Doctor of Philosophy, The Ohio State University, 2005, Molecular Genetics

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 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/loxP 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. Although these 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. To further characterize the functions of Smad proteins during mammalian development, Smad4 was conditionally deleted within the mouse utilizing the FSP-Cre driver strain. Utilizing this strain eliminates expression of Smad4 in fibroblastic, mesenchymal cell types. Mice depleted for Smad4 utilizing this driver strain exhibit multiple phenotypes such as deformed digits, hair follicle dysplasia, and overall immune and health deficiencies. Histological analysis of Smad4 mutant hair follicles show irregular, enlarged hair follicles, dermal cysts, hyperkeratosis, and utriculi. Further analysis has revealed a defect in mutant hair follicles entering the catagen stage of the hair cycle. The catagen stage is characterized by an apoptosis-driven regression phase of the hair cycle that results in the remodeling of the hair follicle. In addit (open full item for complete abstract)

Committee: Michael Weinstein (Advisor) Subjects: Biology, Molecular

Doctor of Philosophy, Case Western Reserve University, 2009, Biochemistry

Ski is the most studied member of a family of proteins all sharing a conserved Dachshund homology domain. It has been implicated in oncogenic transformation, myogenic conversion of avian embryo fibroblasts and also many aspects of vertebrate development, especially myogenesis. Ski-/- mice exhibit severe defects in skeletal muscle and die at birth, yet little is know about either the underlying mechanisms or the role of Ski in adult muscle regeneration. In these studies, I used Ski knockout mice and C2C12 myoblast cultures to address these issues, respectively. Detailed analysis of Ski-/- embryos revealed dramatically reduced hypaxial muscles but less affected epaxial muscles. The reduced number of myogenic regulatory factor positive cells in Ski-/- mice suggested an insufficient myogenic cell pool to support muscle formation. However, both the dermomyotomal hypaxial progenitors and myotomal epaxial progenitors formed and committed to myogenic fate appropriately. The hypaxial muscle defect in Ski-/- mice was not caused by abnormal proliferation, terminal differentiation or apoptosis of the myogenic cells either, but due to impaired migration of embryonic hypaxial progenitors. Surprisingly, the normal distribution of fetal/postnatal myogenic progenitors in Ski-/- mice suggested different effects of Ski on the behaviors of embryonic and fetal/postnatal myogenic progenitors. In addition, although not affecting the terminal differentiation of embryonic myogenic cells, Ski was necessary for that of adult satellite-cell derived C2C12 myoblasts as evidenced by impaired myotube formation and reduced induction of genes essential for myogenic differentiation in the absence of Ski. This function was mainly mediated by Ski's ability to form a complex with Six1 and Eya3 and activate Myog transcription through a MEF3 site. It is important in the future to further study mechanisms underlying the contrasting effects of Ski on embryonic, fetal and adult muscle development, to investi (open full item for complete abstract)

Committee: David Samols PhD (Committee Chair); Ed Stavnezer PhD (Advisor); Clemencia Colmenares PhD (Committee Member); Nikki Harter PhD (Committee Member); Lynn Landmesser PhD (Committee Member) Subjects: Biomedical Research