Studies On The Role Of Gene HDCG1 In Hematopoiesis Using A Zebrafish Model

The process of hematopoiesis and angiogenesis is a very complicated process,and its development process is regulated by a series of factors.Mutations or abnormal expression of these regulatory factors are intrinsic molecular causes of blood and vascular diseases.The molecular regulation mechanism of blood cell and vascular differentiation and development remains to be further studied.Therefore,the purpose of this paper is to study the role of candidate gene HDCG1 in hematopoiesis and angiogenesis.Our lab has previously found a high expression level of HDCG1 gene in blood suggesting that HDCG1 may play a role in blood development,but its role in blood cell differentiation and development is unknown.To study the possible involvement of the HDCG1 gene in the process of hematopoiesis and angiogenesis,we established the HDCG1 knockout model of zebrafish by adopting CRISPR/Cas9 gene editing technique,and the knockout model is effective which was proved by Western blot.Next,using the whole-mount in-situ hybridization,OD staining,Sudan black staining,RT-QPCR and other experiments we proved that in HDCG1 knockout homozygous:1)The expression level of erythrocyte marker gatal and hemoglobin gene hbbel were significantly up-regulated during primitive hematopoiesis and definitive hematopoiesis.2)During primitive hematopoiesis,the expression level of myeloid cells markers pu.1 and mpo were almost unchanged,but the expression level of L-plastin,another myeloid cells marker,was significantly down-regulated.The expression level of hematopoietic stem cells markers cmyb and scl were down-regulated,while the level of runxl,another hematopoietic stem cells marker,was almost unchanged.3)During definitive hematopoiesis,the expression of myeloid cells marker mpo and L-plastin were slightly down-regulated,and the expression of lymphoid cells marker rag1 was significantly up-regulated,while the expression of hematopoietic stem cells marker cmyb,scl and runx1 were down-regulated.4)O-Dianisidine staining showed an increase in hemoglobin and Sudan black staining showed a decrease in the number of myeloid granulocytes.5)The expression of kdrl,flila,markers of vascular endothelial cells,were up-regulated and the expression of myod1,a somite marker,was also up-regulated.6)The expression of cdh1 7 and shha,anterior marker and chordal cord marker respectively,were almost unchanged,which suggested that HDCG1 may not be related to the development of anterior kidney and chordal cord.All these results manifest that HDCG1 may regulate the process of primitive hematopoiesis,definitive hematopoiesis and vascular development in zebrafish.To eliminate the possible errors produced by in-situ hybridization assay,a series of blood and vascular fluorescence labeling strains were applied.HDCG1 knockout homozygotes was crossed with TG(gatal:dsred),TG(pu.l:EGFP),TG(mpo:EGFP),TG(corola.EGFP),TG(runx1:EGFP),TG(scl:EGFP),TG(cmyb:EGFP),TG(CD41:EGFP),TG(flila:EGFP)and TG(kdrl.mcherry)respectively,and then the fluorescent signals were evaluated in transgenic strains.The results further support the correctness of the results obtained from in-situ hybridization.To verify these consequences are indeed caused by the loss of HDCG1 gene,HDCG1 mRNA was injected into the HDCG1 homozygous mutants embryonic when at one cell stage.Overexpression of HDCG1 indeed rescued the mutant phenotypes caused by knockout of HDCG1 gene.In conclusion,HDCG1 may be a key regulator in primitive hematopoiesis,definitive hematopoiesis and vascular development of zebrafish.What is the mechanism of HDCG1 regulating the primitive hematopoiesis,definitive hematopoiesis and angiogenesis of zebrafish?Previous studies have reported that wntl6 regulates the development of hematopoietic stem cells through regulating the Notch signaling pathway by interacting with dld/dlc.Is the HDCG1 gene also related to the Notch signaling pathway?Consequently,whole-mount in-situ hybridization was developed to detect the expression level of Notch signaling pathway molecules in HDCG1 knockout homozygous.The results showed that the expression levels of wntl6,did and dlc were up-regulated in HDCG1 homozygous mutants.All these results suggest that HDCG1 gene may regulate the differentiation and development of hematopoietic stem cells through Notch signaling pathway.To further study whether HDCG1 gene regulates the differentiation and development of hematopoietic stem cells through other signaling pathways too,the whole transcriptome of HDCG1 homozygous mutants was analyzed.The samples of embryos at 24hpf stage were collected and the transcriptome sequencing was carried out,HDCG1-/-embryos as experimental group and wild type embryos as control.By analyzing the results of transcriptome sequencing,we found that the expression of Notch/Wnt/Jak-Stat/Fgf/TGF? signal pathway,hematopoiesis,angiogenesis and hemoglobin related factors were affected to varying degrees after the HDCG1 gene knockout.To verify the results of transcriptome sequencing,RT-QPCR assay was conducted,and consistent results were obtained,which suggests that HDCG1 does affect multiple signaling pathways and the expression of genes associated with blood vessel development.To identify direct target genes regulated by HDCG1,wild type embryos were chose to do ChIP assays.The final purified chromatin was sent to the company for ChIP-Seq analysis of the whole genome.From the results we analyzed,we found that HDCG1 might bind to the promoter of 75 genes,14 of which were related to hematopoiesis and angiogenesis,and they are egfl6,igsf8,slc25a38b,isml,kmt2a,st6galnac2,phkgla,smfn,chstl,stat5b,cenpk,aggf1,uvrag,and fam210b.To confirm the results of ChIP-Seq,then assays such as ChIP,ChIP-PCR and ChIP-QPCR were done,and the results are in agreement with that of ChIP-Seq.Combined results of transcriptome sequencing with ChIP-Seq,five possible candidate target genes with significantly altered expression were screened:egf16,igsf8,stat5b,aggf1 and ism1.We analyzed the expression of the five possible candidate target genes by RT-PCR and RT-QPCR.The results showed that the expression levels of aggfl and igsf8 gene in HDCG1 homozygous mutants were significantly up-regulated while the expression levels of ism1,stat5b and egfl6 were down-regulated,which were consistent with the results of transcriptional sequencing.Aggfl is shown to be the earliest known regulator for differentiation of hemangioblasts,which plays an important role in the process of mesodermal differentiation into hemangioblasts.Aggfl might be a direct target gene of HDCG1,besides,HDCG1 may be a factor regulating the differentiation and development of hematopoietic stem cells at an earlier stage than aggf1.Moreover,a large number of studies have shown that stat5b is a master regulator of hematopoiesis,and it is related to the occurrence of many malignant blood diseases.Fortunately,our ChIP results showed it may also be a direct target gene of HDCG1 gene.Therefore,we will focus on the molecular mechanism of HDCG1 regulating the process of hematopoiesis and angiogenesis through aggfl and stat5b.The elucidation of its mechanism may be helpful to improve the theory of blood vascular development regulation mechanism and to provide a possible therapeutic target site for the treatment of the diseases caused by the development of hemangioblasts.