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| <?xml version="1.0" encoding="UTF-8"?><!DOCTYPE article PUBLIC "-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD v1.1d1 20130915//EN" "JATS-archivearticle1.dtd"><article article-type="research-article" dtd-version="1.1d1" xmlns:xlink="http://www.w3.org/1999/xlink"><front><journal-meta><journal-id journal-id-type="nlm-ta">elife</journal-id><journal-id journal-id-type="hwp">eLife</journal-id><journal-id journal-id-type="publisher-id">eLife</journal-id><journal-title-group><journal-title>eLife</journal-title></journal-title-group><issn publication-format="electronic">2050-084X</issn><publisher><publisher-name>eLife Sciences Publications, Ltd</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="publisher-id">03728</article-id><article-id pub-id-type="doi">10.7554/eLife.03728</article-id><article-categories><subj-group subj-group-type="display-channel"><subject>Research article</subject></subj-group><subj-group subj-group-type="heading"><subject>Developmental biology and stem cells</subject></subj-group><subj-group subj-group-type="heading"><subject>Genomics and evolutionary biology</subject></subj-group></article-categories><title-group><article-title>Divergent mechanisms regulate conserved cardiopharyngeal development and gene expression in distantly related ascidians</article-title></title-group><contrib-group><contrib contrib-type="author" equal-contrib="yes" id="author-15319"><name><surname>Stolfi</surname><given-names>Alberto</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="other" rid="par-2"/><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/></contrib><contrib contrib-type="author" equal-contrib="yes" id="author-15647"><name><surname>Lowe</surname><given-names>Elijah K</given-names></name><xref ref-type="aff" rid="aff2"/><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="other" rid="par-7"/><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/></contrib><contrib contrib-type="author" id="author-15648"><name><surname>Racioppi</surname><given-names>Claudia</given-names></name><xref ref-type="aff" rid="aff3"/><xref ref-type="fn" rid="con4"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/></contrib><contrib contrib-type="author" id="author-15649"><name><surname>Ristoratore</surname><given-names>Filomena</given-names></name><xref ref-type="aff" rid="aff3"/><xref ref-type="fn" rid="con5"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-15651"><name><surname>Brown</surname><given-names>C Titus</given-names></name><xref ref-type="aff" rid="aff2"/><xref ref-type="aff" rid="aff4"/><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="other" rid="par-3"/><xref ref-type="other" rid="par-7"/><xref ref-type="fn" rid="con6"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-3898"><name><surname>Swalla</surname><given-names>Billie J</given-names></name><xref ref-type="aff" rid="aff5"/><xref ref-type="aff" rid="aff6"/><xref ref-type="corresp" rid="cor2">*</xref><xref ref-type="other" rid="par-4"/><xref ref-type="other" rid="par-7"/><xref ref-type="fn" rid="con3"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-15308"><name><surname>Christiaen</surname><given-names>Lionel</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor3">*</xref><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-5"/><xref ref-type="other" rid="par-6"/><xref ref-type="other" rid="par-8"/><xref ref-type="fn" rid="con7"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/></contrib><aff id="aff1"><institution content-type="dept">Center for Developmental Genetics, Department of Biology</institution>, <institution>New York University</institution>, <addr-line><named-content content-type="city">New York</named-content></addr-line>, <country>United States</country></aff><aff id="aff2"><institution content-type="dept">Department of Computer Science and Engineering</institution>, <institution>Michigan State University</institution>, <addr-line><named-content content-type="city">East Lansing</named-content></addr-line>, <country>United States</country></aff><aff id="aff3"><institution content-type="dept">Cellular and Developmental Biology Laboratory</institution>, <institution>Stazione Zoologica Anton Dohrn</institution>, <addr-line><named-content content-type="city">Napoli</named-content></addr-line>, <country>Italy</country></aff><aff id="aff4"><institution content-type="dept">Department of Microbiology and Molecular Genetics</institution>, <institution>Michigan State University</institution>, <addr-line><named-content content-type="city">East Lansing</named-content></addr-line>, <country>United States</country></aff><aff id="aff5"><institution content-type="dept">Department of Biology</institution>, <institution>University of Washington</institution>, <addr-line><named-content content-type="city">Seattle</named-content></addr-line>, <country>United States</country></aff><aff id="aff6"><institution content-type="dept">Friday Harbor Laboratories</institution>, <institution>University of Washington</institution>, <addr-line><named-content content-type="city">Friday Harbor</named-content></addr-line>, <country>United States</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Buckingham</surname><given-names>Margaret</given-names></name><role>Reviewing editor</role><aff><institution>Institut Pasteur</institution>, <country>France</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>ctb@msu.edu</email> (CTB);</corresp><corresp id="cor2"><label>*</label>For correspondence: <email>bjswalla@u.washington.edu</email> (BJS);</corresp><corresp id="cor3"><label>*</label>For correspondence: <email>lc121@nyu.edu</email> (LC)</corresp><fn fn-type="con" id="equal-contrib"><label>†</label><p>These authors contributed equally to this work</p></fn></author-notes><pub-date date-type="pub" publication-format="electronic"><day>10</day><month>09</month><year>2014</year></pub-date><pub-date pub-type="collection"><year>2014</year></pub-date><volume>3</volume><elocation-id>e03728</elocation-id><history><date date-type="received"><day>18</day><month>06</month><year>2014</year></date><date date-type="accepted"><day>05</day><month>09</month><year>2014</year></date></history><permissions><copyright-statement>© 2014, Stolfi et al</copyright-statement><copyright-year>2014</copyright-year><copyright-holder>Stolfi et al</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This article is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="elife03728.pdf"/><abstract><object-id pub-id-type="doi">10.7554/eLife.03728.001</object-id><p>Ascidians present a striking dichotomy between conserved phenotypes and divergent genomes: embryonic cell lineages and gene expression patterns are conserved between distantly related species. Much research has focused on Ciona or Halocynthia spp. but development in other ascidians remains poorly characterized. In this study, we surveyed the multipotent myogenic B7.5 lineage in Molgula spp. Comparisons to the homologous lineage in Ciona revealed identical cell division and fate specification events that result in segregation of larval, cardiac, and pharyngeal muscle progenitors. Moreover, the expression patterns of key regulators are conserved, but cross-species transgenic assays uncovered incompatibility, or ‘unintelligibility’, of orthologous cis-regulatory sequences between Molgula and Ciona. These sequences drive identical expression patterns that are not recapitulated in cross-species assays. We show that this unintelligibility is likely due to changes in both cis- and trans-acting elements, hinting at widespread and frequent turnover of regulatory mechanisms underlying otherwise conserved aspects of ascidian embryogenesis.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03728.001">http://dx.doi.org/10.7554/eLife.03728.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.03728.002</object-id><title>eLife digest</title><p>When two species have features that look similar, this may be because the features arise by the same processes during development. Other features may look similar yet develop by different mechanisms. ‘Developmental system drift’ refers to the process where a physical feature remains unaltered during evolution, but the underlying pathway that controls its development is changed. However, to date, there have been only a few experimental studies that support this idea.</p><p>Ascidians—also commonly known as sea squirts—are vase-like marine creatures, which start off as tadpole-like larvae that swim around until they find a place to settle down and attach themselves. Once attached, the sea squirts lose the ability to swim and start feeding, typically by filtering material out of the seawater. Sea squirts and their close relatives are the invertebrates (animals without backbones) that are most closely related to all vertebrates (animals with backbones), including humans. Furthermore, although different species of sea squirt have almost identical embryos, their genomes are very different.</p><p>Stolfi et al. have now studied whether developmental system drift may have occurred during the evolution of ascidians, by analyzing different species of sea squirt named Molgula and Ciona. Stolfi et al. compared the genomes of Molgula and Ciona and studied the expression of genes in the cells that give rise to the heart and the muscles of the head. As an embryo develops, specific genes are switched on or off, and these patterns of gene activation were broadly identical in the two species of sea squirt examined.</p><p>Enhancers are sequences of DNA that control when and how a gene is switched on. Given the similarities between the development of heart and head muscle cells in the different sea squirts, Stolfi et al. looked to see if the mechanisms of gene expression, and therefore the enhancers, were also conserved. Unexpectedly, this was not the case. When enhancers from Molgula were introduced into Ciona (and vice versa), these sequences were unable to switch on gene expression—thus enhancers from one sea squirt species could not function in the other.</p><p>Stolfi et al. conclude that the developmental systems may have drifted considerably during evolution of the sea squirts, in spite of their nearly identical embryos. This reinforces the view that different paths can lead to the formation of similar physical features.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03728.002">http://dx.doi.org/10.7554/eLife.03728.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>development</kwd><kwd>evolution</kwd><kwd>DSD</kwd><kwd>ascidians</kwd><kwd>tunicates</kwd><kwd>cardiopharyngeal mesoderm</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd>other</kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000057</institution-id><institution>National Institute of General Medical Sciences</institution></institution-wrap></funding-source><award-id>R01GM096032</award-id><principal-award-recipient><name><surname>Christiaen</surname><given-names>Lionel</given-names></name></principal-award-recipient></award-group><award-group id="par-2"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000001</institution-id><institution>National Science Foundation</institution></institution-wrap></funding-source><award-id>NSF-1161835</award-id><principal-award-recipient><name><surname>Stolfi</surname><given-names>Alberto</given-names></name></principal-award-recipient></award-group><award-group id="par-3"><funding-source><institution-wrap><institution>Agriculture and Food Research Initiative</institution></institution-wrap></funding-source><award-id>2010-65205-20361</award-id><principal-award-recipient><name><surname>Brown</surname><given-names>C Titus</given-names></name></principal-award-recipient></award-group><award-group id="par-4"><funding-source><institution-wrap><institution>ASSEMBLE MARINE</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Swalla</surname><given-names>Billie J</given-names></name></principal-award-recipient></award-group><award-group id="par-5"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000968</institution-id><institution>American Heart Association</institution></institution-wrap></funding-source><award-id>10SDG4310061</award-id><principal-award-recipient><name><surname>Christiaen</surname><given-names>Lionel</given-names></name></principal-award-recipient></award-group><award-group id="par-6"><funding-source><institution-wrap><institution>New York Cardiac Center</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Christiaen</surname><given-names>Lionel</given-names></name></principal-award-recipient></award-group><award-group id="par-7"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000001</institution-id><institution>National Science Foundation</institution></institution-wrap></funding-source><award-id>DBI-0939454</award-id><principal-award-recipient><name><surname>Lowe</surname><given-names>Elijah K</given-names></name><name><surname>Brown</surname><given-names>C Titus</given-names></name><name><surname>Swalla</surname><given-names>Billie J</given-names></name></principal-award-recipient></award-group><award-group id="par-8"><funding-source><institution-wrap><institution>New York University College of Arts and Science</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Christiaen</surname><given-names>Lionel</given-names></name></principal-award-recipient></award-group><funding-statement>The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.</funding-statement></funding-group><custom-meta-group><custom-meta><meta-name>elife-xml-version</meta-name><meta-value>2</meta-value></custom-meta><custom-meta specific-use="meta-only"><meta-name>Author impact statement</meta-name><meta-value>Embryos of distantly related sea squirt species are nearly identical but develop according to different gene regulatory mechanisms.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="s1" sec-type="intro"><title>Introduction</title><p>Transcriptional regulatory networks are indispensable for the generation of the diverse germ layers, anatomical structures, and cell types of multicellular organisms (<xref ref-type="bibr" rid="bib58">Levine and Davidson, 2005</xref>). The impact of <italic>cis</italic>-regulatory DNA sequence changes on the evolution of development is undeniable but not yet fully understood (<xref ref-type="bibr" rid="bib116">Wray, 2007</xref>; <xref ref-type="bibr" rid="bib113">Wittkopp and Kalay 2011</xref>). Phenotypic differences between species have often been traced to differences in expression of orthologous genes. In turn, this differential expression can be attributed to changes in <italic>cis</italic>-regulatory modules (or ‘enhancers’), which may alter their binding by sequence-specific transcription factors (TFs) (<xref ref-type="bibr" rid="bib94">Sucena and Stern, 2000</xref>; <xref ref-type="bibr" rid="bib29">Gompel et al., 2005</xref>; <xref ref-type="bibr" rid="bib76">Prud'homme et al., 2006</xref>; <xref ref-type="bibr" rid="bib67">Miller et al., 2007</xref>; <xref ref-type="bibr" rid="bib88">Shirangi et al., 2009</xref>).</p><p>In contrast, gene expression patterns may be identical <italic>in spite</italic> of differences in the <italic>cis</italic>-regulatory DNA sequences controlling their transcription. For instance, orthologous enhancers might be interchangeable between different species without conspicuous DNA sequence similarity (<xref ref-type="bibr" rid="bib62">Maduro and Pilgrim, 1996</xref>; <xref ref-type="bibr" rid="bib61">Ludwig et al., 1998</xref>; <xref ref-type="bibr" rid="bib75">Piano et al., 1999</xref>; <xref ref-type="bibr" rid="bib79">Romano and Wray, 2003</xref>; <xref ref-type="bibr" rid="bib71">Oda-Ishii et al., 2005</xref>). This is often attributed to the conservation of gene regulatory networks (GRNs) and the flexibility of TF binding site distribution in a given enhancer, which contribute to conservation of enhancer function (<xref ref-type="bibr" rid="bib59">Ludwig et al., 2000</xref>; <xref ref-type="bibr" rid="bib71">Oda-Ishii et al., 2005</xref>; <xref ref-type="bibr" rid="bib35">Hare et al., 2008</xref>; <xref ref-type="bibr" rid="bib112">Weirauch and Hughes, 2010</xref>). This cryptic regulatory turnover is termed ‘developmental system/systems drift’ (DSD) (<xref ref-type="bibr" rid="bib106">True and Haag, 2001</xref>). This term broadly applies to the divergence in the molecular or morphogenetic basis for the development of identical homologous characters.</p><p>However, gene expression patterns that appear conserved between species may also be the output of divergent regulatory networks (<xref ref-type="bibr" rid="bib60">Ludwig et al., 2005</xref>). In such cases, one species' <italic>trans</italic> environment may be unable to interpret the other species' enhancer, even if the expression patterns controlled by the orthologous enhancers are identical between the two species. This particularly acute manifestation of DSD is thought to contribute to developmental defects in interspecies hybrids (<xref ref-type="bibr" rid="bib101">Takano, 1998</xref>), contributing to reproductive isolation and speciation as <italic>cis/trans</italic> combinations become so incompatible as to be lethal (<xref ref-type="bibr" rid="bib75a">Porter and Johnson, 2002</xref>). There is ample evidence that DSD is pervasive in metazoan evolution (<xref ref-type="bibr" rid="bib48">Kiontke et al., 2007</xref>; <xref ref-type="bibr" rid="bib108">Verster et al., 2014</xref>), but it has been difficult to study due to its cryptic nature.</p><p>Sea squirts, or ascidians, are well suited for investigating the evolution of gene regulation in development (<xref ref-type="bibr" rid="bib82">Satoh, 2013</xref>). Species on the opposite branches of the ascidian tree, estimated to have diverged ∼520 million years apart, have extremely conserved embryos that share identical stereotyped cell divisions up until the late gastrula stage and beyond (<xref ref-type="bibr" rid="bib96">Swalla, 2006</xref>; <xref ref-type="bibr" rid="bib56">Lemaire, 2009</xref>). This allows one to unequivocally infer homology between specific embryonic cells, gene expression patterns, and GRNs. This is in contrast to the striking lack of non-coding sequence conservation, revealed by bioinformatic alignment of genomic sequences from disparate ascidian species. This means that any two randomly selected ascidian species are likely to be more genetically divergent from one another than humans are from fish, which share several highly conserved non-coding sequences (<xref ref-type="bibr" rid="bib114">Woolfe et al., 2004</xref>). This dichotomy between near-identical embryos and un-alignable <italic>cis</italic>-regulatory sequences represents a singular paradox in the study of evolution and developmental biology today (<xref ref-type="bibr" rid="bib55">Lemaire, 2011</xref>).</p><p>Modern molecular tools for embryo manipulation and visualization are available for several ascidian species, including a protocol for transfection <italic>en masse</italic> by electroporation of plasmid DNA into fertilized eggs of the solitary ascidian <italic>Ciona intestinalis</italic> (<xref ref-type="bibr" rid="bib19">Corbo et al., 1997</xref>; <xref ref-type="bibr" rid="bib15">Christiaen et al., 2009b</xref>). This experimental tractability has allowed researchers to begin addressing the ascidian embryological paradox. Regulation of the <italic>Otx</italic> gene is a prime example, as enhancers upstream of the <italic>Otx</italic> genes from <italic>C. intestinalis</italic> and the distantly related <italic>Halocynthia roretzi</italic> do not show any DNA sequence similarity but are broadly functional in cross-species assays. This was shown to be due to conservation of <italic>cis</italic>-regulatory logic in spite of a reshuffling of functional TF binding sites (<xref ref-type="bibr" rid="bib71">Oda-Ishii et al., 2005</xref>). On the other hand, comparisons of <italic>C. intestinalis</italic> and <italic>H. roretzi</italic> development have also revealed clear examples of acute DSD in regulation of the <italic>Brachyury</italic> gene (<xref ref-type="bibr" rid="bib100">Takahashi et al., 1999</xref>), cell signaling upstream of pigment cell differentiation (<xref ref-type="bibr" rid="bib22">Darras and Nishida, 2001</xref>; <xref ref-type="bibr" rid="bib1">Abitua et al., 2012</xref>), secondary tail muscle specification (<xref ref-type="bibr" rid="bib47">Kim and Nishida, 2001</xref>; <xref ref-type="bibr" rid="bib104">Tokuoka et al., 2007</xref>; <xref ref-type="bibr" rid="bib40">Hudson et al., 2007</xref>; <xref ref-type="bibr" rid="bib41">Hudson and Yasuo, 2008</xref>), and embryonic marginal zone patterning (<xref ref-type="bibr" rid="bib102">Takatori et al., 2010</xref>; <xref ref-type="bibr" rid="bib39">Hudson et al., 2013</xref>).</p><p>These few examples suggest that considerable DSD may have occurred during the evolution of ascidians, even with their presupposed highly constrained mode of embryogenesis. To get a better idea of how DSD may have shaped ascidian evolution, it was necessary to identify species comparable to <italic>Ciona</italic> spp. in their experimental tractability but phylogenetically distant enough as to maximize the genetic differences between them.</p><p><italic>Molgula</italic> spp. belong to the order Stolidobranchia like <italic>H. roretzi,</italic> but produce embryos that are more comparable in size and developmental rate to those of <italic>C. intestinalis,</italic> a member of the order Phlebobranchia (<xref ref-type="fig" rid="fig1">Figure 1A</xref>). The genus is also remarkable for containing several species that have independently evolved anural development (<xref ref-type="bibr" rid="bib7">Berrill, 1931</xref>; <xref ref-type="bibr" rid="bib97">Swalla and Jeffery, 1990</xref>; <xref ref-type="bibr" rid="bib32">Hadfield et al., 1995</xref>; <xref ref-type="bibr" rid="bib44">Jeffery et al., 1999</xref>; <xref ref-type="bibr" rid="bib38">Huber et al., 2000</xref>). These anural, or ‘tail-less’, <italic>Molgula</italic> species produce immotile larvae lacking the differentiated structures that are required for swimming (<xref ref-type="bibr" rid="bib97">Swalla and Jeffery, 1990</xref>; <xref ref-type="bibr" rid="bib52">Kusakabe et al., 1996</xref>), and some species even appear to bypass the larval stage altogether (<xref ref-type="bibr" rid="bib98">Tagawa et al., 1997</xref>; <xref ref-type="bibr" rid="bib63">Maliska and Swalla, 2010</xref>). Thus, <italic>Molgula</italic> species comprise a unique group in which to study body plan and life cycle evolution.<fig-group><fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.03728.003</object-id><label>Figure 1.</label><caption><title>The B7.5 lineage in <italic>M. occidentalis</italic>.</title><p>(<bold>A</bold>) Diagram comparing <italic>M. occidentalis</italic> (top) and <italic>C. intestinalis</italic> (bottom) embryogenesis at 24°C. Embryos were stained with Alexa Fluor dye-conjugated phalloidin to visualize cell outlines and DAPI to visualize cell nuclei. (<bold>B</bold>) Diagram of mVISTA (<xref ref-type="bibr" rid="bib28">Frazer et al., 2004</xref>; <ext-link ext-link-type="uri" xlink:href="http://genome.lbl.gov/vista/">genome.lbl.gov/vista/</ext-link>) alignment of <italic>M. oculata Mesp</italic> (<italic>Moocul.Mesp</italic>) locus to orthologs in <italic>M. occulta, M. occidentalis,</italic> and <italic>C. intestinalis</italic>. Shaded peaks indicate sequence conservation above 70% over 100-bp windows (blue = protein-coding, pink = non-coding). Arrows indicate direction of transcription of protein-coding genes. Non-coding sequences upstream of <italic>Mesp</italic> are only conserved between <italic>M. oculata</italic> and <italic>M. occulta. M. occidentalis</italic> and <italic>C. intestinalis</italic> show considerable divergence even in protein-coding sequences. Note that microsynteny with <italic>SLC5A-related</italic> gene supports the orthology of these sequences among the Molgulids. (<bold>C</bold>) In situ hybridization (ISH) for <italic>Moocci.Mesp</italic> in 110-cell stage embryo (vegetal view), showing mRNA detection (green) in B7.5 blastomeres. Nuclei were counterstained with DAPI (blue). Staging is given by hours post-fertilization (hpf). (<bold>D</bold>) Vegetal view of a 110-cell stage embryo electoporated with <italic>Moocci.Mesp>GFP</italic> reporter construct. Reporter gene expression was detected by ISH for <italic>GFP</italic> transcripts (green). Nuclei were stained with DAPI (blue). (<bold>E</bold>) Lateral view of a mid-tailbud stage embryo electroporated with <italic>Moocci.Mesp>Histone2B::GFP</italic> reporter construct. GFP fluorescence reveals B7.5 descendants on left side of embryo: two trunk ventral cells (TVCs) and two anterior tail muscles (ATMs). (<bold>F</bold>) Diagram of B7.5 lineage divisions from 110-cell stage to mid-tailbud stage, inferred from previous <italic>C. intestinalis</italic> studies. Cells are named according to Conklin's method (<xref ref-type="bibr" rid="bib18">Conklin, 1905</xref>). The lineage is bilaterally symmetric, but only cells on the left side are indicated and named. Relative staging given for <italic>M. occidentalis (Mo.occi)</italic> and <italic>C. intestinalis (Ci.inte)</italic>. 110-cell and late gastrula: vegetal view. Initial tailbud: dorsal view. Mid-tailbud: lateral view. Anterior pole is on the left in all images and illustrations.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03728.003">http://dx.doi.org/10.7554/eLife.03728.003</ext-link></p></caption><graphic xlink:href="elife03728f001"/></fig><fig id="fig1s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03728.004</object-id><label>Figure 1—figure supplement 1.</label><caption><title>Alignment of 5′ flanking sequences from <italic>Mesp</italic> orthologs.</title><p>Top: diagram of mVISTA (<xref ref-type="bibr" rid="bib28">Frazer et al., 2004</xref>; <ext-link ext-link-type="uri" xlink:href="http://genome.lbl.gov/vista/">genome.lbl.gov/vista/</ext-link>) alignment of <italic>M. occidentalis Mesp</italic> (<italic>Moocci.Mesp</italic>) locus to orthologs in (from top to bottom) <italic>M. oculata, M. occulta,</italic> and <italic>C. intestinalis.</italic> Bottom: diagram of mVISTA alignment of <italic>C. intestinalis Mesp</italic> (<italic>Moocci.Mesp</italic>) locus to orthologs in (from top to bottom) <italic>M. oculata, M. occulta,</italic> and <italic>M. occidentalis.</italic> Blue-shaded peaks indicate protein-coding sequence conservation above 70% in 100-bp windows. Arrows indicate direction of transcription of protein-coding genes <italic>Slc5a-related</italic> and <italic>Mesp</italic>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03728.004">http://dx.doi.org/10.7554/eLife.03728.004</ext-link></p></caption><graphic xlink:href="elife03728fs001"/></fig><fig id="fig1s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03728.005</object-id><label>Figure 1—figure supplement 2.</label><caption><title>The B7.5 lineage of the tail-less species <italic>M. occulta.</italic></title><p>(<bold>A</bold>) In situ hybridization for <italic>M. occulta Mesp (Mooccu.Mesp)</italic> in 110-cell st/early gastrula embryo, showing expression (green) in the B7.5 blastomeres. Image is taken from a vegetal view, anterior to the top. (<bold>B</bold>) Lateral view of a transiently-transfected <italic>M. occulta</italic> mid-tailbud-equivalent stage embryo electroporated with a <italic>Moocul.Hand-r</italic> reporter construct (<italic>Moocul.Hand-r>H2B::GFP)</italic>. The reporter proteins (Histone2B::GFP, green) label the cell nuclei descendant of the trunk ventral cells (TVCs), namely the secondary TVCs (STVCs) and the first heart precursors (FHPs). Due to leader/trailer mosaic retention of the electroporated plasmid (<xref ref-type="bibr" rid="bib90">Stolfi and Christiaen, 2012</xref>), the daughters of the leader cell (‘leader STVC’ and ‘leader HFP’) are more strongly labeled than the daughters of the trailer cell. Some staining is also seen in what may be anterior endoderm or A7.6 mesoderm, other known territories of <italic>Hand-r</italic> expression in <italic>M. occulta</italic> and other ascidian species. Anterior is to the bottom left.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03728.005">http://dx.doi.org/10.7554/eLife.03728.005</ext-link></p></caption><graphic xlink:href="elife03728fs002"/></fig><fig id="fig1s3" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03728.006</object-id><label>Figure 1—figure supplement 3.</label><caption><title><italic>Moocci.Mesp</italic> in situ hybridization at tailbud stage.</title><p>In situ hybridization for <italic>Moocci.Mesp</italic> in initial/early tailbud embryo, showing no expression at this stage. Image is a coronal view with anterior to the left.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03728.006">http://dx.doi.org/10.7554/eLife.03728.006</ext-link></p></caption><graphic xlink:href="elife03728fs003"/></fig></fig-group></p><p>In order to realize the full potential of Molgulid ascidians as model organisms for molecular and comparative developmental genetics on par with <italic>Ciona spp.,</italic> we sequenced and assembled the genomes of three <italic>Molgula</italic> species: the tailed species <italic>M. occidentalis</italic> and <italic>M. oculata,</italic> and the tail-less <italic>M. occulta.</italic> Moreover, we adapted the electroporation protocol to <italic>M. occidentalis</italic> embryos. To illustrate the power of comparative approaches between <italic>Molgula</italic> and <italic>Ciona</italic>, we characterized the B7.5 lineage in <italic>M. occidentalis</italic>. In both <italic>C. intestinalis</italic> and <italic>H. roretzi,</italic> this lineage is descended from the B7.5 pair of blastomeres of the early gastrula embryo and gives rise to the anterior tail muscles of the larva, and to the heart and atrial siphon/pharyngeal muscles of the adult (<xref ref-type="bibr" rid="bib36">Hirano and Nishida, 1997</xref>; <xref ref-type="bibr" rid="bib23">Davidson and Levine, 2003</xref>; <xref ref-type="bibr" rid="bib91">Stolfi et al., 2010</xref>). A growing body of research has focused on the molecular basis of B7.5 lineage development in <italic>C. intestinalis</italic> (reviewed in <xref ref-type="bibr" rid="bib105">Tolkin and Christiaen, 2012</xref>; <xref ref-type="bibr" rid="bib20">Cota et al., 2013</xref>), some of which have guided the discovery of vertebrate heart developmental mechanisms (<xref ref-type="bibr" rid="bib43">Islas et al., 2012</xref>). Given its detailed characterization in <italic>C. intestinalis</italic>, we chose the B7.5 lineage for an in-depth comparative analysis. Our analysis revealed a remarkable degree of conservation of the clonal topology and gene expression patterns (together referred to as the ‘ontogenetic motif’) underlying ascidian cardiopharyngeal mesoderm development (<xref ref-type="bibr" rid="bib111">Wang et al., 2013</xref>), but also uncovered divergent regulatory mechanisms underlying conserved gene expression profiles between <italic>Ciona</italic> and <italic>Molgula</italic>.</p></sec><sec id="s2" sec-type="results"><title>Results</title><sec id="s2-1"><title>De novo sequencing of <italic>Molgula spp.</italic> genomes</title><p>Genomes of three <italic>Molgula</italic> species (<italic>M. occidentalis, M. oculata,</italic> and <italic>M. occulta</italic>) were sequenced using next-generation sequencing technology and assembled. A common metric for judging the quality of a genome assembly is the contig N50 length, which is determined such that 50% of the assembly is contained in contigs of this length or greater. We used the contig N50 length to select the best assembly for each species given the varying ‘k’ parameter (length of k-mer overlap). A ‘k’ of 39 yields the best assembly for both <italic>M. occidentalis</italic> and <italic>M. occulta</italic>. The best ‘k’ for <italic>M. oculata</italic> was 61. <italic>M. occidentalis</italic>, <italic>M. occulta</italic>, and <italic>M. oculata</italic> N50 lengths were approximately 26.3 kb, 13 kb, and 34 kb, respectively (<xref ref-type="table" rid="tbl1">Table 1</xref>).<table-wrap id="tbl1" position="float"><object-id pub-id-type="doi">10.7554/eLife.03728.007</object-id><label>Table 1.</label><caption><p>Genome assembly statistics</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03728.007">http://dx.doi.org/10.7554/eLife.03728.007</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th>Species</th><th>N50</th><th>Mean contig length</th><th>Total</th><th>Total number of base pairs</th><th>CEGMA C<sup>1</sup></th><th>CEGMA P<sup>2</sup></th></tr></thead><tbody><tr><td><italic>M. occidentalis</italic></td><td>26,298</td><td>5072</td><td>51,761</td><td>262,547,660</td><td>81.45</td><td>96.77</td></tr><tr><td><italic>M. occulta</italic></td><td>13,011</td><td>3233</td><td>58,489</td><td>189,110,562</td><td>77.42</td><td>98.79</td></tr><tr><td><italic>M. oculata</italic></td><td>34,042</td><td>6270</td><td>25,497</td><td>159,886,716</td><td>89.92</td><td>99.19</td></tr></tbody></table><table-wrap-foot><fn><p>The contig N50 length, mean contig length, total number of contigs, total number of base pairs and CEGMA scores were collected for each draft assembly. The CEGMA scores is a metric of completeness measured against highly Conserved eukaryotic genes. Alignments of 70% or greater of the protein length are called complete (C<sup>1</sup>) and all other statistically significant alignments are called partial (P<sup>2</sup>).</p></fn></table-wrap-foot></table-wrap></p><p>In addition to N50 lengths, we also used CEGMA (Core Eukaryotic Genes Mapping Approach) scores, in order to evaluate the assemblies' representative completeness (<xref ref-type="bibr" rid="bib74">Parra et al., 2007</xref>). CEMGA reports scores for complete and partial alignments to a subset of core eukaryotic genes. An alignment is considered ‘complete’ if at least 70% of a given protein model aligns to a contig in the assembly, while a partial alignment indicates that a statistically significant portion of the protein model aligns. The partial alignment scores are ∼97% or higher for all assemblies. <italic>M. oculata</italic> has the best complete alignment score at ∼90%. <italic>M. occidentalis</italic> and <italic>M. occulta</italic> have complete alignment scores of 81% and 77% respectively (<xref ref-type="table" rid="tbl1">Table 1</xref>). These scores indicate that our assemblies contain at least partial sequences for the vast majority of protein-coding genes in the genomes of these species.</p><p>Various factors make it unreliable to predict genome size and gene density based on assembly metrics alone (<xref ref-type="bibr" rid="bib8">Bradnam et al., 2013</xref>). Of the handful of sequences we isolated and analyzed, we found that the sizes of introns and upstream regulatory regions were roughly comparable to those from their <italic>Ciona</italic> orthologs. This suggests that the <italic>Molgula</italic> genomes may be as compact as the <italic>C. intestinalis</italic> genome (i.e., ∼150–170 Mb, ∼16,000 genes, <xref ref-type="bibr" rid="bib53">Laird, 1971</xref>; <xref ref-type="bibr" rid="bib89">Simmen et al., 1998</xref>; <xref ref-type="bibr" rid="bib84">Satou et al., 2008</xref>).</p><p>Our sequencing efforts revealed extreme genetic divergence not only between <italic>Ciona</italic> and <italic>Molgula,</italic> as expected, but even within the Molgulids. For example, we used BLAST to identify the <italic>Molgula</italic> orthologs of <italic>C. intestinalis Mesp (Ciinte.Mesp,</italic> as per the proposed tunicate gene nomenclature rules, see <xref ref-type="bibr" rid="bib93">Stolfi et al., 2014</xref>)<italic>. Ciinte.Mesp</italic> is the sole ortholog of vertebrate genes coding for MesP and Mesogenin bHLH transcription factor family members (<xref ref-type="bibr" rid="bib83">Satou et al., 2004</xref>)<italic>.</italic> VISTA alignment shows high sequence similarity between sequences 5′ upstream of the <italic>Mesp</italic> genes from the closely related <italic>M. oculata</italic> and <italic>M. occulta</italic> (<xref ref-type="fig" rid="fig1">Figure 1B</xref>)<italic>.</italic> However, there is no conservation of <italic>Mesp</italic> DNA sequences, coding or non-coding, between <italic>M. oculata/occulta</italic> and <italic>M. occidentalis</italic>, nor between <italic>C. intestinalis</italic> and any of the three <italic>Molgula</italic> species (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>). In previous phylogenetic surveys, <italic>M. occidentalis</italic> has been placed as an early-branching <italic>Molgula</italic> species, often grouped together in a subfamily with species ascribed to the genera <italic>Eugyra</italic> and <italic>Bostrichobranchus</italic> instead (<xref ref-type="bibr" rid="bib32">Hadfield et al., 1995</xref>; <xref ref-type="bibr" rid="bib38">Huber et al., 2000</xref>; <xref ref-type="bibr" rid="bib107">Tsagkogeorga et al., 2009</xref>). Our sequencing results support the view that <italic>M. occidentalis</italic> is highly diverged from other <italic>Molgula</italic> spp.</p></sec><sec id="s2-2"><title>Expression of the <italic>M. occidentalis Mesp</italic> gene marks the B7.5 cells</title><p><italic>Ciinte.Mesp</italic> specifies the B7.5 cells as the sole progenitors of the cardiopharyngeal lineage (<xref ref-type="bibr" rid="bib83">Satou et al., 2004</xref>; <xref ref-type="bibr" rid="bib25">Davidson et al., 2005</xref>; <xref ref-type="bibr" rid="bib36">Hirano and Nishida, 1997</xref>; <xref ref-type="bibr" rid="bib91">Stolfi et al., 2010</xref>). We performed RNA in situ hybridization (ISH) for <italic>M. occidentalis Mesp (Moocci.Mesp)</italic> and found that this gene is also expressed only in the B7.5 cells of <italic>M. occidentalis</italic> embryos (<xref ref-type="fig" rid="fig1">Figure 1C</xref>). These cells are unequivocally identified due to the perfect conservation of early embryonic cell cleavage patterns in all ascidians. ISH for the <italic>M. occulta Mesp</italic> gene <italic>(Mooccu.Mesp)</italic> also revealed conserved expression in this tail-less species (<xref ref-type="fig" rid="fig1s2">Figure 1—figure supplement 2A</xref>).</p><p>We successfully adapted the <italic>Ciona</italic> electroporation protocol for simultaneous transfection of reporter gene plasmids into hundreds of synchronized <italic>M. occidentalis</italic> embryos (<xref ref-type="fig" rid="fig1">Figure 1D,E</xref>). We were also able to electroporate <italic>M. occulta</italic> embryos (<xref ref-type="fig" rid="fig1s2">Figure 1—figure supplement 2B</xref>). However, only <italic>M. occidentalis</italic> was routinely available to us for in vivo studies, so we focused our experiments on this species. Development of <italic>M. occidentalis</italic> embryos was optimal at 24°C and faster than that of <italic>C. intestinalis</italic> (<xref ref-type="fig" rid="fig1">Figure 1A</xref>). Using electroporation-based transfection, we determined that an ∼1.1 kb genomic DNA fragment upstream of <italic>Moocci.Mesp</italic> is able to drive expression of fused reporter genes specifically in the B7.5 cells with no ‘leaky’ expression in other cells as is commonly observed in <italic>C. intestinalis</italic> (<xref ref-type="fig" rid="fig1">Figure 1D</xref>; <xref ref-type="bibr" rid="bib90">Stolfi and Christiaen, 2012</xref>).</p><p>This faithful recapitulation of <italic>Moocci.Mesp</italic> expression and the persistence of GFP allows for visualization of the descendants of B7.5 long after endogenous <italic>Moocci.Mesp</italic> transcription has ceased (<xref ref-type="fig" rid="fig1s3">Figure 1—figure supplement 3</xref>; <xref ref-type="bibr" rid="bib25">Davidson et al., 2005</xref>). At the tailbud stage, we find that each B7.5 blastomere gives rise to four grand-daughter cells (<xref ref-type="fig" rid="fig1">Figure 1E</xref>). The two anterior B7.5 grand-daughter cells on either side of the bilaterally symmetric embryo migrate anteriorly and are termed the trunk ventral cells (TVCs) due to their final position in the <italic>C. intestinalis</italic> and <italic>H. roretzi</italic> embryos (<xref ref-type="bibr" rid="bib69">Nishida, 1987</xref>). Their posterior sister cells remain in the tail and become anterior tail muscles (ATMs). As far as we can tell, B7.5 lineage ontogeny is perfectly conserved between <italic>M. occidentalis</italic> and <italic>C. intestinalis</italic> (<xref ref-type="fig" rid="fig1">Figure 1F</xref>).</p></sec><sec id="s2-3"><title>Cardiopharyngeal mesoderm gene expression</title><p>Our focus shifted to the TVCs, which in <italic>H. roretzi</italic> and <italic>C. intestinalis</italic> have been shown to be multipotent cardiopharyngeal progenitor cells that give rise to the heart and pharyngeal muscles of the atrial siphon (<xref ref-type="bibr" rid="bib36">Hirano and Nishida, 1997</xref>; <xref ref-type="bibr" rid="bib91">Stolfi et al., 2010</xref>). We performed a small ISH screen for orthologs of transcription factors that are expressed in <italic>C. intestinalis</italic> TVCs (<xref ref-type="bibr" rid="bib83">Satou et al., 2004</xref>; <xref ref-type="bibr" rid="bib25">Davidson et al., 2005</xref>; <xref ref-type="bibr" rid="bib23">Davidson and Levine, 2003</xref>; <xref ref-type="bibr" rid="bib12">Christiaen et al., 2008</xref>; <xref ref-type="bibr" rid="bib6">Beh et al., 2007</xref>)<italic>.</italic> These include the orthologs of conserved cardiac regulators <italic>Ets1/2, GATA4/5/6,</italic> and <italic>NK4/Nkx2.5/tinman</italic> (<xref ref-type="bibr" rid="bib21">Cripps and Olson, 2002</xref>) (<xref ref-type="fig" rid="fig2">Figure 2</xref>).<fig-group><fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.03728.008</object-id><label>Figure 2.</label><caption><title>Expression of conserved TVC/heart markers in <italic>M. occidentalis</italic> embryos.</title><p>In situ hybridization (ISH) in <italic>M. occidentalis</italic> embryos for (<bold>A</bold> and <bold>A′</bold>) <italic>Moocci.Ets.b</italic>, (<bold>B</bold> and <bold>B′</bold>) <italic>Moocci.Foxf,</italic> (<bold>C</bold> and <bold>C′</bold>) <italic>Moocci.Hand-related (Moocci.Hand-r)</italic>, (<bold>D</bold> and <bold>D′</bold>) <italic>Moocci.Gata4/5/6,</italic> (<bold>E</bold> and <bold>E′</bold>) <italic>Moocci.Nk4,</italic> (<bold>F</bold> and <bold>F′</bold>) <italic>Moocci.Rhod/f</italic>. ISH was performed on initial tailbud (<bold>A</bold>–<bold>F</bold>) and mid tailbud (<bold>A′</bold>–<bold>F′</bold>) stage embryos. Solid arrowheads indicate definitive expression in TVCs. Dotted arrowheads indicate potential expression of <italic>Moocci.Foxf</italic> in initial tailbud, obscured by strong epidermal expression. Dotted outline indicates probable position of TVCs, not visible due to lack of mRNA hybridization signal. Initial tailbud embryos were imaged ventrally or dorsally, while tailbud embryos were imaged laterally.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03728.008">http://dx.doi.org/10.7554/eLife.03728.008</ext-link></p></caption><graphic xlink:href="elife03728f002"/></fig><fig id="fig2s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03728.009</object-id><label>Figure 2—figure supplement 1.</label><caption><title><italic>Ets.b</italic> expression in B7.5 of <italic>M. occidentalis</italic>.</title><p>In situ hybridization (ISH) for <italic>Moocci.Ets.b</italic> in 110-cell st/early gastrula embryo, showing expression (green) in the B7.5 blastomeres. Nuclei are counterstained with DAPI (blue). Embryo was imaged from a vegetal view, with anterior to the top.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03728.009">http://dx.doi.org/10.7554/eLife.03728.009</ext-link></p></caption><graphic xlink:href="elife03728fs004"/></fig><fig id="fig2s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03728.010</object-id><label>Figure 2—figure supplement 2.</label><caption><title>In situ hybridizations reveal TVC gene expression in <italic>M. occidentalis</italic>.</title><p>Mid-tailbud <italic>M. occidentalis</italic> embryos electroporated with <italic>Moocci.Mesp>nls::lacZ,</italic> assayed with β-galactosidase immunodetection (IHC, green) couple to in situ hybridization (ISH, red) for (<bold>A</bold>) <italic>Moocci.Foxf,</italic> (<bold>B</bold>) <italic>Moocci.Hand-r,</italic> (<bold>C</bold>) <italic>Moocci.Gata4/5/6,</italic> and (<bold>D</bold>) <italic>Moocci.Nk4</italic>. Expression of these genes was detected in the TVCs, with the possible exception of <italic>Nk4</italic>. All embryos are oriented with anterior to the left.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03728.010">http://dx.doi.org/10.7554/eLife.03728.010</ext-link></p></caption><graphic xlink:href="elife03728fs005"/></fig><fig id="fig2s3" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03728.011</object-id><label>Figure 2—figure supplement 3.</label><caption><title><italic>Hand-r</italic> and <italic>FoxF</italic> co-expression reveals TVCs of <italic>M. occulta</italic>.</title><p>Coronal view of <italic>M. occulta</italic> embryos at (<bold>A</bold>–<bold>C</bold>) 6.5 hpf (equivalent to the initial tailbud stage of tailed <italic>M. oculata</italic>) and (<bold>D</bold>–<bold>I</bold>) 7.5 hpf (equivalent to the mid-tailbud stage). Embryos were assayed for expression of <italic>Mooccu.Hand-r</italic> (green—<bold>A</bold>, <bold>D</bold>, <bold>G</bold>) and <italic>Mooccu.Foxf</italic> (red—<bold>B</bold>, <bold>E</bold>) or <italic>Mooccu.Aldh1a</italic> (red—<bold>H</bold>) by double in situ hybridization. Merged green and red channels show co-expression in TVCs (<bold>C</bold>, <bold>F</bold>, <bold>I</bold>). <italic>Mooccu.Foxf</italic> expression in epidermis seen in <bold>F</bold> suggests position of TVCs in ventro-lateral regions of the trunk similar to what is observed in <italic>M. occidentalis</italic> and <italic>C. intestinalis</italic>. <italic>Mooccu.Aldh1a</italic>-expressing cells immediately posterior to TVCs are likely the anterior tail muscles. Anterior is to the left in all panels.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03728.011">http://dx.doi.org/10.7554/eLife.03728.011</ext-link></p></caption><graphic xlink:href="elife03728fs006"/></fig><fig id="fig2s4" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03728.012</object-id><label>Figure 2—figure supplement 4.</label><caption><title><italic>Aldh1a</italic> expression in <italic>M. occidentalis</italic> embryo.</title><p>In situ hybridization for <italic>Moocci.Aldh1a</italic> showing expression in TVCs and ATMs in a mid-tailbud embryo.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03728.012">http://dx.doi.org/10.7554/eLife.03728.012</ext-link></p></caption><graphic xlink:href="elife03728fs007"/></fig></fig-group></p><p>In <italic>M. occidentalis, Ets.b</italic> expression is initiated in B7.5 blastomeres (<xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1</xref>), is maintained in their daughter cells the cardiopharyngeal founders and in the TVCs during their migration (<xref ref-type="fig" rid="fig2">Figure 2A</xref>). This profile is similar to the expression of <italic>C. intestinalis Ets.b (Ciinte.Ets.b)</italic>, previously named <italic>Ets/pointed2</italic> or <italic>Ets1/2</italic> (see <xref ref-type="supplementary-material" rid="SD2-data">Supplementary file 2</xref> for list of old and new gene name correspondences). In <italic>C. intestinalis,</italic> Ets.b mediates the FGF/MAPK-dependent induction of TVCs in part by the activation of key regulators such as <italic>Foxf, Hand-related (Hand-r,</italic> also known as <italic>Hand-like</italic> or <italic>NoTrlc),</italic> and <italic>Gata4/5/6</italic> (also known as <italic>GATA-a)</italic> prior to the onset of TVC migration (<xref ref-type="bibr" rid="bib24">Davidson et al., 2006</xref>; <xref ref-type="bibr" rid="bib6">Beh et al., 2007</xref>). In <italic>M. occidentalis,</italic> orthologs of <italic>Foxf</italic> and <italic>Hand-r</italic> are also activated in the TVCs shortly before and throughout their migration away from the ATMs (<xref ref-type="fig" rid="fig2">Figure 2B,C</xref>). <italic>Moocci.Gata4/5/6</italic> expression was detected in migrating TVCs but not before migration (<xref ref-type="fig" rid="fig2">Figure 2D</xref>). This is slightly different from <italic>Ciinte.Gata4/5/6,</italic> which is expressed in <italic>C. intestinalis</italic> TVCs prior to migration (<xref ref-type="bibr" rid="bib14">Christiaen et al., 2010</xref>; <xref ref-type="bibr" rid="bib77">Ragkousi et al., 2011</xref>). Expression of <italic>Moocci.Foxf</italic> and <italic>Moocci.Gata4/5/6</italic> in surrounding epidermis and endoderm, respectively, is identical to the expression domains of their orthologs in <italic>C. intestinalis</italic> and sometimes obscured TVC expression. However, double ISH/immunohistochemical detection (ISH/IHC) of <italic>Moocci.Mesp</italic> promoter-driven reporter gene clearly shows transcripts in the migrating B7.5-derived TVCs (<xref ref-type="fig" rid="fig2s2">Figure 2—figure supplement 2A–C</xref>).</p><p><italic>Foxf</italic> and <italic>Hand-r</italic> are also expressed in the TVCs of tail-less <italic>M. occulta</italic> embryos. Because these embryos do not extend a tail and lack functional larval tail muscles, it is unclear whether their TVCs move away from the posterior pole of the embryo (<xref ref-type="fig" rid="fig2s3">Figure 2—figure supplement 3A–F</xref>). However, their TVCs occupy the ventro-lateral regions of the trunk (<xref ref-type="fig" rid="fig1s2">Figure 1—figure supplement 2B</xref>), similar to what is observed in <italic>M. occidentalis,</italic> suggesting they might be migrating. We cannot draw any further conclusions about the TVCs of <italic>M. occulta,</italic> but have not found any evidence for fundamental molecular or morphological differences relative to <italic>M. occidentalis.</italic></p><p>Notably, the ATMs appear to be specified in <italic>M. occulta,</italic> as revealed by expression of <italic>Aldh1a</italic> (also known as <italic>Raldh2,</italic> <xref ref-type="fig" rid="fig2s3">Figure 2—figure supplement 3G–I</xref>)<italic>,</italic> which encodes the rate-limiting enzyme for retinoic acid (RA) synthesis<italic>.</italic> Therefore, although the larval muscles of <italic>M. occulta</italic> do not differentiate (<xref ref-type="bibr" rid="bib97">Swalla and Jeffery, 1990</xref>; <xref ref-type="bibr" rid="bib52">Kusakabe et al., 1996</xref>)<italic>,</italic> their ATMs may still be required for RA-mediated embryonic patterning like in <italic>C. intestinalis</italic> (<xref ref-type="bibr" rid="bib68">Nagatomo and Fujiwara, 2003</xref>) and probably also in <italic>M. occidentalis</italic> (<xref ref-type="fig" rid="fig2s4">Figure 2—figure supplement 4</xref>).</p><p>Unexpectedly, we could not detect unequivocal expression of the sole <italic>M. occidentalis</italic> ortholog of <italic>NK4/Nkx2.5/tinman (Moocci.Nk4)</italic> in the TVCs (<xref ref-type="fig" rid="fig2">Figure 2E</xref>). Expression in the ventral ectoderm mirrored that seen in <italic>C. intestinalis</italic>, indicating that the probe used was functional. It is possible that expression of <italic>Nk4</italic> in the TVCs of <italic>M. occidentalis</italic> is below the threshold of reliable detection by ISH. Our ISH data are not entirely incompatible with <italic>Moocci.Nk4</italic> being expressed in the TVCs at very low levels (<xref ref-type="fig" rid="fig2s2">Figure 2—figure supplement 2D</xref>).</p><p>Conserved TVC gene expression patterns were not limited to TF-coding genes. The small GTPase-coding <italic>Rhod/f</italic> gene was found to be transcriptionally upregulated in <italic>M. occidentalis</italic> TVCs just prior to migration (<xref ref-type="fig" rid="fig2">Figure 2F</xref>). In <italic>C. intestinalis,</italic> Rhod/f was identified as an effector of cytoskeleton dynamics in TVC migration and a direct transcriptional target of Foxf and activated Ets.b (<xref ref-type="bibr" rid="bib12">Christiaen et al., 2008</xref>). This suggests that the conservation of B7.5 development in ascidians extends to the interface between transcriptional regulators and cellular effectors of cell migration.</p></sec><sec id="s2-4"><title>Specification of heart and pharyngeal muscles from common progenitors</title><p>In <italic>C. intestinalis,</italic> heart and atrial siphon (pharyngeal) muscle precursors are derived from TVCs in a two-step process involving asymmetric cell divisions (<xref ref-type="bibr" rid="bib91">Stolfi et al., 2010</xref>; <xref ref-type="bibr" rid="bib111">Wang et al., 2013</xref>). First, each TVC undergoes an asymmetric cell division along the medial/lateral (M/L) axis to produce a medial First Heart Precursor (FHP) and a lateral Secondary TVC (STVC). Each STVC then also undergoes a M/L asymmetric division to give rise to a medial Second Heart Precursor (SHP) and a lateral atrial siphon muscle founder cell (ASMF). In <italic>C. intestinalis,</italic> this step-wise segregation of heart vs pharyngeal muscle fate is coordinated by (1) activation of <italic>Tbx1/10</italic> in the STVCs (<xref ref-type="bibr" rid="bib111">Wang et al., 2013</xref>) and (2) Tbx1/10-dependent activation of <italic>Collier/Olf/EBF (Ciinte.Ebf,</italic> previously named <italic>COE</italic>) in the ASMFs (<xref ref-type="bibr" rid="bib91">Stolfi et al., 2010</xref>; <xref ref-type="bibr" rid="bib111">Wang et al., 2013</xref>; <xref ref-type="bibr" rid="bib78">Razy-Krajka et al., 2014</xref>). The shared clonality of heart and pharyngeal muscles and similarity of associated TF expression patterns point to a single evolutionary origin for this cardiopharyngeal ‘ontogenetic motif’ in the last common ancestor of tunicates and vertebrates (<xref ref-type="bibr" rid="bib111">Wang et al., 2013</xref>).</p><p>In <italic>M. occidentalis,</italic> we found that the segregation of heart and pharyngeal muscle fates occurs in identical fashion to that of <italic>C. intestinalis,</italic> albeit on a faster timescale<italic>.</italic> At 6 hr post-fertilization (hpf) the first asymmetric division of the TVCs begins, with each TVC giving rise to a smaller, more ventral/medial cell and a larger, more dorsal/lateral cell. At 7.5 hpf, the resulting STVCs also undergo an asymmetric division along the same axis (<xref ref-type="fig" rid="fig3">Figure 3A</xref>). The end result is a cluster consisting of two larger cells (presumptive ASMFs) lateral to four smaller cells (presumptive heart precursors), on either side of the embryo (<xref ref-type="fig" rid="fig3">Figure 3B</xref>).<fig-group><fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.03728.013</object-id><label>Figure 3.</label><caption><title>Specification of heart vs atrial siphon muscle precursors in <italic>M. occidentalis</italic>.</title><p>(<bold>A</bold>) First and second asymmetric division of TVCs in <italic>M. occidentalis</italic> embryos. B7.5 lineage was visualized by electroporation of <italic>Moocci.Mesp>H2B::GFP</italic> (green). The first division occurs at ∼6 hpf at 24°C, and the second division occurs at ∼7.5 hpf at 24°C. (<bold>B</bold>) Result of two asymmetric divisions of TVCs on left side of embryo electroporated with <italic>Moocci.Mesp>H2B::GFP</italic> (green). At 8 hpf at 24°C, a cluster of 6 cells derived from the TVCs is located in the ventro-lateral region of the trunk. From top to bottom: 2 atrial siphon muscle founder cells (ASMFs), 2 second heart precursors (SHPs), and 2 first heart precursors (FHPs). (<bold>C</bold>) In situ hybridization (ISH, red) for <italic>Moocci.Tbx1/10</italic> + β-galactosidase immunodetection (IHC, green) in embryos electoporated with <italic>Moocci.Mesp>nls::lacZ</italic>. <italic>Moocci.Tbx1/10</italic> nascent transcripts are detected as two dots in the nuclei of Secondary TVCs (STVCs), between the first and second asymmetric divisions. (<bold>D</bold>) ISH + IHC for <italic>Moocci.Ebf</italic> (red) in embryos electoporated with <italic>Moocci.Mesp>nls::lacZ</italic> (green), revealing <italic>Moocci.EBF</italic> expression in ASMFs after the second asymmetric division. (<bold>E</bold>) ISH for <italic>Moocci.Ebf</italic> in a swimming larva, viewed dorsally, revealing <italic>Moocci.Ebf+</italic> migrating atrial siphon muscle precursors (ASMPs, arrowheads). (<bold>F</bold>) Lateral view of a <italic>M. occidentalis</italic> juvenile (>100 hpf) electroporated with <italic>Moocci.Mesp>H2B::GFP</italic>. GFP + nuclei reveal contributions of B7.5 lineage to atrial siphon muscle-derived pharyngeal muscles (PhMs) and heart. (<bold>G</bold>) Diagram of the TVC divisions giving rise to the pharyngeal muscles and heart of the adult. (<bold>H</bold>) Ventral view of a <italic>M. occidentalis</italic> embryo electroporated with <italic>Moocci.Mesp>H2B::GFP,</italic> just after the first asymmetric division of the TVCs. (<bold>H′</bold>) Inset from (<bold>H</bold>) is focused on the distance between FHPs on either side of the embryo (mean = 57.6 μm, n = 9). (<bold>I</bold>) Ventrolateral view of a <italic>C. intestinalis</italic> embryo electroporated with <italic>Ciinte.Mesp>H2B::GFP</italic>, right after the first asymmetric division. (<bold>I′</bold>) The distance between FHPs from either side is very small (mean = 7.5 μm, n = 14) since they contact each other at the midline to form a single cluster of cells. (<bold>J</bold>) Double ISH for <italic>Moocci.Dlx.a</italic> (green) and <italic>Moocci.Ebf</italic> (red) in a <italic>M. occidentalis</italic> larva, viewed dorsally. <italic>Moocci.Dlx.a</italic> marks the siphon primordia, while <italic>Moocci.Ebf</italic> marks siphon muscle precursors. Arrowheads point to atrial siphon muscle precursors (ASMPs), which have migrated dorsally but do not encounter an atrial siphon primordium around which to encircle. In contrast, oral siphon muscle precursors form a ring around the oral siphon primordium (OSP). Other <italic>Moocci.Ebf+</italic> cells seen are neurons or neuronal precursors in the central nervous system (cns). (<bold>K</bold>) Double ISH for <italic>Ciinte.Dlx.a</italic> (green) and <italic>Ciinte.Ebf</italic> (red) in a <italic>C. intestinalis</italic> larva viewed dorsolaterally. Arrowhead indicates <italic>Ciinte.Ebf+</italic> ASMPs on one side of the embryo encircling one of two bilaterally paired atrial siphon primordia (ASP). The same configuration of <italic>Ciinte.Ebf+</italic> muscle precursors is seen encircling the OSP.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03728.013">http://dx.doi.org/10.7554/eLife.03728.013</ext-link></p></caption><graphic xlink:href="elife03728f003"/></fig><fig id="fig3s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03728.014</object-id><label>Figure 3—figure supplement 1.</label><caption><title>Mosaic transgene labeling of <italic>M. occidentalis</italic> suggests bilateral origin of juvenile heart.</title><p>(<bold>A</bold>) Dorso-lateral view of a metamorphosing juvenile (>100 hpf) developing from embryo electroporated with <italic>Moocci.Mesp>H2B::GFP.</italic> H2B::GFP labels nuclei of right side of and subset of atrial siphon/body-wall muscles. (<bold>B</bold>) Magnified view of boxed area in <bold>A</bold>. Mosaicism of H2B::GFP-labeling of juvenile tissues is interpreted as reflecting left/right mosaic uptake and/or retention of <italic>Moocci.Mesp>H2B::GFP</italic> plasmid during embryonic stages.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03728.014">http://dx.doi.org/10.7554/eLife.03728.014</ext-link></p></caption><graphic xlink:href="elife03728fs008"/></fig><fig id="fig3s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03728.015</object-id><label>Figure 3—figure supplement 2.</label><caption><title>Delayed atrial siphon primordium specification in <italic>M. occidentalis</italic>.</title><p>(<bold>A</bold>) Dorsal view of hatched larva (13 hpf) stained with Alexa Fluor 546 phalloidin, showing oral siphon primordium (OSP) as a distinct structure, while the atrial siphon primordium (ASP) has not formed yet. (<bold>B</bold>) Dorsal view of metamorphosing juvenile (>72 hpf) stained with Alexa Fluor 546 phalloidin showing fully formed ASP as a rosette of apically constricted cells. OSP is out of the plane of view. (<bold>C</bold>) Dorsal view of <italic>M. oculata</italic> hatched larva stained with Alexa Fluor 633 phalloidin, showing formation of both OSP and ASP at this stage.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03728.015">http://dx.doi.org/10.7554/eLife.03728.015</ext-link></p></caption><graphic xlink:href="elife03728fs009"/></fig><fig id="fig3s3" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03728.016</object-id><label>Figure 3—figure supplement 3.</label><caption><title>Cardiopharyngeal development in <italic>C. intestinalis</italic> vs. <italic>M. occidentalis.</italic></title><p>Schematic diagram comparing cardiopharyngeal development in <italic>C. intestinalis</italic> (top) and <italic>M. occidentalis</italic> (bottom) embryos. The B7.5 cell lineage, which is identical in the two species, is shown in the middle, and each progenitor type is color-coded to match the embryo diagrams. Dashed line in left-most drawings indicates embryonic midline. Although both FHPs and SHPs contribute to the juvenile heart, the relative contributions of their descendants have yet to be elucidated, in either <italic>Molgula</italic> or <italic>Ciona.</italic> Refer to text for other details.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03728.016">http://dx.doi.org/10.7554/eLife.03728.016</ext-link></p></caption><graphic xlink:href="elife03728fs010"/></fig></fig-group></p><p>Gene expression during heart vs pharyngeal muscle segregation is also conserved. Namely, <italic>Moocci.Tbx1/10</italic> is specifically activated in the STVCs immediately following the first asymmetric division (<xref ref-type="fig" rid="fig3">Figure 3C</xref>), while <italic>Moocci.Ebf</italic> is specifically activated in the ASMFs immediately following the second asymmetric division (<xref ref-type="fig" rid="fig3">Figure 3D</xref>). <italic>Moocci.Ebf</italic> expression is maintained in ASMFs and their daughter cells, the atrial siphon muscle precursors (ASMPs) during their migration to the dorsal regions of the now swimming larva (<xref ref-type="fig" rid="fig3">Figure 3E</xref>). This step-wise progression of gene expression, cell division, and migration are virtually identical to those observed in <italic>C. intestinalis</italic>, demonstrating deep evolutionary conservation of the cardiopharyngeal ontogenetic motif in ascidians.</p><p>We allowed embryos electroporated with <italic>Moocci.Mesp>H2B::GFP</italic> to settle and undergo metamorphosis from the larval to the juvenile stage. Visualization of H2B::GFP revealed contributions of the B7.5 lineage to the heart and pharyngeal muscles of juveniles (<xref ref-type="fig" rid="fig3">Figure 3F</xref>). Thus, the post-metamorphic tissues derived from B7.5 are identical to those previously reported for <italic>C. intestinalis</italic> and <italic>H. roretzi</italic> (<xref ref-type="bibr" rid="bib36">Hirano and Nishida, 1997</xref>; <xref ref-type="bibr" rid="bib91">Stolfi et al., 2010</xref>). A summary of the TVC divisions and their post-metamorphic fates is shown in <xref ref-type="fig" rid="fig3">Figure 3G</xref>. Based on the perfectly conserved parallels to <italic>C. intestinalis</italic>, we hypothesize that specification of ASM vs heart fate in <italic>Molgula</italic> occurs through a conserved Tbx1/10-Ebf-dependent regulatory cascade.</p><p>In summary, the cardiopharyngeal mesoderm of <italic>Molgula</italic> and <italic>Ciona</italic> display unequivocally homologous ontogenies, as revealed by nearly identical cell divisions, cell fate choices, and gene expression patterns. These data indicate that the key developmental features of the B7.5 lineage evolved at the base of the tunicate radiation.</p></sec><sec id="s2-5"><title>Species-specific differences in TVC migration and heart primordium formation</title><p>Despite perfect conservation of B7.5 lineage cell divisions and cell fates between <italic>M. occidentalis</italic> and <italic>C. intestinalis</italic>, we noticed a striking species-specific difference in TVC behavior (summarized in <xref ref-type="fig" rid="fig3">Figure 3</xref>, <xref ref-type="fig" rid="fig3s3">Figure 3—figure supplement 3</xref>). Namely, in <italic>M. occidentalis,</italic> the TVCs appear to migrate along a more lateral path than their counterparts in <italic>C. intestinalis.</italic> As a result, the TVCs on one side of the embryo do not contact those on the other side, resulting in two discrete heart precursor clusters during the tailbud stage (<xref ref-type="fig" rid="fig3">Figure 3H</xref>). In <italic>C. intestinalis</italic>, the TVCs converge towards the ventral midline prior to dividing, such that the resulting FHPs and SHPs from the two sides contact each other as soon as they are born, forming a single cluster of heart precursors (<xref ref-type="fig" rid="fig3">Figure 3I</xref>; <xref ref-type="bibr" rid="bib23">Davidson and Levine, 2003</xref>). However, observation of juveniles electroporated with <italic>Mocci.Mesp>H2B::GFP</italic> showed some individuals with hearts that were only half-labeled with H2B::GFP, likely due to left/right transgene mosaicism (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1</xref>). This indicates that heart precursors from both sides of the embryo ultimately contribute to a single heart, as they do in <italic>C. intestinalis.</italic> Therefore, we assume this naturally occurring <italic>cardia bifida</italic> is only a temporary configuration of a single embryonic heart primordium. This odd configuration has not been previously described in the embryos of other ascidians and may represent a <italic>Molgula-</italic>specific feature.</p></sec><sec id="s2-6"><title>Differences in ASMP behavior related to heterochrony of atrial siphon formation</title><p>We also noticed a difference in ASM precursor behavior. In <italic>C. intestinalis</italic>, migrating ASMPs from either side of the embryo migrate dorsally, forming rings around a bilateral pair of atrial siphon primordia (ASPs) on the corresponding side (<xref ref-type="bibr" rid="bib91">Stolfi et al., 2010</xref>). The ASPs are placode-like rosettes of molecularly and morphologically distinct ectodermal cells that arise through a retinoic acid/Hox1- and FGF/MAPK-dependent program and are proposed to be homologous to vertebrate otic placodes (<xref ref-type="bibr" rid="bib110">Wada et al., 1998</xref>; <xref ref-type="bibr" rid="bib66">Mazet et al., 2005</xref>; <xref ref-type="bibr" rid="bib51">Kourakis and Smith, 2007</xref>; <xref ref-type="bibr" rid="bib80">Sasakura et al., 2012</xref>). In juveniles of <italic>C. intestinalis</italic> and other phlebobranch ascidians, there are initially two atrial siphons, one on either side of the animal, that eventually fuse into a single one at the end of the ‘1<sup>st</sup> Ascidian’ stage (<xref ref-type="bibr" rid="bib11">Chiba et al., 2004</xref>; <xref ref-type="bibr" rid="bib50">Kourakis et al., 2010</xref>). In contrast, all stolidobranch ascidians initially specify a single ASP, bypassing the two-siphon fusion process (<xref ref-type="bibr" rid="bib64">Manni et al., 2004</xref>; <xref ref-type="bibr" rid="bib30">Grave, 1926</xref>; <xref ref-type="fig" rid="fig3s2">Figure 3—figure supplement 2A–C</xref>). Indeed, we observed the formation of a single ASP in <italic>M. occidentalis</italic> late in metamorphosis and in the larvae of <italic>M. oculata</italic> (<xref ref-type="fig" rid="fig3s2">Figure 3—figure supplement 2A,B</xref>). This flexibility in atrial siphon development in ascidians is an example of DSD at the morphogenetic level. It is believed that the dual primordium condition is ancestral and that the specification of a single primordium is a derived, Stolidobranchia-specific trait (<xref ref-type="bibr" rid="bib50">Kourakis et al., 2010</xref>).</p><p>We found that <italic>M. occidentalis</italic> ASMPs, marked by <italic>Ebf</italic> expression, migrate dorsally but remain as a cluster, unlike the characteristic rings of cells stretched around the invaginations of the ASPs, as observed in <italic>C. intestinalis</italic> (<xref ref-type="fig" rid="fig3">Figure 3J,K</xref>). On the other hand, oral siphon muscle precursors form a ring of <italic>Moocci.Ebf+</italic> cells around the presumptive oral siphon primordium (<xref ref-type="fig" rid="fig3">Figure 3J</xref>). ISH for the siphon primordium marker <italic>Dlx.a</italic> reveals that only the oral siphon primordium is specified at this stage in <italic>M. occidentalis</italic> larvae<italic>.</italic> In <italic>C. intestinalis,</italic> both oral and atrial siphon primordia expressing <italic>Dlx.a</italic> are specified by the larval stage and are encircled by <italic>Ebf+</italic> siphon muscle precursors (<xref ref-type="fig" rid="fig3">Figure 3K</xref>). Thus, we conclude that this difference in ASMP ring formation is related to the heterochrony of atrial siphon formation between <italic>M. occidentalis</italic> and <italic>C. intestinalis.</italic></p></sec><sec id="s2-7"><title>Divergence of <italic>Mesp cis</italic>-regulatory sequence function between <italic>M. occidentalis</italic> and <italic>C. intestinalis</italic></title><p>Given the obvious parallels between <italic>C. intestinalis</italic> and <italic>M. occidentalis</italic> cardiopharyngeal development, we expected transcriptional regulatory mechanisms to also be highly conserved between the two species. We tested this assumption by electroporating <italic>C. intestinalis</italic> reporter constructs into <italic>M. occidentalis</italic> embryos, and vice-versa. We observed that a <italic>Ciinte.Mesp</italic> reporter construct (<xref ref-type="bibr" rid="bib25">Davidson et al., 2005</xref>), when electroporated into <italic>M. occidentalis</italic> embryos, drives relatively weak reporter gene expression in B7.5 with substantial leaky expression in other tissues (<xref ref-type="fig" rid="fig4">Figure 4A</xref>, <xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1</xref>). Conversely, the <italic>Moocci.Mesp</italic> enhancer fails to drive any reporter gene expression when electroporated into <italic>C. intestinalis</italic> embryos (<xref ref-type="fig" rid="fig4">Figure 4B</xref>), despite recapitulating robust B7.5-specific expression in <italic>M. occidentalis</italic> embryos (<xref ref-type="fig" rid="fig1">Figure 1D,E</xref>).<fig-group><fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.03728.017</object-id><label>Figure 4.</label><caption><title>Developmental system drift of <italic>Mesp</italic> regulation between <italic>C. intestinalis</italic> and <italic>M. occidentalis</italic>.</title><p>(<bold>A</bold>) <italic>M. occidentalis</italic> tailbud embryo electroporated with a <italic>Ciinte.Mesp>H2B::mCherry</italic> reporter construct. Weak reporter gene expression was observed in the B7.5 lineage and occasionally in other territories including B-line mesenchyme and tail muscle cells, and A-line neural plate derivatives. (<bold>B</bold>) <italic>C. intestinalis</italic> tailbud embryo electroporated with <italic>Moocci.Mesp>H2B::GFP</italic> reporter. No fluorescence was seen in any cells, indicating complete lack of activity of <italic>Moocci.Mesp</italic> enhancer in wild-type <italic>C. intestinalis</italic> embryos. (<bold>C</bold>) In situ hybridization (ISH) for <italic>Moocci.Tbx6-r.a</italic>, (<bold>D</bold>) <italic>Moocci.Tbx6-r.b</italic>, (<bold>E</bold>) <italic>Moocci.Lhx3/4.a</italic>, and (<bold>F</bold>) <italic>Moocci.Lhx3/4.b</italic> in 110-cell stage embryos. (<bold>G</bold>) Double ISH in 110-cell stage embryo reveals co-expression of <italic>Moocci.Lhx3/4b</italic> (green) and <italic>Moocci.Tbx6-r.b</italic> (red) exactly in the B7.5 cells of <italic>M. occidentalis</italic>. (<bold>G′</bold>) Magnified view of inset in (<bold>G</bold>). (<bold>H</bold>) Double ISH for <italic>Moocci.Lhx3/4.a</italic> (green) and (<bold>I</bold>) <italic>Moocci.Lhx3/4.b</italic> (red) in a mid-tailbud embryo. <italic>Moocci.Lhx3/4.a</italic> but not <italic>Moocci.Lhx3/4.b</italic> is expressed in motor ganglion neurons (arrowhead).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03728.017">http://dx.doi.org/10.7554/eLife.03728.017</ext-link></p></caption><graphic xlink:href="elife03728f004"/></fig><fig id="fig4s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03728.018</object-id><label>Figure 4—figure supplement 1.</label><caption><title>Weak and leaky expression of <italic>Ciinte.Mesp</italic> reporter in <italic>M. occidentalis</italic> embryos.</title><p>In situ hybridization for mCherry mRNA (green) in a <italic>M. occidentalis</italic> early gastrula stage embryo electroporated with <italic>Ciinte.Mesp>mCherry.</italic> Nuclei counterstained with DAPI (blue). Embryo is viewed vegetally, anterior to the top. Expression in B7.5 blastomeres (solid arrowheads) was observed in 42% of embryos. Ectopic expression in other B-line cells (hollow arrowhead) was seen in 24% of embryos. Ectopic expression in A-line neural precursors (hollow double arrowhead) was seen in 8% of embryos. In contrast, in situ hybridization revealed that <italic>Moocci.Mesp</italic> reporter construct is expressed in the TVCs in 60% of electroporated <italic>M. occidentalis</italic> embryos, with 0% embryos showing any ectopic reporter gene expression (data not shown, see <xref ref-type="fig" rid="fig1">Figure 1D</xref> for example). n = 50 embryos for each construct.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03728.018">http://dx.doi.org/10.7554/eLife.03728.018</ext-link></p></caption><graphic xlink:href="elife03728fs011"/></fig><fig id="fig4s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03728.019</object-id><label>Figure 4—figure supplement 2.</label><caption><title>Configuration of Lhx3/4 protein domains and <italic>Tbx6-r</italic> locus in <italic>M. occidentalis</italic>.</title><p>(<bold>A</bold>) Schematic of <italic>Moocci.Lhx3/4.b</italic> and <italic>Moocci.Lhx3/4.a</italic> proteins. LIM domains (LD1 and LD2) are in yellow, and the homeodomain (HD) is in green. <italic>Moocci.Lhx3/4.a</italic> retains a more extensive C-terminus including a highly conserved motif (highlighted in red) of unknown function. Alignment to <italic>Lhx3/4</italic> orthologs from <italic>C. intestinalis</italic> (Ciinte) and humans (H.sapi.) is shown in inset. (<bold>B</bold>) Schematic representing the <italic>Tbx6-related</italic> locus in <italic>M. occidentalis,</italic> showing head-to-head configuration of <italic>Tbx6-r.a</italic> and <italic>Tbx6-r.b.</italic> Exons are represented by thick blocks. <italic>Tbx6-r.a</italic> is encoded by 6 exons, while <italic>Tbx6-r.b</italic> is encoded by only 2 exons. The region corresponding to the <italic>Moocci.Tbx6-r.b</italic> driver used in this study is shown in periwinkle.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03728.019">http://dx.doi.org/10.7554/eLife.03728.019</ext-link></p></caption><graphic xlink:href="elife03728fs012"/></fig><fig id="fig4s3" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03728.020</object-id><label>Figure 4—figure supplement 3.</label><caption><title>Divergent <italic>Molgula</italic> Lhx3/4.b homeodomains are not predicted to have altered DNA binding specificities.</title><p>(<bold>A</bold>) Alignment of homeodomains (HDs) from a set of <italic>Molgula</italic> Lhx3/4 family proteins, with HD recognition positions highlighted in yellow. HD recognition positions are invariant while intervening sequence is highly diverged between Lhx3/4.a and Lhx3/4.b. (<bold>B</bold>) Logos and matrices for predicted homeodmain specificities for Moocci.Lhx3/4.a (top) and Moocci.Lhx3/4.b (bottom), generated by the Homeodomain Specificity Prediction web page (<ext-link ext-link-type="uri" xlink:href="http://stormo.wustl.edu/cgi-bin/flyhd/hd_pred.cgi">http://stormo.wustl.edu/cgi-bin/flyhd/hd_pred.cgi</ext-link>; <xref ref-type="bibr" rid="bib70">Noyes et al., 2008</xref>). The two predicted binding specificities are identical to one another, due to perfect conservation of the HD recognition positions.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03728.020">http://dx.doi.org/10.7554/eLife.03728.020</ext-link></p></caption><graphic xlink:href="elife03728fs013"/></fig></fig-group></p><p>These data suggest acute DSD of transcriptional regulatory mechanisms underlying otherwise identical <italic>Mesp</italic> expression patterns. More specifically, the <italic>trans</italic>-regulatory environment of the B7.5 blastomeres has diverged between <italic>Molgula</italic> and <italic>Ciona,</italic> and compensatory changes in the respective <italic>Mesp cis</italic>-regulatory sequences must have rendered these unable to function adequately outside of that milieu.</p></sec><sec id="s2-8"><title><italic>Tbx6-related</italic> and <italic>Lhx3/4</italic> gene expression patterns in <italic>M. occidentalis</italic></title><p>The drift of <italic>Mesp</italic> regulation between <italic>M. occidentalis</italic> and <italic>C. intestinalis</italic> prompted us to investigate potential species-specific differences in upstream <italic>trans-</italic>acting regulators<italic>.</italic> In <italic>C. intestinalis, Mesp</italic> is transcriptionally activated downstream of two zygotically expressed TFs: Tbx6-related.b (Ciinte.Tbx6-r.b) and Lhx3/4 (Ciinte.Lhx3/4) (<xref ref-type="bibr" rid="bib83">Satou et al., 2004</xref>; <xref ref-type="bibr" rid="bib25">Davidson et al., 2005</xref>; <xref ref-type="bibr" rid="bib13">Christiaen et al. 2009a</xref>). <italic>Ciinte.Tbx6-r.b</italic> is directly activated in the posterior mesodermal lineages by the maternal determinant Macho-1/Zic-related.a (Ciinte.Zic-r.a) (<xref ref-type="bibr" rid="bib117">Yagi et al., 2004</xref>), while <italic>Ciinte.Lhx3/4</italic> is activated in the vegetal pole in response to stabilized maternal Beta-catenin (<xref ref-type="bibr" rid="bib81">Satou et al., 2001</xref>). Since the expression domains of Ciinte.Tbx6-r.b and Ciinte.Lhx3/4 overlap only in B7.5, activation of <italic>Ciinte.Mesp</italic> is achieved through the synergistic interaction between Ciinte.Tbx6-r.b and Ciinte.Lhx3/4 proteins at the <italic>Ciinte.Mesp</italic> promoter in these cells (<xref ref-type="bibr" rid="bib13">Christiaen et al., 2009a</xref>).</p><p>In each of the three <italic>Molgula</italic> genome assemblies<italic>,</italic> we identified two <italic>Tbx6</italic>-r genes of uncertain orthology relationships to the four <italic>Tbx6-r</italic> genes in the <italic>C. intestinalis</italic> genome (<xref ref-type="bibr" rid="bib93">Stolfi et al., 2014</xref>). In all three <italic>Molgula</italic> genomes, the two <italic>Tbx6-r</italic> genes are arranged in head-to-head configuration with ∼2 kb separating the transcript start sites (<xref ref-type="fig" rid="fig4s2">Figure 4—figure supplement 2B</xref>). Despite the uncertain phylogeny of tunicate <italic>Tbx6-r</italic> genes, we named these genes <italic>Tbx6-r.a</italic> and <italic>Tbx6-r.b</italic> in <italic>Molgula.</italic> In <italic>M. occidentalis,</italic> both <italic>Tbx6-r.a</italic> and <italic>Tbx6-r.b</italic> are expressed in a broad posterior swath of the pre-gastrula embryo in a manner similar to the expression pattern of <italic>Tbx6-r</italic> genes in <italic>C. intestinalis</italic> (<xref ref-type="fig" rid="fig4">Figure 4C,D</xref>; <xref ref-type="bibr" rid="bib117">Yagi et al., 2004</xref>)<italic>.</italic></p><p>We also identified two <italic>Lhx3/4</italic> genes in each <italic>Molgula</italic> genome<italic>.</italic> This was unexpected because no <italic>Lhx3/4</italic> duplications have been identified in any ascidian species (<xref ref-type="bibr" rid="bib13">Christiaen et al., 2009a</xref>; <xref ref-type="bibr" rid="bib49">Kobayashi et al., 2010</xref>). We named these genes <italic>Lhx3/4.a</italic> and <italic>Lhx3/4.b.</italic> In all three <italic>Molgula</italic> species, <italic>Lhx3/4.a</italic> is the more conserved paralog, while <italic>Lhx3/4.b</italic> is more divergent, lacking a well-conserved C-terminal motif (<xref ref-type="fig" rid="fig4s2">Figure 4—figure supplement 2A</xref>). However, the amino acid sequence changes are not predicted to change the DNA-binding preference of the homeodomain (<xref ref-type="fig" rid="fig4s3">Figure 4—figure supplement 3</xref>; <xref ref-type="bibr" rid="bib70">Noyes et al., 2008</xref>). In each of the three <italic>Molgula</italic> species’ genome assemblies, the two <italic>Lhx3/4</italic> genes were found to be located on separate contigs.</p><p>In <italic>M. occidentalis, Lhx3/4.b</italic> is expressed in vegetal cells of the early embryo, overlapping with <italic>Tbx6-r</italic> genes only in the B7.5 cells (<xref ref-type="fig" rid="fig4">Figure 4F,G</xref>). In contrast, <italic>Moocci.Lhx3/4.a</italic> is not zygotically expressed in the early embryo, but is expressed in larval motor neurons (<xref ref-type="fig" rid="fig4">Figure 4E,H</xref>), where <italic>Moocci.Lhx3/4.b</italic> is not transcriptionally active (<xref ref-type="fig" rid="fig4">Figure 4I</xref>). In both <italic>C. intestinalis</italic> and <italic>H. roretzi,</italic> transcription of a single <italic>Lhx3/4</italic> gene from separate proximal and distal promoters results in two distinct transcript variants. Early vegetal cells express one transcript variant while motor neurons express the other transcript variant (<xref ref-type="bibr" rid="bib109">Wada et al., 1995</xref>; <xref ref-type="bibr" rid="bib81">Satou et al., 2001</xref>; <xref ref-type="bibr" rid="bib13">Christiaen et al., 2009a</xref>; <xref ref-type="bibr" rid="bib49">Kobayashi et al., 2010</xref>). Since this pleiotropic expression profile appears to be strictly partitioned between the two <italic>Lhx3/4</italic> paralogs in <italic>M. occidentalis</italic>, this constitutes a clear example of regulatory sub-functionalization following gene duplication (<xref ref-type="bibr" rid="bib72">Ohno, 1970</xref>; <xref ref-type="bibr" rid="bib33">Hahn, 2009</xref>).</p><p>In summary, <italic>M. occidentalis</italic> embryos express <italic>Tbx6-r</italic> and <italic>Lhx3/4</italic> genes in early B7.5 blastomeres, where they could potentially synergize to activate <italic>Moocci.Mesp</italic> following a <italic>trans</italic>-acting logic shared with <italic>C. intestinalis.</italic> The next set of experiments sought to test this potentially conserved logic.</p></sec><sec id="s2-9"><title>Moocci.Tbx6-r.b and Moocci.Lhx3/4 proteins are conserved enough to activate <italic>Ciinte.Mesp</italic></title><p>We asked whether <italic>cis/trans</italic> co-evolution between <italic>Mesp</italic> and Tbx6-r and Lhx3/4 could account for incompatibility of <italic>Mesp</italic> regulatory sequences between <italic>M. occidentalis</italic> and <italic>C. intestinalis.</italic> We previously demonstrated that in <italic>C. intestinalis,</italic> overexpression of Tbx6-r.b in the vegetal pole cells of the embryo, using the <italic>cis-</italic>regulatory sequences from the <italic>Foxd.b</italic> gene (<xref ref-type="bibr" rid="bib87">Shi and Levine, 2008</xref>), results in ectopic <italic>Mesp</italic> activation in these cells due to expanded overlap of Tbx6-r.b and Lhx3/4 (<xref ref-type="bibr" rid="bib13">Christiaen et al., 2009a</xref>). Similarly, overexpression of Lhx3/4 in the posterior pole of the embryo, using the <italic>cis-</italic>regulatory sequences from <italic>Tbx6-r.b</italic>, is sufficient to cause ectopic <italic>Mesp</italic> expression throughout this territory. We used these gain-of-function assays in <italic>C. intestinalis</italic> with the orthologous <italic>M. occidentalis</italic> proteins to test potential conservation of function in synergistic activation of <italic>Mesp</italic> expression.</p><p>Overexpression of Moocci.Tbx6-r.b but not Moocci.Tbx6-r.a was sufficient to activate ectopic <italic>Ciinte.Mesp</italic> reporter construct expression in vegetal pole cells (<xref ref-type="fig" rid="fig5">Figure 5A–C</xref>). In this regard, Moocci.Tbx6-r.b function seemed comparable to that of Ciinte.Tbx6-r.b (<xref ref-type="fig" rid="fig5">Figure 5D</xref>). Conversely, overexpression of either Lhx3/4.b or Lhx3/4.a from <italic>M. occidentalis</italic> in the posterior <italic>C. intestinalis</italic> embryo using the <italic>Ciinte.Tbx6-r.b</italic> promoter caused ectopic expression of both endogenous <italic>Ciinte.Mesp</italic> (<xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1</xref>) and <italic>Ciinte.Mesp</italic> reporter construct (<xref ref-type="fig" rid="fig5">Figure 5E–G</xref>). These experiments revealed that both <italic>M. occidentalis</italic> Lhx3/4 proteins are conserved enough to activate low levels of <italic>Ciinte.Mesp</italic> expression (presumably by synergizing with endogenous Ciinte.Tbx6-r.b)<italic>.</italic> However, neither was as effective as Ciinte.Lhx3/4 in activating <italic>Ciinte.Mesp</italic> (<xref ref-type="fig" rid="fig5">Figure 5H</xref>). These data suggested that <italic>cis/trans</italic> or <italic>trans/trans</italic> co-evolution involving the highly divergent Moocci.Lhx3/4.b paralog may only partially account for the incompatibility of <italic>Mesp cis</italic>-regulatory logic between the two species.<fig-group><fig id="fig5" position="float"><object-id pub-id-type="doi">10.7554/eLife.03728.021</object-id><label>Figure 5.</label><caption><title>Divergence of <italic>Mesp</italic> regulation due to changes in <italic>cis</italic> and <italic>trans</italic>.</title><p>(<bold>A</bold>) Wild-type <italic>C. intestinalis</italic> gastrula embryo showing expression of <italic>Foxd.b</italic> reporter (red, <xref ref-type="bibr" rid="bib87">Shi and Levine, 2008</xref>) in vegetal pole cells and <italic>Ciinte.Mesp</italic> reporter (green) in the B7.5 cells. (<bold>B</bold>) Electroporation of <italic>Ciinte.Foxd.b>Moocci.Tbx6-r.a</italic> has no effect on <italic>Ciinte.Mesp</italic> reporter expression. (<bold>C</bold>) Electroporation of <italic>Ciinte.Foxd.b>Moocci.Tbx6-r.b</italic> results in ectopic <italic>Ciinte.Mesp</italic> reporter expression in the vegetal pole in 58% of embryos (n = 100). (<bold>D</bold>) This is indistinguishable from the effect of <italic>Ciinte.Foxd.b>Ciinte.Tbx6-r.b</italic>, which results in ectopic <italic>Ciinte.Mesp</italic> reporter activation in 62% of embryos (n = 100). (<bold>E</bold>) Wild-type <italic>C. intestinalis</italic> gastrula embryo showing expression of <italic>Tbx6-r.b</italic> reporter (<xref ref-type="bibr" rid="bib13">Christiaen et al., 2009a</xref>, red) in all B-line cells and <italic>Ciinte.Mesp</italic> reporter (green) in the B7.5 cells. (<bold>F</bold>) Electroporation of <italic>Ciinte.Tbx6-r.b>Moocci.Lhx3/4.b</italic> results in ectopic <italic>Ciinte.Mesp</italic> reporter expression in other B-line cells in 17% of embryos (n = 100) (<bold>G</bold>) <italic>Ciinte.Tbx6-r.b>Moocci.Lhx3/4.a</italic> results in ectopic <italic>Ciinte.Mesp</italic> reporter expression in 29% of embryos (n = 100). (<bold>H</bold>) <italic>Ciinte.Tbx6-r.b>Ciinte.Lhx3/4</italic> induces ectopic <italic>Ciinte.Mesp</italic> reporter expression in 88% of embryos (n = 100). (<bold>I</bold>) <italic>C. intestinalis</italic> tailbud embryo showing no expression of <italic>Moocci.Mesp</italic> reporter, as expected. (<bold>J</bold>) <italic>Moocci.Mesp</italic> reporter expression is not rescued by electroporation of <italic>Ciinte.Foxd.b>Moocci.Tbx6-r.a</italic> but (<bold>K</bold>) electroporation of <italic>Ciinte.Foxd.b>Moocci.Tbx6-r.b</italic> is sufficient to activate <italic>Moocci.Mesp</italic> reporter in 39% of embryos (n = 100). (<bold>K</bold>) <italic>Moocci.Mesp</italic> reporter is not transactivated upon electroporation of <italic>Foxd.b>Ciinte.Tbx6-r.b</italic>. (<bold>L</bold>) Wild-type <italic>C. intestinalis</italic> tailbud embryo, showing no expression of electroporated <italic>Moocci.Mesp</italic> reporter. (<bold>M</bold>) Electroporation of <italic>Ciinte.Tbx6-r.b>Moocci.Lhx3/4.b</italic> or (<bold>N</bold>) <italic>Ciinte.Tbx6-r.b>Ciinte.Lhx3/4</italic> is not sufficient to activate co-electroporated <italic>Moocci.Mesp</italic> reporter expression. (<bold>O</bold>–<bold>R</bold>) ISH showing <italic>Moocci.Mesp</italic> expression (green, arrowheads) in 110-cell stage <italic>M. occidentalis</italic> embryos. Ectopic <italic>Moocci.Mesp</italic> was not observed upon overexpression of any Lhx3/4 orthologs in the posterior embryo using the <italic>Moocci.Tbx6-r.b</italic> driver (<bold>P</bold>–<bold>R</bold>). All gastrula stage embryos are viewed vegetally, with anterior to the top of the image. All tailbud embryos are viewed laterally, anterior to the left. <italic>M. occidentalis</italic> embryo nuclei were visualized by staining with DAPI (blue).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03728.021">http://dx.doi.org/10.7554/eLife.03728.021</ext-link></p></caption><graphic xlink:href="elife03728f005"/></fig><fig id="fig5s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03728.022</object-id><label>Figure 5—figure supplement 1.</label><caption><title>Lhx3/4 proteins from <italic>M. occidentalis</italic> are sufficient to activate ectopic expression of endogenous <italic>Mesp</italic> in <italic>C. intestinalis</italic>.</title><p>In situ hybridization for <italic>Ciinte.Mesp</italic> in 110-cell stage <italic>C. intestinalis</italic> embryos. (<bold>A</bold>) Expression of <italic>Ciinte.Mesp</italic> is restricted to the B7.5 blastomeres (solid arrowheads) in control embryos. (<bold>B</bold>) Ectopic <italic>Ciinte.Mesp</italic> expression is seen in a few cells (hollow arrowheads) in 39% (n = 100) of embryos electroporated with <italic>Ciinte.Tbx6-r.b>Moocci.Lhx3/4.b.</italic> (<bold>C</bold>) Ectopic <italic>Ciinte.Mesp</italic> expression is seen in a few cells (hollow arrowheads) in 74% (n = 100) of embryos electroporated with <italic>Ciinte.Tbx6-r.b>Moocci.Lhx3/4.a</italic>. (<bold>D</bold>) Ectopic <italic>Ciinte.Mesp</italic> expression is seen in a broad posterior swath of cells in 86% (n = 50) of embryos electroporated with <italic>Ciinte.Tbx6-r.b>Ciinte.Lhx3/4.</italic> All embryos viewed vegetally with the anterior to the top.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03728.022">http://dx.doi.org/10.7554/eLife.03728.022</ext-link></p></caption><graphic xlink:href="elife03728fs014"/></fig></fig-group></p></sec><sec id="s2-10"><title>Moocci.Tbx6-r.b but not Moocci.Lhx3/4 can activate <italic>Moocci.Mesp</italic> reporter expression in <italic>C. intestinalis</italic> embryos</title><p>Having established that <italic>M. occidentalis</italic> proteins can activate ectopic <italic>Ciinte.Mesp</italic> expression, we next asked whether these could be sufficient to activate <italic>Moocci.Mesp</italic> reporter expression in <italic>C. intestinalis</italic> embryos. Indeed, <italic>Moocci.Mesp</italic> reporter activation in the vegetal pole was observed upon overexpression of Moocci.Tbx6-r.b (<xref ref-type="fig" rid="fig5">Figure 5J</xref>), but not Moocci.Tbx6-r.a (<xref ref-type="fig" rid="fig5">Figure 5I</xref>), consistent with their effects on <italic>Ciinte.Mesp</italic> activation<italic>.</italic> Surprisingly, overexpression of Ciinte.Tbx6-r.b was not sufficient to activate <italic>Moocci.Mesp</italic> reporter (<xref ref-type="fig" rid="fig5">Figure 5K</xref>). These results hint at <italic>cis/trans</italic> co-evolution between <italic>Moocci.Mesp cis-</italic>regulatory sequences and Moocci.Tbx6-r.b, even though the latter retains enough functional information to activate <italic>Ciinte.Mesp.</italic></p><p>In stark contrast to the rescue by Moocci.Tbx6-r.b, <italic>Moocci.Mesp</italic> reporter expression was never observed in <italic>C. intestinalis</italic> embryos upon overexpression of Moocci.Lhx3/4.b or Ciinte.Lhx3/4 in the posterior pole (<xref ref-type="fig" rid="fig5">Figure 5L–N</xref>). This indicates that a functional modification extending beyond <italic>cis/trans</italic> co-evolution accounts for the lack of <italic>Moocci.Mesp</italic> reporter activity in <italic>C. intestinalis</italic> embryos.</p></sec><sec id="s2-11"><title>Moocci.Lhx3/4.b overexpression is not sufficient to activate ectopic <italic>Moocci.Mesp</italic> in <italic>M. occidentalis</italic> embryos</title><p>The heterologous experiments in <italic>C. intestinalis</italic> suggested that <italic>Moocci.Mesp</italic> regulation could be independent of Moocci.Lhx3/4.b. To test this hypothesis, we recapitulated the experiments in <italic>M. occidentalis</italic> embryos. We isolated <italic>cis-</italic>regulatory sequences from the <italic>Moocci.Tbx6-r.b</italic> gene (<xref ref-type="fig" rid="fig4s2">Figure 4—figure supplement 2</xref>) and used this driver to overexpress <italic>Lhx3/4</italic> coding sequences in the posterior pole of <italic>M. occidentalis</italic> embryos. All Lhx3/4 proteins tested failed to induce ectopic <italic>Moocci.Mesp</italic> activation (<xref ref-type="fig" rid="fig5">Figure 5O–R</xref>). These data suggest that <italic>Moocci.Mesp</italic> is not responsive to Moocci.Lhx3/4.b, even in permissive <italic>Moocci.Tbx6-r.b-</italic>expressing cells. This differs markedly from the results of equivalent experiments in <italic>C. intestinalis,</italic> indicating major changes to the <italic>cis-</italic>regulatory logic governing identical <italic>Mesp</italic> spatiotemporal expression patterns in <italic>Ciona</italic> and <italic>Molgula.</italic></p></sec><sec id="s2-12"><title>Conserved requirement for MAPK activation in specification of migratory TVCs</title><p>Given the <italic>cis-</italic>regulatory re-wiring of <italic>Mesp</italic> regulation<italic>,</italic> we extended our analysis to search for other instances of acute DSD between <italic>Molgula</italic> and <italic>Ciona</italic>. We shifted our focus to the next major cell fate decision in the B7.5 lineage, namely the fate choice between TVCs and ATMs. In <italic>C. intestinalis,</italic> the FGF/MAPK pathway, mediated in part by MEK-dependent phosphorylation and activation of Ets.b, is both necessary and sufficient for TVC specification. Inhibition of the FGF/MAPK/Ets pathway converts TVCs to ATMs, inhibiting cell migration and expression of TVC markers (<xref ref-type="bibr" rid="bib24">Davidson et al., 2006</xref>; <xref ref-type="bibr" rid="bib12">Christiaen et al., 2008</xref>; <xref ref-type="bibr" rid="bib115">Woznica et al., 2012</xref>). Conversely, constitutive activation of the pathway induces ectopic TVCs at the expense of ATMs.</p><p>We ought to test the requirement of the MAPK pathway for TVC induction in <italic>M. occidentalis.</italic> We treated embryos with the MEK small molecule inhibitor U0126 prior to the estimated time window of TVC specification (∼5 hpf at 22°C). U0126-treated embryos displayed inhibited TVC migration relative to DMSO-treated control embryos (<xref ref-type="fig" rid="fig6">Figure 6A–D</xref>). Moreover, expression of <italic>Moocci.Foxf</italic> and <italic>Moocci.Hand-r</italic> in the TVCs was severely downregulated by U0126 treatment relative to controls (<xref ref-type="fig" rid="fig6">Figure 6A–D</xref>). These data suggest a conserved requirement for MAPK pathway activity in TVC induction and expression of important TVC regulators in <italic>M. occidentalis.</italic><fig-group><fig id="fig6" position="float"><object-id pub-id-type="doi">10.7554/eLife.03728.025</object-id><label>Figure 6.</label><caption><title>Mutual unintelligibility of orthologous <italic>Foxf</italic> enhancers in spite of shared requirement for the MAPK pathway in TVC fate specification.</title><p>In situ hybridization (ISH, red) + β-galactosidase immunodetection (IHC, green) in embryos electoporated with <italic>Moocci.Mesp>nls::lacZ</italic> and treated with DMSO (control treatment), showing normal TVC induction in 85% of embryos (n = 49) and TVC-localized expression of (<bold>A</bold>) <italic>Moocci.Foxf</italic> and (<bold>B</bold>) <italic>Moocci.Hand-r</italic> in 96% (n = 25) and 100% (n = 23) of embryos, respectively. Treatment with MEK inhibitor U0126 inhibited TVC induction and migration in 66% of embryos (n = 128). Treatment with U0126 also abolished TVC-specific (<bold>C</bold>) <italic>Moocci.Foxf</italic> expression in 91% of embryos (n = 64) and (<bold>D</bold>) <italic>Moocci.Hand-r</italic> expression in 91% of embryos (n = 64). (<bold>E</bold>) Wild-type <italic>M. occidentalis</italic> tailbud embryo with B7.5 lineage labeled by electroporation of <italic>Moocci.Mesp>H2B::GFP</italic>. Note two TVCs and two ATMs. (<bold>F</bold>) TVC induction and migration was enhanced upon electroporation with <italic>Moocci.Mesp>Ciinte.Ets.b::VP16,</italic> which converted ATMs into TVC-like cells in 30% of electroporated embryos (n = 200). (<bold>G</bold>) A fragment from −1517 to −425 upstream of the <italic>Moocci.Foxf</italic> start codon (<italic>Moocci.Foxf[TVC])</italic>, fused to the minimal promoter (−230/+21) from <italic>Moocci.Foxf</italic> and GFP <italic>(bpMoFF>GFP)</italic>, recapitulates expression in the TVCs of <italic>M. occidentalis</italic>. (<bold>H</bold>) This same exact construct is silent in <italic>C. intestinalis</italic> embryos. (<bold>I</bold>) Conversely, a published TVC enhancer from the <italic>Ciinte.Foxf</italic> gene (<xref ref-type="bibr" rid="bib6">Beh et al., 2007</xref>) fused to <italic>bpMoFF>GFP</italic> is silent in <italic>M. occidentalis</italic> embryos. (<bold>J</bold>) This same construct is strongly expressed in the TVCs of <italic>C. intestinalis</italic>. All panels represent lateral views.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03728.025">http://dx.doi.org/10.7554/eLife.03728.025</ext-link></p></caption><graphic xlink:href="elife03728f006"/></fig><fig id="fig6s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03728.026</object-id><label>Figure 6—figure supplement 1.</label><caption><title>Alignment of <italic>Foxf</italic> coding and non-coding sequences.</title><p>Diagram of mVISTA alignment of <italic>M. oculata Foxf</italic> (<italic>Moocul.Foxf</italic>) locus aligned to orthologs in (from top to bottom) <italic>M. occulta, M. occidentalis,</italic> and <italic>C. intestinalis.</italic> Blue-shaded peaks indicate protein-coding sequence conservation above 70% in 100-bp windows. Red-shaded peaks indicate non-coding sequence conservation above 70% in 100-bp windows. Sequence conservation between the Roscovite Molgulids (<italic>M. oculata</italic> and <italic>M. occulta</italic>) and <italic>M. occidentalis</italic> is limited to protein-coding portions, and conservation between <italic>Molgula</italic> and <italic>Ciona</italic> is limited to a narrow portion of the sequence coding for the Foxf DNA-binding domain. Only the first exon of <italic>Foxf</italic> is shown.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03728.026">http://dx.doi.org/10.7554/eLife.03728.026</ext-link></p></caption><graphic xlink:href="elife03728fs015"/></fig><fig id="fig6s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03728.027</object-id><label>Figure 6—figure supplement 2.</label><caption><title>Moocci.Ets.b and Ciinte.Ets.b are functionally very similar.</title><p><italic>C. intestinalis</italic> embryos co-electroporated with <italic>Ciinte.Mesp>H2B::GFP</italic> (green)<italic>, Ciinte.Foxf>mCherry</italic> (red)<italic>,</italic> and various <italic>Ciinte.Mesp-</italic>driven perturbation constructs. Percentages represent the frequency of the phenotype seen in the representative image displayed for each condition. Overexpression of full-length Ets.b proteins from both <italic>C. intestinalis</italic> and <italic>M. occidentalis</italic> results ectopic <italic>Foxf+</italic> TVCs at the expense of ATMs. The same conversion of ATMs to TVCs is seen for overexpression of Ets.b::VP16 fusions. Conversely, Ets.b::WRPW fusions repress TVC induction and <italic>Ciinte.Foxf</italic> reporter expression. As expected from the lack of sequence divergence predicted to affect DNA-binding specificities, Ets.b proteins from both species appear to be equally suited to trans-activating <italic>Ciinte.Foxf</italic> in <italic>C. intestinalis</italic> embryos. n = 100 for all conditions.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03728.027">http://dx.doi.org/10.7554/eLife.03728.027</ext-link></p></caption><graphic xlink:href="elife03728fs016"/></fig><fig id="fig6s3" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03728.028</object-id><label>Figure 6—figure supplement 3.</label><caption><title>Ets.b proteins are not sufficient to transactivate <italic>Moocci.Foxf</italic> reporter construct activation in <italic>C. intestinalis</italic> embryos.</title><p><italic>C. intestinalis</italic> embryos co-electroporated with <italic>Ciinte.Mesp>H2B::GFP</italic> (green)<italic>, Moocci.Foxf>mCherry</italic> (red)<italic>,</italic> and various <italic>Ciinte.Mesp-</italic>driven perturbation constructs. Activation of <italic>Moocci.Foxf</italic> reporter construct was impervious in <italic>C. intestinalis</italic> TVCs even upon overexpression of Ets.b full-length or VP16 fusions from either <italic>C. intestinalis</italic> or <italic>M. occidentalis</italic>. n = 100 for all conditions.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03728.028">http://dx.doi.org/10.7554/eLife.03728.028</ext-link></p></caption><graphic xlink:href="elife03728fs017"/></fig></fig-group></p><p>We also tested the potential involvement of Ets factors in mediating the MAPK-dependent activation of the TVC program. To do so, we expressed a constitutively active fusion of the Ciinte.Ets.b DNA-binding domain to a VP16 transactivation domain (Ciinte.Ets.b::VP16, <xref ref-type="bibr" rid="bib24">Davidson et al., 2006</xref>), in <italic>M. occidentalis</italic> B7.5 cells using the <italic>Moocci.Mesp</italic> driver. Electroporation of <italic>Moocci.Mesp>Ciinte.Ets.b::VP16</italic> induced ectopic TVCs at the expense of ATMs, in 31% of transfected embryos (<xref ref-type="fig" rid="fig6">Figure 6E,F</xref>). In sum, our results are consistent with a conserved function for MAPK-activated Ets.b upstream of the cardiopharyngeal gene regulatory network in <italic>M. occidentalis</italic>.</p></sec><sec id="s2-13"><title><italic>Foxf</italic> TVC enhancers are mutually unintelligible in cross-species assays</title><p>Given the conservation of the MAPK/Ets pathway in ascidian TVC specification, we reasoned that <italic>Foxf cis-</italic>regulatory sequences should be compatible in cross-species assays. We identified a sequence upstream of the <italic>Moocci.Foxf</italic> gene that is sufficient to drive TVC-specific expression in <italic>M. occidentalis</italic> (<xref ref-type="fig" rid="fig6">Figure 6G</xref>, <xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1</xref>)<italic>.</italic> However, this sequence is completely non-functional when tested in <italic>C. intestinalis</italic> (<xref ref-type="fig" rid="fig6">Figure 6H</xref>). Likewise, a previously identified <italic>Ciinte.Foxf</italic> TVC enhancer (<xref ref-type="fig" rid="fig6">Figure 6J</xref>; <xref ref-type="bibr" rid="bib6">Beh et al., 2007</xref>) is incapable of driving TVC-specific expression in <italic>M. occidentalis</italic> embryos (<xref ref-type="fig" rid="fig6">Figure 6I</xref>).</p><p>Thus, to borrow a term from the field of linguistics, these enhancers are said to be ‘mutually unintelligible’, since the enhancer from <italic>M. occidentalis</italic> cannot be interpreted by embryos of <italic>C. intestinalis</italic>, and the enhancer from <italic>C. intestinalis</italic> cannot be interpreted by <italic>M. occidentalis</italic>. This mutual unintelligibility is due to differences between the enhancers and not the promoters, since we used the same <italic>Moocci.Foxf</italic> basal promoter in both constructs. Although the <italic>Ciinte.Foxf</italic> enhancer is able to interact with and potentiate transcription from <italic>Moocci.Foxf</italic> promoter, nucleotide sequence is poorly conserved around <italic>Foxf</italic> proximal promoter regions, even between <italic>M. occidentalis</italic> and <italic>M. occulta/oculata</italic> (<xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1</xref>).</p><p>We asked whether the observed unintelligibility was indicative of changes in Ets.b protein properties between <italic>C. intestinalis</italic> and <italic>M. occidentalis</italic> embryo<italic>.</italic> At first glance, differences in DNA binding domain amino acid sequences between the Ets.b orthologs do not predict a change in binding site preference (<xref ref-type="bibr" rid="bib26">Donaldson et al., 1996</xref>). Indeed, full-length Moocci.Ets.b and Moocci.Ets.b::VP16 were just as effective as their <italic>C. intestinalis</italic> counterparts to induce ectopic TVCs when expressed in the <italic>C. intestinalis</italic> B7.5 lineage (<xref ref-type="fig" rid="fig6s2">Figure 6—figure supplement 2</xref>). Moreover, <italic>C. intestinalis</italic> TVC induction was abolished by expression of the constitutive repressor form (Moocci.Ets.b::WRPW). However, neither full-length Moocci.Ets.b nor Moocci.Ets.b::VP16 was sufficient to induce activation of <italic>Moocci.Foxf</italic> reporter in <italic>C. intestinalis</italic> embryos (<xref ref-type="fig" rid="fig6s3">Figure 6—figure supplement 3</xref>). These surprising results suggest that, even though TVC induction and TVC-specific <italic>Foxf</italic> expression depends upon Ets.b-mediated MAPK activity in <italic>M. occidentalis</italic> embryos, there are profound species-specific differences in the regulatory mechanisms acting in parallel to MAPK/Ets.b upstream of <italic>Foxf</italic>, reflected in the mutual unintelligibility of the orthologous enhancers.</p></sec><sec id="s2-14"><title>Extensive <italic>cis-</italic>regulatory unintelligibility between <italic>C. intestinalis</italic> and <italic>M. occidentalis</italic></title><p>Since investigation into both <italic>Mesp</italic> and <italic>Foxf</italic> regulation revealed divergent <italic>cis</italic>-regulatory logic underlying identical gene expression patterns, we asked whether these examples constituted a general trend between <italic>C. intestinalis</italic> and <italic>M. occidentalis</italic>. Testing several other candidate enhancers, we identified other examples of <italic>cis</italic>-regulatory unintelligibility. For instance, <italic>Moocci.Tbx6-r.b</italic> and <italic>Mooci.Hand-r</italic> upstream sequences recapitulated their endogenous activity in <italic>M. occidentalis</italic> embryos (<xref ref-type="fig" rid="fig7">Figure 7B,E</xref>), but these sequences were conspicuously silent in the B7.5 lineage in <italic>C. intestinalis</italic> embryos (<xref ref-type="fig" rid="fig7">Figure 7C,F</xref>). Notably, activity in other tissues seemed normal: <italic>Moocci.Tbx6-r.b</italic> reporter expression was robust and precise in <italic>C. intestinalis</italic> primary tail muscle cells (<xref ref-type="fig" rid="fig7">Figure 7A,C</xref>) and <italic>Moocci.Hand-r</italic> reporter recapitulated expression in the <italic>C. intestinalis</italic> A7.6 and anterior endoderm lineages (<xref ref-type="fig" rid="fig7">Figure 7D,F</xref>). In the case of <italic>Hand-r,</italic> this partial intelligibility is due to the modular organization of its <italic>cis</italic>-regulatory sequences, which in <italic>C. intestinalis</italic> is comprised of discrete enhancers controlling expression in each of the distinct domains (<xref ref-type="bibr" rid="bib23">Davidson and Levine, 2003</xref>; <xref ref-type="bibr" rid="bib115">Woznica et al., 2012</xref>).<fig-group><fig id="fig7" position="float"><object-id pub-id-type="doi">10.7554/eLife.03728.023</object-id><label>Figure 7.</label><caption><title>Cross-species reporter construct assays reveal multiple cases of <italic>cis</italic>-regulatory unintelligibility.</title><p>(<bold>A</bold>) <italic>C. intestinalis</italic> embryo electroporated with <italic>Ciinte.Tbx6-r.b>GFP</italic> reporter construct (<xref ref-type="bibr" rid="bib13">Christiaen et al., 2009a</xref>), which drives GFP expression in tail muscles and the B7.5 lineage cells (arrowheads), recapitulating endogenous <italic>Ciinte.Tbx6-r.b</italic> expression. (<bold>B</bold>) <italic>M. occidentalis</italic> embryo electroporated with <italic>Moocci.Tbx6-r.b>GFP</italic> reporter construct, which recapitulates expression in tail muscle cells including B7.5 lineage cells (arrowheads). (<bold>C</bold>) <italic>C. intestinalis</italic> embryo electroporated with <italic>Moocci.Tbx6-r.b>GFP,</italic> which drives expression in B-line tail muscle and mesenchyme cells but is excluded from the B7.5 lineage. (<bold>D</bold>) <italic>C. intestinalis</italic> embryo electroporated with <italic>Ciinte.Hand-r>H2B::GFP</italic> reporter (<xref ref-type="bibr" rid="bib23">Davidson and Levine, 2003</xref>), which drives H2B::GFP expression in anterior endoderm, A7.6 lineage, and TVCs (arrowheads), recapitulating endogenous <italic>Ciinte.Hand-r</italic> expression. (<bold>E</bold>) <italic>M. occidentalis</italic> embryo electroporated with <italic>Moccci.Hand-r>H2B::GFP</italic> construct, which recapitulates the same expression pattern. (<bold>F</bold>) <italic>C. intestinalis</italic> embryo electroporated with <italic>Moocci.Hand-r>H2B::GFP,</italic> which drives expression in endoderm and A7.6 lineage, but not in B7.5. (<bold>G</bold>) <italic>M. occidentalis</italic> embryo electroporated with <italic>Ciinte.Ebf</italic> neuron-specific YFP (green) and H2B::mCherry (red) reporter constructs electroporated (<xref ref-type="bibr" rid="bib92">Stolfi and Levine, 2011</xref>), which drive very weak expression in a limited subset of motor ganglion neurons (green and red). (<bold>H</bold>) <italic>C. intestinalis</italic> embryo electroporated with a <italic>Moocci.Ebf>YFP</italic> reporter, which drives robust reporter gene expression in several brain, motor ganglion, and nerve cord neurons. (<bold>I</bold>) <italic>C. intestinalis</italic> embryos electroporated with <italic>Ciinte.Sox1/2/3</italic> (left) and <italic>Moocci.Sox1/2/3</italic> (right) <italic>H2B::mCherry</italic> reporter constructs, both of which recapitulate <italic>Sox1/2/3</italic> expression in ectoderm. Panels <bold>A</bold>–<bold>F</bold> are lateral views of tailbud embryos, panels <bold>G</bold> is a dorsal view of a tailbud embryo, panel <bold>H</bold> is a dorso-lateral view of a hatched larva, and panel <bold>I</bold> shows vegetal views of mid-gastrula stage embryos. Anterior is to the right, except in Panel <bold>I</bold>, in which anterior is to the top.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03728.023">http://dx.doi.org/10.7554/eLife.03728.023</ext-link></p></caption><graphic xlink:href="elife03728f007"/></fig><fig id="fig7s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03728.024</object-id><label>Figure 7—figure supplement 1.</label><caption><title>Asymmetric intelligibility between <italic>Molgula</italic> and <italic>Ciona Hand-r</italic> TVC enhancers.</title><p>(<bold>A</bold>) β-galactosidase immunodetection on <italic>M. occidentalis</italic> late tailbud embryo electroporated with <italic>Ciinte.Hand-r>nls::lacZ.,</italic> showing reporter gene expression in TVC descendents (heart precursors and atrial siphon muscle founder cells) on one side of the embryo. (<bold>B</bold>) <italic>M. occidentalis</italic> embryo electroporated with <italic>Moocul.Hand-r>H2B::GFP,</italic> showing recapitulation of endogenous <italic>Moocci.Hand-r</italic> expression in endoderm, A7.6 lineage, and TVCs. (<bold>C</bold>) <italic>C. intestinalis</italic> embryo electroporated with <italic>Moocul.Hand-r>H2B::GFP</italic> showing slight expression in A7.6 lineage and B-line mesenchyme cells, but no expression in TVCs (hollow arrowheads).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03728.024">http://dx.doi.org/10.7554/eLife.03728.024</ext-link></p></caption><graphic xlink:href="elife03728fs018"/></fig></fig-group></p><p>Curiously, we found that the <italic>Ciinte.Hand-r</italic> reporter can drive reporter gene expression in <italic>M. occidentalis</italic> TVCs (<xref ref-type="fig" rid="fig7s1">Figure 7—figure supplement 1</xref>). Thus, unlike the case of <italic>Foxf,</italic> there is an asymmetric intelligibility of <italic>Hand-r</italic> TVC enhancers between <italic>M. occidentalis</italic> and <italic>C. intestinalis</italic>. Moreover, a <italic>M. oculata Hand-r</italic> TVC enhancer is functional in <italic>M. occidentalis</italic> but not in <italic>C. intestinalis</italic> (<xref ref-type="fig" rid="fig7s1">Figure 7—figure supplement 1</xref>)<italic>.</italic> Taken together, these data suggest that differences in enhancer logic may have accumulated over the course of the deep evolutionary history between <italic>Molgula</italic> and <italic>Ciona</italic> but not between <italic>M. occidentalis</italic> and <italic>M. oculata,</italic> and that some enhancers may have evolved asymmetrically in the two branches, retaining greater pan-ascidian ‘intelligibility’ in one or the other.</p><p>Finally, we found that acute DSD was not restricted to <italic>cis</italic>-regulatory sequences controlling gene expression in the B7.5 lineage. For instance, a <italic>Ciinte.Ebf</italic> driver that has been shown to recapitulate neuronal <italic>Ebf</italic> expression (<xref ref-type="bibr" rid="bib92">Stolfi and Levine, 2011</xref>) was only very weakly active in a subset of <italic>M. occidentalis</italic> motor ganglion neurons (<xref ref-type="fig" rid="fig7">Figure 7G</xref>). However, the orthologous sequence from <italic>M. occidentalis</italic> recapitulated robust reporter gene expression in several <italic>C. intestinalis Ebf+</italic> neurons throughout the central nervous system (<xref ref-type="fig" rid="fig7">Figure 7H</xref>). In contrast, expression of a <italic>Moocci.Sox1/2/3</italic> reporter construct in the ectoderm of <italic>C. intestinalis</italic> embryos was found to be indistinguishable from that of the <italic>C. intestinalis</italic> ortholog (<xref ref-type="fig" rid="fig7">Figure 7I</xref>), suggesting that some <italic>cis-</italic>regulatory sequences may retain mutual intelligibility between <italic>Molgula</italic> and <italic>Ciona</italic> without sequence conservation, as is the case for various <italic>Otx</italic> enhancers between <italic>C. intestinalis</italic> and <italic>H. roretzi</italic> (<xref ref-type="bibr" rid="bib71">Oda-Ishii et al., 2005</xref>)<italic>.</italic> In sum, our preliminary cross-species enhancer activity screen suggests that DSD was rampant during ascidian evolution, resulting in mutual unintelligibility of <italic>cis</italic>-regulatory mechanisms underlying otherwise identical expression patterns of orthologous genes.</p></sec></sec><sec id="s3" sec-type="discussion"><title>Discussion</title><sec id="s3-1"><title><italic>Molgula spp.</italic> as model organisms for molecular developmental genetics</title><p>By sequencing the genomes of three <italic>Molgula</italic> species and adapting the electroporation protocol for routine transfection of <italic>M. occidentalis</italic> embryos<italic>,</italic> we have further expanded the reach of ascidian molecular genetics. We have demonstrated the usefulness of <italic>M. occidentalis</italic> as a species amenable to medium-throughput molecular perturbations and assays. While <italic>Ciona/Halocynthia</italic> comparisons have yielded fundamental insights into chordate development, the lack of a reliable electroporation protocol for transfection of <italic>Halocynthia</italic> eggs has hampered comparative studies on a larger scale. On the other hand, eggs from species such as <italic>Phallusia mammilata</italic> can be electroporated, but this species is much more closely related to <italic>Ciona</italic>. Thus, <italic>Molgula</italic> species may significantly contribute to understanding the extreme conservation of ascidian embryogenesis in spite of vast genetic differences and deep evolutionary histories.</p><p>We have shown that de novo sequencing and assembly of Molgulid genomes using next-generation technologies is relatively efficient and cost-effective, largely due to their compact nature. The sequence information obtained can be used to quickly generate a comprehensive set of molecular tools. We predict that a large number of ascidian genomes will be as compact and efficiently sequenced and assembled as these, unlocking the experimental potential of many ascidian species representing several different families (<xref ref-type="bibr" rid="bib107">Tsagkogeorga et al., 2009</xref>).</p></sec><sec id="s3-2"><title>Similarities and differences between <italic>Molgula</italic> and <italic>Ciona</italic> cardiopharyngeal regulatory mechanisms</title><p>In our survey of B7.5 development, we observed that the cell division events and cell fate choices in the lineage are perfectly conserved between <italic>Ciona</italic> and <italic>Molgula,</italic> demonstrating the deep evolutionary origins of the cardiopharyngeal ontogenetic motif in tunicates<italic>.</italic> Regulatory gene expression patterns in the lineage are also nearly identical, the only potential difference being <italic>Nk4,</italic> which was not confidently detected in <italic>M. occidentalis</italic> TVCs.</p><p>NK4 proteins are intimately linked to cardiac development throughout bilateria (<xref ref-type="bibr" rid="bib2">Akazawa and Komuro, 2005</xref>). Moreover, <italic>Nk4</italic> is expressed in <italic>C. intestinalis</italic> TVCs where it functions as a regulator of heart precursor fate (<xref ref-type="bibr" rid="bib23">Davidson and Levine, 2003</xref>; <xref ref-type="bibr" rid="bib111">Wang et al., 2013</xref>). However, there are distinct early and late phases of <italic>Nk4</italic> expression in <italic>C. intestinalis</italic> heart development. <italic>Nk4</italic> is expressed in the TVCs during the mid-tailbud stage, downregulated during the late tailbud and larval stages, and only re-activated in the differentiating heart of metamorphosing juveniles (<xref ref-type="bibr" rid="bib23">Davidson and Levine, 2003</xref>). This points to distinct early (pre-metamorphic) and late (post-metamorphic) roles for <italic>Nk4</italic> in <italic>C. intestinalis</italic>.</p><p>In <italic>C. intestinalis,</italic> early <italic>Nk4</italic> expression does not appear to be sufficient for heart fate, as it is expressed in the TVCs prior to the segregation of heart and pharyngeal muscle fates (<xref ref-type="bibr" rid="bib23">Davidson and Levine, 2003</xref>; <xref ref-type="bibr" rid="bib6">Beh et al., 2007</xref>; <xref ref-type="bibr" rid="bib12">Christiaen et al., 2008</xref>, <xref ref-type="bibr" rid="bib14">2010</xref>; <xref ref-type="bibr" rid="bib77">Ragkousi et al., 2011</xref>; <xref ref-type="bibr" rid="bib111">Wang et al., 2013</xref>). Second, this early expression may not be strictly required for heart fate either, as the first heart precursors (FHPs) do not adopt a pharyngeal muscle fate upon dominant-negative Nk4 overexpression like the second heart precursor (SHPs) do (<xref ref-type="bibr" rid="bib111">Wang et al., 2013</xref>). Therefore, Nk4 acts as partially redundant mechanism to specify heart precursors in <italic>C. intestinalis</italic>, complementing parallel mechanisms of heart/pharyngeal muscle cell fate choice (e.g., activation of <italic>Tbx1/10</italic> in the STVCs and <italic>Ebf</italic> in the ASMFs).</p><p>It is possible that <italic>Moocci.Nk4</italic> is only activated during metamorphosis, an early role for it being dispensable in <italic>M. occidentalis</italic> TVCs. This might be the case if the complementary mechanism for pharyngeal muscle fate choice is more robust than in <italic>Ciona.</italic> In this way, the divergence of parallel regulatory mechanisms may have contributed to the loss of a key regulator like <italic>Nk4</italic> from the early cardiopharyngeal mesoderm GRN of <italic>Molgula</italic>. A much more detailed investigation will be required to determine a possible role for Nk4 in the <italic>Molgula</italic> heart development.</p></sec><sec id="s3-3"><title>Species-specific differences in B7.5 lineage morphogenetic events</title><p><italic>M. occidentalis</italic> and <italic>C. intestinalis</italic> also display differences in certain morphogenetic behaviors of B7.5 derivatives (summarized in <xref ref-type="fig" rid="fig3">Figure 3</xref>, <xref ref-type="fig" rid="fig3s3">Figure 3—figure supplement 3</xref>). First, <italic>M. occidentalis</italic> TVCs migrate more laterally and do not meet at the ventral midline, resulting in a temporary condition of <italic>cardia bifida</italic>. A similar condition can be induced in <italic>C. intestinalis</italic> embryos by perturbing endoderm development (<xref ref-type="bibr" rid="bib77">Ragkousi et al., 2011</xref>). Since <italic>M. occidentalis</italic> and <italic>C. intestinalis</italic> tailbud embryos look quite different in their shape, it will be interesting to assess endoderm formation and its relationship with TVC migration trajectory in <italic>Molgula.</italic></p><p>Secondly, the ASM precursors in <italic>M. occidentalis</italic> migrate away from the heart progenitors but do not encounter atrial siphon primordia to encircle in the dorsal part of the embryo. This process is uncoupled in <italic>C. intestinalis Hox1</italic> mutants, which fail to specify the ASPs. In these mutant larvae, the ASMPs still appear to migrate dorsally but do not form rings of muscle (<xref ref-type="bibr" rid="bib80">Sasakura et al., 2012</xref>). It remains to be determined how the <italic>M. occidentalis</italic> ASMPs migrate to the general vicinity of the future atrial siphon opening and remain localized there until the later specification and differentiation of the siphon.</p><p>These two major morphogenetic differences prompted us to rethink our assumptions regarding the interdependence of certain processes in ascidian cardiopharyngeal development, as identified in <italic>C. intestinalis.</italic> For instance, one might assume that the convergence of <italic>Ciona</italic> TVCs at the ventral midline to be important for the asymmetric cell divisions and fate choices segregating heart from ASM precursors. Similarly, one might assume the atrial siphon primordia to be the source of a cue required for the dorsal migration of ASMPs. In <italic>M. occidentalis,</italic> these processes have been naturally uncoupled. These observations serve to emphasize that sampling diverse taxa provides insights into the modularity of specific developmental processes.</p></sec><sec id="s3-4"><title>Developmental system drift hidden beneath the surface of highly conserved ascidian embryos</title><p>From our initial survey of a handful of enhancers from <italic>C. intestinalis, M. occidentalis</italic>, and <italic>M. oculata</italic>, we encountered several instances of either mutual unintelligibility, or asymmetric intelligibility of enhancers. Qualitative cross-species assays of <italic>M. occidentalis</italic> and <italic>C. intestinalis cis</italic>-regulatory sequences indicated that specific orthologous pairs of enhancers may be mutually intelligible (e.g., <italic>Sox1/2/3</italic> ectodermal enhancer) while others are mutually unintelligible (e.g., <italic>Foxf TVC</italic> enhancer<italic>).</italic> Some orthologous gene pairs appear to be regulated by a mix of intelligible and unintelligible tissue-specific enhancers (e.g., <italic>Hand-r).</italic> The observation that cross-species intelligibility due to DSD occurs in a tissue-specific manner is consistent with the widely documented modularity of <italic>cis</italic>-regulatory elements controlling complex gene expression (<xref ref-type="bibr" rid="bib4">Arnone and Davidson, 1997</xref>; <xref ref-type="bibr" rid="bib57">Levine, 2010</xref>).</p><p>These results add to the mounting evidence suggesting that acute and pervasive DSD may have occurred over the course of ascidian evolution, obfuscated by the identical cell lineages and highly conserved gene expression patterns of ascidian embryos (<xref ref-type="bibr" rid="bib95">Swalla, 2004</xref>). The multiple examples of <italic>cis-</italic>regulatory unintelligibility we identified were rather unexpected given (a) the extremely conserved pattern of expression of orthologous genes from <italic>Molgula</italic> and <italic>Ciona</italic> (<xref ref-type="bibr" rid="bib99">Takada et al., 2002</xref>) and (b) previous observations of mutual intelligibility of enhancers between <italic>C. intestinalis</italic> and <italic>H. roretzi</italic> (e.g., <italic>Otx</italic>), and between <italic>C. intestinalis</italic> and the more closely related <italic>C. savignyi</italic> (<xref ref-type="bibr" rid="bib45">Johnson et al., 2004</xref>; <xref ref-type="bibr" rid="bib9">Brown et al., 2007</xref>)<italic>.</italic> Large-scale, quantitative cross-species assays and detailed GRN studies will illuminate factors that may contribute to conservation or divergence in regulatory mechanisms.</p></sec><sec id="s3-5"><title>Evolution of different activating inputs for <italic>Mesp</italic> expression in ascidians</title><p><italic>Cis</italic>-regulatory unintelligibility can arise due to different underlying factors. We have presented evidence that, in the case of <italic>Mesp</italic>, this results in part from a change in an upstream activating input. In <italic>C. intestinalis,</italic> Lhx3/4 is an activator of <italic>Mesp,</italic> while in <italic>M. occidentalis, Mesp</italic> activation appears to be independent of Lhx3/4.b, despite the conserved expression pattern of this gene in vegetal pole cells. Regulation of <italic>Mesp</italic> in other ascidian species will need to be investigated in order to establish whether Lhx3/4-dependent activation of <italic>Mesp</italic> in ascidians is ancestral or derived. On the other hand, both <italic>C. intestinalis</italic> and <italic>M. occidentalis</italic> have retained a conserved role for Tbx6-r in regulating <italic>Mesp,</italic> a regulatory connection that may have arisen in the last common ancestor of tunicates and vertebrates (<xref ref-type="bibr" rid="bib118">Yasuhiko et al., 2006</xref>; <xref ref-type="bibr" rid="bib25">Davidson et al., 2005</xref>). In hindsight, it appears the weak expression of <italic>Ciinte.Mesp</italic> reporter constructs observed in the B7.5 cells of <italic>M. occidentalis</italic> embryos is not indicative of GRN conservation. This expression instead appears to be an artifact attributed in part to the conserved, overlapping expression profiles of Moocci.Tbx6-r.b and Moocci.Lhx3/4.b, even if this overlap is not instructive for endogenous <italic>Moocci.Mesp</italic> activation.</p><p>We can only speculate what is the connection, if any, between the re-wiring of <italic>Mesp</italic> regulation and the <italic>Molgula-</italic>specific duplication and sub-functionalization of <italic>Lhx3/4.</italic> Interestingly, in <italic>C. intestinalis</italic> and <italic>H. roretzi</italic>, the early and late functions of Lhx3/4 are partitioned between two transcript variants of the same locus, not between different loci. This substitution of alternate isoforms with gene duplicates has been documented for a few genes in teleost fish (<xref ref-type="bibr" rid="bib3">Altschmied et al., 2002</xref>; <xref ref-type="bibr" rid="bib119">Yu et al., 2003</xref>). Although allowing for some degree of sub-functionalization of protein function, alternative transcription/splicing does not completely relax the constraints imposed by pleiotropy. The Lhx3/4 variants in <italic>C. intestinalis</italic> and <italic>H. roretzi</italic> still share the bulk of their amino acid sequence, differing only at the portion of the N-terminus encoded by the alternate first exons (<xref ref-type="bibr" rid="bib13">Christiaen et al., 2009a</xref>; <xref ref-type="bibr" rid="bib49">Kobayashi et al., 2010</xref>). In <italic>Molgula</italic> spp. on the other hand, the two paralogs are considerably diverged from one another throughout their coding sequences, even though the divergence does not appear to alter DNA sequence-binding specificity. Pleiotropy has been hypothesized to both promote and suppress evolution, more specifically DSD (<xref ref-type="bibr" rid="bib34">Hansen, 2003</xref>). Investigating the full breadth of Lhx3/4 functions in multiple ascidian species will be required in order to determine the relationship between Lhx3/4 duplication and re-wiring of <italic>Mesp</italic> regulation.</p></sec><sec id="s3-6"><title>Multiple cases of cardiopharyngeal mesoderm enhancer unintelligibility point to differences in B7.5/TVC <italic>trans</italic> environments</title><p>The mutual unintelligibility of orthologous <italic>Foxf</italic> enhancers was unexpected, given that both <italic>M. occidentalis</italic> and <italic>C. intestinalis</italic> depend upon a shared MAPK/Ets-based switch for TVC fate induction. This discrepancy could be due to species-specific requirements for additional activating inputs, and/or silencing by species-specific repressors. Our observation that <italic>cis-trans</italic> complementation with Moocci.Ets.b::VP16 does not rescue the activity of the <italic>Moocci.Foxf</italic> enhancer in <italic>C. intestinalis</italic> embryos suggests that the latter possibility may be the case. It is thought that co-evolution between an enhancer and its <italic>trans</italic> environment is critical in order for it to faithfully direct a conserved pattern of gene expression, even if the overarching GRN is broadly conserved (<xref ref-type="bibr" rid="bib54">Landry et al., 2005</xref>). Therefore, the incompatibility of B7.5 lineage-specific enhancers between <italic>M. occidentalis</italic> and <italic>C. intestinalis</italic> may reflect key differences in the <italic>trans</italic>-regulatory milieus of this lineage in the two species. Future comparative expression profiling of <italic>M. occidentalis</italic> and <italic>C. intestinalis</italic> B7.5 lineage cells will shed light on the nature of these differences.</p></sec><sec id="s3-7"><title>Concluding remarks</title><p>Even though the word ‘drift’ implies a greater role for chance over natural selection, adaptive processes are very likely to play a role in DSD. True and Haag hypothesized as much in their seminal introduction to the concept of DSD, predicting that <italic>cis/trans</italic> co-evolution and other compensatory changes should result in enhancers resembling species-specific ‘Rube Goldberg machines’ that give superficially similar outputs but are functionally non-interchangeable (<xref ref-type="bibr" rid="bib106">True and Haag, 2001</xref>). Models incorporating selection, pleiotropy, and compensation (SPC) have refined this idea by ascribing ‘drift’ in one biological process to adaptation in another (<xref ref-type="bibr" rid="bib54">Landry et al., 2005</xref>; <xref ref-type="bibr" rid="bib46">Johnson and Porter, 2007</xref>; <xref ref-type="bibr" rid="bib73">Pavlicev and Wagner, 2012</xref>; <xref ref-type="bibr" rid="bib65">Martinez et al., 2014</xref>). For instance, if the same pleiotropic gene is involved in both processes, any mutations in that gene that are adaptive for the process under selection will require some compensatory changes simply to maintain the same output of the other process. This may occur with or without deleterious intermediate states, with the latter occurring through a process termed ‘pseudocompensation’ (<xref ref-type="bibr" rid="bib31">Haag, 2007</xref>).</p><p>We speculate that a high frequency of compensatory changes, required for the rapidly evolving ascidians to accommodate the constraints imposed by their invariant embryonic cell lineages and highly compact genomes, has given rise to a preponderance of cross-species <italic>cis</italic>-regulatory unintelligibility, following the DSD/SPC model. This perfect storm of intrinsic factors may be the key to explaining the dichotomy observed between highly conserved embryos and divergent <italic>cis</italic>-regulatory structure/function in ascidians.</p></sec></sec><sec id="s4" sec-type="materials|methods"><title>Materials and methods</title><sec id="s4-1"><title>Genomic DNA library preparation and sequencing</title><p>Genomic DNA was phenol/chloroform extracted from dissected gonads of <italic>Molgula occulta</italic> (Kupffer) and <italic>Molgula oculata</italic> (Forbes) adults from Roscoff, France, and a <italic>Molgula occidentalis</italic> (Traustedt) adult from Panacea, Florida, USA (Gulf Specimen Marine Lab). Genomic DNA was sheared using an M220 Focused-ultrasonicator (Covaris, Woburn, MA). Sequencing libraries were prepared using KAPA HiFi Library Preparation Kit (KAPA Biosystems, Wilmington, MA) indexed with DNA barcoded adapters (BioO, Austin, TX). Size selection was performed using Agencourt (Beckman–Coulter, Brea, CA) AMPure XP purification beads (300–400 bp fragments), or Sage Science (Beverly, MA) Pippin Prep (650–750 bp and 875–975 bp fragments). For <italic>M. occulta</italic> and <italic>M. occidentalis</italic> libraries, 6 PCR cycles were used. For <italic>M. oculata</italic> libraries, 8 cycles were used for the 300–400 bp library, and 10 cycles were used for the 650–750 and 875–975 bp libraries. Libraries of different species but same insert size ranges were multiplexed for sequencing in three 100 × 100 PE lanes on a HiSeq 2000 sequencing system (Illumina, San Diego, CA) at the Genomics Sequencing Core Facility, Center for Genomics and Systems Biology at New York University (New York, NY). Thus, each lane was dedicated to a mix of species, specifically barcoded libraries of a given insert size range. Raw sequencing reads were deposited as a BioProject at NCBI under the ID# PRJNA253689.</p></sec><sec id="s4-2"><title>Genome sequence assembly</title><p>All genomes were assembled on Michigan State University High Performance Computing Cluster (<ext-link ext-link-type="uri" xlink:href="http://contact.icer.msu.edu">http://contact.icer.msu.edu</ext-link>). Prior to assembly, read quality was examined using FastQC v0.10.1 (<xref ref-type="bibr" rid="bib5">Babraham Bioinformatics, 2014</xref>). Reads were then quality trimmed on both the 5′ and 3′ end using seqtk trimfq (<ext-link ext-link-type="uri" xlink:href="https://github.com/lh3/seqtk">https://github.com/lh3/seqtk</ext-link>) which uses Phred algorithm to determine the quality of a given base pair. Seqtk trimfq only trims bases, so no reads were discarded. Each library per species was then abundance filtered using 3-pass digital normalization to remove repetitive and erroneous reads (<xref ref-type="bibr" rid="bib10">Brown et al., 2012</xref>; <xref ref-type="bibr" rid="bib86">Schwarz et al., 2013</xref>; <xref ref-type="bibr" rid="bib37">Howe et al., 2014</xref>). Genome assembly was done using Velvet v1.2.08 (<xref ref-type="bibr" rid="bib121">Zerbino and Birney, 2008</xref>) with k-mer overlap length (‘k’) ranging from 19 to 69 and scaffolding was done by Velvet, by default. Velvet does not produce separate files for contigs and scaffolds; because Velvet scaffolded conservatively, contigs dominated the assemblies so we refer to both contigs and scaffolds as contigs. CEGMA scores were then computed to evaluate genome completeness (<xref ref-type="bibr" rid="bib74">Parra et al., 2007</xref>).</p><p>The latest versions of three species' genome assemblies have been deposited on the ANISEED (<underline>A</underline>scidian <underline>N</underline>etwork for <italic><underline>I</underline>n <underline>S</underline>itu</italic> <underline>E</underline>xpression and <underline>E</underline>mbryological <underline>D</underline>ata) database for browsing and BLAST searching at <ext-link ext-link-type="uri" xlink:href="http://www.aniseed.cnrs.fr/">http://www.aniseed.cnrs.fr/</ext-link> (<xref ref-type="bibr" rid="bib103">Tassy et al., 2010</xref>). Scripts for genome assembly and CEGMA analysis can be found in the following github repository: <ext-link ext-link-type="uri" xlink:href="https://github.com/elijahlowe/molgula_genome_assemblies.git">https://github.com/elijahlowe/molgula_genome_assemblies.git</ext-link>.</p></sec><sec id="s4-3"><title>Molecular cloning of <italic>Molgula</italic> sequences</title><p>Putative orthologs of <italic>Ciona intestinalis</italic> (Linnaeus) and <italic>Halocynthia roretzi</italic> (Drasche) protein-coding genes were initially identified by TBLASTN. Identified sequences were aligned to each other to support orthology relationships within <italic>Molgula</italic>, and then queried by BLASTP against the NCBI non-redundant protein sequence database to further support orthology relationships to known tunicate and vertebrate proteins. cDNAs were cloned by RT-PCR, or by 5′ and/or 3′ RACE (SMARTer RACE kit, Clontech, Mountain View, CA). The template cDNA libraries were prepared from total RNA extracted from embryos of various stages. Upstream regulatory sequences were cloned by PCR from genomic DNA. For non-RACE PCRs, we used Phusion high-fidelity polymerase (New England Biolabs, Ipswich, MA). Refer to <xref ref-type="supplementary-material" rid="SD1-data">Supplementary file 1</xref> for detailed sequence information. Genes are named according to the proposed unified nomenclature system for tunicate genetic elements (<xref ref-type="bibr" rid="bib93">Stolfi et al., 2014</xref>). Refer to <xref ref-type="supplementary-material" rid="SD2-data">Supplementary file 2</xref> for table with previously used gene names and the corresponding new proposed gene symbols and names.</p></sec><sec id="s4-4"><title>Electroporation of plasmid DNA into embryos</title><p><italic>M. occidentalis</italic> gravid adults were obtained from Gulf Specimen Marine Lab between March and December. Gravid adults were rare between the winter months from December to March. Animals were kept in a small aquarium with circulating artificial sea water at 24°C until needed. All subsequent manipulations using eggs and embryos were performed between 20°C and 24°C, though 24°C appeared to be the optimal temperature for both dechorionation and embryonic development.</p><p>Individual gonads were dissected in filtered TAPS-buffered artificial sea water (T-ASW) to release eggs and sperm. Eggs were allowed to undergo germinal vesicle breakdown for 10 min and then pooled along with sperm to fertilize for 5 min. Eggs were rinsed thoroughly and dechorionated in solution (168 μl 10N NaOH, 1.2 ml 2.5% pronase E, 0.3 g sodium thioglycolate in 40 ml T-ASW) with constant pipetting for 8–10 min (modified after <xref ref-type="bibr" rid="bib16">Christiaen et al., 2009c</xref>). Viewing under a high-powered dissecting microscope was often necessary to verify removal of the thin, translucent chorion that becomes wrapped tightly around the egg upon immersion in dechorionation solution. After confirming chorion removal, eggs were quickly rinsed by the passage through multiple dishes of T-ASW.</p><p>Electroporation conditions and procedures were identical to those previously described for <italic>C. intestinalis</italic> (<xref ref-type="bibr" rid="bib15">Christiaen et al., 2009b</xref>). Unless otherwise specifically noted, all fluorescent reporter genes were fused at the N-terminus to unc-76 tags (<xref ref-type="bibr" rid="bib27">Dynes and Ngai, 1998</xref>), to ensure distribution throughout the whole cytosol, electroporated typically at ∼50 μg to 90 μg per 700 μl electroporation solution, while nuclei were visualized with histone2B::fluorescent protein constructs electroporated at ∼5 μg to 25 μg per electroporation unless otherwise specifically noted. Perturbation constructs were electroporated at ∼35 μg to 100 μg per electroporation. To image juveniles, larvae were allowed to settle and metamorphose in plastic Petri dishes or on glass coverslips in Petri dishes for several days with frequent changes of T-ASW.</p><p>Fertilization of <italic>M. occulta</italic> eggs was performed as previously described (<xref ref-type="bibr" rid="bib97">Swalla and Jeffery, 1990</xref>). Embryos were dechorionated and electroporated as described for <italic>M. occidentalis</italic> embryos, using a custom-built electroporation machine (<xref ref-type="bibr" rid="bib120">Zeller et al., 2006</xref>). <italic>C. intestinalis</italic> (Type A ‘<italic>robusta</italic>’) adults were collected in San Diego, CA (M-Rep).</p></sec><sec id="s4-5"><title>MEK1/2 inhibitor U0126 treatment</title><p>Embryos were treated in 10 μM U0126 (Cell Signaling Technology, Danvers, MA) in T-ASW at least 30 min prior to the targeted MAPK signaling event.</p></sec><sec id="s4-6"><title>In situ hybridization</title><p>Fluorescent in situ hybridization assays were performed as previously described, with modifications (<xref ref-type="bibr" rid="bib6">Beh et al., 2007</xref>; <xref ref-type="bibr" rid="bib42">Ikuta and Saiga, 2007</xref>; <xref ref-type="bibr" rid="bib17">Christiaen et al., 2009d</xref>). Embryos were fixed in MEM-3.2% to 4% paraformaldehyde buffers for at least 2 hr. Pre-hybridization proteinase K concentrations ranged from 0.25 μg/ml (110-cell stage) to 1 μg/ml (tailbud) and 5 μg/ml (larvae) for <italic>Molgula spp.</italic> embryos. The <italic>Ciinte.Ebf</italic> probe was synthesized from template plasmid from the <italic>C. intestinalis</italic> Gene Collection Release 1 (<xref ref-type="bibr" rid="bib85">Satou et al., 2002</xref>). See <xref ref-type="supplementary-material" rid="SD1-data">Supplementary file 1</xref> for more detailed probe template sequence information.</p></sec><sec id="s4-7"><title>Imaging</title><p>Embryos were counterstained with DAPI and/or Phalloidin conjugates (LifeTechnologies/Thermo Fisher Scientific, Waltham, MA). Images were taken using a combination of different Leica Microsystems (Wetzlar, Germany) microscopes: TCS SP8 X confocal microscope, TCS SP5 confocal microscope, and DM2500 epifluorescence microscope.</p></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>The authors are greatly indebted to Paul Scheid, members of the NYU CGSB Genomics Sequencing Core facility, Rodoniki Athanasiadou and the Gresham Lab for help in planning and performing library preparation and sequencing. We thank ASSEMBLE MARINE for funding the MoEvoDevo project in 2013. We thank the Station Biologique faculty and staff, especially Stéphane Hourdez, Sophie Booker, and Xavier Bailly for their help in carrying out our studies at Roscoff. We would also like to thank Nadine Peyriéras and her research group for sharing of reagents, equipment, and expertise for <italic>M. occulta</italic> and <italic>M. oculata</italic> imaging. We are grateful to Wei Wang for subcloning the full-length <italic>Ciinte.Ets.b</italic> sequence. Work in the laboratory of LC is supported by grants from the National Institute of General Medical Sciences (R01GM096032), the American Heart Association (10SDG4310061), and by the New York Cardiac Center and New York University College of Arts and Sciences. AS is supported by the National Science Foundation Postdoctoral Research Fellowship in Biology (under grant NSF-1161835). EL, BJS, and CTB are supported by the National Science Foundation under Cooperative Agreement No. DBI-0939454. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation. This project was supported by Agriculture and Food Research Initiative Competitive Grant 2010-65205-20361 to CTB.</p></ack><sec sec-type="additional-information"><title>Additional information</title><fn-group content-type="competing-interest"><title>Competing interests</title><fn fn-type="conflict" id="conf1"><p>The authors declare that no competing interests exist.</p></fn></fn-group><fn-group content-type="author-contribution"><title>Author contributions</title><fn fn-type="con" id="con1"><p>AS, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con2"><p>EKL, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con3"><p>BJS, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con4"><p>CR, Conception and design, Acquisition of data, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con5"><p>FR, Conception and design, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con6"><p>CTB, Conception and design, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con7"><p>LC, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn></fn-group></sec><sec sec-type="supplementary-material"><title>Additional files</title><supplementary-material id="SD1-data"><object-id pub-id-type="doi">10.7554/eLife.03728.029</object-id><label>Supplementary file 1.</label><caption><p>DNA sequences of probes, enhancers, promoters, protein-coding cDNAs, in situ hybridization probe templates, primers, etc used in this study.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03728.029">http://dx.doi.org/10.7554/eLife.03728.029</ext-link></p></caption><media mime-subtype="docx" mimetype="application" xlink:href="elife03728s001.docx"/></supplementary-material><supplementary-material id="SD2-data"><object-id pub-id-type="doi">10.7554/eLife.03728.030</object-id><label>Supplementary file 2.</label><caption><p>Table of newly proposed tunicate gene names and symbols (<xref ref-type="bibr" rid="bib93">Stolfi et al., 2014</xref>), and their aliases and synonyms.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03728.030">http://dx.doi.org/10.7554/eLife.03728.030</ext-link></p></caption><media mime-subtype="xlsx" mimetype="application" xlink:href="elife03728s002.xlsx"/></supplementary-material><sec sec-type="datasets"><title>Major dataset</title><p>The following dataset was generated:</p><p><related-object content-type="generated-dataset" source-id="http://www.ncbi.nlm.nih.gov/bioproject/PRJNA253689" source-id-type="uri" id="dataro1"><collab collab-type="author">Stolfi A</collab>, <collab collab-type="author">Lowe EK</collab>, <collab collab-type="author">Racioppi C</collab>, <collab collab-type="author">Ristoratore F</collab>, <collab collab-type="author">Brown CT</collab>, <collab collab-type="author">Swalla BJ</collab>, <collab collab-type="author">Christiaen LC</collab>, <year>2014</year><x>, </x><source>Molgula occidentalis, M. occulta, and M. oculata genome samples</source><x>, </x><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/bioproject/PRJNA253689">http://www.ncbi.nlm.nih.gov/bioproject/PRJNA253689</ext-link><x>, </x><comment>BioProject - 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An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see <ext-link ext-link-type="uri" xlink:href="http://elifesciences.org/review-process">review process</ext-link>). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.</p></boxed-text><p>Thank you for sending your work entitled “Identical cell lineages and gene expression patterns conceal developmental system drift in ascidian evolution” for consideration at <italic>eLife.</italic> Your article has been favorably evaluated by Diethard Tautz (Senior editor), a Reviewing editor, and 3 reviewers, one of whom, Michael Eisen, has agreed to reveal their identity.</p><p>The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.</p><p>The paper represents an important contribution to the field of evolutionary biology and genome regulation. The experimental work is well executed. Before publication, the following points should be addressed by the authors:</p><p>1) As the authors outline in the Introduction, the lack of non-coding sequence conservation between distantly related ascidian species is established. Accordingly, while the authors reinforce and extend this, the extent to which their findings of divergent regulatory mechanisms are “unexpected” might be toned down.</p><p>2) The authors observe that Nk4 seems to be absent from the Molgula B7.5 lineage. The authors should discuss further how loss of key regulators could boost divergence in regulatory mechanisms. Don't the largely conserved gene expression profiles suggest that there may not be major changes in the trans-regulatory milieu as proposed in the Discussion?</p><p>3) The authors include Molgula occulta in their study. Can they draw more conclusions about this species? What happens to the equivalent of ATM cells in this tail-less species?</p><p>4) The differences in initial heart and AS formation between Ciona (single heart and bilateral siphon primordia) and Molgula (bifid heart and single AS primordia) are intriguing. Is it possible that this reflects a limited initial distribution of the B7.5 lineage around the circumference of the ascidian embryo?</p><p>5) Is the expression of regulators of the B7.5 lineage conserved at other sites of expression? For example, the authors mention that Foxf and Gata4/5/6 expression in surrounding tissue obscures TVC cells in Mogula. Is this also the case in Ciona?</p><p>6) Can the authors comment on the extent to which non-coding sequence divergence applies to the proximal promoters of ascidian genes with conserved expression patterns? Are these elements more conserved than enhancers?</p><p>7) More speculatively, can the authors make any conclusions about synteny from their genome sequence data? It would be interesting to know if the turnover of regulatory mechanisms is associated with as breakdown of synteny, as conserved gene order has been proposed to result from conservation of enhancer activity embedded in neighbouring genes.</p><p>8) It would help to have summary gene network schema highlighting similarities and differences between ascidian genera.</p><p>[Editors’ note: further minor revisions were requested prior to acceptance.]</p><p>There is one minor issue that we would like you to consider addressing prior to publication: concerning the use of “identical” in the title and abstract, presumably it implies that as far as the authors compared, the different species the lineages and expression patterns were the same (but Nk4 expression differences noted...). “Similar” may be more accurate and the reviewers and editor prefer that you substitute this term in the title, abstract, and where relevant in the text.</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.03728.032</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p>The most visible proposed change has been in the title from “Identical cell lineages and gene expression patterns conceal developmental system drift in ascidian evolution” to “Identical cardiopharyngeal lineages and expression patterns conceal developmental system drift in ascidian evolution”. We decided to change it in order to convey the notion that our study was nearly entirely focused on the cardiopharyngeal lineage. The original title had contained this key lineage information, but this was abandoned in haste at the moment of submission due to title size restrictions. We have now found a way to fit this back into the title. We hope you accept our reasoning for this title change.</p><p><italic>1) As the authors outline in the Introduction, the lack of non-coding sequence conservation between distantly related ascidian species is established. Accordingly, while the authors reinforce and extend this, the extent to which their findings of divergent regulatory mechanisms are “unexpected” might be toned down</italic>.</p><p>We generally agree with the reviewers’ point and have altered the wording in the text in order to downplay our prior assumptions, which were proven to be misguided. However, we have also attempted to more clearly explain in the Introduction the distinctions between the qualitatively different manifestations of cryptic regulatory divergence described in the literature which have been broadly classified under the term developmental system drift (DSD).</p><p>At face value, the finding that divergent non-coding sequences underlie divergent regulatory mechanisms does not seem unexpected. However, we and other ascidian researchers assumed that <italic>cis-</italic>regulatory mechanisms must be highly conserved throughout the ascidians, in order to explain the remarkable conservation of invariant cell lineages and gene expression patterns between the very distantly related model species <italic>C. intestinalis</italic> and <italic>H. roretzi.</italic> Furthermore, the only in-depth comparative analysis of <italic>cis-</italic>regulatory sequence function between these two species revealed strong conservation of enhancer logic <italic>in spite</italic> of radically divergent sequences (<xref ref-type="bibr" rid="bib71">Oda-Ishii 2005</xref>), mirroring the findings of studies in other groups of animals (<xref ref-type="bibr" rid="bib59">Ludwig et al. 2000</xref>; <xref ref-type="bibr" rid="bib35">Hare et al. 2008</xref>; <xref ref-type="bibr" rid="bib79">Romano & Wray 2003</xref>; <xref ref-type="bibr" rid="bib62">Maduro & Pilgrim 1996</xref>; <xref ref-type="bibr" rid="bib75">Piano et al. 1999</xref>).</p><p>Examples of divergent regulatory mechanisms between <italic>Ciona</italic> and <italic>Halocynthia</italic> in the literature have been mostly limited to differences in extracellular signaling cues, not <italic>cis</italic>-regulatory logic (<xref ref-type="bibr" rid="bib22">Darras & Nishida 2001</xref>; <xref ref-type="bibr" rid="bib1">Abitua et al. 2012</xref>; <xref ref-type="bibr" rid="bib107">Tokuoka et al. 2007</xref>; <xref ref-type="bibr" rid="bib40">Hudson et al. 2007</xref>; <xref ref-type="bibr" rid="bib39">Hudson et al. 2013</xref>; <xref ref-type="bibr" rid="bib102">Takatori et al. 2010</xref>). Thus, when we began the present study, we were biased to expect no significant divergence in <italic>cis-</italic>regulatory mechanisms between <italic>Molgula</italic> and <italic>Ciona,</italic> especially not in those controlling the highly conserved expression patterns of genes such as <italic>Mesp, Foxf,</italic> and <italic>Hand-related.</italic> We were obviously wrong in our expectations, but we believe these were somewhat justified at the onset of our investigation.</p><p><italic>2) The authors observe that Nk4 seems to be absent from the Molgula B7.5 lineage</italic>. <italic>The authors should discuss further how loss of key regulators could boost divergence in regulatory mechanisms. Don't the largely conserved gene expression profiles suggest that there may not be major changes in the trans-regulatory milieu as proposed in the Discussion?</italic></p><p>As we have mentioned in the text, we cannot definitively conclude that <italic>Nk4</italic> is absent from the Molgula B7.5 lineage. We have created a new section of the Discussion dedicated to outlining more clearly both the caveats of our <italic>Nk4</italic> expression analysis and the hypothetical scenarios involving possible loss of (early) <italic>Nk4</italic> expression in <italic>M. occidentalis</italic> TVCs.</p><p>The reviewers suggest that the loss of key regulators could boost divergence in regulatory mechanisms. We generally agree with this statement. However, the converse is just as likely. For instance, it is possible that in <italic>Molgula,</italic> there is no need for the partially redundant function of Nk4 to promote SHP fate, because the complementary mechanism for pharyngeal muscle fate choice may be more robust than in <italic>Ciona.</italic> In this scenario, the divergence of a parallel (or upstream) regulatory mechanism may have prompted the loss of a key regulator. We have explicitly added these points in the Discussion.</p><p><italic>3) The authors include Molgula occulta in their study</italic>. <italic>Can they draw more conclusions about this species? What happens to the equivalent of ATM cells in this tail-less species?</italic></p><p>As far as we can tell, the primary larval muscle cells (including the ATMs) of <italic>M. occulta</italic> are specified but do not differentiate, partly because <italic>M. occulta</italic> have lost certain larval muscle structural genes (<xref ref-type="bibr" rid="bib52">Kusakabe et al. 1996</xref>). There is no evidence that there are any other fundamental differences in B7.5 lineage development between <italic>M. occidentalis</italic> and <italic>M. occulta</italic>.</p><p>We have included additional supplemental figures (<xref ref-type="fig" rid="fig2s3">Figure 2–figure supplement 3</xref>, panels G-I; <xref ref-type="fig" rid="fig2s4">Figure 2–figure supplement 4</xref>) showing <italic>in situ</italic> hybridization assays in <italic>M. occulta</italic> and <italic>M. occidentalis</italic> tailbud-stage embryos for <italic>Aldh1a</italic> (also known as <italic>Raldh2</italic>), which encodes the rate-limiting enzyme for retinoic acid biosynthesis<italic>.</italic> In <italic>C. intestinalis, Aldh1a.a</italic> is expressed in the TVCs and anterior primary muscle cells (including ATMs) and establishes a retinoic acid signaling gradient that plays important roles in patterning, fate specification, and differentiation in different tissues and cell lineages of the embryo (<xref ref-type="bibr" rid="bib68">Nagatomo and Fujiwara 2003</xref>).</p><p>From our data, we can observe that <italic>Aldh1a</italic> expression is conserved in <italic>M. occidentalis</italic> (<xref ref-type="fig" rid="fig2s4">Figure 2–figure supplement 4</xref>). Furthermore, it appears that <italic>M. occulta</italic> embryos have vestigial ATMs that remain in the anterior portion of the abortive tail bud and express <italic>Aldh1a</italic> (<xref ref-type="fig" rid="fig2s3">Figure 2–figure supplement 3G-I</xref>)<italic>.</italic> This suggests that, although the ATM cells do not differentiate, they may still retain a function as an important signaling center for the embryo. We have updated the manuscript to include a brief description and discussion of these new data.</p><p><italic>4) The differences in initial heart and AS formation between Ciona (single heart and bilateral siphon primordia) and Molgula (bifid heart and single AS primordia) are intriguing</italic>. <italic>Is it possible that this reflects a limited initial distribution of the B7.5 lineage around the circumference of the ascidian embryo?</italic></p><p>Since the B7.5 lineage, like most other cell lineages of the ascidian embryo is invariant and always gives rise to a fixed, stereotyped number of cells that is conserved between <italic>Ciona</italic> and <italic>Molgula,</italic> the reviewers are correct in assuming this population of cells is limiting. However, we do not believe there is any connection between the arrangement of the heart precursors and the single- or dual-primordium mode of atrial siphon development. It appears that once the atrial siphon muscle progenitors have separated from the heart precursors, their fates become largely independent from one another.</p><p>It has been well documented that stolidobranch ascidians form a single atrial siphon primordium (<xref ref-type="bibr" rid="bib30">Grave 1926</xref>; Grave 1944), but we have not encountered any bipartite heart primordia previously described in the literature. This probably reflects a <italic>Molgula-</italic>specific condition that evolved well after the divergence of the stolidobranchs and their transition to a single-primordium condition. We see how the readers may be tempted to draw a correlation between the number of heart and atrial siphon primordia, and have modified the text to reinforce the message that the bifid heart is likely a <italic>Molgula-</italic>specific trait, while the single atrial siphon primordium is a pan-stolidobranch character.</p><p><italic>5) Is the expression of regulators of the B7.5 lineage conserved at other sites of expression? For example, the authors mention that Foxf and Gata4/5/6 expression in surrounding tissue obscures TVC cells in Mogula</italic>. <italic>Is this also the case in Ciona?</italic></p><p>Yes, this is the case in <italic>Ciona</italic> (<xref ref-type="bibr" rid="bib7">Beh et al. 2007</xref>)<italic>. Foxf and Gata4/5/6</italic> expression in these tissues is perfectly conserved between <italic>Ciona</italic> and <italic>Molgula.</italic> We have included a line in the text that refers to this conserved expression in other tissues.</p><p><italic>6) Can the authors comment on the extent to which non-coding sequence divergence applies to the proximal promoters of ascidian genes with conserved expression patterns? Are these elements more conserved than enhancers</italic>?</p><p>Annotation of the <italic>Molgula</italic> genomes is still ongoing and thus we are not able to answer this question on a genome-wide scale, but for the few genes we have analyzed in depth, the promoters are no more conserved at the nucleotide sequence level than the enhancers, that is to say, not at all. In fact, even coding sequences are poorly conserved, and alignment is usually only possible between sequences representing the DNA-binding domain or other functional domains. One can see this clearly in the VISTA alignment plots that we have already included in the manuscript (<italic>Mesp,</italic> <xref ref-type="fig" rid="fig1">Figure 1</xref>, and <italic>Foxf,</italic> <xref ref-type="fig" rid="fig6s1">Figure 6-figure supplement 1</xref>)<italic>.</italic></p><p>However, it is clear that the <italic>Moocci.Foxf</italic> proximal promoter is fully functional in <italic>C. intestinalis</italic> embryos. This was demonstrated by generating chimeras between the <italic>Ciinte.Foxf</italic> TVC enhancer and the <italic>Moocci.Foxf</italic> proximal promoter, which worked in <italic>C. intestinalis</italic> but not in <italic>M. occidentalis</italic> to drive TVC gene expression. However, the converse (<italic>Ciinte.Foxf</italic> promoter in <italic>M. occidentalis</italic>) has not been tested. We have added a line highlighting these observations in the text.</p><p><italic>7) More speculatively, can the authors make any conclusions about synteny from their genome sequence data? It would be interesting to know if the turnover of regulatory mechanisms is associated with as breakdown of synteny, as conserved gene order has been proposed to result from conservation of enhancer activity embedded in neighbouring genes</italic>.</p><p>We would not be comfortable making any conclusions about conservation of synteny or co-linearity, given that our assemblies did not use any long-insert library sequence reads, nor have we mapped our assemblies to chromosomes. Therefore, we have not made any changes to the text regarding this topic.</p><p>That being said, in a comparison between the <italic>C. intestinalis</italic> and <italic>C. savignyi</italic> genomes (Hill et al. 2008) observed extensive retention of synteny (genes located on the same chromosome) but an equally extensive loss of co-linearity (genes arranged in the same order and direction on a given chromosome). These findings seem to argue against an association between breakdown of colinearity and regulatory turnover, as regulatory mechanisms are quite conserved between <italic>C. intestinalis</italic> and <italic>C. savignyi</italic> (<xref ref-type="bibr" rid="bib45">Johnson et al. 2004</xref>).</p><p>On the other hand, tunicate genomes are quite compact (Dehal et al. 2002; Denoeud et al. 2010) and their cis-regulatory organization appears relatively simple, with all developmentally-important enhancers reported falling within <10 kb immediately 5’ to the transcription start site or within the introns of the regulated gene in question. To our knowledge there have been no reports, in <italic>Ciona</italic>, of enhancers embedded in one gene controlling the transcription of a neighboring gene. If we had to speculate, we would say that the compact nature of tunicate genomes has allowed for a breakdown of colinearity, given the lack of constraints imposed by the long-range enhancers mentioned by the reviewers.</p><p><italic>8) It would help to have summary gene network schema highlighting similarities and differences between ascidian genera</italic>.</p><p>Strictly speaking, we do not have the evidence to infer gene networks operating in the B7.5 lineage of <italic>Molgula.</italic> The only regulatory connections we have directly established are <italic>Tbx6-r.b</italic> → <italic>Mesp</italic> and MAPK signaling → <italic>Foxf/Hand-r. A</italic>dditionally, the B7.5 network of <italic>Ciona</italic> is not substantially better understood. Therefore, we feel it is misleading to draw gene network schema at this stage of our understanding.</p><p>Better understood are the similarities and differences in the cell division events, basic morphogenesis, and gene expression patterns of the lineage. Of these, only the basic morphogenesis shows striking enough differences to warrant a comparative schematic diagram, which has been drawn and included in the revised version of the manuscript as <xref ref-type="fig" rid="fig3s3">Figure 3–figure supplement 3</xref>.</p><p>[Editors’ note: further minor revisions were requested prior to acceptance.]</p><p><italic>There is one minor issue that we would like you to consider addressing prior to publication: concerning the use of “identical” in the title and abstract, presumably it implies that as far as the authors compared, the different species the lineages and expression patterns were the same (but Nk4 expression differences noted...). “Similar” may be more accurate and the reviewers and editor prefer that you substitute this term in the title, abstract, and where relevant in the text</italic>.</p><p>We would like to propose the following modified title, which conveys the main findings perhaps more clearly and accurately:</p><p>“Divergent mechanisms regulate conserved cardiopharyngeal development and gene expression in distantly related ascidians”</p><p>Please note that where we wrote “identical” in the Abstract, referring to cell division and fate specification events, we showed that these are indeed identical between the two species.</p></body></sub-article></article> |