The Spliceosomal Component Sf3b1is Essential for Hematopoietic Stem Cell Formation through the Regulation of the Jak/Stat Signaling Pathway

Hematopoietic stem cells (HSCs) maintain multi-lineage hematopoiesis for the lifetime of an organism. During embryogenesis, HSCs first emerge from specialized hemogenic endothelial cells within the dorsal aorta through a process termed the endothelial-to-hematopoietic transition. While much is known about the role of transcription factors and transcriptional control in HSC specification, it is ill defined how post-transcriptional RNA processing influences this first HSC fate decision. Using zebrafish loss-of-function mutants for the pre-mRNA spliceosomal component sf3b1 (splicing factor 3b, subunit 1), we demonstrated that impairing splicing hindered HSC production, specifically at the endothelial-to-hematopoietic transition. Loss-of-function sf3b1^hi3394 mutants have diminished expression of thehemogenic endothelial and HSC markers runx1 and gata2b, while the pan-endothelial marker kdrl (kinase insert domain receptor like) and aorta-specific markers notch1b and notch3 are normally expressed at 24 hpf (hours post fertilization). These data indicate that although sf3b1 is ubiquitously expressed, hemogenic endothelium and the downstream HSCs are more sensitive to its loss than other endothelial cells. To uncover the mechanism underlying the defect in hemogenic endothelium and HSCs in sf3b1 mutants, we performed RNA-sequencing on purified kdrl:gfp- positiveendothelial cells from sf3b1 mutants and wild-type siblings at 24 hpf. We observed nearly 900 genes were mis-spliced and of those only 144 were differentially expressed. This group was significantly enriched for genes involved in mRNA processing and Jak (Janus kinase)/Stat (Signaling transducer and activator of transcription) signaling. In particular, target genes of the Jak/Stat pathway were significantly enriched in the downregulated gene list and many components of the signaling pathway were mis-spliced including il6st (interleukin 6 signal transducer), the gene encoding the IL6-family common receptor Gp130 (Glycoprotein 130).To determine if mis-splicing of il6st was sufficient to decrease HSC production, we injected antisense morpholinos into wild-type zebrafish to mimic the sf3b1 mutant - specific exon-skipping event in il6st and found significantly diminished HSC levels at 28 hpf. Stat3 is the major transcription factor downstream of Gp130, thus we ubiquitously overexpressed a constitutively-active form of Stat3 and found it could significantly suppress the HSC defects in sf3b1 mutants. Together, these data indicate that Sf3b1-mediated splicing regulation of the Jak/Stat pathway is critical for HSC emergence. Mutations in SF3B1 are prevalent in myelodysplastic syndrome (MDS), a disorder originating in HSC dysfunction. Altered STAT3 signaling is also common in MDS, thus our data suggest that SF3B1 disruptions in HSCs could be contributing to MDS via mis-regulation of JAK/STAT signaling.