Cleavage and Polyadenylation Specificity Factor 1 Is Required for Definitive Hematopoietic Stem Cell Survival In Zebrafish.

Hematopoiesis is a tightly regulated process requiring the differential activation of specific genetic programs. Inactivating mutations of genes involved in normal hematopoiesis have been implicated in bone marrow failure syndromes, myelodysplastic syndromes and hematopoietic neoplasms. Despite recent advances, many genes regulating normal hematopoiesis are still unknown and innovative gene-discovery approaches can benefit our understanding of the molecular mechanisms underlying this complex developmental process. The combined genetic and embryologic strengths of the zebrafish model system are ideal for studying factors regulating hematopoiesis, which is highly conserved with mammals. We have used ENU-mutated zebrafish lines to screen for genes whose inactivation results in the loss of definitive hematopoiesis. Here, we report the identification and analysis of the zebrafish mutant grechetto, caused by a lethal, recessive, inactivating mutation (grcl8a12) of the cleavage and specificity factor 1 (cpsf1) gene. Cpsf1 encodes a protein required for processing the 3’UTR of a subset of pre-mRNAs. Cpsf1 is maternally expressed ubiquitously through the first five days of development including primitive myeloid and erythroid cells and definitive hematopoietic stem cells (HSC). Although grechetto mutants fail to express cspf1 already at 24 hpf, they appear to develop normally until 72 hpf, when they first become morphologically distinct from wild-type siblings with a smaller head and the absence of a protruding jaw. Importantly, primitive hematopoiesis appears normal, as shown by expression of the myeloid and erythroid markers mpx and band3, respectively, at 24 and 48 hpf by whole mount RNA in situ hybridization (WISH). By 120 hpf the phenotype is more marked and embryos have a curved body, cardiac edema, development defects in the jaw and gut and a reduction in the number of iridophores. While many of these affected tissues represent derivatives of the neural crest (NC), the expression of markers of NC specification (sox10 and foxd3 at 15,5 hpf, crestin at 24 hpf), migration (dlx2 at 36 and 48 hpf) and differentiation (pax9a at 48 hpf) are normal. When definitive hematopoiesis was assayed for markers of mature blood cells by WISH at 120 hpf, grechetto mutants exhibit a loss of myeloid (mpx, lysC, l-plastin), erythroid (band3, gata1) and lymphoid cells (rag1, lck). Since myeloid and erythroid cells are normal at 24 hpf, but severely reduced at 120 hpf, we investigated whether the absence of HSC could explain the loss of definitive hematopoiesis in grechetto mutants. Interestingly, WISH staining for the HSC marker c-myb showed that in mutant embryos HSCs are normally specified and represented in the ventral wall of the dorsal aorta at 36 and 48 hpf. Nevertheless, at 72 hpf the numbers of HSCs are reduced in grechetto mutants upon their migration to the caudal hematopoietic tissue (CHT) and by 120 hpf very few remain. To investigate the fate of these HSCs we crossed the grechetto line with the Tg(c-myb:EGFP) reporter line, that expresses EGFP under the control of the c-myb promoter. By whole mount immunostaining, we were able to find increased co-localization of activated caspase3 and TUNEL staining in the c-myb:EGFP positive cells in the CHT in grechetto mutants compared to WT siblings at 72 and 96 hpf, suggesting that HSCs undergo apoptosis during this stage. Furthermore, PI cell cycle profile of sorted c-myb:EGFP+ cells from 96 hpf mutants shows a sub-G1 peak, representing pyknotic cell nuclei undergoing advanced stages of apoptosis. Importantly, both activated caspase3 staining and TUNEL assay did not show increased apoptosis in the tissue immediately surrounding the CHT. These studies show that cpsf1 is not required for primitive hematopoiesis or definitive HSC specification, however, it is essential for HSC survival in the CHT. The fact that grechetto embryos do not undergo generalized apoptotic cell death suggests that, despite ubiquitous cpsf1 expression and the general development defects of grechetto mutants, the prosurvival pathways in the HSC compartment are particularly dependent on Cpsf1 activity.