Mechanisms of TGF-beta family signaling during early vertebrate embryogenesis

Nodal-related proteins of the TGF-beta family play pivotal roles in mesoderm formation in vertebrate embryos. Nodal signaling is mediated by the type II receptors ActRIIA and ActRIIB, the type I receptor ALK4 and the signal transducer Smad2. Nodal signaling also requires the transcription factor FAST-1 and EGF-CFC factors. In my thesis work, I have investigated the mechanisms by which (1) FAST-1 mediates Nodal-regulated transcription and (2) Cripto, an EGF-CFC factor, acts as a permissive co-factor for Nodal signaling.;FAST-1, Smad2 and Smad4 form a transcription factor complex, activin responsive factor (ARF), which binds to a Nodal/Activin-responsive enhancer (ARE) of Xenopus Mix.2 gene. I find that FAST-1 and Smads play distinct roles in the transcriptional regulation of the ARE. FAST-1 is the major sequence-specific, DNA-binding component of the ARF, while Smad4 stabilizes ARF binding to the ARE and activates transcription. Smad2 serves as an adapter that links FAST-1 and Smad4 in response to Nodal signals.;Genetic studies have revealed that EGF-CFC factors, a family of extracellular membrane proteins, are required for Nodal signaling. I find that Nodal activates Smad2 in a Cripto-dependent manner and that Cripto serves as a co-receptor for Nodal. Cripto interacts with ALK4 to permit Nodal binding to the receptor complex, leading to Smad2 activation by ALK4. The CFC motif in Cripto is necessary for Cripto binding to ALK4, while the EGF-like motif is important for Nodal binding to the receptor complex. I also find that Nodal can inhibit Smad1l activation by BMPs in a Cripto-independent manner. Nodal and BMP7 act as mutual antagonists and the inhibition appears to be mediated by heterodimerization between them.;In summary, Nodal-related proteins control mesoderm formation through their spatially and temporally regulated expression. I provide evidence that Nodal signaling elicits developmentally restricted transcriptional responses using temporally regulated signaling components, such as EGF-CFC factors and FAST-1, in conjunction with broadly expressed TGFbeta family signaling components, such as ActRIIB, ALK4, Smad2 and Smad4.