abstract: In somatic cells, the mitotic spindle apparatus is centrosomal and several isoforms of Protein Kinase C (PKC) have been associated with the mitotic spindle, but their role in stabilizing the mitotic spindle is unclear. Other protein kinases such as, Glycogen Synthase Kinase 3â (GSK3â) also have been shown to be associated with the mitotic spindle. In the study in chapter 2, we show the enrichment of active (phosphorylated) PKCæ at the centrosomal region of the spindle apparatus in metaphase stage of 3T3 cells. In order to understand whether the two kinases, PKC and GSK3â are associated with the mitotic spindle, first, the co-localization and close molecular proximity of PKC isoforms with GSK3â was studied in metaphase cells. Second, the involvement of inactive GSK3â in maintaining an intact mitotic spindle was shown. Third, this study showed that addition of a phospho-PKCæ specific inhibitor to cells can disrupt the mitotic spindle microtubules. The mitotic spindle at metaphase in mouse fibroblasts appears to be maintained by PKCæ acting through GSK3â. The MAPK pathway has been implicated in various functions related to cell cycle regulation. MAPKK (MEK) is part of this pathway and the extracellular regulated kinase (ERK) is its known downstream target. GSK3â and PKCæ also have been implicated in cell cycle regulation. In the study in chapter 3, we tested the effects of inhibiting MEK on the activities of ERK, GSK3â, PKCæ, and á-tubulin. Results from this study indicate that inhibition of MEK did not inhibit GSK3â and PKCæ enrichment at the centrosomes. However, the mitotic spindle showed a reduction in the pixel intensity of microtubules and also a reduction in the number of cells in each of the M-phase stages. A peptide activation inhibitor of ERK was also used. Our results indicated a decrease in mitotic spindle microtubules and an absence of cells in most of the M-phase stages. GSK3â and PKCæ enrichment were however not inhibited at the centrosomes. Taken together, the kinases GSK3â and PKCæ may not function as a part of the MAPK pathway to regulate the mitotic spindle. / Dissertation/Thesis / Ph.D. Molecular and Cellular Biology 2012

Although the mechanisms underlying the divergence of the first cell types in the mouse, the trophectoderm (TE) and the inner cell mass (ICM) have received considerable attention, the upstream signals stimulating their divergence are not well understood. The work presented here examines the roles that morphogenetic factors such as cell adhesion and polarization play in the development of these cell types. I show here that in embryos completely lacking both maternal and zygotic E-cadherin, the normal epithelial morphology of outer cells is disrupted but individual cells still initiate TE and ICM-like fates. A larger proportion of cells than normal expressed TE markers like Cdx2 (a homeodomain containing transcription factor), suggesting that formation of an organized epithelium is not necessary for TE-specific gene expression. Individual cells in such embryos still generate an apical-like domain that correlates with elevated Cdx2 expression. I also show that repolarization can occur in isolated early ICMs from both wild type and Cdx2 mutant embryos, indicating that Cdx2 is not required to initiate polarity. Importantly, I demonstrate a critical role for the Rho-associated kinase ROCK in apical-basal polarization of preimplantation blastomeres. Loss of apical-basal polarization leads to a reduction of Cdx2 expression in outer blastomeres due to activation of Lats1/2 kinase and reduced nuclear Yap1. The influence of polarization upon Lats1/2 kinase is stage-dependent however, as apolar 8-cell blastomeres retain nuclear Yap1. Cell position appears to serve as an additional cue for nuclear localization of Yap and Cdx2 expression from the 8-cell stage to E3.5. Cell polarization plays an additional role in the embryo of maintaining cells in consistently outer or inner positions, thus ensuring that Cdx2 is expressed exclusively in the developing TE. The results of this work demonstrate important links between morphogenesis, cell fate and patterning in the preimplantation embryo. Both cell polarization and cell position act as critical cues to determine gene expression and to pattern this expression within the embryo.

Although the mechanisms underlying the divergence of the first cell types in the mouse, the trophectoderm (TE) and the inner cell mass (ICM) have received considerable attention, the upstream signals stimulating their divergence are not well understood. The work presented here examines the roles that morphogenetic factors such as cell adhesion and polarization play in the development of these cell types. I show here that in embryos completely lacking both maternal and zygotic E-cadherin, the normal epithelial morphology of outer cells is disrupted but individual cells still initiate TE and ICM-like fates. A larger proportion of cells than normal expressed TE markers like Cdx2 (a homeodomain containing transcription factor), suggesting that formation of an organized epithelium is not necessary for TE-specific gene expression. Individual cells in such embryos still generate an apical-like domain that correlates with elevated Cdx2 expression. I also show that repolarization can occur in isolated early ICMs from both wild type and Cdx2 mutant embryos, indicating that Cdx2 is not required to initiate polarity. Importantly, I demonstrate a critical role for the Rho-associated kinase ROCK in apical-basal polarization of preimplantation blastomeres. Loss of apical-basal polarization leads to a reduction of Cdx2 expression in outer blastomeres due to activation of Lats1/2 kinase and reduced nuclear Yap1. The influence of polarization upon Lats1/2 kinase is stage-dependent however, as apolar 8-cell blastomeres retain nuclear Yap1. Cell position appears to serve as an additional cue for nuclear localization of Yap and Cdx2 expression from the 8-cell stage to E3.5. Cell polarization plays an additional role in the embryo of maintaining cells in consistently outer or inner positions, thus ensuring that Cdx2 is expressed exclusively in the developing TE. The results of this work demonstrate important links between morphogenesis, cell fate and patterning in the preimplantation embryo. Both cell polarization and cell position act as critical cues to determine gene expression and to pattern this expression within the embryo.