Epithelial ovarian cancer (EOC) is divided into several histological subtypes characterized by Mullerian-like differentiation and the expression of genes not expressed in normal ovarian surface epithelium. Many of these genes are implicated in female reproductive tract development, including Hoxa7 and Pax8, and it has been proposed that these genes are involved in the differentiation and progression of EOC. The effects of Hoxa7 overexpression (cellular proliferation, morphology, expression of E-cadherin and vimentin) were investigated in cell lines derived from our transgenic mouse models of ovarian cancer. However, Hoxa7 overexpression did not significantly affect any of these parameters. Immunohistochemical analysis revealed that Pax8 often appears to be localized to regions of papillary differentiation in tumours from transgenic tgMISIIR-TAg mice. These results suggest that while Hoxa7 alone does not drive EOC differentiation in vitro, expression of Pax8 may contribute to the differentiated epithelial features of ovarian cancer.

In this research project we investigated a regulatory region upstream of the jing open reading frame containing bHLH-PAS consensus DNA binding sites in order to identify control elements conferring tracheal specificity. Three transgenic fly strains were generated that carried LacZ fusions with genomic sequences from the jing5' regulatory region. Two overlapping fragments (1.5 kb and 2.8 kb) from the jing 5'cis-regulatory region were fused to a lacZ reporter and in vivo expression patterns studied. The 1.5 kb fragment was chosen because it contains three CMEs DNA binding sites for Trh::Tgo and consensus binding sites for the Pou domain transcription factor Drifter and ETS domain transcription factor Pointed. Transgenic embryos carrying the jing1.5-lacZ reporter show expression in the CNS midline, lateral CNS, and in the fusion cells of the trachea. During the early stages of tracheal development (stage 11), jing1.5-lacZ is expressed in the cells adjacent to the tracheal pits. However, despite the widespread distribution of Trh in trachea, jing1.5-lacZ expression was restricted to the fusion cells as confirmed by double labeling of the embryos with anti-beta-gal and the fusion cell-specific antibody, anti-Dysfusion (anti-Dys) and was dependent on trh. trh ectopically activated jing1.5-lacZ in Drosophila embryos in combination with Drifter. Furthermore, the CME sites were required for tracheal jing1.5-lacZ activation. A larger 2.8 kb fragment, encompassing jing1.5 and with no additional CMEs, drove lacZ expression in a larger subset of tracheal cells. jing2.8-lacZ is expressed in all tracheal branches, as early as stage 11. These results suggest that activation of jing in the trachea requires regulators in addition to Trh::Tgo. jing2.8-lacZ reporter is also expressed in the CNS midline, tracheal fusion cells, segmental ectodermal stripes, and posterior spiracles consistent with endogenous jing expression. Previous studies showed that the Jing zinc finger protein is required for both EGFR- and FGFR-dependent tyrosine kinase signaling during tracheal development. Consistent with this, Jing protein is present in the nuclei of most tracheal cells throughout development. We studied its relationship with bHLH-PAS, POD and ETS-domain transcription factors which control FGFR expression during branching in the embryonic Drosophila trachea. The influence of tyrosine kinase signaling on jing1.5-lacZ expression was determined by ectopic expression and mutant analysis. EGFR-mediated signaling activates jing1.5-lacZ expression in surviving midline glia and was required for jing1.5-lacZ tracheal fusion cell expression in rhomboid7M (rho7M) mutant embryos. In addition, genes known to regulate tracheal FGFR expression were required for jing1.5-lacZ activation in the fusion cells. Loss of pointed function and deletion of putative Pnt DNA binding sites in the jing1.5-lacZ reporter was associated with significant reductions in jing1.5-lacZ expression in fusion cells. In combination with reduced jing mRNA in pointed and drifter mutants, these results reveal that jing is regulated by Pnt and Dfr. This is consistent with a reduction in jing1.5-lacZ expression in breathless and branchless mutants which are known to regulate pointed. Furthermore, we show that jing is required for maximal levels of btl expression. In situ hybridization on whole mount embryos revealed that btl transcript levels are significantly reduced in jing loss-of-function backgrounds. Reduction of jing mRNA by RNA interference, specifically in tracheal cells, was associated with reductions in btl mRNA. Finally, we show that expression of ling protein with truncated N-terminal transactivation domains specifically in tracheal cells was associated with reduced btl mRNA. Together, these results suggest that jing is an integral component of the positive feedback mechanism that maintains expression of itself and btl downstream of Trh/Tgo during primary branching. (Abstract shortened by UMI.)

The Drosophila heart is composed of cardioblasts (CBs) and pericardial cells. Together, these cells form a linear heart tube that resembles the early vertebrate heart. Many genes involved in heart development are conserved from fruit flies to mammals. For example, the Drosophila homeobox gene Tinman and its vertebrate homologue Nkx2.5 are both critical for heart development. The secreted protein Slit and its receptor Roundabout (Robo) contribute to heart tube formation by guiding cardioblast alignment and adhesion. Slit null mutant showed severe alignment and migration defects, which include delays, gaps in the heart tube, and failure of lumen formation. Robo loss of function mutant also had severe defects which include, twisted heart tube, missing parts of the heart tube, and aggregation of the CBs. The Slit/Robo complex appears to serve conserved roles across species and in different systems by influencing cell guidance and migration. Previous studies have shown that sarcolemmal membrane associated protein (SLMAP) is expressed early during mammalian cardiac development and it may play role in cardiac function at the level of membrane biology and signal transduction. SLMAP and its homolog in Drosophila DSLMAP have similar structural features which include, a C-terminus transmembrane domain, a coiled-coil region, and a fork head associated domain at the N-terminus. DSLMAP and its mammalian homologue share 28.6% identity over 721 amino acid residues; a higher percentage identity was noted in the forkhead-associated domain (55.9%). Further more, DSLMAP were found to genetically interact with Slit/Robo pathway in the developing nervous system. Here, we show that DSLMAP is expressed in both the CBs and the pericardial cells at early stages of Drosophila heart development. Alterations in DSLMAP expression in the CBs, and the pericardial cells, using a UAS-DSLMAP-RNAi transgene causes a delay in migration of CBs and PCs, and misalignment of the cardiac cells. The up-regulation of DSLMAP in the CBs and the pericardial cells led to delay in migration of the cardiac cells, and to CBs aggregation. Down-regulation of DSLMAP has similar phenotypes to Robo down-regulation in both the CBs and the pericardial cells. In addition, over-expression of DSLMAP or Slit have the same phenotypes in the two cell types. Delayed migration defects are reduced by pericardial expression of UAS-DSLMAP RNAi in Robo down-regulated embryos. Myocardial expression of UAS-DSLMAP transgene suppresses the delayed migration defects seen in Slit over-expression. DSLMAP over-expression in the CBs significantly reduced the phenotypes seen in slit2 loss of function mutant. Over-expression of DSLMAP in the CBs of robo189 heterozygous background showed a significant reduction in the phenotypes observed in robo189 alone. DSLMAP down-regulation in the pericardial cells of slit2 heterozygous background, or robo189 heterozygous background led to a significant decrease in defective embryos and the heart tube was seem to be properly organised. The data suggests that DSLMAP is expressed early in cells that form the Drosophila heart, and its regulated levels are critical for normal heart tube formation. Data also suggests that DSLMAP may genetically interact with the Slit/Robo pathway to ensure proper heart tube formation.

The type-A scavenger receptor (SR-A) plays a major role in the uptake of modified lipoproteins leading to foam cell formation and fatty streak development in artery walls. Our lab previously showed that a peptide known as insulin degrading enzyme (IDE) is able to bind the SR-A cytoplasmic domain in vitro. The present study provides evidence that male low density lipoprotein receptor-null (LDLr-/-) mice on a high fat diet, generate significantly more atherosclerotic lesion in aortic root and the aortic intimal surface when reconstituted with IDE-null (IDE-/-) bone marrow derived cells compared to those reconstituted with wild type bone marrow. Total serum cholesterol and the LDLr cholesterol fraction was also significantly increased in male LDLr-/- mice reconstituted with IDE-/- bone marrow. Macrophages procured from IDE-/- mice showed no difference in uptake of modified LDL compared to wild type cells but may be able to accumulate more cholestertyl ester than wild type cells. Our findings reveal that IDE expression likely exerts an anti-atherogenic effect via cholesterol metabolism, although further in vitro experiments are required to elucidate an exact mechanism.

Hemogen is a murine protein expressed in active sites of hematopoiesis during embryogenesis and in the adult. Hemogen's function has not been clearly elucidated. Evidences suggest Hemogen's role in erythroid differentiation. Hemogen expression coincides with active sites of erythropoiesis. beta-globin expression is a hallmark of erythroid differentiation. Proteomic data suggests that Hemogen associates with MafK which is part of the NF-E2 complex that acts as an activator of beta-globin during erythroid differentiation. To gain insight into Hemogen's mechanistic function during erythroid differentiation, we attempted to decipher Hemogen's associating proteins during erythroid differentiation. We generated a Hemogen antibody functional in Hemogen immunoprecipitation (IP). We performed Hemogen IP and used mass spectrometry to identify putative Hemogen interacting proteins within the Hemogen IP elution. Our immuno-fluorescence microscopy (IFM) images of endogenous Hemogen revealed Hemogen as a nuclear protein within murine erythroleukemia cells before and after differentiation. In line with our IFM images, the majority of candidates identified within our putative list of Hemogen associating proteins are transcription regulators. Hemogen expression progressively increased with erythroid differentiation. Hence, Hemogen may function as a transcriptional regulator during erythroid differentiation.

Atrial Fibrillation (AF), the most common sustained cardiac arrhythmia, is a disease that affects the electrical conductance in the atria. Gap junction channels, composed of connexin (Cx) proteins, allow the electrical signal to propagate between cardiac myocytes. Mutations in two highly expressed cardiac connexin genes, GJA1 (Cx43) and GJA5 (Cx40), have been identified by our group in a cohort of patients with lone AF. To characterize trafficking of a novel somatic frameshift Cx43 mutant and three novel Cx40 mutants, fluorescently tagged connexins were visualized in HeLa cells using confocal microscopy. Electrophysiological studies were performed by a member of our lab to test the function of mutant gap junction channels. The Cx43 frameshift mutant (Cx43-932delC) caused intracellular retention of Cx43, resulting in impaired electrical coupling between paired cells. All three Cx40 mutants (F30L, G311S and R113S) demonstrated normal trafficking and electrical coupling between paired N2A cells. Enhanced hemichannel function was observed for the Cx40-F30L mutant, leading to cell death in Xenopus oocytes. Increased sensitivity to intracellular pH caused closure of gap junction channels composed of Cx40-G311S or Cx40-R113S. Intracellular localization of a previously published Cx40 mutation (P88S) was determined to be retained within the ER and Golgi compartments. Trafficking rescue was observed when P88S was co-expressed with wild-type Cx40 or Cx43. My results and those of our collaborators demonstrate a novel finding that a Cx43 mutation is associated with AF and also further support reported findings on the association of Cx40 mutations with AF.

Vascular progenitor cells (VPCs) facilitate angiogenesis and vascular repair in damaged tissues where inflammatory cytokines coordinate healing through VPC mobilization. VPCs also facilitate tumor angiogenesis and induce angiogenic switching in metastatic sites. VPC binding to extracellular matrix proteins enriched in injured healing tissues and metastatic sites and the participation of integrins in this binding was investigated. VPC binding profiles on fibronectin and laminin of "healthy" controls was different from those of "cancer" patients and patients with acute tissue damage. Specific integrin-mediated binding of cells in VPC clusters were matrix protein-dependent. Patients with oral cancer and lymphoma had elevated plasma vascular endothelial growth factor (VEGF) levels compared to healthy controls. VEGF facilitated VPC clustering on fibronectin and incubating cells from healthy controls with VEGF switched binding profiles from "healthy" to "cancer". This study provided new insights regarding VPC-matrix interactions that help distinguish VPC involvement in vascular repair from vascular-mediated metastasis.

In response to tissue trauma, quiescent satellite cells in adult skeletal muscle undergo proliferative activation and migrate to the site of injury where they differentiate and fuse to mend and replace damaged myofibers. The tightly synchronized expression of myogenic regulatory factors plays a critical role in orchestrating the regenerative process. In recent studies, we identified the transcription factor Pax7 as able to recruit the Wdr5-Ash2L-MLL2 histone methyltransferase complex to the -57.5kb Myf5 enhancer and to activate Myf5 transcription. We also identified Wdr68 as a putative Pax7 binding protein. Wdr68 is a scaffold protein thought to integrate Shh signaling but about which very little is known. Therefore, we set out to characterize a role for Wdr68 with Pax7 in early myogenesis. The interaction of endogenous Pax7 and exogenous Wdr68 was validated by reciprocal co-immunoprecipitation and western blot analysis. Manipulation of Wdr68 levels in primary myoblasts followed by real-time PCR analysis of Pax7 target genes suggests that Wdr68 acts as a negative regulator of Pax7. It has been determined by ChIP experiments that Wdr68 does not bind chromatin at some known genomic Pax7 binding sites. In addition, Wdr68 appears not to function by regulating Pax7 nuclear localization. These experiments provide new insights into the molecular control of Pax7 function during regenerative myogenesis and emphasize its complexity.

The functional redundancy and peri-implantation lethality inherent to members of the CDX family has traditionally limited their investigation. As such, the development by our lab of a conditional null mutant, obliterating CDX activity in the mouse, has opened up an exploration of their additional roles. Along with the loss of caudal tissue and failure of neural tube closure, Cdx1/2 null mutants exhibit abnormal somites, an observation which is echoed by the misregulation of many somitic players including members of the Wnt and Notch signaling pathways upon microarray analysis. The replication of altered expression patterns in situ and identification of consensus CDX response elements (CDRE) in the proximal promoter regions validated many of these genes as potential CDX targets. Characterization of one candidate in particular, DIl1, revealed reduced expression in the absence of CDX and overlapping expression domains with CDX members as well as somitic anomalies in Dil1 null mutants. The identification of two highly conserved putative CDRE in the proximal promoter region, in addition to the ability of CDX2 to occupy the promoter in the vicinity of these sites in vivo and specifically bind the identified motifs in vitro, was consistent with direct regulation. Functional analysis of CDX2 in association with the Dll1 promoter has so far proven inconclusive, but synergistic induction of the wildtype promoter in the presence of TBX6 supports a potential co-regulation. Overall, this thesis presents evidence for delta-like 1(Dll1) as a potential direct target of CDX2. Moreover, these findings support the possibility that CDX may regulate players in the Notch and Wnt signaling pathways and thereby play an important role in somitogenesis.

Pulmonary arterial hypertension (PAH) is a rare and fatal disease caused by excessive remodelling of small pulmonary arterioles. Heterozygous loss-of-function mutations in the bone morphogenetic protein receptor 2 (BMPR2) have recently been identified in a large portion of patients with familial and idiopathic PAH. However, how mutations in this ubiquitously expressed receptor result in such a specific abnormality of the lung microcirculation is unknown. We hypothesized that loss-of-function mutations in BMPR2 lead to PAH by increasing the susceptibility of ECs to apoptosis, particularly within fragile pulmonary arterioles. To examine the consequences of BMPR2 mutations on the development of PAH, we generated mice that ubiquitously over-express an arginine to stop mutation in the receptor's C-terminal domain (BMPR2 R899X) Characterization of these mice revealed a significant increase in right ventricular systolic pressure, an indicator of pulmonary pressure, which was associated with muscularization of small pulmonary arterioles, alveolar septal thickening and pulmonary macrophage infiltration. In addition, a modest increase in apoptosis was detected in these mice. These data suggest that BMPR2 loss-of-function mutations increase the susceptibility of ECs to apoptosis and set the stage for excessive inflammation, possibly leading to spontaneous PAH. This new model will help elucidate the pathophysiological events leading to the development of PAH and provide a unique tool to evaluate novel potential treatments for this disease.