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| <?xml version="1.0" encoding="UTF-8"?><!DOCTYPE article PUBLIC "-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD v1.1d1 20130915//EN" "JATS-archivearticle1.dtd"><article 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">01305</article-id><article-id pub-id-type="doi">10.7554/eLife.01305</article-id><article-categories><subj-group subj-group-type="display-channel"><subject>Research article</subject></subj-group><subj-group subj-group-type="heading"><subject>Developmental biology and stem cells</subject></subj-group></article-categories><title-group><article-title>Deregulated FGF and homeotic gene expression underlies cerebellar vermis hypoplasia in CHARGE syndrome</article-title></title-group><contrib-group><contrib contrib-type="author" id="author-6899"><name><surname>Yu</surname><given-names>Tian</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-6900"><name><surname>Meiners</surname><given-names>Linda C</given-names></name><xref ref-type="aff" rid="aff2"/><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-6901"><name><surname>Danielsen</surname><given-names>Katrin</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="pa1">†</xref><xref ref-type="fn" rid="con4"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-6902"><name><surname>Wong</surname><given-names>Monica TY</given-names></name><xref ref-type="aff" rid="aff3"/><xref ref-type="other" rid="par-3"/><xref ref-type="fn" rid="con3"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-6903"><name><surname>Bowler</surname><given-names>Timothy</given-names></name><xref ref-type="aff" rid="aff4"/><xref ref-type="fn" rid="con5"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-1172"><name><surname>Reinberg</surname><given-names>Danny</given-names></name><xref ref-type="aff" rid="aff5"/><xref ref-type="other" rid="par-5"/><xref ref-type="fn" rid="con6"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-6904"><name><surname>Scambler</surname><given-names>Peter J</given-names></name><xref ref-type="aff" rid="aff6"/><xref ref-type="other" rid="par-4"/><xref ref-type="fn" rid="con7"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-6905"><name><surname>van Ravenswaaij-Arts</surname><given-names>Conny MA</given-names></name><xref ref-type="aff" rid="aff3"/><xref ref-type="fn" rid="con8"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" corresp="yes" id="author-6766"><name><surname>Basson</surname><given-names>M Albert</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff7"/><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-2"/><xref ref-type="fn" rid="con9"/><xref ref-type="fn" rid="conf2"/></contrib><aff id="aff1"><institution content-type="dept">Department of Craniofacial Development and Stem Cell Biology</institution>, <institution>King’s College London</institution>, <addr-line><named-content content-type="city">London</named-content></addr-line>, <country>United Kingdom</country></aff><aff id="aff2"><institution content-type="dept">Department of Radiology, University Medical Center Groningen</institution>, <institution>University of Groningen</institution>, <addr-line><named-content content-type="city">Groningen</named-content></addr-line>, <country>Netherlands</country></aff><aff id="aff3"><institution content-type="dept">Department of Genetics, University Medical Center Groningen</institution>, <institution>University of Groningen</institution>, <addr-line><named-content content-type="city">Groningen</named-content></addr-line>, <country>Netherlands</country></aff><aff id="aff4"><institution content-type="dept">Department of Internal Medicine</institution>, <institution>Montefiore Medical Center</institution>, <addr-line><named-content content-type="city">New York</named-content></addr-line>, <country>United States</country></aff><aff id="aff5"><institution content-type="dept">Department of Biochemistry and Molecular Pharmacology</institution>, <institution>Howard Hughes Medical Institute, New York University School of Medicine</institution>, <addr-line><named-content content-type="city">New York</named-content></addr-line>, <country>United States</country></aff><aff id="aff6"><institution content-type="dept">Molecular Medicine Unit</institution>, <institution>University College London Institute of Child Health</institution>, <addr-line><named-content content-type="city">London</named-content></addr-line>, <country>United Kingdom</country></aff><aff id="aff7"><institution content-type="dept">MRC Centre for Developmental Neurobiology</institution>, <institution>King’s College London</institution>, <addr-line><named-content content-type="city">London</named-content></addr-line>, <country>United Kingdom</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Krumlauf</surname><given-names>Robb</given-names></name><role>Reviewing editor</role><aff><institution>Stowers Institute for Medical Research</institution>, <country>United States</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>albert.basson@kcl.ac.uk</email></corresp><fn fn-type="present-address" id="pa1"><label>†</label><p>Neural Development Unit, University College London Institute of Child Health, London, United Kingdom</p></fn></author-notes><pub-date date-type="pub" publication-format="electronic"><day>24</day><month>12</month><year>2013</year></pub-date><pub-date pub-type="collection"><year>2013</year></pub-date><volume>2</volume><elocation-id>e01305</elocation-id><history><date date-type="received"><day>30</day><month>07</month><year>2013</year></date><date date-type="accepted"><day>09</day><month>11</month><year>2013</year></date></history><permissions><copyright-statement>© 2013, Yu et al</copyright-statement><copyright-year>2013</copyright-year><copyright-holder>Yu et al</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/3.0/"><license-p>This article is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/3.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="elife01305.pdf"/><related-article ext-link-type="doi" id="ra1" related-article-type="commentary" xlink:href="10.7554/eLife.01873"/><abstract><object-id pub-id-type="doi">10.7554/eLife.01305.001</object-id><p>Mutations in <italic>CHD7</italic> are the major cause of CHARGE syndrome, an autosomal dominant disorder with an estimated prevalence of 1/15,000. We have little understanding of the disruptions in the developmental programme that underpin brain defects associated with this syndrome. Using mouse models, we show that <italic>Chd7</italic> haploinsufficiency results in reduced <italic>Fgf8</italic> expression in the isthmus organiser (IsO), an embryonic signalling centre that directs early cerebellar development. Consistent with this observation, <italic>Chd7</italic> and <italic>Fgf8</italic> loss-of-function alleles interact during cerebellar development. CHD7 associates with <italic>Otx2</italic> and <italic>Gbx2</italic> regulatory elements and altered expression of these homeobox genes implicates CHD7 in the maintenance of cerebellar identity during embryogenesis. Finally, we report cerebellar vermis hypoplasia in 35% of CHARGE syndrome patients with a proven <italic>CHD7</italic> mutation. These observations provide key insights into the molecular aetiology of cerebellar defects in CHARGE syndrome and link reduced FGF signalling to cerebellar vermis hypoplasia in a human syndrome.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01305.001">http://dx.doi.org/10.7554/eLife.01305.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.01305.002</object-id><title>eLife digest</title><p>CHARGE syndrome is a rare genetic condition that causes various developmental abnormalities, including heart defects, deafness and neurological defects. In most cases, it is caused by mutations in a human gene called <italic>CHD7</italic>. CHD7 is known to control the expression of other genes during embryonic development, but the molecular mechanisms by which mutations in <italic>CHD7</italic> lead to the neural defects found in CHARGE syndrome are unclear.</p><p>During embryonic development, the neural tube—the precursor to the nervous system—is divided into segments, which give rise to different neural structures. The r1 segment, for example, forms the cerebellum, and the secretion of a protein called FGF8 (short for fibroblast growth factor 8) by a nearby structure called the isthmus organiser has an important role in this process. Since a reduction in FGF8 causes defects similar to those found in CHARGE syndrome, Yu et al. decided to investigate if the FGF signalling pathway was involved in this syndrome.</p><p>Mice should have two working copies of the <italic>Chd7</italic> gene, and mice that lack one of these suffer from symptoms similar to those of humans with CHARGE syndrome. Yu et al. examined the embryos of these mice and found that the isthmus organiser produced less FGF8. Embryos with no working copies of the gene completely lost the r1 segment. The loss of this segment appeared to be caused by changes in the expression of homeobox genes (the genes that determine the identity of brain segments).</p><p>Embryos that did not have any working copies of the <italic>Chd7</italic> gene died early in development, which made further studies impossible. However, embryos that had one working copy of the <italic>Chd7</italic> gene survived, and Yu et al. took advantage of this to study the effects of reduced FGF8 expression on these mice. These experiments showed that mice with just one working copy of the <italic>Fgf8</italic> gene and one working copy of the <italic>Chd7</italic> gene had a small cerebellar vermis. This part of the cerebellum is known to be very sensitive to changes in FGF8 signalling. Yu et al. then used an MRI scanner to look at the cerebellar vermis in patients with CHARGE syndrome, and found that more than half of the patients had abnormal cerebella.</p><p>In addition to confirming that studies on mouse embryos can provide insights into human disease, the work of Yu et al. add defects in the cerebellar vermis to the list of developmental abnormalities associated with CHARGE syndrome. The next step will be to test if any mutations in the human FGF8 gene can contribute to cerebellar defects in CHARGE syndrome, and to investigate if any other developmental defects in CHARGE syndrome are associated with abnormal FGF8 levels.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01305.002">http://dx.doi.org/10.7554/eLife.01305.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>cerebellum</kwd><kwd>CHARGE syndrome</kwd><kwd>CHD7</kwd><kwd>FGF8</kwd><kwd>OTX2</kwd><kwd>GBX2</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd>Human</kwd><kwd>Mouse</kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution>Wellcome Trust</institution></institution-wrap></funding-source><award-id>091475</award-id><principal-award-recipient><name><surname>Basson</surname><given-names>M Albert</given-names></name></principal-award-recipient></award-group><award-group id="par-2"><funding-source><institution-wrap><institution>Medical Research Council</institution></institution-wrap></funding-source><award-id>MR/K022377/1</award-id><principal-award-recipient><name><surname>Basson</surname><given-names>M Albert</given-names></name></principal-award-recipient></award-group><award-group id="par-3"><funding-source><institution-wrap><institution>Fund Nuts-Ohra</institution></institution-wrap></funding-source><award-id>1202-023</award-id><principal-award-recipient><name><surname>Wong</surname><given-names>Monica TY</given-names></name></principal-award-recipient></award-group><award-group id="par-4"><funding-source><institution-wrap><institution>British Heart Foundation</institution></institution-wrap></funding-source><award-id>PG/12/44/29658, RG/15/13/28570</award-id><principal-award-recipient><name><surname>Scambler</surname><given-names>Peter J</given-names></name></principal-award-recipient></award-group><award-group id="par-5"><funding-source><institution-wrap><institution>Howard Hughes Medical Institute</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Reinberg</surname><given-names>Danny</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>Mutations in CHD7, which cause CHARGE syndrome, cause a reduction in FGF8 signalling and subsequent abnormalities in the cerebellar vermis in both mice and humans.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="s1" sec-type="intro"><title>Introduction</title><p>The segmental organisation of the embryonic neural tube is imparted by the action of homeobox genes that show defined expression patterns along its anterior-posterior axis, in combination with growth factors secreted from distinct organising centres (<xref ref-type="bibr" rid="bib21">Kiecker and Lumsden, 2012</xref>). The cerebellum is derived from dorsal rhombomere 1 (r1), the anterior-most segment of the embryonic hindbrain. The survival and patterning of r1 is controlled by Fibroblast Growth Factor 8 (FGF8), secreted from the isthmus organiser (IsO), an organising centre located at the boundary between the embryonic midbrain (mesencephalon, mes) and r1 (reviewed by <xref ref-type="bibr" rid="bib30">Nakamura et al., 2005</xref>; <xref ref-type="bibr" rid="bib27">Martinez et al., 2013</xref>). The IsO forms at the expression boundary of two homeobox genes: <italic>Otx2</italic> (Orthodenticle Homeobox 2) in the anterior neural tube and, <italic>Gbx2</italic> (Gastrulation Brain Homeobox 2), in the posterior neural tube. <italic>Fgf8</italic> expression in the IsO is initiated at early (3–5) somite stages in the mouse embryo, resulting in a stable gene-regulatory network at the IsO, where (1) cross-repressive interactions between <italic>Otx2</italic> and <italic>Gbx2</italic> maintain the IsO, (2) <italic>Otx2</italic> represses <italic>Fgf8</italic> expression, thus restricting it to r1, and (3) <italic>Fgf8</italic> and <italic>Gbx2</italic> contribute to the maintenance of each other’s expression (reviewed by <xref ref-type="bibr" rid="bib20">Joyner et al., 2000</xref>; <xref ref-type="bibr" rid="bib27">Martinez et al., 2013</xref>).</p><p>Studies in the mouse embryo have shown that the level of FGF gene expression and signalling from the IsO has to be tightly controlled to ensure normal cerebellar development. Altered FGF signalling in the mes/r1 region preferentially affects the development of the medial cerebellum, the vermis (<xref ref-type="bibr" rid="bib46">Xu et al., 2000</xref>; <xref ref-type="bibr" rid="bib41">Trokovic et al., 2003</xref>; <xref ref-type="bibr" rid="bib3">Basson et al., 2008</xref>; <xref ref-type="bibr" rid="bib47">Yu et al., 2011</xref>), which is derived from precursors in the most anterior part of r1, closest to the source of FGF8 expression (<xref ref-type="bibr" rid="bib38">Sgaier et al., 2005</xref>). The observation that reduced FGF signalling results in hypoplasia of the cerebellar vermis in mice raises the possibility that reduced FGF signalling might underlie vermis hypoplasia in certain human conditions. However, studies in mice have also found that FGF signalling has many essential roles during development and even small reductions in <italic>Fgf8</italic> expression during embryonic development are incompatible with postnatal survival (<xref ref-type="bibr" rid="bib28">Meyers et al., 1998</xref>). These findings suggest that mutations causing sufficiently reduced <italic>Fgf8</italic> expression or signalling throughout the whole embryo to result in cerebellar defects are unlikely to yield viable offspring. Rather, it seems more likely that a disruption of the mechanisms that regulate local <italic>Fgf8</italic> expression at the IsO will be responsible for cerebellar vermis hypoplasia in humans.</p><p>CHARGE syndrome (MIM#214800) is an autosomal dominant disorder with an estimated prevalence of 1/15,000. Central nervous system defects have been reported in CHARGE (Coloboma of the eye, Heart defects, Atresia of the choanae, Retarded growth and development, Genital anomalies and Ear malformations or deafness) syndrome (<xref ref-type="bibr" rid="bib26">Lin et al., 1990</xref>; <xref ref-type="bibr" rid="bib40">Tellier et al., 1998</xref>; <xref ref-type="bibr" rid="bib4">Becker et al., 2001</xref>; <xref ref-type="bibr" rid="bib18">Issekutz et al., 2005</xref>; <xref ref-type="bibr" rid="bib34">Sanlaville et al., 2006</xref>; <xref ref-type="bibr" rid="bib35">Sanlaville and Verloes, 2007</xref>; <xref ref-type="bibr" rid="bib5">Bergman et al., 2011</xref>), including reports of cerebellar defects in pre-term CHARGE fetuses (<xref ref-type="bibr" rid="bib4">Becker et al., 2001</xref>; <xref ref-type="bibr" rid="bib34">Sanlaville et al., 2006</xref>; <xref ref-type="bibr" rid="bib25">Legendre et al., 2012</xref>). Depending on the clinical selection, 60–90% of the individuals suspected for CHARGE syndrome have de novo<italic>,</italic> heterozygous mutations in the <italic>CHD7</italic> (Chromodomain helicase DNA-binding protein 7, MIM#608892) gene (<xref ref-type="bibr" rid="bib42">Vissers et al., 2004</xref>; <xref ref-type="bibr" rid="bib6">Bilan et al., 2012</xref>; <xref ref-type="bibr" rid="bib19">Janssen et al., 2012</xref>). CHD7 is a member of the SNF2H-like chromatin-remodelling family and has been shown to function as a ‘transcriptional rheostat’ by maintaining appropriate levels of developmental gene expression (<xref ref-type="bibr" rid="bib37">Schnetz et al., 2010</xref>).</p><p>A number of clinical findings led us to hypothesise that some developmental defects in CHARGE syndrome might be caused by insufficient FGF signalling levels. For example, CHARGE syndrome shows significant clinical overlap with 22q11.2 deletion and Kallmann syndromes, both of which have been linked to reduced FGF signalling (<xref ref-type="bibr" rid="bib48">Scambler, 2010</xref>; <xref ref-type="bibr" rid="bib49">Miraoui et al., 2013</xref>; <xref ref-type="bibr" rid="bib50">Corsten-Janssen et al., 2013</xref>; <xref ref-type="bibr" rid="bib31">Randall et al., 2009</xref>). We therefore set out to test the hypothesis that CHD7 is required for normal levels of <italic>Fgf8</italic> expression during development by focusing on the embryonic IsO and cerebellar development.</p></sec><sec id="s2" sec-type="results|discussion"><title>Results and discussion</title><sec id="s2-1"><title>CHD7 regulates <italic>Fgf8</italic> expression levels in the IsO</title><p>We previously reported that mice heterozygous for the <italic>Chd7</italic><sup><italic>XK403</italic></sup> gene-trap allele (henceforth referred to as <italic>Chd7</italic><sup><italic>+/−</italic></sup> mice) phenocopy several aspects of CHARGE syndrome (<xref ref-type="bibr" rid="bib31">Randall et al., 2009</xref>). To determine whether FGF signalling at the IsO was affected by <italic>Chd7</italic> deletion, we first visualised the expression of <italic>Fgf8</italic> in E9.5 embryos by in situ hybridisation. <italic>Fgf8</italic> expression in the IsO appeared reduced in <italic>Chd7</italic><sup><italic>+/−</italic></sup> embryos and was substantially downregulated in <italic>Chd7</italic><sup><italic>−/−</italic></sup> embryos (<xref ref-type="fig" rid="fig1">Figure 1A–C</xref>). Quantitative RT-PCR analysis confirmed that <italic>Fgf8</italic> transcripts were reduced by 20% in <italic>Chd7</italic><sup><italic>+/−</italic></sup> embryos, and by 40% in <italic>Chd7</italic><sup><italic>−/−</italic></sup> embryos (<xref ref-type="fig" rid="fig1">Figure 1D</xref>). Furthermore, <italic>Fgf8</italic> expression was reduced by 80% in <italic>Chd7</italic><sup><italic>+/−</italic></sup><italic>;Fgf8</italic><sup><italic>+/−</italic></sup> embryos, compared to 40% reduction in <italic>Chd7</italic><sup><italic>+/+</italic></sup><italic>;Fgf8</italic><sup><italic>+/−</italic></sup> embryos (<xref ref-type="fig" rid="fig1">Figure 1D</xref>). To ask whether this synergistic genetic interaction between <italic>Chd7</italic> and <italic>Fgf8</italic> loss-of-function alleles translated to defects in FGF signalling, the expression of the FGF target gene <italic>Etv5</italic> was analysed (<xref ref-type="bibr" rid="bib32">Roehl and Nusslein-Volhard, 2001</xref>; <xref ref-type="bibr" rid="bib47">Yu et al., 2011</xref>). Whereas <italic>Etv5</italic> expression was clearly diminished in <italic>Chd7</italic><sup><italic>−/−</italic></sup> embryos compared to wildtype controls (<xref ref-type="fig" rid="fig1">Figure 1E,G</xref>), it did not appear substantially reduced in <italic>Chd7</italic><sup>+/−</sup> embryos (<xref ref-type="fig" rid="fig1">Figure 1F</xref>), an observation confirmed by quantitative RT-PCR (<xref ref-type="fig" rid="fig1">Figure 1H</xref>). However, quantitative analyses showed that <italic>Etv5</italic> expression was reduced by 50% in <italic>Chd7</italic><sup><italic>+/−</italic></sup><italic>;Fgf8</italic><sup><italic>+/−</italic></sup> embryos, compared to wildtype levels in <italic>Chd7</italic><sup><italic>+/−</italic></sup> and <italic>Fgf8</italic><sup><italic>+/−</italic></sup> embryos (<xref ref-type="fig" rid="fig1">Figure 1H</xref>). These data identified CHD7 as an upstream regulator of <italic>Fgf8</italic> in the IsO and revealed a synergistic relationship between the <italic>Chd7</italic> and <italic>Fgf8</italic> genes.<fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.01305.003</object-id><label>Figure 1.</label><caption><title>Reduced <italic>Fgf8</italic> expression and FGF signalling during early cerebellar development in <italic>Chd7</italic>-deficient embryos.</title><p>(<bold>A</bold>–<bold>C</bold>) In situ hybridisation for <italic>Fgf8</italic> at E9.5 shows a <italic>Chd7</italic> gene dosage-dependent reduction in <italic>Fgf8</italic> expression in the mid-hindbrain isthmus organiser (IsO, arrows). Scale bar = 0.5 mm. (<bold>D</bold>) Quantification of <italic>Fgf8</italic> transcript levels in the mes/r1 region of E9.5 embryos. (<bold>E</bold>–<bold>G</bold>) Expression of the FGF-regulated gene <italic>Etv5</italic> in E9.5 mouse embryos visualised by in situ hybridisation. (<bold>H</bold>) Quantification of <italic>Etv5</italic> gene expression in mes/r1 tissue confirms the in situ hybridisation data and indicates a significant reduction in FGF signalling in <italic>Chd7</italic><sup><italic>+/−</italic></sup><italic>;Fgf8</italic><sup><italic>+/−</italic></sup> and <italic>Chd7</italic><sup><italic>−/−</italic></sup> embryos. Data represents mean ± standard error of the mean (SEM) from three individual samples for each genotype. *p<0.05, **p<0.001.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01305.003">http://dx.doi.org/10.7554/eLife.01305.003</ext-link></p></caption><graphic xlink:href="elife01305f001"/></fig></p></sec><sec id="s2-2"><title>Synergistic interactions between <italic>Chd7</italic> and <italic>Fgf8</italic> loss-of-function alleles during development of the cerebellar vermis</title><p>Previous studies have shown that the medial cerebellum, the cerebellar vermis, is most sensitive to perturbations in FGF signalling during development (<xref ref-type="bibr" rid="bib7">Broccoli et al., 1999</xref>; <xref ref-type="bibr" rid="bib46">Xu et al., 2000</xref>; <xref ref-type="bibr" rid="bib41">Trokovic et al., 2003</xref>; <xref ref-type="bibr" rid="bib3">Basson et al., 2008</xref>; <xref ref-type="bibr" rid="bib47">Yu et al., 2011</xref>), hence we predicted that <italic>Chd7</italic> deficiency will predispose embryos to cerebellar vermis defects. Cerebellar size was normal in <italic>Chd7</italic><sup><italic>+/−</italic></sup> and <italic>Fgf8</italic><sup><italic>+/−</italic></sup> animals compared to wildtype littermates (<xref ref-type="fig" rid="fig2">Figure 2A–C</xref>), consistent with the observation that FGF signalling was not substantially reduced in these mutants (<xref ref-type="fig" rid="fig1">Figure 1H</xref>). To accurately compare the sizes of the cerebellar regions between mice, the volumes of cerebellar hemispheres, paravermis and vermis were calculated from surface area measurements taken from serial sections through postnatal day (P)21 cerebella. This analysis confirmed that cerebellar size was not significantly altered in <italic>Chd7</italic><sup><italic>+/−</italic></sup> or <italic>Fgf8</italic><sup><italic>+/−</italic></sup> mice (<xref ref-type="fig" rid="fig2">Figure 2E</xref>). Furthermore, cerebellar foliation in the vermis and hemispheres appeared normal in the mutants (<xref ref-type="fig" rid="fig2">Figure 2A’–C’,A”–C”</xref>). As <italic>Chd7</italic><sup><italic>−/−</italic></sup> embryos die by E11.5, cerebellar development could not be analysed in these mutants. However, <italic>Chd7</italic><sup><italic>+/−</italic></sup><italic>;Fgf8</italic><sup><italic>+/−</italic></sup> animals survive and an analysis of cerebellar size revealed a significant reduction in size owing to vermis aplasia (<xref ref-type="fig" rid="fig2">Figure 2D,D’,E</xref>). The cerebellar hemispheres were of normal size (<xref ref-type="fig" rid="fig2">Figure 2E</xref>) and had normal foliation compared to the controls (<xref ref-type="fig" rid="fig2">Figure 2D”</xref>). Cerebellar vermis aplasia in <italic>Chd7</italic><sup><italic>+/−</italic></sup><italic>;Fgf8</italic><sup><italic>+/−</italic></sup> animals was already present at birth, confirming that defects arose during embryonic development (<xref ref-type="fig" rid="fig2">Figure 2F–I,F’–I’</xref>, red asterisk). We also noted that the posterior midbrain (inferior colliculus), another region that is particularly sensitive to FGF signalling levels, was abnormal in <italic>Chd7</italic><sup><italic>+/−</italic></sup><italic>;Fgf8</italic><sup><italic>+/−</italic></sup> mutants (<xref ref-type="fig" rid="fig2">Figure 2I’</xref>, black asterisk). These observations provided functional evidence for a synergistic <italic>Chd7-Fgf8</italic> interaction and indicated that the potential phenotypic consequences of diminished <italic>Fgf8</italic> expression in <italic>Chd7</italic><sup><italic>+/−</italic></sup> embryos could be revealed by <italic>Fgf8</italic> heterozygosity.<fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.01305.004</object-id><label>Figure 2.</label><caption><title><italic>Chd7</italic> and <italic>Fgf8</italic> loss-of-function alleles interact to cause cerebellar vermis aplasia in the mouse.</title><p>(<bold>A</bold>–<bold>D</bold>) Wholemount views of the mouse cerebellum at P21. The cerebellar vermis is indicated by a double-headed arrow. <italic>Chd7</italic><sup><italic>+/−</italic></sup> animals have normal cerebella, indistinguishable from wildtype and <italic>Fgf8</italic><sup><italic>+/−</italic></sup> control littermates. <italic>Chd7</italic><sup><italic>+/−</italic></sup><italic>;Fgf8</italic><sup><italic>+/−</italic></sup> animals exhibit vermis aplasia (asterisk in <bold>D</bold>). Scale bar = 5 mm. (A’-D’) Cresyl violet-stained sagittal sections through the cerebellar vermis. Note the absence of cerebellar vermis tissue in <italic>Chd7</italic><sup><italic>+/−</italic></sup><italic>;Fgf8</italic><sup><italic>+/−</italic></sup> embryos (<bold>D’</bold>). (<bold>A”</bold>–<bold>D”</bold>) Sagittal sections through cerebellar hemispheres. (<bold>E</bold>) Measurements of cerebellar vermis, paravermis and hemisphere sizes in brains from the indicated genotypes. The data represents the mean of three samples with error bars indicating SEM. **p<0.001. (<bold>F</bold>–<bold>I</bold>) Wholemount views of cerebella at birth (P0), with vermis indicated by arrows. (<bold>F’</bold>–<bold>I’</bold>) Sagittal sections through P0 brains with inferior colliculus (IC) and cerebellum (Cb) indicated. Note the loss of cerebellar vermis (red asterisk) and abnormal IC (black asterisk) in <italic>Chd7</italic><sup><italic>+/−</italic></sup><italic>;Fgf8</italic><sup><italic>+/−</italic></sup> animals (<bold>I’</bold>). Scale bar = 1 mm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01305.004">http://dx.doi.org/10.7554/eLife.01305.004</ext-link></p></caption><graphic xlink:href="elife01305f002"/></fig></p></sec><sec id="s2-3"><title>Deregulated homeobox gene expression and altered r1 identity in the absence of CHD7</title><p>The <italic>Chd7</italic> gene encodes a SNF2H-like chromatin remodelling factor that is characterised by the presence of tandem chromodomains in its N-terminal region. Genome-wide chromatin immunoprecipitation studies in cell lines have shown that CHD7 is recruited to distal gene regulatory elements, presumably through interactions between CHD7 chromodomains and methylated lysine 4 residues in histone 3 (H3K4me), present at regulatory elements (<xref ref-type="bibr" rid="bib36">Schnetz et al., 2009</xref>, <xref ref-type="bibr" rid="bib37">2010</xref>; <xref ref-type="bibr" rid="bib11">Engelen et al., 2011</xref>). The <italic>Drosophila</italic> homologue of the <italic>CHD7</italic> subfamily, <italic>kismet</italic>, was identified as a Trithorax gene and <italic>kismet</italic> mutants have reduced expression of homeotic genes and consequent transformations of body segments to more anterior structures (<xref ref-type="bibr" rid="bib9">Daubresse et al., 1999</xref>). We therefore asked whether CHD7 has a role in maintaining the expression of homeobox genes that impart regional identity in the developing neural tube. The homeobox genes <italic>Otx2</italic> and <italic>Gbx2</italic> influence anterior and posterior identity in the developing embryo, respectively, position the IsO and regulate the levels of <italic>Fgf8</italic> expression (<xref ref-type="bibr" rid="bib7">Broccoli et al., 1999</xref>; <xref ref-type="bibr" rid="bib13">Hidalgo-Sanchez et al., 1999</xref>; <xref ref-type="bibr" rid="bib29">Millet et al., 1999</xref>; <xref ref-type="bibr" rid="bib20">Joyner et al., 2000</xref>; <xref ref-type="bibr" rid="bib12">Heimbucher et al., 2007</xref>). The analysis of <italic>Chd7</italic><sup><italic>−/−</italic></sup> mouse embryos at E8.25 (4ss), shortly after the initiation of <italic>Fgf8</italic> expression in the mes/r1 region revealed <italic>Otx2</italic> upregulation and posterior expansion of its expression (<xref ref-type="fig" rid="fig3">Figure 3A,B</xref>, arrow indicating expanded expression). To investigate how the altered <italic>Otx2</italic> expression domain related spatially to other hindbrain regions, we combined <italic>Otx2</italic> in situ hybridisation with markers to visualise r3+r5 (<italic>Krox20</italic>) and r2 (<italic>Hoxa2</italic>) in the same embryo. Although <italic>Krox20</italic> expression is reduced in <italic>Chd7</italic> mutant embryos (<xref ref-type="bibr" rid="bib2">Alavizadeh et al., 2001</xref>), r3 was still clearly marked, confirming the expansion of <italic>Otx2</italic> expression towards r3 (<xref ref-type="fig" rid="fig3">Figure 3C,D</xref>). Combined <italic>Otx2/Hoxa2</italic> in situ hybridisation experiments suggested that the expansion of <italic>Otx2</italic> expression included most of r1, as indicated by the absence of the <italic>Otx2/Hoxa2</italic>-negative r1 in <italic>Chd7</italic><sup><italic>−/−</italic></sup> embryos (<xref ref-type="fig" rid="fig3">Figure 3E,F</xref>).<fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.01305.005</object-id><label>Figure 3.</label><caption><title><italic>Chd7</italic> loss results in <italic>Otx2</italic> de-repression, loss of rhombomere 1 identity and reduced <italic>Fgf8</italic> expression.</title><p>(<bold>A</bold> and <bold>B</bold>) In situ hybridisation for <italic>Otx2</italic> in 4 somite stage (ss) embryos. Note the posterior expansion of <italic>Otx2</italic> expression in the mutant embryo (arrow in <bold>B</bold>). (<bold>C</bold> and <bold>D</bold>) In situ hybridisation for <italic>Otx2</italic> and <italic>Krox20</italic> to mark the forebrain/mesencephalon and rhombomeres 3 and 5 (r3 and r5), respectively in 6 ss embryos. Note the posterior expansion of <italic>Otx2</italic> (arrow) towards r3. (<bold>E</bold> and <bold>F</bold>) In situ hybridisation for <italic>Otx2</italic> and <italic>Hoxa2,</italic> to mark the forebrain/mesencephalon and r2, respectively in 6 ss embryos. Note the posterior expansion of <italic>Otx2</italic> (arrow) and apparent loss of the <italic>Otx2-Hoxa2</italic>-negative r1 in the <italic>Chd7</italic><sup><italic>−/−</italic></sup> embryo. (<bold>G</bold> and <bold>H</bold>) <italic>Fgf8</italic> in situ hybridisation on 6 ss embryos. Note the initiation of <italic>Fgf8</italic> expression at the correct position in the mutant (<bold>H</bold>), despite posteriorised <italic>Otx2</italic> expression. (<bold>I</bold> and <bold>J</bold>) Side-by-side comparison of <italic>Fgf8</italic> and <italic>Otx2/Hoxa2</italic> expression in 6 ss <italic>Chd7</italic><sup><italic>+/+</italic></sup> and <italic>Chd7</italic><sup><italic>−/−</italic></sup> embryos. Note the posterior expansion of <italic>Otx2</italic> expression (white arrow) and downregulated <italic>Fgf8</italic> expression in the <italic>Chd7</italic><sup><italic>−/−</italic></sup> embryos, compared to wildtype controls. Also note that <italic>Fgf8</italic> expression is initiated at the correct position in the <italic>Chd7</italic><sup><italic>−/−</italic></sup> embryo, with no evidence of a repositioning of the IsO in response to posterior expansion of <italic>Otx2</italic> at this stage of development. (<bold>K</bold> and <bold>L</bold>) In situ hybridisation for <italic>Gbx2</italic> suggesting the loss of r1 identity by E9. (<bold>M</bold>) Summary of regulatory interactions at the IsO in <italic>Chd7</italic><sup><italic>+/+</italic></sup> vs <italic>Chd7</italic><sup><italic>−/−</italic></sup> embryos. The loss of <italic>Otx2</italic> repression and <italic>Gbx2</italic> maintenance by CHD7 are predicted to result in reduced <italic>Fgf8</italic> expression in <italic>Chd7-</italic>deficient embryos. mes = mesencephalon, r1 = rhombomere 1, r2 = rhombomere 2, IsO = isthmus organiser.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01305.005">http://dx.doi.org/10.7554/eLife.01305.005</ext-link></p></caption><graphic xlink:href="elife01305f003"/></fig></p><p>We further confirmed that the posterior expansion of <italic>Otx2</italic> expression in these early <italic>Chd7</italic><sup><italic>−/−</italic></sup> embryos was associated with reduced <italic>Fgf8</italic> expression (<xref ref-type="fig" rid="fig3">Figure 3G,H</xref>), in agreement with previously reported repression of <italic>Fgf8</italic> expression by OTX2 (<xref ref-type="bibr" rid="bib1">Acampora et al., 2001</xref>; <xref ref-type="bibr" rid="bib12">Heimbucher et al., 2007</xref>). A side-by-side comparison of stage-matched embryos indicated that <italic>Fgf8</italic> expression was initiated at the correct position in <italic>Chd7</italic><sup><italic>−/−</italic></sup> embryos (<xref ref-type="fig" rid="fig3">Figure 3I,J</xref>). This observation showed that the posterior expansion of <italic>Otx2</italic> expression was not associated with a re-positioning of the IsO and indicated that <italic>Otx2</italic> was mis-expressed in the anterior hindbrain of <italic>Chd7</italic><sup><italic>−/−</italic></sup> embryos. Therefore, we asked whether the abnormal expansion of <italic>Otx2</italic> expression into the hindbrain just posterior to the IsO affected the identity of r1. Indeed, expression of the homeobox gene <italic>Gbx2</italic>, a marker of r1, was downregulated in <italic>Chd7</italic><sup><italic>−/−</italic></sup> embryos (<xref ref-type="fig" rid="fig3">Figure 3K,L</xref>). These findings are consistent with the known regulatory interactions between <italic>Otx2</italic>, <italic>Gbx2</italic> and <italic>Fgf8</italic> (<xref ref-type="fig" rid="fig3">Figure 3M</xref>) (<xref ref-type="bibr" rid="bib7">Broccoli et al., 1999</xref>; <xref ref-type="bibr" rid="bib29">Millet et al., 1999</xref>). We conclude that CHD7 functions as a key regulator of homeobox gene expression in the early neural tube and that the loss of <italic>Chd7</italic> results in the altered expression of <italic>Otx2</italic> and <italic>Gbx2,</italic> and the concomitant transformation of r1 into a more anterior identity. Interestingly, the effect of <italic>Chd7</italic> mutation on <italic>Otx2</italic> expression appears to be highly context-dependent as <italic>Otx2</italic> is reported to be downregulated in the otic and olfactory regions of <italic>Chd7</italic>-deficient embryos (<xref ref-type="bibr" rid="bib15">Hurd et al., 2010</xref>; <xref ref-type="bibr" rid="bib24">Layman et al., 2011</xref>).</p></sec><sec id="s2-4"><title>CHD7 is associated with <italic>Otx2</italic> and <italic>Gbx2</italic> regulatory elements</title><p>The data presented thus far indicated that CHD7 is required for normal <italic>Otx2</italic> and <italic>Gbx2</italic> gene expression. We therefore sought evidence for CHD7 recruitment to <italic>Otx2</italic> and <italic>Gbx2</italic> regulatory regions. CHD7-associated chromatin was isolated from the mes/r1 region of E9.5 embryos by chromatin immunoprecipitation (ChIP), and genomic DNA fragments (indicated as #1–#10 in <xref ref-type="fig" rid="fig4">Figure 4A</xref>) quantified by qPCR. Specific CHD7 binding was observed at three <italic>Otx2</italic> enhancer elements identified by Kurokawa et al. (<xref ref-type="bibr" rid="bib22">Kurokawa et al., 2004a</xref>, <xref ref-type="bibr" rid="bib23">2004b</xref>). The FM1 enhancer, located ∼71–73 kb upstream (#8 in <xref ref-type="fig" rid="fig4">Figure 4A</xref>), and the FM2 enhancer (#1 in <xref ref-type="fig" rid="fig4">Figure 4A</xref>), located ∼118 kb downstream of <italic>Otx2</italic> are by themselves sufficient to direct gene expression to the forebrain and midbrain after E9, and deletion of both enhancers together results in a smaller rostral brain and expanded r1 (<xref ref-type="bibr" rid="bib33">Sakurai et al., 2010</xref>). DNA fragments within these Otx2 enhancers (#1, #8) were specifically enriched by CHD7 ChIP (<xref ref-type="fig" rid="fig4">Figure 4A</xref>). In addition, CHD7 was also present at the AN enhancer (#9 and #10 in <xref ref-type="fig" rid="fig4">Figure 4A</xref>) that can direct gene expression to the epiblast and anterior neuroectoderm prior to E9.0 (<xref ref-type="bibr" rid="bib23">Kurokawa et al., 2004b</xref>), consistent with the observation that <italic>Otx2</italic> expression was altered in E7.5 embryos (data not shown). We also detected CHD7 association with regions downstream of <italic>Otx2</italic> (#3), and the promotor regions for <italic>Otx2.1</italic> (#6) and <italic>Otx2.2</italic> (#7) transcripts. No specific enrichment was detected at two negative control regions (#2 and #4). These data suggested that CHD7 is recruited to several key <italic>Otx2</italic> regulatory elements in the embryonic mes/r1 region.<fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.01305.006</object-id><label>Figure 4.</label><caption><title>Association of CHD7 with <italic>Otx2</italic> and <italic>Gbx2</italic> regulatory regions in the mes/r1 region.</title><p>(<bold>A</bold>) Genomic map of the mouse <italic>Otx2</italic> locus. The transcriptional start sites of <italic>Otx2.1</italic> and <italic>Otx2.2</italic> transcripts are indicated by arrows and exons by tan-coloured boxes. Positions on chromosome 14 indicated above the diagram are according to the mm9 genome assembly and numbers below the horizontal lines indicate approximate positions relative to the <italic>Otx2.2</italic> transcriptional start site. Known <italic>Otx2</italic> enhancer regions FM1, FM2 and AN are indicated by blue boxes (<xref ref-type="bibr" rid="bib22">Kurokawa et al., 2004a</xref>, <xref ref-type="bibr" rid="bib23">2004b</xref>). The location of DNA fragments amplified by qPCR after ChIP are indicated by rectangular boxes numbered #1–#10. Open boxes indicate negative control regions. ChIP-qPCR data are presented in a graph, with % of input DNA on the Y-axis and amplified region on the X-axis. Results from ChIP reactions using a CHD7-specific antiserum are in magenta and control Ig in turquoise. Error bars indicate standard deviation from reactions performed in triplicate. (<bold>B</bold>) Genomic map of the mouse <italic>Gbx2</italic> locus with the transcriptional start site (TSS) indicated by an arrow and exons by tan-coloured boxes. Positions on chromosome 1 indicated above the diagram are according to the mm9 genome assembly and numbers below the horizontal lines indicate approximate positions relative to the TSS. The location of DNA fragments amplified by qPCR after ChIP are indicated by rectangular boxes numbered #1–#6. Open boxes indicate negative control regions. ChIP-qPCR data are presented in a graph, with % of input DNA on the Y-axis and amplified region on the X-axis. Results from ChIP reactions using a CHD7-specific antiserum are in magenta and control Ig in turquoise. Error bars indicate standard deviation from reactions performed in triplicate.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01305.006">http://dx.doi.org/10.7554/eLife.01305.006</ext-link></p></caption><graphic xlink:href="elife01305f004"/></fig></p><p>A regulatory region 6 kb upstream of zebrafish <italic>Gbx2</italic> capable of driving gene expression in r1, has been described by <xref ref-type="bibr" rid="bib17">Islam et al. (2006)</xref>. ChIP-qPCR experiments identified substantial CHD7 recruitment to a region 5–6.25 kb upstream (#3, #4 and #5 in <xref ref-type="fig" rid="fig4">Figure 4B</xref>) as well as 3.7 kb upstream of <italic>Gbx2</italic> (#1) in mes/r1 tissue. These observations suggested that CHD7 might regulate <italic>Gbx2</italic> expression in r1 by interacting with <italic>Gbx2</italic> regulatory elements. However, further experiments will be required to test whether these regions do indeed control <italic>Gbx2</italic> expression in mouse r1.</p><p>Taken together, our observations support the supposition that homeobox genes represent key CHD7 targets. The mechanisms controlling CHD7 recruitment to regulatory regions and the action whereby CHD7 might affect gene expression in the embryo remain to be elucidated.</p></sec><sec id="s2-5"><title>Cerebellar defects of CHARGE syndrome patients</title><p>Cerebellar defects have been reported in pre-term CHARGE fetuses (<xref ref-type="bibr" rid="bib4">Becker et al., 2001</xref>; <xref ref-type="bibr" rid="bib34">Sanlaville et al., 2006</xref>; <xref ref-type="bibr" rid="bib25">Legendre et al., 2012</xref>). To determine whether cerebellar defects are a common post-natal feature of CHARGE syndrome, we systematically examined cerebellar structure in a cohort of 20 patients with CHARGE syndrome and mutations in the <italic>CHD7</italic> gene. MRI scans revealed cerebellar defects in 55% (11/20) of these patients (<xref ref-type="fig" rid="fig5">Figure 5</xref>; <xref ref-type="table" rid="tbl1">Table 1</xref>). Patients exhibited cerebellar vermis hypoplasia, varying from slight to pronounced hypoplasia (35%, 7/20, <xref ref-type="fig" rid="fig5">Figure 5B,C</xref>) and an anticlockwise rotated vermis (35%, 7/20, <xref ref-type="fig" rid="fig5">Figure 5B,C’</xref>). As a consequence of these abnormalities, fluid-filled spaces surrounding the cerebellum appeared larger. Examples of large foramen of Magendi and fourth ventricle (50%, 10/20) and large subcerebellar cistern (25%, 4/20) are indicated in <xref ref-type="fig" rid="fig5">Figure 5B–D</xref>. Thus, cerebellar defects in CHARGE syndrome have some clinical similarities to Dandy-Walker malformations (vermis hypoplasia and anticlockwise rotated vermis), without the overt posterior fossa enlargement typical of Dandy-Walker malformation (<xref ref-type="bibr" rid="bib10">Doherty et al., 2013</xref>). Two patients with vermis hypoplasia exhibited broad gait or ataxia, consistent with defects that disrupt cerebellar function (<xref ref-type="table" rid="tbl1">Table 1</xref>). Furthermore, 25% (5/20) of the patients had foliation abnormalities (<xref ref-type="fig" rid="fig5">Figure 5D,D’</xref>, <xref ref-type="table" rid="tbl1">Table 1</xref>), implying additional roles for CHD7 during the process of foliation. We conclude that a substantial proportion of patients with CHARGE syndrome present with cerebellar vermis hypoplasia. The incomplete penetrance of cerebellar vermis defects in patients with <italic>CHD7</italic> mutations is consistent with our studies in the mouse, which suggests that mutations in FGF pathway genes are likely to substantially modify the severity of cerebellar defects in CHARGE syndrome.<fig id="fig5" position="float"><object-id pub-id-type="doi">10.7554/eLife.01305.007</object-id><label>Figure 5.</label><caption><title>Representative sagittal MRI scans of CHARGE syndrome patients.</title><p>(<bold>A</bold>) Sagittal T1 scan of patient #18 showing a normal vermis with a normal position, foramen of Magendi (asterisk) and subcerebellar cistern (SC). The orientation of the cerebellum relative to the brainstem is indicated by two parallel white lines. (<bold>B</bold>) Sagittal T1 scan of patient #5 showing pronounced vermis hypoplasia with an anticlockwise rotated axis relative to the axis of the brainstem (arrow), and ensuing large foramen of Magendi (asterisk) and subcerebellar cistern (SC). Cerebellar hemispheres are normal (not shown). (<bold>C</bold> and <bold>C’</bold>) Illustrative sagittal T1 MRI images of patient #3 showing a slightly hypoplastic vermis. The white lines and arrow in <bold>C’</bold> indicate the anticlockwise-rotated axis of the vermis compared to the axis of the brainstem, with ensuing large foramen of Magendi (asterisk) and subcerebellar cistern (SC) indicated in <bold>C</bold>. (<bold>D</bold>) Transverse Inversion Recovery MRI image of patient #10 showing abnormal foliation in the caudal cerebellar hemispheres extending into the cerebellar tonsils (arrow). Also note a wide foramen of Magendi (asterisk). (<bold>D’</bold>) Transverse T2 MRI image of patient #11, with abnormal foliation in the anterior vermis indicated by an arrow. (<bold>E</bold>) Transverse Inversion Recovery image and (<bold>E’</bold>) T2 MRI image of a control patient with normal cerebellum.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01305.007">http://dx.doi.org/10.7554/eLife.01305.007</ext-link></p></caption><graphic xlink:href="elife01305f005"/></fig><table-wrap id="tbl1" position="float"><object-id pub-id-type="doi">10.7554/eLife.01305.008</object-id><label>Table 1.</label><caption><p>Cerebellar findings on MRI scans</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01305.008">http://dx.doi.org/10.7554/eLife.01305.008</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th>Patient</th><th>Sex; age at MRI (y;m)</th><th>Cerebellum</th><th>Suggestive neurological features<xref ref-type="table-fn" rid="tblfn1">*</xref> (age at last examination, y;m)</th><th colspan="2">CHD7 mutation</th></tr></thead><tbody><tr><td>1</td><td>M (1;1)</td><td>Pronounced vermis hypoplasia with anticlockwise rotated axis, large foramen of Magendi and large subcerebellar cistern, fissure vermis</td><td>None (1;1)</td><td>nonsense</td><td>934C>T</td></tr><tr><td>2</td><td>M (0;1)</td><td>Slight caudal vermis hypoplasia with slightly anticlockwise rotated axis, abnormal foliation, large foramen of Magendi, normal subcerebellar cistern</td><td>Ataxic gait (4;4)</td><td>nonsense</td><td>7160C>A</td></tr><tr><td>3</td><td>M (1;0)</td><td>Slight caudal vermis hypoplasia with anticlockwise rotated axis, large foramen of Magendi, large subcerebellar cistern (<xref ref-type="fig" rid="fig5">Figure 5C,C’</xref>)</td><td>None (12;4)</td><td>deletion</td><td>3202-?8994?del</td></tr><tr><td>4</td><td>F (0;3)</td><td>Slight caudal vermis hypoplasia, with anticlockwise rotated axis, large foramen of Magendi, normal subcerebellar cistern</td><td>None (2;2)</td><td>frameshift</td><td>7106delT</td></tr><tr><td>5</td><td>M (5;7)</td><td>Pronounced vermis hypoplasia, with anticlockwise rotated axis, large foramen of Magendi and large subcerebellar cistern (<xref ref-type="fig" rid="fig5">Figure 5B</xref>)</td><td>None (7;10)</td><td>frameshift</td><td>4779delT</td></tr><tr><td>6</td><td>M (0;1)</td><td>Slight caudal vermis hypoplasia, with anticlockwise rotated axis, large foramen of Magendi and large subcerebellar cistern</td><td>None (5;2)</td><td>frameshift</td><td>5680_5681delAG</td></tr><tr><td>7</td><td>F (2;9)</td><td>Slight caudal vermis hypoplasia, with slightly anticlockwise rotated axis, large foramen of Magendi and large subcerebellar cistern</td><td>Broad gait (11;6)</td><td>missense</td><td>3973T>G</td></tr><tr><td>8</td><td>M (1;8)</td><td>Large foramen of Magendi, large fourth ventricle (only on sagittal scans), normal subcerebellar cistern</td><td>None (12;2)</td><td>splice site</td><td>5535-7G>A</td></tr><tr><td>9</td><td>M (2;2)</td><td>Large foramen of Magendi, large fourth ventricle (only on sagittal scans), normal subcerebellar cistern. Abnormal foliation in anterior vermis</td><td>None (6;2)</td><td>nonsense</td><td>3173T>A</td></tr><tr><td>10</td><td>F (1;1)</td><td>Abnormal foliation caudal cerebellar hemispheres and tonsils, large foramen of Magendi (<xref ref-type="fig" rid="fig5">Figure 5D</xref>)</td><td>None (13;0)</td><td>splice site UV</td><td>3340A>T</td></tr><tr><td>11</td><td>F (15;10)</td><td>Abnormal foliation in anterior vermis (<xref ref-type="fig" rid="fig5">Figure 5D</xref>’)</td><td>None (18;0)</td><td>splice site</td><td>3990-1G>C</td></tr><tr><td>12</td><td>M (10;3)</td><td>Abnormal foliation in anterior vermis</td><td>Motor dyspraxia (16;10)</td><td>frameshift</td><td>5564dupC</td></tr><tr><td>13</td><td>M (0;1)</td><td>Normal (indented cranial pons)</td><td>None (0;11)</td><td>frameshift</td><td>1820_1821insTTGT</td></tr><tr><td>14</td><td>F (15;10)</td><td>Normal (large fourth ventricle)</td><td>None (20;6)</td><td>nonsense</td><td>4015C>T</td></tr><tr><td>15</td><td>F (0;1)</td><td>Normal, (split caudal vermis)</td><td>None (5;9)</td><td>nonsense</td><td>7879C>T</td></tr><tr><td>16</td><td>M (0;6)</td><td>Normal</td><td>Broad gait (10;6)</td><td>splice site</td><td>2238+1 G>A</td></tr><tr><td>17</td><td>M (1;10)</td><td>Normal</td><td>None (6;4)</td><td>nonsense</td><td>1480C>T</td></tr><tr><td>18</td><td>F (2;10)</td><td>Normal (<xref ref-type="fig" rid="fig5">Figure 5A</xref>)</td><td>None (17;3)</td><td>frameshift</td><td>7769delA</td></tr><tr><td>19</td><td>M (1;0)</td><td>Normal</td><td>None (16;9)</td><td>nonsense</td><td>1714C>T</td></tr><tr><td>20</td><td>M (6;3)</td><td>Normal</td><td>None (12;10)</td><td>splice site</td><td>2443+5 G>C</td></tr></tbody></table><table-wrap-foot><fn id="tblfn1"><label>*</label><p>all children show motor delay due to vestibular defects.</p></fn></table-wrap-foot></table-wrap></p><p>In summary, this study identifies the chromatin-remodelling factor CHD7 as a key upstream regulator of homeobox gene expression and positional identity in the early neural tube and demonstrates a connection between CHD7 haplo-insufficiency, reduced FGF signalling and cerebellar defects in a human syndrome. We propose that CHD7 remodels chromatin at multiple <italic>Otx2</italic> and <italic>Gbx2</italic> regulatory elements, thereby modifying higher order chromatin architecture and interactions with tissue-specific transcription factors at these loci. Although we cannot completely rule out the possibility that CHD7 also directly fine-tunes <italic>Fgf8</italic> expression in addition to affecting <italic>Otx2</italic> and <italic>Gbx2</italic> expression, the finding that <italic>Fgf8</italic> expression is not substantially changed in the pharyngeal region of <italic>Chd7</italic><sup><italic>−/−</italic></sup> embryos (compare e.g., <xref ref-type="fig" rid="fig3">Figure 3G</xref> with H), suggest that such effects will have to be mediated by CHD7 recruitment to tissue-specific <italic>Fgf8</italic> regulatory elements.</p><p>Our findings predict that mutations and epigenetic alterations of <italic>OTX2</italic> and <italic>GBX2</italic> regulatory regions are likely to contribute to cerebellar hypoplasia in humans and that <italic>OTX2, GBX2</italic> and <italic>FGF8</italic> deregulation might underlie other developmental defects associated with CHARGE syndrome.</p></sec></sec><sec id="s3" sec-type="materials|methods"><title>Materials and methods</title><sec id="s3-1"><title>Animals</title><p>The <italic>Chd7</italic><sup><italic>XK403</italic></sup> and <italic>Fgf8</italic><sup><italic>lacZ/+</italic></sup> loss-of-function alleles were maintained on C57BL/6J and C57BL/6J × DBA/2J F1 backgrounds for these studies (<xref ref-type="bibr" rid="bib16">Ilagan et al., 2006</xref>; <xref ref-type="bibr" rid="bib31">Randall et al., 2009</xref>). Tail DNA preparations were genotyped by PCR as described in the original publications. All animal procedures were approved by the UK Home Office.</p></sec><sec id="s3-2"><title>Histology and volumetric analysis</title><p>Brains were dissected in ice-cold PBS, fixed in 4% paraformaldehyde (PFA) overnight at 4°C, before dehydration and embedding in paraffin wax. Volumetric measurements were carried out on P0 and P21 cerebella. Serial, sagittal 10 μm sections of the cerebellum were dried overnight at 42°C, rehydrated and stained with 0.1% cresyl violet. Images of stained sections were taken and the cerebellar surface area on each section traced and measured by ImageJ. The total volume of each cerebellar region was calculated by multiplying the total surface area of all sections from the same region by the thickness of the sections. Vermis sections were selected as the most medial sections with clearly visible 10 lobules; paravermis sections were adjacent to the vermis sections, with diminishing lobules VIII, IX and X; sections lateral to paravermis were hemisphere sections.</p></sec><sec id="s3-3"><title>Whole-mount in situ hybridisation</title><p>Noon on the day a vaginal plug was observed was defined as embryonic day (E)0.5. Somite-stage embryos were staged more accurately by counting the number of somite pairs. After dissection in ice-cold PBS, embryos were fixed overnight in 4% PFA at 4°C, gradually dehydrated in a methanol series and in situ hybridisation carried out using standard procedures (<xref ref-type="bibr" rid="bib45">Wilkinson et al., 1989b</xref>). Digoxigenin-labelled antisense probes for <italic>Etv5</italic> (<xref ref-type="bibr" rid="bib14">Hippenmeyer et al., 2002</xref>), <italic>Fgf8</italic> (<xref ref-type="bibr" rid="bib8">Crossley and Martin, 1995</xref>), <italic>Gbx2</italic> (<xref ref-type="bibr" rid="bib43">Wassarman et al., 1997</xref>), <italic>Hoxa2</italic> (<xref ref-type="bibr" rid="bib44">Wilkinson et al., 1989a</xref>) and <italic>Otx2</italic> (<xref ref-type="bibr" rid="bib39">Simeone et al., 1993</xref>) were prepared using previously published constructs.</p></sec><sec id="s3-4"><title>Quantitative RT-PCR analyses</title><p>Total RNA was extracted from the mes/r1 region of at least three E9.5 embryos of each genotype using Trizol (Invitrogen, UK) with the addition of 20 µg Ultrapure Glycogen (Life Technologies, UK). A total of 200 ng of RNA was used for first-strand DNA synthesis with the nanoScript Precision RT kit (PrimerDesign Ltd., UK) using random hexamer primers. cDNA synthesis reactions without reverse transcriptase enzyme (no RT) were used as controls for quantitative RT-PCR. Quantitative RT-PCR was performed on a Rotor-Gene Q (Qiagen) using Precision qPCR MasterMix kit with SYBR green (Primerdesign Ltd., UK). All reactions were performed in triplicate. Cq threshold values were determined manually and all were at least 5 Cq values below no RT controls. The Cq values for each sample was normalised to the internal control gene <italic>Ywhaz</italic> (primers provided by Primerdesign) to give the ΔCq value. ΔΔCq values were calculated relative to wildtype samples. The primer sequences used were: <italic>Fgf8</italic>: forward 5′-AGGTCTCTACATCTGCATGAAC-3′, reverse 5′-TGTTCTCCAGCACGATCTCT-3′; <italic>Etv5</italic>: forward 5′-GCAGTTTGTCCCAGATTTTCA-3′, reverse 5′-GCAGCTCCCGTTTGATCTT-3′.</p></sec><sec id="s3-5"><title>Chromatin immunoprecipitation (ChIP)-qPCR</title><p>The embryonic mes/r1 region was dissected from E9.5 CD1 embryos, disrupted by trituration with a 23 G and 25 G needle, fixed for 10 min with 4% PFA, snap-frozen and stored at −80°C until use. After cell lysis and isolation of nuclei, samples were sonicated in a Bioruptor UCD-300 in 10 mM TRIS pH8, 1 mM EDTA, 0.5 mM EGTA, 0.5% N-lauroylsarcosine to 200–500 bp fragment size. Chromatin was immunoprecipiated with antiserum to CHD7 (ab31824, Abcam, UK) and control Ig (Abcam, UK). Complexes were captured with Protein G Dynabeads, washed with modified RIPA buffer (50 mM HEPES pH7.5, 1 mM EDTA, 0.3% Sodium deoxycholate, 1% NP40, 250 mM LiCl), eluted in 50 mM TRIS pH8, 10 mM EDTA, 1% SDS, cross-links reversed by overnight incubation at 65°C and DNA precipitated after phenol-chloroform extraction. Unique DNA fragments were amplified and quantified by qPCR using the primers in <xref ref-type="table" rid="tbl2 tbl3">Tables 2 and 3</xref>. Data were quantified relative to input DNA (% of input).<table-wrap id="tbl2" position="float"><object-id pub-id-type="doi">10.7554/eLife.01305.009</object-id><label>Table 2.</label><caption><p><italic>Otx2</italic> qPCR primers</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01305.009">http://dx.doi.org/10.7554/eLife.01305.009</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th>Region</th><th>Forward</th><th>Reverse</th></tr></thead><tbody><tr><td>#1</td><td>AAACTCACCATAATCCTCCTGCC</td><td>TCCTCCCCTTCTCCTCTAAACAGC</td></tr><tr><td>#2</td><td>CTGCTCTCCTCAACCTTCAGACTC</td><td>TTGCGTGCCTTACCTTACCG</td></tr><tr><td>#3</td><td>CAACCACTCAAGTCAAGCCTATCTG</td><td>TCTTCCTCTGCCTCCCAAGTTC</td></tr><tr><td>#4</td><td>CTGGCTGGTGGCTTCTGATT</td><td>TTAGGTATCGCCAGGTTGCC</td></tr><tr><td>#5</td><td>ACACCAACTTGCTGAACAACA</td><td>TCCAGACTACTAATTAGGTGAAAATGA</td></tr><tr><td>#6</td><td>GAAAACCAAAACCCAAACCACG</td><td>GAATGGAATCCTTAGCAAGCGG</td></tr><tr><td>#7</td><td>AACAGGCTTGTGTCCGTCTACG</td><td>CGCTTTCTCAGCAAATCTCCC</td></tr><tr><td>#8</td><td>CATTTTCTTGCCGTCCTGCC</td><td>AAAGTGTGCCTCCTGTGGTTCC</td></tr><tr><td>#9</td><td>AAAAACACTGGGGAAGAAAGGG</td><td>AAATAAGAGTCAGAAGAGCGGTGC</td></tr><tr><td>#10</td><td>GCTGAATCAAACATGAATGAGCC</td><td>CTGGGAGTAGACAACTGAGACA</td></tr></tbody></table></table-wrap><table-wrap id="tbl3" position="float"><object-id pub-id-type="doi">10.7554/eLife.01305.010</object-id><label>Table 3.</label><caption><p><italic>Gbx2</italic> qPCR primers</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01305.010">http://dx.doi.org/10.7554/eLife.01305.010</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th>Region</th><th>Forward (5’–3’)</th><th>Reverse (5’–3’)</th></tr></thead><tbody><tr><td>#1</td><td>CCCTTGGCTGGCTTTGAAAT</td><td>TCTGCCTTTTGTCCTGGAGA</td></tr><tr><td>#2</td><td>TGAATCCATAGCTTACCCGC</td><td>AGGAACAAAGGGGGAAAGAA</td></tr><tr><td>#3</td><td>CCAGGCTTTCATCTCTCGCA</td><td>ATAGGCCAAGCTAAGCACCC</td></tr><tr><td>#4</td><td>GGGAATGGTGGAATGAATGGC</td><td>TGAGGAGTGTGCTGAAGGGACAAC</td></tr><tr><td>#5</td><td>GTTGGCTGCCCTTTTCTTCA</td><td>ACCTCCATCTCCTCAGGCTA</td></tr><tr><td>#6</td><td>TGTAAACACTCCCTTCCCCGTATC</td><td>CCACCCTAAACCGAAATGCG</td></tr></tbody></table></table-wrap></p></sec><sec id="s3-6"><title>Patients</title><p>All patients included in this study are known at the Dutch expert clinic for CHARGE syndrome located at the University Medical Center Groningen (coordinated by CvRA). All patients had a pathogenic mutation in <italic>CHD7</italic> (<xref ref-type="table" rid="tbl1">Table 1</xref>, see also <ext-link ext-link-type="uri" xlink:href="http://www.CHD7.org">www.CHD7.org</ext-link>). Patients were all evaluated in person by CvRA. Patients and/or parents gave written consent for the collection and analysis of detailed phenotypic information according to national ethical guidelines. Phenotypic information collected also included radiological images. All information is stored in a secure database under a unique patient identification number.</p></sec><sec id="s3-7"><title>MRI scans</title><p>The MRI scans were made at different hospitals. A total of 23 MRI scans could be collected. Only MRI scans that allowed a reliable interpretation of the cerebellum, that is the presence of sagittal and axial images of the cerebellum, were included in this study (n = 20). All cerebellar images were evaluated on visual basis by an experienced neuroradiologist (LCM). MRI images of CHARGE patients were compared with images of age-matched controls. All observations were recorded by MTYW.</p></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>We thank the individuals with CHARGE syndrome and their families for participating in our research. Lies Hoefsloot supervised the diagnostic <italic>CHD7</italic> sequencing of the CHARGE patients. Jorieke van Kammen-Bergman assisted in the collection of cerebral MRI scans. Samantha Martin provided technical assistance and mouse husbandry. We thank Wee-Wei Tee and Shuzo Kaneko for advice on ChIP experiments, Gail Martin, Alex Joyner and John Rubenstein for mouse lines and in situ probes and members of the Basson laboratory and Anthony Graham, Clemens Kiecker and Mohi Ahmed for comments on the manuscript. This work is supported by grants from the Wellcome Trust (091475) and the Medical Research Council (MR/K022377/1) to MAB, Fund Nuts-Ohra (1202–023) to MTYW, and BHF grants PG/12/44/29658 and RG/15/13/28570 to PJS.</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>DR: Reviewing editor, <italic>eLife</italic>.</p></fn><fn fn-type="conflict" id="conf2"><p>The other 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>TY, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con2"><p>LCM, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con3"><p>MTYW, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con4"><p>KD, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con5"><p>TB, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con6"><p>DR, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con7"><p>PJS, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con8"><p>CMAR-A, Conception and design, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con9"><p>MAB, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn></fn-group><fn-group content-type="ethics-information"><title>Ethics</title><fn fn-type="other"><p>Human subjects: Research involving human subjects was carried out in accordance with the Declaration of Helsinki. Informed parental consent was obtained for the anonymous use of the molecular, clinical, and neuroradiological data reported in this manuscript. 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id="SA1"><front-stub><article-id pub-id-type="doi">10.7554/eLife.01305.011</article-id><title-group><article-title>Decision letter</article-title></title-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Krumlauf</surname><given-names>Robb</given-names></name><role>Reviewing editor</role><aff><institution>Stowers Institute for Medical Research</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://elife.elifesciences.org/review-process">review process</ext-link>). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.</p></boxed-text><p>Thank you for sending your work entitled “Deregulated FGF and homeotic gene expression underlies cerebellar vermis hypoplasia in CHARGE syndrome” for consideration at <italic>eLife</italic>. Your article has been favorably evaluated by a Senior editor and 4 reviewers, one of whom is a member of our Board of Reviewing Editors.</p><p>The following individuals responsible for the peer review of your submission have agreed to reveal their identity: Robb Krumlauf (Reviewing editor); Kathleen Millen and Mark Lewandowski (peer reviewers).</p><p>The Reviewing editor and the other 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>There was a uniform opinion among the reviewers that this was an interesting paper thath would be suitable for publication in <italic>eLife</italic> following revisions to address several concerns.</p><p>1) This study formally adds cerebellar malformation to the features of CHARGE, demonstrating that isthmic disruption can cause human cerebellar malformations as has long been demonstrated in mice and other models. However, the authors oversell the connection by claiming (in the Abstract and start of the section on cerebellar defects) that their mouse observations “led us to search for cerebellar defects...”. Rather, several previous clinical reports clearly described the occurrence of vermis deficiency as a non-random association with CHARGE, although the authors' series appears the largest in which an MRI study has been conducted.</p><p>2) An additional major concern is with the phenotypic features of the MRI data in <xref ref-type="table" rid="tbl1">Table 1</xref>. As Barkovich and others have pointed out, Dandy-Walker Variant is not acceptable terminology (see more recent references Barkovivh 2009; Aldinger 2009). Dandy-Walker malformation is a combination of cerebellar hypoplasia and posterior fossa enlargement. These MRI scans show cerebellar vermis hypoplasia. There is not clear evidence of an enlarged posterior fossa.</p><p>3) In the cases shown, the enlarged fourth ventricle is simply filling a normal-sized or perhaps even small posterior fossa. It is an illusion that the cisterna is enlarged. The cerebellum is small, so the space looks large. Other features are noted, but are not visible in the panels presented – in particular the large foramen of Magendi and indented dorsal pons, abnormal foliation in 2 cases. These issues in establishing the connections of cerebellar defects to CHARGE and the interpretation need to be properly dealt with in the text.</p><p>4) An important aspect of the work relates to the conclusion that <italic>Chd7</italic> directs binds regulatory regions of the <italic>Otx2</italic> locus. However there are concerns about the technical aspects of this data. The ChIP-PCR study of <italic>Otx2</italic> in <xref ref-type="fig" rid="fig4">Figure 4</xref> is weak and heavily influenced by a genome-wide ChIP-seq study of murine ES cells described by Schnetz et al. The authors (1) did not provide primer sequences for the regions of <italic>Otx2</italic> analysed (which were chosen from the Schnetz work) and more importantly (2) did not provide any negative control data, for example comparing with other regions of the <italic>Otx2</italic> gene sequence. Although there are some suggestive correlations with other epigenetic marks, there are too few analyses to contextualise the signal they obtained and therefore to interpret it, as they do, as “suggesting a role for CHD7 in directly regulating <italic>Otx2</italic> gene expression through interacting with these elements”. This part of the work would ideally need to be strengthened by conducting a complete PCR-based survey of the <italic>Otx2</italic> gene. Even then, since CHD7 may not bind DNA directly, the conclusions are indirect without a lot more information on co-binding factors. Since this is likely to be beyond the scope of this work, the authors should revise the text to interpret the direct action of <italic>Chd7</italic> on <italic>Otx2</italic> more circumspectly, taking into consideration these potential caveats.</p><p>5) Did they consider undertaking a similar study of <italic>Gbx2</italic>?</p><p>6) The data in <xref ref-type="fig" rid="fig3">Figure 3A,B</xref> do not clearly show differences in <italic>Gbx2</italic> and <italic>Otx2</italic> expression. While it is clear at the later stages the data at E7.5 are not very compelling and add little to the story. They should either be removed, or to clarify the embryonic data at E7.5 the authors should repeat the <italic>Otx2/Gbx2</italic> analysis with embryos that are more closely stage-matched. In particular, the <italic>Otx2</italic>-stained normal embryo is clearly older than the <italic>Chd7</italic> mutant.</p><p>7) The posterior expansion of <italic>Otx2</italic> is an important observation. <xref ref-type="fig" rid="fig3">Figure 3D</xref> shows a 6ss embryo doubly stained for both <italic>Otx2</italic> and <italic>Hoxa2</italic>. The expanded region of label is interpreted to be due to caudally expanded <italic>Otx2</italic> expression, but it could also be due to <italic>Hoxa2</italic> expression rostrally expanded from r2. This could be resolved with two color “in situs”. However, another solution is to stain solely for <italic>Otx2</italic> and place a photo of such a sample as an insert in 2D. Alternatively a more compelling way to illustrate the shifts and a potential transformation would be to employ <italic>Krox20</italic>. It is expressed in r3 and r5. This would indicate that the changes were restricted to r1 and not a truncation of the anterior hindbrain. This general issue needs to be addressed in one of the ways suggested above.</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.01305.012</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p><italic>1) This study formally adds cerebellar malformation to the features of CHARGE, demonstrating that isthmic disruption can cause human cerebellar malformations as has long been demonstrated in mice and other models. However, the authors oversell the connection by claiming (in the Abstract and start of the section on cerebellar defects) that their mouse observations “led us to search for cerebellar defects...”. Rather, several previous clinical reports clearly described the occurrence of vermis deficiency as a non-random association with CHARGE, although the authors' series appears the largest in which an MRI study has been conducted</italic>.</p><p>We have revised the text to clarify our findings in the context of previous reports on cerebellar vermis defects observed in pre-term CHARGE fetuses to avoid giving the impression that we are over-selling our clinical findings.</p><p>In the Abstract we simply state that: “Finally, we report cerebellar vermis hypoplasia in 35% of CHARGE syndrome patients with a proven <italic>CHD7</italic> mutation.”</p><p>And in the text: “Cerebellar defects have been reported in pre-term CHARGE fetuses (<xref ref-type="bibr" rid="bib4">Becker et al., 2001</xref>; <xref ref-type="bibr" rid="bib34">Sanlaville et al., 2006</xref>; <xref ref-type="bibr" rid="bib25">Legendre et al., 2012</xref>). To determine whether cerebellar defects are a common post-natal feature of CHARGE syndrome, we systematically examined cerebellar structure in a cohort of 20 patients with CHARGE syndrome and mutations in the <italic>CHD7</italic> gene.”</p><p><italic>2) An additional major concern is with the phenotypic features of the MRI data in</italic> <xref ref-type="table" rid="tbl1"><italic>Table 1</italic></xref><italic>. As Barkovich and others have pointed out, Dandy-Walker Variant is not acceptable terminology (see more recent references Barkovivh 2009; Aldinger 2009). Dandy-Walker malformation is a combination of cerebellar hypoplasia and posterior fossa enlargement. These MRI scans show cerebellar vermis hypoplasia. There is not clear evidence of an enlarged posterior fossa</italic>.</p><p>We thank the reviewers for pointing these out. We have changed the description by removing references to DWV and posterior fossa enlargement. We amended the text to clarify the relation between the observed cerebellar defects and Dandy-Walker malformations: “Thus, cerebellar defects in CHARGE syndrome have some clinical similarities to Dandy-Walker malformations (vermis hypoplasia and anticlockwise rotated vermis), without the overt posterior fossa enlargement typical of Dandy-Walker malformation (<xref ref-type="bibr" rid="bib10">Doherty et al., 2013</xref>).”</p><p><italic>3) In the cases shown, the enlarged fourth ventricle is simply filling a normal-sized or perhaps even small posterior fossa. It is an illusion that the cisterna is enlarged. The cerebellum is small, so the space looks large. Other features are noted, but are not visible in the panels presented – in particular the large foramen of Magendi and indented dorsal pons, abnormal foliation in 2 cases. These issues in establishing the connections of cerebellar defects to CHARGE and the interpretation need to be properly dealt with in the text</italic>.</p><p>We have amended the text to clarify that the large appearance of the foramen of Magendi and fourth ventricle and large subcerebellar cistern are as a consequence of vermis hypoplasia, making these spaces appear larger: “As a consequence of these abnormalities, fluid-filled spaces surrounding the cerebellum appeared larger. Examples of large foramen of Magendi and fourth ventricle (50%, 10/20) and large subcerebellar cistern (25%, 4/20) are indicated in <xref ref-type="fig" rid="fig5">Figure 5B, C and D</xref>.”</p><p><italic>4) An important aspect of the work relates to the conclusion that</italic> Chd7 <italic>directs binds regulatory regions of the</italic> Otx2 <italic>locus. However there are concerns about the technical aspects of this data. The ChIP-PCR study of</italic> Otx2 <italic>in</italic> <xref ref-type="fig" rid="fig4"><italic>Figure 4</italic></xref> <italic>is weak and heavily influenced by a genome-wide ChIP-seq study of murine ES cells described by Schnetz et al. The authors (1) did not provide primer sequences for the regions of</italic> Otx2 <italic>analysed (which were chosen from the Schnetz work) and more importantly (2) did not provide any negative control data, for example comparing with other regions of the</italic> Otx2 <italic>gene sequence. Although there are some suggestive correlations with other epigenetic marks, there are too few analyses to contextualise the signal they obtained and therefore to interpret it, as they do, as “suggesting a role for CHD7 in directly regulating</italic> Otx2 <italic>gene expression through interacting with these elements”. This part of the work would ideally need to be strengthened by conducting a complete PCR-based survey of the</italic> Otx2 <italic>gene. Even then, since CHD7 may not bind DNA directly, the conclusions are indirect without a lot more information on co-binding factors. Since this is likely to be beyond the scope of this work, the authors should revise the text to interpret the direct action of</italic> Chd7 <italic>on</italic> Otx2 <italic>more circumspectly, taking into consideration these potential caveats</italic>.</p><p>We thank the reviewers for these criticisms, which have allowed us to improve our analysis of CHD7 association with Otx2 regulatory elements significantly. First, we performed an unbiased screen of the Otx2 gene that led to the identification of several regions that show specific CHD7 association, as well as adjacent negative control regions that show no CHD7 binding. Secondly, instead of being led by the Schnetz ES cell data, we tested CHD7 binding around known regulatory regions identified by the Aizawa group to drive Otx2 expression in the embryonic fore- and midbrain after E9.5, referred to as the FM1 and FM2 enhancer regions, as well as an early epiblast/anterior neuroectoderm (AN) enhancer region. We could demonstrate strong, specific association of CHD7 with these regions. All primer sequences used have been included in the revised manuscript.</p><p>We have revised the text to clarify that the association of CHD7 with Otx2 and Gbx2 (see below) regulatory elements do not constitute incontrovertible proof that CHD7 directly regulates these genes. We also include the caveat that the mechanisms whereby CHD7 might be recruited to these regions and the actual role of CHD7 at these elements are not known and fall beyond the scope of the present study.</p><p><italic>5) Did they consider undertaking a similar study of</italic> Gbx2?</p><p>Given the success of our Otx2 ChIP experiments, we also screened the Gbx2 gene, with emphasis on the 10kb region upstream of the gene, as this region likely encompasses an enhancer region that can direct Gbx2 expression to r1, based on studies in zebrafish. We could demonstrate specific CHD7 association with regions upstream of Gbx2. In addition, some of the elements tested represented negative controls with no CHD7 binding. These data are included in the revised manuscript.</p><p><italic>6) The data in</italic> <xref ref-type="fig" rid="fig3"><italic>Figure 3A,B</italic></xref> <italic>do not clearly show differences in</italic> Gbx2 <italic>and</italic> Otx2 <italic>expression. While it is clear at the later stages the data at E7.5 are not very compelling and add little to the story. They should either be removed, or to clarify the embryonic data at E7.5 the authors should repeat the</italic> Otx2/Gbx2 <italic>analysis with embryos that are more closely stage-matched. In particular, the</italic> Otx2<italic>-stained normal embryo is clearly older than the</italic> Chd7 <italic>mutant</italic>.</p><p>We agree with the reviewers that these data do not add much to the story, in particular since the Otx2 enhancers that drive expression at E7.5 are likely different from those that control expression in the mes/r1 region at the stages we deal with in the manuscript. We have therefore removed these data and included additional analyses of E8.25 embryos as requested.</p><p><italic>7) The posterior expansion of</italic> Otx2 <italic>is an important observation.</italic> <xref ref-type="fig" rid="fig3"><italic>Figure 3D</italic></xref> <italic>shows a 6ss embryo doubly stained for both</italic> Otx2 <italic>and</italic> Hoxa2<italic>. The expanded region of label is interpreted to be due to caudally expanded</italic> Otx2 <italic>expression, but it could also be due to</italic> Hoxa2 <italic>expression rostrally expanded from r2. This could be resolved with two color “in situs”. However, another solution is to stain solely for</italic> Otx2 <italic>and place a photo of such a sample as an insert in 2D. Alternatively a more compelling way to illustrate the shifts and a potential transformation would be to employ</italic> Krox20<italic>. It is expressed in r3 and r5. This would indicate that the changes were restricted to r1 and not a truncation of the anterior hindbrain. This general issue needs to be addressed in one of the ways suggested above</italic>.</p><p>We thank the reviewers for these constructive criticisms. We addressed this point in two of the ways suggested. We include pictures of 4ss embryos hybridized with only Otx2 that clearly shows the upregulated, expansion of Otx2 expression. Additionally, we repeated the analysis using Krox20 as a marker for r3+r5 to clearly demonstrate the posterior expansion of Otx2 expression and lack of posterior hindbrain truncation.</p></body></sub-article></article> |