Bone Marrow Transplantation Center, School of Medicine, The First Affiliated Hospital, Zhejiang University, No. 79 Qingchun Road, Hangzhou, 310012, Zhejiang, People's Republic of China.
Liangzhu Laboratory, Zhejiang University Medical Center, 1369 West Wenyi Road, Hangzhou, 311121, People's Republic of China.
Stem Cell Res Ther. 2023 Apr 30;14(1):117. doi: 10.1186/s13287-023-03328-1.
In vertebrates, hematopoietic stem and progenitor cells (HSPCs) emerge from hemogenic endothelium in the floor of the dorsal aorta and subsequently migrate to secondary niches where they expand and differentiate into committed lineages. Glia maturation factor γ (gmfg) is a key regulator of actin dynamics that was shown to be highly expressed in hematopoietic tissue. Our goal is to investigate the role and mechanism of gmfg in embryonic HSPC development.
In-depth bioinformatics analysis of our published RNA-seq data identified gmfg as a cogent candidate gene implicated in HSPC development. Loss and gain-of-function strategies were applied to study the biological function of gmfg. Whole-mount in situ hybridization, confocal microscopy, flow cytometry, and western blotting were used to evaluate changes in the number of various hematopoietic cells and expression levels of cell proliferation, cell apoptosis and hematopoietic-related markers. RNA-seq was performed to screen signaling pathways responsible for gmfg deficiency-induced defects in HSPC initiation. The effect of gmfg on YAP sublocalization was assessed in vitro by utilizing HUVEC cell line.
We took advantage of zebrafish embryos to illustrate that loss of gmfg impaired HSPC initiation and maintenance. In gmfg-deficient embryos, the number of hemogenic endothelium and HSPCs was significantly reduced, with the accompanying decreased number of erythrocytes, myelocytes and lymphocytes. We found that blood flow modulates gmfg expression and gmfg overexpression could partially rescue the reduction of HSPCs in the absence of blood flow. Assays in zebrafish and HUVEC showed that gmfg deficiency suppressed the activity of YAP, a well-established blood flow mediator, by preventing its shuttling from cytoplasm to nucleus. During HSPC initiation, loss of gmfg resulted in Notch inactivation and the induction of Notch intracellular domain could partially restore the HSPC loss in gmfg-deficient embryos.
We conclude that gmfg mediates blood flow-induced HSPC maintenance via regulation of YAP, and contributes to HSPC initiation through the modulation of Notch signaling. Our findings reveal a brand-new aspect of gmfg function and highlight a novel mechanism for embryonic HSPC development.
在脊椎动物中，造血干/祖细胞（HSPCs）从背主动脉底部的血发生内皮中出现，随后迁移到次级龛位，在那里它们扩增并分化为定向谱系。胶质细胞成熟因子 γ（gmfg）是一种关键的肌动蛋白动力学调节剂，在造血组织中高度表达。我们的目标是研究 gmfg 在胚胎 HSPC 发育中的作用和机制。
对我们发表的 RNA-seq 数据进行深入的生物信息学分析，确定 gmfg 是一个与 HSPC 发育有关的关键候选基因。应用缺失和功能获得策略来研究 gmfg 的生物学功能。全胚胎原位杂交、共聚焦显微镜、流式细胞术和 Western blot 用于评估各种造血细胞数量的变化和细胞增殖、细胞凋亡和造血相关标志物的表达水平。进行 RNA-seq 以筛选负责 gmfg 缺乏引起的 HSPC 起始缺陷的信号通路。利用 HUVEC 细胞系评估 gmfg 对 YAP 亚定位的影响。
我们利用斑马鱼胚胎来说明 gmfg 的缺失损害了 HSPC 的起始和维持。在 gmfg 缺陷型胚胎中，血发生内皮和 HSPCs 的数量显著减少，伴随红细胞、髓细胞和淋巴细胞数量减少。我们发现血流调节 gmfg 的表达，gmfg 的过表达可以部分挽救血流缺乏时 HSPC 数量的减少。在斑马鱼和 HUVEC 中的测定表明，gmfg 缺失通过阻止 YAP 从细胞质到细胞核的穿梭，抑制了 YAP 的活性，YAP 是一种公认的血流介质。在 HSPC 起始过程中，gmfg 的缺失导致 Notch 失活，而 Notch 胞内结构域的诱导可以部分恢复 gmfg 缺陷型胚胎中的 HSPC 缺失。
我们得出结论，gmfg 通过调节 YAP 介导血流诱导的 HSPC 维持，并通过调节 Notch 信号通路促进 HSPC 起始。我们的发现揭示了 gmfg 功能的一个全新方面，并强调了胚胎 HSPC 发育的一个新机制。
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