Raphaëlle Grifone

Ming Shao

Audrey Saquet

De-Li Shi
email: de-li.shi@upmc.fr

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

Keywords: RNA-binding protein, Rbm24, cell differentiation, alternative splicing, cytoplasmic polyadenylation, muscle, head sensory organs, lens, inner ear

RNA-binding proteins control the metabolism of RNAs at all stages of their lifetime. They are critically required for the post-transcriptional regulation of gene expression in a wide variety of physiological and pathological processes. Rbm24 is a highly conserved RNA-binding protein that displays strongly regionalized expression patterns and exhibits dynamic changes in subcellular localization during early development. There is increasing evidence that it acts as a multifunctional regulator to switch cell fate determination and to maintain tissue homeostasis.

Dysfunction of Rbm24 disrupts cell differentiation in nearly every tissue where it is expressed, such as skeletal and cardiac muscles, and different head sensory organs, but the molecular events that are affected may vary in a tissue-specific, or even a stage-specific manner. Recent works using different animal models have uncovered multiple post-transcriptional regulatory mechanisms by which Rbm24 functions in key developmental processes. In particular, it represents a major splicing factor in muscle cell development, and plays an essential role in cytoplasmic polyadenylation during lens fiber cell terminal differentiation. Here we review the advances in understanding the implication of Rbm24 during development and disease, by focusing on its regulatory roles in physiological and pathological conditions.

Introduction

RNA-binding proteins (RBPs) play key roles in the post-transcriptional regulation of gene expression in a variety of biological processes. Vertebrate cells express hundreds to thousands of RBPs that display unique binding activity to their RNA targets and specific interaction with other protein partners. They control RNA metabolism at multiple levels, from alternative splicing, to transport, subcellular localization, stability, polyadenylation, and translation [START_REF] Glisovic | RNA-binding proteins and post-transcriptional gene regulation[END_REF]. Thus, protein-RNA interactions are critical for maintaining the homeostasis of protein synthesis during early development and in adult life. Large-scale analyses of protein expression regulation have made the unprecedented finding that protein abundances within the proteome and mRNA levels within the transcriptome are entirely uncoupled in many conditions [START_REF] Vogel | Insights into the regulation of protein abundance from proteomic and transcriptomic analyses[END_REF][START_REF] Liu | On the dependency of cellular protein levels on mRNA abundance[END_REF]. This highlights the importance of additional regulations by the ribonome, which embodies mRNAs and associated RBPs within a rich ribonucleoprotein (RNP) infrastructure in the cell [START_REF] Morris | Systematic analysis of posttranscriptional gene expression[END_REF]. Due to the crucial roles exerted by the processes downstream of transcription in the spatiotemporal control of gene expression, it is not surprising that a growing number of human diseases, such as neurodegenerative disorders and various types of cancers, are associated with RNAs and RBPs [START_REF] Lukong | RNA-binding proteins in human genetic disease[END_REF][START_REF] Castello | RNA-binding proteins in Mendelian disease[END_REF][START_REF] Brinegar | Roles for RNA-binding proteins in development and disease[END_REF][START_REF] Conlon | RNA-binding proteins in neurodegeneration: mechanisms in aggregate[END_REF][START_REF] Pereira | RNA-binding proteins in cancer: old players and new actors[END_REF]. Therefore, RBPs may have the potential to be used as therapeutic targets in many diseased conditions [START_REF] Hong | RNA binding protein as an emerging therapeutic target for cancer prevention and treatment[END_REF][START_REF] Shotwell | The potential of engineered eukaryotic RNA-binding proteins as molecular tools and therapeutics[END_REF][START_REF] Schultz | Understanding and targeting the disease-related RNA binding protein human antigen R (HuR). Wiley Interdiscip[END_REF]. Rbm24 (RNA-binding motif protein 24) is an evolutionarily conserved RBP that contains a single RNA recognition motif (RRM) in its N-terminal region [START_REF] Fetka | Expression of the RNA recognition motif-containing protein SEB-4 during Xenopus embryonic development[END_REF][START_REF] Poon | RNA-binding protein RBM24 is required for sarcomere assembly and heart contractility[END_REF][START_REF] Sun | Characterization of the duck (Anas platyrhynchos) Rbm24 and Rbm38 genes and their expression profiles in myoblast and skeletal muscle tissues[END_REF]. It exhibits strongly restricted tissue-specific expression patterns during vertebrate development [START_REF] Grifone | The RNA-binding protein Rbm24 is transiently expressed in myoblasts and is required for myogenic differentiation during vertebrate development[END_REF], and emerges as an important regulator of cellular differentiation and a potential factor implicated in human disease. Although no mutation of human RBM24 gene has been associated to any disease up today, deficiency in its expression level could be the cause of congenital disorders, such as cardiomyopathy, myopathy or blindness, which affect the normal function of related tissues where crucial roles of this gene have been demonstrated in different animal models. It seems that vertebrate Rbm24 could be involved in nearly all aspects of post-transcriptional regulation.

Most importantly, it functions as a key factor that regulates alternative splicing to establish the contractile function in developing cardiac and skeletal muscles [START_REF] Yang | RBM24 is a major regulator of muscle-specific alternative splicing[END_REF][START_REF] Weeland | Insights into alternative splicing of sarcomeric genes in the heart[END_REF][START_REF] Liu | RNA binding protein 24 deletion disrupts global alternative splicing and causes dilated cardiomyopathy[END_REF], and plays an important role in cytoplasmic polyadenylation (CPA) to ensure the accumulation of crystallin proteins during lens fiber cell terminal differentiation [START_REF] Shao | Rbm24 controls poly(A) tail length and translation efficiency of crystallin mRNAs in the lens via cytoplasmic polyadenylation[END_REF]. There is thus accumulating evidence that Rbm24 acts as a multifaceted regulator to initiate cell differentiation through distinct mechanisms, which may vary in a tissue-specific and even a stage-specific manner. Moreover, Rbm24 presents almost the same characteristics in RNA and protein interactions, and often performs similar functions, as the closely related ancient paralog Rbm38 (RNPC1) in regulating cell cycle progression and apoptosis [START_REF] Ray | Rapid and systematic analysis of the RNA recognition specificities of RNA-binding proteins[END_REF][START_REF] Lucchesi | Modulation of the p53 family network by RNA-binding proteins[END_REF], adding more complexity of its functional implication in various cellular processes. Mechanistic insights on how Rbm24 controls gene expression are beginning to be elucidated using in vivo and in vitro approaches. However, a number of important questions remain unanswered and await further investigation, such as its dynamic subcellular localization and tissue-specific function, the modulation of its activity through interaction with other partners, and its potential implication in human pathologies. In this review, we present recent advances in understanding the regulatory roles of Rbm24 in development and disease.

Rbm24 functional domains

Rbm24 proteins display deep evolutionary conservation. The RRM is located in the N-terminal half and contains two characteristic consensus RNP sequences, RNP1 and RNP2 [START_REF] Afroz | two, three, four! How multiple RRMs read the genome sequence[END_REF]. This domain shows extremely high levels of sequence identity among different species, ranging from nematode to human (Fig. 1). In addition, it is almost identical to the RRM of Rbm38, such that both Rbm24 and Rbm38 prefers similar GU-rich sequences in target mRNAs [START_REF] Ray | Rapid and systematic analysis of the RNA recognition specificities of RNA-binding proteins[END_REF][START_REF] Qian | Structural basis for mRNA recognition by human RBM38[END_REF]. Although the sequence outside the RRM is relatively divergent, at least two conserved domains can be identified in the C-terminal half of vertebrate Rbm24 and Rbm38 [START_REF] Boy | XSEB4R, a novel RNAbinding protein involved in retinal cell differentiation downstream of bHLH proneural genes[END_REF]. In particular, a motif close to the extreme C-terminus, which contains a serine residue (serine 181 in Rbm24 and serine 195 in Rbm38), interacts with eukaryote initiation factor 4E (eIF4E) and disrupts the assembly of translation initiation complex [START_REF] Zhang | Translational repression of p53 by RNPC1, a p53 target overexpressed in lymphomas[END_REF][START_REF] Lucchesi | Disruption of the Rbm38-eIF4E complex with a synthetic peptide Pep8 increases p53 expression[END_REF]. However, at least in several cancer cell lines, phosphorylation of the serine residue by glycogen synthase kinase 3 (GSK3) prevents the interaction with eIF4E and converts Rbm24 or Rbm38 into an activator of mRNA translation [START_REF] Zhang | Glycogen synthase kinase 3 promotes p53 mRNA translation via phosphorylation of RNPC1[END_REF]. A second conserved motif is the extreme C-terminus, whose implication in Rbm24 function is not clear. The C-terminal half of Rbm24 also interacts with a number of other partners, but the exact motif implicated has not been determined and the interaction is often assisted by the RRM. For example, both the N-terminal and C-terminal regions of Rbm24 are required for interaction with the Stk38 kinase [START_REF] Liu | Stk38 modulates Rbm24 protein stability to regulate sarcomere assembly in cardiomyocytes[END_REF]. Although the N-terminal region of zebrafish Rbm24a do not directly interacts with cytoplasmic polyadenylation element-binding protein 1b (Cpeb1b) and cytoplasmic poly(A)-binding protein 1l (Pabpc1l), its absence decreases the capacity of the Cterminal region to interact with these partners [START_REF] Shao | Rbm24 controls poly(A) tail length and translation efficiency of crystallin mRNAs in the lens via cytoplasmic polyadenylation[END_REF]. Thus, Rbm24 displays biochemical and functional conservation with respect to Rbm38, and its functionality in different cellular processes may be regulated through interaction with specific protein partners.

Rbm24 displays restricted expression patterns in developing vertebrate embryos

Another strikingly conserved aspect of vertebrate Rbm24 is the highly restricted developmental expression patterns (Fig. 2). During gastrulation, Xenopus rbm24, also called XSeb4 or MTG1, is mainly expressed in the dorso-lateral mesoderm regions flanking the axial midline, which mostly overlap with the expression domains of MyoD [START_REF] Fetka | Expression of the RNA recognition motif-containing protein SEB-4 during Xenopus embryonic development[END_REF][START_REF] Li | The RNA-binding protein Seb4/RBM24 is a direct target of MyoD and is required for myogenesis during Xenopus early development[END_REF]. During organogenesis, analyses by in situ hybridization and immunofluorescence staining indicate that Rbm24 is expressed in the somites, heart, and head sensory organs in different vertebrate embryos, including zebrafish, Xenopus, chick and mouse [START_REF] Fetka | Expression of the RNA recognition motif-containing protein SEB-4 during Xenopus embryonic development[END_REF][START_REF] Poon | RNA-binding protein RBM24 is required for sarcomere assembly and heart contractility[END_REF][START_REF] Grifone | The RNA-binding protein Rbm24 is transiently expressed in myoblasts and is required for myogenic differentiation during vertebrate development[END_REF][START_REF] Miller | Efficient array-based identification of novel cardiac genes through differentiation of mouse ESCs[END_REF][START_REF] Xu | Global expression profile of highly enriched cardiomyocytes derived from human embryonic stem cells[END_REF][START_REF] Maragh | Identification of RNA binding motif proteins essential for cardiovascular development[END_REF][START_REF] Maragh | Rbm24a and Rbm24b are required for normal somitogenesis[END_REF][START_REF] Cai | Characterization of the transcriptome of nascent hair cells and identification of direct targets of the Atoh1 transcription factor[END_REF][START_REF] Grifone | Expression patterns of Rbm24 in lens, nasal epithelium, and inner ear during mouse embryonic development[END_REF]. Zebrafish genome contains two rbm24 paralogs, rbm24a and rbm24b. The sites of rbm24a expression are identical as those of other vertebrate Rbm24, whereas rbm24b is mainly expressed in the somites and heart, but not in head sensory organs [START_REF] Maragh | Identification of RNA binding motif proteins essential for cardiovascular development[END_REF][START_REF] Maragh | Rbm24a and Rbm24b are required for normal somitogenesis[END_REF]. Within each tissue, Rbm24 is generally expressed in a subset of cells undergoing differentiation. The cellular localization of Rbm24 protein in different tissues has been analyzed in more detail during mouse embryonic development [START_REF] Grifone | The RNA-binding protein Rbm24 is transiently expressed in myoblasts and is required for myogenic differentiation during vertebrate development[END_REF][START_REF] Grifone | Expression patterns of Rbm24 in lens, nasal epithelium, and inner ear during mouse embryonic development[END_REF]. Interestingly, Rbm24 displays dynamic subcellular localization during muscle cell development. In the myotome and in C2C12 cells it is first accumulated in the cytoplasm of MyoD-positive myoblasts entering into the differentiation program, but not in Pax3-positive muscle progenitor cells, while in mature myotubes it is mainly present in the nucleus (Fig. 3). A cytoplasmic localization of Rbm24 protein is also detected in different head sensory organs [START_REF] Grifone | Expression patterns of Rbm24 in lens, nasal epithelium, and inner ear during mouse embryonic development[END_REF]. In the developing lens, it is restricted to differentiating fiber cells in the posterior and equatorial regions; in the otic vesicle, it co-localizes with Myo7A in inner ear hair cells; in the olfactory epithelium, it is expressed in fate-committed neuronal precursors and terminally differentiated olfactory receptor neurons. Thus, the predominant localization of Rbm24 in the cytoplasmic compartment of different cell types closely correlates with their entry into the process of differentiation. Detailed Rbm24 expression and subcellular localization will be further described in sections addressing its tissue-specific function.

Rbm24 regulates muscle cell development through distinct mechanisms

Rbm24 in skeletal myogenesis

The conserved expression of Rbm24 in the paraxial mesoderm and in the somites of vertebrate embryos suggests that it may play a role in skeletal muscle development. Indeed, Xenopus rbm24 (XSeb4) gene has been shown to be a transcriptional target of MyoD and early B cell factor (EBF) family members of transcription factors, and relays the function of these factors during myogenesis [START_REF] Li | The RNA-binding protein Seb4/RBM24 is a direct target of MyoD and is required for myogenesis during Xenopus early development[END_REF][START_REF] Green | EBF proteins participate in transcriptional regulation of Xenopus muscle development[END_REF]. In zebrafish, single knockdown of rbm24a and rbm24b indicates that they are required for somitogenesis, but rbm24a seems to display a predominant activity [START_REF] Maragh | Rbm24a and Rbm24b are required for normal somitogenesis[END_REF]. It is unclear whether rbm24a and rbm24b cooperate in skeletal muscle development, and this question needs to be addressed by inhibiting the function of both genes. In chick embryos, knockdown of Rbm24 in the somites severely alters the expression of muscle-specific myosin, indicating that it is required for myogenic differentiation [START_REF] Grifone | The RNA-binding protein Rbm24 is transiently expressed in myoblasts and is required for myogenic differentiation during vertebrate development[END_REF]. In Rbm24 mutant mice, although no myofiber degeneration can be observed in skeletal muscles, there is a loss of M-bands in sarcomeres, which is accompanied by a reduced inclusion of muscle-specific exons in muscle cells [START_REF] Yang | RBM24 is a major regulator of muscle-specific alternative splicing[END_REF]. This finding suggests that Rbm24 may be involved in skeletal muscle development through regulation of muscle-specific alternative splicing. It is consistent with the function of SUP-12, a Caenorhabditis elegans homolog of vertebrate Rbm24 that regulates alternative splicing of fibroblast growth factor (EGL-15) pre-mRNA in sex myoblast migration [START_REF] Anyanful | The RNA-binding protein SUP-12 controls muscle-specific splicing of the ADF/cofilin pre-mRNA in C. elegans[END_REF][START_REF] Kuroyanagi | The Fox-1 family and SUP-12 coordinately regulate tissue-specific alternative splicing in vivo[END_REF][START_REF] Ohno | Musclespecific splicing factors ASD-2 and SUP-12 cooperatively switch alternative pre-mRNA processing patterns of the ADF/cofilin gene in Caenorhabditis elegans[END_REF]. Thus, Rbm24 may represent an important conserved regulator of muscle-specific alternative splicing events in skeletal muscles. At present, the transcriptional control of muscle cell specification and differentiation has been relatively well documented [START_REF] Buckingham | Myogenic progenitor cells and skeletal myogenesis in vertebrates[END_REF][START_REF] Zammit | Function of the myogenic regulatory factors Myf5, MyoD, Myogenin and MRF4 in skeletal muscle, satellite cells and regenerative myogenesis[END_REF], but the contribution of post-transcriptional regulation to muscle development remains largely unclear [START_REF] Nikonova | Conserved functions of RNA-binding proteins in muscle[END_REF]. However, mis-regulation of several RBPs, including CELF, MBNL and FOX families of proteins, disrupts muscle-specific alternative splicing and has been linked to skeletal muscle disease such as myotonic dystrophy [START_REF] Brinegar | Roles for RNA-binding proteins in development and disease[END_REF][START_REF] Nikonova | Conserved functions of RNA-binding proteins in muscle[END_REF]. Because skeletal muscle is one of the first tissues in which alternative splicing generates abundant contractile proteins from widely expressed genes has been identified [START_REF] Llorian | Decoding muscle alternative splicing[END_REF], these observations provide mechanistic insights into muscle cell differentiation and function.

There are several lines of evidence suggesting that Rbm24 may not only function as a splicing factor, it also regulates skeletal myogenesis through other mechanisms. In vitro studies indicate that Rbm24 promotes myogenic differentiation of C2C12 cells by inducing cell cycle arrest upon binding to target mRNAs encoding cell cycle regulators [START_REF] Miyamoto | RNA-binding proteins Rbm38 and Rbm24 regulate myogenic differentiation via p21-dependent and -independent regulatory pathways[END_REF], and by stabilizing myogenin mRNA through binding to its 3'-untranslated region [START_REF] Jin | RNA-binding motif protein 24 regulates myogenin expression and promotes myogenic differentiation[END_REF]. These functionalities are entirely distinct from the regulation of alternative splicing. Thus, Rbm24 may display multiple or dynamic roles during the process of muscle cell development, which is consistent with its differential subcellular localization in myoblasts and multinucleated myotubes. Further in vivo analyses will be necessary to identify Rbm24-mediated stage-specific post-transcriptional mechanisms that trigger muscle lineage specification and differentiation.

Rbm24 is required for heart development

Loss of Rbm24 severely impairs heart development in zebrafish and mouse embryos [START_REF] Poon | RNA-binding protein RBM24 is required for sarcomere assembly and heart contractility[END_REF][START_REF] Yang | RBM24 is a major regulator of muscle-specific alternative splicing[END_REF][START_REF] Shao | Rbm24 controls poly(A) tail length and translation efficiency of crystallin mRNAs in the lens via cytoplasmic polyadenylation[END_REF][START_REF] Maragh | Identification of RNA binding motif proteins essential for cardiovascular development[END_REF][START_REF] Zhang | Rbm24, a target of p53, is necessary for proper expression of p53 and heart development[END_REF]. Knockdown or knockout of rbm24a in zebrafish is sufficient to cause severe cardiogenic defects [START_REF] Poon | RNA-binding protein RBM24 is required for sarcomere assembly and heart contractility[END_REF][START_REF] Shao | Rbm24 controls poly(A) tail length and translation efficiency of crystallin mRNAs in the lens via cytoplasmic polyadenylation[END_REF][START_REF] Maragh | Identification of RNA binding motif proteins essential for cardiovascular development[END_REF]. Although rbm24a morphants or mutants have a beating heart, blood is only transported back and forth between the ventricle and atrium. This is due to the defective atrioventricular separation, which impairs blood circulation in the whole body [START_REF] Shao | Rbm24 controls poly(A) tail length and translation efficiency of crystallin mRNAs in the lens via cytoplasmic polyadenylation[END_REF]. In Rbm24 homozygous mutant mice, heart malformations, including defective ventricular septum, reduced myocardial compaction, dilated atria and abnormal atrioventricular endocardial cushions, become evident at E10.5, these are followed by growth retardation at subsequent stages and embryonic lethality at E13.5 [START_REF] Yang | RBM24 is a major regulator of muscle-specific alternative splicing[END_REF][START_REF] Zhang | Rbm24, a target of p53, is necessary for proper expression of p53 and heart development[END_REF]. In addition to regulating early heart development, Rbm24 also plays a pivotal role in later cardiac sarcomerogenesis. Both in zebrafish and mice, loss of Rbm24 function causes disrupted Z-discs and sarcomere organization due to a decreased expression of sarcomeric proteins, a phenotype reminiscent of cardiomyopathy [START_REF] Poon | RNA-binding protein RBM24 is required for sarcomere assembly and heart contractility[END_REF][START_REF] Yang | RBM24 is a major regulator of muscle-specific alternative splicing[END_REF][START_REF] Liu | RNA binding protein 24 deletion disrupts global alternative splicing and causes dilated cardiomyopathy[END_REF]. Although no defective alternative splicing is present in Rbm24 heterozygous mutant mice, which are normal and viable, there is an increased sarcomere slack length and a lower myofilament passive stiffness in cardiomyocytes [START_REF] De Groot | Heterozygous loss of Rbm24 in the adult mouse heart increases sarcomere slack length but does not affect function[END_REF]. This raises a possibility that Rbm24 haploinsufficiency may influence disease penetrance in congenital heart disorders.

RNA-seq analyses have provided important insights on Rbm24-mediated posttranscriptional control of cardiac differentiation. Altered splicing of a large number of differentiation-related muscle genes has been identified in mouse embryos lacking Rbm24 function [START_REF] Yang | RBM24 is a major regulator of muscle-specific alternative splicing[END_REF]. A majority of these altered splicing events is also present in zebrafish rbm24a mutants [START_REF] Shao | Rbm24 controls poly(A) tail length and translation efficiency of crystallin mRNAs in the lens via cytoplasmic polyadenylation[END_REF], suggesting a functional conservation of Rbm24-regulated alternative splicing in heart development. Further study on the consequences of Rbm24 deficiency in post-natal heart development using conditional knockout mice indicates a global disruption of alternative splicing events, which mostly affects those genes coding for sarcomere structure proteins involved in muscle contraction, including in particular Ttn [START_REF] Liu | RNA binding protein 24 deletion disrupts global alternative splicing and causes dilated cardiomyopathy[END_REF]. It has been shown that the isoform switch of Ttn in cardiomyocytes is dependent on the function of Rbm20 [START_REF] Guo | RBM20, a gene for hereditary cardiomyopathy, regulates titin splicing[END_REF], which possesses a single central RRM and regulates a large number of heart genes [START_REF] Fochi | The emerging role of the RBM20 and PTBP1 ribonucleoproteins in heart development and cardiovascular diseases[END_REF]. Moreover, mutations of both RBM20 and TTN genes in humans cause dilated cardiomyopathy [START_REF] Brauch | Mutations in ribonucleic acid binding protein gene cause familial dilated cardiomyopathy[END_REF][START_REF] Ware | Role of titin in cardiomyopathy: from DNA variants to patient stratification[END_REF][START_REF] Hey | Pathogenic RBM20-variants are associated with a severe desease expression in male patients with dilated cardiomyopathy[END_REF]. This raises a possibility that human RBM24 may cooperate with RBM20 to regulate cardiac muscular functionality and may represent a new but rare gene associated with heart diseases [START_REF] Gaertner | Screening for mutations in human cardiomyopathy-is RBM24 a new but rare disease gene?[END_REF].

Supporting the cooperation between Rbm24 and Rbm20 in heart development, it has been reported that the two proteins interacts biochemically and bind to the same intronic region to promote the splicing of short Enigma homolog (Enh) splice variants, which encode LIM-less PDZ-LIM proteins that can prevent the hypertrophic growth of cardiomyocytes [START_REF] Ito | RBM20 and RBM24 cooperatively promote the expression of short enh splice variants[END_REF]. Similarly, in Caenorhabditis elegans, SUP-12 and its cofactors regulate muscle-specific alternative splicing through recognition of juxtaposed cis-elements on the target RNA to form a ternary complex [START_REF] Kuroyanagi | The Fox-1 family and SUP-12 coordinately regulate tissue-specific alternative splicing in vivo[END_REF][START_REF] Ohno | Musclespecific splicing factors ASD-2 and SUP-12 cooperatively switch alternative pre-mRNA processing patterns of the ADF/cofilin gene in Caenorhabditis elegans[END_REF][START_REF] Kuwasako | RBFOX and SUP-12 sandwich a G base to cooperatively regulate tissue-specific splicing[END_REF].

Although defective alternative splicing represents an important event caused by loss of Rbm24 during heart development, other post-transcriptional regulatory processes are also affected. In particular, deficiency of Rbm24 in mice leads to aberrant activation of p53dependent apoptosis in heart tissues, whereas overexpression of Rbm24 can inhibit p53 protein expression [START_REF] Zhang | Rbm24, a target of p53, is necessary for proper expression of p53 and heart development[END_REF]. Mechanistically, in vitro studies suggest that Rbm24 prevents eIF4E from binding to p53 mRNA through the eIF4E-binding motif in the absence of phosphorylation at serine 181, thereby interfering with the cap-binding function of eIF4E and repressing p53 expression. In contrast, phosphorylation of this serine residue converts Rbm24 into an activator of p53 expression [START_REF] Zhang | Rbm24, a target of p53, is necessary for proper expression of p53 and heart development[END_REF]. These observations suggest that Rbm24 may be involved in regulating p53 activity during heart development in a phosphorylation-dependent manner. Since p53 induces apoptosis in disease-or aging-related failing heart [START_REF] Morita | Heart failure as an aging-related phenotype[END_REF], Rbm24 may exert a protective role to reduce the incidence of heart failure. Thus, it would be interesting to examine this possibility and to analyze the regulation of Rbm24 activity by phosphorylation during heart development. Consistent with its implication in regulating post-transcriptional processes downstream of alternative splicing, immunofluorescence staining indicates that Rbm24 protein is abundantly accumulated in the cytoplasm of mouse cardiomyocytes at E11.5 [START_REF] Grifone | The RNA-binding protein Rbm24 is transiently expressed in myoblasts and is required for myogenic differentiation during vertebrate development[END_REF]. The distinct roles of Rbm24 during cardiac muscle development imply that it could be involved in various aspects of post-transcriptional regulation. Indeed, RNA immunoprecipitation assay followed by microarray analysis in cardiac myoblast cell lines suggests that Rbm24 binds to, and regulates numerous targets through distinct mechanisms, including mRNA stability, alternative splicing and transcriptional initiation, and mRNA translation [START_REF] Lin | Global profiling of Rbm24 bound RNAs uncovers a multi-tasking RNA binding protein[END_REF]. This highlights the importance of Rbm24-organized RNA regulon in coordinating and stabilizing the expression of structural and functional genes in cardiac muscle [START_REF] Ehler | RBM24: a 'regional business manager' in charge of the maintenance of sarcomeric protein expression 24 h a day?[END_REF].

Dynamic subcellular localization and function of Rbm24

Analysis of Rbm24 subcellular localization provides further support on the possibility of its multiple and dynamic functions during muscle cell development. Within the myotome of mouse embryos at E11.5, Rbm24 protein only accumulates in the cytoplasm of MyoD-positive myoblasts engaged in the differentiation program, but not in Pax3-expressing premyogenic progenitors [START_REF] Grifone | The RNA-binding protein Rbm24 is transiently expressed in myoblasts and is required for myogenic differentiation during vertebrate development[END_REF], which represent a proliferating population of muscle stem cells [START_REF] Relaix | A Pax3/Pax7-dependent population of skeletal muscle progenitor cells[END_REF]. This implies that Rbm24 functions in the cytoplasmic compartment during the differentiation step of muscle development, but its post-transcriptional regulatory roles remain to be explored.

Interestingly, and consistent with its function in regulating alternative splicing of those mRNAs encoding muscle-specific contractile proteins, the localization of Rbm24 protein in myofibers of adult muscles is only restricted to the nucleus. This translocation as a function of cell differentiation state can be also observed in C2C12 cell line expressing Rbm24-GFP. The fusion protein is first expressed in the cytoplasm of mononucleated myoblast cells, and then accumulates in the nucleus of multinucleated myotubes (Fig. 3). These in vivo and in vitro observations suggest that Rbm24 may exert distinct post-transcriptional activities in differentiating myoblasts and terminally differentiated myofibers. Thus, there is a possibility that its regulatory roles on muscle cell differentiation and muscular functionality may be cell-type specific and may depend on its subcellular localization and the presence or absence of its cofactors.

It is likely that the cytoplasmic localization of Rbm24 in myoblasts functions to regulate the stability and/or translation of mRNAs encoding muscle differentiation-promoting factors required for early differentiation steps, such as myogenin [START_REF] Jin | RNA-binding motif protein 24 regulates myogenin expression and promotes myogenic differentiation[END_REF], thereby switching them into the differentiation program. As cells become further differentiated during later steps of myofibrillogenesis, Rbm24 accumulates in the nucleus to promote the expression of muscle structural and contractile proteins through alternative splicing. Consistent with its role in regulating the transition from embryonic cell pluripotency to differentiation, overexpression of Rbm24 is able to induce the specification and trigger the differentiation of mouse embryonic stem cells into cardiomyocytes [START_REF] Zhang | Rbm24 regulates alternative splicing switch in embryonic stem cell cardiac lineage differentiation[END_REF]. Thus, the dynamic subcellular localization of Rbm24 argues that it may regulate multiple steps of myogenic differentiation through distinct posttranscriptional mechanisms. However, the shift of Rbm24 protein from cytoplasm to nucleus during muscle differentiation raises an important question as to how it is shuttling between these subcellular compartments. Whether this depends on the presence or absence of a signal/cofactor that also displays dynamic changes during muscle cell maturation? Interestingly, a similar cytoplasm to nucleus translocation has been observed for MBNL in post-natal skeletal muscle [START_REF] Lin | Failure of MBNL1-dependent post-natal splicing transitions in myotonic dystrophy[END_REF], but its cytoplasmic function during early development is not clear [START_REF] Brinegar | Roles for RNA-binding proteins in development and disease[END_REF]. Therefore, the significance of the dynamic characteristic of RBP subcellular localization during muscle development is intriguing and merits further investigation.

Rbm24 in head sensory organ development

Rbm24 regulates lens fiber cell differentiation

Although much attention has been focused on Rbm24 function in cardiac and skeletal muscle cell differentiation, several recent works have revealed an interesting expression profile and an important role of this protein during the development of vertebrate head sensory organs [START_REF] Shao | Rbm24 controls poly(A) tail length and translation efficiency of crystallin mRNAs in the lens via cytoplasmic polyadenylation[END_REF][START_REF] Cai | Characterization of the transcriptome of nascent hair cells and identification of direct targets of the Atoh1 transcription factor[END_REF][START_REF] Grifone | Expression patterns of Rbm24 in lens, nasal epithelium, and inner ear during mouse embryonic development[END_REF][START_REF] Brastrom | A high-throughput assay for congenital and age-related eye diseases in zebrafish[END_REF][START_REF] Dash | The master transcription factor SOX2, mutated in anophthalmia/microphthalmia, is post-transcriptionally regulated by the conserved RNA-binding protein RBM24 in vertebrate eye development[END_REF]. In particular, it has been shown that Rbm24 is required for lens fiber cell terminal differentiation in zebrafish and in mice, providing evidence for an important regulatory role mediated by RBPs in vertebrate lens development. Indeed, several RBPs have been shown to be required for eye development in humans or animal models [START_REF] Dash | RNA-binding proteins in eye development and disease: implication of conserved RNA granule components[END_REF]. For example, mutations of the Tudor domain RBP TDRD7 in humans cause cataract formation [START_REF] Lachke | Mutations in the RNA granule component TDRD7 cause cataract and glaucoma[END_REF], illustrating a critical implication of post-transcriptional events in lens morphogenesis.

Lens transparency is established by abundant accumulation of crystallin proteins and denucleation in lens fiber cells [START_REF] Cvekl | The cellular and molecular mechanisms of vertebrate lens development[END_REF]. Loss of Rbm24a function in zebrafish directly prevents the efficient translation of crystallin mRNAs into functional proteins, and indirectly affects lens fiber cell denucleation due to impaired blood circulation [START_REF] Shao | Rbm24 controls poly(A) tail length and translation efficiency of crystallin mRNAs in the lens via cytoplasmic polyadenylation[END_REF]. As a consequence, rbm24a mutant embryos develop a severe cataract phenotype (Fig. 4A,B). Detailed examination of Rbm24 protein expression in mouse embryos indicates that it not only shows specific expression in differentiating lens fiber cells, but it is also accumulated in the cytoplasm of these cells [START_REF] Grifone | Expression patterns of Rbm24 in lens, nasal epithelium, and inner ear during mouse embryonic development[END_REF].

During zebrafish development, the expression of rbm24a in the lens placode can be detected around 17 hpf (hours post-fertilization), several hours before the initiation of primary fiber cell differentiation. At early stages of lens development, Rbm24a may participate in the posttranscriptional regulation of several genes involved in the specification of the lens placode, such as Pax6 and Sox2. Consistently, Rbm24a deficiency results in decreased stability of Sox2 mRNA [START_REF] Dash | The master transcription factor SOX2, mutated in anophthalmia/microphthalmia, is post-transcriptionally regulated by the conserved RNA-binding protein RBM24 in vertebrate eye development[END_REF]. As lens fiber cell differentiation proceeds, rbm24a exhibits highly localized expression patterns [START_REF] Shao | Rbm24 controls poly(A) tail length and translation efficiency of crystallin mRNAs in the lens via cytoplasmic polyadenylation[END_REF]. After 24 hpf, it is progressively restricted to the posterior and equatorial regions of the lens mass, where differentiation of primary fiber cells and initial formation of secondary fiber cells take place [START_REF] Greiling | Cell fate and differentiation of the developing ocular lens[END_REF]. This dynamic characteristic of rbm24a expression in lens fiber cells makes it a strong candidate in regulating lens fiber cell terminal differentiation. Indeed, the localization of Rbm24a protein in the cytoplasm of differentiating lens fiber cells before and after nuclear degradation suggests that it should regulate posttranscriptional events other than alternative splicing.

Importantly, The RRM of Rbm24a binds to a wide spectrum of lens-specific mRNAs that encode either small heat shock proteins or lens structural proteins, and the C-terminal region interacts with members of the cytoplasmic polyadenylation element-binding protein (CPEB) and cytoplasmic poly(A)-binding protein (PABPC) families [START_REF] Shao | Rbm24 controls poly(A) tail length and translation efficiency of crystallin mRNAs in the lens via cytoplasmic polyadenylation[END_REF]. CPEB and PABPC are key components of the CPA complex that regulates poly(A) tail elongation of nuclear exported mRNAs [START_REF] Eckmann | Control of poly(A) tail length[END_REF][START_REF] Goss | A) binding proteins: are they created equal? Wiley Interdiscip[END_REF][START_REF] Nicholson | Tales of detailed tails[END_REF]. They are required for translational activation in a variety of processes, including oogenesis and early embryonic development [START_REF] Charlesworth | Specificity factors in cytoplasmic polyadenylation[END_REF][START_REF] Reyes | Cytoplasmic polyadenylation in mammalian oocyte maturation[END_REF]. Loss of Rbm24a specifically reduces the poly(A) tail length of many crystallin mRNAs, which severely prevents the accumulation of lens transparent proteins and causes cataract formation [START_REF] Shao | Rbm24 controls poly(A) tail length and translation efficiency of crystallin mRNAs in the lens via cytoplasmic polyadenylation[END_REF]. The demonstration that Rbm24a may function in the CPA complex is significant in understanding the regulatory mechanism underlying mRNA translational activation to switch and maintain lens fiber cell differentiation. Indeed, when transcriptional contribution declines during denucleation of differentiating lens fiber cells, a post-transcriptional mechanism that promotes the efficient translation of lens-specific mRNAs will be necessary for the production of high amounts of transparent proteins. Zebrafish and mouse Rbm24 is expressed at the right time and place to play a critical role in this process, suggesting that similar post-transcriptional mechanisms control lens development in vertebrates. The importance of post-transcriptional regulation after lens fiber cell denucleation has been also demonstrated for other aspects of lens morphogenesis. Analysis of Tdrd7 function in mice indicates that it is required for maintaining cytoskeletal organization and lens fiber cell morphology by regulating the expression of heat shock protein HSPB1 [START_REF] Barnum | The Tudor-domain protein TDRD7, mutated in congenital cataract, controls the heat shock protein HSPB1 (HSP27) and lens fiber cell morphology[END_REF]. Moreover, members of the PABPC family are ancient paralogs of Rbm24, and they are coexpressed in different tissues. For example, zebrafish Pabpc4 also displays strongly localized expression in the somites and lens primordium during early development [START_REF] Mishima | Translational inhibition by deadenylation-independent mechanisms is central to microRNA-mediated silencing in zebrafish[END_REF]. This correlative evidecne further supports a potential interaction between these RBPs in regulating CPA during lens differentiation. Nevertheless, it remains to be determined how Rbm24 functions precisely within the CPA complex to control mRNA translation in lens fiber cells, and whether it regulates cell differentiation in other tissues through the same mechanism.

Rbm24 and Rbm38 in retinal differentiation

Immunofluorescence staining indicates that mouse Rbm24 protein is expressed in the optic vesicle at E9.5 to E11.5 [START_REF] Dash | The master transcription factor SOX2, mutated in anophthalmia/microphthalmia, is post-transcriptionally regulated by the conserved RNA-binding protein RBM24 in vertebrate eye development[END_REF], but not at more late stages [START_REF] Grifone | Expression patterns of Rbm24 in lens, nasal epithelium, and inner ear during mouse embryonic development[END_REF][START_REF] Dash | The master transcription factor SOX2, mutated in anophthalmia/microphthalmia, is post-transcriptionally regulated by the conserved RNA-binding protein RBM24 in vertebrate eye development[END_REF]. Although both zebrafish and mouse Rbm24 mutants display microphthalmia phenotype [START_REF] Shao | Rbm24 controls poly(A) tail length and translation efficiency of crystallin mRNAs in the lens via cytoplasmic polyadenylation[END_REF][START_REF] Brastrom | A high-throughput assay for congenital and age-related eye diseases in zebrafish[END_REF][START_REF] Dash | The master transcription factor SOX2, mutated in anophthalmia/microphthalmia, is post-transcriptionally regulated by the conserved RNA-binding protein RBM24 in vertebrate eye development[END_REF], this seems to be indirectly caused by the absence of blood supply because rescue of blood circulation in the mutants prevents microphthalmia but not cataract formation [START_REF] Shao | Rbm24 controls poly(A) tail length and translation efficiency of crystallin mRNAs in the lens via cytoplasmic polyadenylation[END_REF]. Thus, it is at present still unclear whether Rbm24 plays a role in retinal development. Nevertheless, XSeb4R (Rbm38) has been shown to regulate retinal neuronal differentiation during Xenopus development. It is expressed in retinoblasts and undifferentiated post-mitotic neurons. Gain-of-function of XSeb4R promotes neural differentiation, whereas loss of function causes an increased number of glial cells [START_REF] Boy | XSEB4R, a novel RNAbinding protein involved in retinal cell differentiation downstream of bHLH proneural genes[END_REF]. The post-transcriptional mechanism in retinogenesis mediated by XSeb4R is not clear. However, given the biochemical and functional conservation between Rbm24 and Rbm38, there is a possibility that it may bind to, and stabilize Sox2 mRNA in the gene regulatory network of the optic vesicle [START_REF] Dash | The master transcription factor SOX2, mutated in anophthalmia/microphthalmia, is post-transcriptionally regulated by the conserved RNA-binding protein RBM24 in vertebrate eye development[END_REF].

Rbm24 is required for inner ear hair cell development

The strongly restricted expression of vertebrate Rbm24 in the inner ear also makes it a potential post-transcriptional regulator of hair cell differentiation and regeneration. In the cristae of the neonatal mouse vestibular system, in situ hybridization analysis shows that the expression of Rbm24 gene in a subset of hair cells is directly regulated by the transcription factor Atoh1 [START_REF] Cai | Characterization of the transcriptome of nascent hair cells and identification of direct targets of the Atoh1 transcription factor[END_REF].

In the embryos at E14.5, Rbm24 protein co-localizes with Myo7A in mechanosensory cells of the auditory and vestibular systems [START_REF] Grifone | Expression patterns of Rbm24 in lens, nasal epithelium, and inner ear during mouse embryonic development[END_REF]. These expression patterns suggest that Rbm24 may function downstream of hair cell fate specification, and may participate in hair cell differentiation or function. Nevertheless, it is at present still unclear whether and how Rbm24 regulates hair cell development in mammalian embryos, and generally, post-transcriptional regulatory mechanisms underlying hair cell development and homeostasis remain largely elusive, mostly due to the lack of functional studies on related gene regulatory networks.

Zebrafish has become particularly attractive for understanding otic development and human diseases affecting hearing function [START_REF] Blanco-Sánchez | Zebrafish models of human eye and inner ear diseases[END_REF][START_REF] Nicolson | The genetics of hair-cell function in zebrafish[END_REF]. Although the precise localization of zebrafish rbm24a expression in inner ear hair cells has not been analyzed in detail, single cell RNA-seq analysis indicates that it dynamically changes from non-cycling progenitors to differentiated hair cells, suggesting that Rbm24a may participate in the differentiation program [START_REF] Lush | scRNA-Seq reveals distinct stem cell populations that drive hair cell regeneration after loss of Fgf and Notch signaling[END_REF]. A recent study has established a functional requirement of Rbm24a for hair cell development [START_REF] Cheng | Loss of Rbm24a causes defective hair cell development in the zebrafish inner ear and neuromasts[END_REF]. Rbm24a deficiency does not affect the specification of the otic vesicle, however, it prevents the differentiation of hair cells in the anterior, lateral and posterior cristae corresponding to the sensory patches for each of the three semicircular canals. This is associated with a disorganization of hair cells and a reduced height of their kinocilia that are involved in the morphogenesis of the hair bundle and in mechanotransduction (Fig. 4C,D). Loss of Rbm24a also impairs the formation of hair cells in the neuromasts of the posterior lateral line system. RNA-seq and qRT-PCR analyses reveal a reduced expression level of several zebrafish orthologs of human deafness genes, such as smpx, gsdmeb and otofa in rbm24a mutants [START_REF] Cheng | Loss of Rbm24a causes defective hair cell development in the zebrafish inner ear and neuromasts[END_REF].

Mutations of these genes in humans have been associated with X-linked deafness-4, autosomal dominant nonsyndromic deafness 5 and sensorineural hearing loss, and autosomal recessive nonsyndromic deafness 9, respectively. Inhibiting their function in zebrafish produces similar defects in the developing ear as observed in rbm24a mutants [START_REF] Chatterjee | Otoferlin deficiency in zebrafish results in defects in balance and hearing: rescue of the balance and hearing phenotype with full-length and truncated forms of mouse otoferlin[END_REF][START_REF] Desmidt | Zebrafish model for nonsyndromic X-linked sensorineural deafness[END_REF]. Consistently, loss of Rbm24a causes hearing and balancing deficits, demonstrating its functional requirement for hair cell differentiation and/or function [START_REF] Cheng | Loss of Rbm24a causes defective hair cell development in the zebrafish inner ear and neuromasts[END_REF]. Since no defective alternative splicing of these genes has been detected by RNA-seq analysis in rbm24a mutants [START_REF] Shao | Rbm24 controls poly(A) tail length and translation efficiency of crystallin mRNAs in the lens via cytoplasmic polyadenylation[END_REF], how Rbm24 regulates their expression remains unclear. Further study on the regulatory hierarchy implicating Rbm24 in the development of inner ear sensory receptors will help to understand the post-transcriptional mechanism operating in this lineage.

Rbm24 and differentiation of olfactory sensory neurons

The functional implication of Rbm24 in the differentiation of sensory neurons within the olfactory epithelium has not been investigated to date. Immunofluorescence staining of mouse embryonic sections indicates that Rbm24 expression is restricted to differentiating neurons [START_REF] Grifone | Expression patterns of Rbm24 in lens, nasal epithelium, and inner ear during mouse embryonic development[END_REF]. At E10.5 and E11.5, it mainly accumulates in the cytoplasm of fate-committed immediate neuronal precursors (INPs) and terminally differentiated olfactory receptor neurons (ORNs) within the nasal pit, suggesting that it may promote neurogenic differentiation by regulating the translation of target mRNAs. At more late stage (E14.5), Rbm24 protein is strongly expressed in basal neural stem cells and weakly expressed in differentiating olfactory neurons. These observations raise the possibility for a role of Rbm24 in maintaining the progenitor population and/or switching their progression toward more committed olfactory neuronal cells. During olfactory epithelium development, it is well established that transcriptional and epigenetic controls of spatiotemporal gene expression determine the formation of olfactory sensory neurons and nonneuronal cell types [START_REF] Sokpor | Transcriptional and epigenetic control of mammalian olfactory epithelium development[END_REF]. However, there is also evidence that mRNAs encoding olfactory receptors in neural lineages are protected from degradation, suggesting a requirement of RBPsmediated post-transcriptional regulatory mechanisms in their stabilization [START_REF] Shum | Identification of novel posttranscriptional features in olfactory receptor family mRNAs[END_REF]. Thus, it is worth to analyze whether and how neuronal differentiation in the olfactory epithelium is affected following loss of Rbm24 function, in order to elucidate the molecular and cellular mechanisms underlying neurogenesis of the olfactory system.

Rbm24 in embryonic germ layer formation

In Xenopus and zebrafish, XSeb4 and rbm24a are expressed as maternal transcripts [START_REF] Fetka | Expression of the RNA recognition motif-containing protein SEB-4 during Xenopus embryonic development[END_REF][START_REF] Poon | RNA-binding protein RBM24 is required for sarcomere assembly and heart contractility[END_REF]. However, their spatial localizations within the cleavage stage embryos and their maternal functions are not clear and await further investigation. Knockdown of XSeb4 by injecting translation-blocking morpholino oligonucleotide into fertilized Xenopus eggs has been shown to inhibit the expression of mesoderm genes at the early gastrula stage, such as the pan-mesoderm marker brachyury [START_REF] Li | The RNA-binding protein Seb4/RBM24 is a direct target of MyoD and is required for myogenesis during Xenopus early development[END_REF]. Nevertheless, it is unclear whether this represents a direct effect or an indirect consequence due to disruption of more upstream events, such as maternal regulators of brachyury gene. Interestingly, several studies have focused on the maternal function of XSeb4R (Rbm38). It has been shown that XSeb4R regulates germ layer formation by binding to the vegetally-localized maternal VegT mRNA and positively regulating its stability and translation [START_REF] Souopgui | The RNA-binding protein XSeb4R: a positive regulator of VegT mRNA stability and translation that is required for germ layer formation in Xenopus[END_REF]. Since VegT is a key T-box transcription factor required for inductive interaction and germ layer specification [START_REF] Carron | Specification of anteroposterior axis by combinatorial signaling during Xenopus development[END_REF], the regulation of its expression by Rbm24-related proteins may be important for the initial formation of endoderm and mesoderm tissues. Maternal XSeb4R transcripts are also enriched in the ectoderm, where the translated protein binds to, and promotes the translation of maternal Sox3 mRNA [START_REF] Bentaya | The RNA-binding protein XSeb4R regulates maternal Sox3 at the posttranscriptional level during maternal-zygotic transition in Xenopus[END_REF]. The importance of maternal XSeb4R functions in promoting germ layer-specific translational activation of maternally stored mRNAs demonstrates a major contribution of post-transcriptional regulation in setting up maternal inputs to trigger zygotic developmental program. On the other hand, the spatiotemporal activation and function of zygotic genes also depends on RBPs-mediated clearance of maternal mRNAs [START_REF] Despic | RNA tales -how embryos read and discard messages from mom[END_REF]. Therefore, whether Rbm24-related proteins may be also implicated in this process merits an investigation.

It is well established that maternal RBPs are critical mediators of translational regulation in the early embryos [START_REF] Christou-Kent | Diversity of RNA-binding proteins modulating post-transcriptional regulation of protein expression in the maturing mammalian oocyte[END_REF]. Before meiotic maturation, the translation of many maternal mRNAs is repressed partly due to the lack of an appropriate length of poly(A) tails [START_REF] Christou-Kent | Diversity of RNA-binding proteins modulating post-transcriptional regulation of protein expression in the maturing mammalian oocyte[END_REF][START_REF] Mendez | Translational control by CPEB: a means to the end[END_REF]. Upon fertilization and during cell-cycle progression, CPA mediated by members of the CPEB and PABPC families plays a critical role in the stabilization and translational activation of oocytestored mRNAs by elongating their poly(A) tails [START_REF] Christou-Kent | Diversity of RNA-binding proteins modulating post-transcriptional regulation of protein expression in the maturing mammalian oocyte[END_REF][START_REF] Mendez | Translational control by CPEB: a means to the end[END_REF][START_REF] Villalba | Cytoplasmic polyadenylation and translational control[END_REF]. Inhibition of CPA prevents transcription of zygotic genes and results in failure of maternal to zygotic transition and delay of developmental progression [START_REF] Winata | Cytoplasmic polyadenylation-mediated translational control of maternal mRNAs directs maternal-to-zygotic transition[END_REF]. Since Rbm24 interacts with Cpeb1b, Pabpc1l and eIF4E [START_REF] Shao | Rbm24 controls poly(A) tail length and translation efficiency of crystallin mRNAs in the lens via cytoplasmic polyadenylation[END_REF][START_REF] Zhang | Rbm24, a target of p53, is necessary for proper expression of p53 and heart development[END_REF], which are conserved mRNA interactors required for the translational activation of maternal mRNAs [START_REF] Christou-Kent | Diversity of RNA-binding proteins modulating post-transcriptional regulation of protein expression in the maturing mammalian oocyte[END_REF], it is potentially involved in the regulation of CPA. Thus, it would be of interest to examine whether maternal Rbm24 regulates the dynamics of CPA for differentially activating or repressing the translation of germ layer-specific maternal mRNAs coding for important mediators of inductive interaction. It is equally important to understand how the spatiotemporal activity of Rbm24 is regulated by other factors in this process.

Potential implication of Rbm24 in disease

A possible tumor suppressor activity of Rbm24 and Rbm38

RBPs regulate gene expression at multiple levels, and they are implicated in a large variety of biological processes through versatile interactions with RNAs. Alterations in protein-RNA interactions have been causally related to the occurrence of various cancers in humans [START_REF] Pereira | RNA-binding proteins in cancer: old players and new actors[END_REF]. Since Rbm24 protein contains a canonical RRM that binds to GU-rich ligand present in a wide spectrum of target mRNAs [START_REF] Ray | Rapid and systematic analysis of the RNA recognition specificities of RNA-binding proteins[END_REF][START_REF] Qian | Structural basis for mRNA recognition by human RBM38[END_REF], it would be not surprising that inappropriate regulation of its expression or function in humans perturbs the homeostasis of protein synthesis and leads to cancer development. There are several lines of evidence that Rbm24 displays a tumor repressive activity. In several cancer cell lines, such as MCF7 and HaCaT cells, both Rbm24 and Rbm38 are transcriptional targets of the tumor suppressor p53, and their overexpression increases the stability of p21 mRNA [START_REF] Jiang | Rbm24, an RNA-binding protein and a target of p53, regulates p21 expression via mRNA stability[END_REF][START_REF] Shu | RNPC1, an RNA-binding protein and a target of the p53 family, is required for maintaining the stability of the basal and stress-induced p21 transcript[END_REF]. Thus, they can function to induce cell cycle arrest and to prevent tumor cell proliferation. However, this p53-induced anti-tumor activity of Rbm24 and Rbm38 needs to be reconciled with their oncogenic potential, which is related to their repressive function on p53 mRNA translation and p53-dependent apoptosis [START_REF] Zhang | Translational repression of p53 by RNPC1, a p53 target overexpressed in lymphomas[END_REF][START_REF] Zhang | Rbm24, a target of p53, is necessary for proper expression of p53 and heart development[END_REF]. Since serine phosphorylation in the eIF4E-binding motif of Rbm24 and Rbm38 converts them into an activator of p53 expression [START_REF] Zhang | Glycogen synthase kinase 3 promotes p53 mRNA translation via phosphorylation of RNPC1[END_REF], it will be intriguing to compare their phosphorylation status between normal and tumor cells, in order to understand how they regulate mRNA translation in pathological processes. Another enigma is the significance of Rbm24 and Rbm38 in preventing p63-mediated tumor suppression because they have been shown to destabilize p63 mRNA in overexpression experiments [START_REF] Zhang | RNPC1, an RNA-binding protein and a target of the p53 family, regulates p63 expression through mRNA stability[END_REF][START_REF] Xu | RNA-binding protein RBM24 regulates p63 expression via mRNA stability[END_REF]. Again, this may be modulated by serine phosphorylation.

An increase in the level of phosphorylated Rbm38 by GSK3ß enhances p63 expression, but this seems to be dependent on its dissociation with the Ago2-miR203 mRNA decay complex, instead of eIF4E [START_REF] Zhang | Serine 195 phosphorylation in the RNAbinding protein Rbm38 increases p63 expression by modulating Rbm38's interaction with the Ago2-miR203 complex[END_REF]. Thus, the functionality of Rbm24 and Rbm38 in tumorigenesis seems to be regulated by the activity of their associated kinases.

In other situations, the tumor suppressor activity of Rbm24 does not seem to be dependent on its phosphorylation. A recent study indicates that in prostate cancer, the microRNA miR-106a-5p promotes cell proliferation by inhibiting Rbm24 expression, and overexpression of wildtype Rbm24 is able to inhibit tumorigenesis [START_REF] Wei | lncRNA HAND2-AS1 Regulates Prostate Cancer Cell Growth Through Targeting the miR-106a-5p/RBM24 Axis[END_REF]. It has been also shown that in nasopharyngeal carcinoma, the expression of Rbm24 is reduced, which leads to the down-regulation of miR-25 that has the ability to suppress cell proliferation by targeting the pro-oncogenic lncRNA MALAT1. Increasing the expression of Rbm24 can suppress cellular proliferation, migration and invasion [START_REF] Hua | RBM24 suppresses cancer progression by upregulating miR-25 to target MALAT1 in nasopharyngeal carcinoma[END_REF]. These observations thus demonstrate an inhibitory effect of Rbm24 in tumorigenesis. A further understanding of the functional consequences of Rbm24 and Rbm38 in regulating cancer-related gene expression should help to define novel therapeutic strategies for modulation of their activity. Moreover, deciphering the detailed mode of RNA-binding by the conserved RRM of Rbm24 and Rbm38 could provide a basis to generate engineered mutants to modulate their interactions with RNA targets [START_REF] Amrane | Backbone-independent nucleic acid binding by splicing factor SUP-12 reveals key aspects of molecular recognition[END_REF]100]. It is also important to understand how the expression and activity of Rbm24 or Rbm38 are dysregulated in cancer cells. In this regard, it has been shown that in hepatocellular carcinoma, the expression of Rbm24 and several cancer-

Rbm24 in congenital disorders and infection diseases

A more plausible implication of Rbm24 in human disease is its ability to promote U1 snRNP recognition of the mutated, but not the wild-type 5' splicing site in IKBKAP gene (inhibitor of κ light polypeptide gene enhancer in B cells, kinase complex-associated protein) in familial dysautonomia (FD), an autosomal recessive disease [106]. The intronic 5' splicing site mutation of IKBKAP gene in FD disrupts the splicing of exon 20, but this produces altered spliced mRNA product only in a tissue-specific manner, affecting essentially sensory and autonomic nervous system [107]. Rbm24 binds to an element downstream of the mutated splicing site and functions as splicing enhancer [106]. Thus, its tissue-specific expression may help to correct the abnormal splicing and to reduce the penetrance of genetic disorders, making it a potential therapeutic target for FD. This observation highlights the importance of regulated Rbm24 activity in maintaining cellular function and homeostasis. Indeed, as presented above, loss of Rbm24 in mice causes cardiomyopathy [START_REF] Poon | RNA-binding protein RBM24 is required for sarcomere assembly and heart contractility[END_REF][START_REF] Yang | RBM24 is a major regulator of muscle-specific alternative splicing[END_REF][START_REF] Liu | RNA binding protein 24 deletion disrupts global alternative splicing and causes dilated cardiomyopathy[END_REF], but overexpression of Rbm24 has been shown to induce cardiac fibrosis in mouse model by promoting collagen synthesis [108]. Moreover, Rbm24 not only regulates the expression of organism's own genes, several in vitro analyses suggest that it can also function as a host factor to participate in the translation, replication and pregenomic RNA packaging of types B and C of hepatitis viruses [109,110,111]. Thus, we are just beginning to perceive the involvement of Rbm24-mediated post-transcriptional events in the etiology of various cellular dysfunctions. It is clear that the increasing association of Rbm24 with various human diseases requires a more comprehensive mechanistic understanding of its posttranscriptional regulatory functions.

Conclusions and perspectives

Rbm24 emerges as a major post-transcriptional regulator in the switch of cell differentiation during vertebrate development. It represents a multifunctional RBP that orchestrates different gene expression circuits in a tissue-specific and stage-specific manner. There is also accumulating evidence that it maintains the homeostasis of protein synthesis in adult tissues. Thus, it is not surprising that dysfunction of Rbm24-mediated post-transcriptional regulation of gene expression affects early development and leads to the occurrence of various diseases.

Many intriguing questions regarding Rbm24 function during development and disease remain unanswered, such as its maternal activity in translational regulation, the mechanism underlying its dynamic subcellular localization and function during muscle cell differentiation, the biochemical mechanism underlying its implication in CPA, and its association with human diseases. At present, no human congenital disorders have been directly linked to Rbm24 mutations, probably due to the lethality resulted from impaired heart development and blood circulation. In this regard, it would be also of interest to identify and understand the function of those cis-and trans-regulatory elements that control the spatiotemporal expression of Rbm24, which may be disrupted in human diseases. In addition, due to the importance of RBP autoregulation to maintain protein homeostasis or to switch cell fate changes [112], it would be important to understand how Rbm24 self-regulates to maintain cellular homeostasis or to initiate cell differentiation during development. Furthermore, vertebrate Rbm24 also interacts and cooperates with other closely related RBPs, such as Rbm20 and Rbm38, in the regulation of cell differentiation and function [START_REF] Miyamoto | RNA-binding proteins Rbm38 and Rbm24 regulate myogenic differentiation via p21-dependent and -independent regulatory pathways[END_REF][START_REF] Ito | RBM20 and RBM24 cooperatively promote the expression of short enh splice variants[END_REF]. Mutations of RBM20 have been clearly associated with human congenital disorders, such as cardiomyopathy [START_REF] Brauch | Mutations in ribonucleic acid binding protein gene cause familial dilated cardiomyopathy[END_REF][START_REF] Hey | Pathogenic RBM20-variants are associated with a severe desease expression in male patients with dilated cardiomyopathy[END_REF], while inappropriate expression of RBM38 is associated with tumorigenesis [START_REF] Zhang | Translational repression of p53 by RNPC1, a p53 target overexpressed in lymphomas[END_REF]. Thus, understanding the mechanisms by which Rbm24 cooperates with other factors in gene expression regulation should help to develop approaches for therapeutic manipulation.

  related genes is reactivated due to increased demethylation of their enhancers [101], suggesting that epigenetic modulation of Rbm24 expression in tumor cells may have the potential to prevent aberrant cell proliferation. 7.2. Interaction between Rbm24 and microRNAs to maintain cellular homeostasis It has become increasingly evident that the interplay between RBPs and microRNAs represents a new level of complexity in the regulation of gene expression in various cellular processes, which either promotes or represses cancer development [102]. As in prostate cancer and nasopharyngeal carcinoma, Rbm24 also interacts with microRNAs in other tissues to maintain cellular homeostasis. The MIR143 host gene (MIR143HG) is a precursor of miR-143 and miR-145, and is overexpressed in colon tissues of patients with Hirschsprung disease (HSCR). Rbm24 forms a negative feedback loop with MIR143HG, and regulates its expression and stability. Thus, an alteration of this balance could lead to defective proliferation or migration of enteric neural crest cells and gangliogenesis in the gut of HSCR patients [103]. Interestingly, the expression of miR-125b-5p is enriched in heart valves. It binds to, and inhibits the expression of, Rbm24 mRNA [104]. Since loss of Rbm24 leads to heart valve defects, this raises a possibility that an imbalance between miR-125b-5p and Rbm24 may disrupt heart development and function. Similarly, miR-222 prevents myogenic differentiation and myoblast fusion. It exerts these effects partially by inhibiting Rbm24 expression [105]. These observations suggest that Rbm24 may function downstream of microRNAs in post-transcriptional regulatory interactions with potential contributions to muscle physiopathology.

Figure 1 .

 1 Figure 1. Rbm24 is a highly conserved RBP. (A) Schematic representation of human RBM24 protein domains. The amino-terminal half contains a canonical RRM with two consensus RNP sequences (RNP1 and RNP2), and the C-terminal region contains two conserved domains (1 and 2) including an eIF4E-binding motif. Amino acid positions are indicated below. (B) Identity in the overall sequence and in the RRM between human RBM24 and Rbm24 proteins from other species. Human RBM24, NM_001143942.2; mouse Rbm24, NM_001081425.1; chick Rbm24, NM_001012863.3; Xenopus laevis Seb4, NM_001087526.1; zebrafish Rbm24a, NM_212865.1; zebrafish Rbm24b, NM_001328141.1; C. elegans SUP-12, NM_076273.5.

Figure 2 .

 2 Figure 2. Conserved and restricted expression patterns of Rbm24 in vertebrate embryos. (A) The expression of rbm24a in a zebrafish embryo at 22 hpf. (B) A chick embryo at HH20 (3 days). (C) A mouse embryo at E11.5. Arrowheads indicate Rbm24 expression in limb muscles. In all vertebrates, Rbm24 is expressed in the somites, heart, lens, and otic vesicle (OV). Scale bars: (A) 100 µm; (B) 1 mm; (C) 1 mm.

Figure 3 .

 3 Figure 3. Dynamic subcellular localization of Rbm24 protein during muscle cell differentiation. (A) Immunofluorescence staining of a mouse embryonic section at E11.5 shows Rbm24 expression in differentiating myoblasts within the myotome (my), but not in Pax3positive premyogenic progenitors. Some Pax3-expressing cells are intermingled with Rbm24positive cells, but they do not yet express Rbm24. (A') Higher magnification shows the punctate cytoplasmic staining of Rbm24 in a myoblast. (B) Immunofluorescence staining of Rbm24 in adult muscles. (B') Higher magnification shows nuclear localization of Rbm24. (C) Localization of Rbm24-GFP in the cytoplasm of myoblasts from C2C12 cells. (D) Strong nuclear and weak cytoplasmic localization of Rbm24-GFP in differentiated myotubes. Nuclei are stained with DAPI. Scale bars: (A) 50 µm; (A') 10 mm; (B) 20 µm; (B') 10 µm; (C) 10 mm; (D) 10 µm.

Figure 4 .

 4 Figure 4. Loss of Rbm24a function impairs lens transparency and affects inner ear hair cell development in zebrafish. (A, B) Histological sections of ocular tissues at the level of optic nerve compares lens differentiation between a wild-type sibling (A) and an rbm24a mutant (B) at 3 dpf (days post-fertilization). The sections were stained by hematoxilin and eosin. Loss of Rbm24a disrupts lens differentiation and causes cataract formation, but has no effect on retina differentiation. The microphthalmia phenotype and defective lens fiber cell denucleation are secondary consequences due to impaired blood circulation. (C, D) Confocal microscopic analyses compare hair cell development and organization of the lateral crista in the zebrafish inner ear from a wild-type sibling (C) and an rbm24a mutant (D) at 3 dpf, under the Tg(pou4f3:GAP-GFP) transgenic background. Scale bars: (A, B), 50 µm; (C, D), 10 µm.

Funding

This work was supported by the Institut de Biologie Paris-Seine (IBPS), the Centre National de la Recherche Scientifique (CNRS), the Sorbonne University, and the National Natural Science Foundation of China (grant number 31871451).

Author contributions

R.G., M.S., and D.L.S. designed research and analyzed data; R.G., M.S., and A.S. performed research; R.G. and D.L.S. wrote the paper.

Conflicts of interest

The authors declare no conflict of interest.


Loss of Rbm24a causes defective hair cell development in the zebrafish inner ear and neuromasts

1

Letter to the Editor Loss of Rbm24 causes defective hair cell development in the zebrafish inner ear and neuromasts Zebrafish presents many advantages for the study of hair cell differentiation, and has become particularly attractive for understanding the development of vertebrate sensory organs. During the past decades, mutagenesis screening in this model has allowed the identification of a large number of genes implicated in otic development, some of which were associated with human diseases affecting hearing function [START_REF] Blanco-Sánchez | Zebrafish models of human eye and inner ear diseases[END_REF][START_REF] Nicolson | The genetics of hair-cell function in zebrafish[END_REF]. These genes essentially encode components of key signaling pathways, transcription factors, or structural constituents. Functional analyses have contributed to our understanding on the transcriptional control of otic placode specification and differentiation (Nicholson, 2017). However, the posttranscriptional mechanism underlying hair cell differentiation and function remains largely elusive, mostly due to lack of mutations affecting related regulatory networks. RNA-binding proteins (RBPs) are implicated in posttranscriptional regulation of gene expression in development and disease [START_REF] Brinegar | Roles for RNA-binding proteins in development and disease[END_REF]. Rbm24 exhibits strongly localized and highly conserved expression pattern in all vertebrate embryos [START_REF] Grifone | The RNA-binding protein Rbm24 is transiently expressed in myoblasts and is required for myogenic differentiation during vertebrate development[END_REF][START_REF] Grifone | Expression patterns of Rbm24 in lens, nasal epithelium, and inner ear during mouse embryonic development[END_REF]. Beside its expression and implication in skeletal and cardiac muscle development [START_REF] Li | The RNA-binding protein Seb4/RBM24 is a direct target of MyoD and is required for myogenesis during Xenopus early development[END_REF][START_REF] Poon | RNA-binding protein RBM24 is required for sarcomere assembly and heart contractility[END_REF][START_REF] Grifone | The RNA-binding protein Rbm24 is transiently expressed in myoblasts and is required for myogenic differentiation during vertebrate development[END_REF][START_REF] Maragh | Rbm24a and Rbm24b are required for normal somitogenesis[END_REF][START_REF] Yang | RBM24 is a major regulator of muscle-specific alternative splicing[END_REF], it is also expressed in a subset of cells within the differentiating head sensory organs. Particularly, Rbm24 protein colocalizes in the cytoplasm with Myo7A in inner and outer cells of the cochlea, as well as in the saccular and utricular maculae, and in semicircular canals of the mouse early embryo [START_REF] Grifone | Expression patterns of Rbm24 in lens, nasal epithelium, and inner ear during mouse embryonic development[END_REF]. This implies that Rbm24 may posttranscriptionally regulate hair cell development and function, but no functional evidence has been reported in vertebrates.

Zebrafish genome contains two rbm24 paralogs, rbm24a and rbm24b. The expression pattern of rbm24a in different tissues is identical as that of rbm24 in other vertebrates. From previous works [START_REF] Poon | RNA-binding protein RBM24 is required for sarcomere assembly and heart contractility[END_REF][START_REF] Maragh | Rbm24a and Rbm24b are required for normal somitogenesis[END_REF], it can be observed that only rbm24a is expressed in the otic vesicle during early stages of development, whereas rbm24b does not show such an expression pattern. We have generated a zebrafish rbm24a mutant line by TALENmediated genome-editing approach (Shao et al. 2020). Heterozygous mutants developed normally, whereas homozygous mutants showed specific loss of function phenotypes, which could be readily distinguished by the appearance of heart edema at 1.5 dpf (days postfertilization) onward, and verified by allele-specific PCR (Fig. S1). Examination of the otic phenotype revealed a progressive occurrence of otic development defects in rbm24a mutants, which were evident at 3 dpf, and characterized by a delayed fusion of semicircular canals in a majority of embryos (arrow in Fig. 1A). In addition, the size of otic vesicles in rbm24a mutants was reduced as development proceeds, but the semicircular canals eventually fused at 4 dpf (Fig. S2). Analyses of the startle response indicated that rbm24a mutant larvae were largely unresponsive to acoustic stimulation (Fig. 1B,C), and displayed balancing deficits. These observations suggest that mutation of rbm24a impairs otic development and causes hearing and balance deficits.

To analyze how loss of Rbm24a function affects otic neural development, we first examined the expression of several representative markers of the otic vesicle by in situ hybridization. The expression of sox2, which specifies sensory fates in the otic vesicle [START_REF] Gou | sox2 and sox3 play unique roles in development of hair cells and neurons in the zebrafish inner ear[END_REF], was not affected in rbm24a mutants (Fig. S3A). The expression patterns of two crista markers, fgf10a and msx3 [START_REF] Thisse | Fast Release Clones: A High Throughput Expression Analysis[END_REF], were also unchanged in rbm24a mutants, despite an obviously reduced size of the otic vesicle (Fig. S3B,C). These results suggest that Rbm24a should function downstream of these factors, and may be not involved in the specification of the otic vesicle.

However, the expression of myo7a, which localizes to sensory hair cells when these cells start the differentiation, and is required for hair bundle integrity [START_REF] Ernest | Mariner is defective in myosin VIIA: a zebrafish model for human hereditary deafness[END_REF], was obviously reduced in the otic vesicle and in different neuromasts in the mutants at 3 dpf (Fig. 1D). This implies that loss of Rbm24a should affect the differentiation of hair cells. We thus addressed this question more precisely using the Tg(pou4f3:GAP-GFP) transgene (Xiao et al. 2005). At 3 dpf, rbm24a mutants exhibited strongly reduced fluorescence intensity both in the otic vesicle and in the posterior lateral line system (Fig. 1E). Examination of the first five neuromasts (L1-L5), without taking into account the differences in fluorescence intensity, revealed that their number was reduced in rbm24a mutants (Fig. 1F). In general, the fifth neuromast (L5) could be observed in a few mutant embryos, and the most posterior neuromasts (L6 and L7) were generally not observed in the mutant embryos. This result suggests that the formation of hair cells in the otic vesicle and the deposition of neuromasts are defective following loss of Rbm24a function.

It remains to be determined how hair cell differentiation in different positions is affected in rbm24a mutants. We set to examine in more detail the formation and organization of hair cells in the otic vesicle and in the first three neuromasts of the posterior lateral line system (Fig. 1G). The size of the three cristae (anterior, lateral and posterior cristae) seemed to be reduced in rbm24a mutants. Since each hair cell within the crista forms one kinocilium that is more accessible for observation, we used this criterion to monitor the defects of hair cell development. This could reflect to some extent the number of hair cells in each crista. Statistical analyses showed a significantly reduced number of kinocilia in the three cristae (Fig. 1H). The height of these kinocilia was also reduced to some extent, particularly in the lateral crista. In the utricular macula of rbm24a mutants, hair cells were not properly aligned, and cell extruding could be observed in a large majority of embryos (Fig. 1G,I). Similar defects have been observed in sox2 morphants with defective maintenance and regeneration of hair cells [START_REF] Millimaki | Sox2 is required for maintenance and regeneration, but not initial development, of hair cells in the zebrafish inner ear[END_REF]. Due to the difficulty to observe saccular macula, we did not detect obvious differences in the cellular organization between WT sibling and rbm24a mutants. In the first three neuromasts that we examined, the cellular organization into rosette was severely disrupted in rbm24a mutants, which was correlated with a reduced number of hair cells (Fig. 1G,J). These phenotypic and quantitative analyses clearly demonstrate that hair cell development is defective in rbm24a mutants, and they are consistent with the expression of vertebrate Rbm24 in the cristae and maculae [START_REF] Grifone | Expression patterns of Rbm24 in lens, nasal epithelium, and inner ear during mouse embryonic development[END_REF]. At present, it is not clear whether Rbm24a is expressed in neuromasts, and thus how its mutation affects neuromast hair cells. We thus performed in situ hybridization to examine more precisely the expression sites of rbm24a at 1 dpf. This revealed that rbm24a transcripts were also localized in the posterior lateral line primordium (pLLP), in addition to anterior and posterior cristae (Fig. 1K). The pLLP forms from the otic vesicle and migrates caudally to periodically deposit neuromasts [START_REF] Chitnis | Building the posterior lateral line system in zebrafish[END_REF]. Thus, the expression of rbm24a in this placode may explain the defects of neuromast hair cell development in rbm24a mutants.

We thus demonstrated for the first time that loss of Rbm24a affected hair cell formation and organization. Both zebrafish rbm24a and mouse Rbm24 show dynamic expression during hair cell differentiation [START_REF] Grifone | Expression patterns of Rbm24 in lens, nasal epithelium, and inner ear during mouse embryonic development[END_REF][START_REF] Lush | scRNA-Seq reveals distinct stem cell populations that drive hair cell regeneration after loss of Fgf and Notch signaling[END_REF], and it may function downstream of transcription factors, such as Atoh1, in hair cell development and regeneration [START_REF] Cai | Characterization of the transcriptome of nascent hair cells and identification of direct targets of the Atoh1 transcription factor[END_REF]. Indeed, it is of interest to note that loss of Rbm24a function has no effect on the expression of several genes coding for transcription factors that are involved in otic vesicle specification, but it affects the expression of hair cell differentiation genes, such myo7a. This is fully consistent with our recent finding showing that Rbm24 protein is localized in the cells where Myo7A is expressed [START_REF] Grifone | Expression patterns of Rbm24 in lens, nasal epithelium, and inner ear during mouse embryonic development[END_REF], and raises the question whether and how Rbm24a posttranscriptionally regulates myo7a whose dysfunction causes the Usher syndrome type 1B [START_REF] Weil | Defective myosin VIIA gene responsible for Usher syndrome type 1B[END_REF].

Besides the specific otic phenotype, our observation of rbm24a expression in the pLLP is also of interest for understanding the developmental mechanisms of vertebrate sensory systems, because the advantages of the zebrafish posterior lateral line system [START_REF] Ghysen | The lateral line microcosmos[END_REF]. Thus, this work could not only provide a useful model to study hearing disorders, it also should open novel avenues for investigating posttranscriptional control in these processes. Along with our present findings demonstrating a critical role of Rbm24 in regulating cytoplasmic polyadenylation during lens differentiation (Shao et al. 2020), the present work further emphasizes a critical role of posttranscriptional regulation in the development of head sensory organs. Given the importance of RBPs in development and diseases, and the highly conserved expression of vertebrate Rbm24 in hair cells, it will be particularly interesting to identify human mutations affecting the cis-regulatory elements that disrupt its inner ear specific expression and lead to hearing disorders.

Fig. 1 .F:

 1 Fig.1. Defective hair cell development in zebrafish rbm24a mutants. A: Confocal differential interference contrast images extracted from Fig. S2 show otic phenotypes at 3 dpf. Notice the delayed fusion of semicircular canals in rbm24a mutants (red arrow). Scale bar: 50 µm. B: Representative graphs from startle response test compare the behaviors of WT siblings and rbm24a mutants at 3 dpf. C: Statistical analyses of the responses to acoustic stimulation. The number in each column indicates total embryos tested in three independent experiments (**, p < 0.01; Student's t-test). D: In situ hybridization analysis shows the reduced expression of myo7aa in the otic vesicle (outline by yellow circle) and neuromasts in rbm24a mutants at 3 dpf. Scale

Acknowledgments

We thank Y.F. Feng for assistance with startle response test and hair cell analysis. This work was supported by the National Key R&D Project of China (grant number 2018YFA0801000), the National Natural Science Foundation of China (grant numbers 31671509, 31900577), the Centre National de la Recherche Scientifique (CNRS), and the Sorbonne University.

Author information

Xiao-Ning Cheng 1 , Jing-Jing Zhang 1 and De-Li Shi 1,2 * 1 Affiliated Hospital of Guangdong Medical University, Zhanjiang 524001, China

Materials and Methods

Zebrafish

Adults of the AB strain were maintained in standard housing systems (Haisheng), and early embryos were cultured at 28.5°C in a temperature-controlled incubator. The rbm24a mutant line was generated using TALEN genome-editing technique as recently described (Shao et al. 2020).

Heterozygous carriers were crossed with the Tg(pou4f3:GAP-GFP) transgenic zebrafish (Xiao et al. 2005). Homozygous mutants could be distinguished by the presence of heart edema at 1.5 dpf onward, and the genotype was subsequently verified by allele-specific PCR using specific primers (Fig. S1) as described previously [START_REF] Xing | Mutational analysis of dishevelled genes in zebrafish reveals distinct functions in embryonic patterning and gastrulation cell movements[END_REF]. To prevent pigment development in some experiments, embryos at 1 dpf were cultured in the presence of 50 mM phenylthiourea (Sigma Aldrich).

Startle response test

WT and mutant embryos at 3 dpf were placed individually in a well of 24-well plate with 1 mL of embryo medium [START_REF] Westerfield | The zebrafish book: a guide for the laboratory use of zebrafish (Danio rerio)[END_REF]. Their behaviors were recorded using the EthoVision XT Version 9.0 System (Noldus, Netherlands) for a short period of 6 seconds. A pure-tone acoustic stimulus (500 Hz, 80 dB) was delivered at 3.5 seconds using the SweepGen software, and the swimming speed of embryos was monitored using an infrared detector. Each embryo was tested 15 times, with an interval of 5 minutes. The experiments were performed using three independent batches of embryos, and the results were expressed as the percentage response manifested by a single embryo.

Quantification of otic vesicle size

WT siblings and mutant embryos were cultured at 28.5°C. At appropriate stage, they were embedded in 1% low melting agarose and individually imaged at the otic vesicle under confocal differential interference contrast mode. The otic vesicle from each image was analyzed using ImageJ software (NIH Image), and the otic size was determined by calculating the number of pixels using at least three embryos in each condition.

In situ hybridization

A short region within the coding sequences of different genes was amplified by PCR from embryonic cDNAs, and cloned in pGEM-T easy vector (Promega). The plasmids were linearized using appropriate enzymes, and antisense probes were synthesized in the presence of digoxygenin-labeled rNTP mix (Roche Diagnostics). Embryos were fixed in 4% paraformaldehyde at 4°C overnight and stored in methanol. In situ hybridization was performed using published protocol [START_REF] Westerfield | The zebrafish book: a guide for the laboratory use of zebrafish (Danio rerio)[END_REF], and BM purple (Roche Diagnostics) was used as a substrate of alkaline phosphatase.

Confocal microscopy

Live embryos were anesthetized in tricaine solution (0.17 mgmL -1 ) and embedded in a cavity microscope slide with 4% methylcellulose. Live Images of whole embryos were acquired using a fluorescence microscope (Leica, M205FA), and hair cells were examined under a confocal microscope (Lecia, SP5 II). Z-stack projections were obtained by using the z-projection function.

Statistical analyses

The data were obtained from at least three different batches of embryos, with representative images shown. Statistical analyses were performed using unpaired Student's t-test and GraphPad Prism 6 software, with p values indicated in both the figures and the corresponding figure legends.