ndr1overexpression in zebrafish blastulas identifies known and novel Nodal target genes. (A) Heatmap of a representative selection of genes induced on ndr1 overexpression across the full range of P values and fold changes. Genes previously identified as Nodal target genes in zebrafish are in bold. (B)In situ hybridisation of wild type and ndr1 mRNA-injected embryos for foxa, klf3, nhsl1b, notum1a and smarcd3 at 50% epiboly showing upregulation in response to ndr1 and absence in MZoep mutant embryos that have no Nodal signaling. Animal views; dorsal to the right. Numbers on each panel indicate the number of embryos showing the phenotype depicted over the total number of embryos analysed. For foxa expression in MZoep mutants the remaining 13/36 embryos showed absent expression except for a patch on one side of the embryo. For nhsl1b and notum1a the remaining 8/26 and 5/37 embryos, respectively, showed reduced expression around the margin. (C) Comparison of all genes represented on the microarray, or ndr1-responsive genes (either up- or down-regulated) to proximal Smad2 binding; comparison was performed for both all and novel ndr1-responsive genes. Compared to all genes on the microarray or those that are down-regulated in response to ndr1, up-regulated ndr1-responsive genes (both all and novel) are significantly associated with Smad2 binding. *P =7 × 10-7; **P =1 × 10-40. (D) Examples of Smad2 binding upstream of known (tbx16 and flh) and novel targets; scale in reads per million reads.

Eomesa and Foxh1 combinatorially regulate early mesendoderm marker expression and are required for endoderm formation. In situ hybridisation of ntla(A-E), gsc(F-L) and sox32(M-S) at 30% epiboly in wild-type embryos, foxh1 morphants, MZeomesa mutants, MZeomesa;foxh1 morphants, MZoep mutants, and in wild type embryos and MZeomesa mutants injected with foxh1 mRNA. Rescued expression of sox32 expression in the ventral margin of MZeomesa mutants injected with foxh1 mRNA is indicated by an asterisk. In situ hybridisation of ttna(T-W) at 24 hpf in wild-type embryos, foxh1 morphants, MZeomesa mutants and MZeomesa;foxh1 morphants showing expression in the heart (arrow heads) and somites. The open arrow heads indicate cardia bifida. Animal views; dorsal to the right. Numbers on each panel indicate the number of embryos showing the phenotype depicted over the total number of embryos analysed. For sox32 expression in MZoep mutants faint staining in the YSL was detected in 11/15 embryos as previously reported [54]. hpf, hours post fertilization.

Eomesa negatively regulates neural marker gene expression. (A) ChIP-seq data showing binding of Eomesa at sphere stage proximal to neural marker genes; scale in reads per million reads. (B)In situ hybridisation of wild type and MZeomesa mutant embryos for stm, tfap2a, vgll4l and zic3 showing upregulation of these genes (lateral view, animal to the top). (C)In situ hybridisation of control injected and eomesa injected embryos for stm, tfap2a, vgll4l and zic3 showing downregulation of these genes (lateral view, animal to the top). ChIP, chromatin immunoprecipitation sequencing.

Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ BMC Biol.