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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">01160</article-id><article-id pub-id-type="doi">10.7554/eLife.01160</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>MEGF8 is a modifier of BMP signaling in trigeminal sensory neurons</article-title></title-group><contrib-group><contrib contrib-type="author" id="author-6297"><name><surname>Engelhard</surname><given-names>Caitlin</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-6298"><name><surname>Sarsfield</surname><given-names>Sarah</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-6299"><name><surname>Merte</surname><given-names>Janna</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-6300"><name><surname>Wang</surname><given-names>Qiang</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-6301"><name><surname>Li</surname><given-names>Peng</given-names></name><xref ref-type="aff" rid="aff2"/><xref ref-type="fn" rid="pa1">†</xref><xref ref-type="fn" rid="con7"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-6307"><name><surname>Beppu</surname><given-names>Hideyuki</given-names></name><xref ref-type="aff" rid="aff3"/><xref ref-type="fn" rid="con8"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-3169"><name><surname>Kolodkin</surname><given-names>Alex L</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-5"/><xref ref-type="fn" rid="con4"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-6303"><name><surname>Sucov</surname><given-names>Henry M</given-names></name><xref ref-type="aff" rid="aff2"/><xref ref-type="other" rid="par-3"/><xref ref-type="other" rid="par-4"/><xref ref-type="fn" rid="con5"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-6028"><name><surname>Ginty</surname><given-names>David D</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-5"/><xref ref-type="fn" rid="con9"/><xref ref-type="fn" rid="conf1"/></contrib><aff id="aff1"><institution content-type="dept">The Solomon H Snyder Department of Neuroscience</institution>, <institution>Howard Hughes Medical Institute, Johns Hopkins University School of Medicine</institution>, <addr-line><named-content content-type="city">Baltimore</named-content></addr-line>, <country>United States</country></aff><aff id="aff2"><institution content-type="dept">Broad CIRM Center for Regenerative Medicine and Stem Cell Research</institution>, <institution>University of Southern California Keck School of Medicine</institution>, <addr-line><named-content content-type="city">Los Angeles</named-content></addr-line>, <country>United States</country></aff><aff id="aff3"><institution content-type="dept">Department of Clinical Laboratory and Molecular Pathology, Graduate School of Medicine and Pharmaceutical Science</institution>, <institution>University of Toyama</institution>, <addr-line><named-content content-type="city">Toyama</named-content></addr-line>, <country>Japan</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>dginty@jhmi.edu</email></corresp><fn fn-type="present-address" id="pa1"><label>†</label><p>Department of Biochemistry, Stanford University, Stanford, United States</p></fn></author-notes><pub-date date-type="pub" publication-format="electronic"><day>17</day><month>09</month><year>2013</year></pub-date><pub-date pub-type="collection"><year>2013</year></pub-date><volume>2</volume><elocation-id>e01160</elocation-id><history><date date-type="received"><day>28</day><month>06</month><year>2013</year></date><date date-type="accepted"><day>16</day><month>08</month><year>2013</year></date></history><permissions><copyright-statement>© 2013, Engelhard et al</copyright-statement><copyright-year>2013</copyright-year><copyright-holder>Engelhard 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="elife01160.pdf"/><abstract><object-id pub-id-type="doi">10.7554/eLife.01160.001</object-id><p>Bone morphogenetic protein (BMP) signaling has emerged as an important regulator of sensory neuron development. Using a three-generation forward genetic screen in mice we have identified Megf8 as a novel modifier of BMP4 signaling in trigeminal ganglion (TG) neurons. Loss of Megf8 disrupts axon guidance in the peripheral nervous system and leads to defects in development of the limb, heart, and left-right patterning, defects that resemble those observed in <italic>Bmp4</italic> loss-of-function mice. <italic>Bmp4</italic> is expressed in a pattern that defines the permissive field for the peripheral projections of TG axons and mice lacking BMP signaling in sensory neurons exhibit TG axon defects that resemble those observed in <italic>Megf8</italic><sup><italic>−/−</italic></sup> embryos. Furthermore, TG axon growth is robustly inhibited by BMP4 and this inhibition is dependent on Megf8. Thus, our data suggest that Megf8 is involved in mediating BMP4 signaling and guidance of developing TG axons.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01160.001">http://dx.doi.org/10.7554/eLife.01160.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.01160.002</object-id><title>eLife digest</title><p>The peripheral nervous system relays information between the brain and spinal cord (the central nervous system) and the rest of the body. During development, neurons of the peripheral nervous system must extend processes (axons) long distances to reach the cells that they will eventually form connections with. Signaling molecules tell neuronal processes which direction to move in, and also tell them when they have reached their intended destination.</p><p>One group of molecules involved in the extension and guidance of neuronal processes are growth factors known as bone morphogenetic proteins (BMPs). These proteins contribute to a range of developmental processes, including the formation of the limbs and the skeleton, as well as various organs. They also help to establish the correct left-right patterning of the embryo, and direct the migration of sensory neurons.</p><p>Now, Engelhard et al. have used a genetic screen to identify additional signaling molecules involved in the development of the peripheral nervous system. They screened mice with a range of mutations, and found that animals with a mutant form of the gene that codes for a protein called MEGF8 closely resembled mice that lacked a member of the BMP family, BMP4. These mutants showed abnormal development of the skeleton and heart, and had six or seven digits on each limb (polydactyly).</p><p>Given the similarities between mice that lacked the gene for BMP4 and those that lacked the gene for MEGF8, Engelhard et al. explored these parallels further, and the results of a series of experiments were consistent with the two proteins being part of the same signaling cascade. In addition to identifying a novel signaling molecule that is involved in the formation of the peripheral nervous system, Engelhard et al. have provided new insights into the mechanisms by which one of the best known developmental signaling cascades is regulated.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01160.002">http://dx.doi.org/10.7554/eLife.01160.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>BMP</kwd><kwd>development</kwd><kwd>trigeminal ganglion</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd>Mouse</kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution>Howard Hughes Medical Institute</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Kolodkin</surname><given-names>Alex L</given-names></name><name><surname>Ginty</surname><given-names>David D</given-names></name></principal-award-recipient></award-group><award-group id="par-2"><funding-source><institution-wrap><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>NS34804</award-id><principal-award-recipient><name><surname>Ginty</surname><given-names>David D</given-names></name></principal-award-recipient></award-group><award-group id="par-3"><funding-source><institution-wrap><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>HL078891</award-id><principal-award-recipient><name><surname>Sucov</surname><given-names>Henry M</given-names></name></principal-award-recipient></award-group><award-group id="par-4"><funding-source><institution-wrap><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>HL070123</award-id><principal-award-recipient><name><surname>Sucov</surname><given-names>Henry M</given-names></name></principal-award-recipient></award-group><award-group id="par-5"><funding-source><institution-wrap><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>MH59199</award-id><principal-award-recipient><name><surname>Kolodkin</surname><given-names>Alex L</given-names></name><name><surname>Ginty</surname><given-names>David D</given-names></name></principal-award-recipient></award-group><funding-statement>The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.</funding-statement></funding-group><custom-meta-group><custom-meta><meta-name>elife-xml-version</meta-name><meta-value>2</meta-value></custom-meta><custom-meta specific-use="meta-only"><meta-name>Author impact statement</meta-name><meta-value>A protein called Megf8 regulates the activity of key signaling molecules involved in the development of the peripheral nervous system.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="s1" sec-type="intro"><title>Introduction</title><p>During development, neurons of the peripheral nervous system (PNS) must project axons over long distances to form connections with their appropriate peripheral targets. To accomplish this task, axons of developing neurons rely on guidance provided by a variety of extracellular cues expressed in the surrounding environment and distal targets. Indeed, as developing axons extend into the periphery, they encounter a plethora of target-derived signals that they must interpret properly in order to accomplish proper axonal guidance, maturation, survival, target innervation, and synapse formation.</p><p>Bone morphogenetic protein (BMP) signaling is an important regulator of sensory neuron development. BMPs are members of the TGFβ superfamily of secreted growth factors and are required for a wide range of developmental functions including gastrulation, mesoderm formation, neural patterning, left-right asymmetry, skeletal and limb development, kidney formation, and heart development (<xref ref-type="bibr" rid="bib32">Zhao, 2003</xref>). In addition to patterning, BMPs also function as axon guidance molecules. BMP7 in the spinal cord roof plate repels axons of developing commissural neurons thus orienting their growth toward the floor plate (<xref ref-type="bibr" rid="bib2">Augsburger et al., 1999</xref>; <xref ref-type="bibr" rid="bib5">Butler and Dodd, 2003</xref>). Moreover, BMP4 is a target-derived cue that augments development of somatosensory neurons in the trigeminal ganglia (TG) and dorsal root ganglia (DRG). BMP4 is expressed in the developing craniofacial region and signals retrogradely to coordinate differential gene expression and patterning along the dorsoventral axis of the trigeminal ganglion (<xref ref-type="bibr" rid="bib12">Hodge et al., 2007</xref>). Furthermore, BMP4 appears to have a trophic effect on TG and DRG neurons as altering the level of BMP4 expression in the skin leads to changes in the peripheral innervation and number of sensory neurons found in both the TG and DRG (<xref ref-type="bibr" rid="bib11">Guha et al., 2004</xref>). Thus, BMP4 signaling contributes to sensory neuron maturation and target innervation; however, the mechanisms by which BMP4 acts on developing sensory neurons to promote axonal growth remain to be elucidated.</p><p>We performed a forward genetic screen in the mouse to identify novel regulators of PNS development (<xref ref-type="bibr" rid="bib20">Merte et al., 2010</xref>). One of the lines that emerged from this screen has a mutation in the gene <italic>Megf8</italic>; loss of Megf8 function leads to defasciculation of the ophthalmic branch of the trigeminal nerve as well as defects in development of the heart, limb, skeleton, and left-right asymmetry. Given these phenotypes, we hypothesized that Megf8 interacts with the BMP4 signaling pathway. Indeed, loss of BMP signaling in trigeminal neurons leads to ophthalmic nerve defects that resemble those observed in <italic>Megf8</italic> loss-of-function mouse lines. Furthermore, TG axon growth is robustly inhibited by BMP4, and this inhibition is dependent on Megf8 expression. Taken together, these results show that Megf8 is a novel mediator of BMP signaling and is required for guidance of developing TG axons by target-derived BMP4.</p></sec><sec id="s2" sec-type="results"><title>Results</title><sec id="s2-1"><title>A forward genetic screen identifies line 687, which exhibits defasciculation of trigeminal ganglion axons</title><p>We performed ENU mutagenesis and a recessive three-generation forward genetic screen in the mouse to identify novel growth and guidance cues for developing PNS axons (<xref ref-type="bibr" rid="bib20">Merte et al., 2010</xref>). One of the mutant mouse lines, Line 687, exhibits severe defasciculation of the ophthalmic branch of the trigeminal nerve. In Line 687 mutants, the two major branches of the ophthalmic nerve initially form properly as tightly bound fascicles; however, as the axons extend into the target field, they prematurely defasciculate within the face (<xref ref-type="fig" rid="fig1">Figure 1A</xref>). We mapped the genetic lesion responsible for the phenotype in Line 687 to a 2.6 Mb region of Chromosome 7 between <italic>rs3715453</italic> and <italic>D7JHMI24</italic> that contains 76 open reading frames (<xref ref-type="fig" rid="fig1">Figure 1B</xref>). Based on bioinformatic analysis of genes within this region, we sequenced the 5′UTRs and coding exons of nine genes: <italic>Tmsb10</italic>, <italic>Dmrtc2</italic>, <italic>Zfp574</italic>, <italic>Zfp526</italic>, <italic>Gsk3a</italic>, <italic>Erf</italic>, <italic>Megf8</italic>, <italic>BC024561</italic>, and <italic>2310004L02Rik</italic>. This analysis revealed a single base pair substitution, 5324T>C, in the gene <italic>m</italic>ultiple <italic>EGF</italic>-like-domains <italic>8</italic> (<italic>Megf8</italic>) (<xref ref-type="fig" rid="fig1">Figure 1C</xref>). Megf8 is a very large putative transmembrane protein, and it is predicted to have a signal peptide, a CUB domain, plexin-semaphorin-integrin (PSI) domains, Kelch domains, EGF/EGF-like domains, and a C-terminal transmembrane domain. The mutation in Line 687 (<italic>Megf8</italic><sup><italic>L1775P</italic></sup>) is predicted to encode a single L>P amino acid substitution (L1775P) in the fourth Kelch domain of Megf8 (<xref ref-type="fig" rid="fig1">Figure 1D</xref>).<fig-group><fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.01160.003</object-id><label>Figure 1.</label><caption><title>Disruption of Megf8 causes defasciculation of the TG ophthalmic nerve.</title><p>(<bold>A</bold>) Whole-mount neurofilament staining of E11.5 control and Line 687 mutant littermates, showing the trigeminal ganglia (TG) and its three main projections: the ophthalmic (OP), maxillary (MX), and mandibular (MD) branches. (<bold>B</bold>) Schematic diagram of the region of Chromosome seven found to contain the Line 687 mutation, the markers used to diagnose linkage, and the frequency of recombination events observed at these markers. ORF, open reading frame. (<bold>C</bold>) Sequence data highlighting the mutation (<italic>Megf8</italic> 5324T>C) observed in Line 687 mutant DNA compared with C57BL/6 wild-type DNA. (<bold>D</bold>) Schematic diagram of Megf8. The Line 687 point mutation induces a single amino acid substitution L1775P in a Kelch domain of Megf8.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01160.003">http://dx.doi.org/10.7554/eLife.01160.003</ext-link></p></caption><graphic xlink:href="elife01160f001"/></fig><fig id="fig1s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01160.004</object-id><label>Figure 1—figure supplement 1.</label><caption><title>Complementation analysis of <italic>Megf8</italic><sup><italic>Trap</italic></sup> and <italic>Megf8</italic><sup><italic>L1775P</italic></sup> alleles.</title><p>(<bold>A</bold>) Schematic diagram of gene trap allele (<italic>Megf8</italic><sup><italic>Trap</italic></sup>). German Gene Trap Consortium cells G037A09 have an intron 2–3 insertion of a secretory trap vector such that the start ATG and signal peptide of Megf8 is captured and fused to a CD2-neomycin fusion protein instead of the remaining endogenous locus. S<sub>D</sub> = splice donor, S<sub>A</sub> = splice acceptor. (<bold>B</bold>) Whole-mount neurofilament staining of E11.5 embryos from an intercross of <italic>Megf8</italic><sup><italic>Trap/+</italic></sup> and <italic>Megf8</italic><sup><italic>L1775P/+</italic></sup> heterozygotes. Resulting <italic>Megf8</italic><sup><italic>L1775P/Trap</italic></sup> embryos have the trigeminal defasciculation phenotype.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01160.004">http://dx.doi.org/10.7554/eLife.01160.004</ext-link></p></caption><graphic xlink:href="elife01160fs001"/></fig></fig-group></p><p>In order to confirm that the Line 687 TG phenotype results from the <italic>Megf8</italic><sup><italic>L1775P</italic></sup> mutation, we performed complementation analysis using a <italic>Megf8</italic> secretory gene-trap line obtained from the German Gene Trap Consortium (clone G037A09, <italic>Megf8</italic><sup><italic>Gt(CD2-neo)GGTC</italic></sup> or <italic>Megf8</italic><sup><italic>Trap</italic></sup>). In this line, a CD2-neomycin secretory trap vector was virally inserted between exons two and three of <italic>Megf8</italic>; this approach results in a null, or hypomorphic, allele of <italic>Megf8</italic> in which a CD2-neomycin fusion protein is brought to the cell surface instead of the endogenous protein (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>). <italic>Megf8</italic><sup><italic>L1775P/Trap</italic></sup> embryos phenocopy the <italic>Megf8</italic><sup><italic>L1775P/L1775P</italic></sup> mutants and show defasciculation of the TG ophthalmic nerve (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>). These results confirm that the Line 687 TG phenotype results from the <italic>Megf8</italic><sup><italic>L1775P</italic></sup> mutation and demonstrate that Megf8 is required for proper growth and guidance of the TG ophthalmic nerve.</p></sec><sec id="s2-2"><title><italic>Megf8</italic> is widely expressed during early embryonic development</title><p>We next examined the expression of <italic>Megf8</italic> by in situ hybridization (ISH) using wild-type embryos. Whole-mount ISH of E8.5–E10.5 embryos showed that <italic>Megf8</italic> is widely expressed, with strong expression in the somites, limb buds, primordial gut, developing eye, and in the pharyngeal arches (<xref ref-type="fig" rid="fig2">Figure 2A–C</xref>). <italic>Megf8</italic> is also highly expressed in the developing nervous system. Sensory neurons of the DRG and TG show strong expression of <italic>Megf8</italic> throughout embryogenesis and into the postnatal period (<xref ref-type="fig" rid="fig2">Figure 2D,E</xref>, <xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1</xref>). <italic>Megf8</italic> is also expressed in the CNS including the developing neuroepithelium (<xref ref-type="fig" rid="fig2">Figure 2F</xref>), postnatal hippocampus, layer 4/5 of the cortex, and the olfactory bulb (<xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1</xref>).<fig-group><fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.01160.005</object-id><label>Figure 2.</label><caption><title><italic>Megf8</italic> is expressed widely during development.</title><p>(<bold>A</bold>–<bold>C</bold>) Whole-mount in situ hybridization (ISH) for <italic>Megf8</italic> at E8.5, E9.5, and E10.5. (<bold>D</bold>) ISH for <italic>Megf8</italic> on E11.5 transverse cryosection shows expression in the DRG. (<bold>E</bold>) ISH for <italic>Megf8</italic> on E11.5 transverse cryosection shows expression in the TG. (<bold>F</bold>) ISH for <italic>Megf8</italic> on E10.5 paraffin sagittal section shows expression in the developing neuroepithelium.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01160.005">http://dx.doi.org/10.7554/eLife.01160.005</ext-link></p></caption><graphic xlink:href="elife01160f002"/></fig><fig id="fig2s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01160.006</object-id><label>Figure 2—figure supplement 1.</label><caption><title><italic>Megf8</italic> is expressed throughout the developing nervous system.</title><p>(<bold>A</bold>) In situ hybridization on coronal cryosections from E11.5-P0 shows strong <italic>Megf8</italic> expression at all time points in the DRG and TG. (<bold>B</bold>–<bold>D</bold>) In situ hybridization on coronal cryosections at P0 shows strong <italic>Megf8</italic> expression in the hippocampus (<bold>B</bold>), layer 4/5 of the cortex (<bold>C</bold>), and in the olfactory bulb (<bold>D</bold>).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01160.006">http://dx.doi.org/10.7554/eLife.01160.006</ext-link></p></caption><graphic xlink:href="elife01160fs002"/></fig></fig-group></p></sec><sec id="s2-3"><title>Megf8 is required for development of the heart, limbs, skeleton, left-right asymmetry, and PNS</title><p>To gain insight into Megf8’s function during development we generated a conditional knockout mouse line using the Cre/LoxP system. We targeted the final exon of <italic>Megf8</italic>, which encodes the transmembrane domain, intracellular C-terminus, and 3′UTR, to generate <italic>Megf8</italic><sup><italic>Flox</italic></sup> conditional mutant mice (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1</xref>). We then crossed <italic>Megf8</italic><sup><italic>Flox</italic></sup> mice with the transgenic <italic>CMV-Cre</italic> line, in which cre recombinase is expressed in all cells, including germ cells (<xref ref-type="bibr" rid="bib26">Schwenk et al., 1995</xref>), to generate a <italic>Megf8</italic> null allele (<italic>Megf8</italic><sup>−</sup>). <italic>Megf8</italic><sup><italic>−/−</italic></sup> embryos phenocopy <italic>Megf8</italic><sup><italic>L1775P/L1775P</italic></sup> embryos and exhibit dramatic defasciculation and undergrowth of the ophthalmic branch of the trigeminal nerve (<xref ref-type="fig" rid="fig3 fig4">Figures 3A and 4H</xref>). In both the <italic>Megf8</italic> null and <italic>Megf8</italic><sup><italic>L1775P/L1775P</italic></sup> lines the maxillary and mandibular branches of the TG are largely unaffected. The maxillary branch shows no defasciculation phenotype although it is slightly undergrown, while the mandibular branch shows no abnormality (<xref ref-type="fig" rid="fig4">Figure 4H</xref>, legend).<fig-group><fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.01160.007</object-id><label>Figure 3.</label><caption><title>Megf8 is required for development of the trigeminal ganglia, limb, skeleton, heart, and left-right asymmetry.</title><p>(<bold>A</bold>) Whole-mount neurofilament staining of E11.5 <italic>Megf8</italic><sup><italic>+/+</italic></sup> and <italic>Megf8</italic><sup><italic>−/−</italic></sup> littermates. The <italic>Megf8</italic><sup><italic>−/−</italic></sup> null mutant phenocopies the point mutant <italic>Megf8</italic><sup><italic>L1775P/L1775P</italic></sup>. (<bold>B</bold>) Whole-mount images of <italic>Megf8</italic><sup><italic>+/+</italic></sup> and <italic>Megf8</italic><sup><italic>−/−</italic></sup> hindlimbs at E13.5 (top) and <italic>Megf8</italic><sup><italic>+/+</italic></sup> and <italic>Megf8</italic><sup><italic>L1775P/L1775P</italic></sup> forelimbs at E16.5 (bottom). (<bold>C</bold>) Alcian blue and alizarin red staining of E16.5 embryonic ribs/sternum. <italic>Megf8</italic><sup><italic>L1775P/L1775P</italic></sup> mutants have a split sternum and delayed ossification of the rib cage. (<bold>D</bold>) Whole-mount images of the heart of freshly fixed E10.5 embryos with dissected pericardial cavity. <italic>Megf8</italic><sup><italic>L1775P/L1775P</italic></sup> have complete left-right inversion of heart looping. Heart is outlined with dotted lines. (<bold>E</bold>) Whole-mount images of E11.5 <italic>Megf8</italic><sup><italic>+/+</italic></sup> and <italic>Megf8</italic><sup><italic>−/−</italic></sup> littermates, showing reversal of embryonic turning and exencephaly in the <italic>Megf8</italic><sup><italic>−/−</italic></sup>. (<bold>F</bold>) Whole-mount images of <italic>Megf8</italic><sup><italic>+/+</italic></sup> and <italic>Megf8</italic><sup><italic>−/−</italic></sup> littermates at E13.5, showing severe edema in the <italic>Megf8</italic><sup><italic>−/−</italic></sup>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01160.007">http://dx.doi.org/10.7554/eLife.01160.007</ext-link></p></caption><graphic xlink:href="elife01160f003"/></fig><fig id="fig3s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01160.008</object-id><label>Figure 3—figure supplement 1.</label><caption><title>Generation of a conditional knock-out mouse line (<italic>Megf8</italic><sup><italic>Flox</italic></sup>).</title><p>(<bold>A</bold>) Schematic diagram of <italic>Megf8</italic> gene targeting strategy. This strategy targeted the final exon of <italic>Megf8</italic>, which is 3.1 kb and comprises the transmembrane domain, intracellular C-terminus, and 3’′ UTR. A targeting vector was designed with an 8.5 kb long arm, a single loxP site located 500 bp upstream of the targeted exon, a frt-Neo-frt-loxP cassette placed 500 bp downstream of the exon, a 2.1 kb short arm, and a DTA selection cassette. Recombination with the endogenous locus resulted in integration of the 5′ loxP and 3′ frt-Neo-frt-loxP sites. Following germline transmission, male carriers were then mated with <italic>FlpE</italic> females to excise the neomycin cassette and generate the conditional allele. (<bold>B</bold>) Western blot of E12.5 TG and DRG lysates from <italic>Megf8</italic><sup><italic>+/+</italic></sup>, <italic>Megf8</italic><sup><italic>+/−</italic></sup>, and <italic>Megf8</italic><sup><italic>−/−</italic></sup> littermates. The blot was probed with rabbit anti-Megf8 and shows a loss of Megf8 protein in <italic>Megf8</italic><sup><italic>−/−</italic></sup> neurons.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01160.008">http://dx.doi.org/10.7554/eLife.01160.008</ext-link></p></caption><graphic xlink:href="elife01160fs003"/></fig><fig id="fig3s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01160.009</object-id><label>Figure 3—figure supplement 2.</label><caption><title><italic>Megf8</italic><sup><italic>L1775P/L1775P</italic></sup> mutants show defects in limb and skeletal development.</title><p>(<bold>A</bold>) Alcian blue staining on E16.5 limbs shows that <italic>Megf8</italic><sup><italic>L1775P/L1775P</italic></sup> mutants have digit duplication as well as duplication of bones in the hand. (<bold>B</bold>) Alizarin red staining on E16.5 limbs shows that <italic>Megf8</italic><sup><italic>L1775P/Trap</italic></sup> mutants have duplication of the bones of the autopodium. (<bold>C</bold>) Alcian blue and alizarin red staining of whole E16.5 embryo skeletons. <italic>Megf8</italic><sup><italic>L1775P/L1775P</italic></sup> mutants have a wider and shorter ribcage, delayed ossification of the ribcage, and a split sternum.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01160.009">http://dx.doi.org/10.7554/eLife.01160.009</ext-link></p></caption><graphic xlink:href="elife01160fs004"/></fig><fig id="fig3s3" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01160.010</object-id><label>Figure 3—figure supplement 3.</label><caption><title>Heart development in <italic>Megf8</italic><sup><italic>L1775P/L1775P</italic></sup> mutant embryos.</title><p>Sections from control (<bold>A</bold>–<bold>C</bold>) and <italic>Megf8</italic><sup><italic>L1775P/L1775P</italic></sup> mutant (<bold>D</bold>–<bold>F</bold>) embryos. (<bold>A</bold> and <bold>E</bold>) Hematoxylin and eosin (H and E) staining of E15.5 control and mutant sections. The arrowhead in the control E15.5 embryo section points to the atrial septum; the arrowhead in the mutant embryo section points to the vestige of the endocardial cushion, above which the atrial septum is missing and below which the atrioventricular valves are poorly formed or atretic. (<bold>B</bold> and <bold>E</bold>) β-galactosidase and nuclear fast red staining of E12.5 embryos, which also carried the <italic>Tie2Cre/R26R</italic> alleles for labeling endothelium and endocardium and derived mesenchymal cells (<xref ref-type="bibr" rid="bib14">Kisanuki et al., 2001</xref>; <xref ref-type="bibr" rid="bib28">Soriano, 1999</xref>). Note the normal endocardial cushion (EC) mesenchyme (arrows). (<bold>C</bold> and <bold>F</bold>) H and E staining of E10.5 control and mutant sections. Note the apparently normal atrioventricular cushion mesenchyme at E10.5 (arrows).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01160.010">http://dx.doi.org/10.7554/eLife.01160.010</ext-link></p></caption><graphic xlink:href="elife01160fs005"/></fig></fig-group><fig-group><fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.01160.011</object-id><label>Figure 4.</label><caption><title>Megf8 is required for development of the PNS.</title><p>(<bold>A</bold> and <bold>D</bold>) Whole-mount neurofilament staining of E11.5 <italic>Megf8</italic><sup><italic>+/+</italic></sup> and <italic>Megf8</italic><sup><italic>−/−</italic></sup> littermates showing the DRG spinal nerves, which are undergrown in the <italic>Megf8</italic><sup><italic>−/−</italic></sup> (arrow). (<bold>B</bold> and <bold>E</bold>) Whole-mount peripherin staining of forelimbs from E13.5 <italic>Megf8</italic><sup><italic>+/+</italic></sup> and <italic>Megf8</italic><sup><italic>−/−</italic></sup> littermates. The radial and ulnar nerves are undergrown in the <italic>Megf8</italic><sup><italic>−/−</italic></sup>embryo. Limbs are outlined with dotted lines. Scale bar (<bold>B</bold> and <bold>E</bold>) represents 500 μm. (<bold>C</bold> and <bold>F</bold>) Whole-mount neurofilament staining of E11.5 <italic>Megf8</italic><sup><italic>+/+</italic></sup> and <italic>Megf8</italic><sup><italic>−/−</italic></sup> littermates. The vagus/glossopharyngeal nerves are defasciculated in the <italic>Megf8</italic><sup><italic>−/−</italic></sup> (arrow). (<bold>G</bold>) Whole-mount neurofilament staining of E11.5 <italic>Megf8</italic><sup><italic>Flox/Flox</italic></sup> and <italic>Wnt1-Cre; Megf8</italic><sup><italic>Flox/Flox</italic></sup> littermates. (<bold>H</bold>) Quantification of ophthalmic branch phenotype for <italic>Megf8</italic><sup><italic>−/−</italic></sup> (KO), <italic>Megf8</italic><sup><italic>L1775P/L1775P</italic></sup> (L1775P), and <italic>Wnt1-Cre; Megf8</italic><sup><italic>Flox/Flox</italic></sup> (cKO) compared to <italic>Megf8</italic><sup><italic>+/+</italic></sup> (WT). Left: the number of branches at the nasociliary branch point was significantly greater for KO, L1775P, and cKO embryos. Right: the ophthalmic branch was undergrown in KO, L1775P, and cKO embryos. Four to seven embryos were analyzed per genotype. Error bars represent mean ± s.e.m. *p<0.05, one-way ANOVA. The relative outgrowth was also measured for the maxillary and mandibular branches of the TG for <italic>Megf8</italic><sup><italic>−/−</italic></sup> embryos (not shown). The maxillary branch was slightly undergrown compared to <italic>Megf8</italic><sup><italic>+/+</italic></sup> (relative outgrowth 0.9, p<0.05) while the mandibular branch was unaffected (relative outgrowth 0.99, p=0.7). To assess defasciculation in the maxillary branch, the relative maxillary area was calculated and no difference was observed between <italic>Megf8</italic><sup><italic>+/+</italic></sup> and <italic>Megf8</italic><sup><italic>−/−</italic></sup> (relative area 0.94, p=0.2).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01160.011">http://dx.doi.org/10.7554/eLife.01160.011</ext-link></p></caption><graphic xlink:href="elife01160f004"/></fig><fig id="fig4s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01160.012</object-id><label>Figure 4—figure supplement 1.</label><caption><title>Conditional deletion of <italic>Megf8</italic> from DRG neurons does not disrupt formation of the radial/ulnar nerves.</title><p>Whole-mount peripherin staining of E13.5 forelimbs from <italic>Wnt1-Cre;Megf8</italic><sup><italic>Flox/+</italic></sup> and <italic>Wnt1-Cre;Megf8</italic><sup><italic>Flox/Flox</italic></sup> littermates. Limbs are outlined with dotted lines. Scale bar represents 500 µm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01160.012">http://dx.doi.org/10.7554/eLife.01160.012</ext-link></p></caption><graphic xlink:href="elife01160fs006"/></fig></fig-group></p><p>Consistent with its widespread expression during embryogenesis, we observed that loss of <italic>Megf8</italic> leads to severe developmental abnormalities in several organ systems. Both <italic>Megf8</italic><sup><italic>−/−</italic></sup> and <italic>Megf8</italic><sup><italic>L1775P/L1775P</italic></sup> embryos display polydactyly in both the forelimbs and hindlimbs; this pre-axial polydactyly is completely penetrant, and embryos show six to seven digits per limb as well as duplication of bones in the hand (<xref ref-type="fig" rid="fig3">Figure 3B</xref>, <xref ref-type="fig" rid="fig3s2">Figure 3—figure supplement 2</xref>). They also exhibit skeletal abnormalities including delayed ossification of the rib cage, a wider and shorter rib cage, and a split sternum (<xref ref-type="fig" rid="fig3">Figure 3C</xref>, <xref ref-type="fig" rid="fig3s2">Figure 3—figure supplement 2</xref>). Furthermore, loss of <italic>Megf8</italic> leads to a disruption of left-right patterning. Approximately one third of <italic>Megf8</italic><sup><italic>−/−</italic></sup> embryos demonstrate a complete left-right inversion of heart looping (<xref ref-type="fig" rid="fig3">Figure 3D</xref>) or embryonic turning (<xref ref-type="fig" rid="fig3">Figure 3E</xref>). Taken together with prior results, these findings indicate that Megf8 is required for development of left-right asymmetry.</p><p>In addition to its role in development of the limb, skeleton, and left-right asymmetry, Megf8 is also required for normal heart development. In addition to the inversion of heart looping observed in some <italic>Megf8</italic><sup><italic>−/−</italic></sup> and <italic>Megf8</italic><sup><italic>L1775P/L1775P</italic></sup> embryos, loss of Megf8 function leads to a complete, or nearly complete, absence of the mitral and tricuspid valves, swollen atria, transposition of the outflow tract, pulmonary stenosis, and atrial and ventricular septal defects (<xref ref-type="fig" rid="fig3s3">Figure 3—figure supplement 3</xref>). These phenotypes were seen in all embryos at E14.5 or later, and they lead to severe peripheral edema (<xref ref-type="fig" rid="fig3">Figure 3F</xref>) and embryonic lethality by age E16.5. Analysis at earlier stages revealed apparently normal endocardial cushion mesenchymal cells (<xref ref-type="fig" rid="fig3s3">Figure 3—figure supplement 3</xref>), which implies that the terminal pathologies result from improper remodeling events after E12.5. Conditional mutation of the <italic>Megf8</italic><sup><italic>Flox</italic></sup> allele using the <italic>Mesp1-Cre</italic> line (<xref ref-type="bibr" rid="bib24">Saga et al., 1999</xref>), which is active in all mesoderm of the heart, did not cause any heart phenotypes (data not shown), implying that early heart and embryo laterality defects might be responsible for the constellation of later heart defects. These findings are consistent with a second mouse line harboring a <italic>Megf8</italic> point mutation (<italic>Megf8</italic><sup><italic>C193R</italic></sup>) that was identified in an independent forward genetic screen and which was shown to have polydactyly and defects in left-right patterning and heart development (<xref ref-type="bibr" rid="bib31">Zhang et al., 2009</xref>).</p><p>Because <italic>Megf8</italic> is expressed strongly throughout the nervous system, we next investigated the function of Megf8 in the developing PNS, in addition to its role in the TG. In <italic>Megf8</italic><sup><italic>−/−</italic></sup> embryos, spinal nerves are shorter at E11.5 than in wild type (<xref ref-type="fig" rid="fig4">Figure 4A,D</xref>), and by E13.5 the radial and ulnar nerves have not fully extended into the limbs, appearing shorter and less branched (<xref ref-type="fig" rid="fig4">Figure 4B,E</xref>). At E13.5 the limbs appear immature in <italic>Megf8</italic><sup><italic>−/−</italic></sup> embryos relative to wild type littermates, which may contribute to the radial and ulnar nerve phenotypes. <italic>Megf8</italic><sup><italic>−/−</italic></sup> embryos also display defects in the developing vagus and glossopharyngeal nerves. At E11.5 these nerves are slightly defasciculated and several wayward axons branch away from the main fiber bundle (<xref ref-type="fig" rid="fig4">Figure 4C,F</xref>).</p><p>Overall, these findings demonstrate that Megf8 is required for proper axonal extension in several areas of the PNS, including the TG, DRG, and vagus/glossopharyngeal nerves (<xref ref-type="table" rid="tbl1">Table 1</xref>). Given the broad expression of <italic>Megf8</italic> in a wide range of tissues during development, we next asked whether these defects, and in particular the TG defect, are due to a cell autonomous function of Megf8 in sensory neurons. To address this question, we crossed the conditional <italic>Megf8</italic><sup><italic>Flox</italic></sup> line with the transgenic <italic>Wnt1-Cre</italic> line (<xref ref-type="bibr" rid="bib6">Danielian et al., 1998</xref>) in which cre recombinase is expressed in neural crest-derived cells, including sensory neurons. <italic>Wnt1-Cre</italic>-mediated conditional deletion of <italic>Megf8</italic> leads to defasciculation of the ophthalmic branch of the trigeminal nerve, phenocopying <italic>Megf8</italic><sup><italic>−/−</italic></sup> and <italic>Megf8</italic><sup><italic>L1775P/L1775P</italic></sup> mutant embryos (<xref ref-type="fig" rid="fig4">Figure 4G</xref>). Although all <italic>Wnt1-Cre;Megf8</italic><sup><italic>Flox/Flox</italic></sup> embryos showed defasciculation of the ophthalmic branch, the severity of the phenotype was more variable than in <italic>Megf8</italic><sup><italic>−/−</italic></sup> and <italic>Megf8</italic><sup><italic>L1775P/L1775P</italic></sup> embryos (<xref ref-type="fig" rid="fig4">Figure 4H</xref>), and in a few embryos the ophthalmic branch defasciculated completely around the eye and failed to project into the periphery (data not shown). These findings suggest that Megf8 mediates guidance of developing TG axons in a cell autonomous manner. <italic>Wnt1-Cre;Megf8</italic><sup><italic>Flox/Flox</italic></sup> embryos do not exhibit a defect in radial/ulnar nerve development, however, suggesting that Megf8’s role in spinal nerve axon guidance is non-cell autonomous (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1</xref>). Of note, <italic>Wnt1-Cre;Megf8</italic><sup><italic>Flox/Flox</italic></sup> embryos do not exhibit the limb immaturity seen in <italic>Megf8</italic><sup><italic>−/−</italic></sup> embryos. Thus, one possibility is that the radial/ulnar nerve phenotype observed in <italic>Megf8</italic><sup><italic>−/−</italic></sup> embryos is secondary to an overall delay in limb development. An alternative explanation is that Megf8 may be required in spinal motor neurons, and that a loss of motor neuron axon guidance in <italic>Megf8</italic><sup><italic>−/−</italic></sup> embryos causes a subsequent disruption in DRG sensory axon guidance; this indirect effect of Megf8 on DRG sensory neuron axon guidance would not be evident in <italic>Wnt1-Cre;Megf8</italic><sup><italic>Flox/Flox</italic></sup> embryos.<table-wrap id="tbl1" position="float"><object-id pub-id-type="doi">10.7554/eLife.01160.013</object-id><label>Table 1.</label><caption><p>Summary of <italic>Megf8</italic><sup><italic>−/−</italic></sup> phenotypes and implicated BMPs</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01160.013">http://dx.doi.org/10.7554/eLife.01160.013</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th>Phenotype</th><th><italic>Megf8</italic><sup><italic>-/-</italic></sup></th><th colspan="2">BMP implicated</th></tr></thead><tbody><tr><td>Trigeminal nerve (V1) defasciculation</td><td>100% (10/10)</td><td>n.d.</td><td/></tr><tr><td>Trigeminal patterning</td><td>n.d.</td><td>BMP4</td><td>(<xref ref-type="bibr" rid="bib12">Hodge et al., 2007</xref>)</td></tr><tr><td>Polydactyly</td><td>100% (9/9)</td><td>BMP4, BMP7</td><td>(<xref ref-type="bibr" rid="bib7">Dudley et al., 1995</xref>; <xref ref-type="bibr" rid="bib8">Dunn et al., 1997</xref>)</td></tr><tr><td>Reversed heart looping</td><td>33% (7/21)</td><td>BMP4</td><td>(<xref ref-type="bibr" rid="bib9">Fujiwara et al., 2002</xref>)</td></tr><tr><td>Reversed embryonic turning (E11.5)</td><td>33% (5/15)</td><td>BMP4</td><td>(<xref ref-type="bibr" rid="bib9">Fujiwara et al., 2002</xref>)</td></tr><tr><td>Edema (E13.5+)</td><td>100% (6/6)</td><td/><td/></tr><tr><td>DRG spinal nerves undergrown (E11.5)</td><td>100% (10/10)</td><td>BMP4</td><td>(<xref ref-type="bibr" rid="bib11">Guha et al., 2004</xref>)</td></tr><tr><td>Radial/ulnar nerves undergrown (E13.5)</td><td>100% (2/2)</td><td>BMP4</td><td>(<xref ref-type="bibr" rid="bib11">Guha et al., 2004</xref>)</td></tr><tr><td>Vagus defasciculation</td><td>90% (9/10)</td><td>n.d.</td><td/></tr><tr><td>Exencephaly</td><td>36% (16/45)</td><td/><td/></tr><tr><td>Disrupted <italic>BMP4</italic> expression around V1</td><td>100% (4/4)</td><td/><td/></tr></tbody></table><table-wrap-foot><fn><p>Phenotypes, stages of observation and penetrances of <italic>Megf8</italic><sup><italic>−/−</italic></sup> mutants are listed. V1 refers to the ophthalmic branch of the trigeminal nerve. <italic>Bmp</italic> loss-of-function lines known to display similar phenotypes are noted.</p></fn></table-wrap-foot></table-wrap></p></sec><sec id="s2-4"><title><italic>Megf8</italic><sup><italic>−/−</italic></sup> phenotypes resemble <italic>Bmp</italic> loss-of-function lines</title><p>Our analysis demonstrates that Megf8 is required for development of the limb, skeleton, heart, left-right asymmetry, and PNS. The spectrum of phenotypes seen in <italic>Megf8</italic><sup><italic>−/−</italic></sup> embryos is strikingly similar to that observed when BMP signaling is disrupted (<xref ref-type="table" rid="tbl1">Table 1</xref>). BMPs are members of the TGF-β superfamily of extracellular ligands and have been implicated in a wide range of developmental functions. We noted that loss of BMP4, and to a lesser extent BMP2 and BMP7, results in similar defects as those observed in the <italic>Megf8</italic> mutants. <italic>Bmp4</italic> and <italic>Bmp7</italic> loss-of-function mouse lines exhibit pre-axial polydactyly (<xref ref-type="bibr" rid="bib7">Dudley et al., 1995</xref>; <xref ref-type="bibr" rid="bib18">Luo et al., 1995</xref>; <xref ref-type="bibr" rid="bib8">Dunn et al., 1997</xref>; <xref ref-type="bibr" rid="bib27">Selever et al., 2004</xref>). <italic>Bmp4</italic> null embryos also display disrupted left-right patterning and loss of normal rightward heart looping (<xref ref-type="bibr" rid="bib9">Fujiwara et al., 2002</xref>). Loss of BMP4, or its receptor BMPR2, results in a wide range of heart defects, including atrial septal defects, ventricular septal defects, atrioventricular septal defects, and abnormal positioning or septation of the outflow tract (<xref ref-type="bibr" rid="bib13">Jiao et al., 2003</xref>; <xref ref-type="bibr" rid="bib17">Liu et al., 2004</xref>; <xref ref-type="bibr" rid="bib4">Beppu et al., 2009</xref>); several of these cardiac phenotypes are exacerbated by a concomitant loss of BMP2 or BMP7 (<xref ref-type="bibr" rid="bib23">Rivera-Feliciano and Tabin, 2006</xref>; <xref ref-type="bibr" rid="bib10">Goldman et al., 2009</xref>). Furthermore, BMP4 signaling is required for dorsoventral patterning of the TG (<xref ref-type="bibr" rid="bib12">Hodge et al., 2007</xref>), and modifying BMP4 expression in the skin leads to changes in the peripheral innervation and survival of sensory neurons (<xref ref-type="bibr" rid="bib11">Guha et al., 2004</xref>). Taken together, our findings demonstrate that loss of either BMP4 or Megf8 leads to a similar spectrum of defects in the limb, left-right asymmetry, and heart, and that both BMP4 and Megf8 are required for normal development of the TG. Based on these functional similarities, we hypothesized that Megf8 interacts with, or is a component of, the BMP4 signaling pathway, and that this interaction is required for axon guidance and peripheral target innervation by TG sensory neurons.</p></sec><sec id="s2-5"><title><italic>Bmp4</italic> is expressed in peripheral targets in a manner that defines a permissive field for trigeminal nerve axonal projections</title><p>To address the hypothesis that Megf8 and BMP4 cooperate to regulate growth and guidance of TG axons, we began by assessing the spatial relationship between <italic>Bmp4</italic> expression and developing TG axons. To do so, we utilized a <italic>Bmp4</italic><sup><italic>lacZ</italic></sup> mouse line in which a <italic>lacZ</italic> reporter cassette was inserted into the <italic>Bmp4</italic> locus (<xref ref-type="bibr" rid="bib16">Lawson et al., 1999</xref>). Using whole-mount β-galactosidase and neurofilament co-staining, we were able to examine the expression pattern of <italic>Bmp4</italic> relative to developing TG axons. At E10.5, <italic>Bmp4</italic> is expressed strongly in the eye and also in the dorsal portion of the face above the eye (<xref ref-type="fig" rid="fig5">Figure 5A</xref>). These two areas of BMP4 expression flank a narrow corridor through which the developing ophthalmic nerve projects; this expression pattern suggests that BMP4 may act as a repulsive guidance cue for developing TG axons. One day later, at E11.5, the TG axons have extended into their peripheral targets and have bifurcated into frontal and nasociliary nerves. At this stage, <italic>Bmp4</italic> expression is striking; as the ophthalmic branch extends beyond the eye, the nasociliary nerve branches in a region devoid of <italic>Bmp4</italic> expression and then immediately projects nasally as a tightly bundled fascicle through an area of strong <italic>Bmp4</italic> expression while the axons of the frontal branch extend rostrally. Remarkably, the pattern of <italic>Bmp4</italic> expression is disrupted in <italic>Megf8</italic><sup><italic>−/−</italic></sup> embryos. In the absence of Megf8, <italic>Bmp4</italic> expression is lost at the point of defasciculation of the ophthalmic branch into the frontal and nasociliary branches (<xref ref-type="fig" rid="fig5">Figure 5B</xref>; <xref ref-type="table" rid="tbl1">Table 1</xref>) suggesting that axonal innervation of the target region is necessary for proper expression of <italic>Bmp4</italic> and that the absence of this BMP signaling leads to defasciculation of the nasociliary branch of the ophthalmic nerve. An alternative explanation is that Megf8 is required in cells of the craniofacial target tissues to regulate <italic>Bmp4</italic> expression. ISH shows that <italic>Megf8</italic> is expressed in craniofacial tissues as well as the TG, although <italic>Megf8</italic> expression is far more robust in neurons of the TG (<xref ref-type="fig" rid="fig2">Figure 2</xref>). Interestingly, in <italic>Megf8</italic><sup><italic>−/−</italic></sup> embryos <italic>Bmp4</italic> expression is disrupted exclusively in the area around the ophthalmic branch but is intact throughout the rest of the embryo (<xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1</xref>).<fig-group><fig id="fig5" position="float"><object-id pub-id-type="doi">10.7554/eLife.01160.014</object-id><label>Figure 5.</label><caption><title>BMP signaling is required for proper extension of the TG ophthalmic nerve.</title><p>(<bold>A</bold>) Whole-mount β-galactosidase and neurofilament (NFM) co-staining on <italic>Bmp4</italic><sup><italic>lacZ/+</italic></sup> embryos at E10.5 and E11.5, showing the relationship between <italic>Bmp4</italic> expression and the developing TG nerve. (<bold>B</bold>) Whole-mount β-galactosidase and neurofilament co-staining on E11.5 embryos: <italic>Bmp4</italic><sup><italic>lacZ/+</italic></sup><italic>;Megf8</italic><sup><italic>+/+</italic></sup> (left) and <italic>Bmp4</italic><sup><italic>lacZ/+</italic></sup><italic>;Megf8</italic><sup><italic>−/−</italic></sup> (center) littermates, as well as <italic>Bmp4</italic><sup><italic>lacZ/+</italic></sup><italic>;Wnt1-Cre/Megf8</italic><sup><italic>Flox/Flox</italic></sup> (right). <italic>Bmp4</italic><sup><italic>lacZ</italic></sup> expression is lost at the location where defasciculation of the TG ophthalmic nerve occurs in <italic>Megf8</italic><sup><italic>−/−</italic></sup> and <italic>Wnt1-Cre;Megf8</italic><sup><italic>Flox/Flox</italic></sup> embryos. The top row shows the whole head with all three TG branches. The bottom row is an enlargement of the top row showing the ophthalmic branch. The area of perturbed <italic>Bmp4</italic> expression is outlined. The disruption of <italic>Bmp4</italic><sup><italic>lacZ</italic></sup> expression was fully penetrant and observed in all <italic>Megf8</italic><sup><italic>−/−</italic></sup> (n = 4) and <italic>Wnt1-Cre;Megf8</italic><sup><italic>Flox/Flox</italic></sup> (n = 3) embryos assessed. (<bold>C</bold>) Whole-mount neurofilament staining of E11.5 <italic>Bmpr2</italic><sup><italic>Flox/+</italic></sup> and <italic>Wnt1-Cre;Bmpr2</italic><sup><italic>Flox/−</italic></sup> littermates.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01160.014">http://dx.doi.org/10.7554/eLife.01160.014</ext-link></p></caption><graphic xlink:href="elife01160f005"/></fig><fig id="fig5s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01160.015</object-id><label>Figure 5—figure supplement 1.</label><caption><title><italic>Bmp4</italic> expression in <italic>Megf8</italic><sup><italic>−/−</italic></sup> embryo compared to wild-type littermate.</title><p>Whole-mount β-galactosidase and neurofilament co-staining on E11.5 embryos: <italic>Bmp4</italic><sup><italic>lacZ/+</italic></sup><italic>;Megf8</italic><sup><italic>+/+</italic></sup> (left) and <italic>Bmp4</italic><sup><italic>lacZ/+</italic></sup><italic>;Megf8</italic><sup><italic>−/−</italic></sup> (right) littermates. <italic>Bmp4</italic><sup><italic>lacZ</italic></sup> expression is lost at the location where defasciculation of the TG ophthalmic nerve occurs in <italic>Megf8</italic><sup><italic>−/−</italic></sup> embryos but is intact throughout the rest of the embryo. (<bold>A</bold>) Presentation of the whole embryo. (<bold>B</bold>) DRG and spinal nerves. (<bold>C</bold>) Forelimbs and hindlimbs.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01160.015">http://dx.doi.org/10.7554/eLife.01160.015</ext-link></p></caption><graphic xlink:href="elife01160fs007"/></fig><fig id="fig5s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01160.016</object-id><label>Figure 5—figure supplement 2.</label><caption><title><italic>Bmpr2</italic> and <italic>Megf8</italic> are expressed throughout the developing TG.</title><p>In situ hybridization on serial transverse cryosections at E12.5 shows strong expression of <italic>Bmpr2</italic> (<bold>A</bold>–<bold>C</bold>) and <italic>Megf8</italic> (<bold>D</bold>–<bold>F</bold>) throughout the developing TG. Representative images are shown for ophthalmic (<bold>A</bold> and <bold>D</bold>), maxillary (<bold>B</bold> and <bold>E</bold>), and mandibular (<bold>C</bold> and <bold>F</bold>) lobes of the TG.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01160.016">http://dx.doi.org/10.7554/eLife.01160.016</ext-link></p></caption><graphic xlink:href="elife01160fs008"/></fig></fig-group></p></sec><sec id="s2-6"><title>Mice lacking BMP signaling in TG sensory neurons exhibit axon guidance defects similar to those of <italic>Megf8</italic><sup><italic>−/−</italic></sup> embryos</title><p>To directly test the idea that BMP4 is required for the growth and guidance of TG axons, we next sought to assess development of the trigeminal nerve in the absence of BMP4 signaling. Because <italic>Bmp4</italic> null embryos die too early in development for our analysis, we used mice lacking the BMP receptor, BMPR2, in cells of the neural crest lineage. We utilized mice harboring one mutant BMP receptor conditional allele (<italic>Bmpr2</italic><sup><italic>Flox</italic></sup>) and one null allele (<italic>Bmpr2</italic><sup><italic>−</italic></sup>) (<xref ref-type="bibr" rid="bib3">Beppu et al., 2005</xref>) crossed to <italic>Wnt1-Cre</italic> mice to eliminate the receptor from TG sensory neurons. BMPR2, one of several BMP receptors, is preferentially activated by BMP ligands and not by other TGF-β superfamily members (<xref ref-type="bibr" rid="bib21">Miyazono et al., 2010</xref>). <italic>Bmpr2</italic> is robustly expressed in all three lobes of the developing TG, as is <italic>Megf8</italic> (<xref ref-type="fig" rid="fig5s2">Figure 5—figure supplement 2</xref>). Interestingly, the ophthalmic branch of the trigeminal nerve in E11.5 <italic>Wnt1-Cre;Bmpr2</italic><sup><italic>Flox/−</italic></sup> embryos is stunted and branches prematurely in a manner reminiscent of the phenotype seen in <italic>Megf8</italic> loss-of-function lines (<xref ref-type="fig" rid="fig5">Figure 5C</xref>). Furthermore, the maxillary and mandibular branches of the trigeminal nerve are unaffected in the <italic>Wnt1-Cre;Bmpr2</italic><sup><italic>Flox/−</italic></sup> embryos, as in <italic>Megf8</italic> null lines. These results demonstrate that BMP signaling is required specifically for development of the ophthalmic branch of the trigeminal nerve, and they suggest that both Megf8 and BMPR2 are necessary to mediate this signaling.</p></sec><sec id="s2-7"><title>BMP4 inhibits TG axon outgrowth, and this inhibition is dependent on <italic>Megf8</italic> expression</title><p>We next asked whether BMP4 and Megf8 functionally interact to mediate growth and guidance of TG axons. To test this idea, we used an in vitro TG explant assay in which TG explants were cultured in a collagen gel and exposed to increasing amounts of bath applied BMP4. Remarkably, BMP4 robustly inhibits the outgrowth of wild-type TG axons, and maximal doses inhibit axon outgrowth by more than 50% (<xref ref-type="fig" rid="fig6">Figure 6A,C</xref>). We then asked whether loss of <italic>Megf8</italic> would alter the response of TG axons to BMP4 by co-culturing explants from <italic>Megf8</italic><sup><italic>−/−</italic></sup> embryos alongside littermate controls. <italic>Megf8</italic><sup><italic>−/−</italic></sup> explants exhibit significantly greater axon outgrowth in the presence of BMP4, but not under control culture conditions (<xref ref-type="fig" rid="fig6">Figure 6B,D</xref>). These results demonstrate that <italic>Megf8</italic><sup><italic>−/−</italic></sup> axons are less sensitive to the inhibitory effects of BMP4 and that the response of TG neurons to BMP4 is partially dependent on <italic>Megf8</italic> expression. Together with our in vivo findings, these results support a model in which Megf8 functions within TG sensory axons, allowing them to respond to intermediate target-derived BMP4 and thus guide these axons to their appropriate peripheral target regions in the face.<fig id="fig6" position="float"><object-id pub-id-type="doi">10.7554/eLife.01160.017</object-id><label>Figure 6.</label><caption><title>Megf8 mediates the inhibition of TG axon growth by BMP4.</title><p>(<bold>A</bold>) E12.5 wild-type TG explants cultured in 0–100 ng/ml BMP4. (<bold>B</bold>) E12.5 TG explants from <italic>Megf8</italic><sup><italic>+/−</italic></sup> and <italic>Megf8</italic><sup><italic>−/−</italic></sup> littermates cultured side by side in 0–50 ng/ml BMP4. (<bold>C</bold>) Quantification of (<bold>A</bold>): axonal outgrowth of wild-type TG explants in the presence of 0–100 ng/ml BMP4. Increasing doses of BMP4 caused robust inhibition of axon outgrowth (**p<0.05, Student’s <italic>t</italic> test). Three to four explants were quantified for each concentration of BMP4. Error bars represent mean ± SEM. (<bold>D</bold>) Quantification of (<bold>B</bold>): relative axon outgrowth of TG explants from control (<italic>Megf8</italic><sup><italic>+/+</italic></sup> or <italic>Megf8</italic><sup><italic>+/-</italic></sup>) and <italic>Megf8</italic><sup><italic>-/-</italic></sup> littermates in the presence of 0–50 ng/ml BMP4. BMP4 inhibition of axon outgrowth is partially lost in <italic>Megf8</italic><sup><italic>−/−</italic></sup> explants (*p<0.05, Student’s <italic>t</italic> test). The experiment was repeated three times, using three to four explants per BMP4 concentration in each experiment; each bar represents results from at least 10 explants. Error bars represent mean ± SEM. Scale bar represents 20 μm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01160.017">http://dx.doi.org/10.7554/eLife.01160.017</ext-link></p></caption><graphic xlink:href="elife01160f006"/></fig></p></sec></sec><sec id="s3" sec-type="discussion"><title>Discussion</title><p>Forward genetics provides a powerful approach to identify new axon guidance cues and also novel components of signaling pathways already known to regulate axon growth and guidance. In this study, we used an unbiased three-generation forward genetic screen to identify the gene <italic>Megf8</italic> as a novel regulator of PNS development. Our findings strongly suggest that Megf8 regulates the response of target-derived BMP4 on TG axons during development and that functional Megf8 signaling is necessary for proper TG axon growth and target innervation.</p><sec id="s3-1"><title>Megf8 regulates development of the limb, skeleton, heart, left-right asymmetry, and PNS</title><p>Analysis of <italic>Megf8</italic><sup><italic>L1775P/L1775P</italic></sup> and <italic>Megf8</italic><sup><italic>−/−</italic></sup> embryos demonstrates that Megf8 functions broadly during embryogenesis and is required for the development of several organ systems. Loss of Megf8 function leads to pre-axial polydactyly, skeletal defects, disruption of left-right patterning, and severe heart defects, all of which together result in embryonic lethality (present study, and <xref ref-type="bibr" rid="bib31">Zhang et al., 2009</xref>). Interestingly, mutations in <italic>Megf8</italic> were recently identified in a small subset of children with Carpenter syndrome, which is an autosomal-recessive multiple-congenital-malformation disorder. Mutations were identified in several domains of <italic>Megf8</italic>, including the Kelch domain adjacent to the Kelch domain harboring the L1775P point mutation, and resulted in thoracic skeletal defects, limb defects such as pre-axial polydactyly, congenital heart defects, and profound lateralization defects including transposition of the great arteries, dextrocardia, and complete situs inversus (<xref ref-type="bibr" rid="bib29">Twigg et al., 2012</xref>). These phenotypes mirror those seen in our <italic>Megf8</italic> loss-of-function lines and reinforce the vital role for <italic>Megf8</italic> throughout embryogenesis.</p><p>Megf8 is also required for development of the peripheral nervous system, including the growth and guidance of the TG, spinal, and vagus/glossopharyngeal nerves. <italic>Megf8</italic><sup><italic>−/−</italic></sup> embryos exhibit profound defasciculation of the ophthalmic branch of the TG, undergrown and underbranched spinal nerves, and defasciculation of the vagus/glossopharyngeal nerves. <italic>Megf8</italic> is strongly expressed in sensory neurons throughout embryogenesis, and conditional deletion of <italic>Megf8</italic> in neural crest-derived cells, including sensory neurons (<italic>Wnt1-Cre;Megf8</italic><sup><italic>Flox/Flox</italic></sup>), leads to a defect in the TG ophthalmic nerve that phenocopies the <italic>Megf8</italic> null and <italic>Megf8</italic><sup><italic>L1775P/L1775P</italic></sup> lines. These results strongly suggest that Megf8 functions cell autonomously in TG neurons to regulate axon guidance and innervation of peripheral targets.</p></sec><sec id="s3-2"><title>Megf8 is a novel modifier of BMP4 signaling in trigeminal ganglion neurons</title><p>The phenotypes observed in <italic>Megf8</italic><sup><italic>L1775P</italic></sup> and <italic>Megf8</italic><sup><italic>−</italic></sup> mouse lines show a striking resemblance to those observed in mice lacking BMP4 signaling (<xref ref-type="table" rid="tbl1">Table 1</xref>). Like the <italic>Megf8</italic><sup><italic>−/−</italic></sup> and <italic>Megf8</italic><sup><italic>L1775P/L1775P</italic></sup> embryos, loss of BMP4 function leads to pre-axial polydactyly (<xref ref-type="bibr" rid="bib8">Dunn et al., 1997</xref>), loss of left-right patterning including reversed heart looping (<xref ref-type="bibr" rid="bib9">Fujiwara et al., 2002</xref>), and severe heart defects (<xref ref-type="bibr" rid="bib13">Jiao et al., 2003</xref>; <xref ref-type="bibr" rid="bib17">Liu et al., 2004</xref>). BMP4 signaling has recently been implicated in PNS development, where it is required by neurons in the TG for specifying positional identity (<xref ref-type="bibr" rid="bib12">Hodge et al., 2007</xref>) and in both the TG and DRG for peripheral target innervation and survival (<xref ref-type="bibr" rid="bib11">Guha et al., 2004</xref>).</p><p>Given these phenotypic similarities, we hypothesized that Megf8 is a component of the BMP4 signaling pathway and specifically regulates the response of TG neurons to target-derived BMP4. We assessed <italic>Bmp4</italic> expression during E10.5–E11.5, which is the period when TG axons extend into their peripheral target fields and found that <italic>Bmp4</italic> expression appears to define the permissible field for ophthalmic nerve target innervation suggesting that BMP4 may function as a repulsive guidance cue. Consistent with these findings, conditional deletion of the BMP receptor, <italic>Bmpr2</italic>, in sensory neurons using <italic>Wnt1-Cre;Bmpr2</italic><sup><italic>Flox/−</italic></sup> mice results in defasciculation of the ophthalmic nerve and phenocopies the <italic>Megf8</italic> mutant lines. Thus, axons of TG ophthalmic sensory neurons require both <italic>Megf8</italic> and BMP signaling to correctly navigate towards their target field. Remarkably, in each of the <italic>Megf8</italic> and <italic>Bmpr2</italic> loss-of-function lines, the TG maxillary and mandibular branches are largely unaffected despite widespread expression of Megf8 and BMPR2 within the TG ganglion, which demonstrates that Megf8 and BMPR2 function selectively in neurons of the TG ophthalmic branch to mediate axon guidance. We suggest that Megf8 is a mediator or a key component of the BMPR2 signaling pathways that control growth and guidance of ophthalmic nerve axons.</p><p>The expression pattern of <italic>Bmp4</italic> relative to the developing ophthalmic nerve suggests that BMP4 acts as an inhibitory cue to prevent developing TG axons from innervating inappropriate targets. This hypothesized role for BMP4 is supported by our in vitro findings, in which BMP4 treatment of TG explant cultures resulted in robust inhibition of axon outgrowth. This inhibition was partially lost in <italic>Megf8</italic><sup><italic>−/−</italic></sup> explants, indicating that TG neurons require <italic>Megf8</italic> expression to mediate their maximal response to the inhibitory effects of BMP4. These findings suggest that Megf8 is necessary for BMP4 signaling in sensory neurons and that this signaling provides an important inhibitory cue to guide developing TG axons. In addition, our finding that <italic>Bmp4</italic> expression is disrupted in <italic>Megf8</italic><sup><italic>−/−</italic></sup> embryos at the site of defasciculation of the TG ophthalmic branch suggests a possible non-cell autonomous role for Megf8 in regulating <italic>Bmp4</italic> expression, wherein <italic>BMP4</italic> expression is dependent on signaling from incoming TG axons, which is disrupted by defasciculation in the <italic>Megf8</italic><sup><italic>−/−</italic></sup> embryo. Alternatively, BMP4 may be directly regulated by Megf8 expressed in cells of the craniofacial region. Given that <italic>Bmp4</italic> expression is preserved in all other areas of the <italic>Megf8</italic><sup><italic>−/−</italic></sup> embryos as well as our in vitro findings that show Megf8 is required in TG neurons to mediate the inhibitory effects of exogenous BMP4, we think it is unlikely that Megf8 predominantly regulates BMP4 signaling through modifying <italic>BMP4</italic> expression. Future studies will address the Megf8 mechanism of action in developing neurons and, in particular, whether it is a mediator of expression or axonal localization of BMPR2, BMP4 binding to BMPR2, or BMP4–BMPR2 signaling in axons.</p><p>Taken together, our findings support a model in which Megf8 is a mediator of BMP4 signaling in TG axons. <italic>Megf8</italic> is expressed in TG sensory neurons where it acts cell autonomously to promote signaling in response to target-derived BMP4. Megf8-mediated BMP4 signaling, which is inhibitory for TG axons, likely enables these axons to remain fasciculated en route to their appropriate targets in the face. Disruption of this signaling, either through loss of Megf8 or other components of the BMP4 pathway, leads to a corresponding loss of inhibition and premature defasciculation of the nerve. Thus, BMP4 signaling is required for TG axon guidance and Megf8 is a novel component of BMP4 signaling in TG sensory neurons. Given the numerous phenotypic similarities between <italic>Megf8</italic> and <italic>Bmp</italic> loss-of-function mouse lines, including defects in the limb, heart, and left-right patterning, we propose that Megf8 mediates signaling by BMP4 and, perhaps, other TGFβ family members throughout the embryo during development.</p></sec></sec><sec id="s4" sec-type="materials|methods"><title>Materials and methods</title><sec id="s4-1"><title>Whole-mount neurofilament staining</title><p>Isolated embryos were fixed overnight in 4% paraformaldehyde/PBS at 4°C. All subsequent washes and incubations were performed at room temperature on an orbital shaker. After fixation, embryos were washed in PBS (three 10 min washes) and dehydrated as follows: 50% methanol/PBS (1 hr), 80% methanol/PBS (2 hr), and 100% methanol (overnight). Endogenous peroxidase activity was quenched by incubating the embryos overnight in a solution of 3% hydrogen peroxide, 70% methanol, and 20% DMSO. The embryos were next washed in five 45 min incubations in TNT (10 mM Tris-base, 154 mM NaCl, 0.1% Triton X-100) and then incubated for 3 days in primary antibody solution, which consisted of 2H3 anti-neurofilament ascites antibody (3.8 mg/ml stock, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) diluted 1:5000 in TNT with 0.02% sodium azide, 4% milk, 5% DMSO, and 2% sheep serum (Millipore, Billerica, MA). Following primary antibody, embryos were washed in TNT (five time, 45 min each), and then incubated for 2 days in secondary sheep anti-mouse IgG HRP-conjugated antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted 1:250 in TNT with 0.02% sodium azide, 4% BSA, 5% DMSO, and 2% sheep serum. The embryos were washed in TBS for four 45 min incubations followed by one overnight incubation. The following morning, the horseradish peroxidase reactions were developed with diaminobenzidine, washed in five incubations of TBS for 5 min each, and dehydrated through methanol as done previously (50%, 80%, 100% methanol). To visualize the staining, embryos were cleared in 1:2 benzyl alcohol/benzyl benzoate.</p></sec><sec id="s4-2"><title>Quantification of trigeminal nerve phenotype</title><p>In order to assess the defasciculation and outgrowth of the three trigeminal nerve branches, images of representative embryos stained with the whole-mount neurofilament assay were analyzed using ImageJ software (<xref ref-type="bibr" rid="bib1">Abramoff et al., 2004</xref>). The ophthalmic branch defasciculation was assessed by counting the number of branches that emerged from the branch point of the nasociliary nerve distal to the eye. The outgrowth of all three trigeminal nerve branches (ophthalmic, maxillary, and mandibular) was quantified by measuring the distance the branch travelled into the peripheral tissue after exiting the TG. This distance was then divided by the outgrowth measured in a littermate control in order to calculate the relative outgrowth of the nerve. In order to assess for possible defasciculation in the maxillary nerve, the area of the maxillary branch was measured beginning at the point of exit from the TG and extending to its innervation of peripheral tissues. This area was then divided by the area measured in a littermate control to calculate the relative area of the maxillary branch.</p></sec><sec id="s4-3"><title>RNA in situ hybridization</title><p>Digoxigenin (DIG)-labeled cRNA probes were used for in situ hybridization. To generate probes directed against <italic>Megf8</italic>, 1.1 kb and 2.5 kb ApaI fragments from MGC clone 5369271 (Thermo Scientific, Pittsburgh, PA) were cloned into pBK-CMV (Agilent, Santa Clara, CA). A probe for <italic>Bmpr2</italic> was amplified using gene-specific PCR primers (Allen Institute for Brain Science, Seattle, WA) and cDNA template prepared from P4 mouse brain. The resulting fragment was cloned into pCRII-TOPO (Life Technologies, Carlsbad, CA). Embryos were fixed in 4% paraformaldehyde/PBS overnight at 4°C. For whole-mount preparations, embryos were then dehydrated into methanol and in situ hybridization was performed as described in <xref ref-type="bibr" rid="bib22">Parr et al. (1993)</xref> with modifications (<xref ref-type="bibr" rid="bib15">Knecht et al., 1995</xref>). For sectioned tissue, after fixation embryos were washed in PBS, cryoprotected in 30% sucrose, embedded and frozen in Tissue-Tek OCT (Sakura Finetek USA, Inc., Torrance, CA), and serially sectioned at 20 μm. In situ hybridization was then performed on sectioned tissue as described previously (<xref ref-type="bibr" rid="bib25">Schaeren-Wiemers and Gerfin-Moser, 1993</xref>). To assess the developing neuroepithelium, in situ hybridization was performed on formalin-fixed, paraffin-embedded tissue.</p></sec><sec id="s4-4"><title>Skeletal analysis</title><p>Cartilage and bone were stained with alcian blue 8GX (Sigma-Aldrich, St. Louis, MO) and alizarin red S (Sigma-Aldrich) as previously described (<xref ref-type="bibr" rid="bib19">McLeod, 1980</xref>).</p></sec><sec id="s4-5"><title>Whole-mount peripherin staining on limb</title><p>E13.5 embryos were eviscerated and fixed in 4% paraformaldehyde/PBS overnight at 4°C. The forelimbs and hindlimbs were then separated and immunostained with rabbit anti-peripherin (Millipore, Billerica, MA, 1:2000) as described in <xref ref-type="bibr" rid="bib30">Wickramasinghe et al. (2008)</xref>.</p></sec><sec id="s4-6"><title>Whole-mount β-galactosidase and neurofilament double staining</title><p>Isolated embryos were fixed in 4% paraformaldehyde/PBS at 4°C for 30 min. After fixation, the embryos were washed three times with X-gal rinse solution (100 mM sodium phosphate pH7.3, 2 mM MgCl<sub>2</sub>, 0.01% sodium deoxycholate, and 0.02% NP40) and then stained overnight at room temperature in X-gal staining solution (5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, and 1 mg/ml X-gal in X-gal rinse solution). They were then post-fixed in 4% paraformaldehyde/PBS at 4°C overnight to improve the stability of the β-galactosidase stain. Following β-galactosidase staining and post-fixation, the embryos were immunostained according to the whole-mount neurofilament protocol outlined above.</p></sec><sec id="s4-7"><title>Trigeminal explant assay and axon growth analysis</title><p>Trigeminal ganglia (TG) were dissected from E12.5 embryos. The dorsal half of each TG was isolated in order to select for neurons that give rise to the ophthalmic branch. Explants were then cultured in collagen droplets in Neurobasal media (Life Technologies, Carlsbad, CA) supplemented with B27, 20 ng/ml NGF, and 0–100 ng/ml BMP4 (R&D Systems, Minneapolis, MN). After 40 hr in culture, the explants were fixed in 4% paraformaldehyde overnight at 4°C. They were then immunostained for neurofilament as follows. After fixation, explants were washed in PBS (three 15 min washes), permeabilized in 0.25% Triton X-100/PBS for 1 hr at room temperature, incubated overnight at room temperature with 2H3 anti-neurofilament ascites antibody (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) diluted 1:5000 in blocking buffer (5% sheep serum, 5% BSA, and 0.25% Triton X-100 in PBS), washed in 0.25% Triton X-100/PBS (five 15 min washes), incubated overnight at room temperature with secondary sheep anti-mouse IgG HRP-conjugated antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted 1:250 in blocking buffer, washed in 0.25% Triton X-100/PBS (five 15 min washes), and finally developed with diaminobenzidine to visualize staining. Images of the explants were quantified using ImageJ software (<xref ref-type="bibr" rid="bib1">Abramoff et al., 2004</xref>). In order to determine the axon outgrowth, we quantified the explant’s total area and then subtracted the small portion of this area that comprised the cell bodies; the remaining area represented the total axon outgrowth for a given explant. The relative axon growth was calculated by dividing an explant’s total axon outgrowth by the average outgrowth for control explants (control genotype and 0 ng/ml BMP4 treatment).</p></sec><sec id="s4-8"><title>Mice and genotyping</title><p>The <italic>Bmp4</italic><sup><italic>lacZ</italic></sup> (<xref ref-type="bibr" rid="bib16">Lawson et al., 1999</xref>), BMP receptor (<italic>Bmpr2</italic>) conditional knockout (<xref ref-type="bibr" rid="bib3">Beppu et al., 2005</xref>), <italic>Wnt1-Cre</italic> (<xref ref-type="bibr" rid="bib6">Danielian et al., 1998</xref>), and <italic>Mesp1-Cre</italic> (<xref ref-type="bibr" rid="bib24">Saga et al., 1999</xref>) mouse lines were generated and genotyped as previously described. The Line 687 point mutant (<italic>Megf8</italic><sup><italic>L1775P</italic></sup>) encodes a restriction enzyme-sensitive polymorphism and was genotyped by amplification of an approximately 600 bp fragment using the primers 5′-GGCAAGGGATGTGGTCATTAAG and 5′-CTTTTCTCCAAACCAGGCATGGAAA, followed by SacI digestion. The <italic>Megf8</italic><sup><italic>Trap</italic></sup> line was genotyped by PCR using the primers 5′-GACCACCAGCCTGAGTGCAAAA and 5′-TCGAGCACAGCTGCGCAAGGAA. The <italic>Megf8</italic><sup><italic>Flox</italic></sup> allele was genotyped using the primers 5′-AAGCCTGGATGCAAGGGCAGAT and 5′-CATAGGGCCACCATCAGCTCAT. The <italic>Megf8</italic><sup><italic>−</italic></sup> allele was genotyped using the primers 5′-AAGCCTGGATGCAAGGGCAGAT and 5′-CATAGGGCCACCATCAGCTCAT.</p></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>We thank members of the Ginty laboratory for discussions throughout the course of this study and for reading and commenting on the manuscript. We thank Brigid Hogan (Duke University) for providing the <italic>Bmp4</italic><sup><italic>LacZ</italic></sup> mice.</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>CE, 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>SS, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con3"><p>QW, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con4"><p>ALK, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con5"><p>HMS, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con6"><p>JM, Conception and design, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con7"><p>PL, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con8"><p>HB, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con9"><p>DDG, Senior author, designed the project and oversaw all aspects of the science and the preparation of the manuscript, Conception and design, Analysis and interpretation of data, Drafting or revising the article</p></fn></fn-group><fn-group content-type="ethics-information"><title>Ethics</title><fn fn-type="other"><p>Animal experimentation: This study was done in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols of the Johns Hopkins University School of Medicine. The protocols were approved by the Animal Care and Use Committee of the Johns Hopkins University School of Medicine (Protocol Numbers: MO11M10 and MO09M412).</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>Abramoff</surname><given-names>MD</given-names></name><name><surname>Magalhaes</surname><given-names>PJ</given-names></name><name><surname>Ram</surname><given-names>SJ</given-names></name></person-group><year>2004</year><article-title>Image processing with image J</article-title><source>Biophotonics Int</source><volume>11</volume><fpage>36</fpage><lpage>42</lpage></element-citation></ref><ref id="bib2"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Augsburger</surname><given-names>A</given-names></name><name><surname>Schucharddt</surname><given-names>A</given-names></name><name><surname>Hoskins</surname><given-names>S</given-names></name><name><surname>Dodd</surname><given-names>J</given-names></name><name><surname>Butler</surname><given-names>S</given-names></name></person-group><year>1999</year><article-title>BMPs as mediators of roof plate repulsion of commissural neurons</article-title><source>Neuron</source><volume>24</volume><fpage>127</fpage><lpage>41</lpage><pub-id pub-id-type="doi">10.1016/S0896-6273(00)80827-2</pub-id></element-citation></ref><ref id="bib3"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Beppu</surname><given-names>H</given-names></name><name><surname>Lei</surname><given-names>H</given-names></name><name><surname>Bloch</surname><given-names>K</given-names></name><name><surname>Li</surname><given-names>E</given-names></name></person-group><year>2005</year><article-title>Generation of a floxed allele of the mouse BMP type II receptor gene</article-title><source>Genesis</source><volume>41</volume><fpage>133</fpage><lpage>7</lpage><pub-id pub-id-type="doi">10.1002/gene.20099</pub-id></element-citation></ref><ref id="bib4"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Beppu</surname><given-names>H</given-names></name><name><surname>Malhotra</surname><given-names>R</given-names></name><name><surname>Beppu</surname><given-names>Y</given-names></name><name><surname>Lepore</surname><given-names>J</given-names></name><name><surname>Parmacek</surname><given-names>M</given-names></name><name><surname>Bloch</surname><given-names>K</given-names></name></person-group><year>2009</year><article-title>BMP type II receptor regulates positioning of outflow tract and remodeling atrioventricular cushion during cardiogenesis</article-title><source>Dev Biol</source><volume>331</volume><fpage>167</fpage><lpage>75</lpage><pub-id pub-id-type="doi">10.1016/j.ydbio.2009.04.032</pub-id></element-citation></ref><ref id="bib5"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Butler</surname><given-names>S</given-names></name><name><surname>Dodd</surname><given-names>J</given-names></name></person-group><year>2003</year><article-title>A role for BMP heterodimers in roof plate-mediated repulsion of commissural axons</article-title><source>Neuron</source><volume>38</volume><fpage>389</fpage><lpage>401</lpage><pub-id pub-id-type="doi">10.1016/S0896-6273(03)00254-X</pub-id></element-citation></ref><ref id="bib6"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Danielian</surname><given-names>P</given-names></name><name><surname>Muccino</surname><given-names>D</given-names></name><name><surname>Rowitch</surname><given-names>D</given-names></name><name><surname>Michael</surname><given-names>S</given-names></name><name><surname>McMahon</surname><given-names>A</given-names></name></person-group><year>1998</year><article-title>Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase</article-title><source>Curr Biol</source><volume>8</volume><fpage>1323</fpage><lpage>6</lpage><pub-id pub-id-type="doi">10.1016/S0960-9822(07)00562-3</pub-id></element-citation></ref><ref id="bib7"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Dudley</surname><given-names>A</given-names></name><name><surname>Lyons</surname><given-names>K</given-names></name><name><surname>Robertson</surname><given-names>E</given-names></name></person-group><year>1995</year><article-title>A requirement for bone morphogenetic protein-7 during development of the mammalian kidney and eye</article-title><source>Genes Dev</source><volume>9</volume><fpage>2795</fpage><lpage>807</lpage><pub-id pub-id-type="doi">10.1101/gad.9.22.2795</pub-id></element-citation></ref><ref id="bib8"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Dunn</surname><given-names>NR</given-names></name><name><surname>Winnier</surname><given-names>G</given-names></name><name><surname>Hargett</surname><given-names>L</given-names></name><name><surname>Schrick</surname><given-names>J</given-names></name><name><surname>Fogo</surname><given-names>A</given-names></name><name><surname>Hogan</surname><given-names>B</given-names></name></person-group><year>1997</year><article-title>Haploinsufficient phenotypes in <italic>Bmp4</italic> heterozygous null mice and modification by mutations in <italic>Gli3</italic> and <italic>Alx4</italic></article-title><source>Dev Biol</source><volume>188</volume><fpage>235</fpage><lpage>47</lpage><pub-id pub-id-type="doi">10.1006/dbio.1997.8664</pub-id></element-citation></ref><ref id="bib9"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Fujiwara</surname><given-names>T</given-names></name><name><surname>Dehart</surname><given-names>D</given-names></name><name><surname>Sulik</surname><given-names>K</given-names></name><name><surname>Hogan</surname><given-names>B</given-names></name></person-group><year>2002</year><article-title>Distinct requirements for extra-embryonic and embryonic bone morphogenetic protein 4 in the formation of the node and primitive streak and coordination of left-right asymmetry in the mouse</article-title><source>Development</source><volume>129</volume><fpage>4685</fpage><lpage>96</lpage></element-citation></ref><ref id="bib10"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Goldman</surname><given-names>D</given-names></name><name><surname>Donley</surname><given-names>N</given-names></name><name><surname>Christian</surname><given-names>J</given-names></name></person-group><year>2009</year><article-title>Genetic interaction between Bmp2 and Bmp4 reveals shared functions during multiple aspects of mouse organogenesis</article-title><source>Mech Dev</source><volume>126</volume><fpage>117</fpage><lpage>27</lpage><pub-id pub-id-type="doi">10.1016/j.mod.2008.11.008</pub-id></element-citation></ref><ref id="bib11"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Guha</surname><given-names>U</given-names></name><name><surname>Gomes</surname><given-names>W</given-names></name><name><surname>Samantra</surname><given-names>J</given-names></name><name><surname>Gupta</surname><given-names>M</given-names></name><name><surname>Rice</surname><given-names>F</given-names></name><name><surname>Kessler</surname><given-names>J</given-names></name></person-group><year>2004</year><article-title>Target-derived BMP signaling limits sensory neuron number and the extent of peripheral innervation in vivo</article-title><source>Development</source><volume>131</volume><fpage>1175</fpage><lpage>86</lpage><pub-id pub-id-type="doi">10.1242/dev.01013</pub-id></element-citation></ref><ref id="bib12"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hodge</surname><given-names>L</given-names></name><name><surname>Klassen</surname><given-names>M</given-names></name><name><surname>Han</surname><given-names>B</given-names></name><name><surname>Yiu</surname><given-names>G</given-names></name><name><surname>Hurrell</surname><given-names>J</given-names></name><name><surname>Howell</surname><given-names>A</given-names></name><etal/></person-group><year>2007</year><article-title>Retrograde BMP signaling regulates trigeminal sensory neuron identities and the formation of precise face maps</article-title><source>Neuron</source><volume>55</volume><fpage>572</fpage><lpage>86</lpage><pub-id pub-id-type="doi">10.1016/j.neuron.2007.07.010</pub-id></element-citation></ref><ref id="bib13"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Jiao</surname><given-names>K</given-names></name><name><surname>Kulessa</surname><given-names>H</given-names></name><name><surname>Tompins</surname><given-names>K</given-names></name><name><surname>Zhou</surname><given-names>Y</given-names></name><name><surname>Batts</surname><given-names>L</given-names></name><name><surname>Baldwin</surname><given-names>HS</given-names></name><etal/></person-group><year>2003</year><article-title>An essential role of Bmp4 in the atrioventricular sepation of the mouse heart</article-title><source>Genes Dev</source><volume>17</volume><fpage>2362</fpage><lpage>7</lpage><pub-id pub-id-type="doi">10.1101/gad.1124803</pub-id></element-citation></ref><ref id="bib14"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kisanuki</surname><given-names>YY</given-names></name><name><surname>Hammer</surname><given-names>RE</given-names></name><name><surname>Miyazaki</surname><given-names>J</given-names></name><name><surname>Williams</surname><given-names>SC</given-names></name><name><surname>Richardson</surname><given-names>JA</given-names></name><name><surname>Yanagisawa</surname><given-names>M</given-names></name></person-group><year>2001</year><article-title>Tie2-Cre transgenic mice: a new model for endothelial cell-lineage analysis in vivo</article-title><source>Dev Biol</source><volume>230</volume><fpage>230</fpage><lpage>42</lpage><pub-id pub-id-type="doi">10.1006/dbio.2000.0106</pub-id></element-citation></ref><ref id="bib15"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Knecht</surname><given-names>A</given-names></name><name><surname>Good</surname><given-names>P</given-names></name><name><surname>Dawid</surname><given-names>I</given-names></name><name><surname>Harland</surname><given-names>R</given-names></name></person-group><year>1995</year><article-title>Dorsal-ventral patterning and differentiation of noggin-induced neural tissue in the absence of mesoderm</article-title><source>Development</source><volume>121</volume><fpage>1927</fpage><lpage>36</lpage></element-citation></ref><ref id="bib16"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lawson</surname><given-names>K</given-names></name><name><surname>Dunn</surname><given-names>NR</given-names></name><name><surname>Roelen</surname><given-names>B</given-names></name><name><surname>Zeinstra</surname><given-names>L</given-names></name><name><surname>Davis</surname><given-names>A</given-names></name><name><surname>Wright</surname><given-names>C</given-names></name><etal/></person-group><year>1999</year><article-title>Bmp4 is required for the generation of primordial germ cells in the mouse embryo</article-title><source>Genes Dev</source><volume>13</volume><fpage>424</fpage><lpage>36</lpage><pub-id pub-id-type="doi">10.1101/gad.13.4.424</pub-id></element-citation></ref><ref id="bib17"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Liu</surname><given-names>W</given-names></name><name><surname>Selever</surname><given-names>J</given-names></name><name><surname>Wang</surname><given-names>D</given-names></name><name><surname>Lu</surname><given-names>M-F</given-names></name><name><surname>Moses</surname><given-names>K</given-names></name><name><surname>Schwartz</surname><given-names>R</given-names></name><etal/></person-group><year>2004</year><article-title>Bmp4 signaling is required for outflow-tract septation and branchial-arch artery remodeling</article-title><source>Proc Natl Acad Sci USA</source><volume>101</volume><fpage>4489</fpage><lpage>94</lpage><pub-id pub-id-type="doi">10.1073/pnas.0308466101</pub-id></element-citation></ref><ref id="bib18"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Luo</surname><given-names>G</given-names></name><name><surname>Hofmann</surname><given-names>C</given-names></name><name><surname>Bronckers</surname><given-names>A</given-names></name><name><surname>Sohocki</surname><given-names>M</given-names></name><name><surname>Bradley</surname><given-names>A</given-names></name><name><surname>Karsentry</surname><given-names>G</given-names></name></person-group><year>1995</year><article-title>BMP-7 is an inducer of nephorogenesis, and is also required for eye development and skeletal patterning</article-title><source>Genes Dev</source><volume>9</volume><fpage>2808</fpage><lpage>20</lpage><pub-id pub-id-type="doi">10.1101/gad.9.22.2808</pub-id></element-citation></ref><ref id="bib19"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>McLeod</surname><given-names>M</given-names></name></person-group><year>1980</year><article-title>Differential staining of cartilage and bone in whole mouse fetuses by alcian blue and alizarin red S</article-title><source>Teratology</source><volume>22</volume><fpage>229</fpage><lpage>301</lpage><pub-id pub-id-type="doi">10.1002/tera.1420220306</pub-id></element-citation></ref><ref id="bib20"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Merte</surname><given-names>J</given-names></name><name><surname>Wang</surname><given-names>Q</given-names></name><name><surname>Vander Kooi</surname><given-names>C</given-names></name><name><surname>Sarsfield</surname><given-names>S</given-names></name><name><surname>Leahy</surname><given-names>D</given-names></name><name><surname>Kolodkin</surname><given-names>A</given-names></name><etal/></person-group><year>2010</year><article-title>A forward genetic screen in mice identifies Sema3A<sup>K108N</sup>, which binds to Neuropilin-1 but cannot signal</article-title><source>J Neurosci</source><volume>30</volume><fpage>5767</fpage><lpage>75</lpage><pub-id pub-id-type="doi">10.1523/JNEUROSCI.5061-09.2010</pub-id></element-citation></ref><ref id="bib21"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Miyazono</surname><given-names>K</given-names></name><name><surname>Kamiya</surname><given-names>Y</given-names></name><name><surname>Morikawa</surname><given-names>M</given-names></name></person-group><year>2010</year><article-title>Bone morphogenetic protein receptors and signal transduction</article-title><source>J Biochem</source><volume>147</volume><fpage>35</fpage><lpage>51</lpage><pub-id pub-id-type="doi">10.1093/jb/mvp148</pub-id></element-citation></ref><ref id="bib22"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Parr</surname><given-names>BA</given-names></name><name><surname>Shea</surname><given-names>MJ</given-names></name><name><surname>Vassileva</surname><given-names>G</given-names></name><name><surname>McMahon</surname><given-names>AP</given-names></name></person-group><year>1993</year><article-title>Mouse <italic>Wnt</italic> genes exhibit discrete domains of expression in the early embryonic CNS and limb buds</article-title><source>Development</source><volume>119</volume><fpage>247</fpage><lpage>61</lpage></element-citation></ref><ref id="bib23"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Rivera-Feliciano</surname><given-names>J</given-names></name><name><surname>Tabin</surname><given-names>C</given-names></name></person-group><year>2006</year><article-title>Bmp2 instructs cardiac progenitors to form the heart-valve-inducing field</article-title><source>Dev Biol</source><volume>295</volume><fpage>580</fpage><lpage>8</lpage><pub-id pub-id-type="doi">10.1016/j.ydbio.2006.03.043</pub-id></element-citation></ref><ref id="bib24"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Saga</surname><given-names>Y</given-names></name><name><surname>Miyagawa-Tomita</surname><given-names>S</given-names></name><name><surname>Takagi</surname><given-names>A</given-names></name><name><surname>Kitajima</surname><given-names>S</given-names></name><name><surname>Miyazaki</surname><given-names>J</given-names></name><name><surname>Inoue</surname><given-names>T</given-names></name></person-group><year>1999</year><article-title>MesP1 is expressed in the heart precursor cells and required for the formation of a single heart tube</article-title><source>Development</source><volume>126</volume><fpage>3437</fpage><lpage>47</lpage></element-citation></ref><ref id="bib25"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Schaeren-Wiemers</surname><given-names>N</given-names></name><name><surname>Gerfin-Moser</surname><given-names>A</given-names></name></person-group><year>1993</year><article-title>A single protocol to detect transcripts of various types and expression levels in neural tissue and cultured cells: in situ hybridization using digoxigenin-labelled cRNA probes</article-title><source>Histochemistry</source><volume>100</volume><fpage>431</fpage><lpage>40</lpage><pub-id pub-id-type="doi">10.1007/BF00267823</pub-id></element-citation></ref><ref id="bib26"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Schwenk</surname><given-names>F</given-names></name><name><surname>Baron</surname><given-names>U</given-names></name><name><surname>Rajewsky</surname><given-names>K</given-names></name></person-group><year>1995</year><article-title>A cre-transgenic mouse strain for the ubiquitous deletion of loxP-flanked gene segments including deletion in germ cells</article-title><source>Nucleic Acids Res</source><volume>23</volume><fpage>5080</fpage><lpage>1</lpage><pub-id pub-id-type="doi">10.1093/nar/23.24.5080</pub-id></element-citation></ref><ref id="bib27"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Selever</surname><given-names>J</given-names></name><name><surname>Liu</surname><given-names>W</given-names></name><name><surname>Lu</surname><given-names>M</given-names></name><name><surname>Behringer</surname><given-names>R</given-names></name><name><surname>Martin</surname><given-names>J</given-names></name></person-group><year>2004</year><article-title>Bmp4 in limb bud mesoderm regulates digit pattern by controlling AER development</article-title><source>Dev Biol</source><volume>276</volume><fpage>268</fpage><lpage>79</lpage><pub-id pub-id-type="doi">10.1016/j.ydbio.2004.08.024</pub-id></element-citation></ref><ref id="bib28"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Soriano</surname><given-names>P</given-names></name></person-group><year>1999</year><article-title>Generalized lacZ expression with the ROSA26 Cre reporter strain</article-title><source>Nat Genet</source><volume>21</volume><fpage>70</fpage><lpage>1</lpage><pub-id pub-id-type="doi">10.1038/5007</pub-id></element-citation></ref><ref id="bib29"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Twigg</surname><given-names>S</given-names></name><name><surname>Lloyd</surname><given-names>D</given-names></name><name><surname>Jenkins</surname><given-names>D</given-names></name><name><surname>Elcioglu</surname><given-names>N</given-names></name><name><surname>Cooper</surname><given-names>C</given-names></name><name><surname>Al-Sannaa</surname><given-names>N</given-names></name><etal/></person-group><year>2012</year><article-title>Mutations in multidomain protein MEGF8 identify a Carpenter syndrome subtype associated with defective lateralization</article-title><source>Am J Hum Genet</source><volume>91</volume><fpage>897</fpage><lpage>905</lpage><pub-id pub-id-type="doi">10.1016/j.ajhg.2012.08.027</pub-id></element-citation></ref><ref id="bib30"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wickramasinghe</surname><given-names>SR</given-names></name><name><surname>Alvania</surname><given-names>R</given-names></name><name><surname>Ramanan</surname><given-names>N</given-names></name><name><surname>Wood</surname><given-names>J</given-names></name><name><surname>Mandai</surname><given-names>K</given-names></name><name><surname>Ginty</surname><given-names>D</given-names></name></person-group><year>2008</year><article-title>Serum response factor mediates NGF-dependent target innervation by embryonic DRG sensory neurons</article-title><source>Neuron</source><volume>58</volume><fpage>532</fpage><lpage>45</lpage><pub-id pub-id-type="doi">10.1016/j.neuron.2008.03.006</pub-id></element-citation></ref><ref id="bib31"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Zhang</surname><given-names>Z</given-names></name><name><surname>Alpert</surname><given-names>D</given-names></name><name><surname>Francis</surname><given-names>R</given-names></name><name><surname>Chatterjee</surname><given-names>B</given-names></name><name><surname>Yu</surname><given-names>Q</given-names></name><name><surname>Tansey</surname><given-names>T</given-names></name><etal/></person-group><year>2009</year><article-title>Massively parallel sequencing identifies the gene Megf8 with ENU-induced mutation causing heterotaxy</article-title><source>Proc Natl Acad Sci USA</source><volume>106</volume><fpage>3219</fpage><lpage>24</lpage><pub-id pub-id-type="doi">10.1073/pnas.0813400106</pub-id></element-citation></ref><ref id="bib32"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Zhao</surname><given-names>G-Q</given-names></name></person-group><year>2003</year><article-title>Consequences of knocking out BMP signaling in the mouse</article-title><source>Genesis</source><volume>35</volume><fpage>43</fpage><lpage>56</lpage><pub-id pub-id-type="doi">10.1002/gene.10167</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.01160.018</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://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 “MEGF8 is a modifier of BMP signaling in trigeminal sensory neurons” for consideration at <italic>eLife</italic>. Your article has been favorably evaluated by a Senior editor, a Reviewing editor, and 2 reviewers.</p><p>The following individuals responsible for the peer review of your submission have agreed to reveal their identity: Marianne Bronner (Reviewing editor); Samantha Butler and Bill Snider (peer reviewers).</p><p>The Reviewing editor and the two 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>This is a straightforward and nicely done study. By screening for peripheral nerve defects after ENU mutagenesis, the authors have identified a mutation in the <italic>Megf8</italic> gene. Mutation of the human homologue (Carpenter syndrome) is associated with dramatic developmental abnormalities. Here, the authors describe the cloning of <italic>Megf8</italic> as well as the careful and thorough characterization of its loss-of-function. Megf8 appears to be a novel receptor with critical roles in axon guidance, the establishment of left-right asymmetry and limb, skeletal, and heart development. Because the mouse and human phenotypes bear similarity to loss of BMP4 signaling, the authors performed a comprehensive analysis of the requirement for Megf8 in development in comparison to the requirement for <italic>Bmp4</italic>.</p><p>As a result of their findings, the authors propose an important mechanistic link between Megf8 and BMP signaling: that Megf8 is required to interpret BMP4 as a repellent. <italic>Bmp4</italic> expression surrounds the ophthalmic branch of the trigeminal ganglion (TG) and apparently keeps the nerve fasciculated. In the absence of either Megf8 or BmprII, this branch is severely defasciculated. Moreover, loss of Megf8 results in the loss of <italic>Bmp4</italic> expression flanking the ophthalmic branch, in the region where the most profound defasciculation defects are observed. Taken together, these studies identify a novel putative regulator of BMP signaling and raise important questions about the non-autonomous control of <italic>Bmp4</italic> expression by Megf8.</p><p>Major comments:</p><p>1) This paper provides a mechanistic explanation for the defasciculation defect observed in <italic>Megf8</italic> mutants, but does not explain the foreshortening of many nerves, including their focus in this paper: the ophthalmic branch (Figure4H)? If removing the response to a repellent, wouldn't one predict that the axons might grow longer, as seen in <xref ref-type="fig" rid="fig6">Figure 6</xref>?</p><p>2) The phenotype observed in <xref ref-type="fig" rid="fig5">Figure 5B</xref> is very interesting, suggesting that Megf8 is required to stabilize or maintain <italic>Bmp4</italic> expression. Is this phenotype also fully penetrant (add to <xref ref-type="table" rid="tbl1">Table 1</xref>?)? Could the authors provide some quantification of <italic>Megf8</italic> mutants to more clearly assess the extent to which <italic>Bmp4</italic> activity staining is lost in the region surrounding the ophthalmic nerve compared to controls? Importantly, the authors barely discuss this very striking result in the Discussion! Doesn't it suggest that the loss of Megf8 has a non-autonomous negative effect on <italic>Bmp4</italic> expression? If so, how mechanistically do they think a membrane bound receptor is suppressing <italic>Bmp4</italic> expression?</p><p>3) Is the interaction between <italic>Megf8</italic> and BMP4 the likely mechanistic explanation for the other phenotypes? In which case, why is <italic>Bmp4</italic> expression not lost in other affected organs (<xref ref-type="fig" rid="fig5">Figure 5</xref>)?</p><p>4) The authors are careful to assay only the response of the ophthalmic branch of the TG nerve to BMP4 stimulation in vitro. Did they try a similar in vitro assay with the maxillary and mandibular branches? It would be interesting to know whether these branches are impervious to BMP4 in vitro as suggested by the results in vivo.</p><p>5) Similarly, presumably other classes of neurons are affected, as there are also abnormalities of spinal roots although the spinal root abnormalities were not analyzed in detail.</p><p>6) Although the link to BMP signaling is intriguing, the current evidence is rather indirect and based primarily on their one in vitro experiment. A simple way to provide strong evidence for a direct effect on BMP signaling in vivo would be to assess Phospho-Smad staining levels in mutant versus wild type trigeminal ganglia. The authors should attempt this experiment.</p><p>7) In the Discussion, it is critical that the authors add further comment on how they think Megf8 regulates BMP4 signaling.</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.01160.019</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p><italic>1) This paper provides a mechanistic explanation for the defasciculation defect observed in</italic> Megf8 <italic>mutants, but does not explain the foreshortening of many nerves, including their focus in this paper: the ophthalmic branch (</italic><xref ref-type="fig" rid="fig4"><italic>Figure 4H</italic></xref><italic>)? If removing the response to a repellent, wouldn't one predict that the axons might grow longer, as seen in</italic> <xref ref-type="fig" rid="fig6"><italic>Figure 6</italic></xref>?</p><p>There are two possibilities for the foreshortening observed in the ophthalmic branch of the trigeminal nerve. One possibility is that the foreshortening is secondary to the defasciculation. In other words, individual ophthalmic axons grow to the same length (or longer) in <italic>Megf8</italic> mutants, but because the axons aberrantly extend into surrounding tissues the overall path of the nerve appears shorter. The other possibility is that in addition to its role in mediating BMP4 inhibitory cues, Megf8 also facilitates the growth of ophthalmic axons into the periphery and thus these axons are undergrown in <italic>Megf8</italic> mutants. This second possibility is consistent with the phenotype observed in the spinal nerves, which are undergrown in <italic>Megf8</italic> null embryos when compared to controls (<xref ref-type="fig" rid="fig4">Figure 4A,D</xref>). In addition, the maxillary branch of the trigeminal nerve is slightly undergrown in <italic>Megf8</italic> mutants compared to control (<xref ref-type="fig" rid="fig4">Figure 4</xref> legend). Taken together, these results suggest a role for Megf8 in promoting axon growth of TG and DRG sensory neurons. However, our data do not determine whether this occurs through modification of BMP4 signaling.</p><p><italic>2) The phenotype observed in</italic> <xref ref-type="fig" rid="fig5"><italic>Figure 5B</italic></xref> <italic>is very interesting, suggesting that Megf8 is required to stabilize or maintain Bmp4 expression. Is this phenotype also fully penetrant (add to</italic> <xref ref-type="table" rid="tbl1"><italic>Table 1</italic></xref><italic>?)? Could the authors provide some quantification of</italic> Megf8 <italic>mutants to more clearly assess the extent to which</italic> Bmp4 <italic>activity staining is lost in the region surrounding the ophthalmic nerve compared to controls? Importantly, the authors barely discuss this very striking result in the Discussion! Doesn't it suggest that the loss of Megf8 has a non-autonomous negative effect on</italic> Bmp4 <italic>expression? If so, how mechanistically do they think a membrane bound receptor is suppressing</italic> Bmp4 <italic>expression</italic>?</p><p>The disruption of <italic>Bmp4-lacZ</italic> expression around the TG ophthalmic branch is fully penetrant in <italic>Megf8</italic><sup><italic>-/-</italic></sup> embryos, and this point is now made in the Table in the revised paper. Interestingly, <italic>Bmp4-lacZ</italic> expression is normal in all other parts of the embryo. This can be appreciated in <xref ref-type="fig" rid="fig5s1">Figure 5–figure supplement 1</xref>, which shows the <italic>Bmp4-lacZ</italic> expression pattern throughout <italic>Megf8</italic><sup><italic>-/-</italic></sup> and <italic>Megf8</italic><sup><italic>+/+</italic></sup> embryos and includes images of the whole embryo, DRG, and limb.</p><p>We have provided commentary on the changes in the <italic>Bmp4-lacZ</italic> expression pattern in the <italic>Megf8</italic><sup><italic>-/-</italic></sup> embryo and the implications of these changes in the revised manuscript. Our findings suggest that the loss of BMP4 expression at the site of defasciculation of the TG ophthalmic branch could result from two possible mechanisms. Either BMP4 expression is dependent on signaling from incoming TG axons (which is disrupted by defasciculation in the <italic>Megf8</italic><sup><italic>-/-</italic></sup> embryo) or Megf8 expressed in the craniofacial target region regulates BMP4 expression. This is an interesting point, as we know from our ISH results that <italic>Megf8</italic> is expressed in TG neurons as well as in the developing craniofacial area. Because neural crest cells contribute to this developing craniofacial area, the <italic>Wnt1-Cre</italic>; <italic>Megf8</italic><sup><italic>f/f</italic></sup> embryo unfortunately does not allow us to distinguish between these two possibilities.</p><p><italic>3) Is the interaction between</italic> Megf8 <italic>and BMP4 the likely mechanistic explanation for the other phenotypes? In which case, why is</italic> Bmp4 <italic>expression not lost in other affected organs (</italic><xref ref-type="fig" rid="fig5"><italic>Figure 5</italic></xref><italic>)</italic>?</p><p>Indeed, given the phenotypic similarities between <italic>Megf8</italic> mutant and <italic>BMP4</italic> loss of function lines, we hypothesize that the functional interaction between Megf8 and BMP4 is the mechanistic explanation for the other phenotypes observed in <italic>Megf8</italic> mutants, including polydactyly, disruption of left-right patterning, and heart defects. As we have shown, <italic>BMP4</italic> expression in <italic>Megf8</italic> null embryos is only disrupted around the TG ophthalmic branch; it is intact in all other areas of the embryo including the DRG, heart, and limbs. These findings suggest that Megf8 modifies BMP4 signaling by acting on the BMP4 ligand/receptor complex or downstream components of the signaling pathway, not through modifying expression of <italic>BMP4</italic> itself. Indeed, this is consistent with our in vitro findings (<xref ref-type="fig" rid="fig6">Figure 6</xref>), in which we found that in the presence of exogenous BMP4 ligand, <italic>Megf8</italic> null axons respond poorly to BMP4 as an inhibitory cue. If Megf8’s role in BMP4 signaling is primarily to modify <italic>BMP4</italic> expression, one would expect that <italic>Megf8</italic> null axons would still be inhibited by exogenous BMP4 ligand, unlike our results in <xref ref-type="fig" rid="fig6">Figure 6</xref>. In fact, we were surprised to find that BMP4 expression is disrupted around the TG ophthalmic nerve. We suggest that this finding (<xref ref-type="fig" rid="fig5">Figure 5</xref>) in conjunction with our in vitro analysis (<xref ref-type="fig" rid="fig6">Figure 6</xref>) indicates that Megf8 modifies BMP4 signaling in TG ophthalmic axons in two ways: 1) Megf8 expressed in the axon is required to mediate the BMP4 inhibitory cue and 2) loss of Meg8 in the axon, loss of Megf8 in the surrounding craniofacial tissue, or loss of normal TG axon extension (secondary to loss of Megf8) disrupts BMP4 expression. This is discussed in the revised manuscript.</p><p><italic>4) The authors are careful to assay only the response of the ophthalmic branch of the TG nerve to BMP4 stimulation</italic> in vitro<italic>. Did they try a similar</italic> in vitro <italic>assay with the maxillary and mandibular branches? It would be interesting to know whether these branches are impervious to BMP4</italic> in vitro <italic>as suggested by the results</italic> in vivo.</p><p>Our initial explant experiments included the entire ganglion and we did see robust inhibition of axon outgrowth by BMP4 (as well as loss of this inhibition in the <italic>Megf8</italic><sup><italic>-/-</italic></sup> explants). However, we decided to focus on the dorsal half of the TG, which contains the ophthalmic lobe and part of the maxillary lobe, so as to enrich for ophthalmic neurons. Given the large amount of maxillary neurons present (roughly 50% of the explant), our results suggest that BMP4 can inhibit maxillary axon outgrowth, although it is unclear if BMP4 inhibits maxillary neurons to the same extent as ophthalmic neurons. We did not attempt a separate analysis of the maxillary and mandibular branches.</p><p><italic>5) Similarly, presumably other classes of neurons are affected, as there are also abnormalities of spinal roots although the spinal root abnormalities were not analyzed in detail</italic>.</p><p><italic>Megf8</italic> null embryos show undergrowth of DRG spinal nerves as well as defasciculation of the vagus and glossopharyngeal nerves. In addition, Megf8 is broadly expressed throughout the developing neuroepithelium (<xref ref-type="fig" rid="fig2">Figure 2</xref>), which suggests that it may play a role in different neuronal populations in the CNS and PNS. However, we have not yet asked whether BMP signaling is disrupted in other classes of neurons. In future experiments it will be important to assess the role of Megf8 in other axon guidance events controlled by BMP signaling, including the ventral axonal trajectory of neurons whose cell bodies reside within the dorsal spinal cord.</p><p><italic>6) Although the link to BMP signaling is intriguing, the current evidence is rather indirect and based primarily on their one</italic> in vitro <italic>experiment. A simple way to provide strong evidence for a direct effect on BMP signaling</italic> in vivo <italic>would be to assess Phospho-Smad staining levels in mutant versus wild type trigeminal ganglia. The authors should attempt this experiment</italic>.</p><p>Phospho-SMAD immunohistochemistry on wild type and <italic>Megf8</italic> null TG was attempted but, unfortunately, we encountered a widespread pattern of very strong staining in virtually all cell types and tissues in the preparations, and very high background. Thus, we were unable to ascertain whether phospho-SMAD expression is intact or diminished in <italic>Megf8</italic> null embryos (which would imply that Megf8 modifies BMP4 signaling via a non-SMAD dependent mechanism), or if we could not observe a difference that was present because of issues of specificity or because this assay would not allow us to detect subtle changes in phospho-SMAD levels. We are therefore not confident that P-SMAD staining provides a specific and reliable quantitative assay for comparing <italic>Megf8</italic><sup><italic>-/-</italic></sup> TG to wild type littermates and have not pursued this direction.</p><p><italic>7) In the Discussion, it is critical that the authors add further comment on how they think Megf8 regulates BMP4 signaling</italic>.</p><p>We have included discussion of mechanisms by which Megf8 regulates BMP signaling in the revised manuscript.</p></body></sub-article></article> |