Defining the functional role of protein N-glycosylation on hematopoietic stem cell production from endothelium

定义蛋白质 N-糖基化对内皮细胞生成造血干细胞的功能作用

• 批准号: 10413509
• 负责人: Dionna Kasper
• 金额: $ 24.9万
• 依托单位: DARTMOUTH COLLEGE
• 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/10413509
• 关键词: Address; Adult; Aorta; Biology; Biosensor; Blood; Blood Vessels; Cardiovascular system; Cell Cycle; Cell physiology; Cells; Data; Defect; Development; Dorsal; Embryo; Embryonic Development; Endothelial Cells; Endothelium; Enzymes; Event; Foundations; Gene Expression; Gene Expression Profile; Gene Silencing; Generations; Genes; Genetic Transcription; Goals; Hematological Disease; Hematopoiesis; Hematopoietic; Hematopoietic Cell Production; Hematopoietic stem cells; In Vitro; Learning; Maintenance; Mammals; Mentors; Mesenchymal; Messenger RNA; Methods; MicroRNAs; Modeling; Molecular; Molecular Target; Neoplasm Metastasis; Optics; Pathway interactions; Phase; Phenocopy; Pluripotent Stem Cells; Post-Translational Protein Processing; Process; Production; Proteins; Regulation; Reporter; Repression; Role; Signal Pathway; Signal Transduction; Specific qualifier value; System; Technology; Testing; Therapeutic; Training; Transgenic Organisms; Untranslated RNA; Uterus; Visualization; Zebrafish; comparative; endothelial stem cell; glycosylation; hemogenic endothelium; in utero; in vivo; inhibitor/antagonist; insight; leukemia; mutant; novel; reconstitution; regenerative therapy; self-renewal; stem cells; tool; transcriptome; zebrafish development

PROJECT SUMMARY Creation of hematopoietic stem/progenitor cells (HSPCs) early in development is a highly dynamic process whereby flat hemogenic endothelial cells (ECs) change cell fate and emerge as rounded HSPCs with self-renewing capacity and multi-lineage potential. Unfortunately, the molecular mechanisms controlling this endothelial-to-hematopoietic transition (EHT) are still not well defined. This is underscored by the inability to mass-produce long-term reconstituting HSPCs for blood-regenerative therapies. Identification of new regulators of HSPC formation has been challenging in mammalian models likely due to the inability to visualize EHT dynamics in utero. Recently, our lab has taken advantage of the transparency and external development of zebrafish embryos to identify microRNA-223 as a novel inhibitor of HSPC formation. Zebrafish embryos lacking miR-223 activity had an increased number of emerging HSPCs, resulting in mature HSPC expansion from the onset and to later stages of hematopoiesis. How miR-223 controls HSPC production from the endothelium at the cellular and molecular levels is currently unknown. miR-223 belongs to the microRNA (miRNA) class of small noncoding RNAs, which provide precision to signaling pathways by post-transcriptionally repressing gene expression of complementary mRNA targets. Comparative transcriptome profiling of zebrafish miR-223 mutant and wildtype endothelial cells revealed N- glycosylation enzymes as candidate miR-223 molecular targets, and embryos treated with N-glycosylation inhibitors phenocopy the HSPC expansion in miR-223 mutants. Thus, the goal of this proposal is to directly test the hypothesis that miR-223 fine tunes N-glycosylation levels to control HSPC formation from the endothelium. The proposed study will develop novel transgenic and mutant approaches in zebrafish to dynamically visualize the cellular events controlled by miR-223 and to validate the N-glycosylation pathway as the direct target of miR-223 during EHT. Altogether, findings from this study will contribute a novel mechanism of HSPC formation, in which a miRNA dependent N-glycome regulates EC to HSPC cell fate transitions.

项目总结 在发育早期创造造血干/祖细胞(HSPC)是一种高度动态的 扁平的血源性内皮细胞改变细胞命运并分化为圆形HSPC的过程 自我更新能力和多谱系潜力。不幸的是，控制这一现象的分子机制 血管内皮细胞向造血细胞的转化(EHT)仍未得到很好的定义。这一点突出表现在无法 批量生产长期重组的HSPC，用于血液再生治疗。识别新的 在哺乳动物模型中，HSPC形成的调节者一直具有挑战性，这可能是由于无法可视化 子宫内EHT的动态变化。最近，我们的实验室利用了透明度和外部发展的优势 对斑马鱼胚胎进行鉴定，以确定microRNA-223是一种新的HSPC形成抑制因子。斑马鱼胚胎 缺乏miR-223活性的HSPC数量增加，导致成熟的HSPC扩张 从造血的开始阶段到后期。MIR-223如何控制HSPC生产 内皮细胞在细胞和分子水平上的作用目前尚不清楚。 MIR-223属于microRNA(MiRNA)类小分子非编码RNA，它为 转录后抑制互补信使核糖核酸靶标基因表达的信号通路。 斑马鱼miR-223突变体和野生型内皮细胞的比较转录组图谱显示N- 作为miR-223候选分子靶点的糖基化酶和N-糖基化处理的胚胎 抑制物在miR-223突变体中表现为HSPC扩增。因此，这项提案的目标是直接测试 假设miR-223微调N-糖基化水平以控制内皮细胞HSPC的形成。 拟议中的研究将在斑马鱼中开发新的转基因和突变方法，以动态显示 MiR-223调控的细胞事件及验证N-糖基化途径作为MIR-223的直接靶点 EHT期间的MIR-223。综上所述，本研究结果将为HSPC的发病机制提供一种新的思路 形成，其中依赖于miRNA的N-糖调节EC到HSPC细胞的命运转变。