Generation and utilization of knockout mice to elucidate the functions of the TGF-β pathway in mammalian endodermal specification and placental development

Ligands of the transforming growth factor beta (TGF-β) superfamily are involved in numerous developmental and disease processes. TGF-β, Activins, and Nodal ligands operate through Smad2 and Smad3 intracellular mediators. Smad2 mutants exhibit early embryonic lethality, while Smad3 mutants are viable, but show a plethora of postnatal phenotypes, including immune dysfunction and skeletal abnormalities. Previously, we have shown that the Smad2 and Smad3 genes function cooperatively during liver morphogenesis. Here we show that Smad2 and Smad3 are required at full dosage for normal embryonic development. Animals lacking one allele of each gene exhibit a variably penetrant phenotype in which structures in the anterior are reduced or lost; additionally, we demonstrate that this craniofacial defect and the previously reported hepatic phenotypes are both due to defects in the definitive endoderm. A reduction of endodermal gene expression and a failure to displace the visceral endoderm occurs despite the formation of a normal foregut pocket. This precedes any defects in anterior patterning and likely causes the abnormalities observed in craniofacial development and hepatogenesis. Furthermore, to circumvent the early lethality of Smad2 deletion and study the spatially and temporally specific functions of the gene, we generated a Smad2 conditional allele using the Cre-loxP system. In addition, we report that Smad2^3loxp, the targeted mutation used to create the Smad2^floxconditional allele, is itself hypomorphic.

To elucidate the interactions of TGF-β signaling with other pathways during murine development, targeted deletion of the Smif gene is created. Smif is a mammalian mRNA decapping protein which specifically interacts with Smad4. While Smif heterozygous animals display no detectable abnormality, homozygous knockout mice are embryonic lethal between E10.5 and E11.5 due to placental failure. In addition, we are able to show that while the trophoblast cell lineages of the placenta are relatively unaffected, the vascularization of the nascent allantois is defective in the mutant embryos. Furthermore, mutant embryos exhibit a reduced number of primordial germ cells, a cell lineage which shares the same precursors with the allantois, implying the early onset of the abnormalities in their common progenitors.