RESUMO
Hematopoietic stem cells (HSCs) guarantee the continuous supply of all blood lineages during life. In response to stress, HSCs are capable of extensive proliferative expansion, whereas in steady state, HSCs largely remain in a quiescent state to prevent their exhaustion. DNA replication is a very complex process, where many factors need to exert their functions in a perfectly concerted manner. Mini-chromosome-maintenance protein 10 (Mcm10) is an important replication factor, required for proper assembly of the eukaryotic replication fork. In this report, we use zebrafish to study the role of mcm10 during embryonic development, and we show that mcm10 specifically regulates HSC emergence from the hemogenic endothelium. We demonstrate that mcm10-deficient embryos present an accumulation of DNA damages in nascent HSCs, inducing their apoptosis. This phenotype can be rescued by knocking down p53. Taken all together, our results show that mcm10 plays an important role in the emergence of definitive hematopoiesis.
Assuntos
Hemangioblastos , Proteínas de Manutenção de Minicromossomo , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Feminino , Apoptose/genética , Proteínas de Ciclo Celular , Células-Tronco HematopoéticasRESUMO
During early vertebrate development, hematopoietic stem and progenitor cells (HSPCs) are produced in hemogenic endothelium located in the dorsal aorta, before they migrate to a transient niche where they expand to the fetal liver and the caudal hematopoietic tissue, in mammals and zebrafish, respectively. In zebrafish, previous studies have shown that the extracellular matrix (ECM) around the aorta must be degraded to enable HSPCs to leave the aortic floor and reach blood circulation. However, the role of the ECM components in HSPC specification has never been addressed. In this study, hapln1b, a key component of the ECM, was specifically expressed in hematopoietic sites in the zebrafish embryo. Gain- and loss-of-function experiments all resulted in the absence of HSPCs in the early embryo, showing that hapln1b is necessary, at the correct level, to specify HSPCs in the hemogenic endothelium. Furthermore, the expression of hapln1b was necessary to maintain the integrity of the ECM through its link domain. By combining functional analyses and computer modeling, we showed that kitlgb interacts with the ECM to specify HSPCs. The findings show that the ECM is an integral component of the microenvironment and mediates the cytokine signaling that is necessary for HSPC specification.
Assuntos
Hematopoese , Peixe-Zebra , Animais , Matriz Extracelular , Células-Tronco Hematopoéticas , Proteínas de Peixe-Zebra/genéticaRESUMO
Understanding the molecular pathways controlling hematopoietic stem cell specification and expansion is a necessary milestone to perform regenerative medicine. Here, we used the zebrafish model to study the role of the ckit signaling pathway in this process. We show the importance of kitb/kitlgb signaling in the specification and expansion of hematopoietic stem cells (HSCs), in the hemogenic endothelium and caudal hematopoietic tissue (CHT), respectively. Moreover, we identified the zebrafish ortholog of Oncostatin M (osm) in the zebrafish genome. We show that the osm/osmr pathway acts upstream of kitb during specification of the hemogenic endothelium, while both pathways act synergistically to expand HSCs in the CHT. Moreover, we found that osm, in addition to its role in promoting HSC proliferation, inhibits HSC commitment to the lymphoid fate. Altogether, our data identified two cytokines, kitlgb and osm, secreted by the vascular niche, that control HSCs during early embryonic development.
Assuntos
Desenvolvimento Embrionário/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Oncostatina M/farmacologia , Fator de Células-Tronco/farmacologia , Peixe-Zebra , Animais , Biomarcadores , Expressão Gênica , Células-Tronco Hematopoéticas/efeitos dos fármacos , Imuno-Histoquímica , Linfócitos/citologia , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Modelos Biológicos , Subunidade beta de Receptor de Oncostatina M/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
In mammals, embryonic hematopoiesis occurs in successive waves, culminating with the emergence of hematopoietic stem cells (HSCs) in the aorta. HSCs first migrate to the fetal liver (FL), where they expand, before they seed the bone marrow niche, where they will sustain hematopoiesis throughout adulthood. In zebrafish, HSCs emerge from the dorsal aorta and colonize the caudal hematopoietic tissue (CHT). Recent studies showed that they interact with endothelial cells (ECs), where they expand, before they reach their ultimate niche, the kidney marrow. We identified tfec, a transcription factor from the mitf family, which is highly enriched in caudal endothelial cells (cECs) at the time of HSC colonization in the CHT. Gain-of-function assays indicate that tfec is capable of expanding HSC-derived hematopoiesis in a non-cell-autonomous fashion. Furthermore, tfec mutants (generated by CRISPR/Cas9) showed reduced hematopoiesis in the CHT, leading to anemia. Tfec mediates these changes by increasing the expression of several cytokines in cECs from the CHT niche. Among these, we found kitlgb, which could rescue the loss of HSCs observed in tfec mutants. We conclude that tfec plays an important role in the niche to expand hematopoietic progenitors through the modulation of several cytokines. The full comprehension of the mechanisms induced by tfec will represent an important milestone toward the expansion of HSCs for regenerative purposes.