Gpr182-mediated calcium mobilization is required for definitive hematopoiesis

Gpr182 介导的钙动员是最终造血所必需的

• 批准号: 10687007
• 负责人: McLean H Williamson
• 金额: $ 4.37万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL GRADUATE SCHOOL OF BIOMEDICAL SCIENCES, LLC
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10687007
• 关键词: Adopted; Aorta; Blood Cells; Blood Platelets; Bone Marrow; Bone Marrow Transplantation; Calcineurin; Calcium; Calmodulin; Calmodulin Pathway; Categories; Cell Lineage; Cell physiology; Cells; Circulation; Clinical; Collection; Cues; Data; Defect; Development; Dorsal; Embryo; Embryonic Development; Endothelial Cells; Endothelium; Erinaceidae; Erythrocytes; Event; Family; Family member; Fertilization; G-Protein-Coupled Receptors; Genes; Genetic Transcription; Genetic study; Goals; Hematologic Neoplasms; Hematopoiesis; Hematopoietic; Hematopoietic Cell Production; Hematopoietic Stem Cell Mobilization; Hematopoietic Stem Cell Specification; Hematopoietic stem cells; Hepatocyte; Hour; Impairment; In Vitro; Individual; Knock-out; Ligands; Link; Lymphoid Cell; Mediating; Modeling; Myeloid Cells; Oligonucleotides; Orphan; Pattern; Platelet-Derived Growth Factor Receptor; Pluripotent Stem Cells; Population; Proteins; Protocols documentation; Regenerative Medicine; Research; Response Elements; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Site; Specific qualifier value; Spleen; Stem Cell Development; T cell response; Testing; Time; Tissues; Transcriptional Regulation; Vascular Endothelial Cell; Vascular Endothelial Growth Factors; Zebrafish; blastomere structure; embryo cell; hematopoietic stem cell differentiation; hemogenic endothelium; improved; in vivo; induced pluripotent stem cell; insight; interest; knock-down; knockout gene; loss of function; mutant; nuclear factors of activated T-cells; overexpression; pharmacologic; receptor; release of sequestered calcium ion into cytoplasm; self renewing cell; sensor; stemness; transcription factor; transdifferentiation; vertebrate embryos

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.

摘要 造血干细胞（HSC）是自我更新的细胞，其在发育过程中产生所有主要的血细胞谱系。 个人的一生。在HSC的胚胎特化过程中，轴向血管必须适当地形成; 胚胎主动脉的血管内皮细胞可大致分为生血内皮细胞（HE）， 和非生血内皮。HSC特异性地从HE转分化并进入循环。长期 已经理解，适当的组织图案化和转录调控是足够的 HSC的规格和生产。最近，人们已经清楚地看到，细胞周围的细胞群， HSC发育的位点--称为HSC微环境--提供了诱导性信号线索， HSC规范要求。在HSC特化中建立的信号分子，如Wingless（Wnt） 和Hedgehog（Hh），激活内皮细胞中G蛋白偶联受体（GPCR）驱动的信号通路。 我们的实验室已经证明，Gpr 182，一种没有目前已知配体的内皮GPCR， 在HSC特化期间细胞内Ca 2+动员。Gpr 182的瞬时损失导致 HSC的前体。这些数据表明，可能需要Gpr 182诱导的钙水平增加， HSC规格。我们的实验室还表明，药物抑制钙传感器和效应器， 蛋白质钙调蛋白和钙调磷酸酶破坏HSC特化。活化T细胞核因子（NFAT） 转录因子家族在钙调磷酸酶下游起作用。几个NFAT家族成员表达于 合适的时间和地点可能影响斑马鱼模型中的HSC规格。但不清楚 这些转录因子是否或如何特异化HSC。虽然体外数据显示 Gpr 182表达和NFAT反应元件活性之间，这两种分子之间没有联系 是在体内制造的。我假设Gpr 182介导的Ca 2 +-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Gpr 182的作用。 通过定义Gpr 182是HSC特化所需的， 评价gpr 182基因敲除对Ca 2+动员的影响， 候选共受体Ramp 2对内皮Ca 2+动员的作用，并确定Gpr 182是否在细胞内起作用。 在HSC前体中自主地。其次，我将确定NFAT转录因子是否是HSC所必需的。 通过用吗啉代寡核苷酸进行功能丧失遗传学研究， 稳定的突变敲除斑马鱼，并评估组成型活性NFAT拯救 由Gpr 182功能丧失引起的造血缺陷。这些研究将推动发展领域 通过建立HSC中Ca 2+动员和NFAT活性的先前未知作用来促进造血 规范.这些发现将更好地为从诱导多能干细胞重编程HSC的努力提供信息。 细胞（iPSC），这一直是再生医学的长期目标。