A genetic screen in zebrafish defines a hierarchical network of pathways required for hematopoietic stem cell emergence

Abstract

Defining the genetic pathways essential for hematopoietic stem cell (HSC) development remains a fundamental goal impacting stem cell biology and regenerative medicine. To genetically dissect HSC emergence in the aorta-gonadmesonephros (AGM) region, we screened a collection of insertional zebrafish mutant lines for expression of the HSC marker, c-myb. Nine essential genes were identified, which were subsequently binned into categories representing their proximity to HSC induction. Using overexpression and loss-of-function studies in zebrafish, we ordered these signaling pathways with respect to each other and to the Vegf, Notch, and Runx programs. Overexpression of vegf and notch is sufficient to induce HSCs in the tbx16 mutant, despite a lack of axial vascular organization. Although embryos deficient for artery specification, such as the phospholipase C gamma-1 (plc␥1) mutant, fail to specify HSCs, overexpression of notch or IntroductionSpecification of definitive hematopoietic stem cells (HSCs) capable of generating the blood cell lineages is a vertebrate-specific process that occurs in the aorta-gonad-mesonephros (AGM) region of the developing embryo. 1 In the mouse, HSCs are located near the ventral endothelium of the dorsal aorta on embryonic day (E) 10, approximately the same time that HSC activity is present in the AGM region. [2][3][4] The proto-oncogene c-myb and the transcription factor runx1 are both excellent markers of these emerging HSCs and are essential for mammalian HSC development. 5 Runx1 is thought to operate very early in HSC specification as the mouse knockout lacks intraaortic hematopoietic clusters. 4 Based on analysis of a point mutant, c-Myb is thought to act in concert with p300 to regulate the proliferation and differentiation of HSCs. 6 Similarly, zebrafish have an AGM-like region in the ventral wall of the dorsal aorta also marked by c-myb and runx1 expression. 5,[7][8][9][10][11] Lineage tracing experiments have shown cells exiting this region and ultimately seeding the kidney and thymus, the sites of definitive hematopoiesis in the zebrafish. 12-14 Consistent with the mouse knockout data, morpholino knockdown of runx1 translation in zebrafish results in the loss of c-myb cells in the aortic region as well as a loss of definitive marker expression in the thymus. 8 Although many studies have focused on the molecular events important for HSC emergence from the AGM, very little is understood about the signals essential for specifying these cells. 15 Some progress has been made through the analysis of several zebrafish mutants. In zebrafish, definitive HSCs are located between and in the axial vasculature, 14 which is developmentally derived from intermediate mesoderm. Mutants with defects in vascular formation and patterning show deficiencies in HSC specification. For example, the cloche (clo) mutant lacks endothelium and thus a vasculature, has no AGM HSCs and does not undergo definitive hematopoiesis. 11 Loss of vasculature organization, such as that in the spadetail (spt) mutant ha...

Hematopoietic defects in rps29 mutant zebrafish depend upon p53 activation

“…Rps29 -/- embryos survive for five days[27], probably due to maternal rps29 protein deposited in the embryo. We previously showed that rps29 -/- embryos have decreased definitive HSC formation, likely the result of a defect in aorta specification[20]. Because ribosomal proteins are mutated in patients with erythroid specific deficiencies, we hypothesized that the zebrafish rps29 mutation might also cause a defect in primitive red blood cell development independent of the definitive HSC defect.…”

“…Our work builds upon the previously characterized HSC and artery defects of the rps29 mutant[20]. We went on to characterize red blood cells in the mutant, as ribosomal protein mutations cause erythroid specific defects in patients with DBA.…”

“…Rps29, a protein in the 40s small subunit of the ribosome important for rRNA processing and ribosome biogenesis[19], was found to be required for definitive hematopoiesis in the zebrafish embryo[20]. The rps29 mutant has decreased HSC markers runx and myb at 36 hours post fertilization (hpf) in the aorta gonad mesonephros (AGM) region.…”

Essential role of c-myb in definitive hematopoiesis is evolutionarily conserved

“…c-myb is part of a complex genetic network whose function is to specify and maintain hematopoietic progenitors and to regulate their differentiation (4). Among vertebrates, most genetic studies of c-myb function have been conducted in the mouse model, primarily because the experimental armamentarium is well-developed but also because no c-myb mutations have yet been described in other species.…”

Whole-exome sequencing and functional studies identify RPS29 as a novel gene mutated in multicase Diamond-Blackfan anemia families

“…The rps29 2/2 mutant zebra fish has key features of the DBA phenotype including significant defects in RBC development, 39 shown by a decrease in Hb levels. We used this as a model to determine whether the RPS29 mutation identified in our DBA cases could rescue the Hb defect.…”