi knockdown driven by <italic>pntP1-GAL4</italic> (this could also indicate that the expression of <italic>btd-GAL4</italic> is likely more close to the endogenous expression pattern of Btd than <italic>pntP1-GAL4</italic> so that the knockdown of Btd driven by <italic>btd-GAL4</italic> is more efficient <italic>pntP1-GAL4</italic>, although we cannot rule out the possibility that the higher RNAi knockdown efficiency is due to the difference of the expression levels of <italic>btd-GAL4</italic> and <italic>pntP1-GAL4</italic>). This Btd RNAi knockdown phenotype could be fully rescued by the expression of <italic>UAS-mSp8</italic> driven by the same <italic>btd-GAL4</italic>. However, the expression of <italic>UAS-btd</italic> driven by <italic>btd-GAL4</italic> could only partially rescue the Btd RNAi knockdown phenotype. The incomplete rescue by <italic>UAS-btd</italic> is most likely because the <italic>UAS-btd</italic> construct contains the target sequence of <italic>UAS-btd RNAi</italic>. In any event, the rescue of the Btd RNAi knockdown phenotypes by the expression of <italic>UAS-mSP8</italic> and <italic>UAS-btd</italic> driven by <italic>btd-GAL4,</italic> together with strong <italic>btd</italic> mutant phenotypes in type II NB lineages (and the failure of PntP1 to induced INP-like cells in btd mutant type I NB lineages), strongly argue that <italic>btd-GAL4</italic> reflects the endogenous expression pattern of <italic>btd</italic>.</p><p><italic>3) Second, the btd-Gal4 construct used by the authors is an insertion in the</italic> btd <italic>gene that actually causes a mutation in the gene (Estella et al., 5929). This is not a serious issue since the authors always use it as heterozygous (the authors should confirm this in the text), but this fact should be explained and the authors should explain the controls that explain why this is not an issue</italic>.</p><p>Thanks for reviewers’ suggestions. We now mentioned in the text that the insertion of GAL4 transgene in the <italic>btd-GAL4</italic> enhancer trap line causes a lethal mutation of <italic>btd</italic> and we only used <italic>btd-GAL4</italic> heterozygous female larvae for phenotypic analyses. We also explained in the text that the generation of INPs are not affected in <italic>btd-GAL4</italic> heterozygous mutant type II NB lineages as shown in <xref ref-type="fig" rid="fig6">Figure 6A-A’</xref> or in <italic>btd-GAL4</italic> homozygous mutant type II NB clones as mentioned above (<xref ref-type="fig" rid="fig6s1">Figure 6–figure supplement 1</xref>). Therefore, <italic>btd-GAL4</italic> heterozygous mutant background would not affect the generation of INP-like cells and thus it should not cause any problems when the <italic>btd-GAL4</italic> line was used for our research.</p><p><italic>4) In some quantifications, the authors have a sample size of n=3. They should quantify at least 6 or 7 samples for each experiment to further support their conclusions.</italic></p><p>We have increased the sample size as the reviewers suggested and did the statistical analyses again based on new sample sizes. We also indicated in the figure legends the samples sizes are the number of brain lobes or the number of type II or type I NB lineages/clones.</p></body></sub-article></article>