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

(1)

HAL Id: hal-02344465

https://hal.archives-ouvertes.fr/hal-02344465

Submitted on 4 Nov 2019

HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Raphaëlle Grifone, Audrey Saquet, Zhigang Xu, De-Li Shi

To cite this version:

Raphaëlle Grifone, Audrey Saquet, Zhigang Xu, De-Li Shi. Expression patterns of Rbm24 in lens,

nasal epithelium, and inner ear during mouse embryonic development. Developmental Dynamics,

Wiley, 2018, 247 (10), pp.1160-1169. �10.1002/dvdy.24666�. �hal-02344465�

(2)

PATTERNS & PHENOTYPES

Expression Patterns of Rbm24 in Lens, Nasal Epithelium, and Inner Ear During Mouse Embryonic Development

Raphaëlle Grifone ,

¹*

Audrey Saquet,

¹

Zhigang Xu,

²

and De-Li Shi

¹

1Sorbonne Universités, UPMC Univ Paris 06, CNRS UMR7622, IBPS-Developmental Biology Laboratory, Paris, France

2School of Life Sciences, Shandong University, Jinan, China

Background:

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 speci

ﬁ

c 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 speci

ﬁ

cally detected in speci

ﬁ

c 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 speci

ﬁ

city and proliferation. Moreover, Rbm24 pro- tein 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.

Developmen- tal Dynamics 247:1160–1169, 2018

. © 2018 Wiley Periodicals, Inc.

Key words:mouse embryogenesis; RNA-binding protein; expression pattern; sensory epithelia

Submitted 3 May 2017; First Decision 13 July 2018; Accepted 17 August 2018; Published online 7 October 2018

Introduction

Rbm24

gene encodes an RNA-binding protein (RBP) harboring in its N-terminal part a single RNA-recognition motif (RRM) that is highly conserved in

C. elegans, Xenopus, mice, and human

(Fetka et al., 2000). RBP in general and RRM-containing proteins in particular, are involved in regulating the metabolism of mRNAs at all stages of their lifetime thus appearing as key regu- lators of post-transcriptional gene expression. By controlling gene expression through the regulation of alternative splicing, transport, localization, and stability of their target RNA, they have been involved in different tissue-speci

ﬁ

c processes during embryonic development and the signi

ﬁ

cance of post- transcriptional mechanisms for the regulation of cell differentia- tion becomes increasingly evident during embryogenesis.

So far, Rbm24 protein has been shown to be involved in car- diac and muscle differentiation during embryogenesis, the two main sites of expression of the gene depicted so far in the embryo.

Rbm24

is indeed expressed in cardiomyocytes as well as in deter- mined myoblasts of the somites and of the limbs in chick and mouse embryos. Loss of function experiments demonstrated the requirement of the Rbm24 protein to trigger skeletal muscle dif- ferentiation (Grifone et al., 2014). A

Rbm24

knock-out mice

model pointed out the role of Rbm24 to enhance embryonic heart morphogenesis and promote

ﬁ

nal sarcomeric maturation of skele- tal and cardiac muscles tissues (Yang et al., 2014). RNA-seq ana- lyses combined to reverse transcriptase-polymerase chain reaction (RT-PCR) indeed demonstrated that Rbm24 regulates alternative splicing, by promoting exon inclusion, of several target RNA involved in heart and muscle development (Yang et al., 2014).

Rbm24-dependent splicing related to exon inclusion have been also highlighted in skeletal muscle and cardiac differentia- tion in vitro (Cardinali et al., 2016; Zhang et al., 2016). This tis- sue speci

ﬁ

c role of Rbm24 in the regulation of cardiac gene expression, sarcomeric assembly, and cardiac contractility seems to be conserved among vertebrates because Rbm24 de

ﬁ

ciency using morpholino oligonucleotides in zebra

ﬁ

sh resulted in a reduction in sarcomeric proteins expression and diminished heart contractility in embryos (Poon et al., 2012).

Whether Rbm24 is a speci

ﬁ

c regulator of striated muscle development or could it play a broader role during embryogene- sis is the question we wanted to adress. To achieve this, we per- formed

in situ

hybridization experiments on different vertebrate embryos models as well as a set of immuno

ﬂ

uorescence analyses on mouse embryo sections at different stages of development. In the present manuscript we describe the accumulation of Rbm24

Article is online at: http://onlinelibrary.wiley.com/doi/10.1002/dvdy.

24666/abstract

*Correspondence to: Raphaëlle Grifone, Sorbonne Universités, UPMC Univ Paris 06, CNRS UMR7622, IBPS-Developmental Biology Laboratory, 9, Quai Saint-Bernard, case 24, 75005 Paris, France. E-mail: raphaelle.

grifone@upmc.fr

1160

DEVELOPMENTAL DYNAMICS 247:1160–1169, 2018 DOI: 10.1002/dvdy.24666

DEVELOPMENTAL DYNAMICS

(3)

protein in developmental territoritories that has not been reported so far: the lens and the olfactory epithelium. Moreover, we describe the accumulation of Rbm24 protein in hair cells of the cochlear and vestibular systems. These results complete a nice study describing by

in situ

hybridization the expression of

Rbm24

in cochlear cells and demonstrating the direct regulation of Rbm24 by Atoh1 in this territory (Cai et al., 2015). Altogether these results provide potential new roles for Rbm24 during verte- brate embryonic development.

Results and Discussion

Rbm24 is Expressed in Nonmuscle Head Territories in Vertebrate Embryos

To analyze whether Rbm24 could be a regulator of other addi- tional developmental processes than skeletal myogenesis and cardiomyogenesis in vertebrates, we performed detailed whole- mount

in situ

hybridization experiments to detect

Rbm24

expression during

Xenopus, chick, and mouse embryogenesis. Of

interest, in addition to the expression in skeletal muscle and car- diac progenitor cells that have been previously described in all three models,

Rbm24

is conservely expressed in eye, ear, and nose anlagen in vertebrate embryos (Fig. 1) (Li et al., 2010; Gri- fone et al., 2014; Maragh et al., 2014). At the level of the eye,

Rbm24

is found to be strongly expressed in the lens of

Xenopus

embryo from stage 26 (Fig. 1A,A

⁰

) to stage 38 (Fig. 1B,B

⁰

) as revealed by whole-mount labeling (Fig. 1A,B) as well as by Vibratome sections of labeled embryos (Fig. 1A

⁰

,B

⁰

).

Rbm24

is similarly strongly expressed in the lens of HH20 chick embryo (Fig. 1C) and of embryonic day (E) 10.5 mouse embryos (Fig. 1E,F).

In the ear anlagen,

Rbm24

is expressed in the otic vesicle of

Xenopus

embyros, at both stages, with a stronger signal in the lateral part of the territory (Fig. 1A,A

⁰

,B). Whereas

Rbm24

is faintly and uniformly expressed in the otic placode of HH20 chick embryo (Fig. 1C), it is strongly activated in the otic vesicle of E9.5 (Fig. 1D) and E10.5 (Fig. 1E) mouse embryos, and more speci

ﬁ

cally in the lateral edge of the placode. Finally, at the level of the nose anlagen,

Rbm24

is, of interest, found to be activated in a profound subregion of the nasal pit in chick and mouse embryos (Fig. 1C,F,G). Our expression analysis demonstrates that three expression sites of

Rbm24

at the head level are conserved in

Xenopus, chick, and mouse embryos. Our data reinforce the

expression data that have been published in zebra

ﬁ

sh embryo where

Rbm24a

gene has been found to be activated in lens and otic vesicle (Maragh et al., 2014).

Rbm24 is Expressed in the Lens Throughout Embryonic Development

Because whole-mount

in situ

hybridization on mouse, chicken, and

Xenopus

embryos revealed the expression of

Rbm24

in the eye, we performed immunostaining on sections of mouse embryos at different stages of development to better examine where Rbm24 protein is accumulating. We performed immunos- taining of Rbm24 on serial sections with antibodies labeling dif- ferent parts of the eye, in combination with Pax6 which is speci

ﬁ

c for the retina, the lens, and the cornea or with Prox1, which is speci

ﬁ

c for the lens. At all stages analyzed, from E10.5 to E14.5, we detected an expression of

Rbm24

in lens speci

ﬁ

- cally. This is consistent with the large scale study identifying Rbm24 as a lens enriched mRNA in mouse (Lachke et al., 2012).

Fig. 1.Expression of Rbm24detected byin situhybridization inXenopusembryos (A,B), chick embryos (C), and mouse embryos (D–G). A,B:

Xenopusembryos, lateral view, anterior toward the right. At the head level, in stage 26 (A) and stage 38 (B)Xenopusembryos,Rbm24is expressed in the optic vesicle (opt) and the otic vesicle (ot) territories. Vibratome transverse cross-sections enable to visualize thatRbm24is expressed in the lateral part of the otic vesicle (A⁰) and in the lens (le) of the developing eye (A⁰and B⁰). A⁰and A⁰are vibratome sections of the embryo imaged in A at the optic and otic level, respectively. B⁰is a vibratome section of the embryo labeled in B.C: HH20 stage chick embryo, lateral view of the head, anterior towards the right.Rbm24is expressed in developing ear (ear) and in the eye with a strong signal in the lens (le). An obvious expression of Rbm24is also detected in the nasal epithelium (ne) territory at the level of the invaginating nasal pit.D: E9.5 mouse embryo, dorsal view at the head level.Rbm24is strongly expressed in otic vesicles (ov).E–G: E10.5 stage embryos in lateral view (E), anterolateral view (F), and anterior view (G).

Rbm24is still expressed in the otic vesicle (ot in E), in the lens of the developing eye (le in E and F), as well as in the nasal epithelium (ne) territory at the level of the invaginating nasal pit (ne in F and G). Note thatRbm24is also expressed in head muscles (hm).

DEVELOPMENTAL DYNAMICS

(4)

At E10.5 when the lens placode has completely invaginated to form a closed lens vesicle, visualized by the immunodetection of Prox1 and Pax6 proteins, Rbm24 protein is found to be expressed in the posterior half of the vesicle that is composed of elongating cells, the primary lens

ﬁ

bers, and is not expressed ventrally in cuboidal cells forming the lens epithelium. Rbm24 is not detected at all in the retina and the cornea visualized by the presence of Pax6 protein (Fig. 2A

–

A

⁰⁰

). At E11.5, Rbm24 protein is still restricted to the elongated primary lens

ﬁ

ber and is not detected in lens epithelium nor in the retina and cornea (Fig. 2B

–

B

⁰⁰

). At later stages, E13.5 and E14.5,

Rbm24

is no lon- ger expressed in differentiated lens

ﬁ

bers but is activated it in the epithelial region just above the lens equator that is known as the germinative zone (Fig. 2C

–

C

⁰⁰

,D

–

D

⁰⁰

). Cells of this region divide, and migrate dorsally, below the equator, initiate their elongation to become the secondary lens

ﬁ

bers that will wrap around the core layer of previously formed

ﬁ

bers.

During development and after birth, these cells are degrading their organelles, including mitochondria and nucleus, and are pressed deeper into the lens by the successive

ﬁ

ber cells to form the transparent tissue that focuses light on the retina (Graw, 2010; Gunhaga, 2011). Ventrally, at E13.5 and E14.5, Rmb24 proteins start to accumulate in the lens epithelium together with Prox1 and Pax6 proteins (Fig. 2C

–

C

⁰⁰

,D

–

D

⁰⁰

). These anterior epi- thelial cells remain mitotically active as a stem cell niche pro- ducing secondary

ﬁ

ber cells (Griep, 2006; Graw, 2010). During early stages of mouse lens development the expression of Rbm24 is similar to that of several transcription factors includ- ing Pax6, Pitx3, Foxe3, Prox1, Sox2, Six3, c-Maf, and L-Maf that are crucial for lens cell speci

ﬁ

cation and primary

ﬁ

ber cell differentiation (Kondoh et al., 2004; Medina-Martinez et al., 2009; Graw, 2010; Gunhaga, 2011). Thus it would be very inter- esting to examine whether

Rbm24

expression could be under the regulation of one or more of these factors at E10.5 and E11.5 stages. Moreover, Fgf and BMP signaling are known to regulate initial differentiation of primary lens

ﬁ

bre and are required for expression of Pax6 and Sox2 (Garcia et al., 2011). Thus, it would be useful to examine whether

Rbm24

expression could be regu- lated by these pathways in the posterior part of the lens.

Finally, the progression of lens differentiation has been shown to be regulated through Src family tyrosine kinase activity (Walker et al., 2002a, 2002b, 2002c). Thus, it would be interest- ing to investigate whether

Rbm24

is part of this regulatory cas- cade. Later on at E13.5 and E14.5, the activation of

Rbm24

expression on the anterior and marginal sides, in mitotically active epithelial cells could be under the dependence of Wnt.

Wnt/ß-catenin pathway genes such as several Fzd receptors and co-receptors are indeed predominantly expressed in these cells and some null mutants have been characterized by an incom- pletely formed anterior epithelium (Stump et al., 2003; de Iongh et al., 2006).

Higher magni

ﬁ

cation observations of Rbm24 immunodetec- tion allow us to examine the subcellular localization of Rbm24 protein in the lens (Fig. 3). Of interest, Rbm24 is exclusively found to accumulate in the cytoplasm of the cells, in a granular pattern, at all levels and stages examined: primary lens

ﬁ

bers (Fig. 3A

–

A

⁰⁰

), cells of the marginal zone (Fig. 3B

–

B

⁰⁰

,C

–

C

⁰⁰

), as well as cells of the lens epithelium (Fig. 3D

–

D

⁰⁰

). Considering the role played by this RNA-binding protein to stabilize speci

ﬁ

c mRNA besides its role to trigger speci

ﬁ

c exon inclusion and intrusion in cardiac and skeletal muscle cells, one can suggest

that Rbm24 is required in the lens to stabilize speci

ﬁ

c mRNA (Yang et al., 2014). Transcriptome analysis in

Rbm24

mutant models would allow identi

ﬁ

cation of speci

ﬁ

c target genes that need to be stabilized throughout lens differentiation.

Altogether these results suggest that Rbm24 protein could be part of the post-transcriptional network governing lens-speci

ﬁ

c gene expression regulation, as it has been shown for other RNA- binding proteins such as Caprin 2 in mouse (Dash et al., 2015).

Finally, a role played by Rbm24 in lens development, as suggested by the microphthalmia brie

ﬂ

y depicted in a

Rbm24

morphant zebra

ﬁ

sh, needs to be extensively monitored in vertebrates (Maragh et al., 2014).

Rbm24 is Expressed in the Sensory Olfactory Epithelium

Because our whole-mount

in situ

hybridization on mouse and chicken embryos revealed the expression of

Rbm24

in the two nasal processes in the anterolateral regions of the head, we per- formed immunostaining on adjacent sections of mouse embryos at E10.5 (Fig. 4A

–

E

⁰⁰

), E11.5 (Fig. 4F

–

K), and E14.5 (Fig. 4L

–

Q) to better examine the accumulation of Rbm24 protein as the devel- opment of the nasal cavity proceeds. At E10.5 when the olfac- tory placode is invaginating to form the nasal pit (Fig. 4A), which continues to deepen inside at E11.5 (Fig. 4F), we can observe that Rbm24 proteins accumulate in cells of the primary olfactory neuroepithelium as visualized by the expression of the neural patterning factor Pax6 (Fig. 4B

–

D,F

–

I).

At these stages of development, the olfactory epithelium is regionalized in an outside-in pattern with sequential domains of cells expressing markers of successively more differentiated stages. Cells at the periphery of the pit are mitotic uncommited neuronal stem cells, expressing Fgf8 and Sox2, while cells in the center of the nasal pit are Neurogenin1 and Mash-1-expressing cells, namely immediate neuronal precursors (INPs), whose prog- eny will give rise to NCAM-positive terminally differentiated olfactory receptor neurons (ORNs) lying at the deepest recess of the pit (Kawauchi et al., 2004,2005; Beites et al., 2005).

Our immunodetection analyses thus reveal an expression domain of Rbm24 encompassing the central two-thirds of the pit at E10.5 and E11.5, containing differentiating INPs and ORNs neurons, we can assume that Rbm24 could play a role in pro- moting the neurogenic differentiation in the vertebrate olfactory epithelium. This role in promoting differentation is consistent with its requirement in regulating the differentiation steps of muscle and cardiac cells, as demonstrated in vitro and in vivo (Grifone et al., 2014; Yang et al., 2014; Cardinali et al., 2016).

Similarly as in lens cells, higher magni

ﬁ

ed views allow us to show that Rbm24 protein is restricted to a heterogenous cytoplasmic accumulation, in neuro-epithelial cells at E10.5 (Fig. 4E

–

E

⁰⁰

) and E11.5 (Fig. 4J,K), thus sugesting a role for this RNA-binding protein to regulate the stabilty or decay of speci

ﬁ

c target RNAs. Of interest, other RNA-binding proteins, such as HuC/D have been shown to accumulate in the cytoplasm of post- mitotic olfactory neurons at this stage in a similar manner as Rbm24, thus suggesting a post-transcriptional requirement for promoting and/or maintaining the differentiation status of these neuronal sensory cells (Maier et al., 2010).

Whether

Rbm24

expression is under the regulation of tran- scription factors present in the same cells and at the same time, such as Lhx2, Hes5, Neurogenin1, NeuroD1, or Sox2 should be

1162 GRIFONE ET AL.

DEVELOPMENTAL DYNAMICS

(5)

further investigated (Beites et al., 2005; Maier and Gunhaga, 2009; Maier et al., 2010; Panaliappan et al., 2018). Finally, because Fgf signaling has been demonstrated to be required for the generation and differentiation of sensory olfactive cells, by counteracting BMP signaling, monitoring

Rbm24

expression in a Fgf or BMP mutant context would be useful (Maier et al., 2010;

Panaliappan et al., 2018).

At a later stage, at E14.5, the olfactory epithelium has elabo- rated several nasal turbinates (Fig. 4L,M) and has matured into different layers composed of speci

ﬁ

c cell types. Stem cells and transit-amplifying cells are found in the basal compartment, close to the basal lamina of the epithelium. On the opposite side, the supporting cells line the apical surface of the olfactory epi- thelium, adjacent to the nasal cavity. Fully differentiated

Fig. 2. A–D⁰⁰: Immunodetection of Rbm24, Prox1, and Pax6 proteins was performed on successive transverse sections of E10.5, E11.5, E13.5, and E14.5 mouse embryos, at the eye level. Prox1 protein labels the lens (le) of the eye at all stages of development analyzed. Pax6 expression allows the vizualisation of the retina (re) as well as the cornea (co) of the eye. Rbm24 expression is detected in the medial part of the lens facing the retinal epithelium at E10.5 as well as at E11.5. At E13.5 and E14.5, Rbm24 protein is found to accumulate in the equatorial (eq) zone of the lens. Scale bars: 100μm.

DEVELOPMENTAL DYNAMICS

(6)

olfactory receptor neurones are located in an intermediate zone between these basal and apical layers, making up the bulk of the epithelium (Beites et al., 2005; Kawauchi et al., 2005). Of inter- est, the olfactory epithelium retains both its epithelial morphol- ogy with layers of progressively differentiating cells and its ablilty to generate olfactory neurons throughout life, olfactory neurons may have a unique capacity to regenerate after trauma.

As presented in Figure 4, Rbm24 is found to be still expressed in the olfactory epithelium at this stage as demonstrated by immunodetection of Rbm24 and Sox2 performed on adjacent sections of mouse embryos (Fig. 4L

–

Q). By comparison with Sox2, whose expression is associated with basal uncommitted stem cells and with apical sustentacular cells, we can assume that Rbm24 is speci

ﬁ

c to basal neural stem cells where it is strongly expressed and to differentiating olfactory neurons where the expression, although weaker, is still clearly detectable (Fig. 4N

–

Q). Rbm24 is not expressed at all in sustentacular cells.

As at earlier stages described above, Rbm24 protein accumu- lates in the cytoplasm of these two populations of neural cells rather than in nuclei, as demonstrated by the higher magni

ﬁ

ca- tion imaging of the epithelium (Fig. 4P,Q). This suggests once again a role for Rbm24 to control, in the cytoplasm of these

cells, the ability of speci

ﬁ

c target mRNAs to be highly translated or not. Because retinoic acid (RA) signaling has been demon- strated to be a key factor to promote the generation and the maintenance of olfactory neural progenitors, where Rbm24 is strongly expressed, and because Raldh2 and 3 are two critical RA-synthesizing enzyme within this neuroepithelium, a control of

Rbm24

expression by RA in progenitor cells could be ana- lyzed using

Raldh2^-/-3^-/-

mutant mice (Paschaki et al., 2013).

Above all, a role for Rbm24 in inducing or maintening the stem/

progenitor population and/or regulating their progression toward more committed olfactory neuronal cells has to be evaluated throughout development using mutant models.

Rbm24 is Expressed in Inner Ear Sensory Epithelia Cells Considering the early expression of Rbm24 in ear placodes in chick and mouse embryos depicted in Figure 1 and due to our great interest in identifying the molecular mechanisms involved in hair cells mediated hearing mechanoelectrical transduction, we examined the expression of

Rbm24

in inner ear of neonate mice (Xu et al., 2008; Wang et al., 2017). Moreover, a nice study has recently demonstrated using RNA-seq and ChIP-seq

Fig. 3. A–D⁰⁰: Observation at high magniﬁcation of Rbm24 immunolocalization in areas of lens framed by a white rectangle in Figure 2. Images in A, B, C, and D correspond to areas framed in E10.5, E11.5, E13.5, and E14.5, respectively. A⁰⁰, B⁰⁰, C⁰⁰, and D⁰⁰are merged images of Rbm24 detection in A, B, C, and D and DAPI staining in A⁰, B⁰, C⁰, and D⁰, respectively. Note that Rbm24 protein accumulates exclusively in the cytoplasm of lens cells in all areas examined and at each developmental stage. Scale bars = 10μm.

1164 GRIFONE ET AL.

DEVELOPMENTAL DYNAMICS

(7)

Fig. 4.Immunodetection of Rbm24 protein and the neuronal cell marker Pax6 in serial sections of the mouse olfactory neuroepithelium at E10.5 (A–E⁰⁰), E11.5 (F–K), and E14.5 (L–Q).A: DAPI immunostaining showing the invaginating nasal pit framed with a white rectangle.B: Immunodetection showing Rbm24 expression in the neurogenic domain of the invaginating nasal neuroepithelium. C,D: Higher magnification of Pax6 (C) and Rbm24 (D) immunodetections on serial sections at the nasal pit level. Rbm24 is expressed in the medial part of the neuroepithelium.E–E⁰: E⁰is a merge image of Rbm24 detection (E⁰) and dapi staining (E⁰) observed at high magnification, in the Rbm24 expressing neuroepithelium. Note that Rbm24 protein accumulates exclusively in the cytoplasm of these neural cells.F–I: Pax6 (F,H) and Rbm24 (G,I) immunodetections in the neurogenic domain of the invaginating nasal neuroepithelium at E11.5 days of development at low (F,G) and higher (H,I) magnifications.J,K: High magnifications of Rbm24 immunodetection and DAPI staining in neuroepithelila cells of the nasal pit showing an exclusive accumulation of Rbm24 in the cytoplasm of the cells. L–Q: Immunodetection of Rbm24 (L,N,P) and Sox2 (M,O,Q) on serial sections of the nasal process of an E14.5 mouse embryo at low (L,M), middle (N,O), and high (P,Q) magnifications. ba, basal; ap, apical; nc, nasal cavity.

DEVELOPMENTAL DYNAMICS

(8)

experiments performed on puri

ﬁ

ed cochlear cells at E17.5, that Rbm24 is a direct target gene of transcription factor Atoh1 and that it is thus activated in inner and outer hair cells in mice (Cai et al., 2015). These results prompted us to examine, using immu- no

ﬂ

uorescence analyses the expression of Rbm24 in inner ear of neonate mice.

As shown in Figure 5, Rbm24 immunoreactivity is detectable in orderly rows of cells similarly to that of Myosin 7A, an unconventional myosin known to be speci

ﬁ

c for hair cells hosted by the cochlear duct and the vestibular system (Fig. 5A,

B,A

⁰

,B

⁰

). More precisely within the cochlea, Rbm24 is found to be expressed in the two types of mechanosensory cells, the inner hair cells (IHCs) and the outer hair cells (OHCs) of the organ of Corti, which transduce mechanical sound vibrations into nerve impulses (Fig. 5F,F

⁰

,G,G

⁰

). OHCs perform an amplify- ing role and IHCs that detect the sound and transmit it to the brain by means of the auditory nerve. These observations are fully in accordance with published results reporting by,

in situ

hybridization, the expression of Rbm24 in IHC and OHCs at E17.5 (Cai et al., 2015).

Fig. 5.Immunodetection of Rbm24 (A–G) and Myosin7A (A⁰–G⁰) in serial sections of inner ears dissected from mouse neonates. Sections are performed through the vestibular system and the cochlear duct. They are perpendicular to the cochlea axis through three spiral passages of the cochlear duct, framed with white rectangles in the low magnification images in A and A⁰. Sections also pass through the saccule, utricule, and semicircular canals regions of the vestibular system, annotated 1, 2, and 3, respectively, in the low magnification images in B and B⁰. B,B⁰: Rbm24 (B) and Myosin7A (named Myo7A) (B⁰) are expressed in the maccula of saccule (arrow in 1), the maccula of the utricle (arrow in 2), as well as in the crista of the semicircular duct (arrowhead). C–D⁰: Higher magnifications of the maccula of the utricle allow visualization of sensory hair cells expressing Rbm24 (C,D) as well as Myosin7A (C⁰,D⁰). E,E⁰: Higher magnifications of the crista of the semicircular duct allow visualization of sensorial hair cells expressing Rbm24 (E) as well as Myosin7A (E⁰). F–G⁰: Higher magnifications of the cochlear duct, at the level of the organ of corti, allow visualization of mechanosensorial hair cells, inner hair cell (IHC), and outer hair cells (OHCs) expressing Rbm24 (F,G) as well as Myosin7A (F⁰,G⁰).

Rbm24 protein is also detected in the neighboring supporting cells. Note that Rbm24 protein accumulation is restricted to the cytoplasm of all the hair cells up to the apical surface, which is its mechanically sensitive organelle, the hair bundle, which consists of dozens of stereocilia. Rbm24 is also found to strongly accumulautes in the kinocilium of these hair cells similarly as Myosin 7A (arrows in C–G and C⁰–G⁰). Scale bars = 10μm.

1166 GRIFONE ET AL.

DEVELOPMENTAL DYNAMICS

(9)

Fig. 6.Immunodetection of Rbm24, Myosin7A, and Sox2 proteins in adjacent sections of the head of an E14.5 mouse embryo at the level of the otolithic organs. Sections are perfomed through two dfferent levels of the vestibular system and presented at low magniﬁcation in A–C and G–I.A–C:

Rbm24, Myosin7A, and Sox2 are expressed in the the saccule (arrowhead). Note the strong accumulation of Rbm24 proteins in muscles masses (m).

The area pointed by the arrowhead in A to C are shown at higher magniﬁcation in D, E, F, respectively.D–F: Rbm24 (D) and Myosin 7A (E) proteins are found to accumulate in the cytoplasm and the stereocilia of the sensory hair cells, whereas Sox2 is restricited to the nuclei of these cells (F).G–I:

Rbm24, Myosin7A, and Sox2 are expressed in the saccule in area 1, the utricle in area 2, and the crista of the semicircular duct in area 3.

J–L: Higher magnification of the saccule in area 1 shows the accumulation of Rbm24 (J) and Myosin7A (K) in the cytoplasm and Sox2 (L) in the nuclei of the hair cells.M–O: Higher magnification of the utricle in area 2 shows the accumulation of Rbm24 (M) and Myosin7A (N) in the cytoplasm and Sox2 (O) in the nuclei of the hair cells.P–R: Higher magnification of the the crista of the semicircular duct in area 3 shows the accumulation of Rbm24 (J) and Myosin7A (K) in the cytoplasm and Sox2 (L) in the nuclei of the hair cells. Scale bars = 200μm in A–C,G–I; 50μm in D–F,J–R.

DEVELOPMENTAL DYNAMICS

(10)

Our immunodetection analyses show that Rbm24 protein is also detected in the neighboring nonstereociliated supporting cells that are myosin7A-negative, such as inner sulcus cells and Deiters

’

cells (Fig. 5F). A closer view of this region allows us to visualize that Rbm24 protein hetereogenously accumulates in the cytoplas- mic compartment, stronglier around the nucleus, and throughout the stereociliary bundles of the hair cells (Fig. 5F,F

⁰

,G,G

⁰

). Besides the cochlea, the vestibular sytem is the sensory part of the inner ear that provides information on motion, equilibrium, and spatial orientation through the utricle and the saccule that detect gravity and linear movement and through the semicircular canals that detect rotational movement (Lim and Brichta, 2016). Within this apparatus, Rbm24 immunoreactivity is detected in sensory hair cells forming the maccula of the utricule, the maccula of the sac- cule as well as the crista of the semicircular canals (Fig. 5B

–

E), similarly as Myosin 7A (Fig. 5B

⁰–

E

⁰

).

As in cochlear cells, Rbm24 protein accumulates in the cyto- plasm of the cell up to the tip of the stereocilia and the kinocilia (Fig. 5C,D,E). Given its strong and speci

ﬁ

c expression pattern in hair cells, examing whether Rbm24 plays a functional role in hair cells is of prime interest. If so, identifying the RNA speci

ﬁ

cally targeted by this RNA-binding protein would increase our knowl- edge of the molecular mechanisms regulating the function of hair cells in the inner ear. Moreover, how Rbm24 expression is tempo- rally and spacially regulated in these differentiated sensory cells should be examined. Whether spec

ﬁ

c transcription factors such as Pax2 or Lhx3 act upstream Rbm24 need to be analyzed in the future (Burton et al., 2004; Hume et al., 2007; Chonko et al., 2013). RNA-seq combined with ChIP-PCR and loss of function analyses have already demonstrated that the transcription factor Atoh1 directly activates the expression of Rbm24 in mouse audi- tory hair cells at E17.5 stage of development (Cai et al., 2015).

From a developmental point of view, we wanted to examine wether

Rbm24

is expressed in the developing inner ear sensory epithelia during embryogenesis. For that purpose we carried out immuno

ﬂ

uorescence analyses on sections of E14.5 mouse embryos at two levels of the ear (Fig. 6A-C,G-I). Of interest, immunodetection of Rbm24, together with Sox2, that labels neu- roepihelial cells and with Myosin7A, that labels hair cells, dem- onstrates that

Rbm24

is already expressed in developing cochlear hair cells (data not shown) as well as in developing ves- tibular hair cells (Fig. 6). Indeed, sections through the ostocyst of an E14.5 embryo clearly show a strong and speci

ﬁ

c accumula- tion of Rbm24 proteins in hair cells of the developing saccule (Fig. 6D,J), the developing utricle (Fig. 6M) as well as the crista from one of the semicircular canals (Fig. 6P) similarly as Myo- sin7A (Fig. 6E,K,N,Q). The transcription factor Sox2 is expressed throughout these neurosensory epithelia (Fig. 6F,L,O,R) as well as other transcription factors such as Pax2, Pax8, and Atoh1, one can suggest that Rbm24 could be under their regulation in these neural cells (Bouchard et al., 2010). Finally, analyzing whether these cells develop normally or not in the absence of Rbm24 protein will shed light on our view on the molecular reg- ulation of the development of inner ear in vertebrates.

Experimental Procedures

Whole-mount In Situ Hybridization

Xenopus

whole-mount

in situ

hybridization was performed using standard protocol (Harland, 1991). Probes for

Xenopus,

chick, and mouse Rbm24 were previously described (Grifone et al., 2014) and were labelled using digoxygenin-11-UTP and appropriate RNA polymerase (Roche). Mouse and chick embryos were

ﬁ

xed in 4% paraformaldehyde and processed for whole- mount

in situ

hybridization as previously described (Grifone et al., 2014). Vibratome sections (100

μ

m) of whole-mount stained embryos were made after inclusions of the embryos in 4% agarose. Sections were then mounted using Kaiser

’

s glycerol gelatin solution (Merck). Stained embryos were pictured using a Leica stereo microscope (MDG41).

Immunohistochemistry on Paraf ﬁ n-embedded Embryo Sections

Mouse embryos were

ﬁ

xed in 4% paraformaldehyde (PFA) for 2 hr, washed in phosphate buffered saline (PBS), dehydrated in ethanol, cleared in xylene, and immersed into warm liquid paraf-

ﬁ

n before being embedded in a paraf

ﬁ

n block. Sections of 10

μ

m thickness were made on a microtome (Leica, RM2125RTS). For immuno-histochemistry, sections were deparaf

ﬁ

nized in xylene, rehydrated in ethanol and rinsed in PBS. Antigen retrieval was performed to unmask the antigen epitope in 10 mM citrate buffer (pH 6.0) at 95

–

100 C for 15 min. Sections are rinced in PBS, blocked for 1 hr in saturation solution (3% bovine serum albumin, 0.1% Triton in PBS) and incubated overnight at 4

C with primary antibodies (Rbm24, ProteinTech, 1/1,000; Prox1, BioLegend, 1/100; Pax6, abcam 1:200; Sox2, ProteinTech, 1/200; Myo7A, Clinisciences, 1/200). After three washes in PBT (0.1% Tween-20 in PBS), sections were incubated for 1 hr in alexa-488 conjugated anti-rabbit (1/400) or alexa-596 conjugated anti-mouse (1/400) secondary antibodies (Interchim), washed in PBT before mounting in Dako Fluorescent Mountig medium. Images were taken using a Zeiss Axioimager apotome.

Acknowledgment

We thank Quentin Tirel for help in immuno

ﬂ

uorescence analyses.

References

Beites CL, Kawauchi S, Crocker CE, Calof AL. 2005. Identiﬁcation and molecular regulation of neural stem cells in the olfactory epithelium. Exp Cell Res 306:309–316.

Bouchard M, de Caprona D, Busslinger M, Xu P, Fritzsch B. 2010.

Pax2 and Pax8 cooperate in mouse inner ear morphogenesis and innervation. BMC Dev Biol 10:89.

Burton Q, Cole LK, Mulheisen M, Chang W, Wu DK. 2004. The role of Pax2 in mouse inner ear development. Dev Biol 272:161–175.

Cai T, Jen HI, Kang H, Klisch TJ, Zoghbi HY, Groves AK. 2015.

Characterization of the transcriptome of nascent hair cells and identiﬁcation of direct targets of the Atoh1 transcription factor. J Neurosci 35:5870–5883.

Cardinali B, Cappella M, Provenzano C, Garcia-Manteiga JM, Lazarevic D, Cittaro D, Martelli F, Falcone G. 2016. MicroRNA-222 regulates muscle alternative splicing through Rbm24 during differentiation of skeletal muscle cells. Cell Death Dis 7:e2086.

Chonko KT, Jahan I, Stone J, Wright MC, Fujiyama T, Hoshino M, Fritzsch B, Maricich SM. 2013. Atoh1 directs hair cell differentiation and survival in the late embryonic mouse inner ear. Dev Biol 381:401–410.

Dash S, Dang CA, Beebe DC, Lachke SA. 2015. Deﬁciency of the RNA binding protein caprin2 causes lens defects and features of Peters anomaly. Dev Dyn 244:1313–1327.

1168 GRIFONE ET AL.

DEVELOPMENTAL DYNAMICS

(11)

de Iongh RU, Abud HE, Hime GR. 2006. WNT/Frizzled signaling in eye development and disease. Front Biosci 11:2442–2464.

Fetka I, Radeghieri A, Bouwmeester T. 2000. Expression of the RNA recognition motif-containing protein SEB-4 during Xenopus embryonic development. Mech Dev 94:283–286.

Graw J. 2010. Eye development. Curr Top Dev Biol 90:343–386.

Griep AE. 2006. Cell cycle regulation in the developing lens. Semin Cell Dev Biol 17:686–697.

Grifone R, Xie X, Bourgeois A, Saquet A, Duprez D, Shi DL. 2014.

The RNA-binding protein Rbm24 is transiently expressed in myoblasts and is required for myogenic differentiation during vertebrate development. Mech Dev 134:1–15.

Garcia CM, Huang J, Madakashira BP, Liu Y, Rajagopal R, Dattilo L, Robinson ML, Beebe DC. 2011. The function of FGF signaling in the lens placode. Dev Biol 351:176–185.

Gunhaga L. 2011. The lens: a classical model of embryonic induc- tion providing new insights into cell determination in early development. Philos Trans R Soc Lond B Biol Sci 366:1193–1203.

Harland RM. 1991. In situ hybridization: an improved whole-mount method for Xenopus embryos. Methods Cell Biol 36:685–695.

Hume CR, Bratt DL, Oesterle EC. 2007. Expression of LHX3 and SOX2 during mouse inner ear development. Gene Expr Patterns 7:798–807.

Kawauchi S, Beites CL, Crocker CE, Wu HH, Bonnin A, Murray R, Calof AL. 2004. Molecular signals regulating proliferation of stem and progenitor cells in mouse olfactory epithelium. Dev Neurosci 26:166–180.

Kawauchi S, Shou J, Santos R, Hebert JM, McConnell SK, Mason I, Calof AL. 2005. Fgf8 expression deﬁnes a morphogenetic center required for olfactory neurogenesis and nasal cavity development in the mouse. Development 132:5211–5223.

Kondoh H, Uchikawa M, Kamachi Y. 2004. Interplay of Pax6 and SOX2 in lens development as a paradigm of genetic switch mechanisms for cell differentiation. Int J Dev Biol 48:819–827.

Lachke SA, Ho JW, Kryukov GV, O’Connell DJ, Aboukhalil A, Bulyk ML, Park PJ, Maas RL. 2012. iSyTE: integrated systems tool for eye gene discovery. Invest Ophthalmol Vis Sci 53:

1617–1627.

Li HY, Bourdelas A, Carron C, Shi DL. 2010. The RNA-binding protein Seb4/RBM24 is a direct target of MyoD and is required for myogenesis during Xenopus early development. Mech Dev 127:

281–291.

Lim R, Brichta AM. 2016. Anatomical and physiological development of the human inner ear. Hear Res 338:9–21.

Maier E, Gunhaga L. 2009. Dynamic expression of neurogenic markers in the developing chick olfactory epithelium. Dev Dyn 238:1617–1625.

Maier E, von Hofsten J, Nord H, Fernandes M, Paek H, Hebert JM, Gunhaga L. 2010. Opposing Fgf and Bmp activities regulate the speciﬁcation of olfactory sensory and respiratory epithelial cell fates. Development 137:1601–1611.

Maragh S, Miller RA, Bessling SL, Wang G, Hook PW, McCallion AS. 2014. Rbm24a and Rbm24b are required for nor- mal somitogenesis. PLoS One 9:e105460.

Medina-Martinez O, Shah R, Jamrich M. 2009. Pitx3 controls multi- ple aspects of lens development. Dev Dyn 238:2193–2201.

Panaliappan TK, Wittmann W, Jidigam VK, Mercurio S, Bertolini JA, Sghari S, Bose R, Patthey C, Nicolis SK, Gunhaga L. 2018. Sox2 is required for olfactory pit formation and olfactory neurogenesis through BMP restriction and Hes5 upregulation. Development 145.

Paschaki M, Cammas L, Muta Y, Matsuoka Y, Mak SS, Rataj-Baniowska M, Fraulob V, Dolle P, Ladher RK. 2013. Retinoic acid regulates olfactory progenitor cell fate and differentiation.

Neural Dev 8:13.

Poon KL, Tan KT, Wei YY, Ng CP, Colman A, Korzh V, Xu XQ. 2012.

RNA-binding protein RBM24 is required for sarcomere assembly and heart contractility. Cardiovasc Res 94:418–427.

Stump RJ, Ang S, Chen Y, von Bahr T, Lovicu FJ, Pinson K, de Iongh RU, Yamaguchi TP, Sassoon DA, McAvoy JW. 2003. A role for Wnt/beta-catenin signaling in lens epithelial differentiation. Dev Biol 259:48–61.

Walker JL, Zhang L, Menko AS. 2002a. A signaling role for the uncleaved form of alpha 6 integrin in differentiating lensﬁber cells.

Dev Biol 251:195–205.

Walker JL, Zhang L, Menko AS. 2002b. Transition between proliferation and differentiation for lens epithelial cells is regulated by Src family kinases. Dev Dyn 224:361–372.

Walker JL, Zhang L, Zhou J, Woolkalis MJ, Menko AS. 2002c. Role for alpha 6 integrin during lens development: Evidence for signaling through IGF-1R and ERK. Dev Dyn 223:273–284.

Wang Y, Li J, Yao X, Li W, Du H, Tang M, Xiong W, Chai R, Xu Z.

2017. Loss of CIB2 causes profound hearing loss and abolishes mechanoelectrical transduction in mice. Front Mol Neurosci 10:401.

Xu Z, Peng AW, Oshima K, Heller S. 2008. MAGI-1, a candidate stereociliary scaffolding protein, associates with the tip-link com- ponent cadherin 23. J Neurosci 28:11269–11276.

Yang J, Hung LH, Licht T, Kostin S, Looso M, Khrameeva E, Bindereif A, Schneider A, Braun T. 2014. RBM24 is a major regulator of muscle-speciﬁc alternative splicing. Dev Cell 31:87–99.

Zhang T, Lin Y, Liu J, Zhang ZG, Fu W, Guo LY, Pan L, Kong X, Zhang MK, Lu YH, Huang ZR, Xie Q, Li WH, Xu XQ. 2016. Rbm24 regulates alternative splicing switch in embryonic stem cell cardiac lineage differentiation. Stem Cells 34:1776–1789.