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Abstract Nodal signaling, mediated through SMAD transcription factors, is necessary for pluripotency maintenance and endoderm commitment. We identified a new motif, termed SMAD complex-associated (SCA), that is bound by SMAD2/3/4 and FOXH1 in human embryonic stem cells (hESCs) and derived endoderm. We demonstrate that two basic helix-loop-helix (bHLH) proteins-HEB and E2A-bind the SCA motif at regions overlapping SMAD2/3 and FOXH1. Furthermore, we show that HEB and E2A associate with SMAD2/3 and FOXH1, suggesting they form a complex at critical target regions. This association is biologically important, as E2A is critical for mesendoderm specification, gastrulation, and Nodal signal transduction in Xenopus tropicalis embryos. Taken together, E proteins are novel Nodal signaling cofactors that associate with SMAD2/3 and FOXH1 and are necessary for mesendoderm differentiation.
Figure 4. e2a and heb are expressed during early development in X. tropicalis. (A) X. tropicalis embryos were analyzed by RTCR for expression of heb and e2a, from blastula stage through early neurula. Ornithine decarboxylase (odc) was used as a loading control. (B) Vegetal endoderm explants of stage 10.5 X. tropicalis express e2a at low levels, but not the mesoderm- specific gene xbra. (C) X. tropicalis embryos were analyzed by in situ hybridization for expression of e2a and heb at early gastrula stage (stage 10.5), at early tailbud stage (stage 22), and at late tailbud stage (32). (D) Bar graphs show the percentage of each motif present in Smad2/3 target regions during gastrulation in X. tropicalis as determined by ChIP-seq.
Figure 5. E2A is essential for gastrulation and gene expression in X. tropicalis. (A, right) X. tropicalis embryos were injected with a translation-blocking MO (e2a MO) directed against e2a, and assayed morphologically at stages 10.5 and 25. These were compared with uninjected controls (shown at left). (B) Blasto- pore lip formation in e2a MO-injected embryos can be restored by subsequent injection of mouse e2a mRNA. Using either in situ hybridization (C) or qRTCR (D), e2a MO-injected embryos were compared with controls for expression of molecular markers. qRTCR results represent at least four biological replicates. (E) e2a morphants and uninjected controls were in- jected in the animal pole at the four-cell stage with 10 pg of Activin mRNA or 40 pg of Xnr1 mRNA, and assayed for the presence of ectopic bottle cells. Representative embryos are shown at stage 11 in animal views.
Supplemental Figure 4 (Supports Figure 5). (A) Injection of 20 ng of HEB MO in both blastomeres at the 2-cell stage does not result in gross morphological defects at gastrulation (stage 10.5) or in early tailbud embryos (stage 22). (B) Co- injection of 10 ng of E2A MO with nuclear β-galactosidase mRNA in one blastomere at the 4-cell stage results in cell-autonomous inhibition of blastopore formation and of xbra expression. Red color represents red-gal substrate staining and marks the injected portion of the embryo. (C) Embryos co-injected with e2a MO and mouse e2a mRNA were compared to uninjected controls for expression of xbra (mesoderm) and sox17β (endoderm). While e2a MO injection led to downregulation of xbra, this was rescued by co-injection with mouse E2A mRNA.
tcf12 (transcription factor 12 (HTF4, helix-loop-helix transcription factors 4)) gene expression in Xenopus tropicalis embryos, NF stage 10.5, as assayed by in situ hybridization, blastoporal view, animal up.
tcf12 ( transcription factor 12 (HTF4, helix-loop-helix transcription factors 4) ) gene expression in Xenopus tropicalis embryos, NF stage 22, as assayed by in situ hybridization, lateral view, anteriorleft, dorsal up.
tcf12 (transcription factor 12 (HTF4, helix-loop-helix transcription factors 4)) gene expression in Xenopus tropicalis embryo, NF stage 32, assayed by in situ hybridization, lateral view, anteriorleft, dorsal up.
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