# In addition to being a marker for muscle connective tissue, Odd skipped-related 2 (OSR2) is expressed in differentiated muscle cells during chick development

Sonya Nassari, Mickael Orgeur, Cedrine Blavet, Sigmar Stricker, Claire

Fournier-Thibault, Delphine Duprez

► To cite this version:

Sonya Nassari, Mickael Orgeur, Cedrine Blavet, Sigmar Stricker, Claire Fournier-Thibault, et al. In addition to being a marker for muscle connective tissue, Odd skipped-related 2 (OSR2) is expressed in differentiated muscle cells during chick development. 2019. hal-02393806

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<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
In addition to being a marker for muscle connective tissue, *Odd skipped-related 2 (OSR2)*
is expressed in differentiated muscle cells during chick development

Sonya Nassari<fnref n="1" />, Mickael Orgeur<fnref n="1" />,<fnref n="2" />, Cédrine Blavet<fnref n="1" />, Sigmar Stricker<fnref n="2" />, Claire Fournier-Thibault<fnref n="1" /># and Delphine Duprez<fnref n="1" />#*

Running title: *OSR2* expression in differentiated muscle cells

Key words: chick, limb, head, muscle, connective tissue, *OSR2*

<footnote n="1">¹ Sorbonne Universités, UPMC Univ Paris 06, CNRS UMR7622, Inserm U1156, IBPS-Developmental Biology Laboratory, F-75005 Paris, France.</footnote>

<footnote n="2">² Institute for Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany</footnote>

<footnote># senior co-authors</footnote>

<footnote>* Author for correspondence</footnote>

<footnote>E-mail [delphine.duprez@upmc.fr](mailto:delphine.duprez@upmc.fr)</footnote>

<footnote>Tel +33 1 44 27 27 53</footnote>
ABSTRACT

The zinc finger transcription factor, Odd skipped-related 2 (OSR2) is a recognized marker of connective tissue in chick embryos. OSR2 gain- and loss-of-function experiments indicate a role in irregular connective tissue differentiation in chick limb undifferentiated cells. Re-investigation of OSR2 transcript location during chick development with in situ hybridization experiments showed that OSR2 was also expressed in differentiated muscle cells in limbs and head. OSR2 expression was also observed in differentiated myotubes in chick foetal myoblast cultures. This shows that in addition to being a marker of connective tissue, OSR2 is also expressed in muscle fibres during chick development.
INTRODUCTION

Connective tissue (CT) is an important component of the body supporting and connecting
organs. During development, reciprocal interactions between CT and myogenic cells are
required to form a functional muscular system (reviewed in Nassari et al., 2017a). In limbs,
CT cells and myogenic cells are intermingled in muscle masses but have different
embryological origins. CT cells originate from lateral plate, while myogenic cells originate
from somites (Bourgeois et al., 2015; Chevallier et al., 1977; Kardon, 1998). The muscle
differentiation program is under the control of the Myogenic Regulatory Factor (MRFs),
including MYF5, MYOD, MRF4 and MYOG (reviewed in Tajbakhsh, 2009). The
differentiation program of limb muscle CT cells is not known (reviewed in Nassari et al.,
2017a). To date, three types of transcription factors have been identified to regulate muscle
formation in a non cell-autonomous manner during limb development: TCF4, a member of
the TCF/LEF family (Kardon et al., 2003; Mathew et al., 2011), the T-box transcription
factors, TBX4 and TBX5 (Hasson et al., 2010) and the zinc finger transcription factors, Odd
skipped-related 1 and 2, OSR1 and OSR2 (Stricker et al., 2012; Vallecillo-García et al.,
2017)

The *odd* gene coding for the zinc finger transcription factor odd-skipped was first
identified in a screen for gene mutations in *Drosophila* as pair-rule genes involved in body
segmentation (Coulter and Wieschaus, 1988; Nüsslein-Volhard and Wieschaus, 1980). In
vertebrates, two odd-skipped-related genes, *Osr1* and *Osr2*, have been identified (Lan et al.,
2001; So and Danielian, 1999). During mouse and chick embryonic development and
organogenesis, *Osr1* and *Osr2* exhibit partially overlapping expression domains in
intermediate and lateral mesoderm and at later stages in mesonephros, branchial arches,
limbs, mandibular and maxillary prominences (Lan et al., 2001; So and Danielian, 1999; Stricker et al., 2006). *Osr1* and *Osr2* genes display functional equivalence during mouse development, indicating that the distinct functions of *Osr* genes rely on their expression domains (Gao et al., 2009). Consistent with *Osr* expression sites, phenotypes of *Osr* mutant mice indicate a role for *Osr1* in heart and urogenital development (James, 2006; Wang et al., 2005) and a role for *Osr2* in palate and tooth development (Lan et al., 2004; Zhang et al., 2009).

*Osr1* and *Osr2* are recognized markers for irregular CT during chick and mouse development (reviewed in Nassari et al., 2017a). During chick limb development, the expression of both genes labels CT cells and is excluded from myogenic cells, labelled with *PAX3* or *MYOD* (Stricker et al., 2006; Stricker et al., 2012). However, *OSR1/2*-positive cells display partial overlap with tendon *SCX*-positive cells and CT *TCF4*-positive cells in limb buds of embryonic day 4, E4, (HH24) chick embryos. However, *OSRs* are not expressed in tendons when they are formed (Orgeur et al., 2017 bioRxiv posted 20 July 2017 doi:10.1101/165837). Moreover, the overexpression of either *OSR1* or *OSR2* promotes irregular CT differentiation, while preventing cartilage, tendon and muscle differentiation in chick limb undifferentiated progenitors (Stricker et al., 2012, Orgeur et al., 2017 bioRxiv posted 20 July 2017 doi:10.1101/165837). Conversely, the blockade of *OSR1* or *OSR2* activity decreases the expression of CT markers, while promoting cartilage marker expression in chick limb undifferentiated progenitors (Stricker et al., 2012). Recently, *Osr1* has been shown to identify a population of embryonic fibro-adipogenic progenitors that has a non cell-autonomous effect on developmental myogenesis (Vallecillo-García et al., 2017). In chick limbs, when the final muscle pattern is set, *OSR2* appears mainly associated with individual muscles, while *OSR1* is expressed in irregular CT within and outside muscles (Nassari et al., 2017b).
In this study, we re-investigated the expression pattern of *OSR2* transcripts during chick development. We showed that in addition to the already known expression in muscle CT, *OSR2* was expressed in myofibres in limbs and head during chick development and in myotubes in muscle cell cultures.
RESULTS AND DISCUSSION

OSR2 has been described as being expressed in muscle CT, a subpopulation of irregular CT
cells during chick limb development (Nassari et al., 2017b; Stricker et al., 2006; Stricker et
al., 2012). We re-investigated OSR2 expression with in situ hybridization experiments to
sections at different developmental stages. We used the chick probe of 361 kb length located
between the nucleotides 127 to 487 of OSR2 gene (Figure 1). This probe has been used
previously for gene expression analysis (Stricker et al., 2006; Stricker et al., 2012). Moreover,
this OSR2 probe did not crossreact/blast with any gene in chicken genomic databases and did
not harbour the zinc finger domain of the OSR2 gene. At E3, corresponding to HH20 (40
somites), OSR1 was expressed in limb mesenchyme, while no OSR2 expression was observed
in chick limb buds (Figure 2A,B). OSR2 was observed in the mesonephros (Figure 2B,
arrow), as previously described (Stricker et al., 2006). Muscle progenitors migrate from the
hypaxial lips of the dermomyotomes towards the forelimb buds from E2 (HH17, 30 somites)
in chick embryos (Chevallier et al., 1977; Tozer et al., 2007). Migrating muscle progenitors
assessed with MYOR expression did not express OSR2 at E3/HH20 (Figure 2B,C), showing
that OSR2 was not expressed in muscle progenitors at this stage. At E5 (HH26), the OSR2
expression domains were similar to that of the myogenic transcription factor MYOD in dorsal
and ventral limb muscle masses (Figure 2D,E), however it was not clear whether they were
expressed in the same cells. From E5 to E8, in addition to being expressed in irregular CT
between cartilage elements in distal limb regions and in dermis regions, OSR2 displayed a
strong expression in developing muscles (Nassari et al., 2017b; Stricker et al., 2012). At E9,
when the final muscle pattern is set, OSR2 expression was mostly expressed in muscles
regions, in addition to feather buds (Figure 2F). Within muscles, OSR2 was expressed in a
subset of MF20-positive muscle fibres (Figure 2G, arrowheads) and in between MF20-
positive cells (Figure 2G, arrows). These results show that in addition to being expressed in irregular and muscle CT during chick limb development, *OSR2* is expressed in differentiated muscle fibres.

Previous studies have highlighted a key role for *OSR2* in the development of specific craniofacial regions (Lan et al., 2004). During craniofacial development of mouse embryos, *Osr2* expression is restricted to specific mesenchymal tissues, including the mesenchyme of the developing palatal shelves and the tongue (Lan et al., 2001). In chick embryos, *OSR2* has been described to be expressed in branchial arches from E3 (Stricker et al., 2006). In order to determine whether *OSR2* was also expressed in differentiated muscle cells during chick craniofacial development, we performed in situ hybridization to head sections of chick embryos at different developmental stages. At E3 (HH20), *OSR1* and *OSR2* were expressed in distinct regions of the first branchial arches outside the muscle regions labelled by *MYOR* (Figure 3A-C). At E4 (HH22), *OSR2* expression was detected in mesenchymal cells of the third branchial arches and did not overlap with that of *MYOR* (Figure 3D-G), which labelled the core of muscle progenitor cells in branchial arches (Grenier et al., 2009). This indicated that *OSR2* was restricted to neural-crest-derived mesenchymal cells in branchial arches before E4. At E7, *OSR2* was strongly expressed in muscle regions in addition to displaying an expression in the head mesenchyme (Figure 3H-J). High magnifications of muscles visualised with transverse (Figure 3K-M) and longitudinal (Figure 3N-P) sections showed that *OSR2* expression was observed in MF20-positive differentiated muscle cells (Figure 3K-P, arrowheads) in addition to muscle CT (Figure 3K-P, arrows).

We also analysed *OSR2* expression *in vitro*, in primary cell cultures of foetal myoblasts isolated from limbs of E10 chicken embryos. Chicken foetal myoblasts were
cultured in differentiation culture medium for 2 days. OSR2 expression was assessed by in situ hybridization (Figure 4A-D) and muscle cell differentiation with MF20 staining (Figure 4A,B,E,F). We observed OSR2 transcripts in MF20-positive cells (Figure 4A-F, arrows), showing that OSR2 was expressed in myotubes in chick foetal myoblast cultures

In addition to being a marker of CT and to promoting CT differentiation from chick mesenchymal stem cells (Nassari et al., 2017b; Stricker et al., 2006; Stricker et al., 2012, Orgeur et al., 2017 bioRxiv posted 20 July 2017 doi:10.1101/165837), we identified OSR2 as being also expressed in myosin-positive differentiated muscle cells during development of chick embryos. OSR2 expression was also observed in myotubes of chick foetal myoblast cultures. This unexpected expression of OSR2 in chick differentiated muscle fibres has never been described before and could bring new insights for OSR2 function during development. It is not clear whether Osr2 displays similar muscle expression in mice. X-gal staining of forelimb sections of E13.5 and E15.5 Osr2LacZ/- embryos did not show any obvious expression in limb muscles (Gao et al., 2011). Moreover, Osr2IresCre mice displayed specific expression (of reporter gene) in mandibular mesenchyme and did not display any obvious expression in muscle areas in branchial arches of E12.5 mice (Lan et al., 2007). Since previous expression analyses rely on genetic tools, double labelling experiments are nevertheless required to exclude an Osr2 expression in myotubes during mouse development.
# MATERIALS AND METHODS

## Chick embryos

Fertilized chick eggs from commercial sources (JA57 strain, Institut de Sélection Animale, Lyon, France) were incubated at 38°C in a humidified incubator until appropriate stages. Embryos were staged according to the number of days *in ovo*. Staging using Hamburger and Hamilton, HH (Hamburger and Hamilton, 1992) or somite numbers was also used for young stages.

## Chick myoblast primary cultures

Primary muscle cell cultures were prepared from skeletal muscles of forelimbs of E10 chick foetuses. Muscle cells were mechanically dissociated and seeded in plastic dishes coated with 0.1% gelatine. Myoblast primary cultures were first incubated in a proliferation medium (2/3 Minimum Essential Eagle Medium (MEM), 1/3 Hanks' salts medium 199, 10% foetal calf serum, 1% penicillin streptomycin and 1% glutamine). At 80% of confluence, differentiation was induced using a differentiation medium (2/3 Minimum Essential Eagle Medium (MEM), 1/3 Hanks' salts medium 199, 2% foetal calf serum, 1% penicillin streptomycin and 1% glutamine).

## In situ hybridization to embryo sections and cells

Embryos were fixed in 4% paraformaldehyde PBS solution, rinsed successively in 4% and 15% sucrose solution, embedded in a gelatine-sucrose solution (7.5% gelatine, 15% sucrose, 50% Phosphate buffer), and frozen in chilled isopentane. Cryostat sections of 10 to 14 µm were collected on Superfrost/Plus slides (CML, France). Primary muscle cell cultures were fixed in 4% paraformaldehyde PBS solution and rinsed with PBS. Sections and cell cultures
were processed for in situ hybridization as previously described (Escot et al., 2013; Nassari et al., 2017b) using digoxigenin-labelled mRNA probes for chick OSR1, chick OSR2 (Nassari et al., 2017b; Stricker et al., 2006), chick MYOR and chick MYOD (Grenier et al., 2009; von Scheven et al., 2006).

## Immunohistochemistry

Differentiated muscle cells were detected on limb and head sections and chick foetal myoblast cultures after in situ hybridization using the monoclonal antibody against sarcomeric myosin heavy chain, MF20 (Developmental Hybridoma Bank, non-diluted supernatant). Immunohistochemistry was processed after in situ hybridization, as previously described (Grenier et al., 2009; Tozer et al., 2007).

## Image capturing

Images of labelled sections and cultured cells were obtained using a Leica DMI6000 B microscope or a Nikon microscope equipped for epifluorescence. Images were processed using Adobe Photoshop software.
## FUNDING

This work was supported by the Fondation pour la Recherche Médicale (FRM) DEQ20140329500, Association Française contre les Myopathies (AFM) N°16826. S.N. and M.O. were part of the MyoGrad International Research Training Group for Myology and received financial support from the AFM (AFM 20150532272) and FRM (FDT20150532272) and from the Université Franco-Allemande (CDF1-06-11 and CT-24-16).

## COMPETING INTERESTS

The authors declare no competing or financial interests.
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FIGURE LEGENDS

**Figure 1**

**Position of the OSR2 probe along the chicken OSR2 gene.** (A) Representation of the OSR2 gene (NM_001170344.1). Position of the OSR2 probe used for *in situ* hybridization running from nucleotide position 127 to 487 on the OSR2 gene. (B) Sequence of the chick OSR2 probe (361 bp).

**Figure 2**

**OSR2 is expressed in CT cells and differentiated muscle cells in limbs. (A-C)** In situ hybridization to adjacent longitudinal limb sections of E3/HH20 chick embryos with OSR1 (A), OSR2 (B) and MYOR (B) probes (blue). OSR1 (A) was expressed in limb mesenchyme, while OSR2 was only detected in mesonephros (B, arrow) and not in muscle progenitors labelled with MYOR (C, arrows). (D,E) In situ hybridization to adjacent transverse limb sections of E5 chick embryos with OSR2 (D) and MYOD (E) probes (blue). OSR2 and MYOD displayed similar and overlapping expression domains in dorsal and ventral muscle masses. (F,G) Transverse forelimb sections of E9 chick embryos were hybridized with OSR2 probe (blue) and then immunostained for myosins using the MF20 antibody (brown). (F) Arrow indicates OSR2 expression in dermis. (G) Arrowheads indicate double OSR2-positive and MF20-positive cells, while arrows point to OSR2-positive and MF20-negative cells. r, radius; u, ulna. For all pictures, dorsal is to the top.
Figure 3

OSR2 is expressed in CT cells and differentiated muscle cells in branchial arches. (A-C)

In situ hybridization to adjacent transverse sections of E3/HH20 chick embryos at the level of the first branchial arches (BA1) with OSR1 (A), OSR2 (B) and MYOR (C) probes (blue). (A-C) Arrows point to the OSR1 and OSR2 expression in mesenchyme excluded from MYOR expression domain that is delineated with dashed lines. (D-G) In situ hybridization to adjacent transverse head sections of E4 chick embryos at the level of the third branchial arches (BA3) with OSR2 (D,E) and MYOR (F,G) probes (blue). (E,G) represent high magnifications of framed regions in (D,F), respectively. At E3 and E4, OSR2 was not detected in the core region of myogenic cells labelled with MYOR but was observed in the mesenchyme of the BA1 and BA3. (H-P) Sagittal head sections of E7 chick embryos were hybridized with OSR2 probe (blue) and then immunostained for myosins using the MF20 antibody (red). (H-J) 3 panels of the same section are shown: OSR2 transcripts (H), MF20 labelling (I) and merged OSR2/MF20 (J). (K-M) and (N-P) are high magnifications of the framed muscle regions in (H-J), respectively. Arrowheads in (K-P) indicate OSR2 expression in MF20-positive cells, while arrows point to OSR2 expression in MF20-negative cells. OSR2 was detected in both muscle CT and myosin-positive differentiated muscle cells. nt, neural tube; hb, hindbrain.

Figure 4

OSR2 is expressed in differentiated muscle cells in vitro. (A-F) Chick foetal myoblasts cultured in differentiation conditions were hybridized with OSR2 probe (black) and immunostained for myosins using the MF20 antibody (green). (A,B) merged of OSR2 expression and MF20 labelling, (C,D) OSR2 transcripts, (E,F) MF20 labelling and DAPI
staining. (B,D,F) represent high magnifications of the boxed regions in (A,C,E), respectively.

Arrows indicate *OSR2* expression in MF20-positive cells.
A

chicken OSR2
(chr2:127,588,554-127,595,208)

<figure><img src="image_2.png" /></figure>

B

cOSR2 probe

ATGGGCAGCAAGGCGCTGCCGGCGCCCATCCCGCTGCACCCGTCCCTGCA_176
GCTCACCAACTACTCCTTCCTCCAGGCCGTCAACACCTTCCCCGCGGCCG_226
TGGACCAGCTGCAAGGGCTGTACGGGCTGAGCGCCGTGCAAACCATGCAC_276
ATGAACCACTGGACGTTGGGCTACCCCGGCGTGCACGAGATCGCCCGCTC_326
CGCCCTCACGGAGATGGCGGCCGCGCAGGGCCTGGTGGACTCGCGCTTCC_376
CCTTCCCCGCGCTGCCCTTCGCCGCGCACCTCTTCCACCCCAAGCAGGGC_426
GCCGCGGCCCACGTCCTCCCGGCGCTGCACAAGGAGCGGCCCCGCTTCGA_476
CTTCGCCAACC _487

<caption>Figure 1</caption>
<figure><img src="image_3.png" /><figcaption>Figure 2</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>Figure 3</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>Figure 4</figcaption></figure>