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Myo10 m1J /m1J mice have a white belly spot and persistent hyaloid vasculature plus a high frequency of exencephaly and other abnormalities. (A) Immunoblot of whole brain lysates from 1 month old mice showing deletion of full-length and headless Myo10. Although bands corresponding to full-length and headless Myo10 are absent in the Myo10 m1J /m1J lysate, it should be noted that the Myo10 antibody shows some non-specific staining, including one band that is slightly larger than headless and another that is near the 100 kDa standard. The signal from the same blot stained with anti-actin (43 kDa) is shown in the red channel as a loading control and has been overlaid onto the black and white Myo10 signal. (B) Total number of pups obtained for each genotype from heterozygous matings showing that Myo10 m1J /m1J homozygotes were obtained at less than half the expected number (n = 28 litters, 174 pups total; mean litter size 6.2). (C) E15.5 embryos showing a Myo10 +/+ control and an exencephalic Myo10 m1J /m1J littermate. (D) Adult Myo10 m1J/m1J mouse showing examples of a white belly spot, a white spot on the back, and syndactyly. (E) Eyecups from 6-week old mice showing a normal eye from a Myo10 +/+ mouse and an example of the pigmented mass of persistent hyaloid vasculature present in the eyes of Myo10 m1J/m1J mice. (F) Rostral coronal sections from E16.5 embryos showing
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Myosin-X (Myo10) is an unconventional myosin best known for its striking localization to the tips of filopodia. Despite the broad expression of Myo10 in vertebrate tissues, its functions at the organismal level remain largely unknown. We report here the generation of KO-first (Myo10 tm1a/tm1a ), floxed (Myo10 tm1c/tm1c ), and KO mice (Myo10 tm1d/tm...
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... of whole brain from a month-old Myo10 m1J/m1J mouse show that neither full-length nor headless Myo10 was detected, demonstrating that the mutation results in complete loss of Myo10 (Fig. 5A). Myo10 +/m1J heterozygotes appeared normal, but when they were mated to one another, pups were obtained in the ratio of 1 Myo10 +/+ : 2.3 Myo10 +/m1J : 0.3 Myo10 m1J/m1J (Fig. 5B). The much lower than expected number of Myo10 m1J/m1J pups (14 Myo10 m1J/m1J nulls versus 48 Myo10 +/+ ) again suggested that loss of Myo10 leads a high ...
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... from a month-old Myo10 m1J/m1J mouse show that neither full-length nor headless Myo10 was detected, demonstrating that the mutation results in complete loss of Myo10 (Fig. 5A). Myo10 +/m1J heterozygotes appeared normal, but when they were mated to one another, pups were obtained in the ratio of 1 Myo10 +/+ : 2.3 Myo10 +/m1J : 0.3 Myo10 m1J/m1J (Fig. 5B). The much lower than expected number of Myo10 m1J/m1J pups (14 Myo10 m1J/m1J nulls versus 48 Myo10 +/+ ) again suggested that loss of Myo10 leads a high level of embry- onic or perinatal lethality. Analysis of embryos (E12.5-E17.5) from heterozygous matings revealed that 22/88 (25%) were Myo10 m1J/m1J homozygotes, indicating that null ...
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... that loss of Myo10 leads a high level of embry- onic or perinatal lethality. Analysis of embryos (E12.5-E17.5) from heterozygous matings revealed that 22/88 (25%) were Myo10 m1J/m1J homozygotes, indicating that null embryos were present in the expected Mendelian percentage. Examination of the 22 null embryos revealed that 73% had exencephaly (Fig. 5C), 18% had craniora- chischisis (a major failure of neural tube closure at the level of the hindbrain and spinal cord), and 9% had a gross developmental defect such as very small size. This demonstrates that a large fraction of Myo10 m1J/m1J embryos have exencephaly and/or other defects that would be incompatible with survival past ...
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... the 14 homozygous Myo10 m1J/m1J pups obtained above, 6/14 were male, 14/14 had a white belly spot, 4/14 also had a white spot on the back, 6/14 had webbed digits on one or both forelimbs (Fig. 5D). Kinked tails were not obvious in Myo10 m1J/m1J mice and were not scored due to the difficulty of defining very mild kinks. Anophthalmia was present in 1/14 of the Myo10 m1J/m1J mice and 3/14 had severe microphthalmia. All 5 of the Myo10 m1J/m1J homozygotes whose eyes were dissected at 6 weeks had persistent hyaloid vasculature, and ...
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... in Myo10 m1J/m1J mice and were not scored due to the difficulty of defining very mild kinks. Anophthalmia was present in 1/14 of the Myo10 m1J/m1J mice and 3/14 had severe microphthalmia. All 5 of the Myo10 m1J/m1J homozygotes whose eyes were dissected at 6 weeks had persistent hyaloid vasculature, and this was pigmented and present bilaterally (Fig. 5E). The average weight at weaning (P21) for Myo10 m1J/m1J pups was 8.5 ± 1.5 g (N = 7), which is slightly smaller than the 11.3 ± 0.7 g for Myo10 +/m1J (N = 38, p = 0.00002) and 11.6 ± 0.5 g for Myo10 +/+ (N = 18; p = 0.00002). Similar results were obtained at P28 (Supplementary Figure 1). Myo10 m1J/m1J homozygotes that survived birth ...
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... that it includes a dense plexus of hyaloid vasculature intermixed with pigment cells. The plexus can be seen most clearly after enlargement on a digital display. To further investigate the defects resulting from loss of Myo10, hematoxylin and eosin stained sections were prepared from the heads of control and Myo10 m1J/m1J embryos at E16.5 (Fig. 5F). Rostral coronal sections show relatively normal development of the brain and cranium in Myo10 m1J/m1J embryos that were not exencephalic, as well as the absence of a cranium and gross defects in brain morphology in an exencephalic embryo. Higher mag- nification views of Myo10 m1J/m1J eyes at E16.5 revealed examples of abnormally ...
Citations
... The MYO10 gene is an integral member of the myosin family, which is involved in various cellular processes such as dynamic actin remodeling, cell migration and adhesion, and filopodia formation 89,90 . Our findings are in accordance with previous studies indicating that the MYO10 gene plays a key role in mediating skin pigmentation through regulating melanosome transportation in the skin [91][92][93] . It was demonstrated that the melanocytes present in the skin control the quantity and types of melanosomes, ultimately determining the coat color 94 . ...
Copy number variations (CNVs) are structural variants consisting of duplications and deletions of DNA segments, which are known to play important roles in the genetics of complex traits in livestock species. However, CNV-based genome-wide association studies (GWAS) have remained unexplored in American mink. Therefore, the purpose of the current study was to investigate the association between CNVs and complex traits in American mink. A CNV-based GWAS was performed with the ParseCNV2 software program using deregressed estimated breeding values of 27 traits as pseudophenotypes, categorized into traits of growth and feed efficiency, reproduction, pelt quality, and Aleutian disease tests. The study identified a total of 10,137 CNVs (6968 duplications and 3169 deletions) using the Affymetrix Mink 70K single nucleotide polymorphism (SNP) array in 2986 American mink. The association analyses identified 250 CNV regions (CNVRs) associated with at least one of the studied traits. These CNVRs overlapped with a total of 320 potential candidate genes, and among them, several genes have been known to be related to the traits such as ARID1B, APPL1, TOX, and GPC5 (growth and feed efficiency traits); GRM1, RNASE10, WNT3, WNT3A, and WNT9B (reproduction traits); MYO10, and LIMS1 (pelt quality traits); and IFNGR2, APEX1, UBE3A, and STX11 (Aleutian disease tests). Overall, the results of the study provide potential candidate genes that may regulate economically important traits and therefore may be used as genetic markers in mink genomic breeding programs.
... Mammalian Myosin VIIa and Myosin XV play roles in maintaining the function of stereocilia: VIIA maintains the integrity of the tip complex to help organize the characteristic 'staircase' organization of inner ear hair cells (Boeda, 2002;Self et al., 1998), where as XV trafficks actin regulators to stereocilia tips that help control the structure's length (Belyantseva et al., 2005;Manor et al., 2011). Mammalian Myosin VIIb is involved in the maintenance and formation of microvilli (Weck et al., 2016), while Myosin X is closely associated with the formation and function of filopodia (Berg and Cheney, 2002;Heimsath et al., 2017). Notably, Myosin X has also been shown to play a role in the trafficking of signaling molecules in cytonemes, as well as in the formation cytonemes (Hall et al., 2021;Snyder et al., 2015). ...
The self-organization of cells during development is essential for the formation of healthy tissues, and requires the coordination of cell activities at local scales. Cytonemes, or signaling filopodia, are dynamic actin-based cellular protrusions that allow cells to engage in contact mediated signaling at a distance. While signaling filopodia have been shown to support several signaling paradigms during development, less is understood about how these protrusions are regulated. We investigated the role of the plus-end directed, unconventional MyTH4-FERM myosins in regulating signaling filopodia during sensory bristle patterning on the dorsal thorax of the fruit fly Drosophila melanogaster. We found that Myosin XV is required for regulating signaling filopodia dynamics and, as a consequence, lateral inhibition more broadly throughout the patterning epithelium. We found that Myosin XV is required for limiting the length and number of signaling filopodia generated by bristle precursor cells. Cells with additional and longer signaling filopodia due to loss of Myosin XV are not signaling competent, due to altered levels of Delta ligand and Notch receptor along their lengths. We conclude that Myosin XV acts to negatively regulate signaling filopodia, as well as promote the ability of signaling filopodia to engage in long-range Notch signaling. Since Myosin XV is present across several vertebrate and invertebrate systems, this may have significance for other long-range signaling mechanisms.
... Myo10's role in filopodia has been widely investigated [2][3][4][5][6][7][8][9][10][11][12][13][14] . Myo10 expression increases dorsal filopodia, and Myo10 overexpression induces many Myo10 tip-localized filopodia 4,15,16 . ...
Myosin 10 (Myo10) is a vertebrate-specific motor protein well known for its role in filopodia formation. Although Myo10-driven filopodial dynamics have been characterized, there is no information about the numbers of Myo10 in filopodia. To better understand molecular stoichiometries and packing restraints in filopodia, we measured Myo10 abundance in these structures. Here we combined SDS-PAGE analysis with epifluorescence microscopy to quantitate HaloTag-labeled Myo10 in U2OS cells. About 6% of total intracellular Myo10 localizes to filopodia, where it tends to be enriched at opposite ends of the cell. Hundreds of Myo10 are found in a typical filopodium, and their distribution across filopodia is log-normal. Some filopodial tips even contain more Myo10 than accessible binding sites on the actin filament bundle. Our estimates of Myo10 molecules in filopodia provide insight into the physics of packing Myo10, its cargo, and other filopodia-associated proteins in narrow membrane deformations in addition to the numbers of Myo10 required for filopodia initiation. Our protocol provides a framework for future work analyzing Myo10 abundance and distribution upon perturbation.
... In this study, we sought to characterize the contribution of TZPs to oocyte development by impairing the structure of TZPs. As a candidate gene, we selected myosin-X (MYO10), a structural component of TZPs (7) known to promote filopodium formation (12,13,14) and potentially involved in the formation or maintenance of TZPs (15). We generated a full knockout mouse of Myo10 in all cell types to completely deplete it from the ovarian follicle (Myo10 −/− full). ...
... To deplete TZPs, we generated full knockout mice lacking the TZP component MYO10 in all cell types (Fig S1A-D). Loss of MYO10 expression in mice was previously characterized as semi-lethal, with some animals dying during embryogenesis whereas others being able to develop until adulthood (14). Similar to this study, we obtained adult Myo10 −/− mice (named Myo10 −/− full thereafter) and most showed phenotypes characteristic of the earlier described Myo10 full knockout, such as the presence of a white spot on the abdomen and webbed fingers (14) (Fig S1B). ...
The oocyte must grow and mature before fertilization, thanks to a close dialogue with the somatic cells that surround it. Part of this communication is through filopodia-like protrusions, called transzonal projections (TZPs), sent by the somatic cells to the oocyte membrane. To investigate the contribution of TZPs to oocyte quality, we impaired their structure by generating a full knockout mouse of the TZP structural component myosin-X (MYO10). Using spinning disk and super-resolution microscopy combined with a machine-learning approach to phenotype oocyte morphology, we show that the lack of Myo10 decreases TZP density during oocyte growth. Reduction in TZPs does not prevent oocyte growth but impairs oocyte-matrix integrity. Importantly, we reveal by transcriptomic analysis that gene expression is altered in TZP-deprived oocytes and that oocyte maturation and subsequent early embryonic development are partially affected, effectively reducing mouse fertility. We propose that TZPs play a role in the structural integrity of the germline–somatic complex, which is essential for regulating gene expression in the oocyte and thus its developmental potential.
... However, in spite of their ability to induce filopodia, expression of either MyoX or active mDia2 fails to rescue microspike formation in these mutants. In line with this notion, loss of MyoX completely abolishes microspikes in B16-F1 cells [31], whereas the knockdown of MyoX in endothelial cells decreases filopodia number only by 50% [38]. ...
Background:
Filopodia are dynamic, finger-like actin-filament bundles that overcome membrane tension by forces generated through actin polymerization at their tips to allow extension of these structures a few microns beyond the cell periphery. Actin assembly of these protrusions is regulated by accessory proteins including heterodimeric capping protein (CP) or Ena/VASP actin polymerases to either terminate or promote filament growth. Accordingly, the depletion of CP in B16-F1 melanoma cells was previously shown to cause an explosive formation of filopodia. In Ena/VASP-deficient cells, CP depletion appeared to result in ruffling instead of inducing filopodia, implying that Ena/VASP proteins are absolutely essential for filopodia formation. However, this hypothesis was not yet experimentally confirmed.
Methods:
Here, we used B16-F1 cells and CRISPR/Cas9 technology to eliminate CP either alone or in combination with Ena/VASP or other factors residing at filopodia tips, followed by quantifications of filopodia length and number.
Results:
Unexpectedly, we find massive formations of filopodia even in the absence of CP and Ena/VASP proteins. Notably, combined inactivation of Ena/VASP, unconventional myosin-X and the formin FMNL3 was required to markedly impair filopodia formation in CP-deficient cells.
Conclusions:
Taken together, our results reveal that, besides Ena/VASP proteins, numerous other factors contribute to filopodia formation.
... This unidirectional organization allows molecular motors, such as myosin-X (MYO10), to walk along filopodia and accumulate at their tips (at ∼600 nm/s) (Kerber et al., 2009). By doing so, MYO10 is thought to transport various proteins to filopodia tips, modulating filopodia function (Jacquemet et al., 2015;Arjonen et al., 2014;Berg and Cheney, 2002;Hirano et al., 2011;Zhang et al., 2004). However, only a very few MYO10 cargoes have been proposed to date, with the netrin DCC receptor (Zhu et al., 2007;Wei et al., 2011), integrins (Zhang et al., 2004;Wei et al., 2011) and VASP (Tokuo and Ikebe, 2004) being the principal ones. ...
... Interestingly, both MYO10 and RAPH1 have been implicated separately as positive regulators of cancer cell migration and invasion in similar contexts (Arjonen et al., 2014;Carmona et al., 2016). In addition, MYO10 and RAPH1 knockout mice share similar phenotypes, such as white belly patches due to defective melanoblast migration (Heimsath et al., 2017;Law et al., 2013). Therefore, it is tempting to speculate that the MYO10-RAPH1 interaction occurring at filopodia tips has strong relevance in health and disease. ...
Myosin-X (MYO10), a molecular motor localizing to filopodia, is thought to transport various cargo to filopodia tips, modulating filopodia function. However, only a few MYO10 cargoes have been described. Here, using GFP-Trap and BioID approaches combined with mass spectrometry, we identified lamellipodin (RAPH1) as a novel MYO10 cargo. We report that the FERM domain of MYO10 is required for RAPH1 localization and accumulation at filopodia tips. Previous studies have mapped the RAPH1 interaction domain for adhesome components to its talin-binding and Ras-association domains. Surprisingly, we find that the RAPH1 MYO10-binding site is not within these domains. Instead, it comprises a conserved helix located just after the RAPH1 pleckstrin homology domain with previously unknown functions. Functionally, RAPH1 supports MYO10 filopodia formation and stability but is not required to activate integrins at filopodia tips. Taken together, our data indicate a feed-forward mechanism whereby MYO10 filopodia are positively regulated by MYO10-mediated transport of RAPH1 to the filopodium tip.
... In the epithelial cells, maintenance of membrane protrusions depends on myosins, which regulate dynamics of actin bundles [32]. Immunostaining for myosin 10 known to be involved in filopodia formation and cell adhesion [33] revealed significant changes in its intracellular distribution. In control ChPl cells, myosin 10 was uniformly concentrated in the vicinity of plasma membrane, whereas in Atp7b -/-ChPl the myosin pattern was disorganized with a weaker staining at the apical membrane and a stronger staining towards the basolateral membrane (Fig 4C). ...
Copper (Cu) has a multifaceted role in brain development, function, and metabolism. Two homologous Cu transporters, Atp7a (Menkes disease protein) and Atp7b (Wilson disease protein), maintain Cu homeostasis in the tissue. Atp7a mediates Cu entry into the brain and activates Cu-dependent enzymes, whereas the role of Atp7b is less clear. We show that during postnatal development Atp7b is necessary for normal morphology and function of choroid plexus (ChPl). Inactivation of Atp7b causes reorganization of ChPl' cytoskeleton and cell-cell contacts, loss of Slc31a1 from the apical membrane, and a decrease in the length and number of microvilli and cilia. In ChPl lacking Atp7b, Atp7a is upregulated but remains intracellular, which limits Cu transport into the brain and results in significant Cu deficit, which is reversed only in older animals. Cu deficiency is associated with down-regulation of Atp7a in locus coeruleus and catecholamine imbalance, despite normal expression of dopamine-β-hydroxylase. In addition, there are notable changes in the brain lipidome, which can be attributed to inhibition of diacylglyceride-to-phosphatidylethanolamine conversion. These results identify the new role for Atp7b in developing brain and identify metabolic changes that could be exacerbated by Cu chelation therapy.
... Indeed, Myo10 knockout mice showed reduced Shh domain in the floor plate associated with patterning defects which highlight the functional relevance of cytonemes in Shh gradient formation and activity [168]. Nevertheless, Myo10 knockout mice did not exhibit phenotypes similar to Shh-deficient mice, such as holoprosencephaly, suggesting that other trafficking mechanisms or different molecular motors are involved in the vesicular trafficking of Shh in the brain [174]. ...
A morphogen is a signaling molecule that induces specific cellular responses depending on its local concentration. The concept of morphogenic gradients has been a central paradigm of developmental biology for decades. Sonic Hedgehog (Shh) is one of the most important morphogens that displays pleiotropic functions during embryonic development, ranging from neuronal patterning to axon guidance. It is commonly accepted that Shh is distributed in a gradient in several tissues from different origins during development; however, how these gradients are formed and maintained at the cellular and molecular levels is still the center of a great deal of research. In this review, we first explored all of the different sources of Shh during the development of the nervous system. Then, we detailed how these sources can distribute Shh in the surrounding tissues via a variety of mechanisms. Finally, we addressed how disrupting Shh distribution and gradients can induce severe neurodevelopmental disorders and cancers. Although the concept of gradient has been central in the field of neurodevelopment since the fifties, we also describe how contemporary leading-edge techniques, such as organoids, can revisit this classical model.
... The knockdown of MyoX has previously been reported to decrease filopodia in HeLa cells (13). Quantification of filopodia in endothelial cells derived from Myo10-null mouse strains subsequently revealed a ~50% decrease in filopodia, strongly suggesting that MyoX promotes the formation of normal filopodia numbers (54). Comparable to VASP, we identified ectopically expressed, EGFP-tagged MyoX as well as endogenous MyoX in prominent clusters at the tips of microspikes in B16-F1 cells (Fig. 4B). ...
Sheet-like membrane protrusions at the leading edge, termed lamellipodia, drive 2D-cell migration using active actin polymerization. Microspikes comprise actin-filament bundles embedded within lamellipodia, but the molecular mechanisms driving their formation and their potential functional relevance have remained elusive. Microspike formation requires the specific activity of clustered Ena/VASP proteins at their tips to enable processive actin assembly in the presence of capping protein, but the factors and mechanisms mediating Ena/VASP clustering are poorly understood. Systematic analyses of B16-F1 melanoma mutants lacking potential candidate proteins revealed that neither inverse BAR-domain proteins, nor lamellipodin or Abi is essential for clustering, although they differentially contribute to lamellipodial VASP accumulation. In contrast, unconventional myosin-X (MyoX) identified here as proximal to VASP was obligatory for Ena/VASP clustering and microspike formation. Interestingly, and despite the invariable distribution of other relevant marker proteins, the width of lamellipodia in MyoX-KO mutants was significantly reduced as compared with B16-F1 control, suggesting that microspikes contribute to lamellipodium stability. Consistently, MyoX removal caused marked defects in protrusion and random 2D-cell migration. Strikingly, Ena/VASP-deficiency also uncoupled MyoX cluster dynamics from actin assembly in lamellipodia, establishing their tight functional association in microspike formation.
... According to the overlapping of differential genes among different skin color groups, the results showed that 19 differential genes belonged to the common differential genes among three groups (Figure 1d, Tables 1 and S6), among which eight genes were reported to be related to pigment production and regulation [8,14,[22][23][24][25][26], including: tyrp1a, slc2a11b, mlana, gch2, loc113060382, loc113079820, loc113068772 and loc113059134. The remaining genes mainly regulate cell proliferation, differentiation and apoptosis, including transcription elongation factor a protein 3, translation initiation factor if-2, RAS and EFhand domain containing, translocation protein Sec62, et al. ...
... Among these 19 genes, the expression level of five genes is the highest in the brown group, and two genes are related to the synthesis and regulation of melanin. The gene loc113068772 involves myosin, which is related to melanosome transport and melanin aggregation [25,26]; tyrp1a involves in dihydroxyindole carboxylic acid oxidase and participates in the production of melanin [8]. The expression levels of the remaining two genes were the highest in the white group, one was not annotated, and the other was related to Sec62. ...
Skin color is an important phenotypic feature of vertebrate fitness under natural conditions. Celestial goldfish, a common goldfish breed in China, mainly shows three kinds of skin colors including white, yellow and brown. However, the molecular genetic basis of this phenotype is still unclear. In this study, high-throughput sequencing was carried out on the back skin tissues of celestial goldfish with different skin colors. About 58.46 Gb of original data were generated, filtered and blasted, and 74,297 mRNAs were obtained according to the reference transcriptome. A total of 4653 differentially expressed genes were screened out among the brown, yellow and white groups, and the expression of melanogenesis related genes in brown goldfish was significantly higher than the other two groups. There are 19 common differentially expressed genes among three groups, of which eight genes are related to pigment production, including tyrp1a, slc2a11b, mlana, gch2, loc113060382, loc113079820, loc113068772 and loc113059134. RT-qPCR verified that the expression patterns of randomly selected differentially expressed transcripts were highly consistent with those obtained by RNA sequencing. GO and KEGG annotation revealed that these differentially expressed genes were mostly enriched in pathways of the production of pigment, including melanogenesis, tyrosine metabolism, Wnt signaling pathway, MAPK signaling pathway etc. These results indicated that the external characteristics of goldfish are consistent with the analysis results at transcriptome level. The results of this study will lay a foundation for further study on the expression characteristics and gene network analysis of pigment related genes.
