The goal of this project is to offer novel insights into heme synthesis and hematopoietic pathways that can improve treatment of congenital and acquired anemia. Despite decades of efforts to understand genetic blood disorders, every year 400,000 newly born babies worldwide face dreadful complications in the absence of effective therapies. Thus, better understanding and knowledge of heme synthesis, iron metabolism, and hematopoietic stem/progenitor revcell (HSPC) biogenesis is required. The zebrafish, with its close synteny to the human genome and its conserved molecular pathways regulating the development of hematopoietic tissues, offers a powerful tool to understand human blood disorders. Based on our Poisson distribution analyses, we hypothesized that the genetic screens for anemic mutants are not at saturation to study hematologic diseases. We employed de novo ethyl nitrosourea (ENU) based genetic screens and identified five unique zebrafish anemic mutants, including sauvignon blanc (sav) and pinotage (pnt). pnt exhibits hypochromic anemia. pnt has a defect in hemoglobinisation while preserving red cell numbers. In contrast, sav shows anemia due to a lack of erythroid cells. sav lacks expression markers for HSPC (scl/tal1), myeloerythroid progenitor (gata-1), erythroid ((-globin) and myeloid (mpo) cells. Expression of vascular markers such as, fli-1 is preserved in sav. These data suggest that the pathophysiology of sav is confined to hematopoietic cell development and not to mesodermal cells. The genetic mapping, positional cloning, loss-of-function, mRNA expression levels, and cRNA over-expression studies have revealed a novel functional gene for the pnt locus. We have identified closely linked (~0 cM) genetic markers flanking the unique sav locus. In order to further characterize the importance of the disrupted genes in pnt and sav mutants and their involvement in genetic blood disorders, we propose: 1) To analyze the biochemical function of the pnt gene in heme synthesis. The pathophysiology of the pnt gene in hypochromic anemia will be ascertained by analyzing mechanisms regulating transport and incorporation of iron into heme or [Fe-S] cluster synthesis using mammalian and other model organisms. 2) To identify the sav gene and its function in hematopoiesis. The positional cloning, loss-of-function, cRNA over-expression studies and mutational analyses will be performed to identify the sav gene. We will perform assays to derive cell intrinsic or extrinsic role of sav in the regulation of hematopoiesis, hematopoietic stem/progenitor cell development, stem cell self-renewal, and cell differentiation. In the long term, the successful outcomes of these projects will lay strong foundations to understand human genetic blood diseases and to discover new opportunities for improved therapies. The potential of zebrafish genetics, expert guidance of mentors and scientific advisors, and the environment of Harvard Medical Community will certainly provide the candidate a unique opportunity to evolve as a successful independent investigator.
The success of this application will advance the field of human genetic blood disorders by identifying novel genes defective in zebrafish genetic mutants, pinotage (pnt) and sauvignon blanc (sav), and establishing their role in iron disorders and hematopoiesis. The expert guidance and environment available to Dr. Shah will help him to successfully complete his project and make him competitive to become an independent investigator. The appeal and the potential of future outcomes of this project satisfy the objectives of NIDDK sponsored Mentored Scientist Development Award (K01) mechanism.
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