Gpr182-mediated calcium mobilization is required for definitive hematopoiesis

摘要 造血干细胞(HSCs)是一种自我更新的细胞，在 个人的一生。在HSCs的胚胎发育过程中，轴向血管系统必须被适当地构筑； 胚胎主动脉血管内皮细胞大致可分为血源性内皮细胞(HE)。 和非血源性内皮细胞。HSCs特异性地从HE转分化并进入循环。它已经有很长时间了 已经理解，适当的组织图案化和转录调控是足够的 HSCs的规格和生产。最近，有一点变得很明显，即周围的细胞群 HSC发育的场所-称为HSC微环境-提供感应信号提示，这些信号提示是 HSC规范所需。HSC规范中建立的信令分子，如无翼(WNT) 和Hedgehog(HH)，激活G蛋白偶联受体(GPCR)驱动的内皮细胞信号通路。 我们的实验室已经证明，GPR182，一种目前没有已知配体的内皮GPCR，调节 HSC规范时细胞内钙离子的动员。GPR182的瞬时损失导致减少 在HSC前体中。这些数据表明，GPR182诱导的钙水平升高可能需要 HSC规范。我们的实验室还表明，钙感受器和效应器的药物抑制 钙调素和钙调神经磷酸酶的蛋白质破坏了HSC的规格。活化T细胞核因子(NFAT) 转录因子家族作用于钙调神经磷酸酶下游。NFAT的几个家庭成员在 在斑马鱼模型中潜在影响HSC规格的正确时间和地点。然而，它是未知的 这些转录因子是否或如何指定HSC。虽然体外数据显示 在GPR182表达和NFAT反应元件活性之间，这两个分子之间没有联系 是在活体内制造的。我推测GPR182介导的钙-钙调神经磷酸酶-NFAT的激活 信令轴是HSC规范所必需的。为了验证这一假设，我将首先评估GPR182的作用 通过定义GPR182是HSC规范所必需的， 评估GPR182基因敲除对钙动员的影响，评估GPR182的作用 候选的共同受体Ramp2对内皮细胞钙离子的动员，并确定GPR182是否发挥细胞- 在HSC前体中自主表达。其次，我将确定HSC是否需要NFAT转录因子 通过对吗啉寡核苷酸进行功能丧失遗传研究来进行规范，生成 稳定的突变基因敲除斑马鱼，并评估成分活性NFAT拯救的潜力 GPR182功能丧失引起的造血缺陷。这些研究将推动发展的领域。 在HSC中建立一个未知的钙动员和NFAT活性的作用来促进造血 规格。这些发现将更好地为从诱导多能干细胞中重新编程HSCs的努力提供信息 细胞(IPSCs)，这一直是再生医学的长期目标。

Abstract Hematopoietic stem cells (HSCs) are self-renewing cells that produce all major blood cell lineages during an individual’s lifetime. During embryonic specification of HSCs, axial vasculature must be appropriately patterned; vascular endothelial cells of the embryonic aorta can be broadly categorized into hemogenic endothelium (HE) and non-hemogenic endothelium. HSCs transdifferentiate specifically from HE and enter circulation. It has long been understood that proper tissue patterning and transcriptional regulation are required for adequate specification and production of HSCs. More recently, it has become clear that cell populations surrounding the sites of HSC development—known as the HSC microenvironment—provide inductive signaling cues that are required for HSC specification. Signaling molecules established in HSC specification, such as Wingless (Wnt) and Hedgehog (Hh), activate G protein-coupled receptor (GPCR)-driven signaling pathways in endothelial cells. Our lab has demonstrated that Gpr182, an endothelial GPCR without a currently known ligand, regulates intracellular Ca2+ mobilization during the time of HSC specification. Transient loss of Gpr182 leads to a reduction in HSC precursors. These data suggest that a Gpr182-induced increase in calcium levels may be required for HSC specification. Our lab has also shown that pharmacological inhibition of the calcium sensor and effector proteins calmodulin and calcineurin disrupts HSC specification. The nuclear factor of activated T cells (NFAT) family of transcription factors act downstream of calcineurin. Several NFAT family members are expressed at the right time and place to potentially influence HSC specification in the zebrafish model. However, it is unknown whether or how these transcription factors HSC specification. While in vitro data have shown a connection between Gpr182 expression and NFAT response element activity, no connection between these two molecules has been made in vivo. I hypothesize that Gpr182-mediated activation of the Ca2+-calcineurin-NFAT signaling axis is required for HSC specification. To test this hypothesis, I will first evaluate the role of Gpr182 activity in Ca2+ mobilization and HSC specification by defining that Gpr182 is required for HSC specification, evaluating the effects of gpr182 gene knockout on Ca2+ mobilization, assessing the contribution of Gpr182 candidate co-receptor Ramp2 to endothelial Ca2+ mobilization, and determining if Gpr182 functions cell- autonomously in HSC precursors. Second, I will determine if NFAT transcription factors are required for HSC specification through performing loss-of-function genetic studies with morpholino oligonucleotides, generating stable mutant knockout zebrafish, and assessing the potential for constitutively active NFAT to rescue hematopoietic defects caused by Gpr182 loss-of-function. These studies will advance the field of developmental hematopoiesis by establishing a previously unknown role for Ca2+ mobilization and NFAT activity in HSC specification. These findings will better inform efforts of reprogramming HSCs from induced pluripotent stem cells (iPSCs), which has been a longstanding goal of regenerative medicine.