Corpus
Pmc
Racine
Corpus
Checkpoint
Pmc
Racine
Pmc
Exploration
Accueil
Racine
Racine

Serveur d'exploration sur le lymphœdème

Attention, ce site est en cours de développement !
Attention, site généré par des moyens informatiques à partir de corpus bruts.
Les informations ne sont donc pas validées.

The hormonal peptide Elabela guides angioblasts to the midline during vasculogenesis

Identifieur interne : 004679 ( Pmc/Corpus ); précédent : 004678; suivant : 004680

The hormonal peptide Elabela guides angioblasts to the midline during vasculogenesis

Auteurs : Christian Sm Helker ; Annika Schuermann ; Cathrin Pollmann ; Serene C. Chng ; Friedemann Kiefer ; Bruno Reversade ; Wiebke Herzog

Source :

RBID : PMC:4468421

Abstract

A key step in the de novo formation of the embryonic vasculature is the migration of endothelial precursors, the angioblasts, to the position of the future vessels. To form the first axial vessels, angioblasts migrate towards the midline and coalesce underneath the notochord. Vascular endothelial growth factor has been proposed to serve as a chemoattractant for the angioblasts and to regulate this medial migration. Here we challenge this model and instead demonstrate that angioblasts rely on their intrinsic expression of Apelin receptors (Aplr, APJ) for their migration to the midline. We further show that during this angioblast migration Apelin receptor signaling is mainly triggered by the recently discovered ligand Elabela (Ela). As neither of the ligands Ela or Apelin (Apln) nor their receptors have previously been implicated in regulating angioblast migration, we hereby provide a novel mechanism for regulating vasculogenesis, with direct relevance to physiological and pathological angiogenesis.

DOI:http://dx.doi.org/10.7554/eLife.06726.001


Url:
DOI: 10.7554/eLife.06726
PubMed: 26017639
PubMed Central: 4468421

Links to Exploration step

PMC:4468421

Le document en format XML

<record>
<TEI>
<teiHeader>
<fileDesc>
<titleStmt>
<title xml:lang="en">The hormonal peptide Elabela guides angioblasts to the midline during vasculogenesis</title>
<author>
<name sortKey="Helker, Christian Sm" sort="Helker, Christian Sm" uniqKey="Helker C" first="Christian Sm" last="Helker">Christian Sm Helker</name>
<affiliation>
<nlm:aff id="aff1">
<institution>University of Muenster</institution>
,
<addr-line>Muenster</addr-line>
,
<country>Germany</country>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Schuermann, Annika" sort="Schuermann, Annika" uniqKey="Schuermann A" first="Annika" last="Schuermann">Annika Schuermann</name>
<affiliation>
<nlm:aff id="aff1">
<institution>University of Muenster</institution>
,
<addr-line>Muenster</addr-line>
,
<country>Germany</country>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Pollmann, Cathrin" sort="Pollmann, Cathrin" uniqKey="Pollmann C" first="Cathrin" last="Pollmann">Cathrin Pollmann</name>
<affiliation>
<nlm:aff id="aff2">
<institution>Max Planck Institute for Molecular Biomedicine</institution>
,
<addr-line>Muenster</addr-line>
,
<country>Germany</country>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Chng, Serene C" sort="Chng, Serene C" uniqKey="Chng S" first="Serene C" last="Chng">Serene C. Chng</name>
<affiliation>
<nlm:aff id="aff3">
<institution content-type="dept">Institute of Medical Biology, Human Genetics and Embryology Laboratory</institution>
,
<institution>A*STAR</institution>
,
<addr-line>Singapore</addr-line>
,
<country>Singapore</country>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Kiefer, Friedemann" sort="Kiefer, Friedemann" uniqKey="Kiefer F" first="Friedemann" last="Kiefer">Friedemann Kiefer</name>
<affiliation>
<nlm:aff id="aff2">
<institution>Max Planck Institute for Molecular Biomedicine</institution>
,
<addr-line>Muenster</addr-line>
,
<country>Germany</country>
</nlm:aff>
</affiliation>
<affiliation>
<nlm:aff id="aff4">
<institution content-type="dept">Cells-in-Motion Cluster of Excellence</institution>
,
<institution>University of Muenster</institution>
,
<addr-line>Muenster</addr-line>
,
<country>Germany</country>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Reversade, Bruno" sort="Reversade, Bruno" uniqKey="Reversade B" first="Bruno" last="Reversade">Bruno Reversade</name>
<affiliation>
<nlm:aff id="aff3">
<institution content-type="dept">Institute of Medical Biology, Human Genetics and Embryology Laboratory</institution>
,
<institution>A*STAR</institution>
,
<addr-line>Singapore</addr-line>
,
<country>Singapore</country>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Herzog, Wiebke" sort="Herzog, Wiebke" uniqKey="Herzog W" first="Wiebke" last="Herzog">Wiebke Herzog</name>
<affiliation>
<nlm:aff id="aff1">
<institution>University of Muenster</institution>
,
<addr-line>Muenster</addr-line>
,
<country>Germany</country>
</nlm:aff>
</affiliation>
<affiliation>
<nlm:aff id="aff2">
<institution>Max Planck Institute for Molecular Biomedicine</institution>
,
<addr-line>Muenster</addr-line>
,
<country>Germany</country>
</nlm:aff>
</affiliation>
<affiliation>
<nlm:aff id="aff4">
<institution content-type="dept">Cells-in-Motion Cluster of Excellence</institution>
,
<institution>University of Muenster</institution>
,
<addr-line>Muenster</addr-line>
,
<country>Germany</country>
</nlm:aff>
</affiliation>
</author>
</titleStmt>
<publicationStmt>
<idno type="wicri:source">PMC</idno>
<idno type="pmid">26017639</idno>
<idno type="pmc">4468421</idno>
<idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4468421</idno>
<idno type="RBID">PMC:4468421</idno>
<idno type="doi">10.7554/eLife.06726</idno>
<date when="????">????</date>
<idno type="wicri:Area/Pmc/Corpus">004679</idno>
<idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">004679</idno>
</publicationStmt>
<sourceDesc>
<biblStruct>
<analytic>
<title xml:lang="en" level="a" type="main">The hormonal peptide Elabela guides angioblasts to the midline during vasculogenesis</title>
<author>
<name sortKey="Helker, Christian Sm" sort="Helker, Christian Sm" uniqKey="Helker C" first="Christian Sm" last="Helker">Christian Sm Helker</name>
<affiliation>
<nlm:aff id="aff1">
<institution>University of Muenster</institution>
,
<addr-line>Muenster</addr-line>
,
<country>Germany</country>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Schuermann, Annika" sort="Schuermann, Annika" uniqKey="Schuermann A" first="Annika" last="Schuermann">Annika Schuermann</name>
<affiliation>
<nlm:aff id="aff1">
<institution>University of Muenster</institution>
,
<addr-line>Muenster</addr-line>
,
<country>Germany</country>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Pollmann, Cathrin" sort="Pollmann, Cathrin" uniqKey="Pollmann C" first="Cathrin" last="Pollmann">Cathrin Pollmann</name>
<affiliation>
<nlm:aff id="aff2">
<institution>Max Planck Institute for Molecular Biomedicine</institution>
,
<addr-line>Muenster</addr-line>
,
<country>Germany</country>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Chng, Serene C" sort="Chng, Serene C" uniqKey="Chng S" first="Serene C" last="Chng">Serene C. Chng</name>
<affiliation>
<nlm:aff id="aff3">
<institution content-type="dept">Institute of Medical Biology, Human Genetics and Embryology Laboratory</institution>
,
<institution>A*STAR</institution>
,
<addr-line>Singapore</addr-line>
,
<country>Singapore</country>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Kiefer, Friedemann" sort="Kiefer, Friedemann" uniqKey="Kiefer F" first="Friedemann" last="Kiefer">Friedemann Kiefer</name>
<affiliation>
<nlm:aff id="aff2">
<institution>Max Planck Institute for Molecular Biomedicine</institution>
,
<addr-line>Muenster</addr-line>
,
<country>Germany</country>
</nlm:aff>
</affiliation>
<affiliation>
<nlm:aff id="aff4">
<institution content-type="dept">Cells-in-Motion Cluster of Excellence</institution>
,
<institution>University of Muenster</institution>
,
<addr-line>Muenster</addr-line>
,
<country>Germany</country>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Reversade, Bruno" sort="Reversade, Bruno" uniqKey="Reversade B" first="Bruno" last="Reversade">Bruno Reversade</name>
<affiliation>
<nlm:aff id="aff3">
<institution content-type="dept">Institute of Medical Biology, Human Genetics and Embryology Laboratory</institution>
,
<institution>A*STAR</institution>
,
<addr-line>Singapore</addr-line>
,
<country>Singapore</country>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Herzog, Wiebke" sort="Herzog, Wiebke" uniqKey="Herzog W" first="Wiebke" last="Herzog">Wiebke Herzog</name>
<affiliation>
<nlm:aff id="aff1">
<institution>University of Muenster</institution>
,
<addr-line>Muenster</addr-line>
,
<country>Germany</country>
</nlm:aff>
</affiliation>
<affiliation>
<nlm:aff id="aff2">
<institution>Max Planck Institute for Molecular Biomedicine</institution>
,
<addr-line>Muenster</addr-line>
,
<country>Germany</country>
</nlm:aff>
</affiliation>
<affiliation>
<nlm:aff id="aff4">
<institution content-type="dept">Cells-in-Motion Cluster of Excellence</institution>
,
<institution>University of Muenster</institution>
,
<addr-line>Muenster</addr-line>
,
<country>Germany</country>
</nlm:aff>
</affiliation>
</author>
</analytic>
<series>
<title level="j">eLife</title>
<idno type="eISSN">2050-084X</idno>
<imprint>
<date when="????">????</date>
</imprint>
</series>
</biblStruct>
</sourceDesc>
</fileDesc>
<profileDesc>
<textClass></textClass>
</profileDesc>
</teiHeader>
<front>
<div type="abstract" xml:lang="en">
<p>A key step in the de novo formation of the embryonic vasculature is the migration of endothelial precursors, the angioblasts, to the position of the future vessels. To form the first axial vessels, angioblasts migrate towards the midline and coalesce underneath the notochord. Vascular endothelial growth factor has been proposed to serve as a chemoattractant for the angioblasts and to regulate this medial migration. Here we challenge this model and instead demonstrate that angioblasts rely on their intrinsic expression of Apelin receptors (Aplr, APJ) for their migration to the midline. We further show that during this angioblast migration Apelin receptor signaling is mainly triggered by the recently discovered ligand Elabela (Ela). As neither of the ligands Ela or Apelin (Apln) nor their receptors have previously been implicated in regulating angioblast migration, we hereby provide a novel mechanism for regulating vasculogenesis, with direct relevance to physiological and pathological angiogenesis.</p>
<p>
<bold>DOI:</bold>
<ext-link ext-link-type="doi" xlink:href="10.7554/eLife.06726.001">http://dx.doi.org/10.7554/eLife.06726.001</ext-link>
</p>
</div>
</front>
<back>
<div1 type="bibliography">
<listBibl>
<biblStruct>
<analytic>
<author>
<name sortKey="Bussmann, J" uniqKey="Bussmann J">J Bussmann</name>
</author>
<author>
<name sortKey="Bos, Fl" uniqKey="Bos F">FL Bos</name>
</author>
<author>
<name sortKey="Urasaki, A" uniqKey="Urasaki A">A Urasaki</name>
</author>
<author>
<name sortKey="Kawakami, K" uniqKey="Kawakami K">K Kawakami</name>
</author>
<author>
<name sortKey="Duckers, Hj" uniqKey="Duckers H">HJ Duckers</name>
</author>
<author>
<name sortKey="Schulte Merker, S" uniqKey="Schulte Merker S">S Schulte-Merker</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Cermak, T" uniqKey="Cermak T">T Cermak</name>
</author>
<author>
<name sortKey="Doyle, El" uniqKey="Doyle E">EL Doyle</name>
</author>
<author>
<name sortKey="Christian, M" uniqKey="Christian M">M Christian</name>
</author>
<author>
<name sortKey="Wang, L" uniqKey="Wang L">L Wang</name>
</author>
<author>
<name sortKey="Zhang, Y" uniqKey="Zhang Y">Y Zhang</name>
</author>
<author>
<name sortKey="Schmidt, C" uniqKey="Schmidt C">C Schmidt</name>
</author>
<author>
<name sortKey="Baller, Ja" uniqKey="Baller J">JA Baller</name>
</author>
<author>
<name sortKey="Somia, Nv" uniqKey="Somia N">NV Somia</name>
</author>
<author>
<name sortKey="Bogdanove, Aj" uniqKey="Bogdanove A">AJ Bogdanove</name>
</author>
<author>
<name sortKey="Voytas, Df" uniqKey="Voytas D">DF Voytas</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Charo, Dn" uniqKey="Charo D">DN Charo</name>
</author>
<author>
<name sortKey="Ho, M" uniqKey="Ho M">M Ho</name>
</author>
<author>
<name sortKey="Fajardo, G" uniqKey="Fajardo G">G Fajardo</name>
</author>
<author>
<name sortKey="Kawana, M" uniqKey="Kawana M">M Kawana</name>
</author>
<author>
<name sortKey="Kundu, Rk" uniqKey="Kundu R">RK Kundu</name>
</author>
<author>
<name sortKey="Sheikh, Ay" uniqKey="Sheikh A">AY Sheikh</name>
</author>
<author>
<name sortKey="Finsterbach, Tp" uniqKey="Finsterbach T">TP Finsterbach</name>
</author>
<author>
<name sortKey="Leeper, Nj" uniqKey="Leeper N">NJ Leeper</name>
</author>
<author>
<name sortKey="Ernst, Kv" uniqKey="Ernst K">KV Ernst</name>
</author>
<author>
<name sortKey="Chen, Mm" uniqKey="Chen M">MM Chen</name>
</author>
<author>
<name sortKey="Ho, Yd" uniqKey="Ho Y">YD Ho</name>
</author>
<author>
<name sortKey="Chun, Hj" uniqKey="Chun H">HJ Chun</name>
</author>
<author>
<name sortKey="Bernstein, D" uniqKey="Bernstein D">D Bernstein</name>
</author>
<author>
<name sortKey="Ashley, Ea" uniqKey="Ashley E">EA Ashley</name>
</author>
<author>
<name sortKey="Quertermous, T" uniqKey="Quertermous T">T Quertermous</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Chng, Sc" uniqKey="Chng S">SC Chng</name>
</author>
<author>
<name sortKey="Ho, L" uniqKey="Ho L">L Ho</name>
</author>
<author>
<name sortKey="Tian, J" uniqKey="Tian J">J Tian</name>
</author>
<author>
<name sortKey="Reversade, B" uniqKey="Reversade B">B Reversade</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Cleaver, O" uniqKey="Cleaver O">O Cleaver</name>
</author>
<author>
<name sortKey="Krieg, Pa" uniqKey="Krieg P">PA Krieg</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Coultas, L" uniqKey="Coultas L">L Coultas</name>
</author>
<author>
<name sortKey="Chawengsaksophak, K" uniqKey="Chawengsaksophak K">K Chawengsaksophak</name>
</author>
<author>
<name sortKey="Rossant, J" uniqKey="Rossant J">J Rossant</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Ferrara, N" uniqKey="Ferrara N">N Ferrara</name>
</author>
<author>
<name sortKey="Carver Moore, K" uniqKey="Carver Moore K">K Carver-Moore</name>
</author>
<author>
<name sortKey="Chen, H" uniqKey="Chen H">H Chen</name>
</author>
<author>
<name sortKey="Dowd, M" uniqKey="Dowd M">M Dowd</name>
</author>
<author>
<name sortKey="Lu, L" uniqKey="Lu L">L Lu</name>
</author>
<author>
<name sortKey="O Shea, Ks" uniqKey="O Shea K">KS O'Shea</name>
</author>
<author>
<name sortKey="Powell Braxton, L" uniqKey="Powell Braxton L">L Powell-Braxton</name>
</author>
<author>
<name sortKey="Hillan, Kj" uniqKey="Hillan K">KJ Hillan</name>
</author>
<author>
<name sortKey="Moore, Mw" uniqKey="Moore M">MW Moore</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Fouquet, B" uniqKey="Fouquet B">B Fouquet</name>
</author>
<author>
<name sortKey="Weinstein, Bm" uniqKey="Weinstein B">BM Weinstein</name>
</author>
<author>
<name sortKey="Serluca, Fc" uniqKey="Serluca F">FC Serluca</name>
</author>
<author>
<name sortKey="Fishman, Mc" uniqKey="Fishman M">MC Fishman</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Gordon, K" uniqKey="Gordon K">K Gordon</name>
</author>
<author>
<name sortKey="Schulte, D" uniqKey="Schulte D">D Schulte</name>
</author>
<author>
<name sortKey="Brice, G" uniqKey="Brice G">G Brice</name>
</author>
<author>
<name sortKey="Simpson, Ma" uniqKey="Simpson M">MA Simpson</name>
</author>
<author>
<name sortKey="Roukens, Mg" uniqKey="Roukens M">MG Roukens</name>
</author>
<author>
<name sortKey="Van Impel, A" uniqKey="Van Impel A">A van Impel</name>
</author>
<author>
<name sortKey="Connell, F" uniqKey="Connell F">F Connell</name>
</author>
<author>
<name sortKey="Kalidas, K" uniqKey="Kalidas K">K Kalidas</name>
</author>
<author>
<name sortKey="Jeffery, S" uniqKey="Jeffery S">S Jeffery</name>
</author>
<author>
<name sortKey="Mortimer, Ps" uniqKey="Mortimer P">PS Mortimer</name>
</author>
<author>
<name sortKey="Mansour, S" uniqKey="Mansour S">S Mansour</name>
</author>
<author>
<name sortKey="Schulte Merker, S" uniqKey="Schulte Merker S">S Schulte-Merker</name>
</author>
<author>
<name sortKey="Ostergaard, P" uniqKey="Ostergaard P">P Ostergaard</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Gore, Av" uniqKey="Gore A">AV Gore</name>
</author>
<author>
<name sortKey="Monzo, K" uniqKey="Monzo K">K Monzo</name>
</author>
<author>
<name sortKey="Cha, Yr" uniqKey="Cha Y">YR Cha</name>
</author>
<author>
<name sortKey="Pan, W" uniqKey="Pan W">W Pan</name>
</author>
<author>
<name sortKey="Weinstein, Bm" uniqKey="Weinstein B">BM Weinstein</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Halpern, Me" uniqKey="Halpern M">ME Halpern</name>
</author>
<author>
<name sortKey="Ho, Rk" uniqKey="Ho R">RK Ho</name>
</author>
<author>
<name sortKey="Walker, C" uniqKey="Walker C">C Walker</name>
</author>
<author>
<name sortKey="Kimmel, Cb" uniqKey="Kimmel C">CB Kimmel</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Helker, Cs" uniqKey="Helker C">CS Helker</name>
</author>
<author>
<name sortKey="Schuermann, A" uniqKey="Schuermann A">A Schuermann</name>
</author>
<author>
<name sortKey="Karpanen, T" uniqKey="Karpanen T">T Karpanen</name>
</author>
<author>
<name sortKey="Zeuschner, D" uniqKey="Zeuschner D">D Zeuschner</name>
</author>
<author>
<name sortKey="Belting, Hg" uniqKey="Belting H">HG Belting</name>
</author>
<author>
<name sortKey="Affolter, M" uniqKey="Affolter M">M Affolter</name>
</author>
<author>
<name sortKey="Schulte Merker, S" uniqKey="Schulte Merker S">S Schulte-Merker</name>
</author>
<author>
<name sortKey="Herzog, W" uniqKey="Herzog W">W Herzog</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Hesselson, D" uniqKey="Hesselson D">D Hesselson</name>
</author>
<author>
<name sortKey="Anderson, Rm" uniqKey="Anderson R">RM Anderson</name>
</author>
<author>
<name sortKey="Beinat, M" uniqKey="Beinat M">M Beinat</name>
</author>
<author>
<name sortKey="Stainier, Dy" uniqKey="Stainier D">DY Stainier</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Hogan, Bm" uniqKey="Hogan B">BM Hogan</name>
</author>
<author>
<name sortKey="Bos, Fl" uniqKey="Bos F">FL Bos</name>
</author>
<author>
<name sortKey="Bussmann, J" uniqKey="Bussmann J">J Bussmann</name>
</author>
<author>
<name sortKey="Witte, M" uniqKey="Witte M">M Witte</name>
</author>
<author>
<name sortKey="Chi, Nc" uniqKey="Chi N">NC Chi</name>
</author>
<author>
<name sortKey="Duckers, Hj" uniqKey="Duckers H">HJ Duckers</name>
</author>
<author>
<name sortKey="Schulte Merker, S" uniqKey="Schulte Merker S">S Schulte-Merker</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Hwang, Wy" uniqKey="Hwang W">WY Hwang</name>
</author>
<author>
<name sortKey="Fu, Yf" uniqKey="Fu Y">YF Fu</name>
</author>
<author>
<name sortKey="Reyon, D" uniqKey="Reyon D">D Reyon</name>
</author>
<author>
<name sortKey="Maeder, Ml" uniqKey="Maeder M">ML Maeder</name>
</author>
<author>
<name sortKey="Tsai, Sq" uniqKey="Tsai S">SQ Tsai</name>
</author>
<author>
<name sortKey="Sander, Jd" uniqKey="Sander J">JD Sander</name>
</author>
<author>
<name sortKey="Peterson, Rt" uniqKey="Peterson R">RT Peterson</name>
</author>
<author>
<name sortKey="Yeh, Jrj" uniqKey="Yeh J">JRJ Yeh</name>
</author>
<author>
<name sortKey="Joung, Jk" uniqKey="Joung J">JK Joung</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Jao, Le" uniqKey="Jao L">LE Jao</name>
</author>
<author>
<name sortKey="Wente, Sr" uniqKey="Wente S">SR Wente</name>
</author>
<author>
<name sortKey="Chen, W" uniqKey="Chen W">W Chen</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Kawakami, K" uniqKey="Kawakami K">K Kawakami</name>
</author>
<author>
<name sortKey="Takeda, H" uniqKey="Takeda H">H Takeda</name>
</author>
<author>
<name sortKey="Kawakami, N" uniqKey="Kawakami N">N Kawakami</name>
</author>
<author>
<name sortKey="Kobayashi, M" uniqKey="Kobayashi M">M Kobayashi</name>
</author>
<author>
<name sortKey="Matsuda, N" uniqKey="Matsuda N">N Matsuda</name>
</author>
<author>
<name sortKey="Mishina, M" uniqKey="Mishina M">M Mishina</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Kimmel, Cb" uniqKey="Kimmel C">CB Kimmel</name>
</author>
<author>
<name sortKey="Ballard, Ww" uniqKey="Ballard W">WW Ballard</name>
</author>
<author>
<name sortKey="Kimmel, Sr" uniqKey="Kimmel S">SR Kimmel</name>
</author>
<author>
<name sortKey="Ullmann, B" uniqKey="Ullmann B">B Ullmann</name>
</author>
<author>
<name sortKey="Schilling, Tf" uniqKey="Schilling T">TF Schilling</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Larson, Jd" uniqKey="Larson J">JD Larson</name>
</author>
<author>
<name sortKey="Wadman, Sa" uniqKey="Wadman S">SA Wadman</name>
</author>
<author>
<name sortKey="Chen, E" uniqKey="Chen E">E Chen</name>
</author>
<author>
<name sortKey="Kerley, L" uniqKey="Kerley L">L Kerley</name>
</author>
<author>
<name sortKey="Clark, Kj" uniqKey="Clark K">KJ Clark</name>
</author>
<author>
<name sortKey="Eide, M" uniqKey="Eide M">M Eide</name>
</author>
<author>
<name sortKey="Lippert, S" uniqKey="Lippert S">S Lippert</name>
</author>
<author>
<name sortKey="Nasevicius, A" uniqKey="Nasevicius A">A Nasevicius</name>
</author>
<author>
<name sortKey="Ekker, Sc" uniqKey="Ekker S">SC Ekker</name>
</author>
<author>
<name sortKey="Hackett, Pb" uniqKey="Hackett P">PB Hackett</name>
</author>
<author>
<name sortKey="Essner, Jj" uniqKey="Essner J">JJ Essner</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Lawson, Nd" uniqKey="Lawson N">ND Lawson</name>
</author>
<author>
<name sortKey="Vogel, Am" uniqKey="Vogel A">AM Vogel</name>
</author>
<author>
<name sortKey="Weinstein, Bm" uniqKey="Weinstein B">BM Weinstein</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Lawson, Nd" uniqKey="Lawson N">ND Lawson</name>
</author>
<author>
<name sortKey="Weinstein, Bm" uniqKey="Weinstein B">BM Weinstein</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Nasevicius, A" uniqKey="Nasevicius A">A Nasevicius</name>
</author>
<author>
<name sortKey="Ekker, Sc" uniqKey="Ekker S">SC Ekker</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Nicoli, S" uniqKey="Nicoli S">S Nicoli</name>
</author>
<author>
<name sortKey="Tobia, C" uniqKey="Tobia C">C Tobia</name>
</author>
<author>
<name sortKey="Gualandi, L" uniqKey="Gualandi L">L Gualandi</name>
</author>
<author>
<name sortKey="De Sena, G" uniqKey="De Sena G">G De Sena</name>
</author>
<author>
<name sortKey="Presta, M" uniqKey="Presta M">M Presta</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Ober, Ea" uniqKey="Ober E">EA Ober</name>
</author>
<author>
<name sortKey="Olofsson, B" uniqKey="Olofsson B">B Olofsson</name>
</author>
<author>
<name sortKey="Makinen, T" uniqKey="Makinen T">T Makinen</name>
</author>
<author>
<name sortKey="Jin, Sw" uniqKey="Jin S">SW Jin</name>
</author>
<author>
<name sortKey="Shoji, W" uniqKey="Shoji W">W Shoji</name>
</author>
<author>
<name sortKey="Koh, Gy" uniqKey="Koh G">GY Koh</name>
</author>
<author>
<name sortKey="Alitalo, K" uniqKey="Alitalo K">K Alitalo</name>
</author>
<author>
<name sortKey="Stainier, Dy" uniqKey="Stainier D">DY Stainier</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Odenthal, J" uniqKey="Odenthal J">J Odenthal</name>
</author>
<author>
<name sortKey="Haffter, P" uniqKey="Haffter P">P Haffter</name>
</author>
<author>
<name sortKey="Vogelsang, E" uniqKey="Vogelsang E">E Vogelsang</name>
</author>
<author>
<name sortKey="Brand, M" uniqKey="Brand M">M Brand</name>
</author>
<author>
<name sortKey="Van Eeden, Fj" uniqKey="Van Eeden F">FJ van Eeden</name>
</author>
<author>
<name sortKey="Furutani Seiki, M" uniqKey="Furutani Seiki M">M Furutani-Seiki</name>
</author>
<author>
<name sortKey="Granato, M" uniqKey="Granato M">M Granato</name>
</author>
<author>
<name sortKey="Hammerschmidt, M" uniqKey="Hammerschmidt M">M Hammerschmidt</name>
</author>
<author>
<name sortKey="Heisenberg, Cp" uniqKey="Heisenberg C">CP Heisenberg</name>
</author>
<author>
<name sortKey="Jiang, Yj" uniqKey="Jiang Y">YJ Jiang</name>
</author>
<author>
<name sortKey="Kane, Da" uniqKey="Kane D">DA Kane</name>
</author>
<author>
<name sortKey="Kelsh, Rn" uniqKey="Kelsh R">RN Kelsh</name>
</author>
<author>
<name sortKey="Mullins, Mc" uniqKey="Mullins M">MC Mullins</name>
</author>
<author>
<name sortKey="Warga, Rm" uniqKey="Warga R">RM Warga</name>
</author>
<author>
<name sortKey="Allende, Ml" uniqKey="Allende M">ML Allende</name>
</author>
<author>
<name sortKey="Weinberg, Es" uniqKey="Weinberg E">ES Weinberg</name>
</author>
<author>
<name sortKey="Nusslein Volhard, C" uniqKey="Nusslein Volhard C">C Nüsslein-Volhard</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Pauli, A" uniqKey="Pauli A">A Pauli</name>
</author>
<author>
<name sortKey="Norris, Ml" uniqKey="Norris M">ML Norris</name>
</author>
<author>
<name sortKey="Valen, E" uniqKey="Valen E">E Valen</name>
</author>
<author>
<name sortKey="Chew, Gl" uniqKey="Chew G">GL Chew</name>
</author>
<author>
<name sortKey="Gagnon, Ja" uniqKey="Gagnon J">JA Gagnon</name>
</author>
<author>
<name sortKey="Zimmerman, S" uniqKey="Zimmerman S">S Zimmerman</name>
</author>
<author>
<name sortKey="Mitchell, A" uniqKey="Mitchell A">A Mitchell</name>
</author>
<author>
<name sortKey="Ma, J" uniqKey="Ma J">J Ma</name>
</author>
<author>
<name sortKey="Dubrulle, J" uniqKey="Dubrulle J">J Dubrulle</name>
</author>
<author>
<name sortKey="Reyon, D" uniqKey="Reyon D">D Reyon</name>
</author>
<author>
<name sortKey="Tsai, Sq" uniqKey="Tsai S">SQ Tsai</name>
</author>
<author>
<name sortKey="Joung, Jk" uniqKey="Joung J">JK Joung</name>
</author>
<author>
<name sortKey="Saghatelian, A" uniqKey="Saghatelian A">A Saghatelian</name>
</author>
<author>
<name sortKey="Schier, Af" uniqKey="Schier A">AF Schier</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Santoro, Mm" uniqKey="Santoro M">MM Santoro</name>
</author>
<author>
<name sortKey="Samuel, T" uniqKey="Samuel T">T Samuel</name>
</author>
<author>
<name sortKey="Mitchell, T" uniqKey="Mitchell T">T Mitchell</name>
</author>
<author>
<name sortKey="Reed, Jc" uniqKey="Reed J">JC Reed</name>
</author>
<author>
<name sortKey="Stainier, Dy" uniqKey="Stainier D">DY Stainier</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Scott, Ic" uniqKey="Scott I">IC Scott</name>
</author>
<author>
<name sortKey="Masri, B" uniqKey="Masri B">B Masri</name>
</author>
<author>
<name sortKey="D Amico, La" uniqKey="D Amico L">LA D'Amico</name>
</author>
<author>
<name sortKey="Jin, Sw" uniqKey="Jin S">SW Jin</name>
</author>
<author>
<name sortKey="Jungblut, B" uniqKey="Jungblut B">B Jungblut</name>
</author>
<author>
<name sortKey="Wehman, Am" uniqKey="Wehman A">AM Wehman</name>
</author>
<author>
<name sortKey="Baier, H" uniqKey="Baier H">H Baier</name>
</author>
<author>
<name sortKey="Audigier, Y" uniqKey="Audigier Y">Y Audigier</name>
</author>
<author>
<name sortKey="Stainier, Dy" uniqKey="Stainier D">DY Stainier</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Shalaby, F" uniqKey="Shalaby F">F Shalaby</name>
</author>
<author>
<name sortKey="Rossant, J" uniqKey="Rossant J">J Rossant</name>
</author>
<author>
<name sortKey="Yamaguchi, Tp" uniqKey="Yamaguchi T">TP Yamaguchi</name>
</author>
<author>
<name sortKey="Gertsenstein, M" uniqKey="Gertsenstein M">M Gertsenstein</name>
</author>
<author>
<name sortKey="Wu, Xf" uniqKey="Wu X">XF Wu</name>
</author>
<author>
<name sortKey="Breitman, Ml" uniqKey="Breitman M">ML Breitman</name>
</author>
<author>
<name sortKey="Schuh, Ac" uniqKey="Schuh A">AC Schuh</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Sumoy, L" uniqKey="Sumoy L">L Sumoy</name>
</author>
<author>
<name sortKey="Keasey, Jb" uniqKey="Keasey J">JB Keasey</name>
</author>
<author>
<name sortKey="Dittman, Td" uniqKey="Dittman T">TD Dittman</name>
</author>
<author>
<name sortKey="Kimelman, D" uniqKey="Kimelman D">D Kimelman</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Verma, A" uniqKey="Verma A">A Verma</name>
</author>
<author>
<name sortKey="Bhattacharya, R" uniqKey="Bhattacharya R">R Bhattacharya</name>
</author>
<author>
<name sortKey="Remadevi, I" uniqKey="Remadevi I">I Remadevi</name>
</author>
<author>
<name sortKey="Li, K" uniqKey="Li K">K Li</name>
</author>
<author>
<name sortKey="Pramanik, K" uniqKey="Pramanik K">K Pramanik</name>
</author>
<author>
<name sortKey="Samant, Gv" uniqKey="Samant G">GV Samant</name>
</author>
<author>
<name sortKey="Horswill, M" uniqKey="Horswill M">M Horswill</name>
</author>
<author>
<name sortKey="Chun, Cz" uniqKey="Chun C">CZ Chun</name>
</author>
<author>
<name sortKey="Zhao, B" uniqKey="Zhao B">B Zhao</name>
</author>
<author>
<name sortKey="Wang, E" uniqKey="Wang E">E Wang</name>
</author>
<author>
<name sortKey="Miao, Rq" uniqKey="Miao R">RQ Miao</name>
</author>
<author>
<name sortKey="Mukhopadhyay, D" uniqKey="Mukhopadhyay D">D Mukhopadhyay</name>
</author>
<author>
<name sortKey="Ramchandran, R" uniqKey="Ramchandran R">R Ramchandran</name>
</author>
<author>
<name sortKey="Wilkinson, Ga" uniqKey="Wilkinson G">GA Wilkinson</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Westerfield, M" uniqKey="Westerfield M">M Westerfield</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Zeng, Xx" uniqKey="Zeng X">XX Zeng</name>
</author>
<author>
<name sortKey="Wilm, Tp" uniqKey="Wilm T">TP Wilm</name>
</author>
<author>
<name sortKey="Sepich, Ds" uniqKey="Sepich D">DS Sepich</name>
</author>
<author>
<name sortKey="Solnica Krezel, L" uniqKey="Solnica Krezel L">L Solnica-Krezel</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Zhang, S" uniqKey="Zhang S">S Zhang</name>
</author>
<author>
<name sortKey="Cao, Z" uniqKey="Cao Z">Z Cao</name>
</author>
<author>
<name sortKey="Tian, H" uniqKey="Tian H">H Tian</name>
</author>
<author>
<name sortKey="Shen, G" uniqKey="Shen G">G Shen</name>
</author>
<author>
<name sortKey="Ma, Y" uniqKey="Ma Y">Y Ma</name>
</author>
<author>
<name sortKey="Xie, H" uniqKey="Xie H">H Xie</name>
</author>
<author>
<name sortKey="Liu, Y" uniqKey="Liu Y">Y Liu</name>
</author>
<author>
<name sortKey="Zhao, C" uniqKey="Zhao C">C Zhao</name>
</author>
<author>
<name sortKey="Deng, S" uniqKey="Deng S">S Deng</name>
</author>
<author>
<name sortKey="Yang, Y" uniqKey="Yang Y">Y Yang</name>
</author>
<author>
<name sortKey="Zheng, R" uniqKey="Zheng R">R Zheng</name>
</author>
<author>
<name sortKey="Li, W" uniqKey="Li W">W Li</name>
</author>
<author>
<name sortKey="Zhang, N" uniqKey="Zhang N">N Zhang</name>
</author>
<author>
<name sortKey="Liu, S" uniqKey="Liu S">S Liu</name>
</author>
<author>
<name sortKey="Wang, W" uniqKey="Wang W">W Wang</name>
</author>
<author>
<name sortKey="Dai, L" uniqKey="Dai L">L Dai</name>
</author>
<author>
<name sortKey="Shi, S" uniqKey="Shi S">S Shi</name>
</author>
<author>
<name sortKey="Cheng, L" uniqKey="Cheng L">L Cheng</name>
</author>
<author>
<name sortKey="Pan, Y" uniqKey="Pan Y">Y Pan</name>
</author>
<author>
<name sortKey="Feng, S" uniqKey="Feng S">S Feng</name>
</author>
<author>
<name sortKey="Zhao, X" uniqKey="Zhao X">X Zhao</name>
</author>
<author>
<name sortKey="Deng, H" uniqKey="Deng H">H Deng</name>
</author>
<author>
<name sortKey="Yang, S" uniqKey="Yang S">S Yang</name>
</author>
<author>
<name sortKey="Wei, Y" uniqKey="Wei Y">Y Wei</name>
</author>
</analytic>
</biblStruct>
</listBibl>
</div1>
</back>
</TEI>
<pmc article-type="research-article">
<pmc-dir>properties open_access</pmc-dir>
<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 pub-type="epub">2050-084X</issn>
<publisher>
<publisher-name>eLife Sciences Publications, Ltd</publisher-name>
</publisher>
</journal-meta>
<article-meta>
<article-id pub-id-type="pmid">26017639</article-id>
<article-id pub-id-type="pmc">4468421</article-id>
<article-id pub-id-type="publisher-id">06726</article-id>
<article-id pub-id-type="doi">10.7554/eLife.06726</article-id>
<article-categories>
<subj-group subj-group-type="display-channel">
<subject>Short Report</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>Human Biology and Medicine</subject>
</subj-group>
</article-categories>
<title-group>
<article-title>The hormonal peptide Elabela guides angioblasts to the midline during vasculogenesis</article-title>
</title-group>
<contrib-group>
<contrib id="author-28584" contrib-type="author">
<name>
<surname>Helker</surname>
<given-names>Christian SM</given-names>
</name>
<xref ref-type="aff" rid="aff1">1</xref>
<xref ref-type="corresp" rid="cor1">*</xref>
<xref ref-type="author-notes" rid="equal-contrib">†</xref>
<xref ref-type="fn" rid="con1"></xref>
<xref ref-type="fn" rid="conf1"></xref>
</contrib>
<contrib id="author-28585" contrib-type="author">
<name>
<surname>Schuermann</surname>
<given-names>Annika</given-names>
</name>
<xref ref-type="aff" rid="aff1">1</xref>
<xref ref-type="author-notes" rid="equal-contrib">†</xref>
<xref ref-type="fn" rid="con2"></xref>
<xref ref-type="fn" rid="conf1"></xref>
</contrib>
<contrib id="author-28586" contrib-type="author">
<name>
<surname>Pollmann</surname>
<given-names>Cathrin</given-names>
</name>
<xref ref-type="aff" rid="aff2">2</xref>
<xref ref-type="fn" rid="con3"></xref>
<xref ref-type="fn" rid="conf1"></xref>
</contrib>
<contrib id="author-28587" contrib-type="author">
<name>
<surname>Chng</surname>
<given-names>Serene C</given-names>
</name>
<xref ref-type="aff" rid="aff3">3</xref>
<xref ref-type="fn" rid="con5"></xref>
<xref ref-type="fn" rid="conf1"></xref>
</contrib>
<contrib id="author-33806" contrib-type="author">
<name>
<surname>Kiefer</surname>
<given-names>Friedemann</given-names>
</name>
<xref ref-type="aff" rid="aff2">2</xref>
<xref ref-type="aff" rid="aff4">4</xref>
<xref ref-type="other" rid="par-4"></xref>
<xref ref-type="fn" rid="con4"></xref>
<xref ref-type="fn" rid="conf1"></xref>
</contrib>
<contrib id="author-28355" contrib-type="author">
<name>
<surname>Reversade</surname>
<given-names>Bruno</given-names>
</name>
<xref ref-type="aff" rid="aff3">3</xref>
<xref ref-type="other" rid="par-3"></xref>
<xref ref-type="fn" rid="con6"></xref>
<xref ref-type="fn" rid="conf1"></xref>
</contrib>
<contrib id="author-28001" contrib-type="author">
<name>
<surname>Herzog</surname>
<given-names>Wiebke</given-names>
</name>
<xref ref-type="aff" rid="aff1">1</xref>
<xref ref-type="aff" rid="aff2">2</xref>
<xref ref-type="aff" rid="aff4">4</xref>
<xref ref-type="corresp" rid="cor2">*</xref>
<xref ref-type="other" rid="par-1"></xref>
<xref ref-type="other" rid="par-2"></xref>
<xref ref-type="fn" rid="con7"></xref>
<xref ref-type="fn" rid="conf1"></xref>
</contrib>
<aff id="aff1">
<label>1</label>
<institution>University of Muenster</institution>
,
<addr-line>Muenster</addr-line>
,
<country>Germany</country>
</aff>
<aff id="aff2">
<label>2</label>
<institution>Max Planck Institute for Molecular Biomedicine</institution>
,
<addr-line>Muenster</addr-line>
,
<country>Germany</country>
</aff>
<aff id="aff3">
<label>3</label>
<institution content-type="dept">Institute of Medical Biology, Human Genetics and Embryology Laboratory</institution>
,
<institution>A*STAR</institution>
,
<addr-line>Singapore</addr-line>
,
<country>Singapore</country>
</aff>
<aff id="aff4">
<label>4</label>
<institution content-type="dept">Cells-in-Motion Cluster of Excellence</institution>
,
<institution>University of Muenster</institution>
,
<addr-line>Muenster</addr-line>
,
<country>Germany</country>
</aff>
</contrib-group>
<contrib-group>
<contrib id="author-1203" contrib-type="editor">
<name>
<surname>Whitfield</surname>
<given-names>Tanya T</given-names>
</name>
<role>Reviewing editor</role>
<aff>
<institution>University of Sheffield</institution>
,
<country>United Kingdom</country>
</aff>
</contrib>
</contrib-group>
<author-notes>
<corresp id="cor1">
<label>*</label>
For correspondence:
<email>Christian.Helker@mpi-bn.mpg.de</email>
(CSMH);</corresp>
<corresp id="cor2">
<email>wiebke.herzog@mpi-muenster.mpg.de</email>
(WH)</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>27</day>
<month>5</month>
<year>2015</year>
</pub-date>
<pub-date pub-type="collection">
<year>2015</year>
</pub-date>
<volume>4</volume>
<elocation-id>e06726</elocation-id>
<history>
<date date-type="received">
<day>27</day>
<month>1</month>
<year>2015</year>
</date>
<date date-type="accepted">
<day>22</day>
<month>5</month>
<year>2015</year>
</date>
</history>
<permissions>
<copyright-statement>© 2015, Helker et al</copyright-statement>
<copyright-year>2015</copyright-year>
<copyright-holder>Helker 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="elife-06726.pdf"></self-uri>
<abstract>
<p>A key step in the de novo formation of the embryonic vasculature is the migration of endothelial precursors, the angioblasts, to the position of the future vessels. To form the first axial vessels, angioblasts migrate towards the midline and coalesce underneath the notochord. Vascular endothelial growth factor has been proposed to serve as a chemoattractant for the angioblasts and to regulate this medial migration. Here we challenge this model and instead demonstrate that angioblasts rely on their intrinsic expression of Apelin receptors (Aplr, APJ) for their migration to the midline. We further show that during this angioblast migration Apelin receptor signaling is mainly triggered by the recently discovered ligand Elabela (Ela). As neither of the ligands Ela or Apelin (Apln) nor their receptors have previously been implicated in regulating angioblast migration, we hereby provide a novel mechanism for regulating vasculogenesis, with direct relevance to physiological and pathological angiogenesis.</p>
<p>
<bold>DOI:</bold>
<ext-link ext-link-type="doi" xlink:href="10.7554/eLife.06726.001">http://dx.doi.org/10.7554/eLife.06726.001</ext-link>
</p>
</abstract>
<abstract abstract-type="executive-summary">
<title>eLife digest</title>
<p>The circulatory system enables blood to move around the body and deliver substances including nutrients and oxygen to the cells that need them. In the embryos of animals with a backbone, blood flows from the heart through the aorta into branching smaller vessels to the cells. The blood then gets collected by progressively bigger vessels and flows back to the heart via the cardinal vein. The cells that make up these blood vessels develop from cells called angioblasts—but first, during development these angioblasts must move to the place where the vessels will form.</p>
<p>A protein called Vascular endothelial growth factor (VEGF) had been suggested to help guide and align the angioblasts as the embryo develops. Now, Helker, Schuermann et al. have examined developing zebrafish embryos using new technologies. This revealed that VEGF is in fact not essential for the dorsal aorta and cardinal vein to develop. Instead, the angioblasts only move to the correct part of the embryo if they can produce the Apelin receptor protein, which forms part of a signaling pathway.</p>
<p>There are two hormones—called Apelin and Elabela—that can bind to and activate the Apelin receptor. Helker, Schuermann et al. show that Elabela alone is needed to guide the angioblasts to the right part of the embryo during blood vessel development. However, in embryos where there is not enough Elabela, the Apelin hormone can compensate for this deficiency and the first blood vessels will later develop correctly. Future research will address whether this signaling pathway not only guides angioblasts to establish a circulatory system, but also guides blood vessel growth. As blood vessel growth is very relevant to human disease, identifying the mechanisms that regulate it will have an impact on biomedical research.</p>
<p>
<bold>DOI:</bold>
<ext-link ext-link-type="doi" xlink:href="10.7554/eLife.06726.002">http://dx.doi.org/10.7554/eLife.06726.002</ext-link>
</p>
</abstract>
<kwd-group kwd-group-type="author-keywords">
<title>Author keywords</title>
<kwd>angioblast migration</kwd>
<kwd>endothelial cell migration</kwd>
<kwd>Vegf</kwd>
<kwd>dorsal aorta</kwd>
<kwd>chemoattractant</kwd>
<kwd>notochord</kwd>
</kwd-group>
<kwd-group kwd-group-type="research-organism">
<title>Research organism</title>
<kwd>Zebrafish</kwd>
</kwd-group>
<funding-group>
<award-group id="par-1">
<funding-source>
<institution-wrap>
<institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100003484</institution-id>
<institution>State of Northrhine Westphalia</institution>
</institution-wrap>
</funding-source>
<award-id>Northrhine Westphalia Return Fellowship</award-id>
<principal-award-recipient>
<name>
<surname>Herzog</surname>
<given-names>Wiebke</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/10.13039/501100004869</institution-id>
<institution>Westfälische Wilhelms-Universität Münster</institution>
</institution-wrap>
</funding-source>
<award-id>Cells in Motion cluster of Excellence, CiM - EXC 1003 / FF-2013-14</award-id>
<principal-award-recipient>
<name>
<surname>Herzog</surname>
<given-names>Wiebke</given-names>
</name>
</principal-award-recipient>
</award-group>
<award-group id="par-3">
<funding-source>
<institution-wrap>
<institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100001348</institution-id>
<institution>Agency for Science, Technology and Research (A*STAR)</institution>
</institution-wrap>
</funding-source>
<award-id>Strategic Positioning Fund on Genetic Orphan Diseases</award-id>
<principal-award-recipient>
<name>
<surname>Reversade</surname>
<given-names>Bruno</given-names>
</name>
</principal-award-recipient>
</award-group>
<award-group id="par-4">
<funding-source>
<institution-wrap>
<institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100004189</institution-id>
<institution>Max-Planck-Gesellschaft</institution>
</institution-wrap>
</funding-source>
<principal-award-recipient>
<name>
<surname>Kiefer</surname>
<given-names>Friedemann</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>2.3</meta-value>
</custom-meta>
<custom-meta specific-use="meta-only">
<meta-name>Author impact statement</meta-name>
<meta-value>Elabela-Apelin receptor signaling is a novel pathway that regulates angioblast migration, instead of the proposed migration regulation through Vascular endothelial growth factor.</meta-value>
</custom-meta>
</custom-meta-group>
</article-meta>
</front>
<body>
<sec id="s1">
<title>Main text</title>
<p>In the vertebrate embryo, the formation of the large axial vessels, namely the dorsal aorta (DA) and the cardinal vein (CV), establishes a first circulatory loop and thereby the core of the developing cardiovascular system. Angioblasts are initially specified in the lateral plate mesoderm and migrate between the somites towards the midline, where they coalesce and assemble the DA and the CV underneath the notochord (NC) (
<xref ref-type="fig" rid="fig1">Figure 1A,B–F</xref>
,
<xref ref-type="other" rid="media1">Video 1</xref>
).
<fig id="fig1" position="float" orientation="portrait">
<object-id pub-id-type="doi">10.7554/eLife.06726.003</object-id>
<label>Figure 1</label>
<caption>
<title>Angioblast migration to the midline is not regulated by Vegf.</title>
<p>(
<bold>A</bold>
–
<bold>F</bold>
) Schematic (
<bold>A</bold>
) and confocal (
<bold>B</bold>
–
<bold>F</bold>
) in vivo time-lapse imaging of angioblasts (green) and their migration in a
<italic>Tg(fli1a:EGFP)</italic>
<sup>
<italic>y1</italic>
</sup>
embryo, injected with
<italic>H2B-mCherry</italic>
mRNA (purple, all nuclei), dorsal views at indicated time points. Arrows indicate the initial migration to the midline, and the coalescing into the dorsal aorta (DA). (
<bold>G</bold>
–
<bold>J</bold>
) Loss of Vegf-signaling components by
<italic>vegfaa/vegfab</italic>
double morpholino (MO) injection (
<bold>H</bold>
) or in
<italic>vegf receptor 2</italic>
(
<italic>kdrl</italic>
) mutants (
<bold>J</bold>
) does not affect angioblast migration. Confocal projections of
<italic>Tg(fli1a:EGFP)</italic>
<sup>
<italic>y1</italic>
</sup>
embryos in dorsal views at 17 hpf. For each condition, n > 15.</p>
<p>
<bold>DOI:</bold>
<ext-link ext-link-type="doi" xlink:href="10.7554/eLife.06726.003">http://dx.doi.org/10.7554/eLife.06726.003</ext-link>
</p>
<p>
<supplementary-material content-type="local-data" id="SD1-data">
<object-id pub-id-type="doi">10.7554/eLife.06726.004</object-id>
<label>Figure 1—source movie 1.</label>
<caption>
<title>Time-lapse movie showing angioblast (green) migration to the midline analyzed using
<italic>Tg(fli1a:EGFP)
<sup>y1</sup>
</italic>
embryos injected with
<italic>H2B-mCherry</italic>
mRNA (purple, all nuclei).</title>
<p>
<bold>DOI:</bold>
<ext-link ext-link-type="doi" xlink:href="10.7554/eLife.06726.004">http://dx.doi.org/10.7554/eLife.06726.004</ext-link>
</p>
</caption>
<media mime-subtype="avi" mimetype="video" xlink:href="elife-06726-fig1-data1.avi" orientation="portrait" id="d35e480" position="anchor"></media>
</supplementary-material>
</p>
</caption>
<graphic xlink:href="elife-06726-fig1"></graphic>
<p content-type="supplemental-figure">
<fig id="fig1s1" specific-use="child-fig" orientation="portrait" position="anchor">
<object-id pub-id-type="doi">10.7554/eLife.06726.005</object-id>
<label>Figure 1—figure supplement 1.</label>
<caption>
<title>Inhibition of Vegfa signaling resulted in normal angioblast migration to the midline, but impaired sprouting angiogenesis.</title>
<p>(
<bold>A</bold>
–
<bold>J</bold>
) Vegfa signaling was impaired using either ligand MOs (
<italic>vegfaa/vegfab</italic>
MO), chemical inhibition of Vegfr2 signaling (2.5 μM SLKB1002 [
<xref rid="bib34" ref-type="bibr">Zhang et al., 2011</xref>
]) or mutations in the
<italic>vegfr2</italic>
gene (
<italic>kdrl</italic>
). Confocal projections of
<italic>Tg(fli1a:EGFP)</italic>
<sup>
<italic>y1</italic>
</sup>
embryos in dorsal views at 17 hpf (
<bold>A</bold>
,
<bold>B</bold>
,
<bold>C</bold>
,
<bold>G</bold>
,
<bold>H</bold>
,
<bold>I</bold>
), 24 hpf (
<bold>F</bold>
,
<bold>J</bold>
,
<bold>K</bold>
,
<bold>L</bold>
) and 28 hpf (
<bold>D</bold>
,
<bold>E</bold>
). At 17 hpf, angioblasts in control embryos as well as Vegfa signaling deficient embryos arrived at the midline (
<bold>A</bold>
,
<bold>B</bold>
,
<bold>C</bold>
,
<bold>G</bold>
,
<bold>H</bold>
,
<bold>I</bold>
). Control embryos showed sprouting of the intersegmental vessels (
<bold>D</bold>
,
<bold>F</bold>
,
<bold>K</bold>
) whereas Vegfa signaling deficient embryos lacked the intersegmental vessels (
<bold>E</bold>
,
<bold>J</bold>
,
<bold>I</bold>
).</p>
<p>
<bold>DOI:</bold>
<ext-link ext-link-type="doi" xlink:href="10.7554/eLife.06726.005">http://dx.doi.org/10.7554/eLife.06726.005</ext-link>
</p>
</caption>
<graphic xlink:href="elife-06726-fig1-figsupp1"></graphic>
</fig>
</p>
<p content-type="supplemental-figure">
<fig id="fig1s2" specific-use="child-fig" orientation="portrait" position="anchor">
<object-id pub-id-type="doi">10.7554/eLife.06726.006</object-id>
<label>Figure 1—figure supplement 2.</label>
<caption>
<title>Abrogation of Shh signaling abolishes
<italic>vegfaa</italic>
expression, but does not influence angioblast migration.</title>
<p>(
<bold>A</bold>
,
<bold>B</bold>
) Embryos treated with 100 μM Cyclopamine (CyA) from 11 hpf to 18 hpf to block Sonic hedgehog signaling showed no defects in medial angioblast migration. Confocal projections of
<italic>Tg(fli1a:EGFP)</italic>
<sup>
<italic>y1</italic>
</sup>
embryos in dorsal views at 18 hpf. c-d)
<italic>vegfaa</italic>
expression in the somites is abolished in embryos treated with 100 μM Cyclopamine (CyA) from 11 hpf to 18 hpf.</p>
<p>
<bold>DOI:</bold>
<ext-link ext-link-type="doi" xlink:href="10.7554/eLife.06726.006">http://dx.doi.org/10.7554/eLife.06726.006</ext-link>
</p>
</caption>
<graphic xlink:href="elife-06726-fig1-figsupp2"></graphic>
</fig>
</p>
</fig>
<fig id="media1" position="anchor">
<label>Video 1.</label>
<caption>
<title>Angioblast migration between 12.5 hpf and 16 hpf observed by time-lapse imaging using
<italic>Tg(fli1a:EGFP)</italic>
<sup>
<italic>y1</italic>
</sup>
to visualize angioblasts.</title>
<p>Angioblasts migrate to the midline in Wt embryos.</p>
<p>
<bold>DOI:</bold>
<ext-link ext-link-type="doi" xlink:href="10.7554/eLife.06726.007">http://dx.doi.org/10.7554/eLife.06726.007</ext-link>
</p>
</caption>
<media xlink:href="elife-06726-media1.avi" mimetype="video" mime-subtype="x-msvideo">
<object-id pub-id-type="doi">10.7554/eLife.06726.007</object-id>
</media>
</fig>
</p>
<p>It has been previously proposed that this process is regulated by Vascular endothelial growth factor A (VEGF-A) (
<xref rid="bib6" ref-type="bibr">Coultas et al., 2005</xref>
;
<xref rid="bib31" ref-type="bibr">Verma et al., 2010</xref>
;
<xref rid="bib10" ref-type="bibr">Gore et al., 2012</xref>
), a master regulator of vascular growth in the embryonic and adult organism. In
<italic>Xenopus</italic>
embryos, transcripts for the
<italic>vegfa</italic>
gene are expressed in the midline and migrating angioblasts were guided towards sites of ectopic Vegfa expression (
<xref rid="bib5" ref-type="bibr">Cleaver and Krieg, 1998</xref>
). Global inactivation of the genes encoding VEGF-A or the corresponding receptor VEGFR2 in mice led to strong vascular defects affecting the whole vascular network including the DA (
<xref rid="bib29" ref-type="bibr">Shalaby et al., 1995</xref>
;
<xref rid="bib7" ref-type="bibr">Ferrara et al., 1996</xref>
). Based on these data it was concluded that in vertebrates VEGF-A signaling regulates angioblast migration to the midline (
<xref rid="bib6" ref-type="bibr">Coultas et al., 2005</xref>
;
<xref rid="bib31" ref-type="bibr">Verma et al., 2010</xref>
;
<xref rid="bib10" ref-type="bibr">Gore et al., 2012</xref>
). To get direct, mechanistic and dynamic insight into angioblast migration and formation of the great vessels, we decided to investigate these processes in zebrafish embryos.</p>
<p>As previously published (
<xref rid="bib23" ref-type="bibr">Nicoli et al., 2008</xref>
), expression analysis of
<italic>vegfaa,</italic>
the gene for the main VEGF-A ortholog in zebrafish, showed the presence of transcripts in the somites between 12 and 15 hr post fertilization (hpf), that is the stage when angioblasts migrate to the midline (see
<xref ref-type="fig" rid="fig1">Figure 1B–F</xref>
). To analyze whether Vegfa signaling, as previously proposed, is required for angioblast migration to the midline, we analyzed loss-of-function zebrafish embryos with deficiencies in the Vegfa signaling pathway resulting from genomic mutations as well as morpholino (MO) -mediated knockdown. To rule out developmental delays, we counted the somites of the embryos and fixed them for confocal analysis, when they had developed 16–17 somites (equivalent to 17 hpf). Surprisingly, neither Vegfa ligand depletion (
<italic>vegfaa/vegfab</italic>
MO) nor a null mutation in the gene encoding the VEGFR2 ortholog (
<italic>kdrl</italic>
) interfered with angioblast migration in zebrafish embryos (
<xref ref-type="fig" rid="fig1">Figure 1H,J</xref>
), although Vegfa signaling dependent phenotypes, like failure to form the intersegmental vessels, could be observed after 24 hpf (
<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>
). Additionally, pharmacological inhibition of Vegfr2 signaling from 6 to 17 hpf did not impair angioblast migration to the midline (
<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>
). As activity of Sonic Hedgehog (Shh) in the midline has been shown to induce
<italic>vegfaa</italic>
expression (
<xref rid="bib20" ref-type="bibr">Lawson et al., 2002</xref>
), we repeated the published experiments and blocked this process by chemical inhibition of Shh signaling using cyclopamine. In line with our previous experiments, angioblast migration was not affected after Shh inhibition (
<xref ref-type="fig" rid="fig1s2">Figure 1—figure supplement 2</xref>
). The sum of these data strongly indicates that Vegfa signaling is dispensable for angioblast specification or migration in zebrafish.</p>
<p>In order to identify the endogenous signal(s) guiding angioblast migration, we further analyzed the expression pattern of factors involved in cell motility and directed cell migration. As previously published (
<xref rid="bib33" ref-type="bibr">Zeng et al., 2007</xref>
), we detected the expression of Apelin, a short secreted peptide encoded by the
<italic>apln</italic>
gene
<italic>,</italic>
by the NC (
<xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1C</xref>
). At the same time, transcripts of its two paralogous receptors
<italic>apelin receptor a</italic>
(
<italic>aplnra</italic>
) and
<italic>apelin receptor b</italic>
(
<italic>aplnrb</italic>
) were present in angioblasts (
<xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1A,B</xref>
; [
<xref rid="bib28" ref-type="bibr">Scott et al., 2007</xref>
;
<xref rid="bib33" ref-type="bibr">Zeng et al., 2007</xref>
]). Using MO-mediated gene knockdown, we observed that both
<italic>aplnra</italic>
and
<italic>aplnrb</italic>
are important for midline migration of angioblasts. While absence of each individual receptor reduced the efficiency of migration towards the midline, double depletion completely abolished this process (
<xref ref-type="fig" rid="fig2">Figure 2A</xref>
,
<xref ref-type="other" rid="media2">Video 2</xref>
). We used TALEN or CRISPR/Cas9-mediated gene editing (
<xref rid="bib15" ref-type="bibr">Hwang et al., 2013</xref>
;
<xref rid="bib16" ref-type="bibr">Jao et al., 2013</xref>
) to introduce mutations in either the
<italic>aplnra</italic>
or the
<italic>aplnrb</italic>
gene (
<xref ref-type="fig" rid="fig2s2">Figure 2—figure supplement 2</xref>
). As our MO-based analysis already indicated an additive effect of both receptor genes, we analyzed the phenotypes of the offspring of
<italic>aplnra</italic>
<sup>
<italic>+/−</italic>
</sup>
<italic>;aplnrb</italic>
<sup>
<italic>+/−</italic>
</sup>
double heterozygous parents. The embryos were all individually scored for their phenotype regarding angioblast midline migration into four categories (normal, mild, strong, stronger;
<xref ref-type="fig" rid="fig2">Figure 2B</xref>
) and then subjected to genotyping. Homozygous mutant embryos phenocopied the MO-induced phenotypes, additional loss of one or two copies of the second receptor increased the severity of the phenotype (
<xref ref-type="fig" rid="fig2">Figure 2C</xref>
). Additionally, we detected
<italic>aplnrb</italic>
transcripts together with the angioblast specification marker
<italic>etv2</italic>
indicating that both genes are expressed very early during angioblast specification. However, when we analyzed
<italic>aplnra/aplnrb</italic>
double deficient embryos, no difference in
<italic>etv2</italic>
expression was detectable by in situ hybridization indicating that Apln receptors only regulate angioblast migration, but not specification (
<xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1E</xref>
).
<fig id="fig2" position="float" orientation="portrait">
<object-id pub-id-type="doi">10.7554/eLife.06726.008</object-id>
<label>Figure 2.</label>
<caption>
<title>Ela/Apelin receptor - signaling guides angioblast migration to the midline.</title>
<p>(
<bold>A</bold>
) Angioblasts have migrated to the midline at 17 hpf in wild type,
<italic>apln</italic>
<sup>
<italic>mu267</italic>
</sup>
mutant or
<italic>apln</italic>
MO injected zebrafish embryos. MO-mediated knockdown of
<italic>apelin receptor a</italic>
or
<italic>b</italic>
partially inhibited midline migration, while simultaneous loss of both
<italic>apelin receptor</italic>
genes completely abolished midline migration of angioblasts. Likewise, embryos with homozygous mutations in the ligand
<italic>ela</italic>
display impaired migration of angioblasts. Arrows indicate aberrant positions. (
<bold>B</bold>
,
<bold>C</bold>
) Mutations in
<italic>apln receptor</italic>
genes impair angioblast migration. Analysis of the offspring of
<italic>aplnra</italic>
<sup>
<italic>+/−</italic>
</sup>
<italic>;aplnrb</italic>
<sup>
<italic>+/−</italic>
</sup>
double heterozygous parents resulted in four phenotypic categories (normal, mild, strong, stronger). (
<bold>B</bold>
) Genotyping of individual embryos revealed an additive effect, while mild phenotypes were observed when one receptor gene was homozygously mutant, phenotypic strength increased with additional loss of functional receptor genes (c; Ra,
<italic>aplnra</italic>
; Rb,
<italic>aplnrb</italic>
). (
<bold>D</bold>
,
<bold>E</bold>
) Ela deficiency can partially be compensated by Apln. Analysis of the offspring of
<italic>apln</italic>
<sup>
<italic>+/−</italic>
</sup>
<italic>;ela</italic>
<sup>
<italic>+/−</italic>
</sup>
double heterozygous parents resulted in four phenotypic categories (normal, mild, strong, severe). (
<bold>D</bold>
) Genotyping of individual embryos revealed a dose dependency, with increasing phenotypic strength correlating with additional loss of
<italic>apln</italic>
alleles in
<italic>ela</italic>
mutant embryos. (
<bold>E</bold>
)
<italic>ela</italic>
<sup>
<italic>−/−</italic>
</sup>
<italic>; apln</italic>
<sup>
<italic>−/−</italic>
</sup>
double mutants phenocopied
<italic>apln receptor</italic>
deficiency. Angioblasts (green) were labeled by
<italic>Tg(fli1a:EGFP)</italic>
<sup>
<italic>y1</italic>
</sup>
expression, scale bars represent 30 μm.</p>
<p>
<bold>DOI:</bold>
<ext-link ext-link-type="doi" xlink:href="10.7554/eLife.06726.008">http://dx.doi.org/10.7554/eLife.06726.008</ext-link>
</p>
<p>
<supplementary-material content-type="local-data" id="SD2-data">
<object-id pub-id-type="doi">10.7554/eLife.06726.009</object-id>
<label>Figure 2—source data 1.</label>
<caption>
<title>Excel table showing the phenotype categories and the number of embryos for each genotype in these phenotypic categories (for the
<italic>apln/ela</italic>
double mutant analysis).</title>
<p>
<bold>DOI:</bold>
<ext-link ext-link-type="doi" xlink:href="10.7554/eLife.06726.009">http://dx.doi.org/10.7554/eLife.06726.009</ext-link>
</p>
</caption>
<media mime-subtype="xlsx" mimetype="application" xlink:href="elife-06726-fig2-data1.xlsx" orientation="portrait" id="d35e970" position="anchor"></media>
</supplementary-material>
</p>
</caption>
<graphic xlink:href="elife-06726-fig2"></graphic>
<p content-type="supplemental-figure">
<fig id="fig2s1" specific-use="child-fig" orientation="portrait" position="anchor">
<object-id pub-id-type="doi">10.7554/eLife.06726.010</object-id>
<label>Figure 2—figure supplement 1.</label>
<caption>
<title>Expression of
<italic>aplnra</italic>
,
<italic>aplnrb</italic>
,
<italic>apln</italic>
and
<italic>ela</italic>
coincides with the formation of the DA and PCV.</title>
<p>(
<bold>A</bold>
–
<bold>D</bold>
) In situ hybridizations detecting expression of
<italic>aplnra</italic>
(
<bold>A</bold>
),
<italic>aplnrb</italic>
(
<bold>B</bold>
),
<italic>apln</italic>
(
<bold>C</bold>
) and
<italic>ela</italic>
(
<bold>D</bold>
) at the indicated time points (12 hpf, 18 hpf shown in dorsal views, 20 hpf in lateral views).
<italic>aplnrs</italic>
are expressed in the migrating angioblasts and continue to be expressed during formation of the DA and PCV. At 12 hpf,
<italic>aplnrb</italic>
is strongly expressed in the angioblasts (prior to migration, arrowheads) and within the lateral plate mesoderm and at the notochord somite boundary.
<italic>aplnra</italic>
expression is strong in the paraxial mesoderm (
<bold>A</bold>
). Both receptors are expressed in the angioblasts at 18 hpf (
<bold>A</bold>
,
<bold>B</bold>
, arrowheads) and in the developing axial vessels at 20 hpf (Arrowheads point to the DA, arrows to the PCV). Prior to angioblast migration
<italic>apln</italic>
and
<italic>ela</italic>
are both expressed by the notochord, with
<italic>ela</italic>
showing a much stronger expression (
<bold>C</bold>
,
<bold>D</bold>
). After angioblasts reached the midline (18 hpf, 20 hpf) only
<italic>apln</italic>
is still expressed by the notochord, whereas notochord derived
<italic>ela</italic>
expression is strongly reduced to absent. (
<bold>E</bold>
) Knockdown of
<italic>aplnr</italic>
does not influence the specification of angioblasts. In situ hybridizations detecting expression of the angioblast marker
<italic>etv2</italic>
at 13.5 hpf (dorsal views).</p>
<p>
<bold>DOI:</bold>
<ext-link ext-link-type="doi" xlink:href="10.7554/eLife.06726.010">http://dx.doi.org/10.7554/eLife.06726.010</ext-link>
</p>
</caption>
<graphic xlink:href="elife-06726-fig2-figsupp1"></graphic>
</fig>
</p>
<p content-type="supplemental-figure">
<fig id="fig2s2" specific-use="child-fig" orientation="portrait" position="anchor">
<object-id pub-id-type="doi">10.7554/eLife.06726.011</object-id>
<label>Figure 2—figure supplement 2.</label>
<caption>
<title>Generation of zebrafish
<italic>aplnra</italic>
,
<italic>aplnrb</italic>
and
<italic>apln</italic>
mutants.</title>
<p>Targeted indel mutations in the
<italic>aplnra</italic>
gene were induced using TALEN mutagenesis, and in the
<italic>aplnrb</italic>
and
<italic>apln</italic>
genes were induced by engineered sgRNA:Cas9. The wild type nucleotide sequences are shown at the top with the TALEN (
<bold>A</bold>
) or CRISPR (
<bold>B</bold>
,
<bold>C</bold>
) target sites highlighted in yellow and the spacer (
<bold>A</bold>
) or PAM (
<bold>B</bold>
,
<bold>C</bold>
) sequences highlighted in red. (
<bold>A</bold>
)
<italic>aplnra</italic>
coding sequence and TALEN mutagenesis target site. An 8 base pair insertion, shown as lower case letters in blue leads to a premature stop and a shortening of the Aplnra protein to 13 AA. (
<bold>B</bold>
)
<italic>aplnrb</italic>
coding sequence and CRISPR mutagenesis target site. A 4 base pair insertion, shown as lower case letters in blue leads to a premature stop and a shortening of the Aplnrb protein to 65 AA. (
<bold>C</bold>
)
<italic>apln</italic>
coding sequence and CRISPR mutagenesis target site. An 1 baise pair loss / 9 base pair insertion, shown as lower case letters in blue causes a frameshift leading to a premature stop codon and a shortening of the Apln protein to 11 AA.</p>
<p>
<bold>DOI:</bold>
<ext-link ext-link-type="doi" xlink:href="10.7554/eLife.06726.011">http://dx.doi.org/10.7554/eLife.06726.011</ext-link>
</p>
</caption>
<graphic xlink:href="elife-06726-fig2-figsupp2"></graphic>
</fig>
</p>
</fig>
<fig id="media2" position="anchor">
<label>Video 2.</label>
<caption>
<title>Angioblast migration between 12.5 hpf and 16 hpf observed by time-lapse imaging using
<italic>Tg(fli1a:EGFP)</italic>
<sup>
<italic>y1</italic>
</sup>
to visualize angioblasts.</title>
<p>No migration to the midline, but minor movements and strong filopodia formation can be visualized in Aplnra/b double deficient embryos (
<italic>aplnra/b</italic>
MO).</p>
<p>
<bold>DOI:</bold>
<ext-link ext-link-type="doi" xlink:href="10.7554/eLife.06726.012">http://dx.doi.org/10.7554/eLife.06726.012</ext-link>
</p>
</caption>
<media xlink:href="elife-06726-media2.avi" mimetype="video" mime-subtype="x-msvideo">
<object-id pub-id-type="doi">10.7554/eLife.06726.012</object-id>
</media>
</fig>
</p>
<p>Next, we analyzed the requirement for the ligand Apln by MO-mediated gene knockdown and, unexpectedly, observed no difference to control MO injected embryos (
<xref ref-type="fig" rid="fig2">Figure 2A</xref>
). To obtain undisputable genetic confirmation of this result, we again used gene editing to introduce mutations in the
<italic>apln</italic>
gene (
<xref ref-type="fig" rid="fig2s2">Figure 2—figure supplement 2</xref>
, most likely resulting in functional null mutants). Consistent with the MO-mediated depletion, homozygous
<italic>apln</italic>
<sup>
<italic>mu267</italic>
</sup>
mutant embryos (
<xref ref-type="fig" rid="fig2">Figure 2A</xref>
) showed no vasculogenesis defects, which indicated that endogenous Apln is not sufficient to regulate angioblast migration to the midline.</p>
<p>Previous studies have identified a requirement for Apln receptors in myocardial development in zebrafish (
<xref rid="bib28" ref-type="bibr">Scott et al., 2007</xref>
;
<xref rid="bib33" ref-type="bibr">Zeng et al., 2007</xref>
) and mice (
<xref rid="bib3" ref-type="bibr">Charo et al., 2009</xref>
), which could not completely be phenocopied by Apln deficiency (
<xref rid="bib28" ref-type="bibr">Scott et al., 2007</xref>
;
<xref rid="bib33" ref-type="bibr">Zeng et al., 2007</xref>
;
<xref rid="bib3" ref-type="bibr">Charo et al., 2009</xref>
). These results led to the proposal that Apln may not be the only ligand for Aplnrs. Recently, a novel peptide hormone named Elabela (Ela, also known as Apela or Toddler) was identified in zebrafish and shown to bind and activate Aplnrs (
<xref rid="bib4" ref-type="bibr">Chng et al., 2013</xref>
;
<xref rid="bib26" ref-type="bibr">Pauli et al., 2014</xref>
). By in situ hybridization we detected
<italic>ela</italic>
expression by the NC (
<xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1D</xref>
, [
<xref rid="bib26" ref-type="bibr">Pauli et al., 2014</xref>
]), consistent with a possible role in attracting angioblasts to the midline. We next analyzed angioblast migration in zebrafish embryos carrying a homozygous null mutation in the
<italic>ela</italic>
gene (
<italic>ela</italic>
<sup>
<italic>br13</italic>
</sup>
). Ela deficiency led to a strong impairment of angioblast migration (
<xref ref-type="fig" rid="fig2">Figure 2A</xref>
), indicating that indeed Ela-Aplnr represent a novel
<italic>bona fide</italic>
ligand–receptor pair and therefore a novel signaling pathway regulating angioblast migration.</p>
<p>Given that the
<italic>ela</italic>
<sup>-/-</sup>
phenotype was not as severe as the depletion of both receptors and that
<italic>apln</italic>
expression increases later in the NC (while
<italic>ela</italic>
expression becomes progressively reduced,
<xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1C,D</xref>
), we analyzed the phenotypes of the offspring of
<italic>apln</italic>
<sup>
<italic>+/−</italic>
</sup>
<italic>;ela</italic>
<sup>
<italic>+/−</italic>
</sup>
double heterozygous parents. The embryos were all individually scored for their phenotype regarding angioblast midline migration into four categories (normal, mild, strong, severe) and then subjected to genotyping. Interestingly a mild phenotype was observed, when either one
<italic>ela</italic>
or both
<italic>apln</italic>
alleles were mutant, a strong phenotype when
<italic>ela</italic>
was homozygously mutant with increasing severity (severe phenotype) upon loss of the
<italic>apln</italic>
alleles (
<xref ref-type="fig" rid="fig2">Figure 2D,E</xref>
). Homozygous
<italic>ela</italic>
<sup>
<italic>−/−</italic>
</sup>
<italic>; apln</italic>
<sup>
<italic>−/−</italic>
</sup>
double mutants phenocopied apln receptor deficiency (compare in
<xref ref-type="fig" rid="fig2">Figure 2A,B</xref>
). The observed dose dependency is indicative, that in this context Ela and Apln might act as novel chemoattractants for angioblast migration to the midline, with Ela acting as the major endogenous attractant and Apln having a minor additive role.</p>
<p>Previous analysis of zebrafish embryos lacking the NC showed, that signals from the midline guide angioblast migration (
<xref rid="bib8" ref-type="bibr">Fouquet et al., 1997</xref>
;
<xref rid="bib30" ref-type="bibr">Sumoy et al., 1997</xref>
). We could show that indeed angioblast migration as well as
<italic>ela</italic>
expression is perturbed in the zebrafish mutants lacking a NC (
<italic>notochord homeobox, noto</italic>
; previous name:
<italic>floating head</italic>
) or exhibiting defective NC precursor differentiation (
<italic>T, brachyury homolog a, ta</italic>
; previous name:
<italic>no tail</italic>
) (
<xref ref-type="fig" rid="fig3">Figure 3</xref>
) (
<xref rid="bib11" ref-type="bibr">Halpern et al., 1993</xref>
;
<xref rid="bib25" ref-type="bibr">Odenthal et al., 1996</xref>
). While angioblasts in
<italic>noto</italic>
<sup>
<italic>tk241</italic>
</sup>
mutant embryos failed to migrate to the midline,
<italic>ta</italic>
<sup>
<italic>b160</italic>
</sup>
angioblasts migrated towards the midline but failed to reach this structure (
<xref ref-type="fig" rid="fig3">Figure 3A</xref>
). In line with these results,
<italic>ela</italic>
expression was strongly diminished in the
<italic>ta</italic>
<sup>
<italic>b160</italic>
</sup>
midline and almost absent in
<italic>noto</italic>
<sup>
<italic>tk241</italic>
</sup>
mutant embryos (
<xref ref-type="fig" rid="fig3">Figure 3B</xref>
). We next wanted to test whether midline
<italic>ela</italic>
expression would be sufficient to restore angioblast migration in
<italic>ta</italic>
<sup>
<italic>b160</italic>
</sup>
mutants. For this purpose, plasmid DNA with a
<italic>shh</italic>
promoter fragment enabling
<italic>ela</italic>
expression in the floorplate was injected together with
<italic>Tol2</italic>
transposase mRNA to obtain mosaic Ela overexpression in the midline. Indeed, Ela overexpression did restore angioblast migration to the midline in
<italic>ta</italic>
<sup>
<italic>b160</italic>
</sup>
embryos (
<xref ref-type="fig" rid="fig3">Figure 3C</xref>
), demonstrating that midline Ela expression is not only necessary, but sufficient to regulate angioblast migration during vasculogenesis.
<fig id="fig3" orientation="portrait" position="float">
<object-id pub-id-type="doi">10.7554/eLife.06726.013</object-id>
<label>Figure 3.</label>
<caption>
<title>Notochord (NC)
<italic>ela</italic>
expression is sufficient to guide angioblasts to the midline.</title>
<p>(
<bold>A</bold>
) Angioblasts fail to migrate to the midline position in the NC mutants
<italic>noto</italic>
and
<italic>ta</italic>
. Confocal projections of
<italic>Tg(fli1a:EGFP)</italic>
<sup>
<italic>y1</italic>
</sup>
embryos in dorsal view at 17 hpf (scale bars: 20 μm). (
<bold>B</bold>
)
<italic>In situ</italic>
hybridization in 17 hpf old zebrafish embryos showing
<italic>ela</italic>
expression from the NC and somites (white arrows). NC deficiency abolishes or strongly reduces
<italic>ela</italic>
expression in
<italic>noto</italic>
or
<italic>ta</italic>
mutant embryos. (
<bold>C</bold>
) Mosaic expression of Ela in the midline, achieved by injection of
<italic>shh:ela</italic>
DNA, led to rescue of angioblast migration in
<italic>ta</italic>
<sup>
<italic>b160</italic>
</sup>
mutant embryos (n = 61 embryos, ctr injected n = 32 embryos). Significance was calculated by chi-square (χ
<sup>2</sup>
= 19.65) equivalent to p < 0.001 (p = 9E-06). Scale bars: 30 μm.</p>
<p>
<bold>DOI:</bold>
<ext-link ext-link-type="doi" xlink:href="10.7554/eLife.06726.013">http://dx.doi.org/10.7554/eLife.06726.013</ext-link>
</p>
</caption>
<graphic xlink:href="elife-06726-fig3"></graphic>
</fig>
</p>
<p>Next, we wanted to analyze the function of Ela-Aplnr signaling more mechanistically. Recently, it was published that Ela does not act as a chemoattractant, but instead acts as a motogen in regulating mesoderm migration during gastrulation (
<xref rid="bib26" ref-type="bibr">Pauli et al., 2014</xref>
). In contrast, the
<italic>aplnr</italic>
deficient (
<italic>aplra/b</italic>
MO injected) angioblasts were still partially motile and did send out filopodia, but failed to migrate to the midline (
<xref ref-type="other" rid="media2">Video 2</xref>
). To test whether Ela does act as a chemoattractant or just enables motility of angioblasts, we overexpressed Ela at ectopic locations in
<italic>noto</italic>
or
<italic>ta</italic>
mutant embryos, as both of these lack endogenous
<italic>ela</italic>
and
<italic>apln</italic>
expression in the NC. We used a heatshock promoter to control timing of expression and labeled Ela overexpressing cells by coexpression of RFP through an internal ribosome entry site (IRES) element (
<xref ref-type="fig" rid="fig4">Figure 4</xref>
). We measured the distance of control (Cherry) or Ela/RFP expressing cells from angioblasts in a 40 μm radius of the red labeled cell and considered an angioblast as attracted, if it was less than 5 μm away from it. While only 20% of the angioblast were found in the vicinity of control cells, more than 52% of the angioblasts were close to Ela overexpressing cells (
<xref ref-type="fig" rid="fig4">Figure 4D,G</xref>
). Together with the observed dose dependency of Ela-Apln, the motility of Aplnr deficient embryos and the rescue of NC mutants by midline Ela expression, our data support the interpretation, that Ela acts as a chemoattractant for angioblasts during midline migration.
<fig id="fig4" orientation="portrait" position="float">
<object-id pub-id-type="doi">10.7554/eLife.06726.014</object-id>
<label>Figure 4.</label>
<caption>
<title>Ela overexpressing cells attract angioblasts.</title>
<p>(
<bold>A</bold>
) F1 embryos from
<italic>noto</italic>
<sup>
<italic>tk241+/−</italic>
</sup>
<italic>or ta</italic>
<sup>
<italic>b160+/−</italic>
</sup>
parents were injected with 250 pg
<italic>Tol2</italic>
mRNA as well as 10 pg of DNA constructs, in which the heatshock promotor was used to drive either
<italic>control (ctr, Cherry)</italic>
or
<italic>ela</italic>
expression. Individual Ela overexpressing cells were labeled by IRES mediated RFP expression. Expression was induced by two consecutive heatshocks (incubation at 39°C) at 12 hpf and 14 hpf for 1 hr each. (
<bold>B</bold>
–
<bold>C′</bold>
) Angioblast migration in
<italic>noto</italic>
<sup>
<italic>tk241−/−</italic>
</sup>
mutant embryos injected with the
<italic>ctr</italic>
(
<bold>B, B′</bold>
) or the
<italic>ela</italic>
overexpression (
<bold>C, C′</bold>
) construct. (
<bold>D</bold>
) Quantification of Ela/RFP or ctr (Cherry) positive cells showed significantly more Ela overexpressing cells attracting angioblasts than ctr cells (n = 10 embryos for
<italic>hs:ela</italic>
, n = 20 embryos for
<italic>hs:ctr</italic>
; p*** = 0.0001). (
<bold>E</bold>
–
<bold>F</bold>
′) Angioblast migration in
<italic>ta</italic>
<sup>
<italic>b160</italic>
</sup>
<sup>
<italic>−/−</italic>
</sup>
mutant embryos injected with the ctr (
<bold>E, E′</bold>
) or the
<italic>ela</italic>
overexpression (
<bold>F, F′</bold>
) construct (
<bold>G</bold>
) Quantification of Ela/RFP or ctr (Cherry) positive cells showed significantly more Ela overexpressing cells attracting angioblasts than ctr cells (n = 8 embryos for
<italic>hs:ela</italic>
, n = 10 embryos for
<italic>hs:ctr</italic>
; p** = 0.0089). Significance was calculated by chi-square test. Maximum intensity projections give an overview of the analyzed embryos. Single planes visualize the closeness of Ela or ctr (Cherry) expressing cells to angioblasts. White arrows point to Ela overexpressing cells with less than 5 μm distance to angioblasts. <5 μm distance between an Ela/ctr positive cell and an angioblast was counted as ‘attractant’; 5–40 μm distance was counted as ‘not attractant’. Scale bars represent 30 μm.</p>
<p>
<bold>DOI:</bold>
<ext-link ext-link-type="doi" xlink:href="10.7554/eLife.06726.014">http://dx.doi.org/10.7554/eLife.06726.014</ext-link>
</p>
</caption>
<graphic xlink:href="elife-06726-fig4"></graphic>
</fig>
</p>
<p>To prove that Ela-Aplnr signaling is directly required in angioblasts and rule out that the observed migration defects were caused by changes in other cell types, we performed transplantation experiments. Mini-ruby injected (red fluorescence) transgenic
<italic>fli1a:EGFP</italic>
positive (angioblasts, green) cells from control MO injected,
<italic>aplnra/b</italic>
MO injected or wild type (WT) donor embryos were transplanted into either WT or
<italic>aplnr</italic>
loss-of-function (
<italic>apnlra/b</italic>
MO injected) host embryos (
<xref ref-type="fig" rid="fig5">Figure 5A</xref>
). Analysis of angioblast migration to the midline at 17 hpf revealed that in average 93.6% of control MO-injected transplanted angioblasts migrated to the midline in a WT host embryo (
<xref ref-type="fig" rid="fig5">Figure 5B,C</xref>
; n = 39 GFP+ angioblasts, 7 embryos). In contrast, only 9.4% of
<italic>aplnra/b</italic>
MO injected angioblasts arrived at the midline, whereas the majority of these cells remained in more lateral positions (
<xref ref-type="fig" rid="fig5">Figure 5B,D</xref>
; n = 62 GFP+ angioblasts, 5 embryos). Transplanted WT angioblasts were perfectly capable to migrate to the midline in an
<italic>aplnr</italic>
deficient environment (average of 81.9%,
<xref ref-type="fig" rid="fig5">Figure 5B,E</xref>
; n = 15 GFP+ angioblasts, 6 embryos), which shows that there is indeed a cell autonomous requirement for Aplnr signaling in angioblasts.
<fig id="fig5" orientation="portrait" position="float">
<object-id pub-id-type="doi">10.7554/eLife.06726.015</object-id>
<label>Figure 5.</label>
<caption>
<title>Cell autonomous requirement for Apelin receptor signaling in angioblasts.</title>
<p>(
<bold>A</bold>
) Experimental design: mini-ruby injected cells from ctr MO (
<bold>C</bold>
)
<italic>aplnra/b</italic>
MO (
<bold>D</bold>
) or WT (
<bold>E</bold>
)
<italic>Tg(fli1a:EGFP)</italic>
<sup>
<italic>y1</italic>
</sup>
embryos were transplanted into WT or
<italic>aplnra/b</italic>
MO host embryos and scored for their migration to the midline. (
<bold>B</bold>
) Quantification: 93.6% of
<italic>ctr</italic>
MO injected (n = 39 GFP + angioblasts, 7 embryos), but only 9.4% of
<italic>aplnra/b</italic>
deficient (
<italic>aplnra</italic>
<italic>/b</italic>
MO injected; n = 62 GFP+ angioblasts, 5 embryos) donor angioblasts migrated to the midline in WT host embryos. In contrast, 81.9% of WT donor angioblasts (n = 15 GFP+ angioblasts, 6 embryos) migrated to the midline in
<italic>aplnra/b</italic>
deficient host embryos. Error bars represent SEM, calculated using the standard deviation of percent of angioblasts in the midline per embryo. Statistical analysis showed significance using 2 way ANOVA and t-test, with p *** = 3.44577E-08 for
<italic>aplnra/b</italic>
MO in WT, and p = 0.232995135 (not significant, n.s.) for WT in
<italic>aplnra/b</italic>
MO. see also
<xref ref-type="supplementary-material" rid="SD3-data">Figure 5—source data 1</xref>
. (
<bold>C</bold>
–
<bold>E</bold>
) Confocal projections showing representative embryos of the transplantation experiments at 17 hpf. Arrows indicate transplanted angioblasts; asterisks label transplanted cells, which are not angioblasts; dashed lines indicate the midline.</p>
<p>
<bold>DOI:</bold>
<ext-link ext-link-type="doi" xlink:href="10.7554/eLife.06726.015">http://dx.doi.org/10.7554/eLife.06726.015</ext-link>
</p>
<p>
<supplementary-material content-type="local-data" id="SD3-data">
<object-id pub-id-type="doi">10.7554/eLife.06726.016</object-id>
<label>Figure 5—source data 1.</label>
<caption>
<title>Statistical analysis and single values for the number of angioblasts and embryos analyzed.</title>
<p>
<bold>DOI:</bold>
<ext-link ext-link-type="doi" xlink:href="10.7554/eLife.06726.016">http://dx.doi.org/10.7554/eLife.06726.016</ext-link>
</p>
</caption>
<media mime-subtype="xlsx" mimetype="application" xlink:href="elife-06726-fig5-data1.xlsx" orientation="portrait" id="d35e1745" position="anchor"></media>
</supplementary-material>
</p>
</caption>
<graphic xlink:href="elife-06726-fig5"></graphic>
</fig>
</p>
<p>Based on the sum of the results presented above, we propose that Ela expression at the embryonic midline provides a chemoattractive signal for Aplnr-positive angioblasts. While Ela and Apln both signal to the Apln receptors, the initial
<italic>ela</italic>
expression at the midline appears stronger than
<italic>apln</italic>
, which peaked at later developmental time points (
<xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1C,D</xref>
). Thus,
<italic>ela</italic>
and
<italic>apln</italic>
may have partially redundant functions, but are required at different stages of vascular morphogenesis. Surprisingly, while Ela-Aplnr signaling was strictly required for the guided migration of angioblasts, this process was not controlled by Vegfa-mediated signaling.</p>
<p>Our studies identified Ela-Aplnr signaling as a novel signaling pathway for angioblasts regulating vascular patterning in the developing vertebrate embryo. As therapeutic intervention with vascular growth is clinically highly relevant and, so far, predominantly focuses on the VEGF-A signaling cascade, the Ela-Aplnr signaling pathway may also represent a novel therapeutic target in human disease.</p>
</sec>
<sec sec-type="materials|methods" id="s2">
<title>Materials and methods</title>
<sec id="s2-1">
<title>Zebrafish husbandry and strains</title>
<p>Zebrafish (
<italic>Danio rerio</italic>
) were maintained in a recirculating aquaculture system under standard laboratory conditions (
<xref rid="bib32" ref-type="bibr">Westerfield, 1993</xref>
). Embryos were staged by hours post fertilization (hpf) at 28.5°C (
<xref rid="bib18" ref-type="bibr">Kimmel et al., 1995</xref>
), for 17 hpf embryos were incubated after gastrulation at 21°C and staged on the following morning by counting somites (16 somites equal 17 hpf [
<xref rid="bib18" ref-type="bibr">Kimmel et al., 1995</xref>
]).</p>
<p>Zebrafish strains used were
<italic>Tg</italic>
(
<italic>fli1a:EGFP</italic>
)
<sup>
<italic>y1</italic>
</sup>
(
<xref rid="bib21" ref-type="bibr">Lawson and Weinstein, 2002</xref>
),
<italic>kdrl</italic>
<sup>
<italic>hu5088</italic>
</sup>
(
<xref rid="bib1" ref-type="bibr">Bussmann et al., 2010</xref>
),
<italic>ta</italic>
<sup>
<italic>b160</italic>
</sup>
(
<xref rid="bib11" ref-type="bibr">Halpern et al., 1993</xref>
),
<italic>noto</italic>
<sup>
<italic>tk241</italic>
</sup>
(
<xref rid="bib25" ref-type="bibr">Odenthal et al., 1996</xref>
) and
<italic>ela</italic>
<sup>
<italic>br13</italic>
</sup>
(
<xref rid="bib4" ref-type="bibr">Chng et al., 2013</xref>
).</p>
</sec>
<sec id="s2-2">
<title>Generation of
<italic>apln</italic>
<sup>
<italic>mu267</italic>
</sup>
,
<italic>aplnra</italic>
<sup>
<italic>mu296</italic>
</sup>
and
<italic>aplnrb</italic>
<sup>
<italic>mu270</italic>
</sup>
mutants</title>
<p>For
<italic>apln</italic>
and
<italic>aplnrb</italic>
Crispr mediated mutagenesis was used. Nls-zCas9-nls mRNA was synthesized as previously described (
<xref rid="bib16" ref-type="bibr">Jao et al., 2013</xref>
).</p>
<p>The target sequences of
<italic>apln</italic>
exon 1 (5′-GAATGTGAAGATCTTGACGC-3′) and
<italic>aplnrb</italic>
exon 1 (5′-CTACATGCTCATCTTCATCC-3′) were cloned into the gRNA expression vector pDR274 as previously described (
<xref rid="bib15" ref-type="bibr">Hwang et al., 2013</xref>
). The
<italic>apln</italic>
sgRNA or
<italic>aplnrb</italic>
sgRNA were transcribed using DraI-digested gRNA expression vector as template and the T7 mMessage mMachine kit (Ambion, Life Technologies, Germany).
<italic>apln</italic>
sgRNA or
<italic>apnrb</italic>
sgRNA and nls-zCas9-nls-encoding mRNA were co-injected into one-cell stage zebrafish embryos. Each embryo was injected with 2 nl of solution containing 12.5 ng/μl sgRNA and 300 ng/μl nls-zCas9-nls mRNA. A HgaI (New England BioLabs, Frankfurt, Germany) restriction site was used for genotyping of
<italic>apln</italic>
<sup>
<italic>mu267</italic>
</sup>
and a FokI (New England BioLabs) restriction site was used for genotyping of
<italic>aplnrb</italic>
<sup>
<italic>mu270</italic>
</sup>
.</p>
<p>
<italic>aplnra</italic>
mutants were generated by TALEN mutagenesis. TALENs were assembled using the Golden Gate method (
<xref rid="bib2" ref-type="bibr">Cermak et al., 2011</xref>
). For targeting the
<italic>aplnra</italic>
locus, a 5′ RVD (NI HD NI HD HD NH NI NH NI HD NI NG NI HD NH NI NG) and a 3′ RVD (HD NI HD NI HD HD HD NI NH NI NH NG HD NI NG NG NI NG NI) were generated. A BsrI (New England BioLabs) restriction site was used for genotyping of
<italic>aplnra</italic>
<sup>
<italic>mu296</italic>
</sup>
. mRNA was generated using the T3 mMessage mMachine Kit (Ambion) and injected using 100 pg of the TALEN mix.</p>
</sec>
<sec id="s2-3">
<title>Microinjections</title>
<p>Microinjections of mRNA or MOs were performed as previously described (
<xref rid="bib22" ref-type="bibr">Nasevicius and Ekker, 2000</xref>
).</p>
<p>mRNA was transcribed using SP6 polymerase (Sp6 mMessage mMachine kit [Ambion]). 100 pg
<italic>H2B-cherry</italic>
mRNA (
<xref rid="bib27" ref-type="bibr">Santoro et al., 2007</xref>
), 2 ng
<italic>aplnra</italic>
MO (
<xref rid="bib28" ref-type="bibr">Scott et al., 2007</xref>
), 0.5 ng
<italic>aplnrb</italic>
MO (
<xref rid="bib33" ref-type="bibr">Zeng et al., 2007</xref>
), 2 ng
<italic>apln</italic>
MO (
<xref rid="bib28" ref-type="bibr">Scott et al., 2007</xref>
) or 2 ng
<italic>vegfaa/</italic>
2.3 ng
<italic>vegfab</italic>
MO (
<xref rid="bib24" ref-type="bibr">Ober et al., 2004</xref>
) were injected. Simultaneous knockdown of
<italic>aplnra</italic>
and
<italic>aplnrb</italic>
was done by coinjection of 2 ng
<italic>aplnra</italic>
MO and 0.5 ng
<italic>aplnrb</italic>
MO (referred to as
<italic>aplnra/b</italic>
MO). 4 ng of standard control MO (Gene tools, Philomath, Oregon) were injected as control.</p>
<p>Plasmid DNA was injected together with
<italic>Tol2</italic>
mRNA (
<xref rid="bib17" ref-type="bibr">Kawakami et al., 2004</xref>
). A 2.2 kb fragment of the
<italic>shh</italic>
promoter (
<xref rid="bib9" ref-type="bibr">Gordon et al., 2013</xref>
) was used to drive either
<italic>rfp</italic>
or
<italic>ela</italic>
expression (
<italic>shh:ctr</italic>
or
<italic>shh:ela</italic>
). The
<italic>heatshock</italic>
promoter (
<italic>hsp70l</italic>
) was used to drive either
<italic>Cherry</italic>
(
<italic>hs:ctr</italic>
[
<xref rid="bib13" ref-type="bibr">Hesselson et al., 2009</xref>
]) or
<italic>ela-IRES-RFP</italic>
(
<italic>hs:ela</italic>
).</p>
</sec>
<sec id="s2-4">
<title>Whole mount in situ hybridization</title>
<p>In situ hybridizations were performed as previously described (
<xref rid="bib12" ref-type="bibr">Helker et al., 2013</xref>
) using the following probes:
<italic>cdh5</italic>
(
<xref rid="bib19" ref-type="bibr">Larson et al., 2004</xref>
),
<italic>etv2</italic>
(
<xref rid="bib12" ref-type="bibr">Helker et al., 2013</xref>
),
<italic>vegfc</italic>
(
<xref rid="bib14" ref-type="bibr">Hogan et al., 2009</xref>
),
<italic>vegfaa</italic>
(
<xref rid="bib20" ref-type="bibr">Lawson et al., 2002</xref>
).</p>
<p>
<italic>apln, aplnra and aplnrb</italic>
probe templates were amplified from cDNA of 19 somite stage old embryos using the following primers:
<italic>apln</italic>
-forward 5′-GAAAGGCCCAAGTCACAGAG-3′ and
<italic>apln</italic>
-reverse 5′-GAGTTCACTATCTGATGTCAAACCA-3′,
<italic>aplnra</italic>
-forward 5′-GAAAGGCCCAAGTCACAGAG-3′ and
<italic>aplnra</italic>
-reverse 5′-GAGTTCACTATCTGATGTCAAACCA-3′,
<italic>aplnrb</italic>
-forward 5′-GAAAGGCCCAAGTCACAGAG-3′ and
<italic>aplnrb</italic>
-reverse 5′-GAGTTCACTATCTGATGTCAAACCA-3′.</p>
<p>The T7 promotor was added to the 5′- end of the reverse primer in a second round of amplification (T7-
<italic>apln</italic>
-reverse 5′-GTAATACGACTCACTATAGGGAGTTCACTATCTGATGTCAAACCA-3′, (T7-
<italic>aplnra</italic>
-reverse 5′-GTAATACGACTCACTATAGGGAGTTCACTATCTGATGTCAAACCA-3′, (T7-
<italic>aplnrb</italic>
-reverse 5′-GTAATACGACTCACTATAGGGAGTTCACTATCTGATGTCAAACCA-3′).</p>
</sec>
<sec id="s2-5">
<title>In vivo time-lapse analysis and confocal microscopy</title>
<p>Embryos were manually dechorionated and mounted in 0.3% agarose (with subsequent removal of agarose from the head/tail region). Medium and agarose were supplemented with 19.2 mg/l Tricaine and 30 mg/l Phenylthiourea. For time-lapse imaging, embryos were kept in a 28.5°C heated chamber surrounding the microscope stage. All fluorescent images were acquired using an upright Leica Sp5 DM 6000 or a Zeiss LSM 780 Confocal microscope. Confocal stacks and confocal movies were assembled using Imaris Software (Bitplane, Switzerland).</p>
</sec>
<sec id="s2-6">
<title>Transplantation experiments</title>
<p>Donor embryos were injected with
<italic>ctr</italic>
MO or
<italic>aplnra/b</italic>
MO and labeled by the injection of 0.1 ng mini-Ruby (Tetramethylrhodamine, Invitrogen/Life Technologies). At sphere stage, cells were removed from donor embryos and transferred to wild type hosts using a glass capillary. Transplanted angioblasts were identified by transgenic EGFP expression together with mini-Ruby stain. By counting the number of EGFP positive donor angioblasts in the midline vs all EGFP positive donor angioblasts (100%), the percentage of donor angioblasts in the midline was determined for each individual embryo.</p>
</sec>
</sec>
</body>
<back>
<sec sec-type="funding-information">
<title>Funding Information</title>
<p>This paper was supported by the following grants:</p>
<list list-type="bullet">
<list-item>
<p>
<funding-source>
<institution-wrap>
<institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100003484</institution-id>
<institution>State of Northrhine Westphalia</institution>
</institution-wrap>
</funding-source>
<award-id>Northrhine Westphalia Return Fellowship</award-id>
to Wiebke Herzog.</p>
</list-item>
<list-item>
<p>
<funding-source>
<institution-wrap>
<institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100004869</institution-id>
<institution>Westfälische Wilhelms-Universität Münster</institution>
</institution-wrap>
</funding-source>
<award-id>Cells in Motion cluster of Excellence, CiM - EXC 1003 / FF-2013-14</award-id>
to Wiebke Herzog.</p>
</list-item>
<list-item>
<p>
<funding-source>
<institution-wrap>
<institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100001348</institution-id>
<institution>Agency for Science, Technology and Research (A*STAR)</institution>
</institution-wrap>
</funding-source>
<award-id>Strategic Positioning Fund on Genetic Orphan Diseases</award-id>
to Bruno Reversade.</p>
</list-item>
<list-item>
<p>
<funding-source>
<institution-wrap>
<institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100004189</institution-id>
<institution>Max-Planck-Gesellschaft</institution>
</institution-wrap>
</funding-source>
to Friedemann Kiefer.</p>
</list-item>
</list>
</sec>
<ack id="ack">
<title>Acknowledgements</title>
<p>We thank Aurelien Courtois and Jeroen Bussman for helpful discussion and suggestions. We thank Arnd Siekmann for the
<italic>vegfaa</italic>
and
<italic>vegfab</italic>
MO and Stefan Schulte-Merker for providing plasmid containing the
<italic>shh </italic>
promoter and the IRES element. We thank Ralf Adams for critical comments on the manuscript. We thank Stefan Volkery for imaging advice and microscope maintenance. Excellent fish husbandry and technical assistance were provided by Reinhild Bußmann and Katja Müller.</p>
</ack>
<sec id="s3" sec-type="additional-information">
<title>Additional information</title>
<fn-group content-type="competing-interest">
<title>
<bold>Competing interests</bold>
</title>
<fn fn-type="COI-statement" id="conf1">
<p>The authors declare that no competing interests exist.</p>
</fn>
</fn-group>
<fn-group content-type="author-contribution">
<title>
<bold>Author contributions</bold>
</title>
<fn fn-type="con" id="con1">
<p>CSMH, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article.</p>
</fn>
<fn fn-type="con" id="con2">
<p>AS, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article.</p>
</fn>
<fn fn-type="con" id="con3">
<p>CP, Analysis and interpretation of data, Drafting or revising the article.</p>
</fn>
<fn fn-type="con" id="con4">
<p>FK, Analysis and interpretation of data, Drafting or revising the article.</p>
</fn>
<fn fn-type="con" id="con5">
<p>SCC, Contributed preliminary data, Drafting or revising the article, Contributed unpublished essential data or reagents.</p>
</fn>
<fn fn-type="con" id="con6">
<p>BR, Drafting or revising the article, Contributed unpublished essential data or reagents.</p>
</fn>
<fn fn-type="con" id="con7">
<p>WH, Conception and design, Analysis and interpretation of data, Drafting or revising the article.</p>
</fn>
</fn-group>
<fn-group content-type="ethics-information">
<title>
<bold>Ethics</bold>
</title>
<fn fn-type="other">
<p>Animal experimentation: All animal experiments were performed in strict accordance with the relevant laws and institutional guidelines the Max Planck Institute for Molecular Biomedicine, Muenster and the Institute of Medical Biology, Singapore. All protocols were approved by animal ethics committees of the state of North Rhine-Westfalia (Germany,# 39.32.7.1) and Singapore, respectively, and all efforts were made to minimize suffering.</p>
</fn>
</fn-group>
</sec>
<ref-list>
<title>References</title>
<ref id="bib1">
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Bussmann</surname>
<given-names>J</given-names>
</name>
<name>
<surname>Bos</surname>
<given-names>FL</given-names>
</name>
<name>
<surname>Urasaki</surname>
<given-names>A</given-names>
</name>
<name>
<surname>Kawakami</surname>
<given-names>K</given-names>
</name>
<name>
<surname>Duckers</surname>
<given-names>HJ</given-names>
</name>
<name>
<surname>Schulte-Merker</surname>
<given-names>S</given-names>
</name>
</person-group>
<year>2010</year>
<article-title>Arteries provide essential guidance cues for lymphatic endothelial cells in the zebrafish trunk</article-title>
<source>Development</source>
<volume>137</volume>
<fpage>2653</fpage>
<lpage>2657</lpage>
<pub-id pub-id-type="doi">10.1242/dev.048207</pub-id>
<pub-id pub-id-type="pmid">20610484</pub-id>
</element-citation>
</ref>
<ref id="bib2">
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Cermak</surname>
<given-names>T</given-names>
</name>
<name>
<surname>Doyle</surname>
<given-names>EL</given-names>
</name>
<name>
<surname>Christian</surname>
<given-names>M</given-names>
</name>
<name>
<surname>Wang</surname>
<given-names>L</given-names>
</name>
<name>
<surname>Zhang</surname>
<given-names>Y</given-names>
</name>
<name>
<surname>Schmidt</surname>
<given-names>C</given-names>
</name>
<name>
<surname>Baller</surname>
<given-names>JA</given-names>
</name>
<name>
<surname>Somia</surname>
<given-names>NV</given-names>
</name>
<name>
<surname>Bogdanove</surname>
<given-names>AJ</given-names>
</name>
<name>
<surname>Voytas</surname>
<given-names>DF</given-names>
</name>
</person-group>
<year>2011</year>
<article-title>Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting</article-title>
<source>Nucleic Acids Research</source>
<volume>39</volume>
<fpage>e82</fpage>
<pub-id pub-id-type="doi">10.1093/nar/gkr218</pub-id>
<pub-id pub-id-type="pmid">21493687</pub-id>
</element-citation>
</ref>
<ref id="bib3">
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Charo</surname>
<given-names>DN</given-names>
</name>
<name>
<surname>Ho</surname>
<given-names>M</given-names>
</name>
<name>
<surname>Fajardo</surname>
<given-names>G</given-names>
</name>
<name>
<surname>Kawana</surname>
<given-names>M</given-names>
</name>
<name>
<surname>Kundu</surname>
<given-names>RK</given-names>
</name>
<name>
<surname>Sheikh</surname>
<given-names>AY</given-names>
</name>
<name>
<surname>Finsterbach</surname>
<given-names>TP</given-names>
</name>
<name>
<surname>Leeper</surname>
<given-names>NJ</given-names>
</name>
<name>
<surname>Ernst</surname>
<given-names>KV</given-names>
</name>
<name>
<surname>Chen</surname>
<given-names>MM</given-names>
</name>
<name>
<surname>Ho</surname>
<given-names>YD</given-names>
</name>
<name>
<surname>Chun</surname>
<given-names>HJ</given-names>
</name>
<name>
<surname>Bernstein</surname>
<given-names>D</given-names>
</name>
<name>
<surname>Ashley</surname>
<given-names>EA</given-names>
</name>
<name>
<surname>Quertermous</surname>
<given-names>T</given-names>
</name>
</person-group>
<year>2009</year>
<article-title>Endogenous regulation of cardiovascular function by apelin-APJ</article-title>
<source>American Journal of Physiology. Heart and Circulatory Physiology</source>
<volume>297</volume>
<fpage>H1904</fpage>
<lpage>H1913</lpage>
<pub-id pub-id-type="doi">10.1152/ajpheart.00686.2009</pub-id>
<pub-id pub-id-type="pmid">19767528</pub-id>
</element-citation>
</ref>
<ref id="bib4">
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Chng</surname>
<given-names>SC</given-names>
</name>
<name>
<surname>Ho</surname>
<given-names>L</given-names>
</name>
<name>
<surname>Tian</surname>
<given-names>J</given-names>
</name>
<name>
<surname>Reversade</surname>
<given-names>B</given-names>
</name>
</person-group>
<year>2013</year>
<article-title>ELABELA: a hormone essential for heart development signals via the apelin receptor</article-title>
<source>Developmental Cell</source>
<volume>27</volume>
<fpage>672</fpage>
<lpage>680</lpage>
<pub-id pub-id-type="doi">10.1016/j.devcel.2013.11.002</pub-id>
<pub-id pub-id-type="pmid">24316148</pub-id>
</element-citation>
</ref>
<ref id="bib5">
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Cleaver</surname>
<given-names>O</given-names>
</name>
<name>
<surname>Krieg</surname>
<given-names>PA</given-names>
</name>
</person-group>
<year>1998</year>
<article-title>VEGF mediates angioblast migration during development of the dorsal aorta in Xenopus</article-title>
<source>Development</source>
<volume>125</volume>
<fpage>3905</fpage>
<lpage>3914</lpage>
<pub-id pub-id-type="pmid">9729498</pub-id>
</element-citation>
</ref>
<ref id="bib6">
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Coultas</surname>
<given-names>L</given-names>
</name>
<name>
<surname>Chawengsaksophak</surname>
<given-names>K</given-names>
</name>
<name>
<surname>Rossant</surname>
<given-names>J</given-names>
</name>
</person-group>
<year>2005</year>
<article-title>Endothelial cells and VEGF in vascular development</article-title>
<source>Nature</source>
<volume>438</volume>
<fpage>937</fpage>
<lpage>945</lpage>
<pub-id pub-id-type="doi">10.1038/nature04479</pub-id>
<pub-id pub-id-type="pmid">16355211</pub-id>
</element-citation>
</ref>
<ref id="bib7">
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Ferrara</surname>
<given-names>N</given-names>
</name>
<name>
<surname>Carver-Moore</surname>
<given-names>K</given-names>
</name>
<name>
<surname>Chen</surname>
<given-names>H</given-names>
</name>
<name>
<surname>Dowd</surname>
<given-names>M</given-names>
</name>
<name>
<surname>Lu</surname>
<given-names>L</given-names>
</name>
<name>
<surname>O'Shea</surname>
<given-names>KS</given-names>
</name>
<name>
<surname>Powell-Braxton</surname>
<given-names>L</given-names>
</name>
<name>
<surname>Hillan</surname>
<given-names>KJ</given-names>
</name>
<name>
<surname>Moore</surname>
<given-names>MW</given-names>
</name>
</person-group>
<year>1996</year>
<article-title>Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene</article-title>
<source>Nature</source>
<volume>380</volume>
<fpage>439</fpage>
<lpage>442</lpage>
<pub-id pub-id-type="doi">10.1038/380439a0</pub-id>
<pub-id pub-id-type="pmid">8602242</pub-id>
</element-citation>
</ref>
<ref id="bib8">
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Fouquet</surname>
<given-names>B</given-names>
</name>
<name>
<surname>Weinstein</surname>
<given-names>BM</given-names>
</name>
<name>
<surname>Serluca</surname>
<given-names>FC</given-names>
</name>
<name>
<surname>Fishman</surname>
<given-names>MC</given-names>
</name>
</person-group>
<year>1997</year>
<article-title>Vessel patterning in the embryo of the zebrafish: guidance by notochord</article-title>
<source>Developmental Biology</source>
<volume>183</volume>
<fpage>37</fpage>
<lpage>48</lpage>
<pub-id pub-id-type="doi">10.1006/dbio.1996.8495</pub-id>
<pub-id pub-id-type="pmid">9119113</pub-id>
</element-citation>
</ref>
<ref id="bib9">
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Gordon</surname>
<given-names>K</given-names>
</name>
<name>
<surname>Schulte</surname>
<given-names>D</given-names>
</name>
<name>
<surname>Brice</surname>
<given-names>G</given-names>
</name>
<name>
<surname>Simpson</surname>
<given-names>MA</given-names>
</name>
<name>
<surname>Roukens</surname>
<given-names>MG</given-names>
</name>
<name>
<surname>van Impel</surname>
<given-names>A</given-names>
</name>
<name>
<surname>Connell</surname>
<given-names>F</given-names>
</name>
<name>
<surname>Kalidas</surname>
<given-names>K</given-names>
</name>
<name>
<surname>Jeffery</surname>
<given-names>S</given-names>
</name>
<name>
<surname>Mortimer</surname>
<given-names>PS</given-names>
</name>
<name>
<surname>Mansour</surname>
<given-names>S</given-names>
</name>
<name>
<surname>Schulte-Merker</surname>
<given-names>S</given-names>
</name>
<name>
<surname>Ostergaard</surname>
<given-names>P</given-names>
</name>
</person-group>
<year>2013</year>
<article-title>Mutation in vascular endothelial growth factor-C, a ligand for vascular endothelial growth factor receptor-3, is associated with autosomal dominant milroy-like primary lymphedema</article-title>
<source>Circulation Research</source>
<volume>112</volume>
<fpage>956</fpage>
<lpage>960</lpage>
<pub-id pub-id-type="doi">10.1161/CIRCRESAHA.113.300350</pub-id>
<pub-id pub-id-type="pmid">23410910</pub-id>
</element-citation>
</ref>
<ref id="bib10">
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Gore</surname>
<given-names>AV</given-names>
</name>
<name>
<surname>Monzo</surname>
<given-names>K</given-names>
</name>
<name>
<surname>Cha</surname>
<given-names>YR</given-names>
</name>
<name>
<surname>Pan</surname>
<given-names>W</given-names>
</name>
<name>
<surname>Weinstein</surname>
<given-names>BM</given-names>
</name>
</person-group>
<year>2012</year>
<article-title>Vascular development in the zebrafish</article-title>
<source>Cold Spring Harbor Perspectives in Medicine</source>
<volume>2</volume>
<fpage>a006684</fpage>
<pub-id pub-id-type="doi">10.1101/cshperspect.a006684</pub-id>
<pub-id pub-id-type="pmid">22553495</pub-id>
</element-citation>
</ref>
<ref id="bib11">
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Halpern</surname>
<given-names>ME</given-names>
</name>
<name>
<surname>Ho</surname>
<given-names>RK</given-names>
</name>
<name>
<surname>Walker</surname>
<given-names>C</given-names>
</name>
<name>
<surname>Kimmel</surname>
<given-names>CB</given-names>
</name>
</person-group>
<year>1993</year>
<article-title>Induction of muscle pioneers and floor plate is distinguished by the zebrafish no tail mutation</article-title>
<source>Cell</source>
<volume>75</volume>
<fpage>99</fpage>
<lpage>111</lpage>
<pub-id pub-id-type="doi">10.1016/S0092-8674(05)80087-X</pub-id>
<pub-id pub-id-type="pmid">8402905</pub-id>
</element-citation>
</ref>
<ref id="bib12">
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Helker</surname>
<given-names>CS</given-names>
</name>
<name>
<surname>Schuermann</surname>
<given-names>A</given-names>
</name>
<name>
<surname>Karpanen</surname>
<given-names>T</given-names>
</name>
<name>
<surname>Zeuschner</surname>
<given-names>D</given-names>
</name>
<name>
<surname>Belting</surname>
<given-names>HG</given-names>
</name>
<name>
<surname>Affolter</surname>
<given-names>M</given-names>
</name>
<name>
<surname>Schulte-Merker</surname>
<given-names>S</given-names>
</name>
<name>
<surname>Herzog</surname>
<given-names>W</given-names>
</name>
</person-group>
<year>2013</year>
<article-title>The zebrafish common cardinal veins develop by a novel mechanism: lumen ensheathment</article-title>
<source>Development</source>
<volume>140</volume>
<fpage>2776</fpage>
<lpage>2786</lpage>
<pub-id pub-id-type="doi">10.1242/dev.091876</pub-id>
<pub-id pub-id-type="pmid">23698350</pub-id>
</element-citation>
</ref>
<ref id="bib13">
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Hesselson</surname>
<given-names>D</given-names>
</name>
<name>
<surname>Anderson</surname>
<given-names>RM</given-names>
</name>
<name>
<surname>Beinat</surname>
<given-names>M</given-names>
</name>
<name>
<surname>Stainier</surname>
<given-names>DY</given-names>
</name>
</person-group>
<year>2009</year>
<article-title>Distinct populations of quiescent and proliferative pancreatic beta-cells identified by HOTcre mediated labeling</article-title>
<source>Proceedings of the National Academy of Sciences of USA</source>
<volume>106</volume>
<fpage>14896</fpage>
<lpage>14901</lpage>
<pub-id pub-id-type="doi">10.1073/pnas.0906348106</pub-id>
</element-citation>
</ref>
<ref id="bib14">
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Hogan</surname>
<given-names>BM</given-names>
</name>
<name>
<surname>Bos</surname>
<given-names>FL</given-names>
</name>
<name>
<surname>Bussmann</surname>
<given-names>J</given-names>
</name>
<name>
<surname>Witte</surname>
<given-names>M</given-names>
</name>
<name>
<surname>Chi</surname>
<given-names>NC</given-names>
</name>
<name>
<surname>Duckers</surname>
<given-names>HJ</given-names>
</name>
<name>
<surname>Schulte-Merker</surname>
<given-names>S</given-names>
</name>
</person-group>
<year>2009</year>
<article-title>Ccbe1 is required for embryonic lymphangiogenesis and venous sprouting</article-title>
<source>Nature Genetics</source>
<volume>41</volume>
<fpage>396</fpage>
<lpage>398</lpage>
<pub-id pub-id-type="doi">10.1038/ng.321</pub-id>
<pub-id pub-id-type="pmid">19287381</pub-id>
</element-citation>
</ref>
<ref id="bib15">
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Hwang</surname>
<given-names>WY</given-names>
</name>
<name>
<surname>Fu</surname>
<given-names>YF</given-names>
</name>
<name>
<surname>Reyon</surname>
<given-names>D</given-names>
</name>
<name>
<surname>Maeder</surname>
<given-names>ML</given-names>
</name>
<name>
<surname>Tsai</surname>
<given-names>SQ</given-names>
</name>
<name>
<surname>Sander</surname>
<given-names>JD</given-names>
</name>
<name>
<surname>Peterson</surname>
<given-names>RT</given-names>
</name>
<name>
<surname>Yeh</surname>
<given-names>JRJ</given-names>
</name>
<name>
<surname>Joung</surname>
<given-names>JK</given-names>
</name>
</person-group>
<year>2013</year>
<article-title>Efficient genome editing in zebrafish using a CRISPR-Cas system</article-title>
<source>Nature Biotechnology</source>
<volume>31</volume>
<fpage>227</fpage>
<lpage>229</lpage>
<pub-id pub-id-type="doi">10.1038/nbt.2501</pub-id>
</element-citation>
</ref>
<ref id="bib16">
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Jao</surname>
<given-names>LE</given-names>
</name>
<name>
<surname>Wente</surname>
<given-names>SR</given-names>
</name>
<name>
<surname>Chen</surname>
<given-names>W</given-names>
</name>
</person-group>
<year>2013</year>
<article-title>Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system</article-title>
<source>Proceedings of the National Academy of Sciences of USA</source>
<volume>110</volume>
<fpage>13904</fpage>
<lpage>13909</lpage>
<pub-id pub-id-type="doi">10.1073/pnas.1308335110</pub-id>
</element-citation>
</ref>
<ref id="bib17">
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Kawakami</surname>
<given-names>K</given-names>
</name>
<name>
<surname>Takeda</surname>
<given-names>H</given-names>
</name>
<name>
<surname>Kawakami</surname>
<given-names>N</given-names>
</name>
<name>
<surname>Kobayashi</surname>
<given-names>M</given-names>
</name>
<name>
<surname>Matsuda</surname>
<given-names>N</given-names>
</name>
<name>
<surname>Mishina</surname>
<given-names>M</given-names>
</name>
</person-group>
<year>2004</year>
<article-title>A transposon-mediated gene trap approach identifies developmentally regulated genes in zebrafish</article-title>
<source>Developmental Cell</source>
<volume>7</volume>
<fpage>133</fpage>
<lpage>144</lpage>
<pub-id pub-id-type="doi">10.1016/j.devcel.2004.06.005</pub-id>
<pub-id pub-id-type="pmid">15239961</pub-id>
</element-citation>
</ref>
<ref id="bib18">
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Kimmel</surname>
<given-names>CB</given-names>
</name>
<name>
<surname>Ballard</surname>
<given-names>WW</given-names>
</name>
<name>
<surname>Kimmel</surname>
<given-names>SR</given-names>
</name>
<name>
<surname>Ullmann</surname>
<given-names>B</given-names>
</name>
<name>
<surname>Schilling</surname>
<given-names>TF</given-names>
</name>
</person-group>
<year>1995</year>
<article-title>Stages of embryonic development of the zebrafish</article-title>
<source>Developmental Dynamics</source>
<volume>203</volume>
<fpage>253</fpage>
<lpage>310</lpage>
<pub-id pub-id-type="doi">10.1002/aja.1002030302</pub-id>
<pub-id pub-id-type="pmid">8589427</pub-id>
</element-citation>
</ref>
<ref id="bib19">
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Larson</surname>
<given-names>JD</given-names>
</name>
<name>
<surname>Wadman</surname>
<given-names>SA</given-names>
</name>
<name>
<surname>Chen</surname>
<given-names>E</given-names>
</name>
<name>
<surname>Kerley</surname>
<given-names>L</given-names>
</name>
<name>
<surname>Clark</surname>
<given-names>KJ</given-names>
</name>
<name>
<surname>Eide</surname>
<given-names>M</given-names>
</name>
<name>
<surname>Lippert</surname>
<given-names>S</given-names>
</name>
<name>
<surname>Nasevicius</surname>
<given-names>A</given-names>
</name>
<name>
<surname>Ekker</surname>
<given-names>SC</given-names>
</name>
<name>
<surname>Hackett</surname>
<given-names>PB</given-names>
</name>
<name>
<surname>Essner</surname>
<given-names>JJ</given-names>
</name>
</person-group>
<year>2004</year>
<article-title>Expression of VE-cadherin in zebrafish embryos: a new tool to evaluate vascular development</article-title>
<source>Developmental Dynamics</source>
<volume>231</volume>
<fpage>204</fpage>
<lpage>213</lpage>
<pub-id pub-id-type="doi">10.1002/dvdy.20102</pub-id>
<pub-id pub-id-type="pmid">15305301</pub-id>
</element-citation>
</ref>
<ref id="bib20">
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Lawson</surname>
<given-names>ND</given-names>
</name>
<name>
<surname>Vogel</surname>
<given-names>AM</given-names>
</name>
<name>
<surname>Weinstein</surname>
<given-names>BM</given-names>
</name>
</person-group>
<year>2002</year>
<article-title>sonic hedgehog and vascular endothelial growth factor act upstream of the Notch pathway during arterial endothelial differentiation</article-title>
<source>Developmental Cell</source>
<volume>3</volume>
<fpage>127</fpage>
<lpage>136</lpage>
<pub-id pub-id-type="doi">10.1016/S1534-5807(02)00198-3</pub-id>
<pub-id pub-id-type="pmid">12110173</pub-id>
</element-citation>
</ref>
<ref id="bib21">
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Lawson</surname>
<given-names>ND</given-names>
</name>
<name>
<surname>Weinstein</surname>
<given-names>BM</given-names>
</name>
</person-group>
<year>2002</year>
<article-title>In vivo imaging of embryonic vascular development using transgenic zebrafish</article-title>
<source>Developmental Biology</source>
<volume>248</volume>
<fpage>307</fpage>
<lpage>318</lpage>
<pub-id pub-id-type="doi">10.1006/dbio.2002.0711</pub-id>
<pub-id pub-id-type="pmid">12167406</pub-id>
</element-citation>
</ref>
<ref id="bib22">
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Nasevicius</surname>
<given-names>A</given-names>
</name>
<name>
<surname>Ekker</surname>
<given-names>SC</given-names>
</name>
</person-group>
<year>2000</year>
<article-title>Effective targeted gene 'knockdown' in zebrafish</article-title>
<source>Nature Genetics</source>
<volume>26</volume>
<fpage>216</fpage>
<lpage>220</lpage>
<pub-id pub-id-type="doi">10.1038/79951</pub-id>
<pub-id pub-id-type="pmid">11017081</pub-id>
</element-citation>
</ref>
<ref id="bib23">
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Nicoli</surname>
<given-names>S</given-names>
</name>
<name>
<surname>Tobia</surname>
<given-names>C</given-names>
</name>
<name>
<surname>Gualandi</surname>
<given-names>L</given-names>
</name>
<name>
<surname>De Sena</surname>
<given-names>G</given-names>
</name>
<name>
<surname>Presta</surname>
<given-names>M</given-names>
</name>
</person-group>
<year>2008</year>
<article-title>Calcitonin receptor-like receptor guides arterial differentiation in zebrafish</article-title>
<source>Blood</source>
<volume>111</volume>
<fpage>4965</fpage>
<lpage>4972</lpage>
<pub-id pub-id-type="doi">10.1182/blood-2007-10-118166</pub-id>
<pub-id pub-id-type="pmid">18326814</pub-id>
</element-citation>
</ref>
<ref id="bib24">
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Ober</surname>
<given-names>EA</given-names>
</name>
<name>
<surname>Olofsson</surname>
<given-names>B</given-names>
</name>
<name>
<surname>Makinen</surname>
<given-names>T</given-names>
</name>
<name>
<surname>Jin</surname>
<given-names>SW</given-names>
</name>
<name>
<surname>Shoji</surname>
<given-names>W</given-names>
</name>
<name>
<surname>Koh</surname>
<given-names>GY</given-names>
</name>
<name>
<surname>Alitalo</surname>
<given-names>K</given-names>
</name>
<name>
<surname>Stainier</surname>
<given-names>DY</given-names>
</name>
</person-group>
<year>2004</year>
<article-title>Vegfc is required for vascular development and endoderm morphogenesis in zebrafish</article-title>
<source>EMBO Rep</source>
<volume>5</volume>
<fpage>78</fpage>
<lpage>84</lpage>
<pub-id pub-id-type="doi">10.1038/sj.embor.7400047</pub-id>
<pub-id pub-id-type="pmid">14710191</pub-id>
</element-citation>
</ref>
<ref id="bib25">
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Odenthal</surname>
<given-names>J</given-names>
</name>
<name>
<surname>Haffter</surname>
<given-names>P</given-names>
</name>
<name>
<surname>Vogelsang</surname>
<given-names>E</given-names>
</name>
<name>
<surname>Brand</surname>
<given-names>M</given-names>
</name>
<name>
<surname>van Eeden</surname>
<given-names>FJ</given-names>
</name>
<name>
<surname>Furutani-Seiki</surname>
<given-names>M</given-names>
</name>
<name>
<surname>Granato</surname>
<given-names>M</given-names>
</name>
<name>
<surname>Hammerschmidt</surname>
<given-names>M</given-names>
</name>
<name>
<surname>Heisenberg</surname>
<given-names>CP</given-names>
</name>
<name>
<surname>Jiang</surname>
<given-names>YJ</given-names>
</name>
<name>
<surname>Kane</surname>
<given-names>DA</given-names>
</name>
<name>
<surname>Kelsh</surname>
<given-names>RN</given-names>
</name>
<name>
<surname>Mullins</surname>
<given-names>MC</given-names>
</name>
<name>
<surname>Warga</surname>
<given-names>RM</given-names>
</name>
<name>
<surname>Allende</surname>
<given-names>ML</given-names>
</name>
<name>
<surname>Weinberg</surname>
<given-names>ES</given-names>
</name>
<name>
<surname>Nüsslein-Volhard</surname>
<given-names>C</given-names>
</name>
</person-group>
<year>1996</year>
<article-title>Mutations affecting the formation of the notochord in the zebrafish, Danio rerio</article-title>
<source>Development</source>
<volume>123</volume>
<fpage>103</fpage>
<lpage>115</lpage>
<pub-id pub-id-type="pmid">9007233</pub-id>
</element-citation>
</ref>
<ref id="bib26">
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Pauli</surname>
<given-names>A</given-names>
</name>
<name>
<surname>Norris</surname>
<given-names>ML</given-names>
</name>
<name>
<surname>Valen</surname>
<given-names>E</given-names>
</name>
<name>
<surname>Chew</surname>
<given-names>GL</given-names>
</name>
<name>
<surname>Gagnon</surname>
<given-names>JA</given-names>
</name>
<name>
<surname>Zimmerman</surname>
<given-names>S</given-names>
</name>
<name>
<surname>Mitchell</surname>
<given-names>A</given-names>
</name>
<name>
<surname>Ma</surname>
<given-names>J</given-names>
</name>
<name>
<surname>Dubrulle</surname>
<given-names>J</given-names>
</name>
<name>
<surname>Reyon</surname>
<given-names>D</given-names>
</name>
<name>
<surname>Tsai</surname>
<given-names>SQ</given-names>
</name>
<name>
<surname>Joung</surname>
<given-names>JK</given-names>
</name>
<name>
<surname>Saghatelian</surname>
<given-names>A</given-names>
</name>
<name>
<surname>Schier</surname>
<given-names>AF</given-names>
</name>
</person-group>
<year>2014</year>
<article-title>Toddler: an embryonic signal that promotes cell movement via Apelin receptors</article-title>
<source>Science</source>
<volume>343</volume>
<fpage>1248636</fpage>
<pub-id pub-id-type="doi">10.1126/science.1248636</pub-id>
<pub-id pub-id-type="pmid">24407481</pub-id>
</element-citation>
</ref>
<ref id="bib27">
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Santoro</surname>
<given-names>MM</given-names>
</name>
<name>
<surname>Samuel</surname>
<given-names>T</given-names>
</name>
<name>
<surname>Mitchell</surname>
<given-names>T</given-names>
</name>
<name>
<surname>Reed</surname>
<given-names>JC</given-names>
</name>
<name>
<surname>Stainier</surname>
<given-names>DY</given-names>
</name>
</person-group>
<year>2007</year>
<article-title>Birc2 (cIap1) regulates endothelial cell integrity and blood vessel homeostasis</article-title>
<source>Nature Genetics</source>
<volume>39</volume>
<fpage>1397</fpage>
<lpage>1402</lpage>
<pub-id pub-id-type="doi">10.1038/ng.2007.8</pub-id>
<pub-id pub-id-type="pmid">17934460</pub-id>
</element-citation>
</ref>
<ref id="bib28">
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Scott</surname>
<given-names>IC</given-names>
</name>
<name>
<surname>Masri</surname>
<given-names>B</given-names>
</name>
<name>
<surname>D'Amico</surname>
<given-names>LA</given-names>
</name>
<name>
<surname>Jin</surname>
<given-names>SW</given-names>
</name>
<name>
<surname>Jungblut</surname>
<given-names>B</given-names>
</name>
<name>
<surname>Wehman</surname>
<given-names>AM</given-names>
</name>
<name>
<surname>Baier</surname>
<given-names>H</given-names>
</name>
<name>
<surname>Audigier</surname>
<given-names>Y</given-names>
</name>
<name>
<surname>Stainier</surname>
<given-names>DY</given-names>
</name>
</person-group>
<year>2007</year>
<article-title>The g protein-coupled receptor agtrl1b regulates early development of myocardial progenitors</article-title>
<source>Developmental Cell</source>
<volume>12</volume>
<fpage>403</fpage>
<lpage>413</lpage>
<pub-id pub-id-type="doi">10.1016/j.devcel.2007.01.012</pub-id>
<pub-id pub-id-type="pmid">17336906</pub-id>
</element-citation>
</ref>
<ref id="bib29">
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Shalaby</surname>
<given-names>F</given-names>
</name>
<name>
<surname>Rossant</surname>
<given-names>J</given-names>
</name>
<name>
<surname>Yamaguchi</surname>
<given-names>TP</given-names>
</name>
<name>
<surname>Gertsenstein</surname>
<given-names>M</given-names>
</name>
<name>
<surname>Wu</surname>
<given-names>XF</given-names>
</name>
<name>
<surname>Breitman</surname>
<given-names>ML</given-names>
</name>
<name>
<surname>Schuh</surname>
<given-names>AC</given-names>
</name>
</person-group>
<year>1995</year>
<article-title>Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice</article-title>
<source>Nature</source>
<volume>376</volume>
<fpage>62</fpage>
<lpage>66</lpage>
<pub-id pub-id-type="doi">10.1038/376062a0</pub-id>
<pub-id pub-id-type="pmid">7596435</pub-id>
</element-citation>
</ref>
<ref id="bib30">
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Sumoy</surname>
<given-names>L</given-names>
</name>
<name>
<surname>Keasey</surname>
<given-names>JB</given-names>
</name>
<name>
<surname>Dittman</surname>
<given-names>TD</given-names>
</name>
<name>
<surname>Kimelman</surname>
<given-names>D</given-names>
</name>
</person-group>
<year>1997</year>
<article-title>A role for notochord in axial vascular development revealed by analysis of phenotype and the expression of VEGR-2 in zebrafish flh and ntl mutant embryos</article-title>
<source>Mechanisms of Development</source>
<volume>63</volume>
<fpage>15</fpage>
<lpage>27</lpage>
<pub-id pub-id-type="doi">10.1016/S0925-4773(97)00671-0</pub-id>
<pub-id pub-id-type="pmid">9178253</pub-id>
</element-citation>
</ref>
<ref id="bib31">
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Verma</surname>
<given-names>A</given-names>
</name>
<name>
<surname>Bhattacharya</surname>
<given-names>R</given-names>
</name>
<name>
<surname>Remadevi</surname>
<given-names>I</given-names>
</name>
<name>
<surname>Li</surname>
<given-names>K</given-names>
</name>
<name>
<surname>Pramanik</surname>
<given-names>K</given-names>
</name>
<name>
<surname>Samant</surname>
<given-names>GV</given-names>
</name>
<name>
<surname>Horswill</surname>
<given-names>M</given-names>
</name>
<name>
<surname>Chun</surname>
<given-names>CZ</given-names>
</name>
<name>
<surname>Zhao</surname>
<given-names>B</given-names>
</name>
<name>
<surname>Wang</surname>
<given-names>E</given-names>
</name>
<name>
<surname>Miao</surname>
<given-names>RQ</given-names>
</name>
<name>
<surname>Mukhopadhyay</surname>
<given-names>D</given-names>
</name>
<name>
<surname>Ramchandran</surname>
<given-names>R</given-names>
</name>
<name>
<surname>Wilkinson</surname>
<given-names>GA</given-names>
</name>
</person-group>
<year>2010</year>
<article-title>Endothelial cell-specific chemotaxis receptor (ecscr) promotes angioblast migration during vasculogenesis and enhances VEGF receptor sensitivity</article-title>
<source>Blood</source>
<volume>115</volume>
<fpage>4614</fpage>
<lpage>4622</lpage>
<pub-id pub-id-type="doi">10.1182/blood-2009-10-248856</pub-id>
<pub-id pub-id-type="pmid">20086248</pub-id>
</element-citation>
</ref>
<ref id="bib32">
<element-citation publication-type="book">
<person-group person-group-type="author">
<name>
<surname>Westerfield</surname>
<given-names>M</given-names>
</name>
</person-group>
<year>1993</year>
<source>The Zebrafish Book</source>
<publisher-name>Oxford University Press, Oxford, UK</publisher-name>
</element-citation>
</ref>
<ref id="bib33">
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Zeng</surname>
<given-names>XX</given-names>
</name>
<name>
<surname>Wilm</surname>
<given-names>TP</given-names>
</name>
<name>
<surname>Sepich</surname>
<given-names>DS</given-names>
</name>
<name>
<surname>Solnica-Krezel</surname>
<given-names>L</given-names>
</name>
</person-group>
<year>2007</year>
<article-title>Apelin and its receptor control heart field formation during zebrafish gastrulation</article-title>
<source>Developmental Cell</source>
<volume>12</volume>
<fpage>391</fpage>
<lpage>402</lpage>
<pub-id pub-id-type="doi">10.1016/j.devcel.2007.01.011</pub-id>
<pub-id pub-id-type="pmid">17336905</pub-id>
</element-citation>
</ref>
<ref id="bib34">
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Zhang</surname>
<given-names>S</given-names>
</name>
<name>
<surname>Cao</surname>
<given-names>Z</given-names>
</name>
<name>
<surname>Tian</surname>
<given-names>H</given-names>
</name>
<name>
<surname>Shen</surname>
<given-names>G</given-names>
</name>
<name>
<surname>Ma</surname>
<given-names>Y</given-names>
</name>
<name>
<surname>Xie</surname>
<given-names>H</given-names>
</name>
<name>
<surname>Liu</surname>
<given-names>Y</given-names>
</name>
<name>
<surname>Zhao</surname>
<given-names>C</given-names>
</name>
<name>
<surname>Deng</surname>
<given-names>S</given-names>
</name>
<name>
<surname>Yang</surname>
<given-names>Y</given-names>
</name>
<name>
<surname>Zheng</surname>
<given-names>R</given-names>
</name>
<name>
<surname>Li</surname>
<given-names>W</given-names>
</name>
<name>
<surname>Zhang</surname>
<given-names>N</given-names>
</name>
<name>
<surname>Liu</surname>
<given-names>S</given-names>
</name>
<name>
<surname>Wang</surname>
<given-names>W</given-names>
</name>
<name>
<surname>Dai</surname>
<given-names>L</given-names>
</name>
<name>
<surname>Shi</surname>
<given-names>S</given-names>
</name>
<name>
<surname>Cheng</surname>
<given-names>L</given-names>
</name>
<name>
<surname>Pan</surname>
<given-names>Y</given-names>
</name>
<name>
<surname>Feng</surname>
<given-names>S</given-names>
</name>
<name>
<surname>Zhao</surname>
<given-names>X</given-names>
</name>
<name>
<surname>Deng</surname>
<given-names>H</given-names>
</name>
<name>
<surname>Yang</surname>
<given-names>S</given-names>
</name>
<name>
<surname>Wei</surname>
<given-names>Y</given-names>
</name>
</person-group>
<year>2011</year>
<article-title>SKLB1002, a novel potent inhibitor of VEGF receptor 2 signaling, inhibits angiogenesis and tumor growth in vivo</article-title>
<source>Clinical Cancer Research</source>
<volume>17</volume>
<fpage>4439</fpage>
<lpage>4450</lpage>
<pub-id pub-id-type="doi">10.1158/1078-0432.CCR-10-3109</pub-id>
<pub-id pub-id-type="pmid">21622720</pub-id>
</element-citation>
</ref>
</ref-list>
</back>
<sub-article id="SA1" article-type="article-commentary">
<front-stub>
<article-id pub-id-type="doi">10.7554/eLife.06726.017</article-id>
<title-group>
<article-title>Decision letter</article-title>
</title-group>
<contrib-group>
<contrib contrib-type="editor">
<name>
<surname>Whitfield</surname>
<given-names>Tanya T</given-names>
</name>
<role>Reviewing editor</role>
<aff>
<institution>University of Sheffield</institution>
,
<country>United Kingdom</country>
</aff>
</contrib>
</contrib-group>
</front-stub>
<body>
<boxed-text position="float" orientation="portrait">
<p>eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see
<ext-link ext-link-type="uri" xlink:href="http://elifesciences.org/review-process">review process</ext-link>
). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.</p>
</boxed-text>
<p>Thank you for sending your work entitled "The hormonal peptides Elabela and Apelin guide angioblasts to the midline during vasculogenesis" for consideration at
<italic>eLife</italic>
. Your article has been evaluated by Janet Rossant (Senior editor), Tanya Whitfield (Reviewing Editor), and two additional reviewers.</p>
<p>The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.</p>
<p>While the reviewers have all found the work interesting, all three have concerns over the extent that the data support your conclusions, including the challenges that you propose to existing literature. The full reviews are appended below, but a summary of the main points that must be addressed is:</p>
<p>1) Data should be supplied to demonstrate the efficacy of each of the morpholinos used, especially (in the case of the Vegf study) when the authors are proposing that knockdown has no effect on angioblast migration. The authors should also show whether any of the morpholinos cause systemic non-specific effects, such as developmental delay. If mutants are available, it would be helpful to repeat the analysis in mutants.</p>
<p>2) Additional, and quantitative, data are needed to support the assertion that Ela acts as an instructive chemoattractant in this context and not simply as a motogen. As two of the reviewers have pointed out, the dataset is currently too small to be able to reach this conclusion. Further data should be supplied to illustrate this point.</p>
<p>It is vitally important to provide strong support for the two points above, as these are the proposed advances in the understanding of angioblast migration over previously published literature.</p>
<p>3) The relative contributions of Apln and Ela to angioblast migration should be clarified, both in the Results section and in the Abstract and Impact Statement.</p>
<p>4) A reduction in Shh signalling should be demonstrated to support the cyclopamine data.</p>
<p>5) Take care to acknowledge the findings of previously published work e.g.
<xref rid="bib26" ref-type="bibr">Pauli et al., 2014</xref>
, and previously published expression patterns, appropriately.</p>
<p>6) Improve quantitation of all experiments: provide cell counts to support image panels; state n numbers wherever % values are shown, and ensure that appropriate statistical tests have been applied.</p>
<p>7) Correct typos, formatting and gene nomenclature throughout.</p>
<p>Reviewer #1:</p>
<p>This is an interesting and well-written short report examining the role of the Apelin receptor signalling pathway in angioblast migration in the zebrafish embryo.</p>
<p>The study challenges the current VEGF model for angioblast migration to the midline, and also proposes that in this context, the ligand Elabela functions as a chemoattractant rather than a motogen, again challenging previous interpretations in the published literature. Although the authors present some interesting preliminary observations to support these challenges, the data need further analysis and quantitation to strengthen the authors' conclusions.</p>
<p>The paper relies on morpholino (MO) knockdown for a number of experiments. Given the current scepticism in the field surrounding the use of MOs, it is particularly important to demonstrate that the MO experiments are justified and rigorous. The authors give references to previously published work for each MO they use, but nevertheless it would be helpful to show data (e.g. antibody or RNA splicing data demonstrating specific knockdown), or give a statement, to comment on the specificity and efficacy of each MO.</p>
<p>Confidence in the MO data is especially important given that the main phenotype that is shown for many of the experiments (a failure of angioblasts to migrate to the midline) could also be explained by a generalised developmental delay. It is therefore important to demonstrate that the MOs do not cause any general developmental delay (e.g. by showing a brightfield image of the whole embryo, and providing quantitative information about, for example, somite number).</p>
<p>The challenge to the VEGF model, a major conclusion of the manuscript, is based on just two data panels (
<xref ref-type="fig" rid="fig1">Figure 1C</xref>
), with no accompanying quantitation. For the
<italic>vegfaa</italic>
MO experiment, notwithstanding the caveats mentioned above for MO use, there do appear to be a few green cells beyond the midline, but the picture is so heavily cropped that it is not possible to assess this properly. The authors should provide some quantitation, together with further information to show that the MO is indeed disrupting VEGF signalling, to strengthen their conclusions for this experiment. The authors state that
<italic>vegfaa</italic>
is the 'main ortholog' in zebrafish, but it would also be helpful to know whether there are any other
<italic>vegf</italic>
genes or isoforms expressed at the relevant time and place, and whether these are affected by
<italic>vegfaa</italic>
MO knockdown or not. The mutant VEGFR (
<italic>kdrl</italic>
) analysis is more convincing, but again, are there other genes or isoforms that might function redundantly with the
<italic>kdrl</italic>
receptor?</p>
<p>The second challenge to existing literature, that Elabela (in this context) acts a chemoattractant rather than a motogen, is also supported only weakly, being based on a single supplementary figure panel (Figure 3–figure supplement 1D) showing one (or possibly two) green angioblasts in an ectopic dorsal position near to the ectopic Elabela-expressing (red) cells. This needs some further examples and quantitiation to support the authors' conclusion.</p>
<p>Quantitation:</p>
<p>The % data shown in
<xref ref-type="fig" rid="fig2 fig3 fig4">Figures 2C, 3C and 4B</xref>
must be accompanied by corresponding n numbers, preferably shown directly on the graphs.</p>
<p>
<xref ref-type="fig" rid="fig4">Figure 4B</xref>
. A
<italic>t</italic>
-test would not be recommended for an experiment involving more than one experimental condition. This analysis should be repeated using ANOVA or an equivalent test. The graph should use a bar to show which columns are being compared (presumably each of the experimental situations with the control).</p>
<p>Reviewer #2:</p>
<p>Helker C. et al. demonstrate the essential role for Elabela (Ela)-Apelin (Apln) receptor (Aplnr) signaling in the initial assembly of angioblasts as a vascular cord in the midline. Unexpectedly,
<italic>vegfa</italic>
is not required as a chemoattractant for angioblast migration, while Elabela, a secretary peptide from the notochord, functions as an angioblast chemoattractant. The data has been convincingly demonstrated using several gene mutants (
<italic>krdl</italic>
<sup>-/-</sup>
,
<italic>ela</italic>
<sup>br13-/-</sup>
,
<italic>apln</italic>
<sup>mu267-/-</sup>
, and
<italic>noto</italic>
) and their morphant.</p>
<p>The objectives written and the findings shown in the present manuscript are clear; however, there are several points should be addressed. There are several statements that are not supported by their results in the present study.</p>
<p>Major concerns:</p>
<p>1) Requirement of Apln for angioblast migration:</p>
<p>The authors claim that not only Ela but also Apln guides angioblasts to the midline in the Results section and in the Title. In
<xref ref-type="fig" rid="fig2">Figure 2A</xref>
, the data of
<italic>apln</italic>
<sup>
<italic>mu267-/-</italic>
</sup>
clearly show that Apln is not required for angioblast migration. However, the authors point to the requirement of Apln using the embryos of the
<italic>ela</italic>
<sup>
<italic>-/-</italic>
</sup>
and
<italic>apln</italic>
<sup>
<italic>-/-</italic>
</sup>
. On the contrary, they state the impact of Ela (instead of Apln)-Aplnr signaling in the Impact Statement. This interpretation might puzzle the readers. Therefore, they need to re-interpret the data and re-write the manuscript including the Title.</p>
<p>2) Vegf or Vegfa:</p>
<p>The authors use "Vegf" and "Vegfa" in the Abstract and main text. This inconsistency should be corrected not to mislead the readers. Are other
<italic>vegf</italic>
members except Vegfa are thought to regulate angioblast migration in the previous papers? According to their results, the conclusion might be that Vegfa-Vegfr2 signaling is required for angioblast migration toward the midline. Therefore, they should rewrite the manuscript by keeping the consistency when they use the similar words.</p>
<p>Reviewer #3:</p>
<p>Helker and colleagues provide evidence for new roles of previously identified small peptides Apelin and Elabela in guiding angioblasts towards the embryonic midline during zebrafish embryogenesis. Previous studies implicated Vascular endothelial growth factor in convergence of angioblasts towards the embryonic midline, where they coalesce to build the first embryonic vessels. The authors first present evidence from zebrafish against the requirement for Vegf in angioblast migration towards the midline. Rather, they proposed, Apelin and Elabela, have partially redundant function to guide angioblasts to the midline by acting through the Apelin receptors. Apelin, Apelin GPCR Receptor axis has been previously implicated in cardiac progenitor cell convergence and cell fate specification, by Scott et al., and Zeng et al., in 2007. Elabela acting through Apelin receptors has been shown to be required for gastrulation cell movements and both heart and endoderm development by
<xref rid="bib4" ref-type="bibr">Chng et al., 2013</xref>
and
<xref rid="bib26" ref-type="bibr">Pauli et al., 2014</xref>
. However, neither Apelin nor Elabela have been proposed to affect gastrulation cell movements acting as guidance molecules, with Elabela being considered a motogen. The authors employ morpholino oligonucleotides and mutations to show that Apelin and Elabela expressed in the notochord have partially redundant function in angioblast migration. Moreover,
<italic>apelin</italic>
receptors
<italic>a</italic>
and
<italic>b</italic>
expressed in angioblasts also have redundant function in this process. Therefore the proposed role of Apelin/Elabela/Apelin receptor signaling to guide angioblasts towards the embryonic midline would be a significant advance. However, the evidence in support of these conclusions are not compelling. There are also problems with the results and their interpretations. These problems should be addressed before the manuscript becomes suitable for publication.</p>
<p>The most novel conclusion of this work is that Elabela/Apelin/Apelin receptor axis has an instructive role in angioblast migration, by guiding them to the midline. The key line of evidence is that when cells ectopically expressing Ela were present in Ela mutant: "We found that individual Ela overexpressing cell were indeed capable of attracting angioblast migration to ectopic locations […]. our data therefore demonstrate, that Ela acts as a chemoattractant for angioblasts during midline migration".</p>
<p>The presented data is not compelling. The authors observed occasional overlap between angioblasts and these Ela expressing cells. An alternative interpretation is that Ela expressed in ectopic location increased motility of angioblasts and some of them wondered in ectopic locations.</p>
<p>The conclusion about Vegf signaling not being required in angioblast migration is based on the observation that "neither Vegf ligand depletion (
<italic>vegfaa</italic>
MO) nor a null mutation in the gene encoding the VEGFR2 ortholog (
<italic>kdrl</italic>
) interfered with angioblast migration in zebrafish embryos." However, there is no data showing the degree of loss-of-function for morpholino or for
<italic>kdrl</italic>
mutant.</p>
<p>Likewise, when the authors inhibit
<italic>Shh</italic>
signaling using cyclopamine, no quantitative data is presented documenting the degree of
<italic>Shh</italic>
signaling inhibition. Reduced expression of
<italic>vegf</italic>
is shown by in situ hybridization, but this is not a quantitative results. Such body of evidence is not sufficient to support the authors' conclusion regarding:</p>
<p>Does mutation in
<italic>apelin</italic>
gene affect
<italic>elabela</italic>
expression and vice versa?</p>
<p>Do mutations in
<italic>kdrl</italic>
affect phenotypes of
<italic>apelin</italic>
,
<italic>elabela</italic>
or
<italic>apelin</italic>
receptor single mutants?</p>
<p>The authors conclude that simultaneous knockdown of
<italic>aplnra</italic>
and
<italic>b</italic>
affects angioblast cell movement but not specification, based on morpholino interference. Whereas previously published morpholinos are used, appropriate experiments showing efficiency and specificity should be presented. Given that mutations for these receptors are available, they should be preferably employed.</p>
<p>Similar concerns are regarding
<italic>apelin</italic>
morpholino and
<italic>apelin</italic>
mutant experiments. Does
<italic>apelin</italic>
mutation lead to nonsense mediated degradation of
<italic>apelin</italic>
mRNA?</p>
<p>The authors conclude that angioblast migrate normally in
<italic>apelin</italic>
mutants, however, in
<xref ref-type="fig" rid="fig2">Figure 2</xref>
it appears that ca. 30% of
<italic>apelin</italic>
mutants showed mild angioblast migration phenotype. This along with a strong enhancement of the phenotype of
<italic>elabela</italic>
mutant indicates that
<italic>apelin</italic>
is also required for angioblast migration.</p>
<p>In the Results the authors write that cardiomyocyte deficiency observed in
<italic>aplnr</italic>
deficient embryos could not be phenocopied by loss of
<italic>apelin</italic>
function. This is not correct; Scott et al., and Zeng et al. reported reduction of cardiomyocyte formation in
<italic>apelin</italic>
morphant embryos.</p>
<p>[Editors' note: further revisions were requested prior to acceptance, as described below.]</p>
<p>Thank you for resubmitting your work entitled "The hormonal peptide Elabela guides angioblasts to the midline during vasculogenesis" for further consideration at
<italic>eLife</italic>
. Your revised article has been favorably evaluated by Janet Rossant (Senior editor) and Tanya Whitfield (Reviewing Editor). The manuscript is much improved and most of the reviewers' recommendations have been addressed. Mutant phenotypes have been used to back up the morpholino data, and most of the requested control data have been provided to demonstrate the efficacy of morpholinos and compounds used. Description of relative roles for Apln and Ela in the migration of angioblasts is now much clearer. However, there are still some minor issues concerning the description of the experimental design and quantitation of the data that should be addressed before publication, as outlined below:</p>
<p>1) The lack of a generalised developmental delay (control for somite number) should be stated explicitly in the text as well as in the Materials and methods, so that it can be ruled out as a possible explanation for a delay or failure of angioblasts to migrate to the midline in MO-injected or mutant embryos.</p>
<p>2) The new data supporting the assertion that Ela acts as a chemoattractant and not a motogen are still based on rather low numbers, and indeed the new data are still presented only as a supplementary figure (Figure 3–figure supplement 1). In the text description (Results, seventh paragraph), n numbers (cells and embryos) must be quoted in the text to support the percentage values, in addition to the information provided in the figure.</p>
<p>The chemoattractive effect of Ela appears to be an important conclusion of the manuscript, but is only based on this supplementary figure. If the data are not strong enough to be shown as a main figure, the conclusion should also be toned down: rather than stating that the data 'demonstrate' that Ela acts as a chemoattractant, wording such as 'the data support the interpretation that Ela is acting as a chemoattractant in this context, but further experiments and higher n numbers would be required to test this more rigorously' should be used. In addition, use of the word 'chemoattractant' should be treated with similar caution throughout the manuscript.</p>
<p>3)
<xref ref-type="fig" rid="fig4">Figure 4</xref>
and description and Results. Again, n numbers should be stated along with the percentages here, in the text as well as on the figure.</p>
<p>4) What do the error bars represent in
<xref ref-type="fig" rid="fig4">Figure 4B</xref>
?</p>
</body>
</sub-article>
<sub-article id="SA2" article-type="reply">
<front-stub>
<article-id pub-id-type="doi">10.7554/eLife.06726.018</article-id>
<title-group>
<article-title>Author response</article-title>
</title-group>
</front-stub>
<body>
<p>
<italic>1) Data should be supplied to demonstrate the efficacy of each of the morpholinos used, especially (in the case of the Vegf study) when the authors are proposing that knockdown has no effect on angioblast migration. The authors should also show whether any of the morpholinos cause systemic non-specific effects, such as developmental delay. If mutants are available, it would be helpful to repeat the analysis in mutants</italic>
.</p>
<p>We have added mutant analysis, to complement morpholino data, wherever possible.</p>
<p>We generated new mutants for both Apln receptors as well as double mutants and have provided better controls as well as additional pharmacological studies for the analysis of the role of Vegf signaling.</p>
<p>
<xref ref-type="fig" rid="fig1">Figure 1</xref>
has been changed and is now showing
<italic>vegfaa/vegfab</italic>
double MO injections, so that they cannot compensate for each other.</p>
<p>In addition a new supplemental figure,
<xref ref-type="fig" rid="fig1s1">Figure1–figure supplement 1</xref>
, has been generated (the cyclopamine data are now
<xref ref-type="fig" rid="fig1s2">Figure1–figure supplement 2</xref>
). We are now showing the later phenotypes of Vegf-signaling deficiency, to demonstrate efficiency of the used reagents. Also inhibition of Vegf-signaling by chemical treatment has been added as an alternative to MO generated data.</p>
<p>
<xref ref-type="fig" rid="fig2">Figure 2</xref>
: new panels have been added and show the phenotype-genotype correlation of single and double
<italic>apelin</italic>
receptor mutant embryos.</p>
<p>
<xref ref-type="fig" rid="fig2s2">Figure 2–figure supplement 2</xref>
: generation of the
<italic>aplnr</italic>
mutants has been added.</p>
<p>Any potential effect of developmental delay has been avoided by careful staging of the embryos (i.e. counting of somites to ensure the same developmental stage: now included in the method description).</p>
<p>
<italic>2) Additional, and quantitative, data are needed to support the assertion that Ela acts as an instructive chemoattractant in this context and not simply as a motogen. As two of the reviewers have pointed out, the dataset is currently too small to be able to reach this conclusion. Further data should be supplied to illustrate this point</italic>
.</p>
<p>We have added a new series of experiments, ectopically overexpressing Ela or Cherry in
<italic>noto</italic>
or
<italic>ta</italic>
mutants (to reduce endogenous Ela and Apln in the midline), in which the distance of angioblasts to Ela overexpressing cells (compared to Cherry overexpressing cells) was quantified.</p>
<p>Our data strongly support the role of Ela as a chemoattractant in the context of angioblast migration.</p>
<p>To address this point, a new Figure 3–figure supplement 1 has been added.</p>
<p>
<italic>It is vitally important to provide strong support for the two points above, as these are the proposed advances in the understanding of angioblast migration over previously published literature</italic>
.</p>
<p>
<italic>3) The relative contributions of Apln and Ela to angioblast migration should be clarified, both in the Results section and in the Abstract and Impact Statement</italic>
.</p>
<p>We have changed the manuscript text accordingly.</p>
<p>
<italic>4) A reduction in Shh signalling should be demonstrated to support the cyclopamine data</italic>
.</p>
<p>Block of hedgehog signaling using cyclopamine was originally published in chicken embryos (Incardona JP et al. Development. 1998 Sep;125(18):3553-62).</p>
<p>In zebrafish cyclopamine treatment has been used in many more than 70 publications, most notably it was shown that:</p>
<p>a) 200µM treatment fully phenocopied the
<italic>shh</italic>
mutant
<italic>sonic you</italic>
(
<ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Neumann%20CJ%5BAuthor%5D%26cauthor=true%26cauthor_uid=10518498">Neumann CJ</ext-link>
et al, Development. 1999 Nov;126(21):4817-26).</p>
<p>b) 100µM completely eliminated Hh activity (
<ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Chen%20W%5BAuthor%5D%26cauthor=true%26cauthor_uid=11493557">Chen W</ext-link>
et al., Development. 2001 Jun;128(12):2385-96.</p>
<p>c) 25µM cyclopamine completely abolishes Vegf expression in the somites (as also seen in
<italic>syu</italic>
and
<italic>yot</italic>
mutants) (Lawson ND et al Dev Cell. 2002 Jul;3(1):127-36).</p>
<p>As this analysis of cyclopamine effects has therefore been done extensively and we have used 100µM (far more than N. Lawson, who demonstrated the effect on Vegf expression), we have not added in situs on other downstream markers. We have changed the wording, to state the previously published result more clearly.</p>
<p>However, we have added an alternative experiment showing the requirement for Vegf signaling by treatment with a Vegf receptor 2 inhibitor, hoping that this will be provide a meaningful alternative (see
<xref ref-type="fig" rid="fig1s1">Figure 1–figure supplement 1</xref>
).</p>
<p>
<italic>5) Take care to acknowledge the findings of previously published work e.g.</italic>
<xref rid="bib26" ref-type="bibr">
<italic>Pauli et al., 2014</italic>
</xref>
<italic>, and previously published expression patterns, appropriately</italic>
.</p>
<p>We have revised the manuscript and added the appropriate references accordingly,</p>
<p>
<italic>6) Improve quantitation of all experiments: provide cell counts to support image panels; state n numbers wherever % values are shown, and ensure that appropriate statistical tests have been applied</italic>
.</p>
<p>We have added the n-numbers (cells and embryos) for all quantifications, provided source files, and added statistical analysis as requested.</p>
<p>
<italic>7) Correct typos, formatting and gene nomenclature throughout</italic>
.</p>
<p>We have edited the manuscript carefully, with special attention to these issues. It sometimes remains non-intuitive for the reader, that the nomenclature rules are not the same for mouse and zebrafish, so we have chosen zebrafish nomenclature, unless specifically referring to mouse data. As an example: "VEGF" would be the correct mouse specific spelling (see
<ext-link ext-link-type="uri" xlink:href="http://www.informatics.jax.org/mgihome/nomen/gene.shtml">http://www.informatics.jax.org/mgihome/nomen/gene.shtml</ext-link>
), "Vegf" would be correct for zebrafish (see ZFIN at
<ext-link ext-link-type="uri" xlink:href="https://wiki.zfin.org/display/general/ZFIN+Zebrafish+Nomenclature+Guidelines">https://wiki.zfin.org/display/general/ZFIN+Zebrafish+Nomenclature+Guidelines</ext-link>
).</p>
<p>
<italic>Reviewer #1</italic>
:</p>
<p>
<italic>The study challenges the current VEGF model for angioblast migration to the midline, and also proposes that in this context, the ligand Elabela functions as a chemoattractant rather than a motogen, again challenging previous interpretations in the published literature. Although the authors present some interesting preliminary observations to support these challenges, the data need further analysis and quantitation to strengthen the authors' conclusions</italic>
.</p>
<p>
<italic>The paper relies on morpholino (MO) knockdown for a number of experiments. Given the current scepticism in the field surrounding the use of MOs, it is particularly important to demonstrate that the MO experiments are justified and rigorous. The authors give references to previously published work for each MO they use, but nevertheless it would be helpful to show data (e.g. antibody or RNA splicing data demonstrating specific knockdown), or give a statement, to comment on the specificity and efficacy of each MO</italic>
.</p>
<p>As discussed above, we have now provided mutant data to complement MO data, wherever possible, for the Vegf studies we have used chemical inhibition of Vegfr2 as an alternative.</p>
<p>
<italic>Confidence in the MO data is especially important given that the main phenotype that is shown for many of the experiments (a failure of angioblasts to migrate to the midline) could also be explained by a generalised developmental delay. It is therefore important to demonstrate that the MOs do not cause any general developmental delay (e.g. by showing a brightfield image of the whole embryo, and providing quantitative information about, for example, somite number)</italic>
.</p>
<p>We had already staged all embryos according to somite numbers, but failed to state this in the manuscript, we have changed the Methods section accordingly.</p>
<p>
<italic>The challenge to the VEGF model, a major conclusion of the manuscript, is based on just two data panels (</italic>
<xref ref-type="fig" rid="fig1">
<italic>Figure 1C</italic>
</xref>
<italic>), with no accompanying quantitation. For the</italic>
vegfaa
<italic>MO experiment, notwithstanding the caveats mentioned above for MO use, there do appear to be a few green cells beyond the midline, but the picture is so heavily cropped that it is not possible to assess this properly. The authors should provide some quantitation, together with further information to show that the MO is indeed disrupting VEGF signalling, to strengthen their conclusions for this experiment. The authors state that</italic>
vegfaa
<italic>is the 'main ortholog' in zebrafish, but it would also be helpful to know whether there are any other</italic>
vegf
<italic>genes or isoforms expressed at the relevant time and place, and whether these are affected by</italic>
vegfaa
<italic>MO knockdown or not. The mutant VEGFR (</italic>
kdrl
<italic>) analysis is more convincing, but again, are there other genes or isoforms that might function redundantly with the</italic>
kdrl
<italic>receptor</italic>
?</p>
<p>
<italic>vegfaa</italic>
MO experiments have been replaced by combined knockdown of
<italic>vegfaa</italic>
and
<italic>vegfab</italic>
. In addition we show as a supplemental figure an analysis of all Vegf-signaling deficient embryos at a later developmental time point, where they all display the previously described defects in Vegf-dependent sprouting angiogenesis.</p>
<p>
<italic>The second challenge to existing literature, that Elabela (in this context) acts a chemoattractant rather than a motogen, is also supported only weakly, being based on a single supplementary figure panel (Figure 3–figure supplement 1D) showing one (or possibly two) green angioblasts in an ectopic dorsal position near to the ectopic Elabela-expressing (red) cells. This needs some further examples and quantitiation to support the authors' conclusion</italic>
.</p>
<p>As described above a new set of experiments with full quantification has been added as Figure 3–figure supplement 1.</p>
<p>Quantitation:</p>
<p>
<italic>The % data shown in</italic>
<xref ref-type="fig" rid="fig2 fig3 fig4">
<italic>Figures 2C, 3C and 4B</italic>
</xref>
<italic>must be accompanied by corresponding n numbers, preferably shown directly on the graphs</italic>
.</p>
<p>We have added the n numbers in each graph.</p>
<p>
<xref ref-type="fig" rid="fig4">
<italic>Figure 4B</italic>
</xref>
<italic>. A</italic>
t
<italic>-test would not be recommended for an experiment involving more than one experimental condition. This analysis should be repeated using ANOVA or an equivalent test. The graph should use a bar to show which columns are being compared (presumably each of the experimental situations with the control)</italic>
.</p>
<p>We have done an ANOVA analysis as well as the
<italic>t</italic>
-test and added the bars.</p>
<p>
<italic>Reviewer #2</italic>
:</p>
<p>
<italic>Major concerns</italic>
:</p>
<p>
<italic>1) Requirement of Apln for angioblast migration</italic>
:</p>
<p>
<italic>The authors claim that not only Ela but also Apln guides angioblasts to the midline in the Results section and in the Title. In</italic>
<xref ref-type="fig" rid="fig2">
<italic>Figure 2A</italic>
</xref>
<italic>, the data of</italic>
apln
<sup>mu267-/-</sup>
<italic>clearly show that Apln is not required for angioblast migration. However, the authors point to the requirement of Apln using the embryos of the</italic>
ela
<sup>-/-</sup>
<italic>and</italic>
apln
<sup>-/-</sup>
.
<italic>On the contrary, they state the impact of Ela (instead of Apln)-Aplnr signaling in the Impact Statement. This interpretation might puzzle the readers. Therefore, they need to re-interpret the data and re-write the manuscript including the Title</italic>
.</p>
<p>We have clarified the inconsistencies, and re-written the manuscript accordingly.</p>
<p>
<italic>2) Vegf or Vegfa</italic>
:</p>
<p>
<italic>The authors use "Vegf" and "Vegfa" in the Abstract and main text. This inconsistency should be corrected not to mislead the readers. Are other</italic>
vegf
<italic>members except Vegfa are thought to regulate angioblast migration in the previous papers? According to their results, the conclusion might be that Vegfa-Vegfr2 signaling is required for angioblast migration toward the midline. Therefore, they should rewrite the manuscript by keeping the consistency when they use the similar words</italic>
.</p>
<p>We now always specify Vegfa, and have re-written the manuscript accordingly.</p>
<p>
<italic>Reviewer #3</italic>
:</p>
<p>
<italic>Helker and colleagues provide evidence for new roles of previously identified small peptides Apelin and Elabela in guiding angioblasts towards the embryonic midline during zebrafish embryogenesis. Previous studies implicated Vascular endothelial growth factor in convergence of angioblasts towards the embryonic midline, where they coalesce to build the first embryonic vessels. The authors first present evidence from zebrafish against the requirement for Vegf in angioblast migration towards the midline. Rather, they proposed, Apelin and Elabela, have partially redundant function to guide angioblasts to the midline by acting through the Apelin receptors. Apelin, Apelin GPCR Receptor axis has been previously implicated in cardiac progenitor cell convergence and cell fate specification, by Scott et al., and Zeng et al., in 2007. Elabela acting through Apelin receptors has been shown to be required for gastrulation cell movements and both heart and endoderm development by</italic>
<xref rid="bib4" ref-type="bibr">
<italic>Chng et al., 2013</italic>
</xref>
<italic>and</italic>
<xref rid="bib26" ref-type="bibr">
<italic>Pauli et al., 2014</italic>
</xref>
<italic>. However, neither Apelin nor Elabela have been proposed to affect gastrulation cell movements acting as guidance molecules, with Elabela being considered a motogen. The authors employ morpholino oligonucleotides and mutations to show that Apelin and Elabela expressed in the notochord have partially redundant function in angioblast migration. Moreover,</italic>
apelin receptors a
<italic>and</italic>
b
<italic>expressed in angioblasts also have redundant function in this process. Therefore the proposed role of Apelin/Elabela/Apelin receptor signaling to guide angioblasts towards the embryonic midline would be a significant advance. However, the evidence in support of these conclusions are not compelling. There are also problems with the results and their interpretations. These problems should be addressed before the manuscript becomes suitable for publication</italic>
.</p>
<p>
<italic>The most novel conclusion of this work is that Elabela/Apelin/Apelin receptor axis has an instructive role in angioblast migration, by guiding them to the midline. The key line of evidence is that when cells ectopically expressing Ela were present in Ela mutant: "We found that individual Ela overexpressing cell were indeed capable of attracting angioblast migration to ectopic locations" […] our data therefore demonstrate, that Ela acts as a chemoattractant for angioblasts during midline migration"</italic>
.</p>
<p>
<italic>The presented data is not compelling. The authors observed occasional overlap between angioblasts and these Ela expressing cells. An alternative interpretation is that Ela expressed in ectopic location increased motility of angioblasts and some of them wondered in ectopic locations</italic>
.</p>
<p>As described above, we have now added a new figure, in which distances between angioblast and control or
<italic>ela</italic>
expressing cells were measured. This analysis now shows a clear attractant role of Ela. By using
<italic>noto</italic>
or
<italic>ta</italic>
mutants, we have abolished
<italic>ela</italic>
and
<italic>apln</italic>
expression in the midline and could therefore score all angioblasts and not only those at very ectopic locations.</p>
<p>
<italic>The conclusion about Vegf signaling not being required in angioblast migration is based on the observation that "neither Vegf ligand depletion (</italic>
vegfaa
<italic>MO) nor a null mutation in the gene encoding the VEGFR2 ortholog (</italic>
kdrl
<italic>) interfered with angioblast migration in zebrafish embryos." However, there is no data showing the degree of loss-of-function for morpholino or for</italic>
kdrl
<italic>mutant</italic>
.</p>
<p>As described above this has been added, in
<xref ref-type="fig" rid="fig1s1">Figure 1–figure supplement 1</xref>
.</p>
<p>
<italic>Likewise, when the authors inhibit Shh signaling using cyclopamine, no quantitative data is presented documenting the degree of Shh signaling inhibition. Reduced expression of</italic>
vegf
<italic>is shown by in situ hybridization, but this is not a quantitative results. Such body of evidence is not sufficient to support the authors' conclusion regarding</italic>
:</p>
<p>The role of Shh (and cyclopamine treatment) on regulating Vegfaa has been published previously (with all controls). We regret that we have not expressed that well enough in the previous manuscript and have changed the wording. Additional experiments (chemical inhibtion of Vegfr2) have been added.</p>
<p>
<italic>Does mutation in</italic>
apelin
<italic>gene affect</italic>
elabela
<italic>expression and vice versa</italic>
?</p>
<p>
<italic>Do mutations in</italic>
kdrl
<italic>affect phenotypes of</italic>
apelin
<italic>,</italic>
elabela
<italic>or</italic>
apelin receptor
<italic>single mutants</italic>
?</p>
<p>
<italic>The authors conclude that simultaneous knockdown of</italic>
aplnra
<italic>and</italic>
b
<italic>affects angioblast cell movement but not specification, based on morpholino interference. Whereas previously published morpholinos are used, appropriate experiments showing efficiency and specificity should be presented. Given that mutations for these receptors are available, they should be preferably employed</italic>
.</p>
<p>We have by now generated mutant in the
<italic>aplnr</italic>
genes and were now able to analyze also double deficient embryos. Our mutant analysis confirms our morpholino generated data.</p>
<p>
<italic>Similar concerns are regarding</italic>
apelin
<italic>morpholino and</italic>
apelin
<italic>mutant experiments. Does</italic>
apelin
<italic>mutation lead to nonsense mediated degradation of</italic>
apelin
<italic>mRNA</italic>
?</p>
<p>
<italic>The authors conclude that angioblast migrate normally in</italic>
apelin
<italic>mutants, however, in</italic>
<xref ref-type="fig" rid="fig2">
<italic>Figure 2</italic>
</xref>
<italic>it appears that ca. 30% of</italic>
apelin
<italic>mutants showed mild angioblast migration phenotype. This along with a strong enhancement of the phenotype of</italic>
elabela
<italic>mutant indicates that</italic>
apelin
<italic>is also required for angioblast migration</italic>
.</p>
<p>
<italic>In the Results the authors write that cardiomyocyte deficiency observed in</italic>
aplnr
<italic>deficient embryos could not be phenocopied by loss of</italic>
apelin
<italic>function. This is not correct; Scott et al., and Zeng et al. reported reduction of cardiomyocyte formation in</italic>
apelin
<italic>morphant embryos</italic>
.</p>
<p>Scott et al claim: "Midline expression of the Agtrl1b ligand Apln suggests that it may provide a signal for migration of the myocardial progenitors. However, injection of three independent
<italic>apln</italic>
MOs (designed against the 5'UTR and the splice donor site in intron 1) caused severe defects in body axis elongation and morphogenesis (
<xref ref-type="fig" rid="fig3">Figure S3D</xref>
), but did not recapitulate the
<italic>grn</italic>
myocardial phenotype, as significant myocardium was formed in the morphants (
<xref ref-type="fig" rid="fig3">Figure S3E</xref>
). The MOs appear to be functional, as coinjection of the 5'UTR-targeted MO was sufficient to reverse the inhibition of cardiomyogenesis caused by
<italic>apln</italic>
RNA levels far exceeding those normally found in vivo (
<xref ref-type="fig" rid="fig3">Figure S3F</xref>
). While exogenous
<italic>apln</italic>
can therefore recapitulate the
<italic>grn</italic>
phenotype, and Apln can act as a ligand to stimulate Agtrl1b activity, an
<italic>apln</italic>
mutant will be required to definitively test whether Apln is the in vivo signal for Agtrl1b during myocardial progenitor development.”</p>
<p>Zeng et al. are not as direct in their statements, but they claim: "However, the amount of cell death did not correlate with the severity of heart loss: we observed a level of cell death in
<italic>agtrl1b</italic>
morphants (where heart was strongly reduced) that was comparable to that in
<italic>apelin</italic>
morphants (with much milder reduction of the heart field,
<xref ref-type="fig" rid="fig4">Figure S4</xref>
and data not shown).”</p>
<p>However, we have changed our wording to a less explicit: “Previous studies have identified a requirement for Apln receptors in myocard development in zebrafish (
<xref rid="bib28" ref-type="bibr">Scott et al., 2007</xref>
;
<xref rid="bib33" ref-type="bibr">Zeng et al., 2007</xref>
) and mice (
<xref rid="bib3" ref-type="bibr">Charo et al., 2009</xref>
), which could not completely be phenocopied by Apln deficiency (
<xref rid="bib3" ref-type="bibr">Charo et al., 2009</xref>
;
<xref rid="bib28" ref-type="bibr">Scott et al., 2007</xref>
;
<xref rid="bib33" ref-type="bibr">Zeng et al., 2007</xref>
).”</p>
<p>[Editors' note: further revisions were requested prior to acceptance, as described below.]</p>
<p>
<italic>1) The lack of a generalised developmental delay (control for somite number) should be stated explicitly in the text as well as in the Materials and methods, so that it can be ruled out as a possible explanation for a delay or failure of angioblasts to migrate to the midline in MO-injected or mutant embryos</italic>
.</p>
<p>We have changed the text accordingly and pointed this out.</p>
<p>
<italic>2) The new data supporting the assertion that Ela acts as a chemoattractant and not a motogen are still based on rather low numbers, and indeed the new data are still presented only as a supplementary figure (Figure 3–figure supplement 1). In the text description (Results, seventh paragraph), n numbers (cells and embryos) must be quoted in the text to support the percentage values, in addition to the information provided in the figure</italic>
.</p>
<p>
<italic>The chemoattractive effect of Ela appears to be an important conclusion of the manuscript, but is only based on this supplementary figure. If the data are not strong enough to be shown as a main figure, the conclusion should also be toned down: rather than stating that the data 'demonstrate' that Ela acts as a chemoattractant, wording such as 'the data support the interpretation that Ela is acting as a chemoattractant in this context, but further experiments and higher n numbers would be required to test this more rigorously' should be used. In addition, use of the word 'chemoattractant' should be treated with similar caution throughout the manuscript</italic>
.</p>
<p>We had discussed already previously, whether we should include this figure into the main manuscript figures and have now added it as
<xref ref-type="fig" rid="fig4">Figure 4</xref>
.
<xref ref-type="fig" rid="fig4">Figure 4</xref>
then has been relabeled to become
<xref ref-type="fig" rid="fig5">Figure 5</xref>
. We have also toned the wording down a little bit (at the position indicated and in the final summary). However, we believe our data not to be based on low numbers as we repeated the same experiment in the 2 notochord mutants and thereby generated separate data sets, each of them alone was statistically significant, so theoretically either of them would have already been sufficient.</p>
<p>
<italic>3)</italic>
<xref ref-type="fig" rid="fig4">
<italic>Figure 4</italic>
</xref>
<italic>and description and Results. Again, n numbers should be stated along with the percentages here, in the text as well as on the figure</italic>
.</p>
<p>We have changed the manuscript in the Results section accordingly. They were already indicated in the figure and stated in the figure legend in b.</p>
<p>
<italic>4) What do the error bars represent in</italic>
<xref ref-type="fig" rid="fig4">
<italic>Figure 4B</italic>
</xref>
?</p>
<p>Now
<xref ref-type="fig" rid="fig5">Figure 5B</xref>
. They actually represent the SEM, however we did not phrase this clearly. We have changed the manuscript accordingly and added the excel sheet used for calculation as a source file. We have referred to these source data in the figure legend of
<xref ref-type="fig" rid="fig5">Figure 5</xref>
.</p>
<p>As an example: transplants of ctr. MO in Wt was analyzed in 7 embryos (with totally 39 cells transplanted), of these in 5 embryos 100% of the transplanted cells migrated to the midline, in 1 embryo 75%, and in one embryo 80%, resulting in an average percentage of 93.57%.</p>
<p>Alternatively, it would also have been possible to calculate total numbers (i.e. 2 cells out of 39 cells did not migrate to the midline), but we wanted to reflect that there was not much variation between the individual embryos analyzed.</p>
</body>
</sub-article>
</pmc>
</record>

Pour manipuler ce document sous Unix (Dilib)

EXPLOR_STEP=$WICRI_ROOT/Wicri/Sante/explor/LymphedemaV1/Data/Pmc/Corpus

HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 004679 | SxmlIndent | more

Ou

HfdSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd -nk 004679 | SxmlIndent | more

Pour mettre un lien sur cette page dans le réseau Wicri

{{Explor lien
   |wiki=    Wicri/Sante
   |area=    LymphedemaV1
   |flux=    Pmc
   |étape=   Corpus
   |type=    RBID
   |clé=     PMC:4468421
   |texte=   The hormonal peptide Elabela guides angioblasts to the midline during vasculogenesis
}}

Pour générer des pages wiki

HfdIndexSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/RBID.i   -Sk "pubmed:26017639" \
       | HfdSelect -Kh $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd   \
       | NlmPubMed2Wicri -a LymphedemaV1
Wicri

This area was generated with Dilib version V0.6.31.
Data generation: Sat Nov 4 17:40:35 2017. Site generation: Tue Feb 13 16:42:16 2024