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Sonic Hedgehog signaling and Gli-1 during embryonic chick myogenesis
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Sonic Hedgehog signaling and Gli-1 during embryonic chick myogenesis Brito b, John Douglas Teixeira a, 1, Ivone de Andrade Rosa a, 1, Jose Yuli Rodrigues Maia de Souza c, Pedro Paulo de Abreu Manso c, Marcelo Pelajo Machado c, Manoel Luis Costa a, Claudia Mermelstein a, * a b c
~o Muscular e Citoesqueleto, Instituto de Ci^ rio de Diferenciaça Laborato encias Biom edicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil ~o e Diferenciaça ~o Celular, Instituto de Ci^ rio de Proliferaça Laborato encias Biom edicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil rio de Patologia, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, RJ, Brazil Laborato
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 November 2018 Accepted 12 November 2018 Available online xxx
The Sonic Hedgehog signaling (Shh) pathway has been implicated in both proliferation of myoblast cells and terminal differentiation of muscle fibers, and contradictory results of these effects have been described. To clarify the role of Shh during myogenesis, we decided to study the effects of recombinant Shh and the distribution of Gli-1 during in vitro and in situ embryonic chick skeletal muscle differentiation at later stages of development. Gli-1 was found in small aggregates near the nucleus in mononucleated myoblasts and in multinucleated myotubes both in vitro and in situ chick muscle cells. Some Gli-1 aggregates colocalized with gamma-tubulin positive-centrosomes. Gli-1 was also found in striations and at the subsarcolemmal membrane in muscle fibers in situ. Recombinant Shh added to in vitro grown muscle cells induced the nuclear translocation of Gli-1, as well as an increase in the number of myoblasts and in the number of nuclei within myotubes. We suggest that Gli-1 aggregates observed in chick muscle cells near the nuclei of myoblasts and myotubes could be a storage site for the rapid cellular redistribution of Gli-1 upon specific signals during muscle differentiation. © 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords: Gli Sonic Hedgehog chick muscle myogenesis
1. Introduction The mature multinucleated skeletal myofiber is formed by the fusion of mononucleated myoblasts during development. Skeletal myogenesis can be described as a two-step process: a proliferation phase, when myoblast cells divide; and a differentiation phase, when post-mitotic myoblasts fuse to form fully contractile multinucleated myofibers. An intricate network of signaling pathways, including Wnt, Notch, bone morphogenetic proteins (BMP) and Sonic Hedgehog (Shh), regulates this complex two-step process. Shh has been implicated in both the proliferative and the differentiation steps of myogenesis [1e5]. Indeed, the graft of Shhsecreting cells into the presumptive first branchial arch region, at 5 somite stage, was able to increase the territory of MyoD expression [6].
* Corresponding author. E-mail address: [email protected] (C. Mermelstein). 1 Both authors contributed equally to this work.
When activated, the Shh signaling pathway triggers a chain of events in target cells, leading to the activation of specific genes by the glioma-associated oncogene homolog (Gli) family of zinc finger transcription factors [7,8]. Upon secretion, Shh binds to the transmembrane receptor proteins, Patched-1 and Patched-2, in target cells [9]. Patched-1 inhibit downstream signaling of the transmembrane protein Smoothened (Smo) via an indirect mechanism not yet described. Patched-1 inhibition of Smo results in the nuclear localization of Gli proteins, which are the terminal effectors of the Shh signaling. In vertebrates, there are three Gli transcription factors (Gli-1, Gli-2 and Gli-3). Gli-1 is the only full-length transcriptional activator whereas Gli-2 and Gli-3 act as either positive or negative regulators. In the absence of Shh, Suppressor of Fused (SUFU) negatively regulates the pathway by directly binding to Gli proteins and anchoring them in the cytoplasm preventing the activation of Gli target genes. Contradictory results have been described regarding the role of the Shh pathway during vertebrate skeletal myogenesis. Different studies have shown that Shh can increase or inhibit myoblasts proliferation and terminal muscle differentiation, depending on the
https://doi.org/10.1016/j.bbrc.2018.11.071 0006-291X/© 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article as: J.D. Teixeira et al., Sonic Hedgehog signaling and Gli-1 during embryonic chick myogenesis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.071
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experimental model [1e5,10e13]. To get further insight into the role of the Shh pathway during myogenesis, we investigated the proliferation or differentiation of embryonic chick muscle cells both in vitro and in situ. We found Gli-1 aggregates in a perinuclear region in myoblasts and myotubes both in vitro and in situ muscle cells. Gli-1 was also found in striations and at the subsarcolemmal membrane in muscle cells in situ. We also studied the effects of recombinant Shh protein in muscle cells using the nuclear translocation of Gli-1 as an indicator of the activation of the Shh pathway. Recombinant Shh added to in vitro grown muscle cells induced an increase in the number of myoblasts, in the number of nuclei within myotubes, as well as the nuclear translocation of Gli1. Interestingly, part of Gli-1 aggregates colocalized with gammatubulin positive-centrosomes.
2.4. Immunofluorescence microscopy and digital image acquisition Cultured cells were fixed with 4% paraformaldehyde diluted in 0.1 M phosphate buffer (pH 7.4) and incubated with primary antibodies overnight at 4 C. Cells were washed and incubated with secondary antibodies for 1 h at 37 C. Nuclei were labeled with DAPI (0.1 mg/ml in 0.9% NaCl) or NucSpot live cell nuclear stain for 5 min. Slides were mounted in ProLong Gold antifade reagent (Molecular Probes, USA) and examined with either an Axiovert 100 inverted microscope (Carl Zeiss, Germany) or a Spinning Disk Confocal microscope (DSU, Olympus, Japan) with 60x lenses (1.3 NA). Control experiments performed omitting the primary antibodies showed only faint background staining. Some confocal images were digitally processed by Universal live-cell super-resolution microscopy (SRRF) [15].
2. Materials and methods
2.5. Quantification of chick cell cultures
2.1. Ethics statement
Cultures were fixed and double labeled for desmin and DAPI, and merged images were used for counting the presence of nuclei within mononucleated (myoblasts and fibroblasts) or multinucleated cells. The DAPI labeling enables the identification of myoblasts and fibroblasts by their nuclear morphologies and fluorescence intensities: muscle fibroblasts have large, flattened and pale nuclei whereas myoblasts have small, round and bright nuclei. The public domain softwares ImageJ (http://rsb.info.nih.gov/ij/) and CellProfiler (http://cellprofiler.org) were used for quantifications. The fusion index, the number of nuclei in myotubes divided by the total number of nuclei, was also calculated. All data was quantified from fifty randomly chosen microscopic fields collected from three independent experiments.
Experiments using chick embryos (Gallus gallus domesticus) were approved by the Ethics Committee for Animal Care and Use in Scientific Research from the Federal University of Rio de Janeiro (UFRJ) and received the approval number: 055/16.
2.2. Primary chick myogenic cell cultures and treatments All cell culture reagents were purchased from Invitrogen (USA). Primary cultures of myogenic cells were prepared from pectoral muscles of E11 (Embryonic day) chick embryos [14]. Briefly, muscles were excised, finely minced, incubated in 0.1% trypsin at 37 C, and centrifuged at approximately 300g for 5 min. Cells were resuspended in 8-1-0.5 medium (minimum essential medium with the addition of 10% horse serum, 0.05% chick embryo extract, 1% Lglutamine and 1% penicillin-streptomycin). Cells were plated at an initial density of 5 105 cells/35 mm culture dishes onto 22 mmAclar plastic coverslips (Pro-Plastics Inc., USA) previously coated with rat-tail collagen. Cells were grown in 2 ml of 8-1-0.5 medium under humidified 5% CO2 atmosphere at 37 C. The percentage of myoblasts in these cell cultures was calculated by the double labeling of 24-h cultures with both DAPI (nuclear staining) and a polyclonal anti-desmin antibody (applied herein to define a muscle-specific marker) and subsequently counting the number of desmin-positive cells out of the total number of cells in the field. On average, myoblasts represented 80% of each culture and non-myogenic cells 20%. Some 24-h myogenic cultures were treated for the next 2 or 48 h with recombinant Sonic protein (R&D Systems, USA) at the final concentration of 1, 2, 5 or 10 ng.
2.3. Antibodies and fluorescent probes Rabbit monoclonal anti-Gli-1 (ab134906) antibody was from Abcam (UK). Rabbit polyclonal anti-desmin and mouse monoclonal anti-gamma-tubulin antibodies were from Sigma Chemical Co. (USA). Mouse monoclonal anti-desmin antibody (RB-9014) was from Thermo Scientific (USA). Alexa Fluor 488-goat anti-mouse/ rabbit IgG and Alexa Fluor 546-goat anti-mouse/rabbit IgG antibodies and DNA-binding probe DAPI (4,6-Diamino-2-phenylindole dyhydrochloride) were from Molecular Probes (USA). NucSpot live cell nuclear stain was from Biotium Inc. (USA). Peroxidaseconjugated goat anti-rabbit antibodies were obtained from Amersham Biosciences (UK).
2.6. Immunofluorescence of embryonic chick muscle cross-sections Pectoral muscle fragments were isolated from E11 chick embryos, fixed in Carson's formalin-Millonig for 48 h [16] and processed according to standard techniques for paraffin embedding. Sections (5 mm thick) of paraffin blocks were submitted to immunofluorescence, as previously described [17]. Sections were incubated with primary antibodies overnight at 4 C, washed, incubated with fluorescently labeled-secondary antibodies at 37 C for 1 h and counterstaining with the nuclear dye DAPI. Negative controls were processed by omitting the incubation with primary antibodies and showed no significant labeling. All crosssections were analyzed under an LSM 710 confocal microscope (Carl Zeiss, Germany). 2.7. SDS-PAGE and western blot Chick myogenic cells were quickly washed in ice-cold PBS and 100 mL of ice-cold RIPA lysis buffer (150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM b-glycerophosphate, 1 mM Na3VO4, 1 mg/ml leupeptin and 20 mM Tris-HCl, pH 7.4) were added to the cultures, and then cells were scraped off the dish with a plastic cell scraper. Forty micrograms of proteins from samples [18] were solubilized in sample buffer (2% SDS, 10% glycerol, 0.0006% bromophenol blue and Tris-HCl, pH 6.8) and boiled for 5 min. A total protein homogenate was also prepared from pectoralis muscles derived from E11 chick embryos. Equal amounts of protein were loaded on 10% SDSpolyacrylamide gels. Molecular weight was estimated using the protein molecular weight standards Kaleidoscope (Bio-Rad, USA). Proteins were then transferred to PVDF membranes and membranes were washed in 0.1% Tween-TBS and incubated in a blocking solution (5% nonfat dry milk diluted in Tween-TBS) for 1 h.
Please cite this article as: J.D. Teixeira et al., Sonic Hedgehog signaling and Gli-1 during embryonic chick myogenesis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.071
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Fig. 1. Gli-1 expression and distribution in myogenic cell cultures. Chick myogenic cells were stained for Gli-1 (green, A, C, D, E, G and J), gamma-tubulin (red, H and J) and DAPI (blue, B, C, I and J). Gli-1 localizes in aggregates at the perinuclear region of multinucleated myotubes (arrows in A-D) and mononucleated myoblasts (arrowheads in A-C), but not in fibroblasts (asterisks in A-C). In (E) is an inset of image (D) with phase contrast and Gli-1 (green). Arrows in G-J point to the colocalization of Gli-1 and gamma-tubulin in centrosomes in a myotube. Bars ¼ 10 mm. A immunoblot against Gli-1 is shown in F. Protein extract from pectoralis muscle from E11 chick embryos (chick extract) and from 24-, 48- and 72-h myogenic cell cultures were analyzed. One major protein band with 160 kDa (asterisk) was detected in all samples. MWM ¼ molecular weight markers. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Subsequently, membranes were incubated with antibodies (1:500 dilution for anti-Gli-1) overnight at 4 C, washed in Tween-TBS, incubated for 1 h at 37 C with anti-rabbit CY5.5-conjugated secondary antibodies and washed again. The bands were visualized using an Odyssey Infrared Imager (LI-COR Biosciences, USA).
2.8. Statistical analysis Statistical analysis was carried out using the GraphPad Prism software version 5. Results of at least three independent experiments were expressed as mean ± standard deviation. Statistical
Please cite this article as: J.D. Teixeira et al., Sonic Hedgehog signaling and Gli-1 during embryonic chick myogenesis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.071
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significance was defined as *P < 0.05; **P < 0.01; ***P < 0.001 using Mann-Whitney or Dunn's test. 3. Results and discussion 3.1. Gli-1 is found in aggregates near the nuclei in myoblasts and myotubes
Gli-1 predominant isoform has a molecular weight of 160 kDa, but other variants with smaller molecular weights can also be found in different tissues [21]. The low expression of Gli-1 in the early stages of in vitro chick myogenesis (24-h) and its increased expression during the latter stages (48- and 72-h) of muscle development suggest a role for Gli-1 during the differentiation of muscle cells. 3.2. Gli-1 partially colocalizes with centrosomes
The aim of this work was to study the role of Sonic Hedgehog (Shh) signaling pathway during the proliferation and differentiation of chick embryonic skeletal muscle. First, we analyzed the distribution of Gli-1, a key target of the Shh pathway, in chick primary cultures of muscle cells. Myogenic cells were grown for 72 h and labeled with an antibody against Gli-1 (Fig. 1). Embryonic chick myogenic cultures are composed of three main cell phenotypes: mononucleated myoblasts, mononucleated fibroblasts and multinucleated myotubes. Gli-1 was found within the cytoplasm of myoblasts and myotubes (Fig. 1AeE), but not in fibroblasts (Fig. 1AeC). Our results showing the distribution of Gli-1 in primary cultures of muscle cells derived from E11 chick embryos are in accordance with a previous study from Elia and colleagues [19], which showed that primary cultures of muscle cells derived from 3day-old chick embryos express Gli-1. Interestingly, we found a strong labeling of Gli-1 concentrated in small aggregates at the perinuclear region of mononucleated myoblasts and multinucleated myotubes (Fig. 1AeE and Supp. Fig.1). It has been shown that Gli-1 can be sequestered in the cytoplasm near the nucleus by a complex of proteins, including SUFU and human Suppressor-of-Fused (SUFUH) [20]. Importantly, Gli-1 was not found within the nuclei of muscle cells (myoblasts and myotubes) and fibroblasts. Since Gli-1 is a transcription factor, these results suggest that the Shh pathway is not activated in cultured chick myogenic cells. We also analyzed the expression of Gli-1, at the protein level, during chick myogenesis. Chick myogenic cells derived from E11 chick embryos muscles were cultured for 24, 48 and 72 h, then harvested and total cell extracts were analyzed in SDS-PAGE followed by immunoblot against Gli-1 antibody (Fig. 1F). Gli-1 was expressed in all the samples (24, 48 and 72 h), but an increase in its expression was observed in 48- and 72-h cultures (Fig. 1F). Gli-1 was also detected in pectoralis muscle tissue sampled from E11 chick embryos (Fig. 1F). Gli-1 was detected in all the samples (from in vitro and in situ embryonic chick muscle cells) as a major protein band with a molecular weight of 160 kDa (Fig. 1F). The full length
Since Gli-1 was found in a perinuclear region in muscle cells, we decided to analyze whether its localization was somehow related to centrosomes. Gli-1 has been reported to be recruited and stabilized at the centrosome [22]. Centrosomes are organizing center for the control of microtubules network in eukaryotic cells [23] and they are composed of several proteins, including gamma-tubulin. Thus, we cultured chick myogenic cells and double-labeled them with anti-Gli-1 and anti-gamma-tubulin antibodies (Fig. 1GeJ). Fig. 1 shows gamma-tubulin in centrosomes in myotubes from 48-h muscle cultures. Multiple centrosomes were detected in single multinucleated myotubes, as observed by multiple gamma-tubulin positive-dots in the sarcolemma of myotubes (Fig. 1GeJ). An earlier report by Tassin and colleagues [24] described that centrosomes are numerous and scattered in myotubes and they were not associated one to each nucleus but were often clustered near nuclei groups. Besides, they reported that centrosomes were significantly smaller in myotubes than those found in mononucleated myoblast cells. Interestingly, some of Gli-1 immunolabeling was found colocalized with gamma-tubulin positive-dots (Fig. 1GeJ). Gli-1 was also found in regions without gamma-tubulin stain (Fig. 1GeJ). These results suggest that Gli-1 could be redistributed to other regions of the cell from the centrosome. The Gli-1 aggregates observed in chick muscle cells near the nuclei of myoblasts and myotubes could be a storage site for the cellular relocation of Gli-1 upon specific signals. Further experiments are needed to confirm this hypothesis and to study the mechanisms by which Gli-1 could be relocated in muscle cells. 3.3. Gli-1 is found in striations, at the sarcolemma and in aggregates in muscle cross sections To analyze whether these in vitro results at the cellular level are similar to in situ chick muscle cells at the tissue level, we analyzed the distribution of Gli-1 in cross sections of pectoralis muscle isolated from E11 chick embryos. Muscle fragments were fixed, and
Fig. 2. Gli-1 expression in embryonic chick pectoralis muscles. Muscle cross-sections were stained with anti-Gli-1 (green) and anti-desmin antibodies (red) and DAPI (blue). Merged images are shown in D, H and L. Gli-1 localizes at the perinuclear region of mononucleated myoblasts (arrowheads in A and D), at the sarcolemma between adjacent muscle fibers (arrowhead in E) and at striations in muscle fibers (arrowhead in I). Muscle fibers at transverse view (AeD) and longitudinal view (EeL) are shown. Bar ¼ 20 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: J.D. Teixeira et al., Sonic Hedgehog signaling and Gli-1 during embryonic chick myogenesis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.071
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5 mm-thick sections were labeled for Gli-1. Gli-1 was found in three different distributions in muscle cross sections: in small aggregates near the nuclei, at the sarcolemma, in myofibrillar striations (Fig. 2AeL). Interestingly, Gli-1 was not found within the nuclei of muscle cells (myoblasts and myotubes) and fibroblasts at the tissue level in muscles from E11 chick embryos (Fig. 2A-L); confirming the results we found in chick muscle cells grown in culture. We also analyzed the distribution of Gli-1 in somites of 24 hpf zebrafish embryos and the protein was found near the septa, around the nuclei and in a periodic distribution in myofibrils, but it was not found within the nuclei of muscle cells (data not shown). 3.4. Shh induces an increase in myoblast numbers and nuclear translocation of Gli-1 Next, we decided to analyze the effects of ectopic Shh during chick myogenesis. Chick myogenic cells grown in vitro were treated
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with recombinant Shh at 1, 2, 5 or 10 ng for 2 or 48 h and cells were immunolabeled with an antibody against desmin and the nuclear dye DAPI or with an anti-Gli-1 and the nuclear dye NucSpot (Fig. 3AeG). Desmin was used as a muscle cell marker to distinguish the two mononucleated cell phenotypes present in these cell cultures: myoblasts and fibroblasts; and DAPI was used to count the number of nuclei within myotubes and the total number of nuclei (Figs. 3 and 4). Interestingly, myotubes formed after treatment with recombinant Shh were larger than control myotubes and their nuclei were perfectly aligned in parallel lines along the cell's longitudinal axis (Fig. 3AeD). A significant increase in the area occupied by muscle cells was found when cells were treated with 10 ng of Shh, but not with lower concentrations of Shh (Fig. 3AeD and 4A). Since only 10 ng of Shh showed a significant effect in the formation of muscle cells (Fig. 4A), all the following experiments were performed with this concentration. Quantification from desmin/ DAPI immunofluorescence images (Fig. 3AeD) showed that Shh
Fig. 3. Shh induces cell proliferation and Gli-1 nuclear localization in myogenic cell cultures. Chick myogenic cells were treated with recombinant Shh protein (10 ng) and stained with anti-desmin antibodies (red, A and C) and DAPI (blue, B and D), or with anti-Gli-1 (red, E and G) and NucSpot (green, F and G). Untreated cells are shown in A and B and Shh treated-cells in C-G. Note that Shh induces an increase in the size of myotubes (arrow in C), in the number of nuclei (D), in the alignment of nuclei within myotubes (arrow in D) and in nuclear translocation of Gli-1 in myoblasts (arrowheads in E-G). Gli-1 was not found within the nuclei of fibroblast cells after Shh treatment (arrows in E-G). Bar in D ¼ 50 mm and bar in G ¼ 20 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: J.D. Teixeira et al., Sonic Hedgehog signaling and Gli-1 during embryonic chick myogenesis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.071
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Fig. 4. Effects of Shh in myogenic cell cultures. Chick myogenic cells were treated with recombinant Shh protein (10 ng in B-F) and stained with anti-desmin antibodies and DAPI. Cultures were quantified in relation to: the area occupied by muscle cells (A), the number of nuclei within myotubes (B), the number of myoblasts (C), the number of fibroblasts (D), the total number of nuclei (E), and the fusion index (F). *P < 0.05, **P < 0.01, ***P < 0.001, Dunn's test in chart A and Mann-Whitney test in charts B-F.
induced an increase in the number of nuclei within myotubes (Fig. 4B) and in the number of mononucleated myoblasts (Fig. 4C). Interestingly, no significant change was observed in the number of fibroblasts and in the total number of nuclei per microscopic field (Fig. 4D and E). These results show that Shh induced an increase in myoblast proliferation in chick muscle cells grown in vitro, and that at least part of these cells fused to form myotubes, as observed by the increase in the number of nuclei within myotubes (Fig. 4B). These data are in accordance with previous studies showing that Shh induces the proliferation of committed skeletal muscle cells in the chick limb [12]. We also quantified the cell fusion index and no significant differences were found between untreated and Shhtreated cells (Fig. 4F). Two possible explanations arise for these results: (i) Shh induces the proliferation of a specific pool of myoblasts present in chick primary cultures and that another pool is refractory to the proliferative effects of Shh, at least at this embryonic stage (E11 chick embryos); or (ii) Shh induces the proliferation of all myoblast cells but not all of them will became fusogenic. Further experiments are necessary to unravel the possible divergent effect of Shh on different populations of embryonic chick myoblast cells. It is important to point out that we found an increase in the number of muscle cells when using an ectopic source of recombinant Shh and therefore care should be taken when comparing these results with in vivo data. Finally, we decided to test whether recombinant Shh would be able to activate the Shh pathway in muscle cells grown in vitro. Recombinant Shh (10 ng for 2 h) induced the nuclear translocation of Gli-1 in myoblasts from chick myogenic cell cultures (Fig. 3EeG). Gli-1 was found within the nuclei of myoblast cells, but not in fibroblasts or in myotubes (Fig. 3EeG). These results are in accordance with our data showing that recombinant Shh induces the proliferation of myoblasts (Fig. 4C) and reinforce the specific effects
of recombinant Shh in myoblast cells, and not in fibroblasts. Interestingly, treatment of chick muscle cells with recombinant Shh (10 ng) for 24 or 48 h did not induced the nuclear translocation of Gli-1, suggesting that the redistribution of Gli-1, from the cytoplasm to the nuclei, occurs in a short time window (2 h) after exposure of cells to Shh. The collection of our results suggests that the Shh signaling pathway is inactive in muscle cells from E11 chick embryos, but it can be rapidly activated when cells are in contact with ectopic Shh. During the inactive stage of Shh pathway, Gli-1 is found in small aggregates in the perinuclear region of myoblasts and myotubes, suggesting that these aggregates could be storage sites for the rapid redistribution of Gli-1 upon Shh activation. Ectopic Shh induced the proliferation of myoblasts and an increase in the size of myotubes formed. These results could have implications for the understanding of the role of Shh pathway during normal and pathological muscle conditions. Conflicts of interest The authors declare that they have no conflicts of interest concerning this article. Author contributions CM conceived the study. CM, IRA, JB, JDT and MLC designed the experiments and participated in the interpretation of data; IRA and JDT performed the cell culture experiments, the immunoblotting and the immunofluorescence of cultured cells; PPAM, MPM and YRMS performed the immunofluorescence muscle cross sections experiments; CM was responsible for writing the manuscript; All authors read, discussed and approved its final version.
Please cite this article as: J.D. Teixeira et al., Sonic Hedgehog signaling and Gli-1 during embryonic chick myogenesis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.071
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Acknowledgments This work was supported by grants from Conselho Nacional de gico, Fundaça ~o Carlos Chagas Desenvolvimento Científico e Tecnolo Pesquisa do Estado do Rio de Janeiro (FAPERJ), Filho de Apoio a Coordenaç~ ao de Aperfeiçoamento de Pessoal de Nível Superior, ~o do Ca ^ncer/Programa de Oncobiologia. This work is part of Fundaça s-Graduaça ~o em Cie ^ncias JDT's master thesis at the Programa de Po gicas (PCM/UFRJ). Morfolo Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.11.071. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.11.071. References [1] R.L. Johnson, E. Laufer, R.D. Riddle, C. Tabin, Ectopic expression of Sonic Hedgehog alters dorsal-ventral patterning of somites, Cell 79 (1994) 1165e1173. [2] A.E. Münsterberg, J. Kitajewski, D.A. Bumcrot, A.P. McMahon, A.B. Lassar, Combinatorial signaling by sonic hedgehog and wnt family members induces myogenic bHLH gene expression in the somite, Genes Dev. 9 (1995) 2911e2922. [3] M. Maroto, R. Reshef, A.E. Münsterberg, S. Koester, M. Goulding, A.B. Lassar, Ectopic Pax-3 activates MyoD and Myf-S expression in embryonic mesoderm and neural tissue, Cell 89 (1997) 139e148. [4] A.G. Borycki, L. Mendham, C. Emerson, Control of somite patterning by Sonic hedgehog SHH and its downstream signal response genes, Development 125 (1998) 777e790. [5] M.A. Teillet, Y. Watanabe, P. Jeffs, D. Duprez, F. Lapointe, N. Le-Douarin, Sonic Hedgehog is required for survival of both myogenic and chondrogenic somitic lineages, Development 125 (1998) 2019e2030. [6] J.M. Brito, M.A. Teillet, N.M. Le Douarin, Induction of mirror-image supernumerary jaws in chicken mandibular mesenchyme by Sonic Hedgehogproducing cells, Development 135 (2008), 2311-2019. [7] E.H. Villavicencio, D.O. Walterhouse, P.M. Iannaccone, The sonic hedgehogpatched-gli pathway in human development and disease, Am. J. Hum.
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Please cite this article as: J.D. Teixeira et al., Sonic Hedgehog signaling and Gli-1 during embryonic chick myogenesis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.071
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Sonic Hedgehog signaling and Gli-1 during embryonic chick myogenesis Brito b, John Douglas Teixeira a, 1, Ivone de Andrade Rosa a, 1, Jose Yuli Rodrigues Maia de Souza c, Pedro Paulo de Abreu Manso c, Marcelo Pelajo Machado c, Manoel Luis Costa a, Claudia Mermelstein a, * a b c
~o Muscular e Citoesqueleto, Instituto de Ci^ rio de Diferenciaça Laborato encias Biom edicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil ~o e Diferenciaça ~o Celular, Instituto de Ci^ rio de Proliferaça Laborato encias Biom edicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil rio de Patologia, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, RJ, Brazil Laborato
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 November 2018 Accepted 12 November 2018 Available online xxx
The Sonic Hedgehog signaling (Shh) pathway has been implicated in both proliferation of myoblast cells and terminal differentiation of muscle fibers, and contradictory results of these effects have been described. To clarify the role of Shh during myogenesis, we decided to study the effects of recombinant Shh and the distribution of Gli-1 during in vitro and in situ embryonic chick skeletal muscle differentiation at later stages of development. Gli-1 was found in small aggregates near the nucleus in mononucleated myoblasts and in multinucleated myotubes both in vitro and in situ chick muscle cells. Some Gli-1 aggregates colocalized with gamma-tubulin positive-centrosomes. Gli-1 was also found in striations and at the subsarcolemmal membrane in muscle fibers in situ. Recombinant Shh added to in vitro grown muscle cells induced the nuclear translocation of Gli-1, as well as an increase in the number of myoblasts and in the number of nuclei within myotubes. We suggest that Gli-1 aggregates observed in chick muscle cells near the nuclei of myoblasts and myotubes could be a storage site for the rapid cellular redistribution of Gli-1 upon specific signals during muscle differentiation. © 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords: Gli Sonic Hedgehog chick muscle myogenesis
1. Introduction The mature multinucleated skeletal myofiber is formed by the fusion of mononucleated myoblasts during development. Skeletal myogenesis can be described as a two-step process: a proliferation phase, when myoblast cells divide; and a differentiation phase, when post-mitotic myoblasts fuse to form fully contractile multinucleated myofibers. An intricate network of signaling pathways, including Wnt, Notch, bone morphogenetic proteins (BMP) and Sonic Hedgehog (Shh), regulates this complex two-step process. Shh has been implicated in both the proliferative and the differentiation steps of myogenesis [1e5]. Indeed, the graft of Shhsecreting cells into the presumptive first branchial arch region, at 5 somite stage, was able to increase the territory of MyoD expression [6].
* Corresponding author. E-mail address: [email protected] (C. Mermelstein). 1 Both authors contributed equally to this work.
When activated, the Shh signaling pathway triggers a chain of events in target cells, leading to the activation of specific genes by the glioma-associated oncogene homolog (Gli) family of zinc finger transcription factors [7,8]. Upon secretion, Shh binds to the transmembrane receptor proteins, Patched-1 and Patched-2, in target cells [9]. Patched-1 inhibit downstream signaling of the transmembrane protein Smoothened (Smo) via an indirect mechanism not yet described. Patched-1 inhibition of Smo results in the nuclear localization of Gli proteins, which are the terminal effectors of the Shh signaling. In vertebrates, there are three Gli transcription factors (Gli-1, Gli-2 and Gli-3). Gli-1 is the only full-length transcriptional activator whereas Gli-2 and Gli-3 act as either positive or negative regulators. In the absence of Shh, Suppressor of Fused (SUFU) negatively regulates the pathway by directly binding to Gli proteins and anchoring them in the cytoplasm preventing the activation of Gli target genes. Contradictory results have been described regarding the role of the Shh pathway during vertebrate skeletal myogenesis. Different studies have shown that Shh can increase or inhibit myoblasts proliferation and terminal muscle differentiation, depending on the
https://doi.org/10.1016/j.bbrc.2018.11.071 0006-291X/© 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article as: J.D. Teixeira et al., Sonic Hedgehog signaling and Gli-1 during embryonic chick myogenesis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.071
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experimental model [1e5,10e13]. To get further insight into the role of the Shh pathway during myogenesis, we investigated the proliferation or differentiation of embryonic chick muscle cells both in vitro and in situ. We found Gli-1 aggregates in a perinuclear region in myoblasts and myotubes both in vitro and in situ muscle cells. Gli-1 was also found in striations and at the subsarcolemmal membrane in muscle cells in situ. We also studied the effects of recombinant Shh protein in muscle cells using the nuclear translocation of Gli-1 as an indicator of the activation of the Shh pathway. Recombinant Shh added to in vitro grown muscle cells induced an increase in the number of myoblasts, in the number of nuclei within myotubes, as well as the nuclear translocation of Gli1. Interestingly, part of Gli-1 aggregates colocalized with gammatubulin positive-centrosomes.
2.4. Immunofluorescence microscopy and digital image acquisition Cultured cells were fixed with 4% paraformaldehyde diluted in 0.1 M phosphate buffer (pH 7.4) and incubated with primary antibodies overnight at 4 C. Cells were washed and incubated with secondary antibodies for 1 h at 37 C. Nuclei were labeled with DAPI (0.1 mg/ml in 0.9% NaCl) or NucSpot live cell nuclear stain for 5 min. Slides were mounted in ProLong Gold antifade reagent (Molecular Probes, USA) and examined with either an Axiovert 100 inverted microscope (Carl Zeiss, Germany) or a Spinning Disk Confocal microscope (DSU, Olympus, Japan) with 60x lenses (1.3 NA). Control experiments performed omitting the primary antibodies showed only faint background staining. Some confocal images were digitally processed by Universal live-cell super-resolution microscopy (SRRF) [15].
2. Materials and methods
2.5. Quantification of chick cell cultures
2.1. Ethics statement
Cultures were fixed and double labeled for desmin and DAPI, and merged images were used for counting the presence of nuclei within mononucleated (myoblasts and fibroblasts) or multinucleated cells. The DAPI labeling enables the identification of myoblasts and fibroblasts by their nuclear morphologies and fluorescence intensities: muscle fibroblasts have large, flattened and pale nuclei whereas myoblasts have small, round and bright nuclei. The public domain softwares ImageJ (http://rsb.info.nih.gov/ij/) and CellProfiler (http://cellprofiler.org) were used for quantifications. The fusion index, the number of nuclei in myotubes divided by the total number of nuclei, was also calculated. All data was quantified from fifty randomly chosen microscopic fields collected from three independent experiments.
Experiments using chick embryos (Gallus gallus domesticus) were approved by the Ethics Committee for Animal Care and Use in Scientific Research from the Federal University of Rio de Janeiro (UFRJ) and received the approval number: 055/16.
2.2. Primary chick myogenic cell cultures and treatments All cell culture reagents were purchased from Invitrogen (USA). Primary cultures of myogenic cells were prepared from pectoral muscles of E11 (Embryonic day) chick embryos [14]. Briefly, muscles were excised, finely minced, incubated in 0.1% trypsin at 37 C, and centrifuged at approximately 300g for 5 min. Cells were resuspended in 8-1-0.5 medium (minimum essential medium with the addition of 10% horse serum, 0.05% chick embryo extract, 1% Lglutamine and 1% penicillin-streptomycin). Cells were plated at an initial density of 5 105 cells/35 mm culture dishes onto 22 mmAclar plastic coverslips (Pro-Plastics Inc., USA) previously coated with rat-tail collagen. Cells were grown in 2 ml of 8-1-0.5 medium under humidified 5% CO2 atmosphere at 37 C. The percentage of myoblasts in these cell cultures was calculated by the double labeling of 24-h cultures with both DAPI (nuclear staining) and a polyclonal anti-desmin antibody (applied herein to define a muscle-specific marker) and subsequently counting the number of desmin-positive cells out of the total number of cells in the field. On average, myoblasts represented 80% of each culture and non-myogenic cells 20%. Some 24-h myogenic cultures were treated for the next 2 or 48 h with recombinant Sonic protein (R&D Systems, USA) at the final concentration of 1, 2, 5 or 10 ng.
2.3. Antibodies and fluorescent probes Rabbit monoclonal anti-Gli-1 (ab134906) antibody was from Abcam (UK). Rabbit polyclonal anti-desmin and mouse monoclonal anti-gamma-tubulin antibodies were from Sigma Chemical Co. (USA). Mouse monoclonal anti-desmin antibody (RB-9014) was from Thermo Scientific (USA). Alexa Fluor 488-goat anti-mouse/ rabbit IgG and Alexa Fluor 546-goat anti-mouse/rabbit IgG antibodies and DNA-binding probe DAPI (4,6-Diamino-2-phenylindole dyhydrochloride) were from Molecular Probes (USA). NucSpot live cell nuclear stain was from Biotium Inc. (USA). Peroxidaseconjugated goat anti-rabbit antibodies were obtained from Amersham Biosciences (UK).
2.6. Immunofluorescence of embryonic chick muscle cross-sections Pectoral muscle fragments were isolated from E11 chick embryos, fixed in Carson's formalin-Millonig for 48 h [16] and processed according to standard techniques for paraffin embedding. Sections (5 mm thick) of paraffin blocks were submitted to immunofluorescence, as previously described [17]. Sections were incubated with primary antibodies overnight at 4 C, washed, incubated with fluorescently labeled-secondary antibodies at 37 C for 1 h and counterstaining with the nuclear dye DAPI. Negative controls were processed by omitting the incubation with primary antibodies and showed no significant labeling. All crosssections were analyzed under an LSM 710 confocal microscope (Carl Zeiss, Germany). 2.7. SDS-PAGE and western blot Chick myogenic cells were quickly washed in ice-cold PBS and 100 mL of ice-cold RIPA lysis buffer (150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM b-glycerophosphate, 1 mM Na3VO4, 1 mg/ml leupeptin and 20 mM Tris-HCl, pH 7.4) were added to the cultures, and then cells were scraped off the dish with a plastic cell scraper. Forty micrograms of proteins from samples [18] were solubilized in sample buffer (2% SDS, 10% glycerol, 0.0006% bromophenol blue and Tris-HCl, pH 6.8) and boiled for 5 min. A total protein homogenate was also prepared from pectoralis muscles derived from E11 chick embryos. Equal amounts of protein were loaded on 10% SDSpolyacrylamide gels. Molecular weight was estimated using the protein molecular weight standards Kaleidoscope (Bio-Rad, USA). Proteins were then transferred to PVDF membranes and membranes were washed in 0.1% Tween-TBS and incubated in a blocking solution (5% nonfat dry milk diluted in Tween-TBS) for 1 h.
Please cite this article as: J.D. Teixeira et al., Sonic Hedgehog signaling and Gli-1 during embryonic chick myogenesis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.071
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Fig. 1. Gli-1 expression and distribution in myogenic cell cultures. Chick myogenic cells were stained for Gli-1 (green, A, C, D, E, G and J), gamma-tubulin (red, H and J) and DAPI (blue, B, C, I and J). Gli-1 localizes in aggregates at the perinuclear region of multinucleated myotubes (arrows in A-D) and mononucleated myoblasts (arrowheads in A-C), but not in fibroblasts (asterisks in A-C). In (E) is an inset of image (D) with phase contrast and Gli-1 (green). Arrows in G-J point to the colocalization of Gli-1 and gamma-tubulin in centrosomes in a myotube. Bars ¼ 10 mm. A immunoblot against Gli-1 is shown in F. Protein extract from pectoralis muscle from E11 chick embryos (chick extract) and from 24-, 48- and 72-h myogenic cell cultures were analyzed. One major protein band with 160 kDa (asterisk) was detected in all samples. MWM ¼ molecular weight markers. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Subsequently, membranes were incubated with antibodies (1:500 dilution for anti-Gli-1) overnight at 4 C, washed in Tween-TBS, incubated for 1 h at 37 C with anti-rabbit CY5.5-conjugated secondary antibodies and washed again. The bands were visualized using an Odyssey Infrared Imager (LI-COR Biosciences, USA).
2.8. Statistical analysis Statistical analysis was carried out using the GraphPad Prism software version 5. Results of at least three independent experiments were expressed as mean ± standard deviation. Statistical
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significance was defined as *P < 0.05; **P < 0.01; ***P < 0.001 using Mann-Whitney or Dunn's test. 3. Results and discussion 3.1. Gli-1 is found in aggregates near the nuclei in myoblasts and myotubes
Gli-1 predominant isoform has a molecular weight of 160 kDa, but other variants with smaller molecular weights can also be found in different tissues [21]. The low expression of Gli-1 in the early stages of in vitro chick myogenesis (24-h) and its increased expression during the latter stages (48- and 72-h) of muscle development suggest a role for Gli-1 during the differentiation of muscle cells. 3.2. Gli-1 partially colocalizes with centrosomes
The aim of this work was to study the role of Sonic Hedgehog (Shh) signaling pathway during the proliferation and differentiation of chick embryonic skeletal muscle. First, we analyzed the distribution of Gli-1, a key target of the Shh pathway, in chick primary cultures of muscle cells. Myogenic cells were grown for 72 h and labeled with an antibody against Gli-1 (Fig. 1). Embryonic chick myogenic cultures are composed of three main cell phenotypes: mononucleated myoblasts, mononucleated fibroblasts and multinucleated myotubes. Gli-1 was found within the cytoplasm of myoblasts and myotubes (Fig. 1AeE), but not in fibroblasts (Fig. 1AeC). Our results showing the distribution of Gli-1 in primary cultures of muscle cells derived from E11 chick embryos are in accordance with a previous study from Elia and colleagues [19], which showed that primary cultures of muscle cells derived from 3day-old chick embryos express Gli-1. Interestingly, we found a strong labeling of Gli-1 concentrated in small aggregates at the perinuclear region of mononucleated myoblasts and multinucleated myotubes (Fig. 1AeE and Supp. Fig.1). It has been shown that Gli-1 can be sequestered in the cytoplasm near the nucleus by a complex of proteins, including SUFU and human Suppressor-of-Fused (SUFUH) [20]. Importantly, Gli-1 was not found within the nuclei of muscle cells (myoblasts and myotubes) and fibroblasts. Since Gli-1 is a transcription factor, these results suggest that the Shh pathway is not activated in cultured chick myogenic cells. We also analyzed the expression of Gli-1, at the protein level, during chick myogenesis. Chick myogenic cells derived from E11 chick embryos muscles were cultured for 24, 48 and 72 h, then harvested and total cell extracts were analyzed in SDS-PAGE followed by immunoblot against Gli-1 antibody (Fig. 1F). Gli-1 was expressed in all the samples (24, 48 and 72 h), but an increase in its expression was observed in 48- and 72-h cultures (Fig. 1F). Gli-1 was also detected in pectoralis muscle tissue sampled from E11 chick embryos (Fig. 1F). Gli-1 was detected in all the samples (from in vitro and in situ embryonic chick muscle cells) as a major protein band with a molecular weight of 160 kDa (Fig. 1F). The full length
Since Gli-1 was found in a perinuclear region in muscle cells, we decided to analyze whether its localization was somehow related to centrosomes. Gli-1 has been reported to be recruited and stabilized at the centrosome [22]. Centrosomes are organizing center for the control of microtubules network in eukaryotic cells [23] and they are composed of several proteins, including gamma-tubulin. Thus, we cultured chick myogenic cells and double-labeled them with anti-Gli-1 and anti-gamma-tubulin antibodies (Fig. 1GeJ). Fig. 1 shows gamma-tubulin in centrosomes in myotubes from 48-h muscle cultures. Multiple centrosomes were detected in single multinucleated myotubes, as observed by multiple gamma-tubulin positive-dots in the sarcolemma of myotubes (Fig. 1GeJ). An earlier report by Tassin and colleagues [24] described that centrosomes are numerous and scattered in myotubes and they were not associated one to each nucleus but were often clustered near nuclei groups. Besides, they reported that centrosomes were significantly smaller in myotubes than those found in mononucleated myoblast cells. Interestingly, some of Gli-1 immunolabeling was found colocalized with gamma-tubulin positive-dots (Fig. 1GeJ). Gli-1 was also found in regions without gamma-tubulin stain (Fig. 1GeJ). These results suggest that Gli-1 could be redistributed to other regions of the cell from the centrosome. The Gli-1 aggregates observed in chick muscle cells near the nuclei of myoblasts and myotubes could be a storage site for the cellular relocation of Gli-1 upon specific signals. Further experiments are needed to confirm this hypothesis and to study the mechanisms by which Gli-1 could be relocated in muscle cells. 3.3. Gli-1 is found in striations, at the sarcolemma and in aggregates in muscle cross sections To analyze whether these in vitro results at the cellular level are similar to in situ chick muscle cells at the tissue level, we analyzed the distribution of Gli-1 in cross sections of pectoralis muscle isolated from E11 chick embryos. Muscle fragments were fixed, and
Fig. 2. Gli-1 expression in embryonic chick pectoralis muscles. Muscle cross-sections were stained with anti-Gli-1 (green) and anti-desmin antibodies (red) and DAPI (blue). Merged images are shown in D, H and L. Gli-1 localizes at the perinuclear region of mononucleated myoblasts (arrowheads in A and D), at the sarcolemma between adjacent muscle fibers (arrowhead in E) and at striations in muscle fibers (arrowhead in I). Muscle fibers at transverse view (AeD) and longitudinal view (EeL) are shown. Bar ¼ 20 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: J.D. Teixeira et al., Sonic Hedgehog signaling and Gli-1 during embryonic chick myogenesis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.071
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5 mm-thick sections were labeled for Gli-1. Gli-1 was found in three different distributions in muscle cross sections: in small aggregates near the nuclei, at the sarcolemma, in myofibrillar striations (Fig. 2AeL). Interestingly, Gli-1 was not found within the nuclei of muscle cells (myoblasts and myotubes) and fibroblasts at the tissue level in muscles from E11 chick embryos (Fig. 2A-L); confirming the results we found in chick muscle cells grown in culture. We also analyzed the distribution of Gli-1 in somites of 24 hpf zebrafish embryos and the protein was found near the septa, around the nuclei and in a periodic distribution in myofibrils, but it was not found within the nuclei of muscle cells (data not shown). 3.4. Shh induces an increase in myoblast numbers and nuclear translocation of Gli-1 Next, we decided to analyze the effects of ectopic Shh during chick myogenesis. Chick myogenic cells grown in vitro were treated
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with recombinant Shh at 1, 2, 5 or 10 ng for 2 or 48 h and cells were immunolabeled with an antibody against desmin and the nuclear dye DAPI or with an anti-Gli-1 and the nuclear dye NucSpot (Fig. 3AeG). Desmin was used as a muscle cell marker to distinguish the two mononucleated cell phenotypes present in these cell cultures: myoblasts and fibroblasts; and DAPI was used to count the number of nuclei within myotubes and the total number of nuclei (Figs. 3 and 4). Interestingly, myotubes formed after treatment with recombinant Shh were larger than control myotubes and their nuclei were perfectly aligned in parallel lines along the cell's longitudinal axis (Fig. 3AeD). A significant increase in the area occupied by muscle cells was found when cells were treated with 10 ng of Shh, but not with lower concentrations of Shh (Fig. 3AeD and 4A). Since only 10 ng of Shh showed a significant effect in the formation of muscle cells (Fig. 4A), all the following experiments were performed with this concentration. Quantification from desmin/ DAPI immunofluorescence images (Fig. 3AeD) showed that Shh
Fig. 3. Shh induces cell proliferation and Gli-1 nuclear localization in myogenic cell cultures. Chick myogenic cells were treated with recombinant Shh protein (10 ng) and stained with anti-desmin antibodies (red, A and C) and DAPI (blue, B and D), or with anti-Gli-1 (red, E and G) and NucSpot (green, F and G). Untreated cells are shown in A and B and Shh treated-cells in C-G. Note that Shh induces an increase in the size of myotubes (arrow in C), in the number of nuclei (D), in the alignment of nuclei within myotubes (arrow in D) and in nuclear translocation of Gli-1 in myoblasts (arrowheads in E-G). Gli-1 was not found within the nuclei of fibroblast cells after Shh treatment (arrows in E-G). Bar in D ¼ 50 mm and bar in G ¼ 20 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: J.D. Teixeira et al., Sonic Hedgehog signaling and Gli-1 during embryonic chick myogenesis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.071
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Fig. 4. Effects of Shh in myogenic cell cultures. Chick myogenic cells were treated with recombinant Shh protein (10 ng in B-F) and stained with anti-desmin antibodies and DAPI. Cultures were quantified in relation to: the area occupied by muscle cells (A), the number of nuclei within myotubes (B), the number of myoblasts (C), the number of fibroblasts (D), the total number of nuclei (E), and the fusion index (F). *P < 0.05, **P < 0.01, ***P < 0.001, Dunn's test in chart A and Mann-Whitney test in charts B-F.
induced an increase in the number of nuclei within myotubes (Fig. 4B) and in the number of mononucleated myoblasts (Fig. 4C). Interestingly, no significant change was observed in the number of fibroblasts and in the total number of nuclei per microscopic field (Fig. 4D and E). These results show that Shh induced an increase in myoblast proliferation in chick muscle cells grown in vitro, and that at least part of these cells fused to form myotubes, as observed by the increase in the number of nuclei within myotubes (Fig. 4B). These data are in accordance with previous studies showing that Shh induces the proliferation of committed skeletal muscle cells in the chick limb [12]. We also quantified the cell fusion index and no significant differences were found between untreated and Shhtreated cells (Fig. 4F). Two possible explanations arise for these results: (i) Shh induces the proliferation of a specific pool of myoblasts present in chick primary cultures and that another pool is refractory to the proliferative effects of Shh, at least at this embryonic stage (E11 chick embryos); or (ii) Shh induces the proliferation of all myoblast cells but not all of them will became fusogenic. Further experiments are necessary to unravel the possible divergent effect of Shh on different populations of embryonic chick myoblast cells. It is important to point out that we found an increase in the number of muscle cells when using an ectopic source of recombinant Shh and therefore care should be taken when comparing these results with in vivo data. Finally, we decided to test whether recombinant Shh would be able to activate the Shh pathway in muscle cells grown in vitro. Recombinant Shh (10 ng for 2 h) induced the nuclear translocation of Gli-1 in myoblasts from chick myogenic cell cultures (Fig. 3EeG). Gli-1 was found within the nuclei of myoblast cells, but not in fibroblasts or in myotubes (Fig. 3EeG). These results are in accordance with our data showing that recombinant Shh induces the proliferation of myoblasts (Fig. 4C) and reinforce the specific effects
of recombinant Shh in myoblast cells, and not in fibroblasts. Interestingly, treatment of chick muscle cells with recombinant Shh (10 ng) for 24 or 48 h did not induced the nuclear translocation of Gli-1, suggesting that the redistribution of Gli-1, from the cytoplasm to the nuclei, occurs in a short time window (2 h) after exposure of cells to Shh. The collection of our results suggests that the Shh signaling pathway is inactive in muscle cells from E11 chick embryos, but it can be rapidly activated when cells are in contact with ectopic Shh. During the inactive stage of Shh pathway, Gli-1 is found in small aggregates in the perinuclear region of myoblasts and myotubes, suggesting that these aggregates could be storage sites for the rapid redistribution of Gli-1 upon Shh activation. Ectopic Shh induced the proliferation of myoblasts and an increase in the size of myotubes formed. These results could have implications for the understanding of the role of Shh pathway during normal and pathological muscle conditions. Conflicts of interest The authors declare that they have no conflicts of interest concerning this article. Author contributions CM conceived the study. CM, IRA, JB, JDT and MLC designed the experiments and participated in the interpretation of data; IRA and JDT performed the cell culture experiments, the immunoblotting and the immunofluorescence of cultured cells; PPAM, MPM and YRMS performed the immunofluorescence muscle cross sections experiments; CM was responsible for writing the manuscript; All authors read, discussed and approved its final version.
Please cite this article as: J.D. Teixeira et al., Sonic Hedgehog signaling and Gli-1 during embryonic chick myogenesis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.071
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Acknowledgments This work was supported by grants from Conselho Nacional de gico, Fundaça ~o Carlos Chagas Desenvolvimento Científico e Tecnolo Pesquisa do Estado do Rio de Janeiro (FAPERJ), Filho de Apoio a Coordenaç~ ao de Aperfeiçoamento de Pessoal de Nível Superior, ~o do Ca ^ncer/Programa de Oncobiologia. This work is part of Fundaça s-Graduaça ~o em Cie ^ncias JDT's master thesis at the Programa de Po gicas (PCM/UFRJ). Morfolo Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.11.071. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.11.071. References [1] R.L. Johnson, E. Laufer, R.D. Riddle, C. Tabin, Ectopic expression of Sonic Hedgehog alters dorsal-ventral patterning of somites, Cell 79 (1994) 1165e1173. [2] A.E. Münsterberg, J. Kitajewski, D.A. Bumcrot, A.P. McMahon, A.B. Lassar, Combinatorial signaling by sonic hedgehog and wnt family members induces myogenic bHLH gene expression in the somite, Genes Dev. 9 (1995) 2911e2922. [3] M. Maroto, R. Reshef, A.E. Münsterberg, S. Koester, M. Goulding, A.B. Lassar, Ectopic Pax-3 activates MyoD and Myf-S expression in embryonic mesoderm and neural tissue, Cell 89 (1997) 139e148. [4] A.G. Borycki, L. Mendham, C. Emerson, Control of somite patterning by Sonic hedgehog SHH and its downstream signal response genes, Development 125 (1998) 777e790. [5] M.A. Teillet, Y. Watanabe, P. Jeffs, D. Duprez, F. Lapointe, N. Le-Douarin, Sonic Hedgehog is required for survival of both myogenic and chondrogenic somitic lineages, Development 125 (1998) 2019e2030. [6] J.M. Brito, M.A. Teillet, N.M. Le Douarin, Induction of mirror-image supernumerary jaws in chicken mandibular mesenchyme by Sonic Hedgehogproducing cells, Development 135 (2008), 2311-2019. [7] E.H. Villavicencio, D.O. Walterhouse, P.M. Iannaccone, The sonic hedgehogpatched-gli pathway in human development and disease, Am. J. Hum.
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Please cite this article as: J.D. Teixeira et al., Sonic Hedgehog signaling and Gli-1 during embryonic chick myogenesis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.071