Endogenous patterns of TGF beta superfamily signaling during early Xenopusdevelopment

Transforming growth factor beta (TCF beta) superfamily signaling has been i mplicated in patterning of the early Xenopus embryo. Upon ligand stimulatio n, TGF beta 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 activi n-like ligands, induces dorsal mesoderm, Although ectopic expression studie s are consistent with roles for TGF beta signals in early Xenopus embryogen esis, when and where BMP and activin-like signaling pathways are active end ogenously has not been directly examined. In this study, we investigate the temporal and spatial activation of TGF beta superfamily signaling in early Xenopus development by using antibodies specific for the type I receptor-p hosphorylated forms of Smad1/5/8 and Smad2, We find that Smad1/5/8 and two distinct isoforms of Smad2, full-length Smad2 and Smad2 Delta exon3, are ph osphorylated in early embryos, Both Smad1/5/8 and Smad2/Smad2 Delta eson3 a rt activated after, but not before, the mid-blastuta transition (MBT). Endo genous activation of Smad2/Smad2 Delta exon3 requires zygotic transcription , while Smad1/5/8 activation at MBT appears to involve transcription-indepe ndent regulation, We also find that the competence of embryonic cells to re spond to TGF beta superfamily ligands is temporally regulated and may be a determinant of early patterning. Levels of phospho-Smad1/5/8 and of phospho -Smad2/Smad2 Delta exon3 are asymmetrically distributed across both the ani mal-vegetal and dorsoventral axes, The timing of the development of these a symmetries differs for phospho-Smad1/5/8 and for phospho-Smad2/Smad2 Delta exon3, and the spatial distribution of phosphorylation of each Smad changes dramatically as gastrulation begins. We discuss the implications of our re sults for endogenous functions of BMP and activin-like signals as candidate morphogens regulating primary germ layer formation and dorsoventral patter ning of the early Xenopus embryo.