Chapter I: The Role Of Ectodermal Smad4 In Murine Limb Morphogenesis.
Vertebrate limb morphogenesis is coordinated by a complex network of signaling centers that influence proliferation, differentiation and movement of the cells in the developing limb field. Several members of signaling molecules of the Transforming Growth Factor beta (TGF-β) family are shown to be potent regulators of limb morphogenesis. Smad4 is the central mediator of the TGF-beta-related signaling in a variety of developmental contexts. Zygotic inactivation of Smad4 results in lethality at peri-gastrulation stage at around E7.5, precluding the possibility to study the role of Smad4 in post-gastrulation development. Here, we generated a conditional knockout of Smad4 that disrupted BMP signaling in the developing limb ectoderm, as confirmed by reporter assay for Cre activity and Smad4 antibody staining. Ectodermal inactivation of Smad4 signaling leads to severe limb developmental defects including failure to form posterior limb structures, dorsal-ventral defects, interdigital tissue retaining and ectopic tissue growth. Molecular analysis has revealed that mutant limbs failed to initiate and maintain intact apical ectodermal ridge (AER), a thickened epithelial structure required for proliferation and survival of underlying mesenchymal cells. More importantly, early AER defects appear linked to dorsal-ventral defects in mutant limbs. Paradoxically, mutant limbs fail to repress AER at later stage. As a result, interdigital tissue fails to undergo programmed cell death and tissue overgrows in the peripheral limb paddle. Loss of posterior limb structure is the result of reduced proliferation rather than abnormal cell death, probably due to defected posterior AER formation and loss of key gene expression in the posterior limb mesenchyme. In addition to Smad4, ectodermal Smad1 is also involved in limb morphogenesis through both Smad4-dependent and Smad4-independent pathways.
Chapter II: The Role Of Maternal Smad4 In Mouse Preimplantation Development.
In vitro culture experiments involving mouse preimplantation embryos establish the importance of autocrine/paracrine growth regulatory pathways in controlling pre-implantation development. One of the groups of signaling molecules implicated is the Transforming Growth Factor beta (TGF-β) family. Signaling molecules of this superfamily are potent regulators of development and tissue homeostasis. Smad4 is a central mediator of the TGF-beta-related signaling and is important for numerous processes including cellular growth, differentiation, migration, and extracellular matrix production. Mice lacking Smad4 die around peri-gastrulation period due to defects in epiblast proliferation, mesoderm formation, and extraembryonic tissue-mediated early patterning events. Maternal gene products play key roles in shaping the earliest developmental programs in a wide variety of organisms. Relatively little is known about the functions of mammalian maternal gene products. Maternal Smad4 gene products are abundant in unfertilized eggs as well as in cleaving blastomeres of preimplantation mouse embryos. We have conditionally inactivated Smad4 in the female germline of the mouse. Eggs depleted of maternal Smad4 gene products complete meiosis and are fertilized normally. However, preimplantation development is severely compromised in embryos derived from such eggs even when they are fertilized by wild type sperms, demonstrating the importance of maternally derived Smad4 gene products in early stages of mouse development. Molecular experiments have uncovered that maternal Smad4 is required for cell division and survival, cell polarity, and lineage segregation. Therefore, We have identified Smad4 as a novel maternal effect gene during mouse preimplantation development.