Dynamics of pax7 expression during development, muscle regeneration, and in vitro differentiation of satellite cells in rainbow trout (Oncorhynchus mykiss)

Cécile Rallière; Sabrina Jagot; Nathalie Sabin; Jean-Charles Gabillard

doi:10.1371/journal.pone.0300850

Abstract

Essential for muscle fiber formation and hypertrophy, muscle stem cells, also called satellite cells, reside beneath the basal lamina of the muscle fiber. Satellite cells have been commonly identified by the expression of the Paired box 7 (Pax7) due to its specificity and the availability of antibodies in tetrapods. In fish, the identification of satellite cells remains difficult due to the lack of specific antibodies in most species. Based on the development of a highly sensitive in situ hybridization (RNAScope^®) for pax7, we showed that pax7⁺ cells were detected in the undifferentiated myogenic epithelium corresponding to the dermomyotome at day 14 post-fertilization in rainbow trout. Then, from day 24, pax7⁺ cells gradually migrated into the deep myotome and were localized along the muscle fibers and reach their niche in satellite position of the fibres after hatching. Our results showed that 18 days after muscle injury, a large number of pax7⁺ cells accumulated at the wound site compared to the uninjured area. During the in vitro differentiation of satellite cells, the percentage of pax7⁺ cells decreased from 44% to 18% on day 7, and some differentiated cells still expressed pax7. Taken together, these results show the dynamic expression of pax7 genes and the follow-up of these muscle stem cells during the different situations of muscle fiber formation in trout.

Citation: Rallière C, Jagot S, Sabin N, Gabillard J-C (2024) Dynamics of pax7 expression during development, muscle regeneration, and in vitro differentiation of satellite cells in rainbow trout (Oncorhynchus mykiss). PLoS ONE 19(5): e0300850. https://doi.org/10.1371/journal.pone.0300850

Editor: Atsushi Asakura, University of Minnesota Medical School, UNITED STATES

Received: October 24, 2023; Accepted: March 5, 2024; Published: May 8, 2024

Copyright: © 2024 Rallière et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting information files.

Funding: This work was supported by the INRAE PHASE department (project API MuStemMark 2016) and the ANR FishMuSC (ANR-20 CE20-0013-01). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Skeletal muscle consists of multinucleated cells called myofibers that result from the fusion of muscle precursors cells called myoblasts. Myofibers formation (hyperplasia) occurs during the embryonic and fetal period and subsequently is restricted to muscle regeneration in adult mammals, whereas in large growing fish such as rainbow trout, hyperplasia persists throughout the post-larval period [1]. Myoblasts proliferate and differentiate into myocytes, which fuse to form multinucleated myotubes, and mature into functional myofibers [2]. After birth, the myoblasts are derived from the activation and proliferation of adult muscle stem cells, called satellite cells because of their position on the surface of the myofiber beneath the surrounding basal lamina [3]. In mammals and birds, satellite cells have been shown to originate from the undifferentiated myogenic dermomyotome epithelium surrounding the primary myotome and migrate through the deep myotome to reach their final position beneath the basal lamina [4, 5].

Initially, satellite cells were identified by electron microscopy, based on their anatomical location between the myofiber membrane and the basal lamina, in numerous mammalian species, but only in a few fish such as carp [6] and zebrafish [7]. Subsequently, satellite cells have been localized by the expression of many specific genes such as desmin, M-Cadherin, myf-5, etc. [2]. Among these satellite cell markers, the Paired box 7 (Pax7) is the most widely used marker due to its specificity and the availability of antibodies [8]. In mammals, pax7 is expressed in satellite cells and myoblasts, and is essential for satellite cell survival, cell fate and self-renewal [9–11]. In zebrafish, pax7a and pax7b are expressed by satellite cells and are required for white muscle regeneration [7]. In fish, the identification of satellite cells remains difficult due to the lack of specific antibodies in most species. In zebrafish, Pax7 immunolabeling partially recapitulates the pattern of pax7 expression visualized in a transgenic line expressing GFP under the control of the pax7a promoter [12]. Surprisingly, very rare Pax7 positive cells were identified in the white muscle of adult zebrafish [7]. Using a heterologous antibody, Steinbacher et al (2007) were able to localize Pax7 positive cells in the myotome of brown trout embryos [13]. In giant danio (Devario cf. Aequipinnatus), Pax7 immunolabeling shows that almost all the cells (>99%) freshly extracted from white muscle are positive for Pax7, whereas Pax7-positive cells were no longer detected 24h later [14]. In rainbow trout, despite numerous attempts with appropriate controls, we were unable to obtain a specific pax7 signal in muscle or in vitro using the same antibody and protocol as Froehlich et al., (2013). Furthermore, using in situ hybridization, pax7 expression was readily detected in the dermomyotome only up to the end of trout embryo somitogenesis [15, 16]. In the final satellite cell niche, pax7⁺ cells are surrounded by the basal lamina, but the dynamics of basal lamina appearance and migration of pax7⁺ cells to their final niche remain unexplored in rainbow trout.

Based on the development of a highly sensitive in situ hybridization technique [17] (RNAScope^®), this work aims to describe the dynamics of pax7 expression during embryonic and larval stages, muscle regeneration and in vitro satellite cell differentiation in rainbow trout.

Materials and methods

Animals

All the experiments presented in this article were developed in accordance with the current legislation governing the ethical treatment and care of experimental animals (décret no. 2001–464, May 29, 2001), and the muscle regeneration study was approved by the Institutional Animal Care and Use Committee of the INRAE PEIMA (Pisciculture Expérimentale INRAE des Monts d’Arrée, Sizun, Britany, France; DOI: 10.15454/1.5572329612068406E12). Rainbow trout (Oncorhynchus mykiss) for cell culture were reared at the LPGP fish facility (DOI: 10.15454/45d2-bn67) approved by the Ministère de l’Enseignement Supérieur et de la Recherche (authorization no. D35-238-6). Briefly, rainbow trout (O. mykiss) were anesthetized with MS-222 (50 mg/l) and euthanatized with overdose (150mg/ml). To avoid any suffering, animals are handled delicately and raised in conditions that meet their natural needs.

After fertilization, rainbow trout eggs were incubated at 10°C in the dark. Trout embryos at days 14, 19, 21, 24, 26 and 28 post-fertilization were fixed with 4% paraformaldehyde overnight at 4°C and stored in 100% methanol at -20°C until use.

Muscle regeneration experiment

This experiment was carried out at the INRAE facility PEIMA. Briefly, 1530 ± 279 g rainbow trout (O. mykiss) were anesthetized with MS-222 (50 mg/l) and using a sterile 1.2 mm needle, the left side of each fish was injured by a puncture behind to the dorsal fin and above the lateral line. The right side of each fish was used as a control. White muscle samples were collected from both sides (in the injured region and contralateral) at 0, 1, 2, 4, 8, 16, and 30 days post-injury using a sterile scalpel after euthanasia by an overdose of MS-222 (200 mg/l). The obtained samples were properly stored in liquid nitrogen until further processing for gene expression analyses. No infection was detected during the experiment and the survival rate was 100%. Another muscle regeneration experiment was performed on 30 g trout using a 0.45 mm needle at the same site as the first experiment. After fish anesthesia (MS-222 at 200 mg/ml), white muscle samples were collected 18 days after injury and fixed in 4% paraformaldehyde overnight at 4°C and embedded in paraffin.

Trout satellite cell culture

Satellite cells from trout white muscle (200-250g body weight) were cultured as previously described [18]. Briefly, muscle tissue was mechanically and enzymatically digested (collagenase, C9891 and trypsin, T4799) prior to filtration (100μm and 40μm). Cells were seeded on poly-L-lysine and laminin precoated Lab-Tek^™ II Chamber Slide^™ (#154534, ThermoScientific, 8 wells) at a density of 80,000 cells/ml and incubated at 18°C in DMEM (D7777) with 10% fetal bovine serum to stimulate cell proliferation and differentiation. Cells were washed twice with PBS and fixed with ethanol/glycine (pH2) from day 2 to day 7 of culture.

RNA extraction, cDNA synthesis, and quantitative PCR analyses

Total RNA was extracted from 100 mg of muscle using TRI reagent (Sigma-Aldrich, catalog no. T9424), and its concentration was determined using the NanoDrop ND-1000 spectrophotometer. One μg of total RNA was used for reverse transcription (Applied Biosystems kit, catalog no. 4368813). We have previously identified three pax7 genes in the trout genome, pax7a1 (GSONMT00081386001, GI: 100884157), pax7a2 (GSONMT00061433001, GI: 100884158) and pax7b (GSONMT00027288001, GI: 110532152) [19]. Rainbow trout pax7a1 primers (forward, 5’-TGGGACTACGATTTATAGTTCGATTT-3’; and reverse, 5’-TTCTTACTCGCGCAAAGTCC-3’), and pax7a2 primers (forward, 5’-TGGGACTACGATTTTATTGTCTCC-3’; and reverse, 5’-TCGTGCAAAGTCCAGACAAG-3’), were previously designed at exon-exon junctions to avoid amplification of genomic DNA [19]. Unfortunately, we were not able to measure the pax7b expression by qPCR due to technical problems. Secondary structure formation in the predicted PCR product were determined with the mFOLD software. Quantitative PCR analyses were performed with 5 μl of cDNA using SYBR© Green fluorophore (Applied Biosystems), according to the manufacturer’s instructions, with a final concentration of 300 nM of each primer. The PCR program used was as follows: 40 cycles of 95°C for 3 s and 60°C for 30 s. The relative expression of target cDNAs within the sample set was calculated from a serial dilution (1:4–1:256) (standard curve) of a cDNA pool using StepOneTM software V2.0.2 (Applied Biosystems). Real-time PCR data were then normalized to elongation factor-1 alpha (eF1α) gene expression as previously described [20].

In situ hybridization

For the detection of pax7 expression in trout embryos, samples were fixed with 4% paraformaldehyde overnight at 4°C and embedded in paraffin. Cross-sections (7 μm) of muscle were then cut using a microtome (HM355; Microm Microtech, Francheville, France) and in situ hybridization was performed using RNAscope Multiplex Fluorescent Assay v2 (Bio-Techne #323100) according to the manufacturer’s protocol. Sections were briefly baked at 60°C for 1 hour, deparaffinized, and air dried. After 10 min in hydrogen peroxide solution (Bio-Techne #322335), sections were treated with 1X Target Retrieval (#322000; Bio-Techne) for 15 min at 100°C, followed by 25 min with Protease Plus solution (#322331; Bio-Techne) at 40°C. Due to the presence of three pax7 genes in the trout genome [19], we designed a set of probes targeting pax7a1, pax7a2 and pax7b mRNA (see S1 Fig). This probe set was hybridized for 2 hours at 40°C. All steps at 40°C were performed in a Bio-Techne HybEZ II hybridization system (#321720). The RNAscope Multiplex Fluorescent Assay allows simultaneous visualization of up to three RNA targets, with each probe being assigned to a different channel (C1, C2 or C3) with its own amplification steps. For embryo sections, pax7 transcripts were targeted with the fluorescent dyes Opal 520 (#FP1487001KT; Akoya Biosciences). At the end of the in situ hybridization protocol, embryo sections were incubated with a primary antibody against salmon Collagen 1 (#520171; Novotec, France) and then with a secondary antibody conjugated with an Alexa Fluor 594 fluorescent dye (#A11005; ThermoFisher). Nuclei were counterstained with DAPI (0.5μg/ml) and sections were mounted with ProlongGold (P36930, Invitrogen).

Detection of pax7 expression was also performed using the chromogenic RNAscope^® 2.5HD detection reagent RED kit (#322360; Bio-Techne) in sections of trout embryos and white muscle from 4 g and 9 g fish. As previously described, samples were fixed in 4% paraformaldehyde overnight at 4°C, embedded in paraffin and cross sections (7 μm) were made. Pax7 in situ hybridization was performed as described above and chromogenic detection using Fast Red, was performed according to the manufacturer’s protocol. Nuclei were counterstained with DAPI (0.5 μg/ml) and sections were mounted with EcoMount (EM897L, Biocare medical). In this chromogenic RNAscope assay, the red signal can be observed with either a white light or fluorescence microscope.

For multiplex RNAscope in situ hybridization, fixed cells were hybridized using the RNAscope Multiplex Fluorescent Assay v2 (Bio-Techne #323100) according to the manufacturer’s protocols. (see above). Pax7 and myomixer transcripts (MN230110) were detected using the fluorescent dyes Opal 620 (Akoya Biosciences #FP1495001KT) and Opal 520 (Akoya Biosciences #FP1487001KT), respectively. Nuclei were counterstained with DAPI (0.5 μg/ml) and cells were mounted with Mowiol^®.

Wheat germ agglutinin (WGA) conjugated with Alexa 488 (Molecular Probes # W11261) was used to visualize connective tissue and basal lamina [21]. WGA is a lectin-based molecule specifically binds to N-acetyl-D-glucosamine and N-acetylneuraminic acid (sialic acid) residues. After four washes with PBS, sections were stained with WGA (5 μg/ml) for 3 hours at room temperature.

All the images were taken with a Canon digital camera coupled to a Canon 90i microscope.

Automated quantification of pax7⁺ and mmx⁺ cells

To automatically quantify the number of cells expressing pax7, mmx or pax7 and mmx, we adapted a macro-command [22] on Fiji software [23] to quantify puncta corresponding to the RNAscope labeling, per cell. Due to the presence of one or two puncta in some cells with the negative control (DapB probe #504081, Bio-Techne), a cell was considered positive when at least 3 puncta were detected. Cells with more than 3 puncta for pax7 and mmx were considered double labelled. Our quantification method is available on https://gitlab.univ-nantes.fr/SJagot/fijimacro_rnascopecells.

Statistical analyses

Data were analyzed using the non-parametric Kruskal–Wallis rank test followed by the post-hoc Dunn test. All analyses were performed with the R statistical package (version 4.2.1).

Results

Pax7 positive cells were detected in the deep myotome before hatching

To determine the stage of appearance of pax7⁺ cells in the deep myotome, we performed highly sensitive in situ hybridization (RNAscope technology) with pax7 probes on cross sections of 14–28 dpf embryos (Fig 1). Collagen 1 labeling allowed easy localization of the myosepta and the horizontal myoseptum, as well as other embryonic structures such as the notochord. At day 14, pax7⁺ cells were detected in the neural tube and in the undifferentiated myogenic epithelium corresponding to the dermomyotome. At day 19, no pax7⁺ cells were detected in the fully differentiated deep myotome, excepted near to the horizontal septum, where rare pax7⁺ cells were observed. Two days later (D21), the majority of pax7 expressing cells were still localized in the dermomyotome, and very rare pax7⁺ cells appeared scattered throughout the deep myotome. From day 24 to day 28, pax7⁺ cells were readily detected in the deep myotome, while a slight decrease in pax7 expression in the dermomyotome epithelium was observed. In situ hybridization of longitudinal sections of 28-days-old embryos showed that pax7⁺ cells were localized along the muscle fibers of the deep myotome (Fig 2) and not in the myoseptum.

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Fig 1. Pax7 positive cells migrate into the deep myotome before hatching.

Transverse sections (7 μm) of trout embryos were analyzed by in situ hybridization for pax7 (green) and by immunofluorescence for Collagen 1 (red) at days 14, 19, 21, 24, 26, and 28 post fertilization. Asterisks indicate the dermomyotome-like epithelium and arrowheads indicate pax7⁺ cells in the deep myotome. The nuclei are counterstained with DAPI and the scale bar corresponds to 100 μm.

https://doi.org/10.1371/journal.pone.0300850.g001

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Fig 2. Pax7 positive cells are positioned along muscle fibers before hatching.

Pax7 expression was analyzed by chromogenic in situ hybridization for pax7 on longitudinal sections of 28-dfp trout embryos. Arrowheads indicate pax7⁺ cells in the deep myotome, adjacent to muscle fibers. The scale bar corresponds to 50 μm.

https://doi.org/10.1371/journal.pone.0300850.g002

The final location of satellite cells is beneath the basal lamina of the muscle fibers. To determine when pax7⁺ cells reach their niche, we stained the basal lamina with Alexa 488-conjugated wheat germ agglutinin. In Fig 3, pax7⁺ cells were easily detected at day 37, when the basal lamina was very thin and did not completely surround the muscle fibers. At day 47 (100mg), the basal lamina was thicker and surrounded most of the fibers and some of the pax7⁺ cells. At day 112 (4g) and 136 (9g), all pax7⁺ cells were located beneath the basal lamina surrounding the muscle fibers.

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Fig 3. Localization of pax7 positive cells in the muscle stem cell niche within the white muscle of trout.

Transverse sections (7 μm) of trout larvae (37, 47, 112 and 136 days post fertilization) were analyzed by in situ hybridization for pax7 (red) and the extracellular matrix was stained with Alexa 488-conjugated wheat germ agglutinin (green). Arrowheads indicate pax7⁺ cells in the deep myotome. The nuclei are counterstained with DAPI and the scale bar corresponds to 25 μm.

https://doi.org/10.1371/journal.pone.0300850.g003

Pax7 positive cells accumulate at the lesion site during regeneration

In vertebrates, muscle regeneration requires the activation, proliferation and differentiation of satellite cells (pax7⁺). To determine whether pax7⁺ cells accumulate at the site of injury during regeneration, we performed in situ hybridization for pax7 on sections of regenerated muscle at day 18 (Fig 4A). At the site of injury, we observed accumulation of nucleus (DAPI) and extracellular matrix (WGA labeling). In addition, our results clearly showed that pax7⁺ cells accumulated at the site of injury in contrast to the uninjured muscle area, where few pax7⁺ cells were observed.

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Fig 4. Expression of pax7 genes increases during white muscle regeneration in trout.

Localization of pax7⁺ cells (red) in 18-day injured muscle was performed by in situ hybridization (A). The extracellular matrix was stained with wheat germ agglutinin (WGA, green) and the nucleus was stained with DAPI (blue). The asterisks indicate the injury site and the scale bar corresponds to 50 μm. Gene expression profile of pax7a1 (B) and pax7a2 (C) during muscle regeneration in rainbow trout normalized with eF1a expression. Bars represent the standard error and the letters indicate the significant differences between means within the same treatment (control or injured). The asterisk indicates significant differences between treatments at a given point. Statistical significance (p < 0.05) was determined by the Kruskal-Wallis rank test followed by the Dunn test.

https://doi.org/10.1371/journal.pone.0300850.g004

To determine whether the expression of pax7 genes (pax7a1 and pax7a2) are upregulated during muscle regeneration in trout, we examined the kinetics of regeneration at 0, 1, 2, 4, 8, 16, and 30 days after injury. QPCR analysis showed that the expressions of pax7a1 and pax7a2 were upregulated after injury with a maximal expression at day 8 with an increase of 8- and 12-fold, respectively. Thereafter, pax7a1 and pax7a2 expression tended to decrease to near control expression by day 30 post-injury (Fig 4B and 4C).

The proportion of Pax7⁺ cells decreases during in vitro differentiation of myogenic cells

To determine the evolution of the proportion of pax7⁺ cells during in vitro differentiation, we performed in situ hybridization for pax7 and myomixer (mmx) on cultured myogenic cells. Fig 5A shows the presence of mononucleated cells expressing pax7 and few cells expressing mmx at day 2 of culture. At day 7, we observed the presence of multinucleated myotubes with a strong expression of mmx and mononucleated cells expressing pax7⁺. Surprisingly, at day 7 we observed mononucleated cells and myotubes expressing both pax7 and mmx. Careful observation suggests that cells with strong expression (number and intensity of dots) of pax7 had low expression of mmx, while the opposite was observed in myotubes. Quantification of the proportion of pax7⁺, mmx⁺ and pax7⁺/mmx⁺ cells is shown in Fig 5B. On day 2, the culture was composed of 44% pax7⁺ cells, 5% of mmx⁺ cells and 6% of pax7⁺/mmx⁺ cells. The percentage of pax7⁺ cells decreased to 18% on day 7, while the percentage of mmx⁺ and pax7⁺/mmx⁺ cells increased up to 19% and 31%, respectively.

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Fig 5. Pax7 positive cells decrease during in vitro differentiation of myogenic progenitors.

Myogenic progenitors were cultured in culture medium (DMEM, 10% SVF) for 7 days. Illustrative images of pax7 (red) and mmx (green) in situ hybridization results performed at day 2 and day 7 of the culture (A). The scale bar corresponds to 50 μm. The percentage of pax7 and myomixer (mmx) positive cells was determined by double in situ hybridization (B). Different capital letters indicate significant differences between means values of mmx positive cells. Different lowercase letters indicate significant differences between means of pax7 positive cells. Different symbols indicate significant differences between means of pax7/mmx positive cells. Statistical significance (p < 0.05) was determined by Kruskal–Wallis rank test followed by Dunn’s test. The scale bar corresponds to 50 μm.

https://doi.org/10.1371/journal.pone.0300850.g005

Discussion

Adult muscle stem cells (satellite cells, pax7⁺ cells) are essential for fiber growth and muscle regeneration. Although the existence of these cells has been demonstrated in fish, the lack of highly sensitive and specific tools to label satellite cells has limited the study of their function. The aim of this work was to describe the dynamics of satellite cells (pax7⁺) during embryonic and larval stages, regeneration and in vitro differentiation in rainbow trout.

In most fish, the observation of satellite cells in adult muscle is difficult due to their small size and number, and the available tools are not sensitive and specific enough. In this context, we decided to take advantage of the recent improvement in in situ hybridization sensitivity using the novel technology RNAScope^® [17], to localize pax7⁺ cells in rainbow trout muscle. In this species, we have previously identified three pax7 genes (pax7a1, pax7a2 and pax7b) resulting from the salmonid-specific whole-genome duplication [19, 24]. The coding sequences of these 3 genes are highly similar, sharing more than 88% of sequence identity. Due to this high sequence identity, it was not possible to design probe sets specific to each pax7 gene using RNAscope^® technology. For that reason, our probe detects the three pax7 mRNAs as shown in S1 Fig, and thus all the cells expressing at least one pax7 gene. Using this method, we were able to observe a pax7 signal in embryos, white muscle and isolated satellite cells with no background (see S2 Fig).

In fish, pax7 expression has previously been detected by classical in situ hybridization in the dermomyotome of zebrafish [25] and trout [15, 16] until to the end of somitogenesis. Using RNAScope^® technology, our results confirmed the presence of pax7⁺ cells at the periphery of the myotome corresponding to the dermomyotome at day 14, in agreement with our previous data obtained using classical in situ hybridization [15]. Similarly, we have shown that pax7⁺ was not expressed in red and white muscle fibers at up to 17–18 days post-fertilization [15, 26]. Our results showed that pax7⁺ cells gradually appeared in the deep myotome from day 24 post fertilization and were observed scattered throughout the somite, suggesting that pax7⁺ cells directly migrated from the dermomyotome to the deep myotome by moving between superficial muscle fibers. Thus, our results support the data obtained in zebrafish [27] and brown trout [28] showing that pax7⁺ cells also migrate between muscle fibers and not around their ends. Double labeling of pax7 and WGA indicated that in trout, pax7⁺ cells are covered by the basal lamina from day 112 post-fertilization. In zebrafish, due to its high rate of development, Pax7⁺ cells are surrounded by the basal lamina from at least day 6 post-fertilization [7, 12]. In addition, in situ hybridization for pax7 performed in muscle sections clearly showed the presence of pax7⁺ cells scattered in white muscle in juveniles (D136, ~9g), in agreement with data obtained with heterologous antibody in salmon [29] but in contrast to zebrafish, where very few pax7⁺ cells are detected in white muscle [7]. This difference may be due to the persistence of a high rate of muscle hyperplasia in salmon in contrast to zebrafish [30], which should require a high number of pax7⁺ cells.

In mammals, satellite cells are known to be required for muscle hyperplasia and hypertrophy, but also for muscle regeneration after injury. In trout, we have previously shown that mechanical injury induces the resumption of myogenesis as evidenced by the upregulation of myogenin, myomaker and myomixer and the formation of new small fibers [26, 31]. The present results showed that during muscle regeneration two pax7 genes (pax7a1 and pax7a2) showed a peak of expression 8 days after injury, well before the peak of expression of myogenin and the formation of new fibers (D30) [31]. In addition, in situ hybridization with pax7 probes at day 18, showed a large number of pax7⁺ cells at the wound site compared to the uninjured area. These results are consistent with previous work in adult [7] and larval stage [12] zebrafish showing that white muscle regeneration requires activation and proliferation of pax7⁺ cells. In sea bream (Sparus aurata), qPCR analysis during muscle regeneration also showed a slight increase of pax7 gene expression two weeks after muscle injury [32]. These results strongly suggest that in trout pax7⁺ cells are also required for muscle regeneration and that pax7 genes are involved. Indeed, in zebrafish, the two pax7 genes (pax7a and pax7b) have been shown to have distinct functions during regeneration [33], whereas in trout the specific roles, if any, of the pax7 genes remain yet unknown. Indeed, the development of tools to specifically monitor the in situ expression of each pax7 gene are required.

In mammals, pax7 has been shown to be mainly expressed in quiescent and activated satellite cells [2]. Our results showed that 2 days after muscle cell extraction, 50% of the cells expressed pax7 (44% pax7⁺ and 6% pax7⁺/mmx⁺) and were thus myogenic progenitors. In agreement with this result, using an antibody against MyoD, we observed that 60–70% of the extracted muscle cells were positive for MyoD [18]. The small difference may be due to the different markers used and to the fact that the trout in the present study are heavier (250g versus 5-10g). During the cell culture, the percentage of pax7⁺cells decreased from 44% to 18% on day 7, while the percentage of mmx⁺ cells increased from 6% to 19%, in agreement with previous qPCR data [26]. Thus, as expected, the differentiating cells down regulated the expression of pax7 and up regulated mmx, a differentiation marker. This result is consistent with previous works in salmon [34] and sea bream [35], showing that pax7 expression decreased during in vitro differentiation of myogenic cells. Surprisingly, the percentage of cells positive for both pax7 and mmx increased from 5% on day 2 to 30% on day 7 of the culture. Close examination of the images, revealed that the pax7 signal was weaker (fewer dots and lower intensity) in the mmx⁺ cells including myotubes. This result is reminiscent of previous observations in zebrafish showing the presence of up to 20% of myogenic cells expressing both pax7 and myogenin [33] two days after muscle injury. The authors showed that Pax7⁺ cells are able to differentiate and fuse with existing myofibers within 48 hours. In trout, we also observed that proliferative myogenic cells in vitro were able to incorporate BrdU and fuse with myotubes within 24 hours (data not shown). Taken together, these results demonstrate that pax7⁺ cells can rapidly progress through the myogenic program, which may explain the residual expression of pax7 in some differentiated cells expressing myomixer.

In conclusion, the in situ RNAScope^® technology allowed us to localize the pax7⁺ cells with high sensitivity and specificity. We showed that pax7⁺ cells migrate into the deep myotome at the end of segmentation and reach their niche after hatching. In addition, we observed an accumulation of pax7⁺ cells at the wound site, suggesting their requirement for muscle regeneration and that pax7 expression decreased in differentiating myogenic cells. Further work is needed to understand the effect of experimental conditions (fasting, aging, temperature) on the number of satellite cells.

Supporting information

S1 Fig. The pax7 probe set enables simultaneous detection of all three pax7 genes.

We developed a dot-blot hybridization approach in order to determine if the pax7 probe set is able to target the three pax7 genes present in the trout genome (pax7a1, pax7a2, pax7b) (Bio-Techne, Om-pax7b-cust # 575461). First, we transcribed cRNA sens of pax7a1, pax7a2 and pax7b from pax7 synthesized genes (Eurofins Genomics), using appropriate polymerase (Proméga, Riboprobe Systems sp6, # P1420, Riboprobe Systems T7, # P1440). The length of the cRNA is about 900 nt. We then applied 1μl (50ng) of each pax7 cRNA onto a nitrocellulose membrane (Macherey-Nagel) and performed a dot-blot hybridization analysis. After UV fixation (UV crosslinker, Appligene Oncor), blots were incubated for 1 hour at 40°C with 1 drop of pax7 probe set in order to hybridize with cRNA targets. After washing away excess probe, the presence of each pax7 cRNA was detected using a chromogenic RNAscope kit (Bio-Techne, RNAscope 2.5 HD Reagent Kit–RED, # 322350). Red dots indicate that the pax7 probe set recognizes the three pax7 and doesn’t recognize the negative control corresponding to the pax7a1 antisense RNA.

https://doi.org/10.1371/journal.pone.0300850.s001

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S2 Fig. Specific pax7 signal observed in white muscle section and in vitro myogenic cells.

Transverse sections (A, B) of trout white muscle (10g) were analyzed by in situ hybridization for pax7 (red) and extracellular matrix was stained with Alexa 488-conjugated wheat germ agglutinin (green). Arrowheads indicate pax7⁺ cells beneath the basal lamina of the muscle fibers. No signal was observed with the negative probe against a bacterial gene DapB (B). Myogenic progenitors (C, D) cultured for 1 day in culture medium (DMEM, 10% SVF) were analysed by in situ hybridization for pax7 (red). A strong signal was observed in mononucleated cells (C). No signal was observed with the negative probe against a bacterial gene DapB (D). The nuclei are counterstained with DAPI and the scale bar corresponds to 50 μm.

https://doi.org/10.1371/journal.pone.0300850.s002

(TIF)

S1 Raw images. dot-blot hybridization approach.

We transcribed cRNA sens and antisens of pax7a1, pax7a2 and pax7b from pax7 synthesized genes (Eurofins Genomics), using appropriate polymerase (Proméga, Riboprobe Systems sp6, # P1420, Riboprobe Systems T7, # P1440). We then applied each pax7 cRNA onto a nitrocellulose membrane (Macherey-Nagel) and performed a dot-blot hybridization analysis.

https://doi.org/10.1371/journal.pone.0300850.s003

(PDF)

Acknowledgments

We particularly thank A Patinote and C. Duret for trout rearing and egg production and L Goardon of the fish facility PEIMA (Pisciculture Expérimentale INRAE des Monts d’Arrée) for muscle.

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- PubMed/NCBI
- Google Scholar
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- View Article
- Google Scholar
7. Berberoglu MA, Gallagher TL, Morrow ZT, Talbot JC, Hromowyk KJ, Tenente IM, et al. Satellite-like cells contribute to pax7-dependent skeletal muscle repair in adult zebrafish. Dev Biol. 2017;424: 162–180. pmid:28279710
- View Article
- PubMed/NCBI
- Google Scholar
8. Seale P, Sabourin LA, Girgis-Gabardo A, Mansouri A, Gruss P, Rudnicki MA. Pax7 is required for the specification of myogenic satellite cells. Cell. 2000;102: 777–786. pmid:11030621
- View Article
- PubMed/NCBI
- Google Scholar
9. Oustanina S, Hause G, Braun T. Pax7 directs postnatal renewal and propagation of myogenic satellite cells but not their specification. The EMBO Journal. 2004;23: 3430–3439. pmid:15282552
- View Article
- PubMed/NCBI
- Google Scholar
10. Olguin HC, Olwin BB. Pax-7 up-regulation inhibits myogenesis and cell cycle progression in satellite cells: a potential mechanism for self-renewal. Developmental Biology. 2004;275: 375–388. pmid:15501225
- View Article
- PubMed/NCBI
- Google Scholar
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- View Article
- PubMed/NCBI
- Google Scholar
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- View Article
- PubMed/NCBI
- Google Scholar
13. Steinbacher P, Haslett JR, Obermayer A, Marschallinger J, Bauer HC, Sänger AM, et al. MyoD and Myogenin expression during myogenic phases in brown trout: a precocious onset of mosaic hyperplasia is a prerequisite for fast somatic growth. Dev Dyn. 2007;236: 1106–1114. pmid:17315228
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- View Article
- PubMed/NCBI
- Google Scholar
15. Dumont E, Rallière C, Rescan P-Y. Identification of novel genes including Dermo-1, a marker of dermal differentiation, expressed in trout somitic external cells. Journal of Experimental Biology. 2008;211: 1163–1168. pmid:18344491
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- PubMed/NCBI
- Google Scholar
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- PubMed/NCBI
- Google Scholar
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- PubMed/NCBI
- Google Scholar
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- View Article
- PubMed/NCBI
- Google Scholar
19. Seiliez I, Froehlich JM, Marandel L, Gabillard J-C, Biga PR. Evolutionary history and epigenetic regulation of the three paralogous pax7 genes in rainbow trout. Cell Tissue Res. 2015;359: 715–727. pmid:25487404
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- PubMed/NCBI
- Google Scholar
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- View Article
- PubMed/NCBI
- Google Scholar
21. Pena SD, Gordon BB, Karpati G, Carpenter S. Lectin histochemistry of human skeletal muscle. J Histochem Cytochem. 1981;29: 542–546. pmid:6166659
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- PubMed/NCBI
- Google Scholar
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- PubMed/NCBI
- Google Scholar
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- View Article
- PubMed/NCBI
- Google Scholar
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- View Article
- PubMed/NCBI
- Google Scholar
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- View Article
- PubMed/NCBI
- Google Scholar
27. Stellabotte F, Dobbs-McAuliffe B, Fernández DA, Feng X, Devoto SH. Dynamic somite cell rearrangements lead to distinct waves of myotome growth. Development. 2007;134: 1253–1257. pmid:17314134
- View Article
- PubMed/NCBI
- Google Scholar
28. Steinbacher P, Stadlmayr V, Marschallinger J, Sänger A m., Stoiber W. Lateral fast muscle fibers originate from the posterior lip of the teleost dermomyotome. Developmental Dynamics. 2008;237: 3233–3239. pmid:18924233
- View Article
- PubMed/NCBI
- Google Scholar
29. Gotensparre SM, Andersson E, Wargelius A, Hansen T, Johnston IA. Insight into the complex genetic network of tetraploid Atlantic salmon (Salmo salar L.): description of multiple novel Pax-7 splice variants. Gene. 2006;373: 8–15. pmid:16567062
- View Article
- PubMed/NCBI
- Google Scholar
30. Johnston IA, Lee H-T, Macqueen DJ, Paranthaman K, Kawashima C, Anwar A, et al. Embryonic temperature affects muscle fibre recruitment in adult zebrafish: genome-wide changes in gene and microRNA expression associated with the transition from hyperplastic to hypertrophic growth phenotypes. Journal of Experimental Biology. 2009;212: 1781–1793. pmid:19482995
- View Article
- PubMed/NCBI
- Google Scholar
31. Landemaine A, Ramirez-Martinez A, Monestier O, Sabin N, Rescan P-Y, Olson EN, et al. Trout myomaker contains 14 minisatellites and two sequence extensions but retains fusogenic function. J Biol Chem. 2019;294: 6364–6374. pmid:30819805
- View Article
- PubMed/NCBI
- Google Scholar
32. Otero-Tarrazón A, Perelló-Amorós M, Jorge-Pedraza V, Moshayedi F, Sánchez-Moya A, García-Pérez I, et al. Muscle regeneration in gilthead sea bream: Implications of endocrine and local regulatory factors and the crosstalk with bone. Frontiers in Endocrinology. 2023;14. Available: https://www.frontiersin.org/articles/ pmid:36755925
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- PubMed/NCBI
- Google Scholar
33. Pipalia TG, Koth J, Roy SD, Hammond CL, Kawakami K, Hughes SM. Cellular dynamics of regeneration reveals role of two distinct Pax7 stem cell populations in larval zebrafish muscle repair. Disease Models & Mechanisms. 2016; dmm.022251. pmid:27149989
- View Article
- PubMed/NCBI
- Google Scholar
34. Bower NI, Johnston IA. Paralogs of Atlantic salmon myoblast determination factor genes are distinctly regulated in proliferating and differentiating myogenic cells. Am J Physiol Regul Integr Comp Physiol. 2010;298: R1615–R1626. pmid:20375265
- View Article
- PubMed/NCBI
- Google Scholar
35. García de la serrana D, Codina M, Capilla E, Jiménez-Amilburu V, Navarro I, Du S-J, et al. Characterisation and expression of myogenesis regulatory factors during in vitro myoblast development and in vivo fasting in the gilthead sea bream (Sparus aurata). Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology. 2014;167: 90–99. pmid:24157945
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Figures

Abstract

Introduction

Materials and methods

Animals

Muscle regeneration experiment

Trout satellite cell culture

RNA extraction, cDNA synthesis, and quantitative PCR analyses

In situ hybridization

Automated quantification of pax7+ and mmx+ cells

Statistical analyses

Results

Pax7 positive cells were detected in the deep myotome before hatching

Pax7 positive cells accumulate at the lesion site during regeneration

The proportion of Pax7+ cells decreases during in vitro differentiation of myogenic cells

Discussion

Supporting information

S1 Fig. The pax7 probe set enables simultaneous detection of all three pax7 genes.

S2 Fig. Specific pax7 signal observed in white muscle section and in vitro myogenic cells.

S1 Raw images. dot-blot hybridization approach.

Acknowledgments

References

Automated quantification of pax7⁺ and mmx⁺ cells

The proportion of Pax7⁺ cells decreases during in vitro differentiation of myogenic cells