Endogenous patterns of TGFβ superfamily signaling during early Xenopus development

Abstract

Transforming growth factor β (TGFβ) superfamily signaling has been implicated in patterning of the early Xenopus embryo. Upon ligand stimulation, TGFβ receptors phosphorylate Smad proteins at carboxy-terminal SS(V/M)S consensus motifs. Smads 1/5/8, activated by bone morphogenetic protein (BMP) signaling, induce ventral mesoderm whereas Smad2, activated by activin-like ligands, induces dorsal mesoderm. Although ectopic expression studies are consistent with roles for TGFβ signals in early Xenopus embryogenesis, when and where BMP and activin-like signaling pathways are active endogenously has not been directly examined. In this study, we investigate the temporal and spatial activation of TGFβ superfamily signaling in early Xenopus development by using antibodies specific for the type I receptor-phosphorylated forms of Smad1/5/8 and Smad2. We find that Smad1/5/8 and two distinct isoforms of Smad2, full-length Smad2 and Smad2Δexon3, are phosphorylated in early embryos. Both Smad1/5/8 and Smad2/Smad2Δexon3 are activated after, but not before, the mid-blastula transition (MBT). Endogenous activation of Smad2/Smad2Δexon3 requires zygotic transcription, while Smad1/5/8 activation at MBT appears to involve transcription-independent regulation. We also find that the competence of embryonic cells to respond to TGFβ superfamily ligands is temporally regulated and may be a determinant of early patterning. Levels of phospho-Smad1/5/8 and of phospho- Smad2/Smad2Δexon3 are asymmetrically distributed across both the animal-vegetal and dorsoventral axes. The timing of the development of these asymmetries differs for phospho- Smad1/5/8 and for phospho-Smad2/Smad2Δexon3, and the spatial distribution of phosphorylation of each Smad changes dramatically as gastrulation begins. We discuss the implications of our results for endogenous functions of BMP and activin-like signals as candidate morphogens regulating primary germ layer formation and dorsoventral patterning of the early Xenopus embryo.