| Smad proteins are the intracellular mediator proteins for TGF-beta, activin, nodal, and BMP signaling molecules. Smad1, 5, and 8 belong to a sub-class that become activated by bone morphogenetic proteins (BMPs), which can multimerize with Smad4 (co-Smad), whereby this complex can translocate into the nucleus to modulate gene transcription. Previous research has shown that Smad1 is important in the formation of the allantois, while Smad5 has been shown to be critical in the process of angiogenesis. Smad4 has been shown to be equally important in the development of extraembryonic tissues. To further analyze the BMP-responsive Smads, the murine Smad8 gene was disrupted utilizing the Cre/IoxP system. A Smad8 hypomorphic allele was constructed that contains an in-frame deletion of exon three, removing one third of the MH2 domain and a small portion of the linker region. Xenopus injection assays indicated that this Smad8 deletion allele is still functional, but has reduced ventralizing capability compared to the wild-type allele. Although Smad8Deltaexon3/Deltaexon3 embryos are phenotypically normal, homozygotes of another allele of Smad8 (Smad83loxP) that contains a neomycin cassette within intron 3, phenocopy an embryonic brain defect observed in roughly 22% of Smad1+/- embryos analyzed at E11.5. Specifically, these embryos exhibited a drastic reduction of hindbrain and midbrain structures. Phenotypic Smad1+/- and Smad83loxp/3loxp embryos were analyzed by examining the levels of genes important for brain patterning and neural tube formation. Interestingly, the levels of genes that function in patterning the embryonic brain such as Fgf8, En2, as well as Gbx2 remained unchanged, although the levels of Pax3 and Pax6, which function to promote cell fates within the neural tube were shown to be upregulated in these mutant embryos.; Since the level of Pax3 was upregulated significantly in phenotypic Smad1+/- embryos, it was tested whether Pax3 expression might be regulated by Smad1. Smad1 is capable of repressing Pax3 promoter activity in concert with a known co-repressor, SIP1, in melanoma cell lines utilizing reporter assays. Transcriptional inhibition requires DNA binding of SIP1, as a SIP1 mutant lacking DNA binding activity was unable to repress Pax3 expression. Pax3 is upregulated in E9.5 Smad1-/- embryos, demonstrating Smad1 negatively regulates its expression. Although many downstream targets of Pax3 have been identified, knowledge regarding which factors regulate Pax3 expression has been limited. (Abstract shortened by UMI.)... |
