Regulation of Nodal signaling during gastrulation:
During vertebrate embryogenesis, complex transcriptional controls regulate the expression of potent morphogens involved in establishment of the body plan. In recent studies, we have been examining the transcriptional mechanisms by which Nodal signaling and its positive feedback loop are regulated at the spatial and temporal levels. In collaboration with Danny Reinberg's laboratory (UMDNJ-RWJMS), we have found that the transcriptional co-repressor DRAP1 has a remarkably specific role in mesoderm formation and patterning in mouse embryogenesis, which is exerted through negative regulation of the Nodal signaling pathway. Loss of Drap1 function results in an expanded primitive streak and defective mesoderm migration and regional specification, severe defects that are consistent with increased expression of Nodal, and which can be partially suppressed by Nodal heterozygosity.

Consistent with a role for DRAP1 in repressing Nodal signaling, we have utilized our cell culture assay for Nodal signaling to show that DRAP1 overexpression decreases Nodal activity, while RNAi of endogenous DRAP1 results in increased activity. We have also shown that DRAP1 interacts with the winged-helix transcription factor FoxH1, and can inhibit binding of a FoxH1/Smad2/Smad4 transcription factor complex to an Activin/Nodal-response element in nuclear extracts from Activin-treated cells. Thus, we have proposed that DRAP1 regulates Nodal signaling in vivo through an interaction between DRAP1 and FoxH1 that precludes FoxH1/Smad2/Smad4 complex binding to its cognate DNA targets. These results suggest that DRAP1 down-modulates the transcriptional response to Nodal signaling, particularly by attenuation of its positive feedback loop. Thus, Drap1 may represent a key component of a mechanism for limiting the spatial or temporal extent of the response to a long-range morphogenetic signal.

Inhibitory role of Lefty proteins in the Nodal pathway:
Soluble inhibitory factors represent key regulators of all major signaling pathways. During vertebrate embryogenesis, members of the Lefty subclass of TGFβ proteins act as extracellular antagonists of the signaling pathway for Nodal. Genetic and biochemical analyses have shown that Nodal signaling is mediated by type I and II activin receptors, but differs from Activin signaling in that Nodal activity requires EGF-CFC co-receptors, such as mammalian Cripto or Cryptic. Loss-of-function experiments have shown that mouse Lefty1 is required as a midline barrier to restrict Nodal expression to the left side during left-right axis specification, and Lefty2 for limiting Nodal activity during mesoderm formation and left-right patterning. Overexpression experiments in zebrafish and frogs have suggested that Lefty proteins can act as long-range inhibitors for Nodal, possibly through competition for binding to activin receptors.

We have examined the molecular basis for how Lefty proteins can block Nodal activity, and have demonstrated two distinct and unexpected mechanisms by which Lefty proteins can antagonize Nodal activity. First, we have found that Lefty can inhibit signaling by Nodal, but not by Activin or TGFβ, which are EGF-CFC independent, using a novel assay for Lefty activity in mammalian cell culture. This inhibitory activity is dependent upon the activity of EGF-CFC proteins such as Cryptic, which is an essential co-receptor for Nodal and is required for left-right patterning. Secondly, we have shown that Lefty can function through an interaction with Nodal in solution, thereby blocking the ability of Nodal to bind to activin receptors. Furthermore, Lefty can also interact with EGF-CFC proteins such as Cryptic, preventing their ability to form part of a Nodal receptor complex. These observations suggest that the long-range activity of Lefty proteins can block Nodal morphogenetic activity in the absence of receptors, and can also inhibit Nodal specifically at the level of its EGF-CFC co-receptor. Thus, these results provide mechanistic insights into how Lefty proteins can achieve efficient and stringent regulation of a potent signaling factor.

Ongoing studies:
In current work, we are investigating later functions of Cripto during gastrulation stages, using hypomorphic and conditional alleles. We are also pursuing chimera analysis to determine whether the requirement for Cripto is cell-autonomous or non-cell-autonomous. In related studies, we are examining the redundant functions of Cripto and Cryptic in early embryonic development, which has led us to investigation of Nodal signaling activities in peri-implantation development.