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A Comparison of Morphant and Mutant Phenotypes: The Role of Snail2 in Hematopoietic Stem Cell Specification

  • Author(s): Bickers, Cara Elizabeth
  • Advisor(s): Traver, David J
  • et al.

Hematopoietic stem cells (HSCs) supply an organism’s entire blood repertoire for its lifespan. This capacity makes HSCs an ideal source of treatment for hematopoietic diseases including leukemia, lymphoma, and anemia; however, the most common source of these life-saving cells is from an immune-matched donor. Even with a rare positive match, the chance of rejection due to Graft-versus-Host disease too often leads to patient illness and death. A major goal of regenerative medicine is to avoid the need for donor tissue by producing HSCs via in vitro differentiation from a multipotent progenitor. Ultimately, this progenitor population will be a patient-derived induced pluripotent stem cell (iPSC); however, current protocols have not yet successfully differentiated a therapeutic grade HSC from an embryonic stem cell. In vitro derived HSCs still fail to engraft well with full mulitlineage potential. True HSCs are still only produced successfully during embryogenesis. At this time, a specialized population of aortic endothelial cells undergo an endothelial-to-hematopoietic transition (EHT), during which they bud from the ventral floor of the aorta and enter circulation. Before EHT can occur, the precursors of the HSC must be properly instructed by their environment to initiate and sustain the hematopoietic program. This process involves many different embryonic tissues each of which provide specific spatial and temporal signaling cues. Our lack of full understanding of this complex process in the developing embryo is likely the reason for failed in vitro differentiation techniques. Thus, we must look to embryogenesis in model organisms to better instruct our laboratory protocols. In this dissertation, we first review what is known about the dynamic environment of the HSC precursor including the different tissues and signals that are known to instruct HSC identity. We then proceed to our analysis of the role of the zinc-finger transcription factor Snail2 in zebrafish HSC specification. This gene was identified via a genomic study of stromal cell lines known to support hematopoietic stem and progenitor cells (HSPCs) in vitro. Further study in zebrafish identified that loss of snai2 transcript caused a defect in embryonic hematopoiesis. Our goal was to further elucidate the cause of this defect by a combination of both genetic knock-down and knock-out. Our results show that while genetic knock-down via morpholino suggests an essential role of snai2 in the somitic HSC niche, mutant alleles of this gene do not generate a detectable phenotype. This contradiction in our data suggests two things. Either our knock-down approach leads to off-target or toxic effects in the embryo with a downstream consequence of loss of HSC specification, or our mutants possess an innate quality that compensates for the loss of Snail2. Recent studies have in fact shown that certain mutations lead to genetic compensation by other similar factors, abrogating the phenotype of the acute loss-of-function provided by morpholino knock-down. At this point, our studies point more strongly towards the snai2 morpholino causing an off- target effect; however, further work is necessary to fully elucidate the true cause of this dichotomy.

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