Center for Molecular Medicine, Maine Medical Center Research Institute, Scarborough, Maine, United States of America.
PLoS One. 2011 Apr 1;6(4):e18374. doi: 10.1371/journal.pone.0018374.
Development of the hematopoietic and endothelial lineages derives from a common mesodermal precursor, the Flk1(+) hemangioblast. However, the signaling pathways that regulate the development of hematopoietic and endothelial cells from this common progenitor cell remains incompletely understood. Using mouse models with a conditional Spry1 transgene, and a Spry1 knockout mouse, we investigated the role of Spry1 in the development of the endothelial and hematopoietic lineages during development.
METHODOLOGY/PRINCIPAL FINDINGS: Quantitative RT-PCR analysis demonstrates that Spry1, Spry2, and Spry4 are expressed in Flk1(+) hemangioblasts in vivo, and decline significantly in c-Kit(+) and CD41(+) hematopoietic progenitors, while expression is maintained in developing endothelial cells. Tie2-Cre-mediated over-expression of Spry1 results in embryonic lethality. At E9.5 Spry1;Tie2-Cre embryos show near normal endothelial cell development and vessel patterning but have reduced hematopoiesis. FACS analysis shows a reduction of primitive hematopoietic progenitors and erythroblastic cells in Spry1;Tie2-Cre embryos compared to controls. Colony forming assays confirm the hematopoietic defects in Spry1;Tie2-Cre transgenic embryos. Immunostaining shows a significant reduction of CD41 or CD71 and dpERK co-stained cells in Spry1;Tie2-Cre embryos compared to controls, whereas the number of VEC(+) and dpERK co-stained cells is comparable. Compared to controls, Spry1;Tie2-Cre embryos also show a decrease in proliferation and an increase in apoptosis. Furthermore, loss of Spry1 results in an increase of CD41(+) and CD71(+) cells at E9.5 compared with controls.
CONCLUSIONS/SIGNIFICANCE: These data indicate that primitive hematopoietic cells derive from Tie2-expressing hemangioblasts and that Spry1 over expression inhibits primitive hematopoietic progenitor and erythroblastic cell development and expansion while having no obvious effect on endothelial cell development.
造血和内皮谱系的发育源自于一个共同的中胚层前体细胞，即 Flk1(+)血管母细胞。然而，调节造血和内皮细胞从这个共同祖细胞发育的信号通路仍不完全清楚。利用具有条件性 Spry1 转基因的小鼠模型和 Spry1 基因敲除小鼠，我们研究了 Spry1 在胚胎发育过程中对内皮和造血谱系发育的作用。
方法/主要发现：实时定量 RT-PCR 分析表明，Spry1、Spry2 和 Spry4 在体内 Flk1(+)血管母细胞中表达，并在 c-Kit(+)和 CD41(+)造血祖细胞中显著下降，而在发育中的内皮细胞中表达得以维持。Tie2-Cre 介导的 Spry1 过表达导致胚胎致死。在 E9.5 时，Spry1;Tie2-Cre 胚胎显示出正常的内皮细胞发育和血管模式，但造血减少。FACS 分析显示 Spry1;Tie2-Cre 胚胎中的原始造血祖细胞和红系细胞减少。集落形成实验证实 Spry1;Tie2-Cre 转基因胚胎存在造血缺陷。免疫染色显示 Spry1;Tie2-Cre 胚胎中与对照组相比，CD41 或 CD71 和 dpERK 共染色细胞显著减少，而 VEC(+)和 dpERK 共染色细胞数量相当。与对照组相比，Spry1;Tie2-Cre 胚胎还显示增殖减少和凋亡增加。此外，与对照组相比，Spry1;Tie2-Cre 胚胎在 E9.5 时 CD41(+)和 CD71(+)细胞减少。
结论/意义：这些数据表明，原始造血细胞来源于表达 Tie2 的血管母细胞，Spry1 过表达抑制原始造血祖细胞和红系细胞的发育和扩增，而对内皮细胞的发育没有明显影响。
