The Role of Nonmuscle Myosin 2B In Vivo

总结 包括人类和小鼠在内的哺乳动物表达三种非肌肉肌球蛋白2旁系同源物（NM 2A、2B和2C）。 两个选择性剪接的外显子，每个位于ATP和/或肌动蛋白结合区，可以掺入NM 2B（B1和B2插入物）和2C（C1和C2插入物）的重链中，但不能掺入NM 2A中。NM 2A消融的小鼠死亡非常早，在E6.5，在原肠胚形成之前，内脏内胚层形成缺陷。NM 2B消融的小鼠在E14.5死亡，心脏和大脑有重大缺陷，而NM 2C消融的小鼠存活至成年，没有明显的异常。为了了解NM 2A在小鼠早期发育中的具体作用，我们产生了遗传交换小鼠，其中NM 2A表达在内源性NM 2A启动子下被NM 2B的非插入亚型或NM C1的插入亚型（NM 2B和2C在哺乳动物中分别是两种最广泛表达的亚型）取代。 我们以前已经证明，小鼠表达NM 2B的地方NM 2A生存超过原肠胚形成，但死亡的E12.5与胎盘形成的主要缺陷。在这里，我们报告的研究，从小鼠的NM 2A被替换为GFP融合，NM 2C 1-GFP（AC 1 *gfp/AC 1 *gfp）。NM 2C 1与NM 2B相似，可以替代2A支持正常功能内脏内胚层的形成和小鼠胚胎在原肠胚形成后的发育。然而，NM 2C 1，类似于NM 2B，不能支持正常的胎盘血管形成。AC 1 *gfp/AC 1 *gfp胚胎在E10.5死亡，具有发育不良、未扩张的胎盘迷路，其缺乏胎儿和母体血管的混合。在来自AC 1 *gfp/AC 1 *gfp胚胎的尿囊外植体中进一步证实了脉管形成的缺陷。此外，每个NM 2蛋白在迁移细胞中具有重叠和独特的细胞定位。因此，NM 2A是小鼠胚胎发育过程中正常胎盘血管形成所必需的。我们将此归因于NM 2A在调节肌动球蛋白细胞骨架、粘着斑的形成以及提供细胞迁移的持久性中的特定作用。相反，NM 2A在维持内脏内胚层细胞-细胞粘附中的作用及其在支持原肠胚形成中的功能可以被NM 2B或NM 2C 1取代。

Summary Mammals including humans and mice express three nonmuscle myosin 2 paralogs (NM 2A, 2B, and 2C). Two alternatively spliced exons, each at the ATP and/or at actin binding regions, can be incorporated into the heavy chains of NM 2B (B1 and B2 inserts) and 2C (C1 and C2 inserts), but not into NM 2A. Mice ablated for NM 2A die very early, at E6.5, before gastrulation with defects in visceral endoderm formation. Mice ablated for NM 2B die by E14.5 with major defects in the heart and brain, while mice ablated for NM 2C survive to adulthood showing no obvious abnormalities. To understand the specific roles of NM 2A in early mouse development, we have generated genetically swapped mice where NM 2A expression was replaced either by the non-inserted isoform of NM 2B or the inserted isoform of NM C1 (the two most widely expressed isoforms for NM 2B and 2C respectively in mammals) under the endogenous NM 2A promoter. We have previously demonstrated that mice expressing NM 2B in place of NM 2A survive beyond gastrulation but die by E12.5 with major defects in placenta formation. Here we report studies from mice where NM 2A is substituted for by GFP-fused, NM 2C1-GFP (AC1*gfp/AC1*gfp). NM 2C1, similarly to NM 2B, can substitute for 2A in supporting normal functional visceral endoderm formation and mouse embryonic development beyond gastrulation. However, NM 2C1, similarly to NM 2B, cannot support normal placental vascular formation. AC1*gfp/AC1*gfp embryos die by E10.5 with a poorly developed, un-expanded placental labyrinth which lacks intermingling of the fetal and maternal blood vasculatures. The defect in vascular formation is further confirmed in allantois explants from AC1*gfp/AC1*gfp embryos. Additionally, each NM 2 paralog has overlapping as well as a unique cellular localization in migrating cells. Thus, NM 2A is essential for normal placental vascular formation during mouse embryonic development. We attribute this to a specific role for NM 2A in regulating the actomyosin cytoskeleton, the formation of focal adhesions, and for providing persistence in cell migration. In contrast, a role of NM 2A in maintaining visceral endodermal cell-cell adhesions and its function in supporting gastrulation, can be replaced by either NM 2B or NM 2C1.