During early development, a series of regulated cell movements is required to set up the adult body plan of an organism. Collectively referred to as gastrulation, these coordinated cell movements organize the germ layers and establish the major body axes of the embryo. One such coordinated cell movement, epiboly, describes the thinning and spreading of a multilayered cell sheet to cover the embryo during gastrulation. The zebrafish embryo has emerged as a vital model system to study the cellular and molecular mechanisms that drive epiboly. In the zebrafish, the blastoderm undergoes epiboly to engulf the yolk cell and close the blastopore at the vegetal pole. This is achieved through the coordinated movement of the deep cells, which make up the embryo proper, and two extra-embryonic tissues, the enveloping layer and yolk syncytial layer. Epiboly is essential to the development of most organisms; however, the cellular and molecular mechanisms driving epiboly are poorly understood. Here I report the findings of two distinct projects which addressed the cellular and molecular basis for epiboly in the zebrafish. One cellular mechanism thought to be involved in driving epiboly is the removal of yolk cell membrane ahead of the advancing blastoderm margin. Using a combination of drug- and dominant-negative based approaches to inhibit Dynamin, a key component of the endocytic machinery, I demonstrated that marginal yolk cell endocytosis is dispensable for the successful completion of epiboly. Instead, I found that Dynamin primarily acts in the blastoderm where it maintains integrity of the enveloping layer (EVL) during epiboly. Dynamin maintains EVL integrity through regulation of the Ezrin/Radixin/Moesin (ERM) family of proteins and the activity of the small GTPase Rho A. With the goal of identifying genes involved in regulating epiboly, I characterized the calpain family of calcium-dependent cysteine proteases in the zebrafish and examined the developmental expression patterns of these genes. My study provided insight into the evolution of this large gene family. Furthermore, I found that most members of this family are expressed in the early embryo, suggesting that they may play a role in regulating early developmental processes such as epiboly.

During early development, a series of regulated cell movements is required to set up the adult body plan of an organism. Collectively referred to as gastrulation, these coordinated cell movements organize the germ layers and establish the major body axes of the embryo. One such coordinated cell movement, epiboly, describes the thinning and spreading of a multilayered cell sheet to cover the embryo during gastrulation. The zebrafish embryo has emerged as a vital model system to study the cellular and molecular mechanisms that drive epiboly. In the zebrafish, the blastoderm undergoes epiboly to engulf the yolk cell and close the blastopore at the vegetal pole. This is achieved through the coordinated movement of the deep cells, which make up the embryo proper, and two extra-embryonic tissues, the enveloping layer and yolk syncytial layer. Epiboly is essential to the development of most organisms; however, the cellular and molecular mechanisms driving epiboly are poorly understood. Here I report the findings of two distinct projects which addressed the cellular and molecular basis for epiboly in the zebrafish. One cellular mechanism thought to be involved in driving epiboly is the removal of yolk cell membrane ahead of the advancing blastoderm margin. Using a combination of drug- and dominant-negative based approaches to inhibit Dynamin, a key component of the endocytic machinery, I demonstrated that marginal yolk cell endocytosis is dispensable for the successful completion of epiboly. Instead, I found that Dynamin primarily acts in the blastoderm where it maintains integrity of the enveloping layer (EVL) during epiboly. Dynamin maintains EVL integrity through regulation of the Ezrin/Radixin/Moesin (ERM) family of proteins and the activity of the small GTPase Rho A. With the goal of identifying genes involved in regulating epiboly, I characterized the calpain family of calcium-dependent cysteine proteases in the zebrafish and examined the developmental expression patterns of these genes. My study provided insight into the evolution of this large gene family. Furthermore, I found that most members of this family are expressed in the early embryo, suggesting that they may play a role in regulating early developmental processes such as epiboly.

Glioblastoma Multiforme (GBM) is the most common and lethal of human primary central nervous system tumours, with a median survival of 14-16 months despite surgery, radiation and chemotherapy. A reason for this dismal prognosis is insufficient understanding of the ontogeny of GBMs, which are highly heterogeneous at a pathological level. This pathological diversity, between and within GBMs as well as varying grades of gliomas, is not fully explained on the grounds of an oncogenic stimulus. Interaction with the tumour microenvironment, as well as inherent characteristics of the tumour cell of origin are likely a source of this heterogeneity. In this thesis we describe the use of a novel mouse model which integrates Cre-Lox mediated and Tet-regulated gene expression. This system in combination with germline and somatic strategies has enabled us to interrogate how the state in glial development and the region in the brain where transformation occurs influence the process of gliomagenesis. The findings of this thesis suggest that the state of glial development at which a mutation is introduced is an important determinant of gliomagenesis. In support of this, we showed that early progenitors in the radial glial lineage are more susceptible to transformation than those, which have committed to a gliogenic lineage and are presumably further along in the process of differentiation. Highlighting the interplay between genetic alterations and the molecular changes that accompany the process of differentiation. Despite findings that suggest that neurogenic regions of the adult brain are more susceptible to transformation, we show that this is not always the case and instead, transformation is dependent on an interaction between specific combinations of genetic mutations and susceptible cell types regardless of the region of origin. Results from this thesis highlight the need to view the tumourigenic process of gliomas in the context of normal brain development as the cell context of oncogene expression may determine the phenotype and biologic aggressiveness of the tumour. Thus, the results of genetic or epigenetic alterations leading to brain tumours may be quite different in different cells of the hierarchy, suggesting unique treatment targets and strategies depending on the cell of origin.

Glioblastoma Multiforme (GBM) is the most common and lethal of human primary central nervous system tumours, with a median survival of 14-16 months despite surgery, radiation and chemotherapy. A reason for this dismal prognosis is insufficient understanding of the ontogeny of GBMs, which are highly heterogeneous at a pathological level. This pathological diversity, between and within GBMs as well as varying grades of gliomas, is not fully explained on the grounds of an oncogenic stimulus. Interaction with the tumour microenvironment, as well as inherent characteristics of the tumour cell of origin are likely a source of this heterogeneity. In this thesis we describe the use of a novel mouse model which integrates Cre-Lox mediated and Tet-regulated gene expression. This system in combination with germline and somatic strategies has enabled us to interrogate how the state in glial development and the region in the brain where transformation occurs influence the process of gliomagenesis. The findings of this thesis suggest that the state of glial development at which a mutation is introduced is an important determinant of gliomagenesis. In support of this, we showed that early progenitors in the radial glial lineage are more susceptible to transformation than those, which have committed to a gliogenic lineage and are presumably further along in the process of differentiation. Highlighting the interplay between genetic alterations and the molecular changes that accompany the process of differentiation. Despite findings that suggest that neurogenic regions of the adult brain are more susceptible to transformation, we show that this is not always the case and instead, transformation is dependent on an interaction between specific combinations of genetic mutations and susceptible cell types regardless of the region of origin. Results from this thesis highlight the need to view the tumourigenic process of gliomas in the context of normal brain development as the cell context of oncogene expression may determine the phenotype and biologic aggressiveness of the tumour. Thus, the results of genetic or epigenetic alterations leading to brain tumours may be quite different in different cells of the hierarchy, suggesting unique treatment targets and strategies depending on the cell of origin.

Upper airways are lined with a pseudostratified mucociliary epithelium maintained by basal cells. To investigate functional and phenotypic heterogeneity within the human basal cell compartment, we used a combination of limiting dilution assays and surface marker profiling on primary cultures of basal cells with verified progenitor activity. The limiting dilution assay suggested functional heterogeneity in the ability of basal cells to repopulate a filter and maintain a barrier at ALI. The frequency of cells with this activity varied between patient strains and ranged from 0.08%-1% of basal cells. Validation of large-scale comprehensive surface marker profiling on basal cells led to identification of 74 antigens demarking consistent subpopulations. Preliminary functional analyses suggest differences in differentiation potential of some subpopulations. This work supports the idea that the basal cell compartment may be functionally heterogeneous, and provides new molecular tools for interrogation of human basal cells.

Inflammatory bone diseases like rheumatoid arthritis and periodontal disease lead to increased bone loss in the inflamed areas. The multinucleated bone resorbing cells, the osteoclasts, present in these diseases are larger than normal, and these larger osteoclasts (10+ nuclei) resorb more bone and more often than smaller osteoclasts (2-5 nuclei). Thus, the focus of this thesis was to determine if there are differences in mechanisms of osteoclast activation between large and small osteoclasts. Experiments using authentic rabbit osteoclasts and RAW 264.7-derived osteoclasts revealed differences in the expression of a number of activating factors; with large osteoclasts expressing higher levels of activating receptors (RANK, IL-1RI, TNFR1 and integrins αv and β3), as well as enzymes involved in cellular resorption, while small osteoclasts expressed higher levels of an alleged fusion receptor and the inhibitory receptor, IL-1RII. Further studies revealed that large osteoclasts more readily responded to stimulation by IL-1 compared to small osteoclasts and at lower concentrations suggesting this is a result of their higher expression of activating receptors. Differences in responses to the IL-1 isoforms, IL-1α and IL-1β, were also seen in large osteoclasts: IL-1α generated more large osteoclasts over the course of differentiation, while IL-1β induced changes in cell morphology and in the induction of integrin β3 phosphorylation. These observations suggested that differences in osteoclast responses are induced by IL-1α and IL-1β and it led to the hypothesis that there are differences in signaling between large and small osteoclasts. To elucidate differences in signaling mechanisms a signaling pathway microarray was used which revealed higher expression of Vegfa in large compared to small osteoclasts. Osteoclast differentiation with RANKL increased Vegfa gene expression in a time-dependent manner and VEGF-A secretion was elevated in populations enriched for large osteoclasts. Furthermore, mechanistic studies with inhibitors of transcription factors involved in differentiation revealed that RANKL-mediated Vegfa expression in large osteoclasts was regulated by the NF-κB pathway via induction of Hif1α. These results support the hypothesis that signaling differences exist between large and small osteoclasts and implicates VEGF-A in osteoclast hyperactivity in inflammatory conditions.

Biological outputs of insulin/IGF signaling are regulated through essential mediators, such as IRSs, PI3-kinase, and PKB/Akt. These mediators serve critical roles in signal propagation, feedback, and as junctions for crosstalk with other pathways. Abnormal insulin/IGF signaling results in disease, such as obesity, diabetes, and cancer. Given the vital role of this signaling pathway to human health, unraveling its regulatory mechanisms is crucial. Components of this pathway are highly conserved throughout evolution. PTEN, one of the well-defined regulators of this pathway, functions as a lipid phosphatase that negatively regulates insulin/IGF-1 signaling at the PIP3 level, a phosphoinositol that is upregulated by activated PI3-kinase in both Drosophila and mammals. To discover genetic modulators of PTEN in Drosophila, we performed a loss-of-function genetic screen to identify molecules that modify the phenotype elicited by PTEN overexpression in the Drosophila eye. From this screen, we identified a member of the Dbl-family, the guanine nucleotide exchange factor DRhoGEF2, which suppresses the PTEN-overexpression eye phenotype via its effects on dPKB/dAkt activation. By conducting a genetic rescue, we established that PDZ-RhoGEF, a member of the regulator of G-protein signal (RGS)-like domain containing Rho GEFs (RGS-RhoGEFs) subfamily of Dbl-family GEFs, is the mammalian counterpart of DRhoGEF2. PDZ-RhoGEF is essential for cell proliferation and survival through ROCK-dependent activation of IRS/PI3-kinase signaling cascade, which has a major impact on adipose tissue homeostasis. Through an integrative approach, we have demonstrated that DRhoGEF2/PDZ-RhoGEF-dependent signaling has tissue-specific effects on insulin/IGF-signaling throughput in both Drosophila and mammals. Particularly, we have demonstrated the role played by PDZ-RhoGEF in diet related pathology, provides an alternative therapeutic opportunity in disease intervention.

The scaffolding protein anillin plays a crucial role during cytokinesis – the physical separation of daughter cells following chromosome segregation. Anillin binds filamentous F-actin, non-muscle myosin II and septins, and in cell culture models has been shown to restrict actomyosin contractility to the cleavage furrow. Whether anillin also serves this function during the incomplete cytokinesis that occurs in developing germ cells has remained unclear. Localization of anillin to several actin-rich structures in developing male germ cells also suggests potential roles for anillin outside of cytokinesis. In this study, I demonstrate that anillin is required for cytokinesis in dividing Drosophila spermatocytes. In addition, spermatid individualization is defective in anillin-depleted cells, although similarities to another cytokinesis mutant, four wheel drive, suggest this may be a secondary effect of failed cytokinesis. Anillin, septins and myosin II stably associate with the cleavage furrow in wild-type dividing spermatocytes. Anillin is necessary for recruitment of septins to the cleavage furrow, and for maintenance of Rho, F-actin and myosin II at the equator in late stages of cytokinesis. Membrane trafficking appears unaffected in anillin-depleted cells, although, unexpectedly, ectopic expression of one membrane trafficking marker, DE-cadherin-GFP, suppresses the cytokinesis defect. DE-cadherin-GFP recruits β-catenin (armadillo) and α-catenin to the cleavage furrow and stabilizes F-actin at the equator. Taken together, my results suggest that the anillin-septin and cadherin-catenin complexes can serve as alternative means to promote tight physical coupling of F-actin and myosin II to the cleavage furrow and successful completion of cytokinesis.

Amphiphysin (Amph) is a multi-domain protein that has been implicated in synaptic vesicle (SV) endocytosis. In vertebrates, Amph1 associates with SVs and binds to known endocytic proteins, such as dynamin and clathrin. Overexpression of the vertebrate Amph1 SH3 domain is sufficient to inhibit SV endocytosis in the lamprey synapse. However, these in vitro and overexpression studies may not reflect Amph function in vivo. To investigate Amph function in vivo, I used Drosophila melanogaster as a model organism. I discovered that Drosophila Amph was broadly expressed throughout all developmental stages and was also highly expressed in specialized membranes such as the postsynaptic membrane at the larval neuromuscular junction and the t-tubule membranes of muscles. amph mutants were viable and had normal synaptic transmission, results that were inconsistent with a role for Amph in SV endocytosis. However, amph mutants had impaired locomotion, which may reflect a defect in the t-tubule network, a membrane system that is specialized to couple muscle membrane excitation to muscle contraction. To further explore this idea, I undertook a structure-function approach to ask if different Amph functional domains could rescue the t-tubule and locomotory defects observed in amph mutants. Partial rescue was observed for most constructs, suggesting that Amph function was dependent on more than one domain. To further elucidate how Amph functions at the t-tubule network, I used different in vitro methods to investigate novel protein partners for Amph. A GST pull-down approach identified actin as a potential Amph partner, consistent with studies in yeast. However, I could not confirm a direct interaction between Amph and actin in Drosophila. Another candidate partner was the actin-nucleating protein, Wiskott Aldrich Syndrome Protein, WASP. Although WASP and Amph could be coimmunoprecipitated in vitro, WASP was not expressed at the t-tubule membrane, and Wasp mutants had normal t-tubule morphology. Clearly, Amph is essential for normal t-tubule morphology and future work is needed to further define the function of Amph at the t-tubule network.

Upper airways are lined with a pseudostratified mucociliary epithelium maintained by basal cells. To investigate functional and phenotypic heterogeneity within the human basal cell compartment, we used a combination of limiting dilution assays and surface marker profiling on primary cultures of basal cells with verified progenitor activity. The limiting dilution assay suggested functional heterogeneity in the ability of basal cells to repopulate a filter and maintain a barrier at ALI. The frequency of cells with this activity varied between patient strains and ranged from 0.08%-1% of basal cells. Validation of large-scale comprehensive surface marker profiling on basal cells led to identification of 74 antigens demarking consistent subpopulations. Preliminary functional analyses suggest differences in differentiation potential of some subpopulations. This work supports the idea that the basal cell compartment may be functionally heterogeneous, and provides new molecular tools for interrogation of human basal cells.