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Non-muscle Myosin IIA (NMIIA) regulates mouse lens cellular differentiation during development and cataract formation
Date
2025
Authors
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Publisher
University of Delaware
Abstract
The ocular lens is a transparent tissue in the anterior chamber of the eye that is responsible for focusing light onto the retina for clear vision. A common condition with lens aging is cataracts, defined as opacities in the lens. The lens grows throughout our lifetime, making it a unique model system to study tissue morphogenesis & age-dependent diseases. The lens comprises a monolayer of epithelial cells covering the anterior hemisphere and a bulk mass of fiber cells. Lens epithelial cells (LECs) proliferate in the germinative zone and then migrate to the equator. At the equator, the LECs transform from randomly packed cells to precisely aligned and hexagon-shaped meridional row (MR) cells. The MR cells then differentiate into fiber cells that retain the precise alignment, hexagon shape, and packing from their precursor MR cells. The exact mechanisms through which these cell shapes and packing transformations occur at the lens equators are poorly understood. ☐ Non-muscle myosin IIs (NMIIs) are motor proteins that self-associate to form bipolar filaments and pull actin filaments (F-actin) to generate contractile forces. In other systems, actomyosin contractility regulates cell shape changes, ordered packing, migration, and cytokinesis. MYH9-related disease (MYH9-RD) is an autosomal dominant genetic disorder caused by mutations in the MYH9 gene that encodes NMIIA heavy chain. Patients with MYH9 mutations present with bleeding disorders but can also exhibit kidney disease, liver disease, hearing loss, and presenile cataracts. The three most common MYH9 mutations are R702C (located in motor domain, reducing ATP hydrolysis and F-actin translocation), D1424N (located in rod domain, affecting actomyosin interactions), and E1841K (located in rod domain, impairing NMIIA bipolar filament assembly). Full-body genetic knock-in mice with these mutations have been previously generated by Robert S. Adelstein at the National Heart, Blood, and Lung Institute. These knock-in mice provide us with an opportunity to examine the role of NMIIA in lens development, cellular morphogenesis, and cataract formation. ☐ Therefore, I initially evaluated lens transparency in 2-month-old control mice and mice with Myh9 mutations (R702C, D1424N, and E1841K). Mice homozygous for R702C and D1424N mutations do not survive. At 2 months of age, both control and mutant (R702C, D1424N, and E1841K) mice exhibited normal-sized transparent lenses. Next, I examined the 2-month-old mouse lenses for cellular defects by performing lens whole mount and/or immunofluorescence confocal microscopy of lens sections. NMIIAGFP-R702C/+ mice exhibit ordered packing suggesting either endogenous-NMIIA allele is sufficient or NMIIB can compensate for mutant motor domain function. NMIIAD1424N/+, NMIIAE1841K/+, and NMIIAE1841K/E1841K mice exhibit irregular fiber cell organization. As NMIIA is predominantly expressed in LECs, I hypothesized that the fiber cell disorder arises due to the irregular packing of MR cells. I examined MR cell alignment, shape, and packing organization and discovered that NMIIAD1424N/+ mice exhibit mild irregular packing defects. MR cells of NMIIAE1841K/E1841K mice display misalignment, aberrant cell shape, and irregular packing. While 92% of NMIIA+/+ MR cells have six neighbors, ~80% of the NMIIAE1841K/E1841K MR cells have six neighbors and 17% of the NMIIAE1841K/E1841K MR cells have five or seven neighbors. These data suggest that NMIIA rod domain function is critical in forming and/or maintaining the hexagonal packing of MR and fiber cells. ☐ Because NMIIAE1841K/E1841K mice exhibit the most defect in hexagonal packing compared to any other mutants, I focused on the NMIIA-E1841K mutation to further examine the role of NMIIA in MR cell hexagonal patterning. Immunofluorescence microscopy of MR cells demonstrates increased enrichment of NMIIA, N-cadherin, and vinculin at AP-oriented sides of NMIIA+/+ MR cells, but equal distributions on all sides of NMIIAE1841K/E1841K MR cells. Further, F-actin is uniformly distributed around all edges of NMIIA+/+ MR cells but reduced at the AP-oriented edges of NMIIAE1841K/E1841K MR cells. Employing Bayesian Mechanical Inference, we discovered that MR cells in NMIIA+/+ lenses exhibit an anisotropic junctional tension, in which relative tension is more concentrated at the anterior-posteriorly (AP) oriented edges. In contrast, MR cells in NMIIAE1841K/E1841K lenses show isotropic junctional tension on all sides. Together, our data suggests that the NMIIA-E1841K mutation results in altered F-actin, NMIIA, N-cadherin, and vinculin distributions, disrupting the anisotropic orientational pattern of mechanical forces within the tissue, leading to disordered cell packing during mouse lens epithelial cell differentiation. So far, we have introduced a novel function of NMIIA during mouse lens cellular differentiation. ☐ As MYH9 mutations cause cataracts in humans and 2-month-old mutant mice (R702C, D1424N and E1841K) are transparent, we examined cataract incidences in aged control and mutant mice (8 months old) to determine if the mutations cause age-dependent cataracts. Lenses from 8-month-old NMIIAD1424N/+ and NMIIAE1841K/E1841K mice exhibit anterior opacity while lenses from NMIIA+/+, NMIIAGFP-R702C/+ and NMIIAE1841K/+ mice remain transparent. In particular, the NMIIAE1841K/E1841K mice exhibit a rare and highly understudied cataract named anterior polar pyramidal cataracts. Consequently, I used lenses from NMIIAE1841K/E1841K mice as a model to study the mechanisms driving the anterior polar pyramidal cataracts. ☐ First, I examined lens cellular morphology and structure from 8-month-old NMIIA+/+ and NMIIAE1841K/E1841K mice by performing whole mount. In NMIIA+/+ mice, the lens epithelial cells appear as a single layer of tightly connected polygonal flat cells that show apical-basal polarity. In addition, the lens capsule (extracellular matrix) appears as a continuous smooth structure located directly above the lens epithelial cells in the anterior region of the NMIIA+/+ lenses. On the contrary, in the cataract region of the NMIIAE1841K/E1841K lenses, I observe multiple layers of fibroblasts or mesenchymal cells that do not adhere and lack apical-basal polarity. Furthermore, the capsule appears highly irregular in the cataract region of the NMIIAE1841K/E1841K lenses. Gene expression suggests that the NMIIAE1841K/E1841K lenses with cataracts undergo epithelial-to-mesenchymal transition (EMT), in which the cells lose their epithelial marker and upregulate mesenchymal markers, further confirming the cellular phenotype observation. In addition, NMIIAE1841K/E1841K lenses with cataracts exhibit significantly decreased collagen IV (a marker for the lens capsule) expression, while upregulating fibronectin 1 (an extracellular matrix protein; typically not present in the native lens capsule), indicating that the lens capsule structure and composition are being remodeled in the mutant lenses with cataracts. This study uncovers a new role of NMIIA in EMT, thus opening up a new research area for defining the mechanistic basis of how the NMIIA-E1841K mutation results in initiation of EMT. ☐ Overall, my work highlights the function of NMIIA in lens epithelial cell differentiation, in the context of lens development and cataract formation with aging. Further, in addition to lens biology, my research findings provide new directions for investigations in epithelial-to-mesenchymal transition, a key cellular behavior of general importance, that is relevant to the understanding of other pathologies such as cancer.
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Keywords
Cancer, Meridional row cells, Lens epithelial cells, Ocular lens, MYH9-related disease