<?xml version="1.0" encoding="UTF-8" standalone="no"?>
<!DOCTYPE article PUBLIC "-//NLM//DTD Journal Publishing DTD v2.3 20070202//EN" "journalpublishing.dtd">
<article xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink" article-type="research-article">
<front>
<journal-meta>
<journal-id journal-id-type="publisher-id">Front. Ecol. Evol.</journal-id>
<journal-title>Frontiers in Ecology and Evolution</journal-title>
<abbrev-journal-title abbrev-type="pubmed">Front. Ecol. Evol.</abbrev-journal-title>
<issn pub-type="epub">2296-701X</issn>
<publisher>
<publisher-name>Frontiers Media S.A.</publisher-name>
</publisher>
</journal-meta>
<article-meta>
<article-id pub-id-type="doi">10.3389/fevo.2015.00080</article-id>
<article-categories>
<subj-group subj-group-type="heading">
<subject>Genetics</subject>
<subj-group>
<subject>Original Research</subject>
</subj-group>
</subj-group>
</article-categories>
<title-group>
<article-title>Expression of Fox genes in the cephalochordate <italic>Branchiostoma lanceolatum</italic></article-title>
</title-group>
<contrib-group>
<contrib contrib-type="author">
<name><surname>Aldea</surname> <given-names>Daniel</given-names></name>
<uri xlink:href="http://loop.frontiersin.org/people/200158/overview"/>
</contrib>
<contrib contrib-type="author">
<name><surname>Leon</surname> <given-names>Anthony</given-names></name>
<uri xlink:href="http://loop.frontiersin.org/people/245683/overview"/></contrib>
<contrib contrib-type="author" corresp="yes">
<name><surname>Bertrand</surname> <given-names>Stephanie</given-names></name>
<xref ref-type="author-notes" rid="fn001"><sup>&#x0002A;</sup></xref>
<uri xlink:href="http://loop.frontiersin.org/people/185343/overview"/></contrib>
<contrib contrib-type="author" corresp="yes">
<name><surname>Escriva</surname> <given-names>Hector</given-names></name>
<xref ref-type="author-notes" rid="fn001"><sup>&#x0002A;</sup></xref>
<uri xlink:href="http://loop.frontiersin.org/people/163892/overview"/></contrib>
</contrib-group>
<aff><institution>Centre National De La Recherche Scientifique, UPMC universit&#x000E9; Paris 06, UMR 7232, BIOM, Observatoire Oc&#x000E9;anologique de Banyuls sur Mer</institution> <country>Banyuls sur Mer, France</country></aff>
<author-notes>
<fn fn-type="edited-by"><p>Edited by: Naoki Osada, Hokkaido University, Japan</p></fn>
<fn fn-type="edited-by"><p>Reviewed by: Jr-Kai Yu, Academia Sinica, Taiwan; Haruki Ochi, Yamagata University, Japan</p></fn>
<fn fn-type="corresp" id="fn001"><p>&#x0002A;Correspondence: Stephanie Bertrand and Hector Escriva, Laboratoire Arago, Avenue du Fontaul&#x000E9;, 66650 Banyuls-sur-Mer, France <email>stephanie.bertrand&#x00040;obs-banyuls.fr</email>; <email>hescriva&#x00040;obs-banyuls.fr</email></p></fn>
<fn fn-type="other" id="fn002"><p>This article was submitted to Evolutionary and Population Genetics, a section of the journal Frontiers in Ecology and Evolution</p></fn>
</author-notes>
<pub-date pub-type="epub">
<day>28</day>
<month>07</month>
<year>2015</year>
</pub-date>
<pub-date pub-type="collection">
<year>2015</year>
</pub-date>
<volume>3</volume>
<elocation-id>80</elocation-id>
<history>
<date date-type="received">
<day>29</day>
<month>05</month>
<year>2015</year>
</date>
<date date-type="accepted">
<day>07</day>
<month>07</month>
<year>2015</year>
</date>
</history>
<permissions>
<copyright-statement>Copyright &#x000A9; 2015 Aldea, Leon, Bertrand and Escriva.</copyright-statement>
<copyright-year>2015</copyright-year>
<copyright-holder>Aldea, Leon, Bertrand and Escriva</copyright-holder>
<license xlink:href="http://creativecommons.org/licenses/by/4.0/"><p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</p></license>
</permissions>
<abstract><p>Forkhead box (Fox) genes code for transcription factors that play important roles in different biological processes. They are found in a wide variety of organisms and appeared in unicellular eukaryotes. In metazoans, the gene family includes many members that can be subdivided into 24 classes. Cephalochordates are key organisms to understand the functional evolution of gene families in the chordate lineage due to their phylogenetic position as an early divergent chordate, their simple anatomy and genome structure. In the genome of the cephalochordate amphioxus <italic>Branchiostoma floridae</italic>, 32 Fox genes were identified, with at least one member for each of the classes that were present in the ancestor of bilaterians. In this work we describe the expression pattern of 13 of these genes during the embryonic development of the Mediterranean amphioxus, <italic>Branchiostoma lanceolatum</italic>. We found that <italic>FoxK</italic> and <italic>FoxM</italic> genes present an ubiquitous expression while all the others show specific expression patterns restricted to diverse embryonic territories. Many of these expression patterns are conserved with vertebrates, suggesting that the main functions of Fox genes in chordates were present in their common ancestor.</p></abstract>
<kwd-group>
<kwd>Fox genes</kwd>
<kwd>amphioxus</kwd>
<kwd>Evo-Devo</kwd>
<kwd>chordates</kwd>
<kwd>embryonic development</kwd>
</kwd-group>
<contract-sponsor id="cn001">EXOMOD</contract-sponsor>
<counts>
<fig-count count="4"/>
<table-count count="0"/>
<equation-count count="0"/>
<ref-count count="71"/>
<page-count count="12"/>
<word-count count="8516"/>
</counts>
</article-meta>
</front>
<body>
<sec sec-type="intro" id="s1"><title>Introduction</title>
<p>Forkhead box (Fox) transcription factors originated early during evolution and are specific to opisthokonts. They are present in fungi as well as in metazoans (Mazet et al., <xref ref-type="bibr" rid="B32">2006</xref>; Larroux et al., <xref ref-type="bibr" rid="B29">2008</xref>; Shimeld et al., <xref ref-type="bibr" rid="B49">2010a</xref>) in which they play essential roles during embryonic development (Carlsson and Mahlapuu, <xref ref-type="bibr" rid="B9">2002</xref>; Tuteja and Kaestner, <xref ref-type="bibr" rid="B59">2007a</xref>,<xref ref-type="bibr" rid="B60">b</xref>; Benayoun et al., <xref ref-type="bibr" rid="B3">2011</xref>). Fox proteins possess a helix-turn-helix DNA-binding domain called the forkhead domain which corresponds to a conserved region of approximately 110 amino acids (Weigel and Jackle, <xref ref-type="bibr" rid="B63">1990</xref>; Clark et al., <xref ref-type="bibr" rid="B13">1993</xref>). A molecular phylogeny-based classification of the Fox gene family allowed to propose its subdivision into 24 classes (ranged from FoxA to FoxS and including subfamilies that were recently subdivided: FoxJ (FoxJ1 and FoxJ2), FoxL (FoxL1 and FoxL2), and FoxN (FoxN1/4 and FoxN2/3) (Mazet et al., <xref ref-type="bibr" rid="B34">2003</xref>). Many Fox gene losses or duplications occurred in different bilaterian clades, affecting different Fox classes. For example, FoxAB is found in cephalochordates and in the sea urchin but not in tunicates or vertebrates (Tu et al., <xref ref-type="bibr" rid="B58">2006</xref>; Yu et al., <xref ref-type="bibr" rid="B70">2008a</xref>), and families R and S are vertebrate-specific (Wotton and Shimeld, <xref ref-type="bibr" rid="B66">2006</xref>; Shimeld et al., <xref ref-type="bibr" rid="B50">2010b</xref>). Using phylogenetic analyses, it has been proposed that 22 Fox gene families were already present in the bilaterian ancestor (Shimeld et al., <xref ref-type="bibr" rid="B50">2010b</xref>).</p>
<p>Cephalochordates (i.e., amphioxus) belong to the chordate phylum together with tunicates and their sister group, the vertebrates. They present morphological, developmental, and genomic characteristics that are proposed to be very similar to the ancestral state in the chordate clade, making amphioxus a key model system to understand chordate evolution (Bertrand and Escriva, <xref ref-type="bibr" rid="B5">2011</xref>, <xref ref-type="bibr" rid="B6">2014</xref>). Interestingly, it has been shown that amphioxus is the only living bilaterian possessing at least one member of each of the 22 Fox gene families proposed to have been present in Urbilateria (Yu et al., <xref ref-type="bibr" rid="B70">2008a</xref>). Thus, the study of Fox genes in this cephalochordate may shed light on the functional evolutionary history of this transcription factor gene family. Past studies using genomic data from the Caribbean cephalochordate <italic>Branchiostoma floridae</italic> described the presence of 32 Fox genes in this species (Yu et al., <xref ref-type="bibr" rid="B70">2008a</xref>) and the expression pattern of 11 of these genes was previously described: <italic>FoxAa</italic> and <italic>FoxAb</italic> (formerly named AmHNF3-1 and AmHNF3-2, respectively) (Shimeld, <xref ref-type="bibr" rid="B51">1997</xref>), <italic>FoxB</italic> (Mazet and Shimeld, <xref ref-type="bibr" rid="B33">2002</xref>), <italic>FoxC</italic> (Mazet et al., <xref ref-type="bibr" rid="B32">2006</xref>), <italic>FoxD</italic> (Yu et al., <xref ref-type="bibr" rid="B68">2002b</xref>), <italic>FoxE4</italic> (Yu et al., <xref ref-type="bibr" rid="B67">2002a</xref>), <italic>FoxF</italic> (Mazet et al., <xref ref-type="bibr" rid="B32">2006</xref>; Onimaru et al., <xref ref-type="bibr" rid="B38">2011</xref>), <italic>FoxG</italic> (Toresson et al., <xref ref-type="bibr" rid="B56">1998</xref>), <italic>FoxL1</italic> (Mazet et al., <xref ref-type="bibr" rid="B32">2006</xref>), <italic>FoxN1/4a</italic> (Bajoghli et al., <xref ref-type="bibr" rid="B1">2009</xref>), <italic>FoxQ1</italic> and <italic>FoxQ2</italic> (Yu et al., <xref ref-type="bibr" rid="B69">2003</xref>; Mazet et al., <xref ref-type="bibr" rid="B32">2006</xref>). In this work we searched for Fox sequences in the transcriptome of the Mediterranean amphioxus <italic>Branchiostoma lanceolatum</italic>. We found 28 Fox sequences and we describe here the spatiotemporal expression pattern of 13 Fox genes during embryonic development, including seven previously described in <italic>B. floridae</italic> and six for which expression was not known. We show that in <italic>B. lanceolatum</italic> some Fox genes exhibit ubiquitous expression as <italic>FoxK</italic> and <italic>FoxM</italic>, while the others show specific and dynamic expression patterns restricted to diverse embryonic territories. These expression patterns suggest that Fox genes are performing both general and specific functions during amphioxus embryonic development, most of them being probably ancestral in the chordate clade.</p>
</sec>
<sec sec-type="materials and methods" id="s2"><title>Materials and methods</title>
<sec><title>Phylogenetic analysis</title>
<p>All reference sequences, except for <italic>B. lanceolatum</italic>, were obtained from Genbank or from Fritzenwanker et al. (<xref ref-type="bibr" rid="B18">2014</xref>) The multiple alignment was performed only for the conserved Forkhead amino acid domain sequences using the MUSCLE module implemented in MEGA 6 and manually refined in its interface (Tamura et al., <xref ref-type="bibr" rid="B54">2013</xref>). The best fit substitution model for phylogenetic reconstruction was estimated using MEGA 6 (Tamura et al., <xref ref-type="bibr" rid="B53">2011</xref>). Bayesian inference (BI) tree was inferred using MrBayes 3.2 (Ronquist et al., <xref ref-type="bibr" rid="B43">2012</xref>), with the model recommended by MEGA 6 under the Akaike information criterion (RtRev&#x0002B;&#x00393;), at the CIPRES Science Gateway V. 3.1 (Miller et al., <xref ref-type="bibr" rid="B35">2015</xref>). Two independent runs were performed, each with four chains and 1 million generations. A burn-in of 25% was used and a 50 majority-rule consensus tree was calculated for the remaining trees.</p>
</sec>
<sec><title>Cloning and expression study</title>
<p><italic>B. lanceolatum</italic> Fox sequences were recovered from its reference transcriptome (Oulion et al., <xref ref-type="bibr" rid="B39">2012</xref>) by TBLASTN using sequences from <italic>B. floridae</italic> as queries. Specific primers were then designed for RT-PCR amplification from total RNA. Primer sequences are as follow:</p>
<list list-type="simple">
<list-item><p>FoxA_a_5&#x02032; AAGTCGCCGGTGTACGAGATG</p></list-item>
<list-item><p>FoxA_a_3&#x02032; GTATTATAGAGACGAAGGTTG</p></list-item>
<list-item><p>FoxA_b_5&#x02032; CATTTCCTCAGAACAGACATG</p></list-item>
<list-item><p>FoxA_b_3&#x02032; TCCTAAAGACTCCCAACAACA</p></list-item>
<list-item><p>FoxAB_5&#x02032; CAGTGTGAGGTGAACATCATG</p></list-item>
<list-item><p>FoxAB_3&#x02032; CGATTGACAGGTTGATAGAAC</p></list-item>
<list-item><p>FoxB_5&#x02032; ACAACAGGACCCTGACTCGT</p></list-item>
<list-item><p>FoxB_3&#x02032; GCATTCCCTGACGTCTTGA</p></list-item>
<list-item><p>FoxC_5&#x02032; AACCGTCCCGTTTTCCTCATG</p></list-item>
<list-item><p>FoxC_3&#x02032; CAGTTTTGATTCGTAAGGACT</p></list-item>
<list-item><p>FoxD_5&#x02032; ACAGCTGTGGAGTGGACACTT</p></list-item>
<list-item><p>FoxD_3&#x02032; CACGAGACATGTAAGTCTCCG</p></list-item>
<list-item><p>FoxEa_5&#x02032; AACCAACCCCGTACCAGCATG</p></list-item>
<list-item><p>FoxEa_3&#x02032; ATATGACACGGACACTGAACT</p></list-item>
<list-item><p>FoxG_5&#x02032; ACGCACATTAGCACAGTTCG</p></list-item>
<list-item><p>FoxG_3&#x02032; ACTTGACCCTGGCTTGACAC</p></list-item>
<list-item><p>FoxJ1_5&#x02032; TACAGACAACTGTAAACCATG</p></list-item>
<list-item><p>FoxJ1_3&#x02032; TTGTAATGCAGGGTGGGGCCT</p></list-item>
<list-item><p>FoxK_5&#x02032; GGAAGGCGGAGTTGGACAATG</p></list-item>
<list-item><p>FoxK_3&#x02032; CCGGACACGTCCTGCACCTGT</p></list-item>
<list-item><p>FoxM_5&#x02032; AGGAGAGTGTGACAAACCATG</p></list-item>
<list-item><p>FoxM_3&#x02032; TTCTCAGCTATTCAGTAATAC</p></list-item>
<list-item><p>FoxN1/4a_5&#x02032; GCGCACCGAGTATCGTTCTGA</p></list-item>
<list-item><p>FoxN1/4a_3&#x02032; ACATAGGTAGGACTATGTACT</p></list-item>
<list-item><p>FoxN2/3_5&#x02032; CAGTAAACACGAGCAGACATG</p></list-item>
<list-item><p>FoxN2/3_3&#x02032; AGCTGAAGACAATGATGATCC</p></list-item>
</list>
<p>A mix of total mRNA of <italic>B. lanceolatum</italic> extracted from embryos at different developmental stages was used as a template for retro-transcription. Amplification was performed using Advantage 2 Polymerase kit (Clontech) and a touch-down PCR program with annealing temperature ranging from 65 to 40&#x000B0;C. Amplified fragments were cloned using the pGEM-T Easy system (Promega) and sub-cloned in pBluescript II KS&#x0002B; for probe synthesis.</p>
</sec>
<sec><title>Whole mount <italic>In situ</italic> hybridization</title>
<p>Probes were synthesized using the DIG labeling system (Roche) after plasmid linearization with the appropriate enzymes. Ripe animals of <italic>B. lanceolatum</italic> were collected in Argel&#x000E8;s-sur-Mer (France), and gametes were obtained by heat stimulation (Fuentes et al., <xref ref-type="bibr" rid="B21">2004</xref>, <xref ref-type="bibr" rid="B20">2007</xref>). <italic>In vitro</italic> fertilization was undertaken in Petri dishes filled with filtered sea water. Fixation and whole mount <italic>in situ</italic> hybridization were performed as described in Somorjai et al. (<xref ref-type="bibr" rid="B52">2008</xref>).</p>
</sec>
</sec>
<sec sec-type="results" id="s3"><title>Results</title>
<sec><title>Molecular phylogenetic analysis of <italic>B. lanceolatum</italic> fox gene sequences</title>
<p>We looked for Fox gene sequences in the reference transcriptome of <italic>B. lanceolatum</italic> (Oulion et al., <xref ref-type="bibr" rid="B39">2012</xref>). The sequences that were recovered were used to conduct a phylogenetic tree reconstruction presented in Figure <xref ref-type="fig" rid="F1">1</xref>. We showed that <italic>B. lanceolatum</italic> possesses at least 28 Fox genes, each of them being orthologous to one of the 32 genes described in <italic>B. floridae</italic> and corresponding to at least one member of each of the 22 families present in the bilaterian ancestor (Yu et al., <xref ref-type="bibr" rid="B70">2008a</xref>). Specific duplications, that occurred in the cephalochordate clade at least in the ancestor of <italic>B. floridae</italic> and <italic>B. lanceolatum</italic>, gave rise to three members in the FoxQ2 group (<italic>FoxQ2a, FoxQ2b, FoxQ2c</italic>), two members in the FoxN1/4 group (<italic>FoxN1/4a</italic> and <italic>FoxN1/4b</italic>), and two genes in the FoxE group (<italic>FoxEa</italic> and <italic>FoxEc</italic>). We then analyzed the expression pattern during <italic>B. lanceolatum</italic> embryonic development of 13 of these 28 Fox genes corresponding to those showing a higher expression level in the transcriptome (Oulion et al., <xref ref-type="bibr" rid="B39">2012</xref>).</p>
<fig id="F1" position="float">
<label>Figure 1</label>
<caption><p><bold>Phylogenetic analysis of <italic><bold>B. lanceolatum</bold></italic> Fox genes</bold>. Unrooted 50 majority-rule consensus Bayesian inference tree based on the amino acid sequences of the forkhead domain. Posterior probablilities are shown at each node. The different paralogy groups are colored in pink or light blue boxes. Divergent sequences appeared outside these boxes. Only one amphioxus Fox gene, named Fox1 (Yu et al., <xref ref-type="bibr" rid="B70">2008a</xref>), that probably originated by a specific duplication and fast evolutionary rate in cephalochordates, localizes outside these paralogy groups. Abbreviations: Dm, <italic>Drosophila melanogaste</italic>r; Mm, <italic>Mus musculus;</italic> Dr, <italic>Danio rerio;</italic> Ci, <italic>Ciona intestinalis;</italic> Sp, <italic>Strongylocentrotus purpuratus;</italic> Sk, <italic>Saccoglossus kowalevskii;</italic> Nv, <italic>Nematostella vectensis;</italic> Bf, <italic>Branchiostoma floridae;</italic> Bl, <italic>Branchiostoma lanceolatum</italic>. Red stars indicate Bl sequences. Scale bar represents 0.4 amino acid substitution per site.</p></caption>
<graphic xlink:href="fevo-03-00080-g0001.tif"/>
</fig>
</sec>
<sec><title><italic>FoxAa</italic> and <italic>FoxAb</italic></title>
<p><italic>FoxAa</italic> (formerly named <italic>AmHNF3-1</italic>) (Shimeld, <xref ref-type="bibr" rid="B51">1997</xref>) was first expressed at the gastrula stage in the anterior ventral endoderm and in the mesendodermal layer of the dorsal blastoporal lip (Figures <xref ref-type="fig" rid="F2">2A,B</xref>). At the late gastrula stage, we detected transcripts in the axial dorsal mesendoderm corresponding to the presumptive notochord territory, as well as in mesendoderm cells of the archenteron floor (Figures <xref ref-type="fig" rid="F2">2C,D</xref>). Expression in the axial mesoderm and endoderm persisted through mid-late neurula stage (Figures <xref ref-type="fig" rid="F2">2E,F</xref>). Later on, at late neurula stage before the mouth opens, the expression in the notochord was restricted to the most anterior and posterior tips of the embryo, while the endodermal expression was restricted to the middle region of the gut (Figure <xref ref-type="fig" rid="F2">2G</xref>). At the larva stage, the expression at the anterior tip of the notochord and in the tailbud was still observed and we detected a diffuse expression in the gut (Figure <xref ref-type="fig" rid="F2">2H</xref>).</p>
<fig id="F2" position="float">
<label>Figure 2</label>
<caption><p><bold>Expression of <italic><bold>B. lanceolatum FoxAa, FoxAb, FoxAB, and FoxB</bold></italic></bold>. In all the panels except <bold>(B, J, Q, R, T, X)</bold> anterior is to the left. In lateral and blastoporal views dorsal is to the top. <italic>FoxAa</italic> expression pattern <bold>(A&#x02013;H)</bold>. Gastrula lateral <bold>(A)</bold> and blasporal <bold>(B)</bold> views. Late gastrula lateral <bold>(C)</bold> and dorsal <bold>(D)</bold> views. Mid-late neurula lateral <bold>(E)</bold> and dorsal <bold>(F)</bold> views. In the late neurula lateral view <bold>(G)</bold> arrow marks the endodermal expression in the middle region. In the larva stage lateral view <bold>(H)</bold>, the double arrowhead indicates the expression in the anterior tip of the notochord and the arrowhead marks the expression in the tailbud. <italic>FoxAb</italic> expression pattern <bold>(I&#x02013;P</bold>). In the gastrula lateral <bold>(I)</bold> and blastoporal <bold>(J)</bold> views the arrow indicates the expression in the mesendodermal part of the dorsal blastoporal lip. Late gastrula lateral <bold>(K)</bold> and dorsal <bold>(L)</bold> views. In the mid-late neurula lateral <bold>(M)</bold> and dorsal <bold>(N)</bold> views the double arrowhead marks the expression in the cerebral vesicle. In the late neurula lateral view <bold>(O)</bold>, the double arrow marks the expression in the most anterior part of the pharynx. In larva lateral view <bold>(P)</bold> the arrowhead indicates the expression in the tailbud. <italic>FoxAB</italic> expression pattern <bold>(Q&#x02013;V)</bold>. Eight-cell stage <bold>(Q)</bold>. Blastula stage <bold>(R)</bold>. Gastrula lateral <bold>(S)</bold> and blasporal <bold>(T)</bold> views. Late gastrula lateral <bold>(U)</bold> and dorsal <bold>(V)</bold> views. <italic>FoxB</italic> expression pattern <bold>(W&#x02013;D&#x00027;)</bold>. Gastrula lateral <bold>(W)</bold> and blastoporal <bold>(X)</bold> views. Early neurula lateral view <bold>(Y)</bold>. In the early neurula dorsal <bold>(Z)</bold> view the arrowhead indicates the two expression patches in the posterior paraxial mesendoderm. Mid-late neurula lateral <bold>(A&#x00027;)</bold> and dorsal <bold>(B&#x00027;)</bold> views. The double arrowhead marks the expression in the newly formed somites. Late neurula lateral view <bold>(C&#x00027;)</bold>. In larva lateral view <bold>(D&#x00027;)</bold> the arrow indicates the expression in the cerebral vesicle. Scale bar: 10 &#x003BC;m <bold>(A&#x02013;F), (I&#x02013;N)</bold>, <bold>(Q-V)</bold>, <bold>(W-B&#x00027;)</bold>, and 50 &#x003BC;m <bold>(G,H)</bold>, <bold>(O,P)</bold>, <bold>(C&#x00027;,D&#x00027;)</bold>.</p></caption>
<graphic xlink:href="fevo-03-00080-g0002.tif"/>
</fig>
<p><italic>FoxAb</italic> (formerly named <italic>AmHNF3-2</italic>) (Shimeld, <xref ref-type="bibr" rid="B51">1997</xref>) expression was first detected at the gastrula stage as a weak signal in the mesendodermal part of the dorsal blastoporal lip (Figures <xref ref-type="fig" rid="F2">2I,J</xref>). At the late gastrula stage, we detected expression in the central paraxial mesoderm on both sides of the notochord anlagen (Figures <xref ref-type="fig" rid="F2">2K,L</xref>). At the mid-late neurula stage transcripts were detected in the neural tube, including the cerebral vesicle, and in the dorsal part of the endoderm (Figures <xref ref-type="fig" rid="F2">2M,N</xref>). At the late neurula stage, before the mouth opens, <italic>FoxAb</italic> was expressed in the neural tube and in the most anterior part of the pharynx. In the posterior region, expression was detected in the tailbud and in the dorsal midline of the gut (Figure <xref ref-type="fig" rid="F2">2O</xref>). At the larva stage, we observed expression in the pharynx, in the preoral pit, in the club-shaped gland and in the tailbud. At this stage, the expression in the neural tube gets restricted to some neurons and to the posterior part of the cerebral vesicle (Figure <xref ref-type="fig" rid="F2">2P</xref> and Figure <xref ref-type="supplementary-material" rid="SM2">S1A</xref>).</p>
</sec>
<sec><title>FoxAB</title>
<p><italic>FoxAB</italic> transcripts were detected as a weak and ubiquitous signal from the eight-cell stage to the blastula stage (Figures <xref ref-type="fig" rid="F2">2Q,R</xref>). This ubiquitous expression was confirmed by the presence of reads in transcriptome analyses (data not shown). At the gastrula stage we observed a strong specific expression in the dorsal blastoporal lip, the amphioxus putative organizer (Figures <xref ref-type="fig" rid="F2">2S,T</xref>). At the late gastrula stage, expression gets restricted to the presumptive notochord territory (Figures <xref ref-type="fig" rid="F2">2U,V</xref>). No expression could be detected by <italic>in situ</italic> hybridization in later stages.</p>
</sec>
<sec><title>FoxB</title>
<p><italic>FoxB</italic> expression was first detected dorsally, both in the ectoderm and in the mesendoderm, as a weak signal in mid gastrula stage embryos (Figures <xref ref-type="fig" rid="F2">2W,X</xref>). Later on, in early neurula stage embryos, a signal could be observed in the neural plate on either side of the midline, as well as in two patches in the posterior paraxial mesendoderm (Figures <xref ref-type="fig" rid="F2">2Y,Z</xref>). During the late neurula stage, expression was detected in the most posterior paraxial mesoderm that give rise to the newly formed somites and in the neural tube posterior to the cerebral vesicle (Figures <xref ref-type="fig" rid="F2">2A&#x00027;,B&#x00027;</xref>). Then, <italic>FoxB</italic> expression in the mesoderm faded away in late neurulae (Figure <xref ref-type="fig" rid="F2">2C&#x00027;</xref>) and get later restricted to the cerebral vesicle and to some neurons along the neural tube in larvae (Figure <xref ref-type="fig" rid="F2">2D&#x00027;</xref> and Figure <xref ref-type="supplementary-material" rid="SM2">S1B</xref>).</p>
</sec>
<sec><title>FoxC</title>
<p><italic>FoxC</italic> was expressed at the gastrula stage in the dorsal paraxial mesendoderm (Figures <xref ref-type="fig" rid="F3">3A,B</xref>). Later on, at the late gastrula stage, expression was detected in the region that gives rise to the three most anterior somites (Figures <xref ref-type="fig" rid="F3">3C,D</xref>). In mid-late neurulae, the transcripts remained all along the body in the somites and a new expression domain appeared in the anterior endoderm at the level where the first gill slit opens (Figure <xref ref-type="fig" rid="F3">3E</xref>). At the late neurula stage, the expression persisted in the pharynx and somites and was also detected in the club-shaped gland anlagen (Figures <xref ref-type="fig" rid="F3">3F,G</xref>). At the larva stage a diffuse expression was observed in the somites as well as in the preoral pit, in the club-shaped gland and in the first gill slit (Figure <xref ref-type="fig" rid="F3">3H</xref> and Figure <xref ref-type="supplementary-material" rid="SM2">S1C</xref>).</p>
<fig id="F3" position="float">
<label>Figure 3</label>
<caption><p><bold>Expression of <italic><bold>B. lanceolatum FoxC, FoxD, FoxEa, and FoxG</bold></italic></bold>. In all the panels except <bold>(B,J)</bold>, anterior is to the left. In lateral and blastoporal views dorsal is to the top. <italic>FoxC</italic> expression pattern <bold>(A&#x02013;H)</bold>. Gastrula lateral <bold>(A)</bold> and blastoporal <bold>(B)</bold> views. The double arrowhead indicates the expression in the paraxial mesoderm. Late gastrula lateral <bold>(C)</bold> and dorsal <bold>(D)</bold> views. The arrowheads marks the region that will give rise to the three most anterior somites. In mid-late neurula lateral view <bold>(E)</bold> the arrow indicates a new expression domain in the anterior endoderm. Late neurula dorsal <bold>(F)</bold> and lateral <bold>(G)</bold> views. The arrow marks the expression domain in the pharynx. Larva lateral view <bold>(H)</bold>. <italic>FoxD</italic> expression pattern <bold>(I&#x02013;P)</bold>. Gastrula lateral <bold>(I)</bold> and blasporal <bold>(J)</bold> views. Late gastrula lateral <bold>(K)</bold> and dorsal <bold>(L)</bold> views. The arrow indicates the expression in the anterior region of the neural plate and the double arrowhead marks the expression in the paraxial dorsal mesendoderm. Mid-late neurula lateral view <bold>(M)</bold>. Late neurula dorsal <bold>(N)</bold> and lateral <bold>(O)</bold> views. Larva lateral view <bold>(P)</bold>. In <bold>(M, O, P)</bold> the arrows indicate the expression domain in the cerebral vesicle. <italic>FoxE</italic> expression pattern <bold>(Q&#x02013;V)</bold>. Early neurula lateral <bold>(Q)</bold> and dorsal <bold>(R)</bold> views. Mid-late neurula lateral <bold>(S)</bold> and dorsal <bold>(T)</bold> views. Late neurula lateral view <bold>(U)</bold>. Larva lateral view <bold>(V)</bold>. <italic>FoxG</italic> expression pattern <bold>(W&#x02013;B&#x00027;)</bold>. Early neurula lateral <bold>(W)</bold> and dorsal <bold>(Y)</bold> views. Mid-late neurula lateral <bold>(X)</bold> and dorsal <bold>(Z)</bold> views. The arrowhead indicates the expression in the three most anterior somites. In the late neurula stage lateral view <bold>(A&#x00027;)</bold> the arrows mark the neurons within the neural tube. Larva stage lateral view <bold>(B&#x00027;)</bold>. Scale bar: 10 &#x003BC;m <bold>(A&#x02013;E), (I&#x02013;L), (Q&#x02013;T), (W&#x02013;Z)</bold>, and 50 &#x003BC;m <bold>(F&#x02013;H), (N&#x02013;P), (U,V), (A&#x00027;,B&#x00027;)</bold>.</p></caption>
<graphic xlink:href="fevo-03-00080-g0003.tif"/>
</fig>
</sec>
<sec><title>FoxD</title>
<p><italic>FoxD</italic> transcripts were first detected at the gastrula stage in the dorsal blastoporal lip (Figures <xref ref-type="fig" rid="F3">3I,J</xref>). Then, at the late gastrula stage, <italic>FoxD</italic> was expressed in the dorsal axial mesendoderm, in part of the dorsal paraxial mesendoderm as two patches on both sides of the midline and in the anterior region of the neural plate (Figures <xref ref-type="fig" rid="F3">3K,L</xref>). At the mid-late neurula stage, the notochord and the somites, as well as the cerebral vesicle, were labeled (Figure <xref ref-type="fig" rid="F3">3M</xref>). At the late neurula stage, before the mouth opens, transcripts were detected in the paraxial somitic mesoderm, in the notochord, in the cerebral vesicle and in the posterior endoderm (Figures <xref ref-type="fig" rid="F3">3N,O</xref>). A faint labeling was also detected at this stage in the first gill slit and in the club-shaped gland anlagens. At the larva stage, we observed a low expression level in the cerebral vesicle, in the preoral pit, in the club-shaped gland, in the first gill slit, in the notochord and in the posterior part of the gut. We also observed an anterior to posterior gradient of expression in the somites (Figure <xref ref-type="fig" rid="F3">3P</xref> and Figure <xref ref-type="supplementary-material" rid="SM2">S1D</xref>).</p>
</sec>
<sec><title>FoxEa</title>
<p><italic>FoxEa</italic> (formerly named <italic>FoxE4</italic> in <italic>B. floridae</italic>) expression was first detected at early neurula stage in the antero-ventral mesendoderm (Figures <xref ref-type="fig" rid="F3">3Q,R</xref>). Later on, at the mid-late neurula stage, <italic>FoxEa</italic> transcripts were detected ventrally in the endoderm with a higher expression level on the right side of the pharynx (Figures <xref ref-type="fig" rid="F3">3S,T</xref>), and a slight expression domain in the posterior gut was also visible. At the late neurula stage, <italic>FoxEa</italic> transcripts remained ventrally in the pharyngeal endoderm on the right side (Figure <xref ref-type="fig" rid="F3">3U</xref>). Finally, at the larva stage, transcripts were detected in the club-shaped gland (Figure <xref ref-type="fig" rid="F3">3V</xref> and Figure <xref ref-type="supplementary-material" rid="SM2">S1E</xref>).</p>
</sec>
<sec><title>FoxG</title>
<p><italic>FoxG</italic> expression was first observed at the neurula stage in the anterior region of the first somites (Figures <xref ref-type="fig" rid="F3">3W,Y</xref>). At the late neurula stage, <italic>FoxG</italic> was expressed in the anterior ventral region of the three most anterior somites (Figures <xref ref-type="fig" rid="F3">3X,Z</xref>). Later on, in late neurula before the mouth opens, a neural expression appeared in some individual neurons within the neural tube, while the expression observed in the first somites disappeared (Figure <xref ref-type="fig" rid="F3">3A&#x00027;</xref>). This expression persisted in the larva stage embryos in which <italic>FoxG</italic> was also detected in some neurons of the cerebral vesicle (Figure <xref ref-type="fig" rid="F3">3B&#x00027;</xref> and Figure <xref ref-type="supplementary-material" rid="SM2">S1F</xref>).</p>
</sec>
<sec><title>FoxJ1</title>
<p><italic>FoxJ1</italic> showed a dynamic expression pattern. Expression began during gastrulation and was detected in the ectoderm except the ectoderm around the blastopore (Figures <xref ref-type="fig" rid="F4">4A,B</xref>). Later on, at the late gastrula stage, this expression pattern persisted in the ectoderm that give rise to the epidermis (Figures <xref ref-type="fig" rid="F4">4C,D</xref>). At the mid-late neurula stage, we detected transcripts in the neural tube while the expression in the epidermis was completely lost (Figures <xref ref-type="fig" rid="F4">4E,F</xref>). This neural tube expression was no more observed in late neurula stage embryos before the mouth opens (data not show), however at the larva stage we observed expression at the anterior tip of the embryo and in the pharynx at the level of the preoral pit and of the first gill slit (Figure <xref ref-type="fig" rid="F4">4G</xref> and Figure <xref ref-type="supplementary-material" rid="SM2">S1G</xref>).</p>
<fig id="F4" position="float">
<label>Figure 4</label>
<caption><p><bold>Expression of <italic><bold>B. lanceolatum FoxJ1, FoxN1/4a</bold></italic>, and <italic><bold>FoxN2/3</bold></italic></bold>. In all the panels except <bold>(B, H, I, K, Q, R, T)</bold> anterior is to the left. In lateral and blastoporal views dorsal is to the top. <italic>FoxJ1</italic> expression pattern <bold>(A&#x02013;G)</bold>. Gastrula lateral <bold>(A)</bold> and blasporal <bold>(B)</bold> views. Late gastrula lateral <bold>(C)</bold> and dorsal <bold>(D)</bold> views. Mid-late neurula lateral <bold>(E)</bold> and dorsal <bold>(F)</bold> views. In the larva lateral view <bold>(G)</bold> the bracket indicates the pharyngeal region. <italic>FoxN1/4a</italic> expression pattern <bold>(H&#x02013;P)</bold>. Eight-cell stage <bold>(H)</bold>. Blastula stage <bold>(I)</bold>. Gastrula lateral <bold>(J)</bold> and blastoporal views <bold>(K)</bold>. Early neurula lateral <bold>(L)</bold> and dorsal <bold>(M)</bold> views. In the mid-late neurula lateral view <bold>(N)</bold>, the arrowhead, double arrowhead and arrow mark the three main expression domains: at the level of the cerebral vesicle, in the anterior ventral endoderm and in the posterior mesoderm, respectively. Late neurula stage lateral view <bold>(O)</bold>. Larva stage lateral view <bold>(P)</bold>. <italic>FoxN2/3</italic> expression pattern <bold>(Q&#x02013;Y)</bold>. Eight-cell stage <bold>(Q)</bold>. Blastula stage <bold>(R)</bold>. Gastrula lateral <bold>(S)</bold> and blasporal <bold>(T)</bold> views. Late gastrula lateral <bold>(U)</bold> and dorsal <bold>(V)</bold> views. Mid-late neurula lateral view <bold>(W)</bold>. Late neurula lateral <bold>(X)</bold> and dorsal <bold>(Y)</bold> views. The arrow in <bold>(X)</bold> indicates the expression domain in the pharyngeal endoderm. Scale bar: 10 &#x003BC;m <bold>(A&#x02013;F), (H&#x02013;N), (Q&#x02013;W)</bold>, and 50 &#x003BC;m <bold>(G), (O&#x02013;P), (X,Y)</bold>.</p></caption>
<graphic xlink:href="fevo-03-00080-g0004.tif"/>
</fig>
</sec>
<sec><title>FoxK</title>
<p><italic>FoxK</italic> was ubiquitously expressed from the eight-cell stage to the blastula stage (Figures <xref ref-type="supplementary-material" rid="SM3">S2A,B</xref>). At the gastrula stage, the expression became restricted to the mesendoderm (Figures <xref ref-type="supplementary-material" rid="SM3">S2C,D</xref>), and by the late gastrula stage transcripts were detected mostly in the dorsal mesoderm (Figures <xref ref-type="supplementary-material" rid="SM3">S2E,F</xref>). At the mid-late neurula stage, we detected a stronger expression in the most anterior region of the embryo (Figures <xref ref-type="supplementary-material" rid="SM3">S2G,H</xref>). Transcripts were then detected in the whole embryo at the late neurula stage with a stronger expression in the anterior tip (Figures <xref ref-type="supplementary-material" rid="SM3">S2I,J</xref>). Finally, at the larva stage, we observed a ubiquitous expression with a higher level at the anterior tip and in the pharynx (Figure <xref ref-type="supplementary-material" rid="SM3">S2K</xref>).</p>
</sec>
<sec><title>FoxM</title>
<p><italic>FoxM</italic> transcripts were detected ubiquitously during the whole embryonic development, from the eight-cell stage until the mid-late neurula stage except in the epidermis (Figures <xref ref-type="supplementary-material" rid="SM3">S2L&#x02013;S</xref>). Later on, at late neurula stage, <italic>FoxM</italic> expression could not be detected anymore by <italic>in situ</italic> hybridization (Figure <xref ref-type="supplementary-material" rid="SM3">S2T</xref>).</p>
</sec>
<sec><title>FoxN1/4a</title>
<p>Ubiquitous <italic>FoxN1/4a</italic> expression was detected from the eight-cell stage until the blastula stage (Figures <xref ref-type="fig" rid="F4">4H,I</xref>). At the gastrula stage, a signal was detected in the anterior ectoderm (Figures <xref ref-type="fig" rid="F4">4J,K</xref>). Later on, at the early neurula stage, we observed transcripts in the anterior endoderm as well as in the axial central mesoderm (Figures <xref ref-type="fig" rid="F4">4L,M</xref>). At the mid-late neurula stage, we detected three major expression domains: one anterior, at the level of the cerebral vesicle, a second one in the anterior ventral endoderm and a third one in the posterior mesoderm (Figure <xref ref-type="fig" rid="F4">4N</xref>). At the late neurula stage before the mouth opens, we observed expression in the anterior and posterior endoderm (Figure <xref ref-type="fig" rid="F4">4O</xref>). Finally, at the larva stage, we detected expression in the posterior region of the gut and in the anus (Figure <xref ref-type="fig" rid="F4">4P</xref>).</p>
</sec>
<sec><title>FoxN2/3</title>
<p>Ubiquitous expression of <italic>FoxN2/3</italic> was observed from the eight-cell stage (Figure <xref ref-type="fig" rid="F4">4Q</xref>) to the blastula stage (Figure <xref ref-type="fig" rid="F4">4R</xref>). Then, at the gastrula stage, the expression was restricted to the mesendoderm (Figures <xref ref-type="fig" rid="F4">4S,T</xref>). At the late gastrula stage, the expression remained strong in the mesendoderm but started to become lower in the ventral part (Figures <xref ref-type="fig" rid="F4">4U,V</xref>). By the mid-late neurula stage, <italic>FoxN2/3</italic> transcripts were detected in the mesoderm and in the neural tube (Figure <xref ref-type="fig" rid="F4">4W</xref>). At the late neurula stage, before the mouth opens, the expression was mainly detected in the paraxial mesoderm (somites) and in the notochord. A new expression domain also appeared at this stage in the pharyngeal endoderm (Figures <xref ref-type="fig" rid="F4">4X,Y</xref>). At the larva stage, we did not detect any specific signal using <italic>in situ</italic> hybridization.</p>
</sec>
</sec>
<sec sec-type="discussion" id="s4"><title>Discussion</title>
<sec><title>Fox genes expression in cephalochordate species</title>
<p>The complete or partial embryonic expression patterns of <italic>FoxAa, FoxAb, FoxB, FoxC, FoxD, FoxEa, FoxG</italic>, and <italic>FoxN1/4a</italic> were previously described in <italic>B. floridae</italic> and/or <italic>B. belcheri</italic> (Shimeld, <xref ref-type="bibr" rid="B51">1997</xref>; Terazawa and Satoh, <xref ref-type="bibr" rid="B55">1997</xref>; Toresson et al., <xref ref-type="bibr" rid="B56">1998</xref>; Mazet and Shimeld, <xref ref-type="bibr" rid="B33">2002</xref>; Yu et al., <xref ref-type="bibr" rid="B67">2002a</xref>,<xref ref-type="bibr" rid="B68">b</xref>; Mazet et al., <xref ref-type="bibr" rid="B32">2006</xref>; Bajoghli et al., <xref ref-type="bibr" rid="B1">2009</xref>). These genes overwhelmingly show a similar embryonic expression to what we observed in <italic>B. lanceolatum</italic>, as we have previously noticed for other important developmental genes (Somorjai et al., <xref ref-type="bibr" rid="B52">2008</xref>). However, our work brings some new information.</p>
<p>First, in contrast to what has been described in <italic>B. floridae</italic>, we showed that <italic>FoxAa</italic> and <italic>FoxAb</italic> have different expression patterns. Indeed, in <italic>B. floridae, FoxAb in situ</italic> hybridization data showed that it has a similar expression to <italic>FoxAa</italic> at early stages whereas expression was no more detected after the eight somites stage (Shimeld, <xref ref-type="bibr" rid="B51">1997</xref>). Here we showed that although both genes were expressed in the mesendodermal part of the dorsal blastoporal lip at the gastrula stage, the overall expression patterns are consistently different between the two genes and we observed a restricted expression of <italic>FoxAb</italic> from the gastrula to the larva stage. These discrepancies might be explained by the fact that the level of expression of <italic>FoxAb</italic> is very low. Indeed, staining of embryos hybridized to <italic>FoxAb</italic> took very long suggesting a low expression level. Thus, the staining time used in <italic>B. floridae</italic> might have been too short to detect expression in late stage embryos. Moreover, the expression we observed for <italic>FoxAa</italic> in <italic>B. lanceolatum</italic> is different from what was observed in <italic>B. floridae</italic> but similar to what has been described in <italic>B. belcheri</italic> (Terazawa and Satoh, <xref ref-type="bibr" rid="B55">1997</xref>). Indeed, as in <italic>B. belcheri, FoxAa</italic> was not expressed in the central nervous system of <italic>B. lanceolatum</italic>. On the other hand, <italic>FoxAb</italic> showed a very specific expression in the ventral part of the neural tube in neurula stage embryos, which has been proposed to be homologous to the vertebrate floor plate. Vertebrates have three FoxA group paralogous genes that are expressed in the organizer, the notochord, the floor plate and the endoderm (Friedman and Kaestner, <xref ref-type="bibr" rid="B17">2006</xref>). In <italic>Ciona</italic> (Di Gregorio et al., <xref ref-type="bibr" rid="B16">2001</xref>), <italic>Ci-fkh</italic> is also expressed in the notochord, the floor plate and the endoderm. The data we obtained in <italic>B. lanceolatum</italic> suggest that the expression of <italic>FoxA</italic> in the chordate ancestor was similar to what is observed in tunicates and that independent sub-functionalizations occurred in cephalochordates after specific gene duplication and in vertebrates after the two rounds of whole genome duplications.</p>
<p>Concerning <italic>FoxB</italic>, expression in <italic>B. floridae</italic> was first detected in neurulae with five somites (Mazet and Shimeld, <xref ref-type="bibr" rid="B33">2002</xref>). Here we showed that in <italic>B. lanceolatum FoxB</italic> expression could be observed in gastrula embryos in the dorsal posterior mesendoderm and ectoderm. Then, in neurulae, we detected expression in the neural plate similar to <italic>B. floridae</italic>, as well as an expression in the most posterior somites that was not previously described. This expression in the neural plate/neural tube and in the lastly formed somites persisted until the late neurula stage. Interestingly, in amphioxus three different somitic populations have been described (Bertrand et al., <xref ref-type="bibr" rid="B4">2011</xref>). The first, most anterior, population forms under the control of the FGF signal and the two posterior populations forms independently of the FGF signal. Several genes are expressed specifically in these three somitic populations but only one gene, <italic>Mox</italic>,(Minguillon and Garcia-Fernandez, <xref ref-type="bibr" rid="B36">2002</xref>) is expressed in the second and third populations. The present data suggest that <italic>FoxB</italic> also plays a role in the formation of these somitic population since it is also expressed in the two most-posterior somitic populations.</p>
<p>In <italic>B. floridae, FoxC</italic> has been described as being firstly expressed in the mesoderm of neurulae but its expression was described only in one developmental stage (Mazet et al., <xref ref-type="bibr" rid="B32">2006</xref>). Here we showed that expression starts much earlier, at the gastrula stage, in the dorsal paraxial mesendoderm, the presumptive somitic mesoderm territory. Expression persisted in the paraxial mesoderm/somites until the larva stage, and at the late neurula stage we started to observe expression in the club-shaped gland anlagen and at the place where the first gill slit opens. These data suggest a major ancestral role of <italic>FoxC</italic> during somitogenesis which would have been conserved in vertebrates (Kume et al., <xref ref-type="bibr" rid="B27">2001</xref>; Wilm et al., <xref ref-type="bibr" rid="B64">2004</xref>; Wotton et al., <xref ref-type="bibr" rid="B65">2008</xref>) and lost in tunicates in which <italic>FoxC</italic> is expressed in neural and palp cells (Imai et al., <xref ref-type="bibr" rid="B23">2006</xref>).</p>
<p><italic>FoxD</italic> and <italic>FoxEa</italic> expression in <italic>B. lanceolatum</italic> was very similar to previous descriptions in <italic>B. floridae</italic> (Yu et al., <xref ref-type="bibr" rid="B67">2002a</xref>,<xref ref-type="bibr" rid="B68">b</xref>). However we noticed expression in some specific regions of the pharynx in late neurulae and larvae for <italic>FoxD</italic>, and a transient expression in mid-late and late neurula stage embryos in the posterior endoderm for <italic>FoxEa</italic> that were not described in the Caribbean species.</p>
<p><italic>FoxG</italic>, previously known as <italic>Brain Factor 1</italic> (<italic>BF-1</italic>), was described in <italic>B. floridae</italic> as a gene that is ventrally expressed in the cerebral vesicle and in the anterior-most portion of the first somite pair (Toresson et al., <xref ref-type="bibr" rid="B56">1998</xref>). Our results showed a conserved expression pattern in the cerebral vesicle area in <italic>B. lanceolatum</italic>. However, mesoderm expression is not only limited to the first somite pair but the first three somite pairs exhibit the same pattern at the neurula stage suggesting that this gene might play a role during anterior somitogenesis. This result highlights the functional differences between the formation of the anterior somites which is under the control of the FGF signaling pathway and the formation of the most posterior somites which is not FGF-dependent (Bertrand et al., <xref ref-type="bibr" rid="B4">2011</xref>). Moreover, expression is localized in the ventral part of these three most anterior somites which will give rise to the perivisceral coelom, suggesting a function of <italic>FoxG</italic> in the establishment of the somitic compartments.</p>
</sec>
<sec><title><italic>FoxJ1</italic> and the formation of motile cilia</title>
<p><italic>FoxJ1</italic> orthologs were identified in many eumetazoans as well as in sponges (Larroux et al., <xref ref-type="bibr" rid="B28">2006</xref>) and choanoflagellates (King et al., <xref ref-type="bibr" rid="B26">2008</xref>). In vertebrates, <italic>FoxJ1</italic> plays an essential role in the generation of motile cilia and in mediating Left/Right asymmetry (Chen et al., <xref ref-type="bibr" rid="B11">1998</xref>; Brody et al., <xref ref-type="bibr" rid="B8">2000</xref>; Yu et al., <xref ref-type="bibr" rid="B71">2008b</xref>). It has also recently been shown that misexpression of <italic>FoxJ1</italic> from placozoans, echinoderms and platyhelminthes in zebrafish embryos induces the expression of ciliary genes, whereas the inactivation of <italic>FoxJ1</italic> in the flatworm <italic>Schmidtea mediterranea</italic> impairs the normal differentiation of motile cilia, suggesting a conserved function in metazoans (Vij et al., <xref ref-type="bibr" rid="B62">2012</xref>). This conserved function is also supported by the embryonic expression of <italic>FoxJ1</italic> in different phyla (Choi et al., <xref ref-type="bibr" rid="B12">2006</xref>; Tu et al., <xref ref-type="bibr" rid="B58">2006</xref>; Fritzenwanker et al., <xref ref-type="bibr" rid="B18">2014</xref>). In <italic>B. lanceolatum</italic>, we showed that <italic>FoxJ1</italic> is first expressed in the ectoderm of the gastrulae, excluding the blastoporal region and the presumptive neural plate, at the time at which motile cilia start to grow. Then, in neurulae, expression was lost in the epidermis and appeared in the closed neural tube. At the larva stage, expression was restricted to the anterior tip of the animal and to the ciliated preoral pit and first gill slit. This expression pattern suggests that in amphioxus <italic>FoxJ1</italic> might also play a role in the formation of motile cilia. However, other cells, like the epithelial gut cells, also harbor motile cilia and do not express <italic>FoxJ1</italic>, suggesting that other genes might also be implicated in ciliogenesis in these embryonic structures.</p>
</sec>
<sec><title>FoxAB</title>
<p>In <italic>B. lanceolatum, FoxAB</italic> was transiently expressed in the organizer at the gastrula stage and in the presumptive notochord later on. No expression could be detected in mid-neurulae or larvae. FoxAB family genes were described in hemichordates (Fritzenwanker et al., <xref ref-type="bibr" rid="B18">2014</xref>), sea urchin (Tu et al., <xref ref-type="bibr" rid="B58">2006</xref>) and cnidarians and are absent in vertebrates and tunicates, the two other chordate clades (Yu et al., <xref ref-type="bibr" rid="B70">2008a</xref>). In the hemichordate <italic>Saccoglossus kowalevskii, FoxAB</italic> is expressed in the ectoderm and the mouth perforates through the ring expressing this gene in the ventral side (Fritzenwanker et al., <xref ref-type="bibr" rid="B18">2014</xref>). In bryozoans, <italic>FoxAB</italic> also shows an ectodermal expression (Fuchs et al., <xref ref-type="bibr" rid="B19">2011</xref>). Therefore, it is still difficult to propose any scenario for the evolution of the function of FoxAB family genes in bilaterians. <italic>FoxAB</italic> could have been recruited for the patterning of the notochord field in the ancestor of chordates, but the absence of genes of this family in tunicates and vertebrates make this hypothesis unlikely.</p>
</sec>
<sec><title><italic>FoxK</italic> and <italic>FoxM</italic> ubiquitous expression</title>
<p>We detected a ubiquitous expression of <italic>FoxK</italic> starting at the eight-cell stage until the larva stage. In other bilaterians data are scarce. In vertebrates, there are two paralogs in the FoxK family, <italic>FoxK1</italic> and <italic>FoxK2</italic>. In mouse, the study of the function of <italic>FoxK1</italic> during embryonic development was undertaken showing that the gene is involved in myogenic differentiation (Bassel-Duby et al., <xref ref-type="bibr" rid="B2">1994</xref>). In <italic>Ciona intestinalis</italic> (Imai et al., <xref ref-type="bibr" rid="B22">2004</xref>) as in the hemichordate <italic>S. kowalevskii</italic> (Fritzenwanker et al., <xref ref-type="bibr" rid="B18">2014</xref>), the expression of <italic>FoxK</italic> is quite ubiquitous as observed for <italic>B. lanceolatum</italic>. Finally, studies in <italic>Drosophila</italic> have shown that <italic>FoxK</italic> is involved in the differentiation of midgut in the fly embryo (Casas-Tinto et al., <xref ref-type="bibr" rid="B10">2008</xref>). Altogether these data do not allow us to infer any putative ancestral function for FoxK family genes and further studies are required in different animal phyla.</p>
<p><italic>FoxM</italic> expression is also ubiquitous in <italic>B. lanceolatum</italic> and was first detected as early as the eight-cell stage. Then the expression level continuously decreased while development proceeds and became undetectable by <italic>in situ</italic> hybridization at the late neurula stage. In <italic>Xenopus, FoxM1</italic> is maternally expressed and transcripts are thereafter detected in the neuroectoderm (Pohl et al., <xref ref-type="bibr" rid="B40">2005</xref>). Moreover this gene has been shown to be important for early neuronal differentiation (Ueno et al., <xref ref-type="bibr" rid="B61">2008</xref>). In mouse, <italic>FoxM1</italic> is expressed in dividing cells and knock-out animals exhibit embryonic lethal phenotype due to many malformations affecting different organs such as the liver, the heart, the lung, or the vasculature (Kalin et al., <xref ref-type="bibr" rid="B24">2011</xref>). As for FoxK, the data available up to now do not give us any indication on the putative ancestral function of genes belonging to the FoxM family.</p>
</sec>
<sec><title><italic>FoxN1/4a</italic> and <italic>FoxN2/3</italic> expression</title>
<p>In all vertebrates studied so far, <italic>FoxN1</italic> plays an essential role in thymus development (Ma et al., <xref ref-type="bibr" rid="B31">2012</xref>; Neves et al., <xref ref-type="bibr" rid="B37">2012</xref>; Lee et al., <xref ref-type="bibr" rid="B30">2013</xref>; Romano et al., <xref ref-type="bibr" rid="B42">2013</xref>). Moreover, in mammals, <italic>FoxN1</italic> is essential for hair formation whereas it is also expressed in chick during feather development (Darnell et al., <xref ref-type="bibr" rid="B15">2014</xref>). Although mammal and fish FoxN1s are able to activate the expression of hair keratin genes, FoxN1/4 from amphioxus is not because its N-terminal region of the forkhead domain is different compared with vertebrates (Schlake et al., <xref ref-type="bibr" rid="B45">2000</xref>). On the other hand, <italic>FoxN4</italic> is expressed in the nervous system, including retina, during vertebrate development (Danilova et al., <xref ref-type="bibr" rid="B14">2004</xref>; Kelly et al., <xref ref-type="bibr" rid="B25">2007</xref>; Boije et al., <xref ref-type="bibr" rid="B7">2013</xref>). Outside vertebrates, embryonic expression has been described in <italic>S. kowalevskii</italic> (Fritzenwanker et al., <xref ref-type="bibr" rid="B18">2014</xref>) and in a single developmetal stage of <italic>B. floridae</italic> (Bajoghli et al., <xref ref-type="bibr" rid="B1">2009</xref>). In the hemichordate, expression of <italic>FoxN1/4</italic> is ubiquitous during early development and is thereafter observed in the ectoderm. In <italic>B. lanceolatum</italic>, the expression of <italic>FoxN1/4a</italic> was very dynamic with a maternal ubiquitous expression followed by restricted expression in the ectoderm at the gastrula stage, in the endoderm and axial mesoderm in neurulae, in the cerebral vesicle, the pharynx and the posterior somites later on, and, finally, in the posterior gut of the larvae. These data suggest that <italic>FoxN1</italic> and <italic>FoxN4</italic> probably acquired new functions in vertebrates, and analysis of the expression of <italic>FoxN1/4</italic> family genes in tunicates will be needed to better understand this point. Interestingly, the gut of amphioxus larva and adult is considered as a major organ for immunity and <italic>FoxN1/4a</italic> might, as vertebrates <italic>FoxN1</italic>, play a role in the control of immune system function in amphioxus. However, further functional studies are required to test this hypothesis.</p>
<p>In vertebrates, <italic>FoxN3</italic> is important for craniofacial and eye development (Schuff et al., <xref ref-type="bibr" rid="B48">2007</xref>; Samaan et al., <xref ref-type="bibr" rid="B44">2010</xref>; Schmidt et al., <xref ref-type="bibr" rid="B46">2011</xref>). In <italic>Xenopus, FoxN3</italic> is expressed in neural crest and eye field whereas <italic>FoxN2</italic> is expressed early in the eye field and then in branchial arches, retina and vagal ganglion (Schuff et al., <xref ref-type="bibr" rid="B47">2006</xref>). In mouse, <italic>FoxN2</italic> is expressed in craniofacial, limb, nervous system and somitic tissues (Tribioli et al., <xref ref-type="bibr" rid="B57">2002</xref>). In <italic>Ciona intestinalis</italic>, expression of <italic>FoxN2/3</italic> is quite ubiquitous during early development and becomes more intense in the sensory vesicle, the mesenchyme, the notochord and the palps after gastrulation (Imai et al., <xref ref-type="bibr" rid="B22">2004</xref>). In sea urchin <italic>FoxN2/3</italic> is expressed in the non-skeletogenic mesoderm and, later on, in the endoderm and it has been shown that <italic>FoxN2/3</italic> function is important for ingression and for the expression of genes coding for proteins of the skeletal matrix (Rho and Mcclay, <xref ref-type="bibr" rid="B41">2011</xref>). Here, we show that <italic>FoxN2/3</italic> in amphioxus was ubiquitously expressed at early stages. Then, at the gastrula stage, its expression was restricted to the endomesoderm and later on we observed a specific expression in the somites. Altogether, this suggests a conserved role of <italic>FoxN2/3</italic> in the development of mesoderm in deuterostomes, although genes of this family seem to have acquired specific functions in each chordate lineage.</p>
</sec>
</sec>
<sec sec-type="conclusions" id="s5"><title>Conclusions</title>
<p>Analyzing the expression of Fox genes in the Mediterranean amphioxus, <italic>B. lanceolatum</italic> showed us several points. First, as previously described for other gene families (Somorjai et al., <xref ref-type="bibr" rid="B52">2008</xref>), the expression of orthologous genes in different amphioxus species shows a high degree of stasis. However, differences may be found that can easily be explained by variation in experimental sensitivity. And, second, the comparative analyzes of the expression of amphioxus Fox genes with other metazoans and particularly chordates have shown a high degree of conservation for some genes (e.g., <italic>FoxC, FoxD</italic>), but also divergent patterns in others (e.g., <italic>FoxM, FoxN1/4a</italic>). This indicates that Fox genes were necessary for essential functions in metazoans but they were also instrumental for the evolution of new functions. Further studies in amphioxus and other metazoans, and particularly functional studies, will be extremely important in the future to establish the complete picture of Fox genes expression and function and their role in the evolution of animals.</p>
<sec><title>Conflict of interest statement</title>
<p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p>
</sec>
</sec>
</body>
<back>
<ack><p>DA holds a fellowship from CONICYT &#x0201C;Becas Chile.&#x0201D; Part of this study was supported by the EXOMOD grant from the CNRS.</p>
</ack>
<sec sec-type="supplementary-material" id="s6">
<title>Supplementary material</title>
<p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type="uri" xlink:href="http://journal.frontiersin.org/article/10.3389/fevo.2015.00080">http://journal.frontiersin.org/article/10.3389/fevo.2015.00080</ext-link></p>
<supplementary-material xlink:href="DataSheet1.DOCX" id="SM1" mimetype="application/vnd.openxmlformats-officedocument.wordprocessingml.document" xmlns:xlink="http://www.w3.org/1999/xlink"/>
<supplementary-material xlink:href="Image1.PDF" id="SM2" mimetype="application/pdf" xmlns:xlink="http://www.w3.org/1999/xlink"/>
<supplementary-material xlink:href="Image2.PDF" id="SM3" mimetype="application/pdf" xmlns:xlink="http://www.w3.org/1999/xlink"/>
</sec>
<ref-list>
<title>References</title>
<ref id="B1">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Bajoghli</surname> <given-names>B.</given-names></name> <name><surname>Aghaallaei</surname> <given-names>N.</given-names></name> <name><surname>Hess</surname> <given-names>I.</given-names></name> <name><surname>Rode</surname> <given-names>I.</given-names></name> <name><surname>Netuschil</surname> <given-names>N.</given-names></name> <name><surname>Tay</surname> <given-names>B. H.</given-names></name> <etal/></person-group>. (<year>2009</year>). <article-title>Evolution of genetic networks underlying the emergence of thymopoiesis in vertebrates</article-title>. <source>Cell</source> <volume>138</volume>, <fpage>186</fpage>&#x02013;<lpage>197</lpage>. <pub-id pub-id-type="doi">10.1016/j.cell.2009.04.017</pub-id><pub-id pub-id-type="pmid">19559469</pub-id></citation>
</ref>
<ref id="B2">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Bassel-Duby</surname> <given-names>R.</given-names></name> <name><surname>Hernandez</surname> <given-names>M. D.</given-names></name> <name><surname>Yang</surname> <given-names>Q.</given-names></name> <name><surname>Rochelle</surname> <given-names>J. M.</given-names></name> <name><surname>Seldin</surname> <given-names>M. F.</given-names></name> <name><surname>Williams</surname> <given-names>R. S.</given-names></name></person-group> (<year>1994</year>). <article-title>Myocyte nuclear factor, a novel winged-helix transcription factor under both developmental and neural regulation in striated myocytes</article-title>. <source>Mol. Cell Biol.</source> <volume>14</volume>, <fpage>4596</fpage>&#x02013;<lpage>4605</lpage>. <pub-id pub-id-type="pmid">8007964</pub-id></citation>
</ref>
<ref id="B3">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Benayoun</surname> <given-names>B. A.</given-names></name> <name><surname>Caburet</surname> <given-names>S.</given-names></name> <name><surname>Veitia</surname> <given-names>R. A.</given-names></name></person-group> (<year>2011</year>). <article-title>Forkhead transcription factors: key players in health and disease</article-title>. <source>Trends Genet.</source> <volume>27</volume>, <fpage>224</fpage>&#x02013;<lpage>232</lpage>. <pub-id pub-id-type="doi">10.1016/j.tig.2011.03.003</pub-id><pub-id pub-id-type="pmid">21507500</pub-id></citation>
</ref>
<ref id="B4">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Bertrand</surname> <given-names>S.</given-names></name> <name><surname>Camasses</surname> <given-names>A.</given-names></name> <name><surname>Somorjai</surname> <given-names>I.</given-names></name> <name><surname>Belgacem</surname> <given-names>M. R.</given-names></name> <name><surname>Chabrol</surname> <given-names>O.</given-names></name> <name><surname>Escande</surname> <given-names>M. L.</given-names></name> <etal/></person-group>. (<year>2011</year>). <article-title>Amphioxus FGF signaling predicts the acquisition of vertebrate morphological traits</article-title>. <source>Proc. Natl. Acad. Sci. U.S.A.</source> <volume>108</volume>, <fpage>9160</fpage>&#x02013;<lpage>9165</lpage>. <pub-id pub-id-type="doi">10.1073/pnas.1014235108</pub-id><pub-id pub-id-type="pmid">21571634</pub-id></citation>
</ref>
<ref id="B5">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Bertrand</surname> <given-names>S.</given-names></name> <name><surname>Escriva</surname> <given-names>H.</given-names></name></person-group> (<year>2011</year>). <article-title>Evolutionary crossroads in developmental biology: amphioxus</article-title>. <source>Development</source> <volume>138</volume>, <fpage>4819</fpage>&#x02013;<lpage>4830</lpage>. <pub-id pub-id-type="doi">10.1242/dev.066720</pub-id><pub-id pub-id-type="pmid">22028023</pub-id></citation>
</ref>
<ref id="B6">
<citation citation-type="book"><person-group person-group-type="author"><name><surname>Bertrand</surname> <given-names>S.</given-names></name> <name><surname>Escriva</surname> <given-names>H.</given-names></name></person-group> (<year>2014</year>). <article-title>Chordates: The acquisition of an axial backbone</article-title>, <source>The Tree of Life</source>, eds <person-group person-group-type="editor"><name><surname>Vargas</surname> <given-names>P.</given-names></name> <name><surname>Zardoya</surname> <given-names>R.</given-names></name></person-group> (<publisher-loc>Sunderland, MA</publisher-loc>: <publisher-name>Sinauer Associates, Inc</publisher-name>), <fpage>460</fpage>&#x02013;<lpage>468</lpage>.</citation>
</ref>
<ref id="B7">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Boije</surname> <given-names>H.</given-names></name> <name><surname>Shirazi Fard</surname> <given-names>S.</given-names></name> <name><surname>Ring</surname> <given-names>H.</given-names></name> <name><surname>Hallbook</surname> <given-names>F.</given-names></name></person-group> (<year>2013</year>). <article-title>Forkheadbox N4 (FoxN4) triggers context-dependent differentiation in the developing chick retina and neural tube</article-title>. <source>Differentiation</source> <volume>85</volume>, <fpage>11</fpage>&#x02013;<lpage>19</lpage>. <pub-id pub-id-type="doi">10.1016/j.diff.2012.12.002</pub-id><pub-id pub-id-type="pmid">23314287</pub-id></citation>
</ref>
<ref id="B8">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Brody</surname> <given-names>S. L.</given-names></name> <name><surname>Yan</surname> <given-names>X. H.</given-names></name> <name><surname>Wuerffel</surname> <given-names>M. K.</given-names></name> <name><surname>Song</surname> <given-names>S. K.</given-names></name> <name><surname>Shapiro</surname> <given-names>S. D.</given-names></name></person-group> (<year>2000</year>). <article-title>Ciliogenesis and left-right axis defects in forkhead factor HFH-4-null mice</article-title>. <source>Am. J. Respir. Cell Mol. Biol.</source> <volume>23</volume>, <fpage>45</fpage>&#x02013;<lpage>51</lpage>. <pub-id pub-id-type="doi">10.1165/ajrcmb.23.1.4070</pub-id><pub-id pub-id-type="pmid">10873152</pub-id></citation>
</ref>
<ref id="B9">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Carlsson</surname> <given-names>P.</given-names></name> <name><surname>Mahlapuu</surname> <given-names>M.</given-names></name></person-group> (<year>2002</year>). <article-title>Forkhead transcription factors: key players in development and metabolism</article-title>. <source>Dev. Biol.</source> <volume>250</volume>, <fpage>1</fpage>&#x02013;<lpage>23</lpage>. <pub-id pub-id-type="doi">10.1006/dbio.2002.0780</pub-id><pub-id pub-id-type="pmid">12297093</pub-id></citation>
</ref>
<ref id="B10">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Casas-Tinto</surname> <given-names>S.</given-names></name> <name><surname>Gomez-Velazquez</surname> <given-names>M.</given-names></name> <name><surname>Granadino</surname> <given-names>B.</given-names></name> <name><surname>Fernandez-Funez</surname> <given-names>P.</given-names></name></person-group> (<year>2008</year>). <article-title>FoxK mediates TGF-beta signalling during midgut differentiation in flies</article-title>. <source>J. Cell Biol.</source> <volume>183</volume>, <fpage>1049</fpage>&#x02013;<lpage>1060</lpage>. <pub-id pub-id-type="doi">10.1083/jcb.200808149</pub-id><pub-id pub-id-type="pmid">19075113</pub-id></citation>
</ref>
<ref id="B11">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Chen</surname> <given-names>J.</given-names></name> <name><surname>Knowles</surname> <given-names>H. J.</given-names></name> <name><surname>Hebert</surname> <given-names>J. L.</given-names></name> <name><surname>Hackett</surname> <given-names>B. P.</given-names></name></person-group> (<year>1998</year>). <article-title>Mutation of the mouse hepatocyte nuclear factor/forkhead homologue 4 gene results in an absence of cilia and random left-right asymmetry</article-title>. <source>J. Clin. Invest.</source> <volume>102</volume>, <fpage>1077</fpage>&#x02013;<lpage>1082</lpage>. <pub-id pub-id-type="doi">10.1172/JCI4786</pub-id><pub-id pub-id-type="pmid">9739041</pub-id></citation>
</ref>
<ref id="B12">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Choi</surname> <given-names>V. M.</given-names></name> <name><surname>Harland</surname> <given-names>R. M.</given-names></name> <name><surname>Khokha</surname> <given-names>M. K.</given-names></name></person-group> (<year>2006</year>). <article-title>Developmental expression of FoxJ1.2, FoxJ2, and FoxQ1 in Xenopus tropicalis</article-title>. <source>Gene Expr. Patterns</source> <volume>6</volume>, <fpage>443</fpage>&#x02013;<lpage>447</lpage>. <pub-id pub-id-type="doi">10.1016/j.modgep.2005.11.007</pub-id><pub-id pub-id-type="pmid">16461016</pub-id></citation>
</ref>
<ref id="B13">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Clark</surname> <given-names>K. L.</given-names></name> <name><surname>Halay</surname> <given-names>E. D.</given-names></name> <name><surname>Lai</surname> <given-names>E.</given-names></name> <name><surname>Burley</surname> <given-names>S. K.</given-names></name></person-group> (<year>1993</year>). <article-title>Co-crystal structure of the HNF-3/fork head DNA-recognition motif resembles histone H5</article-title>. <source>Nature</source> <volume>364</volume>, <fpage>412</fpage>&#x02013;<lpage>420</lpage>. <pub-id pub-id-type="doi">10.1038/364412a0</pub-id><pub-id pub-id-type="pmid">8332212</pub-id></citation>
</ref>
<ref id="B14">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Danilova</surname> <given-names>N.</given-names></name> <name><surname>Visel</surname> <given-names>A.</given-names></name> <name><surname>Willett</surname> <given-names>C. E.</given-names></name> <name><surname>Steiner</surname> <given-names>L. A.</given-names></name></person-group> (<year>2004</year>). <article-title>Expression of the winged helix/forkhead gene, foxn4, during zebrafish development</article-title>. <source>Brain Res. Dev. Brain Res.</source> <volume>153</volume>, <fpage>115</fpage>&#x02013;<lpage>119</lpage>. <pub-id pub-id-type="doi">10.1016/j.devbrainres.2004.05.014</pub-id><pub-id pub-id-type="pmid">15464224</pub-id></citation>
</ref>
<ref id="B15">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Darnell</surname> <given-names>D. K.</given-names></name> <name><surname>Zhang</surname> <given-names>L. S.</given-names></name> <name><surname>Hannenhalli</surname> <given-names>S.</given-names></name> <name><surname>Yaklichkin</surname> <given-names>S. Y.</given-names></name></person-group> (<year>2014</year>). <article-title>Developmental expression of chicken FOXN1 and putative target genes during feather development</article-title>. <source>Int. J. Dev. Biol.</source> <volume>58</volume>, <fpage>57</fpage>&#x02013;<lpage>64</lpage>. <pub-id pub-id-type="doi">10.1387/ijdb.130023sy</pub-id><pub-id pub-id-type="pmid">24860996</pub-id></citation>
</ref>
<ref id="B16">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Di Gregorio</surname> <given-names>A.</given-names></name> <name><surname>Corbo</surname> <given-names>J. C.</given-names></name> <name><surname>Levine</surname> <given-names>M.</given-names></name></person-group> (<year>2001</year>). <article-title>The regulation of forkhead/HNF-3beta expression in the Ciona embryo</article-title>. <source>Dev. Biol.</source> <volume>229</volume>, <fpage>31</fpage>&#x02013;<lpage>43</lpage>. <pub-id pub-id-type="doi">10.1006/dbio.2000.9964</pub-id><pub-id pub-id-type="pmid">11133152</pub-id></citation>
</ref>
<ref id="B17">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Friedman</surname> <given-names>J. R.</given-names></name> <name><surname>Kaestner</surname> <given-names>K. H.</given-names></name></person-group> (<year>2006</year>). <article-title>The Foxa family of transcription factors in development and metabolism</article-title>. <source>Cell Mol. Life Sci.</source> <volume>63</volume>, <fpage>2317</fpage>&#x02013;<lpage>2328</lpage>. <pub-id pub-id-type="doi">10.1007/s00018-006-6095-6</pub-id><pub-id pub-id-type="pmid">16909212</pub-id></citation>
</ref>
<ref id="B18">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Fritzenwanker</surname> <given-names>J. H.</given-names></name> <name><surname>Gerhart</surname> <given-names>J.</given-names></name> <name><surname>Freeman</surname> <given-names>R. M.</given-names> <suffix>Jr.</suffix></name> <name><surname>Lowe</surname> <given-names>C. J.</given-names></name></person-group> (<year>2014</year>). <article-title>The Fox/Forkhead transcription factor family of the hemichordate <italic>Saccoglossus kowalevskii</italic></article-title>. <source>Evodevo</source> <volume>5</volume>:<fpage>17</fpage>. <pub-id pub-id-type="doi">10.1186/2041-9139-5-17</pub-id><pub-id pub-id-type="pmid">24987514</pub-id></citation>
</ref>
<ref id="B19">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Fuchs</surname> <given-names>J.</given-names></name> <name><surname>Martindale</surname> <given-names>M. Q.</given-names></name> <name><surname>Hejnol</surname> <given-names>A.</given-names></name></person-group> (<year>2011</year>). <article-title>Gene expression in bryozoan larvae suggest a fundamental importance of pre-patterned blastemic cells in the bryozoan life-cycle</article-title>. <source>Evodevo</source> <volume>2</volume>:<fpage>13</fpage>. <pub-id pub-id-type="doi">10.1186/2041-9139-2-13</pub-id><pub-id pub-id-type="pmid">21645327</pub-id></citation>
</ref>
<ref id="B20">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Fuentes</surname> <given-names>M.</given-names></name> <name><surname>Benito</surname> <given-names>E.</given-names></name> <name><surname>Bertrand</surname> <given-names>S.</given-names></name> <name><surname>Paris</surname> <given-names>M.</given-names></name> <name><surname>Mignardot</surname> <given-names>A.</given-names></name> <name><surname>Godoy</surname> <given-names>L.</given-names></name> <etal/></person-group>. (<year>2007</year>). <article-title>Insights into spawning behavior and development of the European amphioxus (<italic>Branchiostoma lanceolatum</italic>)</article-title>. <source>J. Exp. Zool. B Mol. Dev. Evol.</source> <volume>308</volume>, <fpage>484</fpage>&#x02013;<lpage>493</lpage>. <pub-id pub-id-type="doi">10.1002/jez.b.21179</pub-id><pub-id pub-id-type="pmid">17520703</pub-id></citation>
</ref>
<ref id="B21">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Fuentes</surname> <given-names>M.</given-names></name> <name><surname>Schubert</surname> <given-names>M.</given-names></name> <name><surname>Dalfo</surname> <given-names>D.</given-names></name> <name><surname>Candiani</surname> <given-names>S.</given-names></name> <name><surname>Benito</surname> <given-names>E.</given-names></name> <name><surname>Gardenyes</surname> <given-names>J.</given-names></name> <etal/></person-group>. (<year>2004</year>). <article-title>Preliminary observations on the spawning conditions of the European amphioxus (<italic>Branchiostoma lanceolatum</italic>) in captivity</article-title>. <source>J. Exp. Zool. B Mol. Dev. Evol.</source> <volume>302</volume>, <fpage>384</fpage>&#x02013;<lpage>391</lpage>. <pub-id pub-id-type="doi">10.1002/jez.b.20025</pub-id><pub-id pub-id-type="pmid">15287102</pub-id></citation>
</ref>
<ref id="B22">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Imai</surname> <given-names>K. S.</given-names></name> <name><surname>Hino</surname> <given-names>K.</given-names></name> <name><surname>Yagi</surname> <given-names>K.</given-names></name> <name><surname>Satoh</surname> <given-names>N.</given-names></name> <name><surname>Satou</surname> <given-names>Y.</given-names></name></person-group> (<year>2004</year>). <article-title>Gene expression profiles of transcription factors and signaling molecules in the ascidian embryo: towards a comprehensive understanding of gene networks</article-title>. <source>Development</source> <volume>131</volume>, <fpage>4047</fpage>&#x02013;<lpage>4058</lpage>. <pub-id pub-id-type="doi">10.1242/dev.01270</pub-id><pub-id pub-id-type="pmid">15269171</pub-id></citation>
</ref>
<ref id="B23">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Imai</surname> <given-names>K. S.</given-names></name> <name><surname>Levine</surname> <given-names>M.</given-names></name> <name><surname>Satoh</surname> <given-names>N.</given-names></name> <name><surname>Satou</surname> <given-names>Y.</given-names></name></person-group> (<year>2006</year>). <article-title>Regulatory blueprint for a chordate embryo</article-title>. <source>Science</source> <volume>312</volume>, <fpage>1183</fpage>&#x02013;<lpage>1187</lpage>. <pub-id pub-id-type="doi">10.1126/science.1123404</pub-id><pub-id pub-id-type="pmid">16728634</pub-id></citation>
</ref>
<ref id="B24">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Kalin</surname> <given-names>T. V.</given-names></name> <name><surname>Ustiyan</surname> <given-names>V.</given-names></name> <name><surname>Kalinichenko</surname> <given-names>V. V.</given-names></name></person-group> (<year>2011</year>). <article-title>Multiple faces of FoxM1 transcription factor: lessons from transgenic mouse models</article-title>. <source>Cell Cycle</source> <volume>10</volume>, <fpage>396</fpage>&#x02013;<lpage>405</lpage>. <pub-id pub-id-type="doi">10.4161/cc.10.3.14709</pub-id><pub-id pub-id-type="pmid">21270518</pub-id></citation>
</ref>
<ref id="B25">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Kelly</surname> <given-names>L. E.</given-names></name> <name><surname>Nekkalapudi</surname> <given-names>S.</given-names></name> <name><surname>El-Hodiri</surname> <given-names>H. M.</given-names></name></person-group> (<year>2007</year>). <article-title>Expression of the forkhead transcription factor FoxN4 in progenitor cells in the developing <italic>Xenopus laevis</italic> retina and brain</article-title>. <source>Gene Expr. Patterns</source> <volume>7</volume>, <fpage>233</fpage>&#x02013;<lpage>238</lpage>. <pub-id pub-id-type="doi">10.1016/j.modgep.2006.10.003</pub-id><pub-id pub-id-type="pmid">17110173</pub-id></citation>
</ref>
<ref id="B26">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>King</surname> <given-names>N.</given-names></name> <name><surname>Westbrook</surname> <given-names>M. J.</given-names></name> <name><surname>Young</surname> <given-names>S. L.</given-names></name> <name><surname>Kuo</surname> <given-names>A.</given-names></name> <name><surname>Abedin</surname> <given-names>M.</given-names></name> <name><surname>Chapman</surname> <given-names>J.</given-names></name> <etal/></person-group>. (<year>2008</year>). <article-title>The genome of the choanoflagellate Monosiga brevicollis and the origin of metazoans</article-title>. <source>Nature</source> <volume>451</volume>, <fpage>783</fpage>&#x02013;<lpage>788</lpage>. <pub-id pub-id-type="doi">10.1038/nature06617</pub-id><pub-id pub-id-type="pmid">18273011</pub-id></citation>
</ref>
<ref id="B27">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Kume</surname> <given-names>T.</given-names></name> <name><surname>Jiang</surname> <given-names>H.</given-names></name> <name><surname>Topczewska</surname> <given-names>J. M.</given-names></name> <name><surname>Hogan</surname> <given-names>B. L.</given-names></name></person-group> (<year>2001</year>). <article-title>The murine winged helix transcription factors, Foxc1 and Foxc2, are both required for cardiovascular development and somitogenesis</article-title>. <source>Genes Dev.</source> <volume>15</volume>, <fpage>2470</fpage>&#x02013;<lpage>2482</lpage>. <pub-id pub-id-type="doi">10.1101/gad.907301</pub-id><pub-id pub-id-type="pmid">11562355</pub-id></citation>
</ref>
<ref id="B28">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Larroux</surname> <given-names>C.</given-names></name> <name><surname>Fahey</surname> <given-names>B.</given-names></name> <name><surname>Liubicich</surname> <given-names>D.</given-names></name> <name><surname>Hinman</surname> <given-names>V. F.</given-names></name> <name><surname>Gauthier</surname> <given-names>M.</given-names></name> <name><surname>Gongora</surname> <given-names>M.</given-names></name> <etal/></person-group>. (<year>2006</year>). <article-title>Developmental expression of transcription factor genes in a demosponge: insights into the origin of metazoan multicellularity</article-title>. <source>Evol. Dev.</source> <volume>8</volume>, <fpage>150</fpage>&#x02013;<lpage>173</lpage>. <pub-id pub-id-type="doi">10.1111/j.1525-142X.2006.00086.x</pub-id><pub-id pub-id-type="pmid">16509894</pub-id></citation>
</ref>
<ref id="B29">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Larroux</surname> <given-names>C.</given-names></name> <name><surname>Luke</surname> <given-names>G. N.</given-names></name> <name><surname>Koopman</surname> <given-names>P.</given-names></name> <name><surname>Rokhsar</surname> <given-names>D. S.</given-names></name> <name><surname>Shimeld</surname> <given-names>S. M.</given-names></name> <name><surname>Degnan</surname> <given-names>B. M.</given-names></name></person-group> (<year>2008</year>). <article-title>Genesis and expansion of metazoan transcription factor gene classes</article-title>. <source>Mol. Biol. Evol.</source> <volume>25</volume>, <fpage>980</fpage>&#x02013;<lpage>996</lpage>. <pub-id pub-id-type="doi">10.1093/molbev/msn047</pub-id><pub-id pub-id-type="pmid">18296413</pub-id></citation>
</ref>
<ref id="B30">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Lee</surname> <given-names>Y. H.</given-names></name> <name><surname>Williams</surname> <given-names>A.</given-names></name> <name><surname>Hong</surname> <given-names>C. S.</given-names></name> <name><surname>You</surname> <given-names>Y.</given-names></name> <name><surname>Senoo</surname> <given-names>M.</given-names></name> <name><surname>Saint-Jeannet</surname> <given-names>J. P.</given-names></name></person-group> (<year>2013</year>). <article-title>Early development of the thymus in <italic>Xenopus laevis</italic></article-title>. <source>Dev. Dyn.</source> <volume>242</volume>, <fpage>164</fpage>&#x02013;<lpage>178</lpage>. <pub-id pub-id-type="doi">10.1002/dvdy.23905</pub-id><pub-id pub-id-type="pmid">23172757</pub-id></citation>
</ref>
<ref id="B31">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Ma</surname> <given-names>D.</given-names></name> <name><surname>Wang</surname> <given-names>L.</given-names></name> <name><surname>Wang</surname> <given-names>S.</given-names></name> <name><surname>Gao</surname> <given-names>Y.</given-names></name> <name><surname>Wei</surname> <given-names>Y.</given-names></name> <name><surname>Liu</surname> <given-names>F.</given-names></name></person-group> (<year>2012</year>). <article-title>Foxn1 maintains thymic epithelial cells to support T-cell development via mcm2 in zebrafish</article-title>. <source>Proc. Natl. Acad. Sci. U.S.A.</source> <volume>109</volume>, <fpage>21040</fpage>&#x02013;<lpage>21045</lpage>. <pub-id pub-id-type="doi">10.1073/pnas.1217021110</pub-id><pub-id pub-id-type="pmid">23213226</pub-id></citation>
</ref>
<ref id="B32">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Mazet</surname> <given-names>F.</given-names></name> <name><surname>Amemiya</surname> <given-names>C. T.</given-names></name> <name><surname>Shimeld</surname> <given-names>S. M.</given-names></name></person-group> (<year>2006</year>). <article-title>An ancient Fox gene cluster in bilaterian animals</article-title>. <source>Curr. Biol.</source> <volume>16</volume>, <fpage>R314</fpage>&#x02013;<lpage>R316</lpage>. <pub-id pub-id-type="doi">10.1016/j.cub.2006.03.088</pub-id><pub-id pub-id-type="pmid">16682334</pub-id></citation>
</ref>
<ref id="B33">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Mazet</surname> <given-names>F.</given-names></name> <name><surname>Shimeld</surname> <given-names>S. M.</given-names></name></person-group> (<year>2002</year>). <article-title>The evolution of chordate neural segmentation</article-title>. <source>Dev. Biol.</source> <volume>251</volume>, <fpage>258</fpage>&#x02013;<lpage>270</lpage>. <pub-id pub-id-type="doi">10.1006/dbio.2002.0831</pub-id><pub-id pub-id-type="pmid">12435356</pub-id></citation>
</ref>
<ref id="B34">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Mazet</surname> <given-names>F.</given-names></name> <name><surname>Yu</surname> <given-names>J. K.</given-names></name> <name><surname>Liberles</surname> <given-names>D. A.</given-names></name> <name><surname>Holland</surname> <given-names>L. Z.</given-names></name> <name><surname>Shimeld</surname> <given-names>S. M.</given-names></name></person-group> (<year>2003</year>). <article-title>Phylogenetic relationships of the Fox (Forkhead) gene family in the Bilateria</article-title>. <source>Gene</source> <volume>316</volume>, <fpage>79</fpage>&#x02013;<lpage>89</lpage>. <pub-id pub-id-type="doi">10.1016/S0378-1119(03)00741-8</pub-id><pub-id pub-id-type="pmid">14563554</pub-id></citation>
</ref>
<ref id="B35">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Miller</surname> <given-names>M. A.</given-names></name> <name><surname>Schwartz</surname> <given-names>T.</given-names></name> <name><surname>Pickett</surname> <given-names>B. E.</given-names></name> <name><surname>He</surname> <given-names>S.</given-names></name> <name><surname>Klem</surname> <given-names>E. B.</given-names></name> <name><surname>Scheuermann</surname> <given-names>R. H.</given-names></name> <etal/></person-group>. (<year>2015</year>). <article-title>A RESTful API for access to phylogenetic tools via the CIPRES science gateway</article-title>. <source>Evol. Bioinform. Online</source> <volume>11</volume>, <fpage>43</fpage>&#x02013;<lpage>48</lpage>. <pub-id pub-id-type="doi">10.4137/EBO.S21501</pub-id><pub-id pub-id-type="pmid">25861210</pub-id></citation>
</ref>
<ref id="B36">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Minguillon</surname> <given-names>C.</given-names></name> <name><surname>Garcia-Fernandez</surname> <given-names>J.</given-names></name></person-group> (<year>2002</year>). <article-title>The single amphioxus Mox gene: insights into the functional evolution of Mox genes, somites, and the asymmetry of amphioxus somitogenesis</article-title>. <source>Dev. Biol.</source> <volume>246</volume>, <fpage>455</fpage>&#x02013;<lpage>465</lpage>. <pub-id pub-id-type="doi">10.1006/dbio.2002.0660</pub-id><pub-id pub-id-type="pmid">12051829</pub-id></citation>
</ref>
<ref id="B37">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Neves</surname> <given-names>H.</given-names></name> <name><surname>Dupin</surname> <given-names>E.</given-names></name> <name><surname>Parreira</surname> <given-names>L.</given-names></name> <name><surname>Le Douarin</surname> <given-names>N. M.</given-names></name></person-group> (<year>2012</year>). <article-title>Modulation of Bmp4 signalling in the epithelial-mesenchymal interactions that take place in early thymus and parathyroid development in avian embryos</article-title>. <source>Dev. Biol.</source> <volume>361</volume>, <fpage>208</fpage>&#x02013;<lpage>219</lpage>. <pub-id pub-id-type="doi">10.1016/j.ydbio.2011.10.022</pub-id><pub-id pub-id-type="pmid">22057081</pub-id></citation>
</ref>
<ref id="B38">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Onimaru</surname> <given-names>K.</given-names></name> <name><surname>Shoguchi</surname> <given-names>E.</given-names></name> <name><surname>Kuratani</surname> <given-names>S.</given-names></name> <name><surname>Tanaka</surname> <given-names>M.</given-names></name></person-group> (<year>2011</year>). <article-title>Development and evolution of the lateral plate mesoderm: comparative analysis of amphioxus and lamprey with implications for the acquisition of paired fins</article-title>. <source>Dev. Biol.</source> <volume>359</volume>, <fpage>124</fpage>&#x02013;<lpage>136</lpage>. <pub-id pub-id-type="doi">10.1016/j.ydbio.2011.08.003</pub-id><pub-id pub-id-type="pmid">21864524</pub-id></citation>
</ref>
<ref id="B39">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Oulion</surname> <given-names>S.</given-names></name> <name><surname>Bertrand</surname> <given-names>S.</given-names></name> <name><surname>Belgacem</surname> <given-names>M. R.</given-names></name> <name><surname>Le Petillon</surname> <given-names>Y.</given-names></name> <name><surname>Escriva</surname> <given-names>H.</given-names></name></person-group> (<year>2012</year>). <article-title>Sequencing and analysis of the Mediterranean amphioxus (<italic>Branchiostoma lanceolatum</italic>) transcriptome</article-title>. <source>PLoS ONE</source> <volume>7</volume>:<fpage>e36554</fpage>. <pub-id pub-id-type="doi">10.1371/journal.pone.0036554</pub-id><pub-id pub-id-type="pmid">22590565</pub-id></citation>
</ref>
<ref id="B40">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Pohl</surname> <given-names>B. S.</given-names></name> <name><surname>Rossner</surname> <given-names>A.</given-names></name> <name><surname>Knochel</surname> <given-names>W.</given-names></name></person-group> (<year>2005</year>). <article-title>The Fox gene family in <italic>Xenopus laevis</italic>:FoxI2, FoxM1 and FoxP1 in early development</article-title>. <source>Int. J. Dev. Biol.</source> <volume>49</volume>, <fpage>53</fpage>&#x02013;<lpage>58</lpage>. <pub-id pub-id-type="doi">10.1387/ijdb.051977bp</pub-id><pub-id pub-id-type="pmid">15744668</pub-id></citation>
</ref>
<ref id="B41">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Rho</surname> <given-names>H. K.</given-names></name> <name><surname>Mcclay</surname> <given-names>D. R.</given-names></name></person-group> (<year>2011</year>). <article-title>The control of foxN2/3 expression in sea urchin embryos and its function in the skeletogenic gene regulatory network</article-title>. <source>Development</source> <volume>138</volume>, <fpage>937</fpage>&#x02013;<lpage>945</lpage>. <pub-id pub-id-type="doi">10.1242/dev.058396</pub-id><pub-id pub-id-type="pmid">21303847</pub-id></citation>
</ref>
<ref id="B42">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Romano</surname> <given-names>R.</given-names></name> <name><surname>Palamaro</surname> <given-names>L.</given-names></name> <name><surname>Fusco</surname> <given-names>A.</given-names></name> <name><surname>Giardino</surname> <given-names>G.</given-names></name> <name><surname>Gallo</surname> <given-names>V.</given-names></name> <name><surname>Del Vecchio</surname> <given-names>L.</given-names></name> <etal/></person-group>. (<year>2013</year>). <article-title>FOXN1: a master regulator gene of thymic epithelial development program</article-title>. <source>Front. Immunol.</source> <volume>4</volume>:<issue>187</issue>. <pub-id pub-id-type="doi">10.3389/fimmu.2013.00187</pub-id><pub-id pub-id-type="pmid">23874334</pub-id></citation>
</ref>
<ref id="B43">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Ronquist</surname> <given-names>F.</given-names></name> <name><surname>Teslenko</surname> <given-names>M.</given-names></name> <name><surname>Van Der Mark</surname> <given-names>P.</given-names></name> <name><surname>Ayres</surname> <given-names>D. L.</given-names></name> <name><surname>Darling</surname> <given-names>A.</given-names></name> <name><surname>Hohna</surname> <given-names>S.</given-names></name> <etal/></person-group>. (<year>2012</year>). <article-title>MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space</article-title>. <source>Syst. Biol.</source> <volume>61</volume>, <fpage>539</fpage>&#x02013;<lpage>542</lpage>. <pub-id pub-id-type="doi">10.1093/sysbio/sys029</pub-id><pub-id pub-id-type="pmid">22357727</pub-id></citation>
</ref>
<ref id="B44">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Samaan</surname> <given-names>G.</given-names></name> <name><surname>Yugo</surname> <given-names>D.</given-names></name> <name><surname>Rajagopalan</surname> <given-names>S.</given-names></name> <name><surname>Wall</surname> <given-names>J.</given-names></name> <name><surname>Donnell</surname> <given-names>R.</given-names></name> <name><surname>Goldowitz</surname> <given-names>D.</given-names></name> <etal/></person-group>. (<year>2010</year>). <article-title>Foxn3 is essential for craniofacial development in mice and a putative candidate involved in human congenital craniofacial defects</article-title>. <source>Biochem. Biophys. Res. Commun.</source> <volume>400</volume>, <fpage>60</fpage>&#x02013;<lpage>65</lpage>. <pub-id pub-id-type="doi">10.1016/j.bbrc.2010.07.142</pub-id><pub-id pub-id-type="pmid">20691664</pub-id></citation>
</ref>
<ref id="B45">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Schlake</surname> <given-names>T.</given-names></name> <name><surname>Schorpp</surname> <given-names>M.</given-names></name> <name><surname>Maul-Pavicic</surname> <given-names>A.</given-names></name> <name><surname>Malashenko</surname> <given-names>A. M.</given-names></name> <name><surname>Boehm</surname> <given-names>T.</given-names></name></person-group> (<year>2000</year>). <article-title>Forkhead/winged-helix transcription factor Whn regulates hair keratin gene expression: molecular analysis of the nude skin phenotype</article-title>. <source>Dev. Dyn.</source> <volume>217</volume>, <fpage>368</fpage>&#x02013;<lpage>376</lpage>. <pub-id pub-id-type="doi">10.1002/(SICI)1097-0177(200004)217:4&#x0003C;368::AID-DVDY4&#x0003E;3.0.CO;2-Z</pub-id><pub-id pub-id-type="pmid">10767081</pub-id></citation>
</ref>
<ref id="B46">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Schmidt</surname> <given-names>J.</given-names></name> <name><surname>Schuff</surname> <given-names>M.</given-names></name> <name><surname>Olsson</surname> <given-names>L.</given-names></name></person-group> (<year>2011</year>). <article-title>A role for FoxN3 in the development of cranial cartilages and muscles in <italic>Xenopus laevis</italic> (Amphibia: <italic>Anura: Pipidae</italic>) with special emphasis on the novel rostral cartilages</article-title>. <source>J. Anat.</source> <volume>218</volume>, <fpage>226</fpage>&#x02013;<lpage>242</lpage>. <pub-id pub-id-type="doi">10.1111/j.1469-7580.2010.01315.x</pub-id><pub-id pub-id-type="pmid">21050205</pub-id></citation>
</ref>
<ref id="B47">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Schuff</surname> <given-names>M.</given-names></name> <name><surname>Rossner</surname> <given-names>A.</given-names></name> <name><surname>Donow</surname> <given-names>C.</given-names></name> <name><surname>Knochel</surname> <given-names>W.</given-names></name></person-group> (<year>2006</year>). <article-title>Temporal and spatial expression patterns of FoxN genes in <italic>Xenopus laevis</italic> embryos</article-title>. <source>Int. J. Dev. Biol.</source> <volume>50</volume>, <fpage>429</fpage>&#x02013;<lpage>434</lpage>. <pub-id pub-id-type="doi">10.1387/ijdb.052126ms</pub-id><pub-id pub-id-type="pmid">16525939</pub-id></citation>
</ref>
<ref id="B48">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Schuff</surname> <given-names>M.</given-names></name> <name><surname>Rossner</surname> <given-names>A.</given-names></name> <name><surname>Wacker</surname> <given-names>S. A.</given-names></name> <name><surname>Donow</surname> <given-names>C.</given-names></name> <name><surname>Gessert</surname> <given-names>S.</given-names></name> <name><surname>Knochel</surname> <given-names>W.</given-names></name></person-group> (<year>2007</year>). <article-title>FoxN3 is required for craniofacial and eye development of <italic>Xenopus laevis</italic></article-title>. <source>Dev. Dyn.</source> <volume>236</volume>, <fpage>226</fpage>&#x02013;<lpage>239</lpage>. <pub-id pub-id-type="doi">10.1002/dvdy.21007</pub-id><pub-id pub-id-type="pmid">17089409</pub-id></citation>
</ref>
<ref id="B49">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Shimeld</surname> <given-names>S. M.</given-names></name> <name><surname>Boyle</surname> <given-names>M. J.</given-names></name> <name><surname>Brunet</surname> <given-names>T.</given-names></name> <name><surname>Luke</surname> <given-names>G. N.</given-names></name> <name><surname>Seaver</surname> <given-names>E. C.</given-names></name></person-group> (<year>2010a</year>). <article-title>Clustered Fox genes in lophotrochozoans and the evolution of the bilaterian Fox gene cluster</article-title>. <source>Dev. Biol.</source> <volume>340</volume>, <fpage>234</fpage>&#x02013;<lpage>248</lpage>. <pub-id pub-id-type="doi">10.1016/j.ydbio.2010.01.015</pub-id><pub-id pub-id-type="pmid">20096280</pub-id></citation>
</ref>
<ref id="B50">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Shimeld</surname> <given-names>S. M.</given-names></name> <name><surname>Degnan</surname> <given-names>B.</given-names></name> <name><surname>Luke</surname> <given-names>G. N.</given-names></name></person-group> (<year>2010b</year>). <article-title>Evolutionary genomics of the Fox genes: origin of gene families and the ancestry of gene clusters</article-title>. <source>Genomics</source> <volume>95</volume>, <fpage>256</fpage>&#x02013;<lpage>260</lpage>. <pub-id pub-id-type="doi">10.1016/j.ygeno.2009.08.002</pub-id><pub-id pub-id-type="pmid">19679177</pub-id></citation>
</ref>
<ref id="B51">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Shimeld</surname> <given-names>S. M.</given-names></name></person-group> (<year>1997</year>). <article-title>Characterisation of amphioxus HNF-3 genes: conserved expression in the notochord and floor plate</article-title>. <source>Dev. Biol.</source> <volume>183</volume>, <fpage>74</fpage>&#x02013;<lpage>85</lpage>. <pub-id pub-id-type="doi">10.1006/dbio.1996.8481</pub-id><pub-id pub-id-type="pmid">9119116</pub-id></citation>
</ref>
<ref id="B52">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Somorjai</surname> <given-names>I.</given-names></name> <name><surname>Bertrand</surname> <given-names>S.</given-names></name> <name><surname>Camasses</surname> <given-names>A.</given-names></name> <name><surname>Haguenauer</surname> <given-names>A.</given-names></name> <name><surname>Escriva</surname> <given-names>H.</given-names></name></person-group> (<year>2008</year>). <article-title>Evidence for stasis and not genetic piracy in developmental expression patterns of <italic>Branchiostoma lanceolatum</italic> and <italic>Branchiostoma floridae</italic>, two amphioxus species that have evolved independently over the course of 200 Myr</article-title>. <source>Dev. Genes Evol.</source> <volume>218</volume>, <fpage>703</fpage>&#x02013;<lpage>713</lpage>. <pub-id pub-id-type="doi">10.1007/s00427-008-0256-6</pub-id></citation>
</ref>
<ref id="B53">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Tamura</surname> <given-names>K.</given-names></name> <name><surname>Peterson</surname> <given-names>D.</given-names></name> <name><surname>Peterson</surname> <given-names>N.</given-names></name> <name><surname>Stecher</surname> <given-names>G.</given-names></name> <name><surname>Nei</surname> <given-names>M.</given-names></name> <name><surname>Kumar</surname> <given-names>S.</given-names></name></person-group> (<year>2011</year>). <article-title>MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods</article-title>. <source>Mol. Biol. Evol.</source> <volume>28</volume>, <fpage>2731</fpage>&#x02013;<lpage>2739</lpage>. <pub-id pub-id-type="doi">10.1093/molbev/msr121</pub-id><pub-id pub-id-type="pmid">21546353</pub-id></citation>
</ref>
<ref id="B54">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Tamura</surname> <given-names>K.</given-names></name> <name><surname>Stecher</surname> <given-names>G.</given-names></name> <name><surname>Peterson</surname> <given-names>D.</given-names></name> <name><surname>Filipski</surname> <given-names>A.</given-names></name> <name><surname>Kumar</surname> <given-names>S.</given-names></name></person-group> (<year>2013</year>). <article-title>MEGA6: molecular evolutionary genetics analysis version 6.0</article-title>. <source>Mol. Biol. Evol.</source> <volume>30</volume>, <fpage>2725</fpage>&#x02013;<lpage>2729</lpage>. <pub-id pub-id-type="doi">10.1093/molbev/mst197</pub-id><pub-id pub-id-type="pmid">24132122</pub-id></citation>
</ref>
<ref id="B55">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Terazawa</surname> <given-names>K.</given-names></name> <name><surname>Satoh</surname> <given-names>N.</given-names></name></person-group> (<year>1997</year>). <article-title>Formation of the chordamesoderm in the amphioxus embryo: analysis with Brachyury and fork head/HNF-3 genes</article-title>. <source>Dev. Genes Evol.</source> <volume>207</volume>, <fpage>1</fpage>&#x02013;<lpage>11</lpage>. <pub-id pub-id-type="doi">10.1007/s004270050086</pub-id><pub-id pub-id-type="pmid">20607475</pub-id></citation>
</ref>
<ref id="B56">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Toresson</surname> <given-names>H.</given-names></name> <name><surname>Martinez-Barbera</surname> <given-names>J. P.</given-names></name> <name><surname>Bardsley</surname> <given-names>A.</given-names></name> <name><surname>Caubit</surname> <given-names>X.</given-names></name> <name><surname>Krauss</surname> <given-names>S.</given-names></name></person-group> (<year>1998</year>). <article-title>Conservation of BF-1 expression in amphioxus and zebrafish suggests evolutionary ancestry of anterior cell types that contribute to the vertebrate telencephalon</article-title>. <source>Dev. Genes Evol.</source> <volume>208</volume>, <fpage>431</fpage>&#x02013;<lpage>439</lpage>. <pub-id pub-id-type="doi">10.1007/s004270050200</pub-id><pub-id pub-id-type="pmid">9799423</pub-id></citation>
</ref>
<ref id="B57">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Tribioli</surname> <given-names>C.</given-names></name> <name><surname>Robledo</surname> <given-names>R. F.</given-names></name> <name><surname>Lufkin</surname> <given-names>T.</given-names></name></person-group> (<year>2002</year>). <article-title>The murine fork head gene Foxn2 is expressed in craniofacial, limb, CNS and somitic tissues during embryogenesis</article-title>. <source>Mech. Dev.</source> <volume>118</volume>, <fpage>161</fpage>&#x02013;<lpage>163</lpage>. <pub-id pub-id-type="doi">10.1016/S0925-4773(02)00220-4</pub-id><pub-id pub-id-type="pmid">12351180</pub-id></citation>
</ref>
<ref id="B58">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Tu</surname> <given-names>Q.</given-names></name> <name><surname>Brown</surname> <given-names>C. T.</given-names></name> <name><surname>Davidson</surname> <given-names>E. H.</given-names></name> <name><surname>Oliveri</surname> <given-names>P.</given-names></name></person-group> (<year>2006</year>). <article-title>Sea urchin Forkhead gene family: phylogeny and embryonic expression</article-title>. <source>Dev. Biol.</source> <volume>300</volume>, <fpage>49</fpage>&#x02013;<lpage>62</lpage>. <pub-id pub-id-type="doi">10.1016/j.ydbio.2006.09.031</pub-id><pub-id pub-id-type="pmid">17081512</pub-id></citation>
</ref>
<ref id="B59">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Tuteja</surname> <given-names>G.</given-names></name> <name><surname>Kaestner</surname> <given-names>K. H.</given-names></name></person-group> (<year>2007a</year>). <article-title>Forkhead transcription factors II</article-title>. <source>Cell</source> <volume>131</volume>, <fpage>192</fpage>. <pub-id pub-id-type="doi">10.1016/j.cell.2007.09.016</pub-id><pub-id pub-id-type="pmid">17923097</pub-id></citation>
</ref>
<ref id="B60">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Tuteja</surname> <given-names>G.</given-names></name> <name><surname>Kaestner</surname> <given-names>K. H.</given-names></name></person-group> (<year>2007b</year>). <article-title>SnapShot: forkhead transcription factors I</article-title>. <source>Cell</source> <volume>130</volume>, <fpage>1160</fpage>. <pub-id pub-id-type="doi">10.1016/j.cell.2007.09.005</pub-id><pub-id pub-id-type="pmid">17889656</pub-id></citation>
</ref>
<ref id="B61">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Ueno</surname> <given-names>H.</given-names></name> <name><surname>Nakajo</surname> <given-names>N.</given-names></name> <name><surname>Watanabe</surname> <given-names>M.</given-names></name> <name><surname>Isoda</surname> <given-names>M.</given-names></name> <name><surname>Sagata</surname> <given-names>N.</given-names></name></person-group> (<year>2008</year>). <article-title>FoxM1-driven cell division is required for neuronal differentiation in early Xenopus embryos</article-title>. <source>Development</source> <volume>135</volume>, <fpage>2023</fpage>&#x02013;<lpage>2030</lpage>. <pub-id pub-id-type="doi">10.1242/dev.019893</pub-id><pub-id pub-id-type="pmid">18469223</pub-id></citation>
</ref>
<ref id="B62">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Vij</surname> <given-names>S.</given-names></name> <name><surname>Rink</surname> <given-names>J. C.</given-names></name> <name><surname>Ho</surname> <given-names>H. K.</given-names></name> <name><surname>Babu</surname> <given-names>D.</given-names></name> <name><surname>Eitel</surname> <given-names>M.</given-names></name> <name><surname>Narasimhan</surname> <given-names>V.</given-names></name> <etal/></person-group>. (<year>2012</year>). <article-title>Evolutionarily ancient association of the FoxJ1 transcription factor with the motile ciliogenic program</article-title>. <source>PLoS Genet</source> <volume>8</volume>:<fpage>e1003019</fpage>. <pub-id pub-id-type="doi">10.1371/journal.pgen.1003019</pub-id><pub-id pub-id-type="pmid">23144623</pub-id></citation>
</ref>
<ref id="B63">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Weigel</surname> <given-names>D.</given-names></name> <name><surname>Jackle</surname> <given-names>H.</given-names></name></person-group> (<year>1990</year>). <article-title>The fork head domain: a novel DNA binding motif of eukaryotic transcription factors?</article-title> <source>Cell</source> <volume>63</volume>, <fpage>455</fpage>&#x02013;<lpage>456</lpage>. <pub-id pub-id-type="doi">10.1016/0092-8674(90)90439-L</pub-id><pub-id pub-id-type="pmid">2225060</pub-id></citation>
</ref>
<ref id="B64">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Wilm</surname> <given-names>B.</given-names></name> <name><surname>James</surname> <given-names>R. G.</given-names></name> <name><surname>Schultheiss</surname> <given-names>T. M.</given-names></name> <name><surname>Hogan</surname> <given-names>B. L.</given-names></name></person-group> (<year>2004</year>). <article-title>The forkhead genes, Foxc1 and Foxc2, regulate paraxial versus intermediate mesoderm cell fate</article-title>. <source>Dev. Biol.</source> <volume>271</volume>, <fpage>176</fpage>&#x02013;<lpage>189</lpage>. <pub-id pub-id-type="doi">10.1016/j.ydbio.2004.03.034</pub-id><pub-id pub-id-type="pmid">15196959</pub-id></citation>
</ref>
<ref id="B65">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Wotton</surname> <given-names>K. R.</given-names></name> <name><surname>Mazet</surname> <given-names>F.</given-names></name> <name><surname>Shimeld</surname> <given-names>S. M.</given-names></name></person-group> (<year>2008</year>). <article-title>Expression of FoxC, FoxF, FoxL1, and FoxQ1 genes in the dogfish <italic>Scyliorhinus canicula</italic> defines ancient and derived roles for Fox genes in vertebrate development</article-title>. <source>Dev. Dyn.</source> <volume>237</volume>, <fpage>1590</fpage>&#x02013;<lpage>1603</lpage>. <pub-id pub-id-type="doi">10.1002/dvdy.21553</pub-id><pub-id pub-id-type="pmid">18498098</pub-id></citation>
</ref>
<ref id="B66">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Wotton</surname> <given-names>K. R.</given-names></name> <name><surname>Shimeld</surname> <given-names>S. M.</given-names></name></person-group> (<year>2006</year>). <article-title>Comparative genomics of vertebrate Fox cluster loci</article-title>. <source>BMC Genomics</source> <volume>7</volume>:<fpage>271</fpage>. <pub-id pub-id-type="doi">10.1186/1471-2164-7-271</pub-id><pub-id pub-id-type="pmid">17062144</pub-id></citation>
</ref>
<ref id="B67">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Yu</surname> <given-names>J. K.</given-names></name> <name><surname>Holland</surname> <given-names>L. Z.</given-names></name> <name><surname>Jamrich</surname> <given-names>M.</given-names></name> <name><surname>Blitz</surname> <given-names>I. L.</given-names></name> <name><surname>Hollan</surname> <given-names>N. D.</given-names></name></person-group> (<year>2002a</year>). <article-title>AmphiFoxE4, an amphioxus winged helix/forkhead gene encoding a protein closely related to vertebrate thyroid transcription factor-2: expression during pharyngeal development</article-title>. <source>Evol. Dev.</source> <volume>4</volume>, <fpage>9</fpage>&#x02013;<lpage>15</lpage>. <pub-id pub-id-type="doi">10.1046/j.1525-142x.2002.01057.x</pub-id><pub-id pub-id-type="pmid">11868660</pub-id></citation>
</ref>
<ref id="B68">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Yu</surname> <given-names>J. K.</given-names></name> <name><surname>Holland</surname> <given-names>N. D.</given-names></name> <name><surname>Holland</surname> <given-names>L. Z.</given-names></name></person-group> (<year>2002b</year>). <article-title>An amphioxus winged helix/forkhead gene, AmphiFoxD: insights into vertebrate neural crest evolution</article-title>. <source>Dev. Dyn.</source> <volume>225</volume>, <fpage>289</fpage>&#x02013;<lpage>297</lpage>. <pub-id pub-id-type="doi">10.1002/dvdy.10173</pub-id><pub-id pub-id-type="pmid">12412011</pub-id></citation>
</ref>
<ref id="B69">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Yu</surname> <given-names>J. K.</given-names></name> <name><surname>Holland</surname> <given-names>N. D.</given-names></name> <name><surname>Holland</surname> <given-names>L. Z.</given-names></name></person-group> (<year>2003</year>). <article-title>AmphiFoxQ2, a novel winged helix/forkhead gene, exclusively marks the anterior end of the amphioxus embryo</article-title>. <source>Dev. Genes Evol.</source> <volume>213</volume>, <fpage>102</fpage>&#x02013;<lpage>105</lpage>. <pub-id pub-id-type="doi">10.1007/s00427-003-0302-3</pub-id><pub-id pub-id-type="pmid">12632180</pub-id></citation>
</ref>
<ref id="B70">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Yu</surname> <given-names>J. K.</given-names></name> <name><surname>Mazet</surname> <given-names>F.</given-names></name> <name><surname>Chen</surname> <given-names>Y. T.</given-names></name> <name><surname>Huang</surname> <given-names>S. W.</given-names></name> <name><surname>Jung</surname> <given-names>K. C.</given-names></name> <name><surname>Shimeld</surname> <given-names>S. M.</given-names></name></person-group> (<year>2008a</year>). <article-title>The Fox genes of <italic>Branchiostoma floridae</italic></article-title>. <source>Dev. Genes Evol.</source> <volume>218</volume>, <fpage>629</fpage>&#x02013;<lpage>638</lpage>. <pub-id pub-id-type="doi">10.1007/s00427-008-0229-9</pub-id><pub-id pub-id-type="pmid">18773219</pub-id></citation>
</ref>
<ref id="B71">
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Yu</surname> <given-names>X.</given-names></name> <name><surname>Ng</surname> <given-names>C. P.</given-names></name> <name><surname>Habacher</surname> <given-names>H.</given-names></name> <name><surname>Roy</surname> <given-names>S.</given-names></name></person-group> (<year>2008b</year>). <article-title>Foxj1 transcription factors are master regulators of the motile ciliogenic program</article-title>. <source>Nat. Genet.</source> <volume>40</volume>, <fpage>1445</fpage>&#x02013;<lpage>1453</lpage>. <pub-id pub-id-type="doi">10.1038/ng.263</pub-id><pub-id pub-id-type="pmid">19011630</pub-id></citation>
</ref>
</ref-list>
</back>
</article>