Research Project: The Role of TGFβ Ligans in Adrenocortical Tissue Maintenance and Differentiation. Our laboratory is interested in understanding the adrenal-specific role of several ligands of the TGFβ family such as activin, inhibin and TGFβ2. The means by which these ligands function in concert to mediate adrenocortical development and maintenance is uncertain. I have chosen to initially approach this question with mathematical modeling to help bridge the complex and diverse functions of these ligands in adrenal organogenesis. The ensuing basic molecular and developmental science to validate the mathematical model formulations will be carried out subsequently. The mathematical modeling of this interdisciplinary research proposal will guide my experimental methodology, and in turn, my experimental data will validate the mathematical equations. Ultimately, this approach will facilitate my final goal of ascertaining how these various TGF family ligands contribute to adrenocortical development.

Research Project: Understanding the Role of Wnt5a in Epithelial Polarity and Organization During Intestinal Organogenesis. My research project is the study of both intestinal organ development as well as abnormal intestinal growth. The Wnt5a null mouse has obvious defects in processes of villus emergence and intestinal length; further study of this model will likely reveal critical new information not only about intestinal organogenesis, but potentially also leading to further understanding of congenital short bowel syndrome.

Research Project: Regulation of Liver Regeneration Through Interaction of the Growth Hormone and TGF-β Signaling Pathways. Liver regeneration following injury is a unique phenomenon in the body and an understanding of the mechanisms of this process has the potential to address conceptual problems of differentiated somatic cell proliferation and clinical problems related to poor liver regeneration following resection. The balance of signals from both inhibitors and activators of cellular proliferation during liver regeneration is critical for this process. Two such opposing factors implicated in liver regeneration are TGF-beta (a negative regulator) and growth hormone (GH) (a positive regulator). We have recently obtained evidence of an interaction between the GH and TGF-beta pathways that may provide valuable insight into how GH facilitates liver regeneration. I will examine the interaction between JAK2, the main effector of GH signaling, and embryonic liver fodrin (ELF), a critical adaptor protein in TGF-beta signaling, and determine the role of this interaction on hepatocyte proliferation. JAK2 is a kinase that is activated GH and serves to transduce the GH signal inside the cell by phosphorylating the GH receptor as well as additional signaling proteins. ELF is a spectrin that is responsible for the proper localization and signaling of two important intracellular mediators of TGF-beta signaling, Smad3 and Smad4, by facilitating their interaction with the TGF-beta type II receptor and subsequent translocation to the nucleus. It is known that phosphorylation of spectrins can alter their localization and function, and it has previously been shown that GH can suppress TGH-beta signaling, and However, a concrete mechanism for this suppression has not been established and an interaction between the GH and TGF-beta signaling pathways has not been investigated in the context of liver regeneration. I have found that JAK2 binds and subsequently phosphorylates ELF, providing a link between the two signaling pathways. We hypothesize that JAK2 phosphorylates ELF in response to GH in the regenerating liver, thereby altering TGF-beta signaling and facilitating cellular proliferation.

Research Project: The Roles of Wnt6 and Tcf7l2 in Adipocytes. The long-term goal of my research project is to understand how Wnt signaling inhibits adipogenesis. The Wnt/β-catenin pathway stabilizes cytosolic β-catenin, which then accumulates in the nucleus and forms dimers with TCF/LEF transcription factors to activate Wnt target genes. Wnt/β-catenin signaling is important in adipocyte development, as Wnt1, Wnt3a, and Wnt10b inhibit adipogenesis, and dominant-negative TCF4 promotes adipogenesis by blocking Wnt signaling. Wnt6 and Tcf7l2 (TCF4) are highly expressed in murine adipose tissue and Tcf7l2 variants are strongly associated with type 2 diabetes and β-cell function in humans, so I have directed my efforts towards examining their role in adipocyte biology. I have discovered Wnt6 is expressed in preadipocytes, is suppressed during adipocyte differentiation, and is a potent inhibitor of adipogenesis in vitro. Tcf7l2 is expressed in both cultured preadipocytes and adipocytes, suggesting it can function outside of differentiation. I have shown Wnt6 and Tcf7l2 mRNA levels are inversely correlated with body weight and adipose depot mass in mice. Therefore, I hypothesize that Wnt6 and Tcf7l2 are endogenous inhibitors of adipogenesis and Tcf7l2 modulates insulin sensitivity in adipocytes.

Research Project: Regulation of Spinal Interneuron Development by the Olig2-related Transcription Facto Bhlhb5. This project proposes to study how neural stem and progenitor cells give rise to motor neurons (MNs) and interneurons (INs) in the spinal cord. Errors in the process of assigning specific cell-type identity to the progenitor cells that generate neuronal and glial cell types in the CNS during development result in devastating congenital abnormalities. Loss of survival or function of these cell types underlies neurological conditions such as spinal ataxias, spastic disorders, spinal muscular atrophies, ALS, and Kennedy’s disease as well as spinal cord injuries. While a great deal of attention has been focused on describing the formation and function of MNs, recent studies have shown that several classes of excitatory and inhibitory spinal INs that regulate MN activity are critical for normal motor functions. The mechanisms that lead to IN generation in most regions of the CNS, however, are not well understood. In my proposed research, I will examine the function of a newly described transcription factor, Bhlhb5, in the generation of spinal INs. An understanding of how factors such as Bhlhb5 control the differentiation of specific IN cell types should yield insights into developmental mechanisms that lead to proper CNS function as well as causes of congenital abnormalities. In addition, this line of research may help to identify therapeutic targets to ameliorate these defects, and provide a foundation for current and future studies seeking to use stem cell-based therapies to treat neurological injuries and diseases. The experimental approaches proposed span the disciplines of genetics, cellular and molecular biology, biochemistry, and developmental biology to study mechanisms and cellular interactions that permit embryonic stem cells to generate the distinct cell types and functional circuits in the spinal cord necessary for coordinated movement.

Research Project: Structure, Function, and Evolution of a Notch- and EGFR-regulated Eye Enhancer. My project focuses on transcriptional regulation of the Pax2 sparkling enhancer during Drosophila eye morphogenesis. We are taking a highly interdisciplinary approach, incorporating genetics, biochemistry, bioinformatics, and evolutionary biology into our experimental design. I will be identifying novel regulators binding within sparkling, which will provide more information about the regulation of Pax2 expression and cone cell specification during Drosophila eye development. In addition, I will be investigating the role of enhancer structure in gene expression, including how the spatial organization of sparkling affects the pattern of gene expression. This portion of my project will add to our understanding of the structure and function of enhancer elements in vivo.

Research Project: Anterior-Posterior Patterning of the Nephrogenic Mesenchyme by Hox Genes. Hox genes are conserved in all metazoans and are important for anterior-posterior (AP) patterning of the vertebrate body plan, but their mechanistic roles during development are not well defined. The Hox10 and Hox11 paralogous genes are necessary for adult kidney formation, regulating patterning events in the metanephric mesenchyme correctly to promote ureteric bud induction from the Wolffian duct and later branching morphogenesis. Hox11 triple mutants have no ureteric bud induction. We have demonstrated that Hox11 proteins interact with Pax2 and Eya1 to regulate expression of Six2 and Gdnf, the signal necessary to initiate ureteric bud invasion into the mesenchyme. Hox10 triple mutants have misrouted ureters and disrupted branching, but their molecular phenotypes and target genes are not known. Hox11 5-allele mutants also have severe mesenchymal patterning defects. These preliminary data lead to the hypothesis that Hox10 and Hox11 genes regulate the AP patterning of the intermediate mesoderm (IM) to form competent nephrogenic mesenchyme. We will determine downstream targets of the Hox10 genes by examining the molecular phenotype in Hox10 mutant IM and using cell culture reporter analyses to test which genes are directly regulated by the Hox10 genes. We will also compare IM patterning disruptions in Hox10 and Hox11 allelic mutants, using whole mount organ culture to visualize tubulogenesis and molecular markers to show the position of the nephrogenic mesenchyme along the AP body axis, and establish the role by which these genes position the mesenchyme along the AP axis. By defining the molecular disruptions during early branching in the Hox10 mutants, along with analyses of Hox10 and Hox11 tubulogenesis defects, we will develop an understanding of how Hox genes regulate AP patterning of the intermediate mesoderm.

Research Project: Identification of the Role of Hedgehog Signaling in Intestinal Villus Integrity and Smooth Muscle Development. In the developing mammalian gut, Sonic and Indian Hedgehog (Hh) expression in the endoderm-derived epithelium is crucial for the proper patterning of both the epithelial and mesenchymal layers of the stomach, small intestine, and colon. Lack of either Shh or Ihh results in marked developmental abnormalities involving both the epithelial and mesenchymal compartments of the intestine. Our group has taken several coordinated approaches to further elucidate the effects of Hh signaling on the development of the mammalian small intestine. We have developed transgenic models of Hh overexpression and inhibition that have profound adult phenotypes relating to smooth muscle development and maintenance of villus integrity, demonstrating that Hh signaling drives differentiation and expansion of smooth muscle populations in the small intestine. In addition, we have performed microarray analysis on Shh and Ihh-treated E18.5 intestinal mesenchyme to identify specific mesenchymal targets of Hh signaling during intestinal development. Two of the putative Hh targets, Myocardin and IGF-1, have been implicated in the differentiation of smooth muscle. Currently, we are working to completely characterize the development of the adult phenotypes of our transgenic animals and to solidify our understanding of the pathway by which Hh signaling activates Myocardin and IGF-1 to direct smooth muscle development.

Research Project: The Role of Megakaryocytes in the Regulation of Hematopoietic Stem Cell Function. I hypothesize that megakaryocytes are a critical component of the hematopoietic stem cell (HSC) niche that regulates HSC maintenance through cell-cell interactions and secreted factors. This interaction is essential for proper HSC localization and homeostasis. In collaboration with Dr. McCauley’s laboratory in the Dental School, we will test this hypothesis and whether effects of megakaryocytes in HSC maintenance are independent of effects on bone formation. This work could fundamentally change models of the HSC niche by implicating megakaryocytes in HSC regulation for the first time. By focusing on the interaction of cells in different lineages and tissues (hematopoietic and bone), this work could provide new insights into the complex interplay of progenitors from different tissues.

Research Project: The Roles of NFAT:Fos-Jun and NFAT:FOXP Transcription Regulatory Complexes in Pathological Hypertrophic Growth of Cardiomyocytes. Cardiac hypertrophy is an adaptive response of the heart to many forms of cardiac disease induced by pathological stimuli or congenital defects. It is characterized by the growth of individual cardiomyocytes and re-activation of a fetal program of gene expression. Sustained maladaptive hypertrophy leads to heart failure. The goal of the proposed study is to dissecting the molecular basis of maladaptive hypertrophy for effective prevention of pathological hypertrophy without provoking hemodynamic compromise. My hypothesis in this proposed study is that NFAT3:Fos-Jun and NFAT3:FOXP complexes play pivotal roles in maintaining cardiac function and that imbalance of these complexes plays a causal role in pathological cardiac hypertrophy. This hypothesis is based on the function of these transcription factors in cardiac hypertrophy and heart development. The specific aims are: Specific aim1: Investigate cardiac specific NFAT3:Fos-Jun and NFAT3:FOXP protein complexes in cardiomyocytes by BiFC (bimolecular fluorescence complementation) and co-immunoprecipitation analysis. Examine changes in NFAT complexes upon induction of hypertrophy with cardiac agonists and inhibitors that blunt hypertrophy using multicolour BiFC. Specific aim 2: Determine the roles of NFAT3:Fos-Jun and NFAT3:FOXP (1/4) complexes in the regulation of cardiac genes that are significantly altered in hypertrophic and failing heart. Specific aim 3: Determine the roles of NFAT:Fos-Jun and NFAT:FOXP complexes in cardiac hypertrophy by using primary cardiomyocytes and cardiac-specific transgenic mice expressing NFAT3 mutants that cannot interact with Fos-Jun or FOXP.

Research Project: Distraction Enterogenesis: Molecular Mechanisms of Intestinal Growth Control. Short-bowel syndrome (SBS) is a condition where the small intestinal length is less than that required for proper nutrient absorption needed for survival. The condition can occur in pediatric and adult populations, and may be due to congenital processes, or acquired through the loss of large amounts of small intestine due to inflammatory conditions or ischemic events. A novel treatment method under investigation is lengthening of the bowel through distraction forces. Preliminary porcine data has shown promise in that the bowel subjected to distraction forces increased in length, and had a proportionately increased absorptive capacity as well. However, the mechanisms which promote distraction enterogenesis are unknown. Preliminary data showed a significant up-regulation of several factors including Indian hedgehog, desmin and vimentin in these lengthened segments. The increased desmin and vimentin potentially supports a Wnt-mediated signalling pathway, as these factors are exclusively expressed in the mucosa of developing intestine during increased Wnt expression. The marked change in expression of these factors may help drive the formation of the crypt-villus axis. My projects will attempt to quantify the effects of distraction forces by examining certain morphometric parameters including crypt depth, villus height, and mucosal thickness. Functional capacity will also be examined using an Ussing chamber. We will also be examining the optimal ways to apply distraction forces to the bowel in cooperation with the Department of Engineering. After quantifying the effects, expression of growth-related factors will be tested after disrupting iHH expression. Other projects include identifying changes in cytokine expression and extent of regulatory T-cells in mice receiving TPN.

Research Project: An Investigation on the Role of Transcription Factors Ascl1a and Zic 2b, and RNA Binding Protein Lin 28 in Retinal Regeneration. My project aims to understand the molecular mechanisms underlying successful regeneration of an injured retina. These mechanisms may, in part, recapitulate developmental mechanisms of retinogenesis; however because retinal repair is taking place in an adult environment, novel mechanisms specific to adult regeneration are likely to be revealed. We anticipate that this work will impact the ability to maintain and repair a damaged or diseased retina and may also benefit in understanding mechanisms of neural repair in the other CNS areas. Zebrafish are used as a model system for this study because of their robust regenerative powers and the ease of applying molecular genetic techniques to this organism. Our long-term goal is to apply to mammals what we learn from studying regeneration in zebrafish.

Research Project: The Role of Insulin Signaling in the Regulation of Germline Stem Cells. A steady supply of adult stem cells is critical to maintain differentiated cells at the appropriate level of homeostasis. Stem cells can divide asymmetrically so that one daughter cell remains undifferentiated to maintain the stem cell population while the other begins to differentiate. If the stem cell population is depleted, differentiated cells cannot be replaced, while overproliferation of stem cells can cause tumors to develop. An increase in tumor formation and cancer is associated with aging. This research project which studies the effect of insulin signaling on germline stem cells, is at the interface of stem cell biology, aging and cancer. There is a relationship between insulin signaling and aging/longevity that is inversely correlated with reproductive fitness. Conditions which are favorable for increased longevity, for example nutrient deficiency, often result in decreased reproduction. Conversely, increased numbers of progeny are correlated with decreased lifespan. The Drosophila male germline stem cell niche is an ideal model system to use to study stem cell interaction because the niche and germline stem cells are easily identified and the cell cycle pathway has been well-characterized. We are able to follow the stem cell niche both in fixed testes and varying ages as well as live cell imaging. Conclusions reached through studying this pathway may then be used as a basis to study stem cell-niche interactions in other model systems.