Chengyu Liu

Mouse models of MYH9-related disease: mutations in nonmuscle myosin II-A

We have generated 3 mouse lines, each with a different mutation in the nonmuscle myosin II-A gene, Myh9 (R702C, D1424N, and E1841K). Each line develops MYH9-related disease similar to that found in human patients. R702C mutant human cDNA fused with green fluorescent protein was introduced into the first coding exon of Myh9, and D1424N and E1841K mutations were introduced directly into the corresponding exons. Homozygous R702C mice die at embryonic day 10.5-11.5, whereas homozygous D1424N and E1841K mice are viable. All heterozygous and homozygous mutant mice show macrothrombocytopenia with prolonged bleeding times, a defect in clot retraction, and increased extramedullary megakaryocytes. Studies of cultured megakaryocytes and live-cell imaging of megakaryocytes in the BM show that heterozygous R702C megakaryocytes form fewer and shorter proplatelets with less branching and larger buds. The results indicate that disrupted proplatelet formation contributes to the macrothrombocytopenia in mice and most probably in humans. We also observed premature cataract formation, kidney abnormalities, including albuminuria, focal segmental glomerulosclerosis and progressive kidney disease, and mild hearing loss. Our results show that heterozygous mice with mutations in the myosin motor or filament-forming domain manifest similar hematologic, eye, and kidney phenotypes to humans with MYH9-related disease.

Replacement of Nonmuscle Myosin II-B with II-A Rescues Brain but Not Cardiac Defects in Mice

The purpose of these studies was to learn whether one isoform of nonmuscle myosin II, specifically nonmuscle myosin II-A, could functionally replace a second one, nonmuscle myosin II-B, in mice. To accomplish this, we used homologous recombination to ablate nonmuscle myosin heavy chain (NMHC) II-B by inserting cDNA encoding green fluorescent protein (GFP)-NMHC II-A into the first coding exon of the Myh10 gene, thereby placing GFP-NMHC II-A under control of the endogenous II-B promoter. Similar to B-/B- mice, most Ba*/Ba* mice died late in embryonic development with structural cardiac defects and impaired cytokinesis of the cardiac myocytes. However, unlike B-/B- mice, 15 Ba*/Ba* mice of 172 F2 generation mice survived embryonic lethality but developed a dilated cardiomyopathy as adults. Surprisingly none of the Ba*/Ba* mice showed evidence for hydrocephalus that is always found in B-/B- mice. Rescue of this defect was due to proper localization and function of GFP-NMHC II-A in place of NMHC II-B in a cell-cell adhesion complex in the cells lining the spinal canal. Restoration of the integrity and adhesion of these cells prevents protrusion of the underlying cells into the spinal canal where they block circulation of the cerebral spinal fluid. However, abnormal migration of facial and pontine neurons found in NMHC II-B mutant and ablated mice persisted in Ba*/Ba* mice. Thus, although NMHC II-A can substitute for NMHC II-B to maintain integrity of the spinal canal, NMHC II-B plays an isoform-specific role during cytokinesis in cardiac myocytes and in migration of the facial and pontine neurons.

Nonmuscle myosin II isoform and domain specificity during early mouse development

Nonmuscle myosins (NMs) II-A and II-B are essential for embryonic mouse development, but their specific roles are not completely defined. Here we examine the isoforms and their domain specifically in vivo and in vitro by studying mice and cells in which nonmuscle myosin heavy chain (NMHC) II-A is genetically replaced by NMHC II-B or chimeric NMHC IIs that exchange the rod and head domains of NM II-A and II-B. In contrast with the failure of visceral endoderm formation resulting in embryonic day (E)6.5 lethality of A−/A− mice, replacement with NM II-B or chimeric NM IIs restores a normal visceral endoderm. This finding is consistent with NM IIs role in cell adhesion and also confirms an essential, isoform-independent requirement for NM II in visceral endoderm function. The knock-in mice die between E9.5 and 12.5 because of defects in placenta formation associated with abnormal angiogenesis and cell migration, revealing a unique function for NM II-A in placenta development. In vitro results further support a requirement for NM II-A in directed cell migration and focal adhesion formation. These findings demonstrate an isoform-specific role for NM II-A during these processes, making replacement by another isoform, or chimeric NM II isoforms, less successful. The failure of these substitutions is not only related to the different kinetic properties of NM II-A and II-B, but also to their subcellular localization determined by the C-terminal domain. These results highlight the functions of the N-terminal motor and C-terminal rod domains of NM II and their different roles in cell-cell and cell-matrix adhesion.

Conditional Ablation of Nonmuscle Myosin II-B Delineates Heart Defects in Adult Mice

Rationale: Germline ablation of the cytoskeletal protein nonmuscle myosin II (NMII)-B results in embryonic lethality, with defects in both the brain and heart. Tissue-specific ablation of NMII-B by a Cre recombinase strategy should prevent embryonic lethality and permit study of the function of NMII-B in adult hearts. Objective: We sought to understand the function of NMII-B in adult mouse hearts and to see whether the brain defects found in germline-ablated mice influence cardiac development. Methods and Results: We used a loxP/Cre recombinase strategy to specifically ablate NMII-B in the brains or hearts of mice. Mice ablated for NMII-B in neural tissues die between postnatal day 12 and 22 without showing cardiac defects. Mice deficient in NMII-B only in cardiac myocytes (B&agr;MHC/B&agr;MHC mice) do not show brain defects. However, B&agr;MHC/B&agr;MHC mice display novel cardiac defects not seen in NMII-B germline-ablated mice. Most of the B&agr;MHC/B&agr;MHC mice are born with enlarged cardiac myocytes, some of which are multinucleated, reflecting a defect in cytokinesis. Between 6 to 10 months, they develop a cardiomyopathy that includes interstitial fibrosis and infiltration of the myocardium and pericardium with inflammatory cells. Four of 5 B&agr;MHC/B&agr;MHC hearts develop marked widening of intercalated discs. Conclusions: By avoiding the embryonic lethality found in germline-ablated mice, we were able to study the function of NMII-B in adult mice and show that absence of NMII-B in cardiac myocytes results in cardiomyopathy in the adult heart. We also define a role for NMII-B in maintaining the integrity of intercalated discs.

A role for borg5 during trophectoderm differentiation.

Stem cell differentiation is accompanied by a gradual cellular morphogenesis and transcriptional changes. Identification of morphological regulators that control cell behavior during differentiation could shed light on how cell morphogenesis is coupled to transcriptional changes during development. By analyzing cellular behavior during differentiation of mouse embryonic stem cells (ESCs), we uncover a role of Borg5 (binder of Rho guanosine 5′‐triphosphatase 5) in regulating trophectoderm (TE) cell morphogenesis. We report that differentiation of ESCs toward TE is accompanied by enhanced actin protrusion and cell motility that require upregulation of Borg5. Borg5 interacts with both Cdc42 and atypical protein kinase C (aPKC) and functions downstream of Cdc42 to enhance TE cell motility. Borg5 is required for the sorting of differentiating TE to the outside of ESCs in vitro. In developing embryos, Borg5 protein localizes to cell–cell contacts and the cytoplasm after compaction. It exhibits higher levels of expression in outer cells than in inner cells in morula and blastocysts. Reduction of Borg5 disrupts aPKC localization and inhibits blastocyst formation. Since Cdx2 and Borg5 facilitate each others expression as ESCs differentiate toward TE, we propose that cell morphogenesis is coupled with transcriptional changes to regulate TE differentiation. Our studies also demonstrate the utility of ESCs in identifying morphological regulators important for development. STEM Cells 2010;28:1030–1038

Borg5 is required for angiogenesis by regulating persistent directional migration of the cardiac microvascular endothelial cells

Using mouse knockout strategy, the authors uncovered a role for Borg5 in microvascular angiogenesis. In primary mouse cardiac endothelial cells, Borg5 interacts with septin cytoskeleton and colocalizes with perinuclear actomyosin fibers. The data presented suggest that Borg5 and septin regulate the actomyosin activity critical for persistent directional migration.

Conditional deletion of nonmuscle myosin II-A in mouse tongue epithelium results in squamous cell carcinoma

To investigate the contribution of nonmuscle myosin II-A (NM II-A) to early cardiac development we crossed Myh9 floxed mice and Nkx2.5 cre-recombinase mice. Nkx2.5 is expressed in the early heart (E7.5) and later in the tongue epithelium. Mice homozygous for deletion of NM II-A (ANkx/ANkx) are born at the expected ratio with normal hearts, but consistently develop an invasive squamous cell carcinoma (SCC) of the tongue (32/32 ANkx/ANkx) as early as E17.5. To assess reproducibility a second, independent line of Myh9 floxed mice derived from a different embryonic stem cell clone was tested. This second line also develops SCC indistinguishable from the first (15/15). In ANkx/ANkx mouse tongue epithelium, genetic deletion of NM II-A does not affect stabilization of TP53, unlike a previous report for SCC. We attribute the consistent, early formation of SCC with high penetrance to the role of NM II in maintaining mitotic stability during karyokinesis.

Identification and characterization of MYH9 locus for high efficient gene knock-in and stable expression in mouse embryonic stem cells

Targeted integration of exogenous genes into so-called safe harbors/friend sites, offers the advantages of expressing normal levels of target genes and preventing potentially adverse effects on endogenous genes. However, the ideal genomic loci for this purpose remain limited. Additionally, due to the inherent and unresolved issues with the current genome editing tools, traditional embryonic stem (ES) cell-based targeted transgenesis technology is still preferred in practical applications. Here, we report that a high and repeatable homologous recombination (HR) frequency (>95%) is achieved when an approximate 6kb DNA sequence flanking the MYH9 gene exon 2 site is used to create the homology arms for the knockout/knock-in of diverse nonmuscle myosin II (NM II) isoforms in mouse ES cells. The easily obtained ES clones greatly facilitated the generation of multiple NM II genetic replacement mouse models, as characterized previously. Further investigation demonstrated that though the targeted integration site for exogenous genes is shifted to MYH9 intron 2 (about 500bp downstream exon 2), the high HR efficiency and the endogenous MYH9 gene integrity are not only preserved, but the expected expression of the inserted gene(s) is observed in a pre-designed set of experiments conducted in mouse ES cells. Importantly, we confirmed that the expression and normal function of the endogenous MYH9 gene is not affected by the insertion of the exogenous gene in these cases. Therefore, these findings suggest that like the commonly used ROSA26 site, the MYH9 gene locus may be considered a new safe harbor for high-efficiency targeted transgenesis and for biomedical applications.

Mouse Models of Human MYH9-Related Diseases

Point mutations in MYH9, the gene encoding nonmuscle myosin heavy chain IIA (NMHC IIA), underlie autosomal dominant syndromes in humans (incidence 1 in 500,000). The abnormalities can manifest as macrothrombocytopenia, granulocyte inclusions, progressive proteinuric renal disease, cataracts, and sensorineural deafness. To gain insight into the pathological mechanism of MYH9-related diseases in humans, we generated mouse models of three disease-associated mutations, Arg702Cys in the amino-terminal domain of NMHC IIA which controls myosin motor activity, and Asp1424Gln and Glu1841Lys in the carboxyl-terminal rod domain, which regulates filament formation. Heterozygous Asp1424Gln and Glu1841Lys mutant mice produce homozygous mutant offspring at close to normal ratios. By contrast, homozygous Arg702Cys mice die at embryonic day E10.5 to E11.5 which though early, is considerably later in development than knockout NMHC IIA mice (E6.5). These results indicate that the motor domain function of NMHC IIA is critically important during the latter phase of mouse embryonic development. Giant platelets accompanied by decreased platelet counts and prolonged bleeding times are found in adult heterozygous and homozygous mice from all three mutant mouse lines. Bone marrow histology is consistent with failure of platelet release into the circulation. Some adult heterozygotes from all three mouse lines and homozygotes from Asp1424Gln and Glu1841Lys mouse lines have higher urine albumin/creatinine ratios than those of wild type controls. Light and transmission electron microscopy reveals focal segmental glomerulosclerosis with thicker basement membranes and abnormal podocytes. Some of the mutant mice also have lens abnormalities consistent with early cataract formation. Our results show that even heterozygous mutations in the mouse Myh9 gene can reproduce human MYH9-related diseases. These mouse models should be useful in understanding the pathophysiology of human MYH9-related diseases and also in designing and developing therapeutic stratagies.