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Homozygous Myo10 reporter knockout (Myo10 tm2/tm2 ) mouse embryos develop exencephaly. (A) Pigmentation defects in homozygous mutants. Image of an adult Myo10 tm2/tm2 mouse showing white belly spots. (B) Genotype frequency of offspring derived from HET (heterozygous) x HET and HET x HOM (homozygous) matings. HOM offspring were produced at less than expected frequency, indicated by dashed lines and asterices. (C) Two examples of X-gal stained, homozygous Myo10 mutant embryos at E14.5, with (left) and without (right) exencephalus, caused by failure of the cranial neural tube to close. The white arrow on the left indicates everted cranial neural folds, a hallmark of exencephalus. About 1 in 4 (24%) of homozygous Myo10 mutant (Myo10 tm2/tm2 ) embryos developed exencephalus. Neural tube defect is abbreviated NTD. (D) Enlarged view from panel C (yellow square) and skin histological section showing Myo10 expression in the skin and hair placodes (blue spots). (E) Whole-mount X-gal staining. Myo10 is expressed in the head and the first and second branchial arches (labeled 1 and 2, respectively) of the developing embryo (E8.5 and E9.5). (F) X-gal staining and histology (E10.5) reveals expression of Myo10 in the ectoderm and dorsal regions, but not in the neural tube. ht, heart; ov, otic vesicle; s, somite; nt, neural tube; D, dorsal; V, ventral; L, lateral.
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We investigated the physiological functions of Myo10 (myosin X) using Myo10 reporter knockout (Myo10tm2) mice. Full-length (motorized) Myo10 protein was deleted, but the brain-specific headless (Hdl) isoform (Hdl-Myo10) was still expressed in homozygous mutants. In vitro, we confirmed that Hdl-Myo10 does not induce filopodia, but it strongly locali...
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... of homozygous Myo10 reporter knockout (Myo10 tm2/tm2 ) mice. Homozygous, but not heterozygous, Myo10 reporter knockout mice consistently exhibited pigmentation defects, white belly spots ( Fig. 2A). Otherwise, homozygous mutants appeared healthy and fertile. However, mating of heterozygous (HET) mice (HET × HET) or heterozygous and homozygous (HOM) mice (HET × HOM) produced less homozy- gous mutant mice than expected by Mendelian inheritance (Fig. 2B). This discrepancy could be explained by the development of exencephalus, a ...
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... reporter knockout mice consistently exhibited pigmentation defects, white belly spots ( Fig. 2A). Otherwise, homozygous mutants appeared healthy and fertile. However, mating of heterozygous (HET) mice (HET × HET) or heterozygous and homozygous (HOM) mice (HET × HOM) produced less homozy- gous mutant mice than expected by Mendelian inheritance (Fig. 2B). This discrepancy could be explained by the development of exencephalus, a neural tube closure defect, in 24% of Myo10 tm2/tm2 embryos (Fig. 2C). Failure of the neural tube to close causes the neuroepithelium to protrude (exencephaly), which through degeneration progresses to anencephaly 31 . Whole-mount E14.5 Myo10 tm2/tm2 ( Fig. 2C) ...
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... and fertile. However, mating of heterozygous (HET) mice (HET × HET) or heterozygous and homozygous (HOM) mice (HET × HOM) produced less homozy- gous mutant mice than expected by Mendelian inheritance (Fig. 2B). This discrepancy could be explained by the development of exencephalus, a neural tube closure defect, in 24% of Myo10 tm2/tm2 embryos (Fig. 2C). Failure of the neural tube to close causes the neuroepithelium to protrude (exencephaly), which through degeneration progresses to anencephaly 31 . Whole-mount E14.5 Myo10 tm2/tm2 ( Fig. 2C) and Myo10 +/tm2 (not shown for E14.5) embryos were stained with X-gal to visualize Myo10 expression (X-gal becomes intensely blue following ...
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... (Fig. 2B). This discrepancy could be explained by the development of exencephalus, a neural tube closure defect, in 24% of Myo10 tm2/tm2 embryos (Fig. 2C). Failure of the neural tube to close causes the neuroepithelium to protrude (exencephaly), which through degeneration progresses to anencephaly 31 . Whole-mount E14.5 Myo10 tm2/tm2 ( Fig. 2C) and Myo10 +/tm2 (not shown for E14.5) embryos were stained with X-gal to visualize Myo10 expression (X-gal becomes intensely blue following cleavage by β-galactosidase, the enzyme encoded by the reporter gene lacZ). Myo10 expression (X-gal staining) could be clearly detected in the developing skin and hair placodes ( Fig. 2D). At E8.5 ...
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... Myo10 tm2/tm2 ( Fig. 2C) and Myo10 +/tm2 (not shown for E14.5) embryos were stained with X-gal to visualize Myo10 expression (X-gal becomes intensely blue following cleavage by β-galactosidase, the enzyme encoded by the reporter gene lacZ). Myo10 expression (X-gal staining) could be clearly detected in the developing skin and hair placodes ( Fig. 2D). At E8.5 -E9.5, Myo10 was expressed in the first and second branchial arches, as well as in the otic vesicle and somites (Fig. 2E). Myo10 was not detected in the heart (E9.5). Transverse sections of a paraffin embedded and X-gal stained E10.5 Myo10 tm2/tm2 embryo revealed Myo10 expression in the developing epidermis and dorsolaterally ...
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... (X-gal becomes intensely blue following cleavage by β-galactosidase, the enzyme encoded by the reporter gene lacZ). Myo10 expression (X-gal staining) could be clearly detected in the developing skin and hair placodes ( Fig. 2D). At E8.5 -E9.5, Myo10 was expressed in the first and second branchial arches, as well as in the otic vesicle and somites (Fig. 2E). Myo10 was not detected in the heart (E9.5). Transverse sections of a paraffin embedded and X-gal stained E10.5 Myo10 tm2/tm2 embryo revealed Myo10 expression in the developing epidermis and dorsolaterally in the dermis (Fig. ...
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... E8.5 -E9.5, Myo10 was expressed in the first and second branchial arches, as well as in the otic vesicle and somites (Fig. 2E). Myo10 was not detected in the heart (E9.5). Transverse sections of a paraffin embedded and X-gal stained E10.5 Myo10 tm2/tm2 embryo revealed Myo10 expression in the developing epidermis and dorsolaterally in the dermis (Fig. ...
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... into epidermal keratinocytes 22 . If Myo10 was important for melanosome transfer, deletion of full-length Myo10 would be expected to produce a coat color phenotype similar to Myo5a-deficient mice 37 , which have widespread hypopigmentation. Instead, Myo10 tm2/tm2 mice have white belly patches, useful as an indicator of the homozygous genotype ( Fig. 2A), and infrequently a dorsal white patch (Fig. 6A). We con- firmed using histological skin sections and antibodies against DCT (dopachrome tautomerase), a melanocyte marker, that the hair follicles in white patches were not populated with melanocytes (Fig. ...
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... (68% versus 24%) in Myo10 null (Myo10 tm1d/tm1d ) 27 versus Myo10 tm2/tm2 mice 28 . Thus, headless Myo10, which strongly localizes to the plasma membrane (see Figs 3 and 4), may partially compensate for loss of full-length Myo10 during neurulation. X-gal staining for Myo10 expression in Myo10 tm2 embryos appeared negative at the neural fold (see Fig. 2E). However, the expression of headless-Myo10 is prob- ably not reported in Myo10 tm2 mutants since the lacZ reporter gene is located 5′ upstream of headless Myo10 transcripts (see Fig. 1), whereas both full-length and headless Myo10 are expected to be reported by X-gal staining in Myo10 tm1a embryos 27 ...
Similar publications
Waardenburg syndrome is a rare autosomal dominant genetic disorder of neural crest cell migration. It is characterized by congenital sensorineural hearing loss, heterochromia iridis, depigmentation of hair and skin, and increased intercanthal distance. It is subdivided into four subtypes with I and II being most common. These subtypes are categoriz...
Citations
... Experimental depletion of MYO10 from cultured mouse GOCs was associated with a reduction in the number of actin-TZPs, and the natural loss of actin-TZPs during oocyte maturation was accompanied by a reduction on the number of MYO10 foci. On the other hand, mice lacking Myo10 are fertile, although a detailed examination of their reproductive capacity has not been reported [54,55]. Taken together, these results suggest that MYO10 increases the efficiency of the formation or maintenance of actin-TZPs. ...
Granulosa cells of growing ovarian follicles elaborate filopodia-like structures termed transzonal projections (TZPs) that supply the enclosed oocyte with factors essential for its development. Little is known, however, of the mechanisms underlying the generation of TZPs. We show in mouse and human that filopodia, defined by an actin backbone, emerge from granulosa cells in early-stage primary follicles and that actin-rich TZPs become detectable as soon as a space corresponding to the zona pellucida appears. mRNA encoding Myosin10 (MYO10), a motor protein that accumulates at the base and tips of filopodia and has been implicated in their initiation and elongation, is present in granulosa cells and oocytes of growing follicles. MYO10 protein accumulates in foci located mainly between the oocyte and innermost layer of granulosa cells, where it co-localizes with actin. In both mouse and human, the number of MYO10 foci increases as oocytes grow, corresponding to the increase in the number of actin-TZPs. RNAi-mediated depletion of MYO10 in cultured mouse granulosa cell-oocyte complexes is associated with a 52% reduction in the number of MYO10 foci and a 28% reduction in the number of actin-TZPs. Moreover, incubation of cumulus-oocyte complexes in the presence of epidermal growth factor, which triggers a 93% reduction in the number of actin-TZPs, is associated with a 55% reduction in the number of MYO10 foci. These results suggest that granulosa cells possess an ability to elaborate filopodia, which when directed towards the oocyte become actin-TZPs, and that MYO10 increases the efficiency of formation or maintenance of actin-TZPs.
... On the other hand, some of these genes displaying signatures of positive selection in the human lineage have not been completely characterized in their specific function in the inner ear (ATP2B1 and MYO10A). While other members of the myosin family were regarded as having at least one type of human-specific selection [62][63][64][65], an interesting candidate is MYO10A, an unconventional myosin that produces a Waardenburg-like syndrome when deleted in mice [66]. ...
Background
Mammals possess unique hearing capacities that differ significantly from those of the rest of the amniotes. In order to gain insights into the evolution of the mammalian inner ear, we aim to identify the set of genetic changes and the evolutionary forces that underlie this process. We hypothesize that genes that impair hearing when mutated in humans or in mice (hearing loss (HL) genes) must play important roles in the development and physiology of the inner ear and may have been targets of selective forces across the evolution of mammals. Additionally, we investigated if these HL genes underwent a human-specific evolutionary process that could underlie the evolution of phenotypic traits that characterize human hearing.
Results
We compiled a dataset of HL genes including non-syndromic deafness genes identified by genetic screenings in humans and mice. We found that many genes including those required for the normal function of the inner ear such as LOXHD1 , TMC1 , OTOF , CDH23 , and PCDH15 show strong signatures of positive selection. We also found numerous noncoding accelerated regions in HL genes, and among them, we identified active transcriptional enhancers through functional enhancer assays in transgenic zebrafish.
Conclusions
Our results indicate that the key inner ear genes and regulatory regions underwent adaptive evolution in the basal branch of mammals and along the human-specific branch, suggesting that they could have played an important role in the functional remodeling of the cochlea. Altogether, our data suggest that morphological and functional evolution could be attained through molecular changes affecting both coding and noncoding regulatory regions.
... The essential role of MYO10 in filopodia formation led to the suggestion that deletion of this motor would be lethal for a multicellular organism. A large fraction of homozygous Myo10 -/null mutant mouse embryos die due to exencephaly, but a significant number still survive 48,49 . The brains of the surviving Myo10 -/-embryos do not exhibit any gross abnormalities, but it remains to be seen if these animals have defects in cognitive function 48,49 . ...
... A large fraction of homozygous Myo10 -/null mutant mouse embryos die due to exencephaly, but a significant number still survive 48,49 . The brains of the surviving Myo10 -/-embryos do not exhibit any gross abnormalities, but it remains to be seen if these animals have defects in cognitive function 48,49 . The phenotypes of the surviving Myo10 -/mice are consistent with the loss of filopodia, including failed migration of melanocytes resulting in the appearance of a characteristic white belly spot, syndactyly (webbed digits), and abnormalities in the retinal vasculature due to persistent hyaloid vasculature 48,49 . ...
... The brains of the surviving Myo10 -/-embryos do not exhibit any gross abnormalities, but it remains to be seen if these animals have defects in cognitive function 48,49 . The phenotypes of the surviving Myo10 -/mice are consistent with the loss of filopodia, including failed migration of melanocytes resulting in the appearance of a characteristic white belly spot, syndactyly (webbed digits), and abnormalities in the retinal vasculature due to persistent hyaloid vasculature 48,49 . The surprising survival of the Myo10 -/mice and the $50% reduction in filopodia that occurs during retinal angiogenesis in these mutants indicates that cells and tissues have strategies to bypass the loss of MYO10 and can use alternative pathways to make filopodia. ...
Filopodia, microvilli and stereocilia represent an important group of plasma membrane protrusions. These specialized projections are supported by parallel bundles of actin filaments and have critical roles in sensing the external environment, increasing cell surface area, and acting as mechanosensors. While actin-associated proteins are essential for actin-filament elongation and bundling in these protrusions, myosin motors have a surprising role in the formation and extension of filopodia and stereocilia and in the organization of microvilli. Actin regulators and specific myosins collaborate in controlling the length of these structures. Myosins can transport cargoes along the length of these protrusions, and, in the case of stereocilia and microvilli, interactions with adaptors and cargoes can also serve to anchor adhesion receptors to the actin-rich core via functionally conserved motor–adaptor complexes. This review highlights recent progress in understanding the diverse roles myosins play in filopodia, microvilli and stereocilia.
... MYO10 is an unconventional myosin that regulates the formation and elongation of filopodia and other actin-based protrusions that are important for cancer invasion, such as filopodialike long protrusions and invadopodia (16). MYO10 loss in mice leads to severe developmental defects in several collective migrationdependent processes (40)(41)(42), which suggests that MYO10 regulates collective migration in development and cancer. We show that MYO10 is enriched in leader cells across multiple NSCLC cell lines and a patient-derived lung NSCLC cell line, and we further demonstrate that MYO10-driven filopodia are critical for leader-driven lung cancer collective invasion (Fig. 3). ...
Tumor heterogeneity drives disease progression, treatment resistance, and patient relapse, yet remains largely underexplored in invasion and metastasis. Here, we investigated heterogeneity within collective cancer invasion by integrating DNA methylation and gene expression analysis in rare purified lung cancer leader and follower cells. Our results showed global DNA methylation rewiring in leader cells and revealed the filopodial motor MYO10 as a critical gene at the intersection of epigenetic heterogeneity and three-dimensional (3D) collective invasion. We further identified JAG1 signaling as a previously unknown upstream activator of MYO10 expression in leader cells. Using live-cell imaging, we found that MYO10 drives filopodial persistence necessary for micropatterning extracellular fibronectin into linear tracks at the edge of 3D collective invasion exclusively in leaders. Our data fit a model where epigenetic heterogeneity and JAG1 signaling jointly drive collective cancer invasion through MYO10 up-regulation in epigenetically permissive leader cells, which induces filopodia dynamics necessary for linearized fibronectin micropatterning.
... Myo10 -/-mice are semi-lethal with ~60% of homozygous mutants exhibiting exencephaly with embryonic or perinatal lethality. Surviving animals display white belly spots, with a subset of these animals also exhibiting syndactyly (27,29). Exencephaly and syndactyly can be attributed to reduced Bone Morphogenic Protein (BMP) signaling and de-repression, rather than disruption, of SHH signaling (30,31). ...
Morphogens function in concentration-dependent manners to instruct cell fate during tissue patterning. Molecular mechanisms by which these signaling gradients are established and reinforced remain enigmatic. The cytoneme transport model posits that specialized filopodia extend between morphogen-sending and responding cells to ensure that appropriate signal activation thresholds are achieved across developing tissues. How morphogens are transported along and deployed from cytonemes is not known. Herein we show that the actin motor Myosin 10 promotes cytoneme-based transport of Sonic Hedgehog (SHH) morphogen to filopodial tips, and that SHH movement within cytonemes occurs by vesicular transport. We demonstrate that cytoneme-mediated deposition of SHH onto receiving cells induces a rapid signal response, and that SHH cytonemes are promoted by a complex containing a ligand-specific deployment protein and associated co-receptor.
One-Sentence summary
Cytoneme-based delivery of the Sonic Hedgehog activation signal is promoted by Myosin 10 and BOC/CDON co-receptor function.
... Knockout. Myo10 is expressed in the retina and the TM [105][106][107]. Recent RNAi knockdown studies and knockout mouse models have revealed functional roles for Myo10 in the eye. ...
... Two Myo10 knockout mouse models were recently described, and both have posterior chamber ocular phenotypes ( Figure 5) [105,106]. The first knockout model ablated both full-length and headless forms of Myo10 [105]. ...
... A second Myo10 knockout mouse was developed, which selectively knocked out the full-length Myo10 molecule, the actin-binding form, but "headless" Myo10 expression was unaffected [106]. Similar to the complete Myo10 knockout, persistent hyaloid vasculature, white belly spots, and syndactyly were common, but there was a reduced rate of exencephaly (24%) (Figure 5(c)). ...
Cellular communication is an essential process for the development and maintenance of all tissues including the eye. Recently, a new method of cellular communication has been described, which relies on formation of tubules, called tunneling nanotubes (TNTs). These structures connect the cytoplasm of adjacent cells and allow the direct transport of cellular cargo between cells without the need for secretion into the extracellular milieu. TNTs may be an important mechanism for signaling between cells that reside long distances from each other or for cells in aqueous environments, where diffusion-based signaling is challenging. Given the wide range of cargoes transported, such as lysosomes, endosomes, mitochondria, viruses, and miRNAs, TNTs may play a role in normal homeostatic processes in the eye as well as function in ocular disease. This review will describe TNT cellular communication in ocular cell cultures and the mammalian eye in vivo, the role of TNTs in mitochondrial transport with an emphasis on mitochondrial eye diseases, and molecules involved in TNT biogenesis and their function in eyes, and finally, we will describe TNT formation in inflammation, cancer, and stem cells, focusing on pathological processes of particular interest to vision scientists.
... Here we describe a chemotaxis assay using the same principle but using a closed chamber featuring four filling ports. Using this system and time-lapse, phase-contrast microscopy, we developed an assay to image mouse resident peritoneal macrophages migrating in a chemotactic complement C5a gradient 31,34,35,36 . This assay, combined with knockout mouse models, proved instrumental in the investigation of the roles of various Rho GTPases and motor proteins in macrophage morphology, motility, and chemotaxis 31,34,35,36,37 . ...
... Using this system and time-lapse, phase-contrast microscopy, we developed an assay to image mouse resident peritoneal macrophages migrating in a chemotactic complement C5a gradient 31,34,35,36 . This assay, combined with knockout mouse models, proved instrumental in the investigation of the roles of various Rho GTPases and motor proteins in macrophage morphology, motility, and chemotaxis 31,34,35,36,37 . We have also used this approach to image human peripheral blood monocytes migrating on a 2D surface or in a 3D collagen type I matrix 38 . ...
Filopodia are dynamic cell surface protrusions used for cell motility, pathogen infection, and tissue development. The molecular mechanisms determining how and where filopodia grow and retract need to integrate mechanical forces and membrane curvature with extracellular signaling and the broader state of the cytoskeleton. The involved actin regulatory machinery nucleates, elongates, and bundles actin filaments separately from the underlying actin cortex. The refined membrane and actin geometry of filopodia, importance of tissue context, high spatiotemporal resolution required, and high degree of redundancy all limit current models. New technologies are improving opportunities for functional insight, with reconstitution of filopodia in vitro from purified components, endogenous genetic modification, inducible perturbation systems, and the study of filopodia in multicellular environments. In this review, we explore recent advances in conceptual models of how filopodia form, the molecules involved in this process, and our latest understanding of filopodia in vitro and in vivo.
Expected final online publication date for the Annual Review of Cell and Developmental Biology, Volume 39 is October 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Non-syndromic cleft lip with/without cleft palate (nsCL/P) is a highly heritable facial disorder. To date, systematic investigations of the contribution of rare variants in non-coding regions to nsCL/P etiology are sparse. Here, we re-analyzed available whole genome sequence (WGS) data from 211 European case-parent trios with nsCL/P and identified 13,522 de novo mutations (DNMs) in nsCL/P cases, 13,055 of which mapped to non-coding regions. We integrated these data with DNMs from a reference cohort, with results of previous genome-wide association studies (GWAS), and functional and epigenetic datasets of relevance to embryonic facial development. A significant enrichment of nsCL/P DNMs was observed at two GWAS risk loci (4q28.1 (P=8x10⁻⁴) and 2p21 (P=0.02)), suggesting a convergence of both common and rare variants at these loci. We also mapped the DNMs to 810 position weight matrices indicative of transcription factor (TF) binding, and quantified the effect of the allelic changes in silico. This revealed a nominally significant overrepresentation of DNMs (P=0.037), and a stronger effect on binding strength, for DNMs located in the sequence of the core binding region of the TF Musculin (MSC). Notably, MSC is involved in facial muscle development, together with a set of nsCL/P genes located at GWAS loci. Supported by additional results from single-cell transcriptomic data and molecular binding assays, this suggests that variation in MSC binding sites contributes to nsCL/P etiology. Our study describes a set of approaches that can be applied to increase the added value of WGS data.
