Tuberous Sclerosis Complex (TSC) is a pediatric disorder of dysregulated growth and differentiation caused by loss of function mutations in either the TSC1 or TSC2 genes, which regulate mTOR kinase activity. TSC causes refractory epilepsy and intellectual disability but the pathogenesis of the neurological symptoms is not understood. Identifying when and in what cell types mutations in TSC1 or TSC2 lead to neurological dysfunction is the first step to better and more targeted treatments. To study aberrations of early development in TSC, we generated induced pluripotent stem cells using dermal fibroblasts obtained from patients with TSC. During validation, we found that stem cells generated from TSC patients had a very high rate of integration of the reprograming plasmid containing a shRNA against TP53. Loss of one allele of TSC2 in human fibroblasts is sufficient to increase p53 levels and impair stem cell reprogramming. Increased p53 was also observed in TSC2 heterozygous and homozygous mutant human stem cells, suggesting that the interactions between TSC2 and p53 are consistent across cell types and gene dosage. Further, we show that homozygous loss of TSC2 leads to increased mTORC1 in neural progenitors and impaired neural progenitor formation. These results support the contributions of TSC2 heterozygous and homozygous mutant cells to the pathogenesis of TSC and the important role of p53 during reprogramming.

<p> Skeletal muscle generates contractile forces that allow the body to execute movements for walking, speaking and breathing. Although we understand a great deal about the steps of muscle formation, the mechanisms that control muscle size are poorly understood. Even less is known about how muscles interact with skeletal elements, including connective tissue, tendon and bone. This dissertation describes a novel role for Abelson tyrosine-protein kinase 2, a non-receptor tyrosine kinase, during muscle development. First, I characterize the defects in skeletal muscle of <i>abl2</i> mutant mice and show that muscle fibers in the diaphragm and other muscles are extraordinarily long in <i>abl2</i> mutant mice. As a consequence of expansion of the diaphragm muscle, the central tendon of the diaphragm is proportionally reduced in size. Second, I demonstrate that <i>abl2</i> controls muscle size by regulating myoblast proliferation. Third, I show that Abl2 acts in myoblasts to attenuate their proliferation, thereby limiting myoblast fusion and muscle fiber size. Fourth, I show that the exercise endurance of <i> abl2</i> mutant mice is diminished, likely due to the compensatory reduction in size of the diaphragm central tendon. Finally, I provide evidence for signaling between muscle cells and tendon cells that induces tendon cell differentiation. </p><p>

In Schizosaccharomyces pombe cytokinesis requires assembly and constriction of an actomyosin-based contractile ring (CR). Nucleation of F-actin for the CR requires a single essential formin, Cdc12, that localizes to the cell middle upon mitotic onset. The molecular mechanisms dictating its divison site targeting during cytokinesis are unknown. We defined that a Cdc12 N-terminal motif directly binds the F-BAR domain of the scaffolding protein Cdc15 and this interaction is controlled by Cdk1 phosphorylation of Cdc12. Phosphorylation of Cdc12 inhibits binding to the F-BAR Cdc15. cdc12 alleles that cannot bind Cdc15 or with all six Cdk1 sites mutated to phospho-mimetic residues show reduced Cdc12 cell division site accumulation and delayed CR formation. Thus Cdk1 phosphorylation of Cdc12 antagonizes its interaction with Cdc15 and its division site localization, consistent with a general role for Cdk1 in inhibiting cytokinesis until chromosome segregation is complete. The CR is physically linked to the plasma membrane (PM). Cells lacking efr3, which encodes a conserved PM scaffold for the phosphatidylinositol 4-kinase Stt4, build CRs that can slide away from the cell middle in a myosin-V-dependent manner. The Efr3-dependent CR anchoring mechanism is distinct from previously reported pathways dependent on the F-BAR protein Cdc15 and paxillin. In efr3â, the concentrations of several membrane-binding proteins were reduced in the CR and/or on the PM. Our results suggest that proper PM lipid composition is important to stabilize the central position of the CR and resist myosin V-based forces to promote the fidelity of cell division.

In normal epithelial wound repair, cells across an epithelial sheet begin a coordinated process of re-epithelialization within minutes of wounding. These coordinated behaviors are driven by a calcium wave, a rise in cytosolic calcium expanding away from the wound in a wave-like fashion. The calcium wave is evolutionarily conserved and is the earliest detectable wound response. Understanding the mechanisms of calcium influx and propagation may reveal fundamental aspects of wound detection and of cell coordination. We observed multiple, distinct mechanisms of calcium influx and propagation around reproducible wounds in the Drosophila notum. First, extracellular calcium flows directly into cells through micro-tears on the cell surface. We were able to assess the role of micro-tears in calcium dynamics by using pulsed laser ablation, a common wounding method that generates exaggerated micro-tears. Pulsed laser ablation creates a cavitation bubble, which forms and collapses within microseconds of ablation and damages the plasma membranes of cells tens of microns away from the wound. Once inside the cells, our model predicts calcium diffuses to neighboring cells via gap junctions. Next, we observed a larger, wound-induced calcium wave that is driven by an unknown extracellular signal. This signal activates a Gαq mediated signaling cascade and induces calcium release from intracellular ER stores. Our simple, pulsed laser ablation wounding model recapitulates a complex damage profile and reveals multiple patterns of calcium influx and propagation around a single wound. For this reason, this model has the potential to unite previous, and seemingly contradictory, findings regarding calcium dynamics in the wound healing field.

Cytokinesis is the final step in the cell cycle where one cell is physically divided into two. Animal and fungal cells perform cytokinesis with an actin- and myosin-based molecular machine, the contractile ring. A complete list of components in the contractile ring has been determined in the model organism Schizosaccharomyces pombe; however, it remains unknown how these components organize into a functional division apparatus. Here, I have investigated how the F-BAR family of proteins organizes upon the plasma membrane in the contractile ring. I found the Cdc15 F-BAR forms extended linear oligomers that stably bind the plasma membrane in the contractile ring. These oligomers robustly concentrate Cdc15 at the division site and contribute to anchoring the contractile ring in the cell middle. I found the Imp2 F-BAR, similar to previously studied F-BARs, forms oligomerizes in a helical fashion. Despite this fact, Imp2 does not rely upon oligomerization for its contractile ring function. I have also determined the precise spatial organization of 29 components of the contractile ring with super resolution microscopy, and constructed a nanoscale model of the contractile ring that may serve as a template for understanding the ringâs inner mechanics.

<p> Sperm mobility is a major determinant of male fertility in chicken. In spite of low heritability of reproductive traits, sperm mobility has high heritability index which suggests presence of quantitative trait loci (QTLs) governing the trait. Our research focused on three objectives: i) to identify the QTLs affecting low mobility phenotype in chicken, ii) to understand the impact of Sertoli-cells and germ cells interactions in influencing the mobility phenotype and iii) to identify the genes and gene networks differentially expressed in male and female PGCs. To detect the QTLs, genome wide association studies (GWAS) was conducted which revealed the presence of multiple minor alleles influencing the trait and indicated the role of epistasis. The second section of research involved isolation, culture and transfer of primordial germ cells (PGCs) to create high line germ line chimera chicken carrying low line PGCs. We established the culture of chicken PGCs isolated from the embryonic blood in a feeder free culture conditions but could not detect the presence of low line genotype in the semen of transgenic males. Our final study involved RNA-sequencing (RNAseq) of male and female PGCs to identify differentially expressed genes from their transcriptomes. We identified five candidate genes: 3-hydroxy-3-methylglutaryl CoA reductase (HMGCA), germ cell-less (GCL), SWIM (zinc finger SWIM domain containing transcription factor), SLC1A1 (solute carrier family 1 member 1), UBE2R2L (ubiquitin conjugating enzyme) and validated their expression level in male and female PGCs by RT-qPCR. GCL was exclusively expressed in males while SLC1A1 &amp; UBE2R2L were expressed only in female cPGCs. This present study provides novel gender specific germ cell markers in the broiler chicken. These results will help in elucidating the genetic programming of gender specific germ line development in broilers.</p><p>

The Notch and TGF13 signaling pathways have been shown to play important roles in regulating endothelial-to-mesenchymal transition (EndMT) during cardiac morphogenesis. EndMT is the process by which endocardial cells of the atrioventricular canal and the outflow tract repress endothelial cell phenotype and upregulate mesenchymal cell phenotype. EndMT is initiated by inductive signals emanating from the overlying myocardium and inter-endothelial signals and generate the cells that form the heart valves and atrioventricular septum. The Notch and TGFf3 pathway are thought to act in parallel to modulate endothelial phenotype and promote EndMT. Vascular endothelial (VE) cadherin is a key regulator of cardiac endothelial cell phenotype and must be downregulated during EndMT. Accordingly, VE-cadherin expression remains stabilized in the atrioventricular canal and outflow tract of Notchl-deficient mouse embryos, while activation of the Notch or TGFP pathways results in decreased VE-cadherin expression in endothelial cells. However, the downstream target gene(s) that are involved in regulating endothelial cell phenotype and VE-cadherin expression remain largely unknown. In this thesis the transcriptional repressor Slug is demonstrated to be expressed by the mesenchymal cells and a subset of endocardial cells of the atrioventricular canal and outflowtract during cardiac morphogenesis. Slug is demonstrated to be required for cardiac development through its role in regulating EndMT in the cardiac cushion. Data presented in Chapter 6 further suggests that Slug-deficiency in the mouse is compensated for by a increase in Snail expression after embryonic day (E) 9.5, which restores EndMT in the cardiac cushions. Additionally, the Notch pathway, via CSL, directly binds and regulates expression of the Slug promoter, while a close Slug family member, Snail is regulated by the TGFB pathway in endothelial cells. While Notch does not directly regulate Snail expression, Notch and TGFB act synergistically to regulate Snail expression in endothelial cells. It is further demonstrated that Slug is required for Notch mediated EndMT, binds to and represses the VE-cadherin promoter, and induces a motile phenotype. Collectively the data demonstrate that Notch signaling directly regulates Slug, but not Snail, expression and that the combined expression of Slug and Snail are required for cardiac cushion morphogenesis. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

Enterocytes, epithelial cells of the small intestine, exhibit remarkable apical-basal polarity. The apical surfaces of enterocytes display an array of tightly packed microvilli termed the brush border. Microvilli are composed of membrane supported by a linear actin bundle, with the plus ends of the actin filaments at the microvillar tips. Despite the importance of the brush border in nutrient absorption and host defense, the mechanism of brush border assembly is unclear. Because of the central role of actin in microvilli, the goal of this thesis is to provide molecular detail as to how microvillar actin bundles form. A proteomic analysis of the brush border by our laboratory identified two actin nucleators in the brush border: the Arp2/3 complex and Cordon-Bleu (COBL). Small molecule inhibition of the Arp2/3 complex did not have an effect on brush border assembly in Ls174T-W4 (W4) cells, which act as a single cell model of enterocyte polarization and brush border formation. Therefore, this work focused on the linear actin nucleator COBL. We show that COBL localizes to the base of the brush border in mouse small intestine and in W4 cells. COBL is necessary and sufficient to induce microvillar growth using a mechanism that requires functional WH2 domains. COBL functions downstream of the F-BAR domain containing protein syndapin-2, which drives targeting to the apical domain of enterocytes. In the syndapin-2 knockout mouse, COBL enrichment at the apical domain of enterocytes is impaired, and microvilli are significantly shorter as compared to wild-type control mice. In cells that do not normally build microvilli, exogenous COBL drives the aberrant formation of dynamic cytoplasmic actin bundles that grow and shrink over the course of minutes; stabilization of COBL-induced bundles by the actin bundling protein espin leads to robust microvillus-like protrusions. This study provides novel insight on mechanisms that control microvillar growth and thus, the maintenance of intestinal homeostasis. This work also reveals a novel assembly paradigm for actin-based protrusions that do not emerge from a dendritic array.

One of the many ways that the ubiquitously expressed Polycomb Group protein, Yin-Yang1 (Yy1), is believed to regulate gene expression is through direct binding to DNA elements found in promoters or enhancers of target loci. Additionally, YY1 contains diverse domains that enable a plethora of protein-protein interactions, including association with the Oct4/Sox2 pluripotency complex and the Polycomb Group silencing complexes. To elucidate the in vivo role of YY1 during gastrulation, Yy1 was deleted in the epiblast of mouse embryos using Sox2-Cre. Yy1 conditional knockout (cKO) embryos initiate gastrulation, but the primitive streak fails to extend anteriorly. Migration through the streak is severely impaired, and streak descendants fail to downregulate E-Cadherin resulting in an aberrant accumulation of streak cells. Intriguingly, we find an accumulation of Nodal and a concomitant reduction of Nodal antagonists suggesting that YY1 is normally required for proper Nodal regulation. We have observed that definitive endoderm is specified but fails to properly delaminate into the outer layer and mutant embryos also fail to accumulate any axial midline structure. Although anterior neuroectoderm is clearly specified, mesoderm specification is severely restricted. Our results reveal critical requirements of YY1 in several important developmental processes, including epithelial to mesenchymal transition (EMT), Nodal regulation and PRC2 mediated H3K27Me3 of the inactive X-chromosome. Despite the localization of Oct4 and Sox2 transcripts in the neuroectoderm of the Yy1 epiblast cKO and the presence of stable transcripts of both genes in the Yy1 RNAi knock down blastocyst, both proteins are void in these models and Oct4 protein is absent in the peri-implantion Yy1 KO mouse. We believe YY1 is required for stabilization of the Oct4/Sox2 pluripotency complex in vivo. We have identified two endogenous forms of YY1 and we believe these posttranslational modifications of YY1 permit the protein to perform the diverse activities it performs in vivo. For example, in addition to the roles in transcriptional regulation and protein complex stabilization, we have also observed a role in YY1 in epigenetic regulation in vivo, including deposition of histone 3 lysine 27 trimethylation (H3K27Me3) on the inactive X-chromosome in female embryos and a role in imprinted gene expression of the Dlk1/Dio3 locus. Detailed analysis of the peri-implantation lethal Yy1 KO mouse in utero revealed unexpected novel developmental events. A large scale follow up examination of wildtype implantation primarily through analysis of immunohistochemical data and gene expression profiling at the cellular level. We analyzed expression patterns of important developmental genes including Oct4, Sox2, Nanog, Cdx2, Gata6 and Sox17, as well as markers of epithelial biogenesis including ZO1, E-Cadherin and Laminin. Interestingly we identified consistent variances in cell populations within the ICM as well as likely primitive endoderm progenitors that produce Laminin and first appear at the periphery of the ICM. We also identified a novel upregulation of Sox17 specifically at the site of implantation. With these data we compose a staging diagram of peri-implantation embryonic and maternal changes during the elusive window of development. These results are the first to elucidate the role of YY1 during gastrulation and peri- implantation, providing potential in vivo targets of YY1 and highlighting the diversity of function of YY1 in the early embryo. Additionally we have been able to advance molecular knowledge of peri-implantation development, in order to provide a platform from which to analyze other peri-implantation lethal KO mice, as well as to aid biomedical understanding of implantation and implantation failure in mammals.

Autosomal recessive primary microcephaly (MCPH, MIM 251200) is a neurodevelopmental disorder that results from a loss of neural progenitors in the embryonic neocortex. Patients with MCPH have a significantly small brain and exhibit reduced cognition. MCPH is a genetically heterogeneous disease involving mutations in thirteen genes, nine of which centrosome protein-coding genes, one of which is CDK5RAP2. The centrosome is the major microtubule organizing center in all animal cells. While MCPH is a neural stem cell disease, the molecular mechanisms for the disease remains unknown. Proteomic analysis of a mutant in the Drosophila CDK5RAP2 ortholog, centrosomin (cnn), we discovered proteins involved in intermediary metabolism, oxidative stress, and inherited Parkinson's disease that were post-translationally modified in mutant brains relative to wild type brains. These findings led us to discover that cnn mutants have neurological defects, including poor locomotor and flight performance, and are less active. We further demonstrated that cnn and Sas-4 (MCPH6/CPAP in human) mutant cells have elevated reactive oxygen species (ROS) levels, chronically activating the Jun N-terminal kinase (JNK) stress signaling pathway and thus activating FOXO by nuclear localization. The cause for these stress responses appears to be due to a severe deficiency in autophagy induction in MCPH mutant cells. Autophagy is a major catabolic pathway for the degradation of damaged proteins and organelles. Here we show that MCPH proteins are required for autophagy induction and act downstream of mechanistic target of rapamycin (mTOR) kinase, a negative regulator of autophagy. Together these results demonstrate a novel function for MCPH genes in oxidative stress and regulating autophagy. / A Dissertation submitted to the Department of Biomedical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Fall Semester, 2014. / November 7, 2014. / autophagy, centrosome, microcephaly, oxidative stress / Includes bibliographical references. / Timothy Megraw, Professor Directing Dissertation; Yoichi Kato, Committee Member; Branko Stefanovic, Committee Member; Yanchang Wang, Committee Member.