cpsf1 is required for definitive HSC survival in zebrafish

Abstract

A comprehensive understanding of the genes and pathways regulating hematopoiesis is needed to identify genes causally related to bone marrow failure syndromes, myelodysplastic syndromes, and hematopoietic neoplasms. To identify novel genes involved in hematopoiesis, we performed an ethyl-nitrosourea mutagenesis screen in zebrafish (Danio rerio) to search for mutants with defective definitive hematopoiesis. We report the recovery and analysis of the grechetto mutant, which harbors an inactivating mutation in cleavage and polyadenylation specificity factor 1 (cpsf1), a gene ubiquitously expressed and required for 3 untranslated region processing of a subset of pre-mRNAs. IntroductionHematopoiesis is a complex developmental process that results in the production of mature blood cells from HSCs and depends on the highly regulated activation of specific genetic programs. 1 Analysis of critical genetic regulators of normal hematopoiesis has identified that constitutional or acquired inactivating mutations of such genes may contribute to bone marrow failure syndromes, myelodysplastic syndromes, and/or hematopoietic neoplasms. [2][3][4] Despite advances in whole-genome analysis, the underlying genetic events that contribute to many hematopoietic disorders remain unclear, and innovative gene-discovery approaches are needed to deepen our understanding of hematopoietic development in order to design more effective and specific therapeutic approaches to treating hematopoietic diseases.In recent years, the zebrafish (Danio rerio) has emerged as an excellent animal model system for studying the genetic pathways of vertebrate development. 5 The combined genetic and embryologic advantages of the zebrafish are ideal for studying organogenesis and, in particular, hematopoiesis. 6 Zebrafish and mammalian hematopoiesis are very comparable with respect to all cell lineages, and key genetic regulators have been conserved through evolution. 7 The sequential waves of zebrafish hematopoietic cell development correspond well with the distinct waves of blood cell generation observed during mammalian embryogenesis. 7 Embryonic hematopoiesis in the zebrafish is divided into 2 major waves that differ in the specific blood cell lineages that are formed. Primitive hematopoiesis is characterized by the generation of embryonic erythrocytes in the intermediate cell mass 8 and a distinct population of primitive macrophages that arise from the anterior lateral plate mesoderm between 14 and 16 hours postfertilization (hpf). 9 Studies in mutants lacking definitive hematopoiesis indicate that most primitive hematopoietic cells are gradually lost between 8 and 12 days postfertilization (dpf). 10 Definitive hematopoiesis occurs in 2 phases: initially, transient erythromyeloid progenitors form in the caudal hematopoietic tissue (CHT) starting around 24 hpf, 11 and by 30 hpf HSCs begin to form in the ventral wall of the dorsal aorta, a region corresponding to the mammalian aorta-gonad-mesonephros region. 12 These HSCs then migrate to the CHT, and from th...

25 a Zebrafish Model of Myelodysplastic Syndrome Produced Through Tet2 Genomic Editing

“…In the last few years, several mouse models with targeted disruption of Tet2 in the hematopoietic system have been reported, and they have been used to study the role of Tet2-inactivating mutations in normal hematopoietic cell development, the evolution of clonal dominance, and the development of hematopoietic malignancies. Tet2-deficient mice predominantly developed CMML, followed by hematopoietic malignancies, such as MDS and MPN (23)(24)(25)(26). Similar to the results reported in murine models (23)(24)(25)(26), the tet2 m/m zebrafish we studied did not exhibit defects in embryonic hematopoiesis, remained viable and fertile, and developed clonal myelodysplasia of the kidney marrow as they aged, culminating in true MDS with anemia.…”

“…Tet2-deficient mice predominantly developed CMML, followed by hematopoietic malignancies, such as MDS and MPN (23)(24)(25)(26). Similar to the results reported in murine models (23)(24)(25)(26), the tet2 m/m zebrafish we studied did not exhibit defects in embryonic hematopoiesis, remained viable and fertile, and developed clonal myelodysplasia of the kidney marrow as they aged, culminating in true MDS with anemia. At 24 months of age, tet2 zebrafish mutants develop increased percentages of progenitor cells and myelomonocytes and decreased percentages of erythrocyte lineage cells in the kidney marrow.…”

“…The zebrafish has been shown to provide a faithful model of vertebrate hematopoiesis in which both small-molecule and genetic screens have proven particularly advantageous. For example, small-molecule screens conducted in zebrafish embryos have yielded insights directly relevant to pathways regulating both human HSCs and more differentiated hematopoietic cells, leading to the discovery of drugs that augment engraftment, as shown in clinical trials of cord blood cell transplantation in patients (21)(22)(23)(24)(25)(26)(27)(28)(29)(30).…”

SUMO1-activating enzyme subunit 1 is essential for the survival of hematopoietic stem/progenitor cells in zebrafish

“…Thus, the VDA, CHT and kidney of the zebrafish are respectively the functional analogs of the AGM, liver and bone marrow in mice (Chen and Zon, 2009). Using forward genetic analyses, three previously uncharacterized factors [rumba (znf574), haus3 and cpsf1] have recently been found to play an essential role in regulating fetal hematopoiesis in zebrafish (Bolli et al, 2011;Du et al, 2011), confirming that the employment of an unbiased forward genetic approach in zebrafish can complement our current knowledge of mammals.…”

Methionine aminopeptidase 2 is required for HSC initiation and proliferation

“…The results also corroborated with the recent study that mutation of a similarly ubiquitous gene cpsf1 in zebrafish led to specific defects of HSC. 36 The mechanisms of action of metap2 have remained unclear. Although inhibition of metap2 in both zebrafish and human CB significantly reduced CamKII activity, a direct link between CamKII, the noncanonical Wnt pathway, and HSC initiation has yet to be determined.…”