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Nonmuscle Myosin II Isoform and Domain Specificity During Nonmuscle myosin II isoform and domain specificity during early mouse development Aibing Wanga, Xuefei Maa, Mary Anne Contia, Chengyu Liub, Sachiyo Kawamotoa, and Robert S. Adelsteina,1 aLaboratory of Molecular Cardiology, and bTransgenic Mouse Core Facility, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892-1583 Edited* by Thomas D. Pollard, Yale University, New Haven, CT, and approved July 7, 2010 (received for review March 26, 2010) Nonmuscle myosins (NMs) II-A and II-B are essential for embryonic quire the cross-linking properties of myosin could be replaced by mouse development, but their specific roles are not completely another isoform, but those functions dependent on myosin’s defined. Here we examine the isoforms and their domain specifi- motor activity were not substitutable because of a difference in cally in vivo and in vitro by studying mice and cells in which kinetic properties. This hypothesis remains to be further tested, nonmuscle myosin heavy chain (NMHC) II-A is genetically replaced especially in regard to substitution of NM II-A by II-B. Fur- by NMHC II-B or chimeric NMHC IIs that exchange the rod and head thermore, studies using chimeric NM IIs, which contain func- domains of NM II-A and II-B. In contrast with the failure of visceral tional domains from two different isoforms, can provide more endoderm formation resulting in embryonic day (E)6.5 lethality of direct evidence to substantiate this idea and also help to un- A−/A− mice, replacement with NM II-B or chimeric NM IIs restores derstand their domain specificities. a normal visceral endoderm. This finding is consistent with NM II’s To test this hypothesis in vivo and in vitro, we used a genetic- role in cell adhesion and also confirms an essential, isoform- replacement strategy (15, 16) to study NM II in mouse embryos independent requirement for NM II in visceral endoderm function. and in cells isolated from these embryos. We generated the The knock-in mice die between E9.5 and 12.5 because of defects in following four mouse lines (Fig. S1 A–C) in which the Myh9 first placenta formation associated with abnormal angiogenesis and coding exon is disrupted by: (i) cDNA encoding GFP-tagged cell migration, revealing a unique function for NM II-A in placenta human NMHC II-B (GFP-hNMHC II-B, Ab*/Ab* mice); (ii) development. In vitro results further support a requirement for cDNA encoding chimeric GFP-hNMHC II-AB (the N-terminal NM II-A in directed cell migration and focal adhesion formation. domain of NMHC II-A fused to the C-terminal II-B domain, These findings demonstrate an isoform-specific role for NM II-A Aab/Aab mice); (iii) GFP-hNMHC II-BA (the N-terminal do- during these processes, making replacement by another isoform, main of NMHC II-B fused to the C-terminal II-A domain, Aba/ or chimeric NM II isoforms, less successful. The failure of these Aba mice); and (iv) as a control, cDNA encoding mCherry- substitutions is not only related to the different kinetic properties hNMHC II-A (AmCh/AmCh mice) was likewise inserted into the of NM II-A and II-B, but also to their subcellular localization de- same site of the Myh9 locus. Each of these expression cassettes termined by the C-terminal domain. These results highlight the was placed under control of the NMHC II-A promoter. There- functions of the N-terminal motor and C-terminal rod domains of fore, mutant mice or cells lack endogenous NM II-A but express NM II and their different roles in cell-cell and cell-matrix adhesion. knock-in proteins (Fig. S1D). Our results support a critical role for NM II in visceral endoderm development. They reveal cell migration | genetic substitution | placenta development | visceral a unique function for NM II-A in placenta development and endoderm formation | chimeric myosin II support a requirement for NM II-A in directed cell migration and focal adhesion formation in vitro and in vivo. onmuscle myosin II (NM II) is a major cytoskeletal protein Results and Discussion that interacts with actin to contribute to cellular processes, N An Essential but Isoform-Independent Role for NM II in Visceral such as cell migration (1–4), cell adhesion (5–8), and cytokinesis CELL BIOLOGY (9). In mammals there are three NM II isoforms, each composed Endoderm Formation. Thegenerationoffourmutantknock-in SI Materials and Methods of two identical heavy chains and two pairs of light chains. Three mouse lines is described in and shown in separate genes (Myh9, Myh10, Myh14) encode the nonmuscle Fig. S1. All heterozygotes from the different lines are in- distinguishable from their wild-type littermates. They were myosin heavy chains (NMHCs; NMHC II-A, II-B, and II-C), mCh mCh which together with the light chains are referred to as NM II-A, crossed to produce homozygous embryos. Control A /A II-B, and II-C. The three NM II isoforms not only show con- mice are born at the expected Mendelian frequency and are normal, demonstrating that the phenotypes observed in the mu- siderable homology in primary structure, but also have a similar tant mice are not a result of genetic manipulations of the Myh9 molecular structure in that each NM II contains two structurally locus (Table S1). The ratio of the GFP-NM II-B to the endoge- defined regions: a globular region at the N-terminal end har- α nous II-B in MEF cells is 2.8:1, indicative that GFP-NM II-B is boring MgATPase and actin binding activities, and an -helical D fi expressed under control of the NMHC II-A promoter (Fig. S1 , coiled-coil C-terminal tail region that mediates lament assem- right-most lane). Fig. S1E indicates that expression of the chi- bly (10). The in vivo functions of two of the isoforms have been meric NM IIs are similar to GFP-NM II-B in Ab*/Ab* mice. studied following germline ablation, revealing markedly different We first determined whether knock-in NM II-B or chimeric phenotypes: death by embryonic day (E)6.5 because of a failure NM IIs could functionally replace NM II-A, and rescue the cell- in cell-cell adhesion and visceral endoderm formation in the case of NM II-A and lethality by E14.5, resulting from cardiac and brain defects following II-B ablation (7, 11, 12). These results Author contributions: A.W., X.M., and R.S.A. designed research; A.W., X.M., M.A.C., and C.L. suggest that both isoforms are essential for mouse development. performed research; A.W., X.M., M.A.C., S.K., and R.S.A. analyzed data; and A.W., X.M., Because most cells contain more than one isoform, their specific M.A.C., S.K., and R.S.A. wrote the paper. in vivo roles during embryogenesis are unclear. Previous work The authors declare no conflict of interest. has shown that some defects associated with the loss of NM II-B *This Direct Submission article had a prearranged editor. could be rescued in vivo by a motor-impaired II-B or when NM 1To whom correspondence should be addressed. E-mail: [email protected]. fi II-A is expressed from the II-B locus (13, 14). These ndings This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. resulted in the hypothesis that the functions of NM IIs that re- 1073/pnas.1004023107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1004023107 PNAS | August 17, 2010 | vol. 107 | no. 33 | 14645–14650 Downloaded by guest on September 27, 2021 cell adhesion defects of the visceral endoderm associated with II- a similar phenotype based on their appearance, as exemplified by Adeficiency at E6.5. Ab*/Ab*, Aab/Aab,Aba/Aba (collectively Ab*/Ab* embryos. These embryos display pale yolk sacs with referred to as “mutant”) and A+/A+ embryo sections were fewer visible vessels compared with the A+/A+ yolk sacs (Fig. stained with antibodies to NMHC II-A or II-B together with E- 2A). Whole-mount PECAM-1 staining also reveals that Ab*/Ab* − − cadherin (Fig. 1 and Fig. S2). In contrast to A /A embryos, embryos have a less intricate vascular network in the head and which have unidentifiable cell layers and a disorganized visceral trunk regions compared with the well-developed and hierarchi- endoderm marked by GATA4 staining of the nuclei (compare cally organized vascular architecture of A+/A+ counterparts Fig. 1 J with I), all mutant embryos (Fig. 1 E and G; Fig. S2 E, G, (Fig. 2B). These findings suggest that although vasculogenesis I, K) appear normal, with a polarized columnar visceral endo- occurs in the Ab*/Ab* yolk sacs, there is a defect in subsequent derm similar to A+/A+ embryos (Fig. 1 A and C and Fig. S2 A angiogenesis, which results in the impairment of the blood supply and C). Moreover, the cell-cell borders of the visceral endoderm to the embryo. In addition, Ab*/Ab* embryos show growth re- of A+/A+ and mutant embryos contain E-cadherin (arrows in tardation, which becomes more pronounced with age (Fig. S3). − − Fig. 1I and Fig. S2), which is not present in the A /A GATA4- However, this defect in growth is not a result of abnormalities in positive cells (arrows, Fig. 1J). As noted previously (7), wild-type cell proliferation or apoptosis, as shown in Fig. S4A, which shows visceral endoderm expresses NM II-A at the cell-cell boundaries no difference in the BrdU and TUNEL staining between Ab*/Ab* but has no detectable II-B (Fig.
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