Caudal Dorsal Artery Generates Hematopoietic Stem And Progenitor Cells Via The Endothelial To Hematopoietic Transition In Zebrafish

Hematopoietic stem cells?HSCs?are common progenitor cells of various cell components in the blood system.They form the whole blood cells through proliferation and orderly differentiation,and maintain the homeostasis of hematopoietic stem cells and the whole blood cell population through continuous self-renewal.Abnormal formation,maintenance,proliferation and differentiation of hematopoietic stem cells can lead to a variety of diseases of the hematological malignancies and immune system,such as leukemia,anemia,rheumatoid and lupus.Therefore,the normal formation of hematopoietic stem cells plays a vital role in the health of the body.At the same time,the transplantation of hematopoietic stem cells is an important plan for the treatment of hematopoietic diseases.Effective acquisition of hematopoietic stem cells and sufficient amplification are the prerequisites for the realization of the transplantation treatment.The requirement for the realization of this goal is the in-depth understanding of the formation and regulatory mechanism of hematopoietic stem cells.Therefore,the related research on hematopoietic stem cells,especially the origin of hematopoietic stem cells,lineage differentiation and the molecular regulatory mechanism,could significantly advance hematopoietic stem cell transplantation.Through the study of all vertebrate organisms,researchers found that the first blood cells appear from the mouse blood islands in the extraembryonic yolk sac.These blood cells give rise to erythrocytes and macrophages.As embryonic development,the definitive wave hematopoiesis detected in aorta-gonad-mesonephros?AGM?.Hemogenic endothelium cells undergo endothelial hematopoietic transition?EHT?and produce hematopoietic stem cells.Recently,researchers have found that the cerebral blood vessels of mice can also generate hematopoietic stem cells in situ during the E10.5-11.5,and the generation of these hematopoietic stem cells is almost synchronous with the AGM source.In addition to the hematopoietic endothelial cells in the AGM region,embryonic cells in other parts of the embryo can reconstruct the hematopoietic system of adult mice,which makes researchers wonder whether there are other organs or tissues that generate hematopoietic stem cells at a specific time of embryonic development.Zebrafish plays an irreplaceable role in the understanding of the occurrence process and regulatory mechanism of HSC.Researchers used time-lapse imaging and observed HSCs formed by budding from the hemogenic endothelium on the ventral wall of dorsal aorta.Then these HSCs migrate to the caudal hematopoietic tissue?CHT?,thymus and kidney.The caudal hematopoietic tissue is functionally similar to the fetal liver and placenta and serves as a transitory microenvironment supporting the proliferation and differentiation.However,whether CHT can generate HSC is a completely new topic that has not been studied.To detect whether the caudal dorsal artery?CDA?can directly produce HSPCs,we performed time-lapse imaging and found that several kdrl-EGFP⁺cells along the ventral wall of the CDA presented a similar EHT process as that observed in the dorsal aorta?DA?.They eventually produced cells expressing typical HSPC markers,such as CD41-GFP,cmyb-GFP,and runx1-GFP.Careful observation indicated that some of the budding kdrl-EGFP⁺cells began to form obvious holes,which filled quickly,probably the consequence of cell folding during the EHT process.Occasionally,newly emerged kdrl-EGFP⁺cells divided rapidly.The behavior and hematopoietic nature of the transformed kdrl-EGFP⁺cells resembled those observed in the DA,suggesting that the CDA was an additional site of hematopoiesis that was not recognized previously.This conclusion was further validated by examining Tg?kdrl:Dendra2?,in which kdrl-Dendra2⁺cells in the CDA regions were labeled via green-to-red photoconversion.The photo-converted kdrl-Dendra2⁺cells subsequently appeared in various hematopoietic organs,including CHT,thymus and kidney.Time-lapse imaging were performed on CDA about 12 embryonic zebrafish,during which approximately 60hours post fertilization?hpf?occurred after the EHT process of DA.The most frequent EHT events in the CDA were detected from 60 hpf to 84 hpf,and the frequency of these events then decreased in a time-dependent manner(i.e.,5.60±0.68,3.40±0.81,1.50±0.40,0.80±0.58,and 0.0±0.0 budding events per four somites during the periods of 60-84,84-108,108-132,132-156,and 156-180 hpf,respectively.The EHT events in the CDA were less frequent than those in the DA(approximately 3.57±0.57events occurred per four somites in the CDA versus 5.86±0.26 events per four somites in the DA during 15 h observation.Therefore,CDA is spatially independent of DA and generates HSPCs through the EHT process.To further explore the HSPCs generated by CDA,we used Tg?CD41:GFP;coro1a:DsRed?and Tg?kdrl:memCherry;coro1a:Kaede?to track the behavior characteristics of newly generated HSPCs from DA and CDA.In the newly born cells,the coro1a-DsRed signals were co-stained with weak CD41-GFP signals,and the coro1a-Kaede signal was also expressed in the newly budding kdrl-memCherry⁺cells,indicating that we could use the coro1a-kaede to mark the newly generated HSPCs of DA and CDA.Thus,Tg?coro1a:Kaede?was generated,in which individual target cells were clearly labeled by green to red photoconversion.The image data indicated that approximately 30%-40%of the CDA derived coro1a-Kaede⁺cells quickly gave rise to two daughter cells,followed by a second round of division within the next 12 h.Subsequently,some of the cells moved toward the vein and entered the circulation.However,other cells remained in the same space and underwent a third round of division.Occasionally,the cells that moved away reappeared in the CHT niche and proliferated again.Overall,these CDA-derived cells divided frequently to generate progenies with circular morphology and reduced size,displaying spherical HSPC-like behaviors.More than half of the CDA derived coro1a-Kaede⁺cells gave rise to progenies with myeloid-like features.The coro1a-kaede⁺cells budding from DA and CDA were followed up for 60 hours,and showed similar proliferation ability.The progenies from the CDA preferred to stay in the CHT rather than move to the thymus,in contrast to those from the DA.Taken together,these findings demonstrated that the CDA-derived HSPCs were heterogeneous and largely retained in their original environment during the early stages of development.Since the HSPCs produced by DA and CDA are different in space-time and migration tendency,we explore the potential regulatory molecules.Runx1 is an essential transcription factor for the EHT process in DA,so how does Runx1 affect the EHT process in the CDA region?Using runx1^w84x/w84xTg?kdrl:EGFP?embryo,we found that CDA could not complete the EHT process,and some cells would tend to sprout,but eventually shrink back.These results indicated that Runx1 functions essentially in hematopoiesis of the CDA,as observed in the DA.In the process of hematopoiesis,the blood flow to the blood vessel shear stress promoted the production of hematopoietic stem cells.We used the non-blood flow tnnt2a^swu77 zebrafish mutant and found that the EHT process of CDA was inhibited.The above successive occurrence of EHT from the DA to the CDA provided evidence for hematopoietic productive ability along the whole artery.We therefore evaluated the adult hematopoietic features of the artery at different time points by treating Tg(kdrl:CreER^T2;actb2:loxP-DsRed-stop-loxP-EGFP)^s928 embryos with4-hydroxytamoxifen?4-OHT?.Five time points,20 hpf,60 hpf,72 hpf,120 hpf and 9days post fertilization?dpf?were selected for 4-OHT treatment.Lcp1⁺cells in the hematopoietic organs,including the kidney and thymus,were largely GFP positive?⁷6%in the kidney and⁸9%in the thymus?in the fish treated at 20 hpf,and the proportion of GFP⁺Lcp1⁺cells decreased in the fish that were treated with 4-OHT at later stages in a time dependent manner.Therefore,the kdrl⁺artery endothelium contributes to the adult hematopoietic lineage through EHT and their contributive ability reduces gradually.Overall,our present study demonstrated that the whole artery,including both DA and CDA,produced HSPCs via EHT.Our investigation provided an important extension to the previous studies on zebrafish hematopoiesis.