The Role of Nonmuscle Myosins in Development

本研究的目的是了解两个非肌肉肌球蛋白II（NM II）亚型，NM IIA和NM IIC 1之间的差异，通过研究NM IIC 1是否可以在发育过程中功能上取代NM IIA。 为了在小鼠中用NM IIC 1遗传地替换NM IIA，通过将编码NMHC IIC 1-GFP的cDNA插入编码NMHC IIA的基因的第一个编码外显子中来消融非肌肉肌球蛋白重链NM IIA。表达50%野生型NM IIA的杂合AC 1 *-gfp/A+小鼠是正常的。纯合AC 1 *gfp/AC 1 *gfp小鼠在大约E10.5时死亡，表明NM IIC 1可以挽救导致A/A小鼠胚胎日（E）6.5致死的内脏内胚层功能的衰竭。这证实了NM II在支持内脏内胚层功能和随后的原肠胚形成中的亚型独立要求。相比之下，AC 1 *gfp/AC 1 *gfp胚胎在E10.5时的致死性似乎是由于胎盘中的细胞凋亡和异常增加，这显示了血管发生和血管生成的缺陷。 在培养的AC 1 *gfp/AC 1 *gfp MEF细胞中发现细胞迁移、粘着斑形成和肌动球蛋白细胞骨架结构缺陷的异常，有助于解释胎盘缺陷。我们已经模拟了这些胎盘血管缺陷的尿囊外植体，以确认NM IIA和NM II磷酸化在血管发育中的作用。因此，NM IIC 1，这是不正常表达的小鼠，直到E11，可以恢复细胞-细胞粘附和正常的内脏内胚层功能，在E6.5，但不能取代NM IIAs的作用，在细胞骨架组织和胎盘血管形成。 之前，我们在小鼠中模拟了在人NM IIA中发现的3种不同突变（R702 C、D1424 N和E1841 K）。我们报道了所有三个细胞系表型模仿人类MYH 9相关疾病。此外，突变E1841 K纯合的雄性小鼠由于缺乏成熟和能动的精子而表现出不育。 突变睾丸的电子显微镜显示生殖细胞凋亡，多核巨细胞和缺乏晚期细长精子细胞的证据

The purpose of this study is to understand the differences between the two nonmuscle myosin II (NM II) isoforms, NM IIA and NM IIC1 by studying whether NM IIC1 can functionally replace NM IIA during development. To genetically replace NM IIA with NM IIC1 in mice , nonmuscle myosin heavy chain NM IIA was ablated by inserting cDNA encoding NMHC IIC1-GFP into the first coding exon of the gene encoding NMHC IIA. Heterozygous AC1*-gfp/A+ mice, expressing 50% wild-type NM IIA, are normal. Homozygous AC1*gfp/AC1*gfp mice die at approximately E10.5 demonstrating that NM IIC1 can rescue the failure of visceral endoderm function that results in embryonic day (E)6.5 lethality of A−/A− mice. This confirms an isoform-independent requirement for NM II in supporting visceral endoderm function and consequent gastrulation. In contrast, lethality at E 10.5 of the AC1*gfp/AC1*gfp embryos appears to be due to increased apoptosis and abnormalities in the placenta which shows a defect in vasculogenesis and angiogenesis. Abnormalities in cell migration, focal adhesion formation and defects in the actomyosin cytoskeleton structure are found in cultured AC1*gfp/AC1*gfp MEF cells and help to explain the placental defects. We have modeled these placental vascular defects in allantois explants to confirm a role for NM IIA and NM II phosphorylation in vascular development. Thus NM IIC1, which is not normally expressed in mice until E11, can restore cell-cell adhesions and normal visceral endoderm function at E6.5 but cannot replace NM II As roles in cytoskeleton organization and placenta vascular formation. Previously we modeled 3 different mutations that are found in human NM IIA in mice (R702C, D1424N and E1841K). We reported that all three lines phenocopied human MYH9-Related disease. In addition, male mice that are homozygous for the mutation E1841K exhibit infertility due to a lack of mature and motile sperm. Electron microscopy on the mutant testes shows evidence for germ cell apoptosis, multinucleated giant cells and a lack of late stage elongated spermatids