The Hedgehog (Hh) signaling pathway regulates normal development and cell proliferation across the metazoa. Upon its aberrant activation, mammalian Hh signaling can also cause tumor formation. Hh-induced tumors can arise from different tissues and can be locally invasive but rarely metastatic or highly aggressive, as is the case with basal cell carcinoma (BCC) of the skin and the cerebellar tumor medulloblastoma (MB), respectively. Little is known about common cell-intrinsic factors that control the development of such diverse Hh-dependent tumors. The zinc-finger transcription factor Zfx is required for the self-renewal of several stem cell types in both mouse and human, but its role in malignant transformation remains controversial. We found that Zfx is variably required for the development of two distinct Hh-dependent tumors in vivo. Co-deletion of Zfx prevented BCC formation initiated by Hh pathway overactivation in the skin following deletion of the inhibitory receptor Ptch1. Co-deletion of Zfx also delayed development of Hh-dependent MB caused by Ptch1 deletion in vivo. In contrast, Zfx was dispensable for the development of the PTEN-dependent brain tumor glioblastoma, showing that a requirement for Zfx is not generalizable across all cancers. We used genome-wide expression and chromatin binding analysis in a human MB cell line to identify direct, evolutionarily conserved targets of Zfx. These targets included the Hh signal transducer Smoothened (Smo). Smo expression data from Zfx-deficient BCC and MB cells in vivo and in vitro suggest that Zfx may directly regulate Hh pathway activation in some cancers. We identified two additional conserved downstream targets of Zfx, Dis3L and Ube2j1, which were required for optimal growth of human MB cells in vitro. These results identify Zfx as a common cell-intrinsic regulator of diverse Hh-induced tumors. Further investigation of the requirement for Zfx and its conserved downstream target genes, such as Dis3L and Ube2j1, in in vivo models of Hh-dependent BCC and MB could lead to the identification of novel targetable molecules for therapies directed against these malignancies.

The lymphatic vascular system is necessary for physiological regulation of tissue fluid homeostasis and absorption of dietary fat. Lymphatics also function in the inflammatory response and are involved in pathological conditions such as wound healing and cancer. We show that the Notch signaling pathway is a regulator of both developmental and pathological lymphangiogenesis. Notch1 and Notch4 are expressed by the lymphatic endothelium, and Delta-like ligand 4 (Dll4) is the predominantly expressed Notch ligand in the developing lymphatic vessels of the embryonic dermis and pathological lymphatic vessels of the wounded cornea. Dll4 was able to induce Notch activation in human dermal lymphatic endothelial cells (HDLECs), whereas Jagged1 (Jag1) was not. In HDLECs, Notch signaling is activated in response to Vascular Endothelial Growth Factor (VEGF) or Vascular Endothelial Growth Factor-C (VEGF-C) stimulation. In vitro assays demonstrated that Notch activation inhibits HDLEC proliferation, migration, and capillary network formation; these effects were coincident with increased levels of HEY1 and HEY2, biphasic regulation of VEGFR-3, and decreased levels of VEGFR-2. Using genetic intervention of Notch signaling, we demonstrated that Notch regulates developmental sprouting lymphangiogenesis by restricting growth and sprouting of lymphatics in the murine embryonic dermis. Using pharmacological intervention of Notch signaling, we found that Notch restricted pathological sprouting lymphangiogenesis in the corneal suture assay, which models inflammation-induced lymphangiogenesis. However, pharmacological intervention of Notch signaling did not measurably affect pathological sprouting lymphangiogenesis in an orthotopic tumor model of human breast cancer. Our data from analysis of HDLECs, dermis, and sutured cornea support a role for Dll4-driven Notch signaling in restricting sprouting lymphangiogenesis. Lymphatic specification/separation requires a venous endothelial cell to become a lymphatic endothelial cell, and lymphatic valve formation requires a duct endothelial cell to become a valve endothelial cell. Through analysis of genes regulated by Notch in HDLECs, we demonstrated that Notch determines lymphatic endothelial cell fates. Notch inhibits genes critical for lymphatic specification and separation (PROX1, PDPN), and induces genes important for lymphatic valve formation (FNEIIIA, ITGA9, CX37). We conclude that Notch is a context-dependent regulator of lymphangiogenesis. Notch functions in the tip/stalk, venous to lymphatic, and duct endothelial to valve endothelial cell fate decisions in lymphatic vasculature. Given the critical functions of the lymphatic vasculature in multiple physiological and pathological settings, understanding Notch functions in the lymphatic vasculature is critical to design treatments for conditions caused by lymphatic malfunction.

Neurons are divided into functional compartments: the soma houses the DNA and transcriptional machinery, the axons conduct action potentials, presynaptic boutons transmit synaptic signals, and dendrites receive and integrate synaptic information. These functional differences are achieved by localizing different complements of proteins to these compartments; axons contain a high density of voltage-gated channels, presynaptic boutons house the machinery for synaptic vesicle-mediated neurotransmitter release, while dendrites contain ligand-gated and voltage-gated ion channels that generate and integrate postsynaptic responses. These primary compartments are further divided in some cell types, allowing the dendrite to expand its information processing power. For example, in CA1 pyramidal neurons of the hippocampus, a brain region crucial for memory formation, the apical dendrite is divided into a proximal and a distal compartment. These two dendritic compartments receive synaptic inputs from different sources and contain different proteins, making them functionally distinct. While many motifs and molecules have been identified that regulate the trafficking of proteins to axons versus dendrites, little is known about the mechanisms of protein targeting to compartments within a dendrite. We have investigated the regulation of protein composition of the distal dendritic compartment in the CA1 neurons. This distal compartment is enriched in specific ion channels, including the hyperpolarization-activated HCN1 cation channel, which we have focused on because of its striking distal localization and its importance in hippocampal function. Using dissociated and organotypic hippocampal culture systems, we show that HCN1 surface expression is activity dependent. However, the axons that innervate the distal compartment are not required for HCN1 localization. Our data suggest that while activity plays a role in HCN1 channel regulation, it is not sufficient for the distal dendritic targeting. We show that proper distal localization of HCN1 channels requires a non-cell autonomous factor. We provide evidence that the extracellular matrix protein reelin acts as this non-cell autonomous factor regulating ion channel composition of the distal dendrites. Blocking reelin signaling in organotypic culture results in reduction of HCN1 in the dendrites and distal HCN1 levels are reduced in reeler mice. In vivo viral knockdown of dab1, the intracellular signaling partner of reelin, leads to loss of HCN1 and other distally enriched ion channels specifically from the distal dendrites, but does not alter ion channel composition of the proximal dendritic compartment. Viral knockdown of dab1 increases a physiological indicator of HCN channels at the soma, indicating that some of HCN1 channels are redistributed. Blockade of src family kinases that are activated by reelin signaling likewise leads to a loss of distally enriched ion channels. These results define a novel role for reelin signaling in dendritic compartmentalization by regulating ion channel composition.

Fatty acid deposition in non-adipose tissue leads to a cellular dysfunction known as lipotoxicity. Neutral lipid synthesis is known to protect against lipotoxicity but many additional pathways are likely to be integral in this process. In order to identify pathways protective against lipid induced cell death, we performed a genome-wide unsaturated fatty acid (UFA) sensitivity screen in yeast. Of the ~5,500 gene mutants tested, we identified 156 which resulted in sensitivity to growth on media containing palmitoleate. These genes identified many cellular processes, including vesicular trafficking, lipid metabolism and vacuolar protein sorting. Deletion of three members of the GET complex, a complex essential for tail anchored protein insertion into the ER, caused vulnerability to fatty acids. We went on to assess the role of GET3 in cellular lipid metabolism and found that ablation of GET3 results in a defect in vacuolar hydrolysis and a reduction in lipid droplet number; pathways which we hypothesize to be integrally related. Furthermore, a major goal of this study was to find mammalian genes playing an integral role in pathways of lipoprotection. Of the 156 gene deletions found to confer fatty acid sensitivity in yeast, 68 have been conserved in mammals. We demonstrate that two of these mammalian orthologs, ARV1and ASNA1, are vulnerable to fatty acid treatment upon knockdown in the MIN6 pancreatic beta-cell line. These mammalian genes, which were identified through the fatty acid sensitivity screen in yeast, are involved in lipid induced cellular dysfunction in pancreatic beta-cells and, in the case of ARV1, hepatocytes. Therefore, these genes likely play a role in the progression of the lipotoxic diseases; type 2 diabetes and nonalcoholic fatty liver disease.

Characterizing the binding preferences of transcription factors is a major objective in molecular biology. Important processes such as development and responses to environmental stresses are regulated by the interactions between transcription factors and DNA. In this thesis, we address three key issues in the analysis of protein-DNA interactions. First, we demonstrate how transcription factor binding motifs can be inferred from ChIP-seq data by integrating a peak-calling algorithm and a biophysical model of transcription factor specificity. Next, we show that high-resolution DNase I cleavage profiles can provide detailed information about the role that DNA shape plays in protein- DNA recognition. Our analysis reveals the interplay between DNA sequence, methylation status, DNA geometry, and DNase I cleavage. Finally, we construct a model of transcription factor-DNA interaction that allows multiple transcription factors to bind co- operatively and competitively. In addition, the model can also infer transcription factor concentration. As the binding preferences of transcription factors continue to be characterized with a high degree of precision, we anticipate that use of these more realistic models will become more prevalent.

Radial glial progenitor (RGP) cells are neural stem cells that give rise to the majority of neurons, glia, and adult stem cells during cortical development. These cells divide either symmetrically to form two daughter RGP cells or asymmetrically to form a daughter RGP cell or a daughter neuron/neuronal precursor. In between divisions, the nuclei of RGP cells oscillate in coordination with the cell cycle in a form of behavior known as interkinetic nuclear migration (INM). RGP nuclei migrate basally during G1, undergo S phase, and migrate apically during G2 to the apical, ventricular surface (VS). Mitosis only occurs when the nucleus reaches the VS. Two microtubule-associated motor proteins are required to drive nuclear movement: the unconventional kinesin, Kif1a, during G1-specific basal migration and cytoplasmic dynein during G2-specific apical migration. The strict coordination of motor activity, migratory direction, and cell cycle phase is highly regulated and we find that a G2 cell cycle-dependent protein kinase activates two distinct G2-specific mechanisms to recruit dynein to nuclear pores. The activities of these pathways initiate apical nuclear migration and maintain nuclear movement throughout G2. Originally identified in HeLa cells, we find the two G2-specific recruitment pathways (“RanBP2-BicD2” and “Nup133-CENP-F”) are conserved in RGP cells. Disrupting either pathway arrests apical nuclear migration but does not affect G1-dependent basal migration. The “RanBP2-BicD2” pathway initiates early during G2 and is maintained throughout the cell cycle phase while the “Nup133-CENP-F” pathway is activated later in G2. Forced targeting of dynein to the nuclear envelope (NE) restores apical nuclear migration, with nuclei successfully reaching the VS. We also find that the G2/M-specific Cdk1 serves as a master regulator of apical nuclear migration in RGP cells. Pharmacological drug inhibitors of Cdk1 arrest apical migration without any effect on G1-dependent basal migration. Conversely, overactivating Cdk1 causes premature, accelerated apical nuclear migration. Specifically, Cdk1 drives apical nuclear migration through activation of both the “RanBP2-BicD2” and “Nup133-CENP-F” pathways. Cdk1 acts by phosphorylating RanBP2, priming it for BicD2 interaction. Forced targeting of BicD2-dynein to the NE in a RanBP2-independent manner rescues apical nuclear migration in the presence of Cdk1 drug inhibition. Additionally, Cdk1 seems to activate the “Nup133-CENP-F” at the CENP-F level, phosphorylating the protein to trigger nuclear export. INM plays an important role in proper cell cycle progression and we find that arresting nuclei away from the VS prevents mitotic entry, demonstrating that apical nuclear migration to the VS is not just a correlated with cell cycle progression, but is required. When apical migration is restored by forced recruitment of dynein to the NE, mitotic entry is restored as well. In contrast, we find that arresting basal migration by Kif1a does not have a major influence on cell cycle progression. RGP cells still enter S-phase despite remaining close to the VS, revealing that, unlike mitotic entry, S-phase entry is not coupled with nuclear positioning. However, symmetric, proliferative divisions are favored over asymmetric, neurogenic divisions after inhibition of basal migration. We further find that Kif1a and the proteins involved in the two recruitment pathways play additional role later in brain development. After a neurogenic division, the newly-born neuron migrates past the RPG nuclei and they undergo a multipolar morphology. After at least twenty-four hours, the immature neuron then transitions to a bipolar, migratory morphology where it continues migrating towards its final destination along RGP fibers to the cortical plate. We demonstrate that Kif1a and NE dynein recruitment proteins seem to be involved in the multipolar to bipolar transition and RNAi for these proteins prevent further migration by arresting the immature neurons in a multipolar morphology. Kif1a RNAi, in particular, also induced comparable arrest in surrounding control neurons. Further analysis reveal that the multipolar arrest in neurons is independent of the basal nuclear migration arrest in RGP cells. These results identify the control mechanism for NE dynein recruitment in RGP cells to drive apical nuclear migration, the relationship of cell cycle phase progression with nuclear positioning, and the sequential, independent roles of these proteins, particularly Kif1a, in neuronal maturation.

Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2004. / Includes bibliographical references (leaves 79-83). Also available in electronic version. Access restricted to campus users.

Thesis (Ph. D.)--University of Queensland, 2002. / Includes bibliographical references.

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