The vestibular system of the inner ear detects head position using three orthogonally oriented semicircular canals. Vestibular function relies on precise canal shape and orientation, and slight changes can cause vestibular defects. Canals are sculpted from pouches that protrude from the otic vesicle, the simple sphere of epithelium that forms the inner ear. In the center of each pouch, a "fusion plate" forms where cells lose their epithelial morphology and the basement membrane breaks down. The opposing layers of the fusion plate intercalate and are subsequently removed, creating a canal. Proper fusion depends on Netrin-1, which regulates basement membrane breakdown during fusion in mice, although the underlying molecular mechanism is unknown. This dissertation describes our work to better understand the cellular effects of Netrin-1 during canal formation. Although vestibular apparatus structure is shared among species, some developmental events that lead to this structure differ. For example, while fusion plate basement membrane breakdown is conserved, apoptosis is required for fusion in chicks, but not in mice. We used gain-of-function approaches to determine the main cellular effect of Netrin-1 during fusion in chicks and mice. We show that overexpression of Netrin-1 in chicks prevents canal fusion from occurring normally by interfering with apoptosis. On the other hand, we show that ectopic expression of Netrin-1 in mice using a conditional expression allele causes excessive fusion, resulting in canal truncation. This suggests that Netrin-1 may play divergent roles during canal morphogenesis in chicks and mice. To determine if Netrin-1 regulates the basement membrane in other contexts, we created a Netrin-1 conditional null allele. This was necessary because existing Netrin-1 mutants express residual Netrin-1 protein, which could be sufficient to rescue basement membrane defects in other tissues, and because existing mutants die shortly after birth, preventing postnatal analysis. Complete loss of Netrin-1 protein in our newly generated mice does not cause more severe defects in fusion compared to existing Netrin-1 hypomorphs, suggesting that residual Netrin-1 protein does not affect the basement membrane during fusion in Netrin-1 hypomorphs. Future work will determine if complete loss of Netrin-1 affects basement membrane integrity in other tissues.

No description available.

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.

Dynein, a microtubule motor protein complex, plays critical roles in cell-cycle progression in many systems. The dynein accessory factor <i>LIS1</i>, first identified as a causative factor of the human brain disorder Lissencephaly when lost in one copy, is essential for a majority of the cellular activities of dynein. To gain insight into the in vivo functions of LIS1, we characterized a male-sterile allele of the <i>Drosophila</i> homolog of human <i>LIS1</i>. We found defects in centrosome migration and attachments in <i>Lis-1</i> spermatocytes and spermatids. The localization pattern of LIS-1 protein throughout <i>Drosophila</i> spermatogenesis mirrors that of dynein, and dynein recruitment to the nuclear surface and spindle poles is severely reduced in <i>Lis-1</i> male germ cells. We previously identified <i>asunder</i> (<i>asun</i>) as a novel regulator of dynein localization during <i>Drosophila</i> spermatogenesis. We present a model in which <i>Lis-1</i> and <i>asun</i> cooperate to regulate dynein localization and centrosome positioning during <i>Drosophila</i> spermatogenesis. Expression of <i>asun</i> is much higher in <i>Drosophila</i> ovaries than in testes. We therefore sought to determine whether ASUN plays roles in oogenesis. We characterized the female germline phenotypes of flies homozygous for a null allele of <i>asun</i> (<i>asun^d93</i>). <i>asun^d93</i> females lay very few eggs, and a majority of these eggs are ventralized, possibly as a result of mislocalization of <i>gurken</i> transcripts, a dynein-regulated step, within <i>asun^d93</i> oocytes. Dynein localization and dynein-mediated processes are disrupted in <i>asun^d93</i> oocytes. Taken together, our data indicate that <i>asun</i> is a critical regulator of dynein during <i>Drosophila</i> gametogenesis. As <i>asun</i> plays a conserved role in regulating dynein during <i>Drosophila</i> gametogenesis, we sought to identify other proteins that cooperate with <i>asun</i> to perform this function. We utilized a set of publicly available 2nd chromosome deficiency lines to initiate a dominant modifier screen to identify genes that could enhance or suppress the <i>asun</i> male phenotype when lost in one copy. Further testing will be required to identify and characterize the individual genes within these deficiency intervals that cooperate with <i>asun</i> to regulate dynein during <i>Drosophila</i> spermatogenesis.

The role of Four jointed box 1 (FJX1) in colorectal cancer (CRC) is presented in this dissertation. FJX1 was identified as a candidate gene for regulating tumor formation in CRC as it was inhibited in rectal cancers after one week treatment with celecoxib. FJX1 mRNA and protein are upregulated in human CRC, and high expression of FJX1 is associated with poor patient prognosis. Novel FJX1 antibodies and expression vectors were developed and used to characterize recombinant FJX1 in vitro. In vivo, FJX1 promotes tumor formation by increasing tumor cell proliferation and vascularization. In vitro, conditioned media from FJX1 expressing cells promoted endothelial cell capillary tube formation in a HIF1-α dependent manner. In addition to these experimental observations, microarray expression profiling of CRC cells with manipulated FJX1 expression is also presented. In sum, these results support the conclusion that FJX1 is a novel regulator of tumor progression, due in part, to its effect on tumor vascularization.

Tuberous Sclerosis Complex (TSC) is a multi-organ hamartomatous disease caused by loss of function mutations in either the TSC1 or TSC2 genes. Despite involvement of multiple organs such as the kidneys, lungs, and skin, neurological aspects are usually the most severe due to a very high prevalence of cognitive impairment, autism and epilepsy. The protein products of TSC1 and TSC2, hamartin and tuberin respectively, regulate the mTOR kinase signaling pathway. Current models of TSC propose that hamartoma formation is secondary to a loss of heterozygosity at either the TSC1 or TSC2 loci, and subsequent hyperactivation of mTOR Complex 1 (mTORC1). In this dissertation I explore the underlying mechanisms of organ specific pathogenesis in TSC. In the first half of my dissertation, I demonstrate that loss of Tsc1 in the distal convoluted tubule of the kidney results in cystogenesis. Cyst formation in these kidneys is due to a mTORC1 but not mTORC2 dependent process. I then show that cystic changes in these kidneys may be due to ciliary defects. While a loss of heterozygosity has clearly been reported in the kidney and other organ system, second hit mutations in neural lesions have only rarely been identified. Thus, to begin to define the role of the heterozygosity of TSC1 or TSC2 during the pathogenesis of TSC in the brain, we generated induced pluripotent stem cells (iPSC) from patients with TSC. Deep sequencing of these patents revealed that all of our patient derived lines are heterozygous for TSC2 mutations. I then provide evidence that these heterozygous iPSCs are abnormal with increased cell survival and enhanced maintenance of pluripotency. These changes may be due to slight changes in mTORC1 signaling. The work presented in this dissertation increases our understanding of the tissue specific phenotypes and underlying mechanisms of TSC pathogenesis. This research may lead to the identification of new therapeutic targets for TSC and associated comorbidities.

The genome of a eukaryotic cell tightly packed within the nucleus with a high degree of structural organization. Two mechanisms accounting for nuclear structure and the dynamics of subnucler organization in S. cerevisiae are presented within. First, two powerful genetic screens identify requirements for the RSC chromatin-remodeling complex in maintaining nuclear morphology. The major NE-malformations observed in rsc mutants likely result from aberrant transcription and lipid homeostasis. Second, nuclear organization of transcriptional events in response to osmotic stress in S. cerevisiae involves the relocalization of the Hot1 transcription factor to foci that overlap with corresponding target genes. Casein Kinase II negatively regulates Hot1 localization to foci, and also leads to a reduced transcriptional response. These results suggest that the nuclear organization of transcription events impact the stochastic activity of environmentally induced genes. In conclusion, both chromatin organization and transcription events result in dynamic alterations in nuclear structure impacting the output of the genome.

During liver development, precise signaling is required to direct cell fate decisions and architectural establishment. It is hypothesized that the same signaling pathways are important in directing liver regeneration. To examine the signals important in liver development and disease, we genetically manipulated Recombination signaling binding protein J kappa (Rbpj) and Hepatocyte nuclear factor 6 (Hnf6), or Vascular endothelial growth factor (Vegf) within the liver epithelium embryonically in mice. Along with these genetic models, we also examined several non-genetic rodent liver injury models and assessed the protein localization and tissue architecture of both epithelial and endothelial structures in the liver. We find that Rpbj and Hnf6 are together required for the embryonic formation of the intrahepatic bile duct, but dispensable for its regeneration postnatally. In both genetic and non-genetic cholestatic liver injuries in rodent models and in human liver disease, the expression of Sry-related HMG box 9 (Sox9) was found to be a marker of non-proliferative hepatobiliary intermediate cells that may aid the regenerative process. We also determined that the epithelial secretion of Vegf is required for proper endothelial and epithelial cell maturation and tissue architecture. These studies implicate a number of signaling relationships required for proper liver development and regeneration.

Cytoplasmic dynein is a large, multimeric complex that walks along microtubules to perform multiple functions within the cell. This motor is commonly found associated with the dynein-activating complex, dynactin. Dynein is required for a variety of essential functions such as cargo transport, organelle positioning, centrosome assembly and coupling to the nuclear envelope at the G2/M transition, mitotic spindle positioning, and ciliogenesis. Due to the vast number of roles within the cell, dynein complexes are subject to multiple layers of regulation, including binding of accessory proteins, phosphorylation, variations in subunit composition, and subcellular localization. Additionally, dynein roles are often cell cycle dependent. At the G2/M transition, a subpopulation of dynein is found anchored to the nuclear envelope in multiple species. In cultured human cells, this pool facilitates centrosome coupling to the nuclear envelope, centrosome splitting and migration to the presumptive poles, and nuclear envelope breakdown. Bicaudal D2 and Centromere protein F are required for anchoring this pool of dynein on the nuclear envelope. Another cell cycle-dependent role for dynein is primary ciliogenesis during G1 in cultured human cells. Dynein is required for promoting assembly of the primary cilium as well as regulating its length. For both functions, the complete mechanism for regulating dynein remains unclear. Asunder has been previously shown to regulate perinuclear dynein at G2/M in Drosophila spermatogenesis. Additionally, Asunder has been shown to be an essential subunit in the small nuclear RNA-processing complex, Integrator. Herein, I present data showing that Asunder has a conserved role in regulating perinuclear dynein in cultured human cells via its role in the Integrator complex. I additionally report that Integrator is required for primary ciliogenesis in G1-arrested cells. My work supports the model that Integrator independently regulates these cellular events at the RNA-processing level. This works expands our understanding of the mechanisms for dynein anchoring to the nuclear surface at G2/M and ciliogenesis by adding an RNA-processing component.

Cerebellar development is a complex process involving the tightly regulated proliferation, specification, migration, and connectivity of thousands of neurons and glia. Perturbations in signaling pathways important for these processes can have drastic consequences, including medulloblastoma formation. In this dissertation work I have focused on Sonic Hedgehog (Shh) signaling and its role in cerebellar development and formation of medulloblastoma. Our studies have identified a novel contribution of the multipotent hindbrain roof plate cell to diverse lineages in the cerebellum and its susceptibility to oncogenic transformation by deregulated Shh signaling, which leads to medulloblastoma formation. In addition, we have determined a previously unappreciated role for Shh signaling in specialized cerebellar glial cells, which functions to sustain proliferation of neighboring neuronal precursors. Last, we identify a small molecule as a novel and potent Hh pathway antagonist in multiple cell types, including Hh-responsive medulloblastoma cells. My work therefore offers insight into the diverse roles of Shh activity in the cerebellum during development and disease, which can provide insight into brain growth and the development of targeted therapies for disease processes.