The Function of Nonmuscle Myosin Heavy Chains

在脊椎动物中，Myh9、Myh10和Myh14三个基因编码三种不同形式的非肌肉肌球蛋白II重链(NMHC II)。非肌肉肌球蛋白II(NM II)的运动活动位于N端的球状头域，而细丝形成位于C端的杆状域。以前的工作表明，在小鼠中，消融NM II-A会导致E6.5致死，并伴有细胞-细胞黏附缺陷和无法产生合格的内脏内胚层。为了了解NM II-A在发育过程中的功能，我们利用同源重组产生了4个不同的小鼠品系：1-我们用NM II-B取代NM II-A，将编码人NMHC II-B的基因“敲入”到II-A位点，从而去除II-A，并将NMHC II-B置于内源性II-A启动子(Ab*/Ab*小鼠)的控制之下。2-我们通过敲入两个嵌合的NMHC来取代内源性NMII-A，一个编码NMHC II-A的N端运动域与C端II-B杆状结构域融合(Aab/Aab小鼠)，3-编码NMHC II-B的N端结构域与II-A的C端结构域融合(Aba/Aba小鼠)。4-对于对照组小鼠，我们将编码NMHC II-A的基因插入到II-A基因座。用II-B(Ab*/Ab*)代替NM II-A，可以使内脏内胚层正常发育、原肠形成、器官发生和存活到E9-10。这些小鼠表现出胚胎发育延迟，血管系统和心内膜有缺陷。ABA/ABA小鼠的死亡年龄与AB*/AB*小鼠相同，这表明尽管II-A和II-B的运动学性质不同，但II-A和II-B直到E9-10的N-末端运动域是可以互换的。AAB/Aab小鼠在血管系统正常但心脏发育不良和主动脉覆盖室间隔缺陷的情况下存活到E12以后。因此，N端II-A运动域将发育周期从E9-10延长到E13，显示了其对早期胚胎发育的重要性。

In vertebrates three genes, Myh9, Myh10 and Myh14 encode three different isoforms of the nonmuscle myosin II heavy chain (NMHC II). The motor activity of nonmuscle myosin II (NM II) resides in the N-terminal globular head domain and filament formation resides in the C-terminal rod domain. Previous work has shown that ablation of NM II-A in mice results in lethality by E6.5 with defects in cell-cell adhesion and a failure to produce a competent visceral endoderm. To understand the function of NM II-A during development we used homologous recombination to generate 4 different mouse lines:1-we replaced NM II-A with NM II-B by "knocking in" cDNA encoding human NMHC II-B into the II-A locus, thereby ablating II-A and placing NMHC II-B under control of the endogenous II-A promoter (Ab*/Ab* mice). 2-we replaced endogenous NM II-A by knocking in two chimeric NMHCs, one encoding the N-terminal motor domain of NMHC II-A fused to the C-terminal II-B rod domain (Aab/Aab mice), 3-and another encoding the N-terminal domain of NMHC II-B fused to the C-terminal domain of II-A (Aba/Aba mice). 4-For control mice we inserted cDNA encoding NMHC II-A into the II-A locus. Replacing NM II-A with II-B (Ab*/Ab*) allows normal development of the visceral endoderm, gastrulation, organogenesis and survival to E9-10. These mice show a delay in embryonic turning with defects in the vasculature and endocardium. Aba/Aba mice die at the same age as Ab*/Ab* mice indicating that the N-terminal motor domains are interchangeable between II-A and II-B up to E9-10 despite differences in the kinetic properties of the motors. Aab/Aab mice survive beyond E12 with a normal vasculature but with a hypoplastic heart and an aorta overriding a ventricular septal defect. Thus, the N-terminal II-A motor domain has extended the developmental period from E9-10 to E13, displaying its importance for the early embryonic development.