title: Global identification of Smad2 and Eomesodermin targets in zebrafish identifies a conserved transcriptional network in mesendoderm and a novel role for Eomesodermin in repression of ectodermal gene expression.
abstract: BACKGROUND
  Nodal signalling is an absolute requirement for normal mesoderm and endoderm formation in vertebrate embryos, yet the transcriptional networks acting directly downstream of Nodal and the extent to which they are conserved is largely unexplored, particularly in vivo. Eomesodermin also plays a role in patterning mesoderm and endoderm in vertebrates, but its mechanisms of action, and how it interacts with the Nodal signalling pathway are still unclear.
  
  
  RESULTS
  Using a combination of ChIP-seq and expression analysis we identify direct targets of Smad2, the effector of Nodal signalling in blastula stage zebrafish embryos, including many novel target genes. Through comparison of these data with published ChIP-seq data in human, mouse and Xenopus we show that the transcriptional network driven by Smad2 in mesoderm and endoderm is conserved in these vertebrate species. We also show that Smad2 and zebrafish Eomesodermin a (Eomesa) bind common genomic regions proximal to genes involved in mesoderm and endoderm formation, suggesting Eomesa forms a general component of the Smad2 signalling complex in zebrafish. Combinatorial perturbation of Eomesa and Smad2-interacting factor Foxh1 results in loss of both mesoderm and endoderm markers, confirming the role of Eomesa in endoderm formation and its functional interaction with Foxh1 for correct Nodal signalling. Finally, we uncover a novel, role for Eomesa in repressing ectodermal genes in the early blastula.
  
  
  CONCLUSIONS
  Our data demonstrate that evolutionarily conserved developmental functions of Nodal signalling occur through maintenance of the transcriptional network directed by Smad2. This network is modulated by Eomesa in zebrafish which acts to promote mesoderm and endoderm formation in combination with Nodal signalling, whilst Eomesa also opposes ectoderm gene expression. Eomesa therefore regulates the formation of all three germ layers in the early zebrafish embryo.
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Nelson
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Cutty
authors.forename: Marie
authors.initials: M
authors.lastname: Niini
authors.forename: Derek L
authors.initials: DL
authors.lastname: Stemple
authors.forename: Paul
authors.initials: P
authors.lastname: Flicek
authors.forename: Corinne
authors.initials: C
authors.lastname: Houart
authors.forename: Ashley E E
authors.initials: AE
authors.lastname: Bruce
authors.forename: Fiona C
authors.initials: FC
authors.lastname: Wardle
mesh_terms: D000818:Animals; D019521:Body Patterning; D004475:Ectoderm / Q000196:embryology / Q000378:metabolism; D004625:Embryo, Nonmammalian / Q000196:embryology / Q000378:metabolism; D004707:Endoderm / Q000196:embryology / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D053263:Gene Regulatory Networks; D008648:Mesoderm / Q000196:embryology / Q000378:metabolism; D015398:Signal Transduction; D051899:Smad2 Protein / Q000235:genetics* / Q000378:metabolism; D020825:T-Box Domain Proteins / Q000235:genetics* / Q000378:metabolism; D015027:Zebrafish / Q000196:embryology / Q000235:genetics* / Q000378:metabolism; D029961:Zebrafish Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D051899:Smad2 Protein; D020825:T-Box Domain Proteins; D029961:Zebrafish Proteins; C491197:eomesa protein, zebrafish; C497786:smad2 protein, zebrafish