During angiogenesis, nascent blood vessels sprout from pre-existing vasculature and recruit pericytes to induce maturation and vessel quiescence. Perictyes are associated with small vessels and capillaries where they share the basement membrane with the endothelium to provide vascular support. Pericytes are a critical component of the blood-brain barrier and regulate endothelial cell proliferation, vessel diameter, and vascular permeability. Endothelial cells express Notch1, whereas pericytes express both Notch1 and Notch3. Here we show that Notch signaling is essential for pericyte function. Through genetic manipulation and pharmacological tools we show that Notch regulates pericyte recruitment and pericyte/endothelial cell interactions. Notch1^+/-;Notch3^-/- mutant mice display decreased pericyte coverage and altered pericyte association with the retinal vascular plexus. Notch deficiency is associated with vascular anomalies where Notch1^+/-;Notch3-/- mice display retinal arteriovenous malformations (AVM) characterized by dilated vessels, vascular tangles and arteriovenous shunts that are similar to human brain AVMs. Disruption of pericyte/endothelial cell association is accompanied by an increase in vascular density, venule enlargement, and increased vascular permeability observed prior to AVM formation. In the ovary, we show that Jagged is essential for pericyte association with the endothelium where inhibition of Jagged-specific Notch activation results in luteal vessel dilation and hemorrhaging following ovarian hyperstimulation. By in vitro analysis of cultured pericytes we show that Notch1 and Notch3 induce plated derived growth factor receptor-β (PDGFR-β) expression to regulate cell migration. These findings expand the role for Notch in angiogenesis by demonstrating that Notch signaling in pericytes is essential for vascular development and function.

The dual specificity transcription factor Mga contains both a T-box binding domain and a basic helix-loop-helix zipper (bHLHZip) domain. Loss of Mga leads to embryonic lethality by E5.5. In vitro blastocyst culture and embryonic stem (ES) cell culture identify a lack of pluripotent inner cell mass (ICM) derived cells as the cause of embryonic lethality. Loss of Mga leads to increased apoptosis in E4.5 embryos, though there is no decrease in the amount of cell proliferation. Embryos with mutant Mga have fewer pluripotent ICM cells during delayed implantation, though the number of differentiated primitive endoderm cells remained initially stable. Despite the loss of pluripotent cells, there is no change in the pattern of expression of Nanog or Oct4, pluripotent cell markers, or Gata4, a primitive endoderm marker. Expression of Ornithine Decarboxylase (ODC), the rate-limiting enzyme in the synthesis of cellular polyamines, was identified as a possible cause of embryonic lethality based on a similar mutant phenotype as well as the presence of E-box sequences in genetic regulation loci. ODC is expressed at lower levels in the ICM of Mga mutants. Blastocyst and ES cell culture defects were rescued when cultured in the presence of exogenous putrescine, the metabolic product of ODC. These results suggest a mechanism for Mga to influence pluripotent cell survival through interactions with other bHLHZip domain proteins in the regulation of the polyamine pool in pluripotent cells of the embryo.

Immediate Early Genes (IEGs) are expressed upon re-entry of quiescent cells into the cell cycle following serum stimulation. These genes are involved in growth control and differentiation and hence their expression is tightly controlled. Many IEGs are regulated through Serum Response Elements (SREs) in their promoters, which bind Serum Response Factor (SRF). However, many other IEGs do not have SREs in their promoters and their serum regulation is poorly understood. We have identified SRF-independent IEGs in SRF-depleted fibroblasts. One of these, Id1, was examined more closely. We mapped a serum responsive element in the Id1 promoter and find that it is identical to a BMP Responsive Element (BRE). The Id1 BRE is necessary and sufficient for the serum regulation of Id1. Inhibition of the BMP pathway by siRNA depletion of Smad4, treatment with the BMP antagonist noggin, or the BMP receptor inhibitor dorsomorphin blocked serum induction of Id1. Further, BMP2 is sufficient to induce Id1 expression. Given reports that SRC inhibitors can block Id1 expression, we tested the SRC inhibitor, AZD0530, and found that it inhibits the serum activation of Id1. Surprisingly, this inhibition is independent of SRC or its family members. Rather, we show that AZD0530 directly inhibits the BMP type I receptors. Serum induction of the Id1 related gene Id3 also required the BMP pathway. Given these and other findings we conclude that the Id family of IEGs is regulated by BMPs in serum through similar BREs. This represents a second pathway for serum regulation of IEGs.

Recent advances in the field of axon guidance have revealed complex transcription factor codes that regulate neuronal subtype identity and their corresponding axon projections. Retinal axon divergence at the optic chiasm midline is key to the establishment of binocular vision in higher vertebrates. In the visual system of binocular animals, the ipsilaterally and contralaterally projecting retinal ganglion cells are distinguished by the laterality of their axonal projections. Specific axon guidance receptors and their ligands are expressed in retinal ganglion cells (RGCs) and at the chiasm, tightly regulating the development of the ipsilateral (uncrossed) and contralateral (crossed) retinal projections. Though many factors are known, their dysfunction leads to only partial misrouting of RGC axons. Moreover, the complex transcription factor codes that regulate RGC subtype identity are only beginning to be uncovered. Numerous gaps remain in our understanding of how these guidance molecules are transcriptionally regulated and how they are induced by the patterning genes that set up the different domains in which these RGC subtypes reside. An even more elusive question within the field is how the ipsilateral and contralateral RGC subpopulations acquire their different cell fates. In this thesis, I present my work on dissecting out the molecular signatures of the ipsilateral and contralateral RGC populations during embryonic development through gene profiling followed by the functional characterization of one candidate from this screen. In Chapter 2, I developed a cell purification method based on retrograde labeling of these two cell populations from their divergent axonal projections followed by cell sorting. This method can be used in studies requiring purified populations of embryonic RGCs. In Chapter 3, I conducted a microarray screen of purified ipsilateral and contralateral RGCs using the above method. Through subsequent validation of the in vivo expression patterns of select candidates, I identified a number of genes that are differentially expressed in ipsilateral and contralateral RGCs. Subsequent functional characterization of these genes has the potential to uncover novel mechanisms for regulating axon guidance, cell differentiation, fate specification, and other regulatory pathways in ipsilateral and contralateral RGC development and function. The results of this screen also revealed that ipsilateral and contralateral RGC may have distinct developmental origins and utilize different strategies for differentiation. In Chapter 4, I demonstrate a novel role for cyclin D2, one of the above candidates, in the production of ipsilateral RGCs. The G1-active cyclin D2 is highly expressed in the ventral peripheral retina preceding and coincident with the developmental window of ipsilateral RGC genesis. I further found that ipsilateral RGC production is disrupted in the cyclin D2 null mouse. The expression of cyclin D2 in a distinct proliferative zone that has evolutionary significance in ipsilateral RGC production and its subtype-specific requirement during retinal development suggest that cyclin D2 may mark a distinct progenitor pool for ipsilateral RGCs. Thus, these studies offer an important advance in our understanding of neuronal subtype diversification within the retina.

The ability to make transgenic animals has been a great tool for biologists to study living organisms. In C. elegans, the way transgenes are generated makes them problematic in many circumstances, and there is no single, simple, reliable approach that circumvents all of the problems with current methods of introducing transgenes into C. elegans. In Chapter 2, I discuss my attempt to develop a transgenic system in C. elegans using the bacteriophage phiC31 integrase system. I show evidence that phiC31 integrase is active in C. elegans somatic tissue. I have successfully integrated a transgene into the C. elegans genome in single-copy using phiC31 dependent recombination-mediated cassette exchange. However, attempts to repeat phiC31-mediated integration has been unsuccessful. In Chapter 3, I use genetic analysis to test many genes that were reported to be associated with the gamma-secretase complex in a mammalian tissue culture system. The gamma-secretase complex is an important component in the Notch signaling pathway. Not only is the gamma-secretase complex essential in the Notch pathway, it is also implicated in the pathology of familial Alzeheimer's disease (FAD). As gamma-secretase complex components show a Notch loss-of-function phenotype in C. elegans, a reverse genetic approach, using genes encoding proteins that associate with Presenilin was used to identify putative new Notch modulators. Several genes were identified that suppress a glp-1(gf) allele and one gene that suppress a gfp-1(lf) allele. These genes are unlikely to be core components of the Notch signaling pathway.

Cell motility is an essential process that depends on a coherent, cross-linked cytoskeleton that physically coordinates the actions of numerous structural and signaling molecules. In culture, a common feature of cells is the coherent movement of the endoplasmic reticulum and membranous organelles toward the periphery during substrate adhesion and spreading. The actin cross-linking protein, filamin (Fln), has been implicated in the support of three-dimensional cortical actin networks capable of both maintaining cellular integrity and withstanding large forces. Although numerous studies have examined cells lacking one of the multiple Fln isoforms, compensatory mechanisms can mask novel phenotypes only observable by further Fln depletion. Indeed, shRNA-mediated knockdown of FlnA in FlnB-/- mouse embryonic fibroblasts (MEFs) causes a novel endoplasmic spreading deficiency as detected by endoplasmic reticulum markers. Microtubule (MT) extension rates are also decreased but not by peripheral actin flow, because this is also decreased in the Fln-depleted system. Additionally, Fln-depleted MEFs exhibit decreased adhesion stability that leads to increased ruffling of the cell edge, reduced adhesion size, transient traction forces, and decreased stress fibers. FlnA-/- MEFs, but not FlnB-/- MEFs, also show a moderate defect in endoplasm spreading, characterized by initial extension followed by abrupt retractions and stress fiber fracture. FlnA localizes to actin linkages surrounding the endoplasm, adhesions, and stress fibers. Thus I suggest that Flns have a major role in the maintenance of actin-based mechanical linkages that enable endoplasmic spreading and MT extension as well as sustained traction forces and mature focal adhesions. I also report that treatment with the calpain inhibitor N-[N-(N-Acetyl-L-leucyl)-L-leucyl]- L-norleucine (ALLN) restores endoplasmic spreading and focal adhesion (FA) maturation in the absence of Fln. Further, expression of calpain-uncleavable talin, but not full-length talin, also rescues endoplasmic spreading in Fln-depleted cells and indicates a crucial role for stable, mature FAs in endoplasmic spreading. Because FA maturation involves the vimentin intermediate filament (vIF) network, I also examined the role of vIFs in endoplasmic spreading. Wild-type cells expressing a dominant-negative vimentin variant incapable of vIF polymerization exhibit deficient endoplasmic spreading as well as defects in FA maturation. ALLN treatment restores FA maturation despite the lack of vIFs, but does not restore endoplasmic spreading. Consistent with a role for vIFs in endoplasmic spreading, adhesive structures do not contain vIFs when the endoplasm does not spread. Fln-depleted cells also exhibit a microtubule-dependent mistargeting of vIFs. Thus, I propose a model in which cellular force generation and interaction of vIFs with mature FAs are required for endoplasmic spreading. Additionally, I discuss future lines of investigation concerning the role of FlnA in the endoplasmic spreading mechanism as well as mechanosensitive functions of FlnA. Finally, I speculate on a potential application of endoplasmic spreading deficiencies as hallmarks of metastatic breast cancer.

It is well established that the p53 tumor suppressor plays a crucial role in controlling cell proliferation and apoptosis upon various types of stress. There is increasing evidence showing that p53 is also critically involved in various non-canonical pathways, including metabolism, autophagy, senescence and aging. Through a ChIP-on-chip screen, we identified a novel p53 metabolic target, pantothenate kinase-1 (PANK1). PanK1 catalyzes the rate-limiting step for CoA synthesis, and therefore, controls intracellular CoA content; Pank1 knockout mice exhibit defect in β-oxidation and gluconeogenesis in the liver after starvation due to insufficient CoA levels. We demonstrated that PANK1 gene is a direct transcriptional target of p53. Although DNA damage-induced p53 upregulates PanK1 expression, depletion of PanK1 expression does not affect p53-dependent growth arrest or apoptosis. Interestingly, upon glucose starvation, PanK1 expression is significantly reduced in HCT116 p53 (-/-) but not in HCT116 p53 (+/+) cells, suggesting that p53 is required to maintain PanK1 expression under metabolic stress conditions. Moreover, by using p53-mutant mice, we observed that PanK activity and CoA levels are lower in livers of p53-null mice than that of wild-type mice upon starvation. Similar to the case in Pank1 knockout mice, β-oxidation and gluconeogenesis are impaired in p53-null mice. Together, our findings show that p53 is critical in regulating energy homeostasis through transcriptional control of PANK1. Our study on PANK1 led us to the question of how p53 can differentially regulate a diverse array of downstream targets in a context-dependent manner. Studies have shown that p53 acetylation at K120 and K164 lysine residues contribute to p53-mediated apoptosis and growth arrest functions, which was further supported by the 3KR mouse model (K117/161/162R) that mirrors the K120/164R mutations in human p53. These studies also suggest that a potentially large number of p53 targets can still be regulated by p53 in the absence of K120/164 acetylation (K117/161/162R in mouse). To investigate whether additional modifications of p53 can further contribute to promoter-specific transactivation, we conducted a screen using mass spectrometry and identified a novel acetylation site at K101. Our data demonstrated that K101 in human p53, as well as the homologous K98 lysine residue in mouse p53, can be acetylated by acetyltransferase CBP. Acetylation at this novel site does not contribute to p53 stability or DNA-binding capabilities. Ablation of K98 acetylation in mouse p53 alone does not affect the transcriptional activity of p53. However, simultaneous loss of K98 acetylation with the previously characterized K117/161/162 acetylations (4KR98 p53) significantly abrogates p53-mediated activation of TIGAR and MDM2 genes. The 3KR mouse model, although cannot elicit canonical p53-mediated apoptotic and cell cycle arrest responses, still retains the ability to suppress tumor formation. We, therefore, investigated whether other non-canonical targets of p53 could potentially mediate tumor suppression. By RNA-seq profiling of gene expression in cells expressing 3KR p53, we identified TNFRSF14 (tumor necrosis factor receptor superfamily, member 14) as a novel p53 target. The TNFRSF14 receptor has been shown to be frequently mutated in follicular lymphoma and diffuse large B cell lymphoma, and stimulation by its ligand LIGHT leads to cell death in many cancer cells. We report that TNFRSF14 is a novel p53 target that can be activated by 3KR p53. Interestingly, transactivation of TNFRSF14 is defective by 4KR98 p53. Furthermore, LIGHT ligand stimulates cell death in TNFRSF14-expressing cells and cells expressing 3KR p53, but not those expressing 4KR98 p53. Altogether, our findings in these studies underscore the extensive scope of p53 functions and provide new insights into the versatility of non-canonical pathways. Not only does p53 mediate tumor suppression through both canonical and non-canonical downstream effectors, p53 can also contribute to cellular homeostasis and energy balance.

The development of insulin resistance (IR) in the liver is one of the key pathophysiologic events in the development of type 2 diabetes mellitus, but most patients do not become uniformly resistant to the hepatic actions of insulin. Although insulin loses its ability to blunt glucose production, it largely retains its capacity to drive lipogenesis. This "selective IR" results in the characteristic hyperglycemia and dyslipidemia of type 2 diabetes. In this thesis, we take two approaches to better understand the mechanisms underlying selective IR. First, the compensatory chronic hyperinsulinemia (CHI) of insulin resistance downregulates levels of the insulin receptor (InsR). We have therefore modeled CHI in primary hepatocytes to demonstrate that the reduction in InsR number results in insufficient signaling capacity to halt glucose production while still leaving enough residual signaling capacity to promote lipogenesis. That is, the two processes are inherently differentially sensitive to insulin. Second, we hypothesize that FoxO1, a key insulin-inhibited transcription factor, coordinately regulates both hepatic glucose and lipid homeostasis. We have developed a transgenic mouse model heterozygous for a knocked-in allele of DNA binding-deficient FoxO1 and have proceeded to dissect the mechanisms by which FoxO1 differentially regulates glucose and lipid handling. We found that while the former requires FoxO1 to bind to its consensus sequences in target-gene promoters, the latter proceeds via a co-regulatory action of FoxO1. Taken together, these findings reveal novel connections between the glucose and lipid "arms" of the insulin-signaling pathway and how they may go awry in the run-up to diabetes.

We investigated the role of Notch signaling in the vascular microenvironment, with particular attention paid to the vascular consequences of Notch signaling disruption in myeloid cells. We adapted an established in vitro model of angiogenesis to recreate interactions between endothelial sprouts and vascular support cells, including macrophages and vascular pericytes. We found that inflammatory polarization of macrophages increased their ability to foster angiogenesis, and that intact Notch signaling was essential to this phenomenon. We also demonstrated a role for Notch/Jagged1 signaling in the interaction between vascular pericytes and endothelial sprouts, the disruption of which limits the growth and maturation of vessel networks. We have also investigated the role of myeloid Notch signaling in vivo, using a number of developmental and pathological models of angiogenesis. We found that Notch inhibition leads to decreased myeloid cell recruitment to a broad variety of functionally distinct angiogenic sites. Importantly, we observed that myeloid Notch disruption has vascular consequences in both physiological and pathological angiogenesis. Myeloid Notch- inhibited mice exhibit decreased vascular complexity in the deep retinal plexus during development. Additionally, these mice show significantly increased vascular tuft formation in the setting of oxygen-induced retinopathy, suggestive of a heretofore- undescribed role for myeloid Notch signaling in the pathogenesis of this significant human disease. This body of work increases our understanding of the role of Notch signaling both in the dynamics of myeloid cells and in their specific contribution to angiogenesis in multiple disparate contexts. It also contributes to our understanding of a number of key models of human disease, and may prove useful in the development of novel therapies to treat those diseases. Further, we are confident that our new experimental methodology will allow continued fruitful reductive study of the complex intercellular interactions within the vascular microenvironment.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological tumor resulting from the malignant transformation of immature T-cell progenitors. Originally associated with a dismal prognosis, the outcome of T-ALL patients has improved remarkably over the last two decades as a result of the introduction of intensified chemotherapy protocols. However, these treatments are associated with significant acute and long-term toxicities, and the treatment of patients presenting with primary resistant disease or those relapsing after a transient response remains challenging. Oncogenic activation of NOTCH1 signaling plays a central role in the pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL), with mutations on this signaling pathway affecting more than 60% of patients at diagnosis. However the transcriptional regulatory circuitries driving T-cell transformation downstream of NOTCH1 remain incompletely understood. Here we identify HES1, a transcriptional repressor controlled by NOTCH1 as a critical mediator of NOTCH1 induced leukemogenesis strictly required for tumor cell survival. Mechanistically, we demonstrate that HES1 inhibits leukemia cell death by repressing BBC3, the gene encoding the PUMA BH3-only proapototic factor. Finally, we identify perhexiline, a small molecule inhibitor of mitochondrial carnitine palmitoyltransferase-1, as a HES1-signature antagonist drug with robust antileukemic activity against NOTCH1 induced leukemias in vitro and in vivo.