源于斑马鱼尾部主动脉的造血干细胞的谱系分化及调控机制研究

Hematopoietic stem cells (HSCs) are the common ancestors of all blood lineages. Their origin, generation process and underlying molecular mechanisms are heat topics that have been extensively studied for decades. It is now well accepted that HSCs are primarily originated from the hemogenic endothelium of the aorta-gonad-mesonephros (AGM) during lineage-restricted developmental stages. However, whether the AGM is the sole site of HSC production remains under debate. By taking advantage of live imaging and lineage tracing in zebrafish, we discover that the caudal artery (CA) gives rise to a novel wave of HSCs occurring immediately after the dorsal aorta (DA)/AGM wave via endothelial-to-hematopoietic transition (EHT). The CA is located in the caudal hematopoietic tissue (CHT)-the functional ortholog of the fetal liver. Thus, the future study will focus on: 1) Defining the time-window of the CA wave and comparing the EHT process, frequency, and HSC numbers from DA versus CA. 2) Taking advantage of Tg(coro1a:kaeda) that could label single HSC to analyze the CA and DA derived HSCs’ early characteristics, including migration, proliferation, differentiation and so on. 3) Based on the spatial and temporal difference, permanently marking DA and CA-derived HSC in the 4-OHT induced Tg(kdrl:CreERT2; actb2:loxP-Dsred-stop-loxP-GFP) at different time points and heat short induced Tg(hsp70:cre; actb2:loxp-stop-loxp-eGFP;kdrl:memCherry) in different regions, tracing the lineage contribution capability of both waves of HSCs. 4) Distinguishing and sorting out the CA versus DA derived HSCs to perform the transcriptome analysis, digging out the important factors and molecular mechanisms in the HSCs' generation from CA versus DA. 5) Exploring the effect of blood flow in the regulation of DA versus CA-derived HSCs.

AGM/DA是公认的HSC的最初来源，但是否还存在着其他原位产生HSC的组织/器官仍有争议。本研究发现斑马鱼CHT的CA能原位产生HSC，因此CHT存在着一波以往没有被报道的独立于DA的新的HSC发生过程。基于此，本研究将重点关注：1) CA产生HSC的时间窗，并比较CA和DA产生HSC的过程、频率和数目。2)、基于能单细胞标记HSC的Tg(coro1a:kaeda)，深入分析CA与DA来源的HSC初始增殖、迁移及分化潜能的异同。3)、基于Tg(kdrl:CreERT2; actb2:loxP-Dsred-stop-loxP-GFP)等cre-loxp品系，利用DA与CA产生HSC的时间点和位置差别追踪CA与DA来源的HSC谱系分化的异同。4)、分选CA和DA来源的HSC并进行转录组测序，深入研究CA和DA产生HSC的调控机制。5)探讨血流等物理因素调控CA和DA产生HSC的异同。

尾部造血组织(CHT)是斑马鱼一个重要的临时造血器官。在基于活体实时成像研究CHT髓系细胞发育和功能的过程中，申请人课题组发现CHT的kdrl+主动脉内皮细胞在较晚期仍可以通过血管内皮-血液细胞转分化(EHT)产生一波新的血液细胞。进一步细致分析表明这群血液细胞产生的时间窗在已报道的源于背部主动脉(DA)的血液发生之后，且造血能力较弱。但CHT产生血液细胞的能力受血流影响较小。随后通过cre-loxp系统介导的谱系追踪发现这群较晚产生的血液细胞更倾向于分化为淋系细胞。整个研究发现了一群以往没有被关注的新的血液细胞来源，拓展了学界对斑马鱼永久造血时空发育过程的认知，研究成果发表在《Journal of Genetics and Genomics》。在课题执行中，揭示ikzf1-irf4-ccr9信号轴调控淋系细胞早期发育的分子机理。通过CRISPR/Cas9技术创建了ikzf1的突变体斑马鱼品系。初步研究结果表明在ikzf1突变体中，淋系细胞的发育出现明显缺陷。进一步的研究表明，ikzf1突变体中淋系细胞发育缺陷是由于造血干祖细胞向胸腺的迁移和进一步分化障碍所导致。在此过程中，irf4和ccr9是ikzf1发挥作用的重要下游因子。irf4a和ccr9a的突变体斑马鱼表现出类似的淋系细胞向胸腺迁移和发育缺陷的表型。在ikzf1突变体中回补irf4a和ccr9a可以非常有效地拯救其淋系发育缺陷的表型。整个研究在体回答了早期淋系细胞发育的生物学过程和ikzf1调控的分子机理，对于淋系细胞发育缺陷的免疫性疾病的发生原因提供重要的参考信息。成果发表在《Journal of Biological Chemistry》。