Cul4a Promotes Zebrafish Primitive Erythropoiesis Via Upregulating Scl And Gata1 Expression

The development of hematopoietic system is an important process in embryo development.A lot of diseases of blood system are closely related to the abnormal development of hematopoietic.Blood cells originate from hemangioblasts which come from mesoblast during embryonic development.Hemangioblasts give rise to hematopoietic progenitors.The primitive blood cells produced by hematopoietic progenitors take part in primitive hematopoiesis,while hematopoietic stem cells formed by hematopoietic progenitors regulate and control the definitive hematopoiesis to generate mature erythrocytes,myeloid cells and lymphocytes.The normal development process of hematopoiesis is under the adjustment and control of a complicated but sophisticated molecular signal regulation network.Any abnormity of the key molecular modulation could lead to severe developmental defects or serious diseases.It is very important to build up the molecular network in the development of hematopoiesis,thus providing new molecular targets to treat hematopoietic diseases.Part One cul4a regulates zebrafish primitive erythropoiesisZebrafish(Danio rerio)has become a powerful model for investigating hematopoiesis because zebrafish embryonic transparency greatly facilitates the visualization of early hematopoietic development.To assess whether cul4a is involved in the blood cell development in zebrafish,we firstly examined the expression pattern of cul4a during zebrafish embryonic development using WISH(whole-mount in situ hybridization).Using morpholino knockdown technology as well as CRISPR/Cas9(Clustered regularly interspaced short palindromic repeats-associated system 9)technology,we found that cul4a ablation specifically led to abnormal erythropoiesis development of zebrafish instead of myeloid lineages and definitive hematopoiesis.We made the following findings:(1)The expression pattern of cul4a during zebrafish embryonic development via WISH at 1-cell,6-somite,12 hpf(12 hours post fertilization),24 hpf and 48 hpf stages with the cul4a antisense probe indicated that cul4a transcripts were distributed evenly in all blastomeres.It was found to express elusively in two primitive hematopoietic sites,the PLPM(posterior lateral plate mesoderm)at 12 hpf and the ICM(intermediate cell mass)at 24 hpf This spatiotemporal distribution pattern of cul4a expression probably corresponds to the function in primitive hematopoiesis.(2)Our previous study showed that zebrafish cul4 gene is highly conserved in evolution,and embryos injected with cul4a-MO exhibited defects of heart looping,whereas there was no exact biological function of zebrafish cul4b.To examine whether cul4a affect the development of zebrafish's hematopoiesis,we knockdown cul4a in zebrafish embryos.Decreased number of blood cells was observed under brightfield as well as by o-dianisidine staining.As shown in WISH and qRT-PCR(quantitative real-time PCR)assays,the expression of hbbe3,the marker of globin was remarkably downregulated in cul4a morphants.Coinjection of cul4a-MO with cul4a mRNA could efficiently resuced the decreased erythrocytes in cul4a morphants.Then,we detected gatal,the marker of erythroid precursors.Consistent with the scarcity of erythrocytes in cul4a morphants,expression level of gatal was dramatically downregulated by ablation of cul4a.Coinjection of in vitro synthesized cul4a nRNA rescued the decreased expression of gatal in cu14a morphants.qRT-PCR assays and Tg(gatal:EGFP)transgenic embryos further confirmed the reduction of gatal in cul4a morphants and the rescue by cul4a mRNA.To determine whether decreased number of erythrocytes was mediated by downregulation of gatal,we performed rescue experiments using in vitro synthesized gata1 mRNA and found that coiniection of in vitro synthesized gatal mRNA could efficiently block the reduction in the number of erythrocytes caused by cul4a depletion.Moreover,the expression levels of pu.1 and mpo,markers for myeloid progeniors,and runxl and c-myb,markers for definitive hematopoiesis,were not appeared to be affected in cul4a morphants.To further confirm the above observation,we generated cul4a-/-,cul4b-/-,or double knockout mutants using CRISPR/Cas9 technology.Depletion of cul4a,but not cul4b,resulted in a significant reduction in the number of erythrocytes as demonstrated by o-dianisidine staining or hbbe3 expression.Consistently,neither cul4a nor cul4b deletion affect primitive myeloid progenitors or definitive stem cells.In conclusion,our results in this part indicate that Cul4a affects the development of primary erythropoiesis by regulating gatal,but not primitive myeloid progenitors or definitive stem cells.In addition,consistent with previous study,zebrafish cul4b is not involved in the regulation of embryonic development.Part Two Zebrafish Cul4a regulates the primitive erythropoiesis by regulating hemangioblastsThe above results indicate that knockdown of cul4a results in the reduction in the number of red blood cells.To understand the mechanisms responsible for this phenotype,we performed the following analyses:(1)To investigate the effect of cul4a on red blood cell proliferation,we stained zebrafish embryos with antibody against phosphohistone H3,which marks condensed chromosomes in metaphase and early anaphase cells.There was no significant difference in the number of phosphohistone H3-positive cells in the ICM between CoMO and cul4a-MO injected embryos,which indicated that decreased red blood cells led by cul4a depletion was not mediated by cell proliferation.(2)Previous reports demonstrated that off-target effects of morpholino may induce p53 activation and cause apoptosis.We thus detected the level of p53 by Western Blot and qRT-PCR.No significant difference in both protein and mRNA levels were detected between CoMO and cul4a-MO injected embryos.Then,we used TUNEL(terminal transferase-mediated deoxyuridine triphosphate nick-end labeling)assay to examine DNA fragmentations in apoptotic cells.The number of apoptotic cells in the ICM of embryos injected with cul4a-MO was colparable with that in CoMO-injected embryos.Thus,decreased red blood cells led by cul4a knockdown was not due to increased apoptosis.(3)To verify the requirement of cul4a for specification of primitive hematopoietic precursors,we examined the expression of genes that establish the regulatory hierarchy of hemangioblasts and initiation of primitive hematopoiesis.The expression of the earliest hematopoietic marker scl,which are capable of converting mesoderm into hemangioblasts/hematopoietic precursors in zebrafish embryos,were downregulated in cul4a morphants at the PLPM of 6-somite and 24 hpf stages.Importantly,coinjection of cul4a mRNA significantly blocked the reduction in the expression level of scl,both confirmed by WISH and qRT-PCR.It has been shown that zebrafish has two scl isoforms,scl-? and sel-?,which have different effect on the initiation of primitive hematopoiesis.scl-?,but not scl-?,is required for specification of definitive HSC.The fact that lack of cul4a impaired primitive erythropoiesis,but not definitive hematopoiesis,suggesting that Cul4a might regulate the expression of scl-?,but not that of scl-?.To confirm this notion,we used specific primers to distinguish two isoforms.As expected,knockdown of cul4a in zebrafish resulted in a downregulation of scl-?,but not scl-?.we performed rescue experiments with in vitro synthesized scl nRNA.We found that coinjection of in vitro synthesized scl-?mRNA rather than scl-? rescued the decreased primitive erythropoiesis in cul4a morphants.Consistently,the reduced expression of lineage-specific genes gatal and hbbe3 was rescued by coinjection with scl-? mRNA as indicated by WISH and qRT-PCR.We further confirmed that scl and lmo2 expression levels were uniquely decreased in cul4a-/-mutants ald double knockout mutants,but not cul4b-/-mutants.Interestingly,coinjection of scl-? mRNA also blocked the reduction in the expression of lmo2,suggesting that the reduction of lmo2 is caused by the downregulation of scl-?.Collectively,these results imply that Scl/Lmo2 act downstream of Cul4a for primitive hematopoiesis.Cul4a takes part in the differentiation of primary hematopoietic progenitors through the specific regulation of scl-?,and then participates in the development of primitive erythropoiesis.Part Three The molecular mechanism by which cul4a complex regulates scl and gatal expresssionCul4A was reported to activate tbx5 expression in zebrafish development by promoting H3K4 methylation previously.To determine whether Cul4a directly regulate scl,we performed E1-ChIP(whole embryos ehromatin immunoprecipitation)analysis using six pairs of primers covering a region approximately ranging from-2000 bp to+200 bp of scl-? transcription start site.It revealed that the Cul4a occupancy peaked at the region around-1643 bp to-1143 bp of the scl promoter,and H3K4me3,a histone marker for transcription activation,was also enriched in the same region.We next examined the effect of cul4a knockdown on the enrichment of these proteins on the scl-a promoter and found that accompanying a marked reduction in the enrichment of cul4a bound to the scl promoter,the level of H3K4me3 at the scl promoter was also markedly decreased when cul4a was knocked-down.These results showed that Cul4a can bind to scl-a promoter and thereafter promotes H3K4 trimethylation en route to activate scl-a transcription.The results in part one indicated that cul4a promotes primitive erythropoiesis by regulating gatal expression.To test this hypothesis,we used E-ChlP assay to examine whether gatal gene is bound by Cul4a using seven pairs of primers specific for a region located approximately-2000 bp to +200 bp of gatal transcription start site.The E-ChIP assay showed that Cul4a could directly bind to the region at-1059 bp to-538 bp of gatal promoter.H3K4me3 was also bound to the same region.Moreover,the quantitative ChIP assay showed that knockdown of cul4a significantly decreased the binding of cul4a on the gatal promoter.Consistently,the level of H3K4me3 at the gatal promoter was also markedly decreased when cul4a was depleted.In summary,we demonstrated that Cul4a can activate scl-a and gatal transcription by promoting H3K4 trimethylation,and thus promote primitive erythropoiesis.