Expression patterns of Rbm24 in lens, nasal epithelium, and inner ear during mouse embryonic development

International audienceBackground: RNA-binding proteins plays critical roles in several post-transcriptional regulatory processes. The RNA-binding protein, Rbm24, has been shown to be involved in the development of the heart and skeletal muscles by regulating different post-transcriptional processes such as splicing and stabilization of specific target mRNAs. Here, by performing a detailed expression and localization analysis in mice embryos, we show that Rbm24 protein is not only expressed in heart and skeletal muscles as previously reported, but it is also strongly and specifically detected in specific regions of all the head sensory organs during mouse development. Results: Rbm24 expression is indeed found to be activated in the lens, in the sensory olfactory epithelium and in mechanosensory cells of the auditory and vestibular systems. Within these territories, Rbm24 is shown to be restricted to distinct subdomains, potentially regulating cell specificity and proliferation. Moreover, Rbm24 protein is found to be restricted to the cytoplasmic compartment in all these organs, thus providing clues to the posttranscrip-tional activity that it may exert in these cells. Conclusions: Altogether, these results highlight that Rbm24 may potentially function as a novel key regulator for the development of the eye, nasal epithelium, and inner ear in vertebrates

RNA-binding protein Rbm24 as a multifaceted post-transcriptional regulator of embryonic lineage differentiation and cellular homeostasis

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Emerging Roles of RNA-Binding Proteins in Inner Ear Hair Cell Development and Regeneration

International audienceRNA-binding proteins (RBPs) regulate gene expression at the post-transcriptional level. They play major roles in the tissue- and stage-specific expression of protein isoforms as well as in the maintenance of protein homeostasis. The inner ear is a bi-functional organ, with the cochlea and the vestibular system required for hearing and for maintaining balance, respectively. It is relatively well documented that transcription factors and signaling pathways are critically involved in the formation of inner ear structures and in the development of hair cells. Accumulating evidence highlights emerging functions of RBPs in the post-transcriptional regulation of inner ear development and hair cell function. Importantly, mutations of splicing factors of the RBP family and defective alternative splicing, which result in inappropriate expression of protein isoforms, lead to deafness in both animal models and humans. Because RBPs are critical regulators of cell proliferation and differentiation, they present the potential to promote hair cell regeneration following noise- or ototoxin-induced damage through mitotic and non-mitotic mechanisms. Therefore, deciphering RBP-regulated events during inner ear development and hair cell regeneration can help define therapeutic strategies for treatment of hearing loss. In this review, we outline our evolving understanding of the implications of RBPs in hair cell formation and hearing disease with the aim of promoting future research in this field

The Xenopus homologue of Down syndrome critical region protein 6 drives dorsoanterior gene expression and embryonic axis formation by antagonising polycomb group proteins

International audienceMesoderm and embryonic axis formation in vertebrates is mediated by maternal and zygotic factors that activate the expression of target genes. Transcriptional derepression plays an important role in the regulation of expression in different contexts; however, its involvement and possible mechanism in mesoderm and embryonic axis formation are largely unknown. Here we demonstrate that XDSCR6, a Xenopus homologue of human Down syndrome critical region protein 6 (DSCR6, or RIPPLY3), regulates mesoderm and embryonic axis formation through derepression of polycomb group (PcG) proteins. Xdscr6 maternal mRNA is enriched in the endoderm of the early gastrula and potently triggers the formation of dorsal mesoderm and neural tissues in ectoderm explants; it also dorsalises ventral mesoderm during gastrulation and induces a secondary embryonic axis. A WRPW motif, which is present in all DSCR6 homologues, is necessary and sufficient for the dorsal mesoderm- and axis-inducing activity. Knockdown of Xdscr6 inhibits dorsal mesoderm gene expression and results in head deficiency. We further show that XDSCR6 physically interacts with PcG proteins through the WRPW motif, preventing the formation of PcG bodies and antagonising their repressor activity in embryonic axis formation. By chromatin immunoprecipitation, we demonstrate that XDSCR6 releases PcG proteins from chromatin and allows dorsal mesoderm gene transcription. Our studies suggest that XDSCR6 might function to sequester PcG proteins and identify a novel derepression mechanism implicated in embryonic induction and axis formation

The RNA-binding protein Rbm24 is transiently expressed in myoblasts and is required for myogenic differentiation during vertebrate development

International audienceRNA-binding proteins (RBP) contribute to gene regulation through post-transcriptional events. Despite the important roles demonstrated for several RBP in regulating skeletal myogenesis in vitro, very few RBP coding genes have been characterized during skeletal myogenesis in vertebrate embryo. In the present study we report that Rbm24, which encodes the RNA-binding motif protein 24, is required for skeletal muscle differentiation in vivo. We show that Rbm24 transcripts are expressed at all sites of skeletal muscle formation during embryogenesis of different vertebrates, including axial, limb and head muscles. Interestingly, we find that Rbm24 protein starts to accumulate in MyoD-positive myoblasts and is transiently expressed at the onset of muscle cell differentiation. It accumulates in myotomal and limb myogenic cells, but not in Pax3-positive progenitor cells. Rbm24 expression is under the direct regulation by MyoD, as demonstrated by in vivo chromatin immunoprecipitation assay. Using morpholino knockdown approach, we further show that Rbm24 is required for somitic myogenic progenitor cells to differentiate into muscle cells during chick somitic myogenesis. Altogether, these results highlight Rbm24 as a novel key regulator of the myogenic differentiation program during vertebrate development

Autoinhibition of Dishevelled protein regulated by its extreme C terminus plays a distinct role in Wnt/β-catenin and Wnt/planar cell polarity (PCP) signaling pathways

International audienceEdited by Xiao-Fan Wang Dishevelled (Dvl) is a key intracellular signaling molecule that mediates the activation of divergent Wnt pathways. It contains three highly conserved domains known as DIX, PDZ, and DEP, the functions of which have been well characterized in ␤-catenin-dependent canonical and ␤-catenin-independent noncanonical Wnt signaling. The C-terminal region is also highly conserved from invertebrates to vertebrates. However, its function in regulating the activation of different Wnt signals remains unclear. We reported previously that Dvl conformational change triggered by the highly conserved PDZ-binding C terminus is important for the pathway specificity. Here we provide further evidence demonstrating that binding of the C terminus to the PDZ domain results in Dvl autoinhibition in the Wnt signaling pathways. Therefore, the forced binding of the C terminus to the PDZ domain reduces the activity of Dvl in noncanonical Wnt signaling, whereas obstruction of this interaction releases Dvl autoinhibition, impairs its functional interaction with LRP6 in canonical Wnt signaling, and increases its specificity in noncanonical Wnt signaling, which is closely correlated with an enhanced Dvl membrane localization. Our findings highlight the importance of the C terminus in keeping Dvl in an appropriate autoinhibited state, accessible for regulation by other partners to switch pathway specificity. Particularly, the C-terminally tagged Dvl fusion proteins that have been widely used to study the function and cellular localization of Dvl may not truly represent the wild-type Dvl because those proteins cannot be autoinhibited

Rbm24 displays dynamic functions required for myogenic differentiation during muscle regeneration

International audienceAbstract Skeletal muscle has a remarkable capacity of regeneration after injury, but the regulatory network underlying this repair process remains elusive. RNA-binding proteins play key roles in the post-transcriptional regulation of gene expression and the maintenance of tissue homeostasis and plasticity. Rbm24 regulates myogenic differentiation during early development, but its implication in adult muscle is poorly understood. Here we show that it exerts multiple functions in muscle regeneration. Consistent with its dynamic subcellular localization during embryonic muscle development, Rbm24 also displays cytoplasm to nucleus translocation during C2C12 myoblast differentiation. In adult mice, Rbm24 mRNA is enriched in slow-twitch muscles along with myogenin mRNA. The protein displays nuclear localization in both slow and fast myofibers. Upon injury, Rbm24 is rapidly upregulated in regenerating myofibers and accumulates in the myonucleus of nascent myofibers. Through satellite cell transplantation, we demonstrate that Rbm24 functions sequentially to regulate myogenic differentiation and muscle regeneration. It is required for myogenin expression at early stages of muscle injury and for muscle-specific pre-mRNA alternative splicing at late stages of regeneration. These results identify Rbm24 as a multifaceted regulator of myoblast differentiation. They provide insights into the molecular pathway orchestrating the expression of myogenic factors and muscle functional proteins during regeneration.Skeletal muscle has a remarkable capacity of regeneration after injury, but the regulatory network underlying this repair process remains elusive. RNA-binding proteins play key roles in the post-transcriptional regulation of gene expression and the maintenance of tissue homeostasis and plasticity. Rbm24 regulates myogenic differentiation during early development, but its implication in adult muscle is poorly understood. Here we show that it exerts multiple functions in muscle regeneration. Consistent with its dynamic subcellular localization during embryonic muscle development, Rbm24 also displays cytoplasm to nucleus translocation during C2C12 myoblast differentiation. In adult mice, Rbm24 mRNA is enriched in slow-twitch muscles along with myogenin mRNA. The protein displays nuclear localization in both slow and fast myofibers. Upon injury, Rbm24 is rapidly upregulated in regenerating myofibers and accumulates in the myonucleus of nascent myofibers. Through satellite cell transplantation, we demonstrate that Rbm24 functions sequentially to regulate myogenic differentiation and muscle regeneration. It is required for myogenin expression at early stages of muscle injury and for muscle-specific pre-mRNA alternative splicing at late stages of regeneration. These results identify Rbm24 as a multifaceted regulator of myoblast differentiation. They provide insights into the molecular pathway orchestrating the expression of myogenic factors and muscle functional proteins during regeneration

Autoinhibition of Dishevelled protein regulated by its extreme C terminus plays a distinct role in Wnt/β-catenin and Wnt/planar cell polarity (PCP) signaling pathways

Dishevelled (Dvl) is a key intracellular signaling molecule that mediates the activation of divergent Wnt pathways. It contains three highly conserved domains known as DIX, PDZ, and DEP, the functions of which have been well characterized in β-catenin-dependent canonical and β-catenin-independent noncanonical Wnt signaling. The C-terminal region is also highly conserved from invertebrates to vertebrates. However, its function in regulating the activation of different Wnt signals remains unclear. We reported previously that Dvl conformational change triggered by the highly conserved PDZ-binding C terminus is important for the pathway specificity. Here we provide further evidence demonstrating that binding of the C terminus to the PDZ domain results in Dvl autoinhibition in the Wnt signaling pathways. Therefore, the forced binding of the C terminus to the PDZ domain reduces the activity of Dvl in noncanonical Wnt signaling, whereas obstruction of this interaction releases Dvl autoinhibition, impairs its functional interaction with LRP6 in canonical Wnt signaling, and increases its specificity in noncanonical Wnt signaling, which is closely correlated with an enhanced Dvl membrane localization. Our findings highlight the importance of the C terminus in keeping Dvl in an appropriate autoinhibited state, accessible for regulation by other partners to switch pathway specificity. Particularly, the C-terminally tagged Dvl fusion proteins that have been widely used to study the function and cellular localization of Dvl may not truly represent the wild-type Dvl because those proteins cannot be autoinhibited

Mutational analysis of <i>dishevelled</i> genes in zebrafish reveals distinct functions in embryonic patterning and gastrulation cell movements

<div><p>Wnt signaling plays critical roles in dorsoventral fate specification and anteroposterior patterning, as well as in morphogenetic cell movements. Dishevelled proteins, or Dvls, mediate the activation of Wnt/ß-catenin and Wnt/planar cell polarity pathways. There are at least three highly conserved Dvl proteins in vertebrates, but the implication of each Dvl in key early developmental processes remains poorly understood. In this study, we use genome-editing approach to generate different combinations of maternal and zygotic <i>dvl</i> mutants in zebrafish, and examine their functions during early development. Maternal transcripts for <i>dvl2</i> and <i>dvl3a</i> are most abundantly expressed, whereas the transcript levels of other <i>dvl</i> genes are negligible. Phenotypic and molecular analyses show that early dorsal fate specification is not affected in maternal and zygotic <i>dvl2</i> and <i>dvl3a</i> double mutants, suggesting that the two proteins may be dispensable for the activation of maternal Wnt/ß-catenin signaling. Interestingly, convergence and extension movements and anteroposterior patterning require both maternal and the zygotic functions of Dvl2 and Dvl3a, but these processes are more sensitive to Dvl2 dosage. Zygotic <i>dvl2</i> and <i>dvl3a</i> double mutants display mild axis extension defect with correct anteroposterior patterning. However, maternal and zygotic double mutants exhibit most strongly impaired convergence and extension movements, severe trunk and posterior deficiencies, and frequent occurrence of cyclopia and craniofacial defects. Our results suggest that Dvl2 and Dvl3a products are required for the activation of zygotic Wnt/ß-catenin signaling and Wnt/planar cell polarity pathway, and regulate zygotic developmental processes in a dosage-dependent manner. This work provides insight into the mechanisms of Dvl-mediated Wnt signaling pathways during early vertebrate development.</p></div

Analysis of MZ<i>dvl2</i>;MZ<i>dvl3a</i> mutants.

<p>(A) Schema illustrating the strategy to generate female mosaic m<i>dvl2</i>^+(-)/-;<i>dvl3a</i>^-/- adult fish with a new mutant allele (red) in <i>dvl2</i>. MZ<i>dvl2</i>;MZ<i>dvl3a</i> mutant embryos are present at varied proportions in the offspring from crosses between female m<i>dvl2</i>^{+(<i>-</i>)/-};<i>dvl3a</i>^-/- and male <i>dvl2</i>^+/-;<i>dvl3a</i>^-/- fish, depending on the efficiency of germline mutations in the remaining <i>dvl2</i> WT allele. (B) An example of the sequencing chromatogram with both the original mutant allele and a new indel in <i>dvl2</i> locus. (C-F) Lateral (C, D) and dorsal (E, F) views of a representative WT embryo (C, E), and an MZ<i>dvl2</i>;MZ<i>dvl3a</i> mutant (D, F) at 11.5 hpf. Notice that the mutant embryo displays most severely impaired AP axis and convergence of paraxial mesoderm, with strongly widened somites (arrowheads). (G) A WT embryo at 30 hpf. (H) Lateral view of a representative MZ<i>dvl2</i>;MZ<i>dvl3a</i> mutant at 30 hpf shows deficiency of trunk and posterior regions, and cyclopia (arrowhead; see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007551#pgen.1007551.s012" target="_blank">S12 Fig</a>). (I) The phenotype of a Z<i>dvl2</i>;MZ<i>dvl3a</i> mutant with characteristic caudal truncation at 30 hpf. (J) Genotyping of <i>dvl2</i> alleles in MZ<i>dvl2</i>;MZ<i>dvl3a</i> mutant embryos from 7 independent fish pairs. A novel indel (red) along with the original mutation (blue) are present in the mutants. The left and right TALEN targeting sites are indicated in green. Dots are introduced to optimize sequence alignment. (K) Quantitative analyses of the occurrence of MZ<i>dvl2</i>;MZ<i>dvl3a</i> and Z<i>dvl2</i>;MZ<i>dvl3a</i> mutants among offspring from 7 independent fish pairs. Each fish pair was crossed three times, and numbers on the top of each column indicate total embryos scored. Scale bar: (C-F) 400 μm; (G-I) 400 μm.</p