Abstract
BackgroundSmad4 mutant embryos arrest shortly after implantation and display a characteristic shortened proximodistal axis, a significantly reduced epiblast, as well as a thickened visceral endoderm layer. Conditional rescue experiments demonstrate that bypassing the primary requirement for Smad4 in the extra-embryonic endoderm allows the epiblast to gastrulate. Smad4-independent TGF-β signals are thus sufficient to promote mesoderm formation and patterning. To further analyse essential Smad4 activities contributed by the extra-embryonic tissues, and characterise Smad4 dependent pathways in the early embryo, here we performed transcriptional profiling of Smad4 null embryonic stem (ES) cells and day 4 embryoid bodies (EBs).ResultsTranscripts from wild-type versus Smad4 null ES cells and day 4 EBs were analysed using Illumina arrays. In addition to several known TGF-β/BMP target genes, we identified numerous Smad4-dependent transcripts that are mis-expressed in the mutants. As expected, mesodermal cell markers were dramatically down-regulated. We also observed an increase in non-canonical potency markers (Pramel7, Tbx3, Zscan4), germ cell markers (Aire, Tuba3a, Dnmt3l) as well as early endoderm markers (Dpp4, H19, Dcn). Additionally, expression of the extracellular matrix (ECM) remodelling enzymes Mmp14 and Mmp9 was decreased in Smad4 mutant ES and EB populations. These changes, in combination with increased levels of laminin alpha1, cause excessive basement membrane deposition. Similarly, in the context of the Smad4 null E6.5 embryos we observed an expanded basement membrane (BM) associated with the thickened endoderm layer.ConclusionSmad4 functional loss results in a dramatic shift in gene expression patterns and in the endodermal cell lineage causes an excess deposition of, or an inability to breakdown and remodel, the underlying BM layer. These structural abnormalities probably disrupt reciprocal signalling between the epiblast and overlying visceral endoderm required for gastrulation.
Highlights
Smad4 mutant embryos arrest shortly after implantation and display a characteristic shortened proximodistal axis, a significantly reduced epiblast, as well as a thickened visceral endoderm layer
Transcriptional profiling of Smad4 null embryonic stem (ES) cells and embryoid bodies (EBs) To identify developmentally regulated transcripts that are potentially mis-regulated in the absence of Smad4, we analysed ES cells grown in the presence of LIF, or induced to differentiate as EBs in suspension culture
After 4 days of suspension culture the outer cells of EBs are induced to differentiate to form a layer of primitive endoderm, while a sub-population of inner cells express nascent mesodermal markers
Summary
Smad mutant embryos arrest shortly after implantation and display a characteristic shortened proximodistal axis, a significantly reduced epiblast, as well as a thickened visceral endoderm layer. To further analyse essential Smad activities contributed by the extra-embryonic tissues, and characterise Smad dependent pathways in the early embryo, here we performed transcriptional profiling of Smad null embryonic stem (ES) cells and day 4 embryoid bodies (EBs). Members of the TGF-β super-family of secreted growth factors activate a cell surface receptor complex comprised of two distinct transmembrane serine/threonine kinases that, upon ligand binding, phosphorylate members of the downstream receptor-associated Smads (R-Smads) (reviewed by [1]). The closely related R-Smads, Smad and Smad are phosphorylated in response to TGF-β s, Activin and Nodal signals. The phosphorylated RSmads in association with the common mediator Smad, Smad, recruit additional cofactors to form higher order complexes that regulate target gene expression (reviewed by [2]). Smad4-RSmad complexes control a diverse array of biological processes, including cell proliferation, differentiation and cell survival during development and adult tissue homeostasis
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