Organization of the Nerve Terminal by Synaptic Cleft Components
(Hiroshi Nishimune, Ph.D., P.I.)
Preimplantation Embryonic Secreted/Released Proteins: Embryo Quality Predictors
(Lane Christenson, Ph.D., PI)
Genetic Models of Congenital Vascular Malformations
(Jay Vivian, Ph.D., P.I.)
Regulation of Gene Expression in the TH2 Cytokine Locus
(Patrick E. Fields, Ph.D., P.I.)
Germ Cell Development in the atrichosis Mutant Mouse
(T. Rajendra Kumar, Ph.D., P.I.)
Transcriptional Mechanisms of Endothelial Function and Differentiation
(Soumen Paul, Ph.D., P.I.)
Five Beginning Investigator Research Projects are currently funded for a maximum of three years.
Functional analysis of histone demethylase activity in hypoxic cancer cells
(Adam Krieg, Ph.D., Assistant Professor, Obstetrics and Gynechology, P.I.)
Uncontrolled cell growth within a tumor results in hypoxia as the metabolic needs of the cells exceed the ability of the tumor vasculature to provide oxygen and nutrients. In response to the hypoxic microenvironment, cells undergo a massive reprogramming of transcription to promote survival. The Hypoxia Inducible Transcription Factors (HIFs) are primary regulators of the hypoxic response and induce the expression of glycolytic genes, cell migration factors, and angiogenic factors. HIFs also induce expression of several transcriptional regulators, including several histone demethylases, providing a mechanism for the hypoxic cell to extend or fix the expression of pro-survival genes. One of these histone demethylases, JMJD2B, demethylates tri-methylated histone H3 lysine 9 (H3K9me3), a key marker of repressed chromatin structure. Hypoxic induction of JMJD2B may play an important role in activating gene expression to promote tumor growth. Consistent with this hypothesis, forced knock-down of JMJD2B expression reduces growth of tumor xenografts, and is overexpressed in ovarian cancers. In this proposal, the extent to which JMJD2B regulates tumor growth will be tested in vivo using tumor xenograft experiments and in vitro by assaying for cell proliferation, invasion, and angiogenesis as a result of JMJD2B expression (Specific Aim 1). Subsequent biochemical experiments will determine the mechanism of specific target gene regulation in hypoxia, identify new genes regulated by JMJD2B in hypoxia, and characterize the regulation of key pathways of genes important for the tumorigenic phenotype (Specific Aim 2). The experiments described in this proposal will establish the mechanisms utilized by JMJD2B to regulate tumorigenesis, while identifying new pathways to target for enhanced tumor therapies.
Targeting and regulation of O-GlcNAc transferase at M phase
(Chad Slawson, Ph.D., Assistant Professor, Biochemistry and Molecular Biology, P.I.)
O-GlcNAc is the covalent modification of proteins at serine/threonine amino acids by the sugar moiety N-acetylglucosamine. The O-GlcNAc modification is not elongated to more complex oligosaccharide structures and is found only on nuclear and cytoplasmic proteins. The modification is highly dynamic and responds to signals of the extracellular environment such hormones, stress, and nutrients. Previously, I demonstrated that O-GlcNAc is critical for the proper progression of the cell cycle in eukaryotic cells; gain of function of either O-GlcNAc transferase (OGT), the enzyme which adds the modification, or O-GlcNAcase (OGA), the enzyme which removes the modification, causes mitotic exit delays. Furthermore, OGT and OGA can be found in a signaling complex with mitotic kinases such as Aurora Kinase B. The goal of this proposal is to investigate the interactions of OGT with mitotic structures such as the spindle and with mitotic proteins. We hypothesize that OGT forms complexes with various proteins during M phase progression that then targets OGT the spindle and midbody. I plan to use multiple techniques such as using FRAP (Fluorescent Recovery after Photo-bleaching) on GFP-OGT to measure the kinetics of OGT at spindle/midbody, quantitative proteomics to identify OGT targeting proteins, and finally identifying the function of Aurora Kinase B in targeting OGT to mitotic substrates and structures. Our expected contribution is significant because we expect to find insight into the regulation of OGT by mitotic targeting proteins and how these interactions are uncoupled in diseases such as cancer.
Impact of early experience on vulvovaginal sensitivity in adult mouse
(Julie Christianson, Ph.D., Assistant Professor, Anatomy and Cell Biology, P.I.)
Early experience has been shown to have a profound impact on the prevalence of chronic pain, particularly within the viscerosensory system. Preterm neonates are exposed to numerous stressors, including repeated invasive procedures (often without adequate anesthesia) and prolonged periods of maternal separation. Models of neonatal stress in rats, generated by either maternal separation or unpredictable shock, produce visceral hyperalgesia, as do neonatal rats that receive noxious mechanical or chemical irritation of the colon or bladder during the second week of life. The focus of this proposal is to determine how neonatal stress or irritation affects nociceptive processing from the vulva and vagina of adult mice and whether ablation of a select population of sensory neurons can normalize vulvovaginal sensation. Vulvodynia is clinically defined as chronic discomfort or pain of the vulva, often occurring as burning, stinging or soreness, in the absence of specific pathology or neurological disorder. Despite an estimated 15% of women experiencing chronic or evoked vulvovaginal pain lasting at least 3 months, relatively few published studies have investigated changes in vulvovaginal innervation and no animal models exist in the current literature. The experiments in this application are outlined in two specific aims designed to 1) determine how neonatal stress or irritation impacts vulvovaginal sensitivity and the nociceptive phenotype of sensory neurons innervating the affected tissue and 2) employ growth factor-conjugated saporin to selectively silence those fibers responsible for pain sensations as a preclinical test for treating vulvodynia in humans. Successful completion of these studies will not only provide the first published model of vulvodynia, but is also the first step in developing a new class of compounds that alleviates chronic pelvic pain without affecting normal sensations. Considering the high degree of comorbidity between vulvodynia and other chronic pelvic pain syndromes, e.g. endometriosis, irritable bowel syndrome and interstitial cystitis, chemical ablation of this population of nociceptors could also alleviate symptoms of these syndromes, as well.
Molecular mechanism of THM1-mediated renal cystogenesis
(Pamela Tran, Ph.D., Assistant Professor, Anatomy and Cell Biology, P.I.)
Cystic kidney disease (CKD) is proposed to originate from an underlying ciliary defect, though molecular mechanisms remain unclear. We have identified a novel ciliary protein, THM1 (Tetratricopeptide Repeat Containing Hedgehog Modulator 1, also termed TTC21B or IFT139), which negatively regulates Hedgehog (Hh) signaling. Recently, THM1 has been shown to contribute the most pathogenic alleles among ciliary genes to patients with ciliopathies, including Meckel-Gruber Syndrome (MKS), Bardet-Biedl Syndrome (BBS), nephronophthisis (NPHP), Joubert's Syndrome (JS) and Jeune's Asphyxiating Thoracic Disorder (JATD). A major clinical feature of these ciliopathies is CKD, and indeed, genetic deletion of Thm1 during late embryogenesis results in CKD in the adult mouse. While Hh signaling has not been studied extensively in CKD, our analyses of THM1 have led us to investigate a possible role for enhanced Hh activity in renal cystogenesis. In support of this hypothesis, cyst formation in an embryonic culture model of CKD was prevented by genetic deletion of Gli2, the main transcriptional activator of Hh signaling, and by treatment with small molecule Hh inhibitors. The goal of this proposal is to determine whether enhanced Hh activity leads to renal cysts. In the first aim, the effect of paracrine Hh signaling in CKD will be elucidated by conducting a spatial and temporal quantitative assessment of Hh signaling in the Thm1 conditional knock-out mouse. In the second aim, this spatial analysis will be extended by examining the effects of ablating Thm1 in renal tubular epithelial cells versus in renal stromal cells. In the third aim, causality of increased Hh signaling in renal cystogenesis will be explored by downregulating the Hh pathway genetically and pharmacologically in Thm1 conditional knock-out mice. Examining gene expression in a spatial context may provide a tool with which to gain better understanding of the pathogenesis of CKD. Furthermore, these experiments will establish a causal role for Hh signaling in renal cystogenesis and may offer important implications for the design of preventive strategies against CKD.
Role of cytoskeletal protein SPECC1L in facial morphogenesis and facial clefting
(Irfan Saadi, Ph.D., Assistant Professor, Anatomy and Cell Biology, P.I.)
Orofacial clefts are one of the most common birth defects in the U.S., occurring in 1/750 live births. The lifetime cost for medical treatment, educational services and lost productivity averages more than $100,000 per affected person. While the majority of orofacial clefts result in cleft lip with or without cleft palate (CL/P), a small percentage results in oblique facial clefts (ObFC) that extend from the oral cavity to the eye. Although less common, insights into the cellular mechanism of ObFC - first definitively classified by Paul Tessier in 1976 - have remained elusive. We have identified two de novo occurrences of SPECC1L mutations in patients with ObFC. Our studies in zebrafish and fly provide significant insight into SPECC1L function, which thus far had remained unstudied with no scientific publications. Knockdown of a previously uncharacterized zebrafish SPECC1L homolog perturbs cranial neural crest (CNC) and results in a dramatic loss of facial structures, thus extending SPECC1L function in facial morphogenesis to other vertebrates. In addition, knockdown of the sole uncharacterized Drosophila ortholog phenocopies - to an extraordinary extent - known fly mutants in the integrin-signaling pathway that exhibit cell adhesion and migration defects. Furthermore, our cellular and molecular analyses show that SPECC1L is a novel cytoskeletal cross-linking protein that interacts with both the microtubule and actin cytoskeletons. Transient expression of SPECC1L-GFP stabilizes a subset of microtubules, while SPECC1L knockdown causes defective actin cytoskeleton reorganization and impairs cell adhesion and migration. Together with mouse Specc1l expression in the developing facial prominences, these results begin to explain how human ObFC can arise following SPECC1L deficiency. The aim of this proposal is to develop a mouse model to test the pathogenetic mechanism of SPECC1L deficiency in mammalian facial morphogenesis (Aim 1) and to precisely define the cellular (Aim 2) and molecular (Aim 3) role of SPECC1L in CNC cell migration and specification of facial structures.