Exploring in vivo pectoral muscle differentiation in Ross chickens: sustained expression of MMP-2 and MT1-MMP throughout all stages and absence of MMP-9

Exploring in vivo pectoral muscle differentiation in Ross chickens: sustained expression of MMP-2 and MT1-MMP throughout all stages and absence of MMP-9 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Exploring in vivo pectoral muscle differentiation in Ross chickens: sustained expression of MMP-2 and MT1-MMP throughout all stages and absence of MMP-9 Alisson Rodrigo Oliveira, Maria Albertina Miranda Soares, Jose Rosa Gomes This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6339860/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Aug, 2025 Read the published version in Histochemistry and Cell Biology → Version 1 posted 10 You are reading this latest preprint version Abstract There are pertinent studies on the relationship between MMPs and muscle cell differentiation in vitro, but there are currently few studies in vivo exploring MMP expressions in muscle chicken during different stages of differentiation. Therefore, we aimed to investigate in vivo, whether MMP-2, MT1-MMP, and MMP-9 are expressed in pectoral muscle during the stages of chicken development on days E11, E15, and E19. Our results demonstrated that, in contrast to earlier reports in vitro, that primary myotubes are formed on E11 while on E15 and E19 is occurring the secondary muscle waves. MMP-2, on the other hand, appears to be more expressed than MT1-MMP throughout the differentiation process, whereas MMP-9 is not expressed at any point. Additionally, serine was discovered as an unexpected finding in the zymogram analysis. We conclude that during in vivo pectoral muscle development in Ross chickens, MMP-9 is not expressed at any stage of muscle differentiation, underscoring its non-essential role, whereas the expression of MMP-2 and MT1-MMP is crucial, with primary myotubes emerging on day E11 and secondary muscle cells appearing on days E15 and E19, reflecting a distinct timeline for this muscle differentiation in this chicken lineage. chicken muscle cell differentiation MMPs Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Most of our understanding of muscle development derives from experiments conducted on various vertebrate models, including fish, amphibians, mammals, and birds (Dali et al., 2002 ; Yabe and Takada, 2016; Sabillo et al., 2016 ; Garcia et al., 2021 ). However, due to its advantages in handling and developmental timeframe, the chicken embryo is a widely used model for studies investigating muscle development biology, meat production, human and animal pathologies, and bioengineering approaches (Hirst and Marcelle, 2015 ; Possidonio et al., 2014 ; Costa et al., 2021 ; Wachholz et al., 2021 ; Fernández-Costa et al., 2021 ; Malila et al., 2022 ; Sola and Caillot, 2022 ; Wang et al., 2024). Muscle development begins in the pre-embryo phase with the gastrulation process, during which several epiblast cells migrate to different regions, preceding tissue differentiation in vertebrates. During gastrulation, the inward movement of epiblast cells through the primitive streak results in the formation of the ectoderm, mesoderm, and endoderm (Acloque et al., 2011 ). Notably, the mesoderm is the tissue responsible for the development of connective and muscle tissues. The mesoderm is divided into paraxial, intermediate, and lateral regions, which are situated alongside the notochord and the neural tube (Maroto et al., 2012 ). In chickens, the paraxial mesoderm self-organizes, forming somites every 90 minutes in a rostrocaudal direction. Between days 1 and 5 of development, approximately 52 pairs of somites are formed. They consist of a ventral region (sclerotome), responsible for forming most of the skeletal bones, and a dorsal region (dermomyotome), which can differentiate into dermal tissue or skeletal muscles as well (Pourquié, 2004; 2018) in a process referred to as myogenesis. During the differentiation of dermomyotome cells, various signaling pathways play crucial roles, including Notch, Wnt, FGF, BMP, and Sonic Hedgehog (Chal and Pourquié, 2017 ; Sieiro et al., 2016; Scall and Marcelle, 2018). Myogenesis in chickens begins with progenitor cells located in the dermomyotome, marking the primary phase of myogenesis, which is identified between days 3 and 7 of development (the first phase of myogenesis). This phase begins with the expression of specific myogenic determinant genes such as Pax3 + and Pax7+, and these cells are referred to as myocytes. The myocytes migrate from the dorsal region of the somite to the body wall, and through a fusion process, they lead to the formation of the first myotubes. These myotubes develop into primary myofibers that express Myogenin (Myog), MyoD, slow myosin heavy chain (MyHC), and myosin light chain 1 (Chal and Pourquié, 2017 ). The next phase of myogenesis, denominated as the fetal phase, begins on day E8 and is characterized by the preferential expression of Pax7 + in myocytes. Using primary fibers as a scaffold, these myocytes differentiate into secondary or fetal fibers, expressing specific markers such as β-enolase and Nfix. Muscle growth during this phase occurs primarily through the continuous fusion of progenitor myoblasts, which is distinct from adult myogenesis, involving the proliferation of satellite cells (also Pax7) that augment an already differentiated cell population (Chal and Pourquié, 2017 ). Additionally, during development, cells may produce various extracellular components, including enzymes such as matrix metalloproteinases (MMPs). MMPs are zinc-dependent enzymes essential for remodeling extracellular components, playing vital roles in differentiation, proliferation, apoptosis, angiogenesis, tissue repair, and the maintenance of homeostasis (Cui et al., 2017 ; Duong and Erickson, 2004 ). The widely studied MMPs are MMP-2, MMP-9, and MT1-MMP, and some of them have well-established roles in different experimental conditions (Gomes et al., 2011 ; dos Reis and Gomes, 2019; Christensen and Purslow, 2016 ; Chan et al., 2020 ; Moracho et al., 2022 ). However, concerning to stages of chicken muscle differentiation, there is no reports about the expression of MMPs, however those that exist used precursor cells obtained from rats such as the C2C12 cell lineage which is a subclone from a myoblast line established from normal adult C3H mouse leg muscle (Blau et al, 1985 ). The original cell line, C2, was established in primary cultures from the thigh muscle of 2-month-old normal mice, 70 hours after crush injury cell lineage (Yaffe and Saxel, 1977 ). Although it is not a chicken myocyte lineage but cells derived from adult mammalian muscle, the expression of matrix metalloproteinases (MMPs) such as MMP-2, MMP-9 and MT1-MMP has been described during the in vitro differentiation of these cells into muscle cells (Couch and Strittmatter, 1983 , 1984 ; Guérin and Holland, 1995 ; Lewis et al., 2000 ; Chen and Li, 2009; Smith et al., 2020 ). In contrast, in vivo approaches have investigated the processes of muscle morphology and the expression of MMPs in rodent muscles in the context of diseases or following muscle injuries (Fukushima et al., 2007; Zimowska et al., 2008 ; Kumar et al., 2022 ; Chen et al., 2023). Although the importance of this results already reported they were obtained in several experimental conditions that are different from those found during the embryonic stages in vivo, which need to be clarified yet. Thus, the aim of this study was to investigate in vivo the expression of MMP-2, MT1-MMP, and MMP-9 in the pectoral muscle of Ross lineage chickens during the secondary or fetal stages of development. These findings will contribute to a more comprehensive understanding of myogenesis and the roles of MMPs in muscle development during in vivo chicken embryo-fetal development stages. Materials and Methods 2.1 Design of Experiments and Ethical Approval : The calculation of sample size was based on a pilot experiment in which the height of 30 villi from the intestines of two chicken embryos, at 16 and 17 days, was measured (Paiva et al., 2019 ). The linear tool of the ImageJ software, which was downloaded from the public domain ( https://imagej.nih.gov ), was calibrated using a 20-micrometer bar that was present in the images of semi-serial sections. Following that, the measurements were loaded into the Bioestat 5.3 software, which can be downloaded from this link: https://www.mamiraua.org.br/downloads/programas/ . Before the measurements were transformed to square roots, the Shapiro-Wilk test was performed to check the normality of the data. Subsequently, descriptive statistics were calculated and tested using the two-sample Z test to provide sample mean data and standard deviation. This data was then subjected to independent sample testing to determine the sample size for each time point studied. Based on the means and standard deviations of villus height, with a 1:1 relationship between samples, a test power of 80%, and an alpha level of 0.05 for reliability, a sample size of 3 embryos was obtained for each time evaluated (n = 9). All experiments in this work were authorized by the University Committee for Ethics in Animal Research, under protocol number 23.000028800-9. 2.2 Experimental Procedures : Granja Econômica Avícola LTDA (Carambeí-PR, Brazil) kindly provided nine fertilized eggs (ROSS lineage). They were incubated at 37.8°C and 60% relative humidity until E11, E15, and E19 days. To prevent contamination, each embryo was decapitated, and its chest was removed while still in a sterilized state and placed into the laminar flow at each evaluation point. According to subsequent analyses, detailed below, the chest was divided into three pieces and submerged in each of three solutions. 2.3 Morphological Analysis : The chest fragments from each embryo were immersed for 48 hours in a 2% paraformaldehyde/0.1 M phosphate buffer at pH 7.2. They were then dehydrated in alcohol and embedded in paraplast to create three serial sections of 7 µm. Sections were dewaxed and rehydrated before staining with Masson's trichromic solution. Regions of samples exhibiting longitudinal or cross sections areas were selected for posterior analysis according to methods used. 2.4 Immunohistochemistry for MMP-2, MMP-9, MT1-MMP, and actin : For each embryo/time evaluated, slides were prepared with three serial sections of 7 µm. After dewaxing and rehydrating procedures, the sections were subjected to antigen retrieval using a citrate buffer solution at pH 6.0 for 20 minutes at 98°C. Afterward, they were quenched three times with 2% hydrogen peroxide for 10 minutes each to inhibit endogenous peroxidase activity. Slides were washed in water and PBS (pH 7.4) and incubated in PBS containing 3% BSA to block non-specific antigen binding for 1 hour. After washing in PBS again, they were covered with monoclonal primary antibodies (2 µg/ml, purchased from Chemicon, USA) as follows: MMP-9 (1:200), MMP-2 (1:200), and MT1-MMP (1:200) in PBS containing 1% BSA, and incubated overnight. Additionally, sections of cerebellum and incisor tooth, in which the presence of MMP-9 had been previously demonstrated (Gomes et al., 2011 ; Yasmin and Gomes, 2023), were used as positive controls for MMP-9. Sections were then washed in PBS and incubated with secondary antibodies using the DAKO (Universal LSAB kit) for 30 minutes at 37°C in each solution. After washing in PBS, sections were incubated with the DAB reagent (SIGMA) prepared in PBS in the presence of hydrogen peroxide. Negative control staining was performed for each molecule by either omitting the primary antiserum or substituting the primary antiserum with non-immune serum. Actin detection was performed by immunofluorescence as follows: After passing through a 3% BSA solution for 1 hour, the sections were incubated with a primary monoclonal beta-actin antibody (1:500) (ThermoFisher Scientific, USA, MA5-15739) in PBS containing 1% BSA and 0.1% Triton X-100 overnight. After washing in PBS (pH 7.4), they were incubated with a secondary antibody, Alexa Fluor 488 (1:1000) (Life Technologies, Brazil, Lot No. 1832425), in PBS containing 1% BSA for 1 hour, then washed again at room temperature. After this and PBS washing, the sections were incubated with DAPI (2 µg/mL, Sigma-Aldrich, USA) for 30 minutes, washed, and covered with Fluorescence Mounting Medium (DAKO, USA) under coverslips. The Masson's trichromic staining images were taken using an Olympus DP72 microscope and epifluorescence microscopy (Leica, China) for photos of immunofluorescence sections. 2.3 Procedures for the Zymography Assay : Small pieces of chest muscle were taken under sterile conditions for each evaluation, and then washed in PBS, cut into smaller pieces with a surgical blade, and distributed into three wells of a 96-well plate. The plate was then washed once with Dulbecco's Modified Eagle Medium (purchased from SIGMA), which contained 100 µg/ml of the antibiotic gentamicin, without the addition of fetal bovine serum. The fragments were incubated for 24 hours at 37°C to produce MMP-conditioned media (Gomes et al., 2011 ). Following that, the material was collected into a microtube and centrifuged at 2000 rpm for one minute. For the zymography and protease detection tests, 0.5 µg of each sample recovered from the conditioned medium was resuspended, briefly sonicated in Laemmli buffer under non-reducing conditions, and then subjected to SDS-PAGE in a 10% polyacrylamide gel containing 5% gelatin. A positive sample for MMP-9 previously identified (Dos Reis et al., 2020 ) and a sample of medium free of fragments were used as negative controls for the zymography test. Following electrophoresis, the gels were shaken and rinsed twice for thirty minutes in 2.5% Triton X-100. They were then submerged in 1 M CaCl₂ and 1 M Tris-HCl incubation buffer for sixteen hours at 37°C to activate the proteases in the samples. Gels were then stained with Coomassie Blue until the degraded bands were visible, and the molecular weight of the bands was compared to a molecular weight standard obtained from Fermentas Life Science (Burlington, Ontario, Canada). To characterize the protease family, various inhibitors were added to the incubation buffer of other gels. These inhibitors included 0.5 mM N-Ethylmaleimide (NEM) (cysteine inhibitor), 1,10-phenanthroline (PHE), and EDTA (MMP inhibitors); 2 mM phenylmethylsulfonyl fluoride (PMSF) (serine inhibitor); 2 mM benzamidine (trypsin serine inhibitor); and 0.5 mM N-Ethylmaleimide (NEM) (cysteine inhibitor) at 37°C for 16 hours. Gels were then stained with Coomassie Blue until the degraded bands were visible. Results 3.1 The stage of myocyte fusion begins on day 11, while the primary and secondary stages of myotube formation occur on days 15 and 19 during the development of the chicken pectoral muscle. The morphological phases of muscle cell differentiation are shown in detail in Fig. 1 . Initially, on day E11 of development, myocytes with spherical nuclei are observed in a clustered organization (oval circle) with thin cell membrane extensions (arrows) touching the membranes of adjacent myocytes. In Figs. 1 b and 1b1, the fusion process among the myocytes is clearly visible on day E15, producing a syncytium structure (indicated by the oval circle) to generate primary myotubes of varying thicknesses, as shown in Fig. 1 b1. The nuclei of the myotubes assume an elliptical morphology (arrow) with active fusion of myoblasts, while other myocytes are outside the myotubes (tip of arrow). Some transverse striations are visible (dashed lines). This day, E15, is considered the beginning of the secondary phase of pectoral muscle cell differentiation. As seen in Fig. 1 c, by day E19 of development, the myotubes are already very thick, with elongated regions in the cytoplasm showing stronger red staining that we identified as nascent myofibrils (arrows). Figure 1c1 shows transverse striations (dashed lines) running parallel to the cytoplasm of muscle cells, as well as thin blue-stained structures emerging from surrounding tissue and branching out over the muscle cells (arrows), which could be nerve fiber ramifications. 3.2 The distribution of actin in the cytoplasm changes depending on the differentiation stages of muscle cells. Figure 2 shows the immunostaining for actin during the phases of muscle cell differentiation. On day E11, actin is distributed throughout the myoblast cytoplasm during their fusion process (Fig. 2a1 and a2). However, on day E15, although actin can be detected in the cytoplasm of the myotubes, it appears to begin concentrating in specific regions within the cytoplasm of the myotubes, as observed in the images (Fig. 2b1,b3) in a longitudinal section (indicated by white arrows), as well as in the transversal sections (Fig. 2b2,b4), where the strong actin staining appears in the shape of a ring delimiting a core region with less staining (b4 detailed). This core can be interpreted as a long, tubular-shaped structure in a cross-section. On day E19, actin staining is detected in the same longitudinal stick-shaped sections along the cytoplasm of muscle cells, as demonstrated in Fig. 2c1, indicated by white arrows. It is also clear that the actin staining is not continuous in this structure, which is interrupted by small dark gaps between regions of actin staining, as represented by the white arrows in Fig. 2c2, suggesting its association with the myofibril organization. 3.3 MMP-2 and MT1-MMP were expressed at all stages of muscle cell differentiation; however, MMP-9 was not expressed. Figure 3 A shows immunostaining for MMP-2 and MT1-MMP on days E11, E15, and E19 in myocytes (a and d), myotubes (b and e), and muscle cells (c and f), respectively. However, MMP-9 was not detected at any time evaluated, as shown in images g, h, and i, when compared to positive controls in sections of the cerebellum (J) and tooth (k and l), respectively. When the optical density of immunostaining of MMP-2 and MT1-MMP was measured and compared using ANOVA with Tukey's post-hoc test, MMP-2 was significantly (p < 0.05) more expressed on day 15 when compared to MT1-MMP on day 11 and with MMP-2 on day 19 of development, as demonstrated in Fig. 3 B. The zymogram obtained from the conditioned medium of muscle cell fragments from the pectoral muscle samples is shown in Fig. 4 A, where gelatin band degradations with molecular weights below 72 kDa were detected on all days (11, 15, and 19), with the absence of gelatin band degradation at the molecular weight for MMP-9 when compared to that obtained from the control samples. The bands corresponding to pre-, pro-, and active forms of MMP-2 were confirmed after the inhibitory test, the results of which are shown in Fig. 4 B. It was found that EDTA and PMSF completely inhibited the activation of proteases in the conditioned medium. On the other hand, PHE completely inhibited the activity on day 15 and partially on days 11 and 19. Benzamidine and NEM, however, were unable to inhibit the enzymes. These results confirm the expression of MMP-2, but also the presence of serine proteases by the muscle cells during their differentiation process. The band area of gelatin degradation produced by MMP-2 was not significantly different among the times evaluated when tested using ANOVA with Tukey's post-hoc test, as demonstrated in Fig. 4 B. Discussion In vivo, the investigation into muscle differentiation is crucial not only for understanding myopathies and muscle regeneration but also for improving meat production in the poultry industry (Gaglianone et al., 2020 ; Wachholz et al., 2021 ; Orlowski et al., 2021 ; Gus et al., 2024 ). The literature reports that the morphology of myogenesis in chicken development is divided into an embryonic period that spans from days E4 to E7, during which myocytes from the myotome migrate to different body parts and fuse to form primary myotubes. The next stage, termed fetal, begins on day E8, characterized by the formation of a secondary wave of myotubes, where new myocytes fuse to the previously formed myotubes. During this stage, cells actively produce various isoforms of muscle proteins organized into myofibrils (Stockdale, 1992 ., 2001; Chal and Pourquié, 2017 ). On the other hand, findings related to morphological events of muscle differentiation have been derived from in vitro studies using muscle cell precursors from adult muscle lesioned or from the C2C12 cell lineage, which was isolated from the muscle of adult rodents. The C2C12 cell lineage may not be considered an embryonic cell but rather a lineage that already has determination and the potential to differentiate into several types of cells, such as adipocytes, osteocytes, fibroblasts, and also muscle cells. Thus, the differentiation pathway of C2C12 cells depends on specific components added to the culture medium to trigger the differentiation process (Strittmatter, 1984 ; Fahime et al., 2000 ; C). Concurrently, some studies have demonstrated that several MMPs are expressed during muscle differentiation in response to the components of the culture medium (Lluri and Jaworski, 2005 ; Ohtake et al., 2006 ; Cha and Purslow, 2010 ). However, in vitro studies do not replicate the real conditions present in the embryo during its development. Due to the limited information on the relationship between the morphological phases of muscle differentiation and the expression of MMPs related to that, we present here the in vivo results regarding the morphology and expression of MMPs in the differentiation of chicken pectoral muscle during days E11, E15, and E19 of development. These time points were chosen based on previous reports that used primary culture to study different aspects of muscle differentiation from development day E11 (Mermelstein et al., 2006 ; Bagri et al., 2022 ). Our results, while aligning in some aspects with previous reports related to the cellular morphological phases of muscle differentiation, such as fusion of myocytes to form myotubes, differ in other respects when compared to in vitro cell lineages or in vivo studies using chicken. The first difference found relates to the timing of myocyte fusion and the formation of primary myotubes in our study when compared to described previously. While the literature describes that primary myotubes occur around day E7, termed the embryonic phase (Stockdale, 1992 ; Chal et al., 2016), in our in vivo study using the eggs of Ross chicken lineage, we observed that the onset of myocyte fusion, forming the primary wave of myotube differentiation, occurs on day E11, which, according to the same reports, corresponds to the fetal phase where the second wave of myotubes should be in progress. However, in our study, the second wave was observed only on day E15, while on day E19, the myotubes were in an advanced stage of differentiation. Thus, the timing observed within embryonic and fetal periods differs from what has been reported previously (Stockdale, 1992 ; Chal et al., 2016). Taken together, our results on days E11, E15, and E19 are more similar to the myogenesis phases described in mice, as previously reported (Biressi et al., 2007 ), than when compared to those reported in chickens (Stockdale, 1992 ; Chal et al., 2016). This indicates that in the pectoral muscle of Ross chickens, muscle cell differentiation may be more advanced than previously recognized, suggesting that variations in the timing of differentiation processes among different muscles or even between species should not be dismissed. Another relevant aspect discussed in this study is the expression of matrix metalloproteinases (MMPs). It has been reported that MMP-9 activity is detectable at all stages of myoblast differentiation isolated from rats, whereas MMP-2 activity reaches its highest level during myoblast fusion (Zimowska et al., 2008 ). In satellite cells isolated from adult mouse muscle, maintained in vitro with silenced expression of MMP-9, some disruption in their differentiation was observed (Nowak et al., 2028). However, during muscle regeneration in adult animals, it was demonstrated that the expression of MMP-2 and MMP-9 depends on the type of muscle evaluated (slow or fast) as well as the phase of regeneration. Additionally, when C2C12 cells are under normoxic conditions, MMP-9 is required in the earlier phases of myoblast differentiation (Chellini et al., 2023 ). However, other studies show that the expression of MMP-9 in C2C12 cells may respond differently to substrate coating and cyclic mechanical stretching (Cha and Purslow, 2010 ). Therefore, the results for MMP-9 expression obtained from studies using muscle cell precursors isolated from adult rats, such as satellite cells or for the C2C12 lineage, are still controversial and dependent on several differing experimental conditions, thus opposing our findings in the real developmental context of chickens, where MMP-9 was not detected by immunohistochemistry or zymography. This absence raises questions about the plasticity and regulatory mechanisms present during in vivo muscle development that may not exist when cells are studied under in vitro conditions. Nonetheless, our in vivo results confirmed the continuous expression of MMP-2 and MT1-MMP from day E11 up to E19 of the chicken Roos lineage, aligning with previous reports in vitro from precursor cells obtained from adult muscle of mice (Nowak et al., 2028). Although they do not represent the real conditions of muscle differentiation as demonstrated in our in vivo study, the results indicate that MMP-2 and MT1-MMP are important during the differentiation of muscle in vivo as well. Finally, the chicken model proves promising for elucidating the underlying mechanisms of muscle differentiation and MMPs for further investigations that not only expand our understanding of myogenesis but also explore the practical application of this knowledge in improving production conditions to understand muscle health in poultry (Christensen and Purslow, 2016 ) and myopathies (Kumar et al., 2022 ) In conclusion, during in vivo pectoral muscle development in Ross chickens, MMP-9 is not expressed at any stage of muscle differentiation, underscoring its non-essential role, whereas the expression of MMP-2 and MT1-MMP is crucial, with primary myotubes emerging on day E11 and secondary muscle cells appearing on days E15 and E19, reflecting a distinct timeline for muscle differentiation in this species. Declarations C r ediT A uthorship contribution statement : Alisson Rodrigo de Oliveira: experimental methodology, investigation and formal analysis execution. Maria Albertina de Miranda Soares: formal analysis, data curation and original write revision. Jose Rosa Gomes: conceptualization, final writing and edition, formal analysis, data curation, conceptualization, general supervision. Data availability : Data will be made available on request. Acknowledgements : The authors would like to acknowledge for Coordination for the improvement of higher education personnel (CAPES) by the student scholarship provided and to Granja Economica Avicola LTDA (Carambeí-PR-Brazil) that kindly provided the fertilized eggs (ROSS lineage). References Acloque H, Ocaña OH, Matheu A, Rizzoti K, Wise C, Lovell-Badge R, Nieto MA (2011). Reciprocal repression between Sox3 and snail transcription factors defines embryonic territories at gastrulation. Dev Cell 21(3): 546–558. https://doi.org/10.1016/j.devcel.2011.07.005 Bagri KM, Oliveira LF, Pereira MG, Abreu JG, Mermelstein C (2022). The Wnt/Beta-Catenin Pathway and Cytoskeletal Filaments Are Involved in the Positioning, Size, and Function of Lysosomes during Chick Myogenesis. Cells 11(21): 3402. https://doi.org/10.3390/cells11213402 Blau HM, Pavlath GK, Hardeman EC, Chiu CP, Silberstein L, Webster SG, Miller SC, Webster C (1985). Plasticity of the differentiated state. Science 230(4727): 758–766. https://doi.org/10.1126/science.2414846 Cha MC, Purslow PP (2010). The activities of MMP-9 and total gelatinase respond differently to substrate coating and cyclic mechanical stretching in fibroblasts and myoblasts. Cell Biol Int 34(6): 587–591. https://doi.org/10.1042/CBI20090096 Chal J, Pourquié O (2017). Making muscle: skeletal myogenesis in vivo and in vitro. Development. https://doi.org/10.1242/dev.151035 Biressi S, Molinaro M, Cossu G (2007). Cellular heterogeneity during vertebrate skeletal muscle development. Dev Biol 308(2): 281–293. https://doi.org/10.1016/j.ydbio.2007.06.00 Chan ZC, Oentaryo MJ, Lee CW (2020). MMP-mediated modulation of ECM environment during axonal growth and NMJ development. Neurosci Lett 724: 134822. https://doi.org/10.1016/j.neulet.2020.134822 Chellini F, Tani A, Parigi M, Palmieri F, Garella R, Zecchi-Orlandini S, Squecco R, Sassoli C (2023). HIF-1α/MMP-9 Axis Is Required in the Early Phases of Skeletal Myoblast Differentiation under Normoxia Condition In Vitro. Cells 12(24): 2851. https://doi.org/10.3390/cells12242851 Christensen S, Purslow PP (2016). The role of matrix metalloproteinases in muscle and adipose tissue development and meat quality: A review. Meat Sci 119: 138–146. https://doi.org/10.1016/j.meatsci.2016.04.025 Couch CB, Strittmatter WJ (1983). Rat myoblast fusion requires metalloendoprotease activity. Cell https://doi.org/10.1016/0092-8674(83)90516-0 Couch CB, Strittmatter WJ (1984). Specific blockers of myoblast fusion inhibit a soluble and not the membrane-associated metalloendoprotease in myoblasts. J Biol Chem 259(9): 5396–5399. Costa ML, Jurberg AD, Mermelstein C (2021). The Role of Embryonic Chick Muscle Cell Culture in the Study of Skeletal Myogenesis. Front Physiol 12: 668600. https://doi.org/10.3389/fphys.2021.668600 Cui N, Hu M, Khalil RA (2017). Biochemical and Biological Attributes of Matrix Metalloproteinases. Prog Mol Biol Transl Sci. https://doi.org/10.1016/bs.pmbts.2017.02.005 Dali L, Gustin J, Perry K, Domingo CR (2002). Signals that instruct somite and myotome formation persist in Xenopus laevis early tailbud stage embryos. Cells Tissues Organs https://doi.org/10.1159/000064387 Dos Reis CA, de Miranda Soares MA, Gomes JR (2020). Expression of the matrix metalloproteinases 2 and 9 in the rat small intestine during intrauterine and postnatal life. Anat Rec (Hoboken) https://doi.org/10.1002/ar.24314 Duong TD, Erickson CA (2004). MMP-2 plays an essential role in producing epithelial-mesenchymal transformations in the avian embryo. Dev Dyn https://doi.org/10.1002/dvdy.10465 El Fahime E, Torrente Y, Caron NJ, Bresolin MD, Tremblay JP (2000). In vivo migration of transplanted myoblasts requires matrix metalloproteinase activity. Exp Cell Res https://doi.org/10.1006/excr.2000.4962 Fernández-Costa JM, Fernández-Garibay X, Velasco-Mallorquí F, Ramón-Azcón J (2021). Bioengineered in vitro skeletal muscles as new tools for muscular dystrophies preclinical studies. J Tissue Eng https://doi.org/10.1177/2041731420981339 Gaglianone RB, Bloise FF, Ortiga-Carvalho TM, Quirico-Santos T, Costa ML, Mermelstein C (2020). Comparative study of calcium and calcium-related enzymes with differentiation markers in different ages and muscle types in mdx mice. Histol Histopathol 35(2): 203–216. https://doi.org/10.14670/HH-18-145 Garcia P, Wang Y, Viallet J, Macek J, Jilkova Z (2021). The Chicken Embryo Model: A Novel and Relevant Model for Immune-Based Studies. Front Immunol https://doi.org/10.3389/fimmu.2021.791081 Gomes JR, Omar NF, dos Santos Neves J, Narvaes EA, Novaes PD (2011). Immunolocalization and activity of the MMP-9 and MMP-2 in odontogenic region of the rat incisor tooth after post shortening procedure. J Mol Histol https://doi.org/10.1007/s10735-011-9318-6 Guérin CW, Holland PC (1995). Synthesis and secretion of matrix-degrading metalloproteases by human skeletal muscle satellite cells. Dev Dyn https://doi.org/10.1002/aja.1002020109 Gus S, Huang Q, Sun C, Wen C, Yang N (2024). Transcriptomic and epigenomic insights into pectoral muscle fiber formation at the late embryonic development in pure chicken lines. Poultry Sci 103(8): 103882. https://doi.org/10.1016/j.psj.2024.103882 Hirst CE, Marcelle C (2015). The avian embryo as a model system for skeletal myogenesis. Results Probl Cell Differ. https://doi.org/10.1007/978-3-662-44608-9_5 Kumar L, Bisen M, Khan A, Kumar P, Patel SKS (2022). Role of Matrix Metalloproteinases in Musculoskeletal Diseases. Biomedicines https://doi.org/10.3390/biomedicines10102477 Lewis MP, Tippett HL, Sinanan AC, Morgan MJ, Hunt NP (2000). Gelatinase-B (matrix metalloproteinase-9; MMP-9) secretion is involved in the migratory phase of human and murine muscle cell cultures. J Muscle Res Cell Motil https://doi.org/10.1023/a:1005670507906 Lluri G, Jaworski DM (2005). Regulation of TIMP-2, MT1-MMP, and MMP-2 expression during C2C12 differentiation. Muscle Nerve https://doi.org/10.1002/mus.20383 Malila Y, Thanatsang KV, Sanpinit P, Arayamethakorn S, Soglia F, Zappaterra M, Bordini M, Sirri F, Rungrassamee W, Davoli R, Petracci M (2022). Differential expression patterns of genes associated with metabolisms, muscle growth and repair in Pectoralis major muscles of fast- and medium-growing chickens. PLOS ONE 17(10): e0275160. https://doi.org/10.1371/journal.pone.0275160 Maroto M, Bone RA, Dale JK (2012). Somitogenesis. Development (Cambridge, England) https://doi.org/10.1242/dev.069310 Mermelstein CS, Andrade LR, Portilho DM, Costa ML (2006). Desmin filaments are stably associated with the outer nuclear surface in chick myoblasts. Cell Tissue Res 323(2): 351–357. https://doi.org/10.1007/s00441-005-0063-6 Moracho N, Learte AI, Muñoz-Sáez E, Marchena MA, Cid MA, Arroyo AG, Sánchez-Camacho C (2022). Emerging roles of MT-MMPs in embryonic development. Dev Dyn 251(2): 240–275. https://doi.org/10.1002/dvdy.398 Nowak E, Gawor M, Ciemerych MA, Zimowska M (2018). Silencing of gelatinase expression delays myoblast differentiation in vitro. Cell Biol Int 42(3): 373–382. https://doi.org/10.1002/cbin.10914 Ohtake Y, Tojo H, Seiki M (2006). Multifunctional roles of MT1-MMP in myofiber formation and morphostatic maintenance of skeletal muscle. J Cell Sci https://doi.org/10.1242/jcs.03158 Orlowski SK, Dridi S, Greene ES, Coy CS, Velleman SG, Anthony NB (2021). Histological Analysis and Gene Expression of Satellite Cell Markers in the Pectoralis Major Muscle in Broiler Lines Divergently Selected for Percent 4-Day Breast Yield. Front Physiol 12: 712095. https://doi.org/10.3389/fphys.2021.712095 Paiva NH, Gomes JR, Loddi MM, Dos Reis CA, de Miranda Soares MA (2019). Spatial and temporal changes in cell proliferation in the chick jejunum during the folding of the ridges into zigzags. Acta Histochem https://doi.org/10.1016/j.acthis.2019.02.005 Possidonio AC, Soares CP, Portilho DM, Midlej V, Benchimol M, Butler-Browne G, Costa ML, Mermelstein C (2014). Differences in the expression and distribution of flotillin-2 in chick, mice and human muscle cells. PLOS ONE 9(8): e103990. https://doi.org/10.1371/journal.pone.0103990 Sabillo A, Ramirez J, Domingo CR (2016). Making muscle: Morphogenetic movements and molecular mechanisms of myogenesis in Xenopus laevis. Semin Cell Dev Biol https://doi.org/10.1016/j.semcdb.2016.02.006 Scaal M, Marcelle C (2018). Chick muscle development. Int J Dev Biol https://doi.org/10.1387/ijdb.170312cm Smith LR, Kok HJ, Zhang B, Chung D, Spradlin RA, Rakoczy KD, Lei H, Boesze-Battaglia K, Barton ER (2020). Matrix Metalloproteinase 13 from Satellite Cells is Required for Efficient Muscle Growth and Regeneration. Cell Physiol Biochem 54(3): 333–353. https://doi.org/10.33594/000000223 Stockdale FE (1992). Myogenic cell lineages. Dev Biol 154(2): 284–298. https://doi.org/10.1016/0012-1606(92)90068-r Sola B, Caillot M (2022). L ’ embryon de poule - Un modèle préclinique alternatif en cancérologie [The hen embryo: An alternative preclinical model in cancer]. Médecine et Sciences https://doi.org/10.1051/medsci/2022123 Strittmatter WJ (1984). Specific blockers of myoblast fusion inhibit a soluble and not the membrane-associated metalloendoprotease in myoblasts. J Biol Chem 259(9): 5396–5399. Wachholz GE, Rengel BD, Vargesson N, Fraga LR (2021). From the Farm to the Lab: How Chicken Embryos Contribute to the Field of Teratology. Front Genet https://doi.org/10.3389/fgene.2021.666726 Yaffe D, Saxel O (1977). Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle. Nature 270(5639): 725–727. https://doi.org/10.1038/270725a0 Fidler Y, Gomes JR (2023). Effects of a Single Dose of X-Ray Irradiation on MMP-9 Expression and Morphology of the Cerebellum Cortex of Adult Rats. Cerebellum 22(2): 240–248. https://doi.org/10.1007/s12311-022-01386-4 Zimowska M, Brzoska E, Swierczynska M, Streminska W, Moraczewski J (2008). Distinct patterns of MMP-9 and MMP-2 activity in slow and fast twitch skeletal muscle regeneration in vivo. Int J Dev Biol https://doi.org/10.1387/ijdb.072331mz Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 05 Aug, 2025 Read the published version in Histochemistry and Cell Biology → Version 1 posted Editorial decision: Revision requested 07 May, 2025 Reviews received at journal 23 Apr, 2025 Reviews received at journal 15 Apr, 2025 Reviewers agreed at journal 09 Apr, 2025 Reviewers agreed at journal 02 Apr, 2025 Reviewers agreed at journal 01 Apr, 2025 Reviewers invited by journal 01 Apr, 2025 Editor assigned by journal 01 Apr, 2025 Submission checks completed at journal 31 Mar, 2025 First submitted to journal 30 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6339860","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":444879002,"identity":"5af6f71c-2dc1-4ea6-8a16-caef40fdbbe2","order_by":0,"name":"Alisson Rodrigo Oliveira","email":"","orcid":"","institution":"University of Ponta Grossa","correspondingAuthor":false,"prefix":"","firstName":"Alisson","middleName":"Rodrigo","lastName":"Oliveira","suffix":""},{"id":444879003,"identity":"0c525fca-bf27-4fee-9304-a5efe0f4ed34","order_by":1,"name":"Maria Albertina Miranda Soares","email":"","orcid":"","institution":"University of Ponta Grossa","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"Albertina Miranda","lastName":"Soares","suffix":""},{"id":444879004,"identity":"557f155a-bbe0-4cb0-a044-f989388b5de5","order_by":2,"name":"Jose Rosa Gomes","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9klEQVRIiWNgGAWjYBACNvbm4z8kIGzGB0CCh4+QFj6eYwkSUC3MBiAtbIS0yEnkKEjAbAQzCGphk8hhMLDcUyun297+rPJrjp0MGwPzw0c38GnheXsgQeLZcWOzM2fMbstuSwY6jM3YOAefFva8hAMSB44lbruRw3ZbchszUAsPmzReLQw5hg1gLfefPyuW3FZPhBaOHGMGiQM1QFsYzBg/bjtMhBaeY2lALQeAfskxlmbcdpyHjZmAX+Tbm48xSxyokzM7fvzhx5/bqu352ZsfPsanBQSYJRgOQxg8YJKAchBg/MBQB2H8IEL1KBgFo2AUjDwAAER8R0OEIqvNAAAAAElFTkSuQmCC","orcid":"","institution":"University of Ponta Grossa","correspondingAuthor":true,"prefix":"","firstName":"Jose","middleName":"Rosa","lastName":"Gomes","suffix":""}],"badges":[],"createdAt":"2025-03-30 18:23:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6339860/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6339860/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00418-025-02405-1","type":"published","date":"2025-08-05T15:57:21+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81124328,"identity":"0d4b30c6-bc8d-4d3f-8838-3bc87a991e1a","added_by":"auto","created_at":"2025-04-22 13:36:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":7636684,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative Masson-stained images of cells of pectoral muscle samples of chicken at days 11 (a), 15 (b), and 19 (c) in the differentiation process. (a) The circle represents myocyteswith esferic nuclei, while the arrows represent cell membrane extensions. (b) The circle represents myocytesundergoing fusion in a syncytium configuration, while the dashed lines in (b1) indicate transversal striation. The brackets indicate myotubes in growth. (c) The arrows indicate red staining where are the nascent myofibrils, while in (c1) dashed lines indicate transversal striations, and arrows may indicate neural fiber extensions growing on muscle cells. Scale bars: 20μm\u003c/p\u003e","description":"","filename":"FIG1.png","url":"https://assets-eu.researchsquare.com/files/rs-6339860/v1/6e334dcc485a58c263669e75.png"},{"id":81124323,"identity":"9d3c8553-c2f8-4d70-85db-51fa064e3673","added_by":"auto","created_at":"2025-04-22 13:36:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4482937,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative immunohistochemistry for actin, separated by DAPI in blue on days 11, 15 and 19 (a, b, c), actin in green at days 11 (a1), 15 ( b1, b2), and 19 (c1), and merged at days 11 (a2), 15 (b3, b4), and 19 (c2) during muscle cell differentiation. Image a1 and a2 shows the distribution of actin in myocytes cytoplasms on day 11. Actin in myyotubes is shown in longitudinal in images b1, b3 (white arrows) and in cross sections in b2, b4 ( cycle and white arrow) on day 15 and in longitudinal sections in images c1,c2 (white arrows) on day 19, respectively. The cycle shows actin in myotubes organizing to generate future muscular fascicles. The arrows indicate the presence of actin in growing myofibrils. In c2, arrows indicate actin staining between gaps along with in myofibrils. Scale bars: 20μm\u003c/p\u003e","description":"","filename":"FIG2.png","url":"https://assets-eu.researchsquare.com/files/rs-6339860/v1/29dae7ac6268e74d167d42fe.png"},{"id":81124330,"identity":"6f5e10c3-4a60-41de-8a12-09117fe7e019","added_by":"auto","created_at":"2025-04-22 13:36:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3923649,"visible":true,"origin":"","legend":"\u003cp\u003eA) Representative immunohistochemical images of MMP-2 and MT1-MMP expression on the cell membrane expansion (arrows) on day 11 (a and d), and in myotubes and muscle cells on days 15 and 19 respectively (arrows) MMP-9 was not detected in any stage of differentiation evaluated (g, h, i) when compared to positive control samples in molecular layer (j) and white substance (k) in cerebellum and in axonal nervous fibers (l) of incisor sections. (B) shows optical density of immunostaining of MMP-2 and MT1-MMP was measured and compared using ANOVA with Tukey's post-hoc test, MMP-2 was significantly (p \u0026lt; 0.05) more expressed on day 15 when compared to MT1-MMP on day 11 and with MMP-2 on day 19 of development. Scale bars: 20μm\u003c/p\u003e","description":"","filename":"FIG3.png","url":"https://assets-eu.researchsquare.com/files/rs-6339860/v1/47cad5fdc86c4bbbdf8fd7a2.png"},{"id":81124325,"identity":"0f05cdda-f486-4627-b109-52c74cf61c89","added_by":"auto","created_at":"2025-04-22 13:36:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2260545,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative images of zymogram results. (A) shows the protease bands at a level of 60 kDa in the conditioned medium from chest muscle obtained on days 11, 15, and 19 of chicken development. (B) shows the inhibitory tests for samples, indicating that the bands observed in are composed principally of MMP-2, which was inhibited by PHE and EDTA but also by serinase as a result obtained by PMSF. (C) shows band area of gelatin degradation produced by MMP-2 was not significantly different among the times evaluated when tested using ANOVA with Tukey's post-hoc test.\u003c/p\u003e","description":"","filename":"FIG4.png","url":"https://assets-eu.researchsquare.com/files/rs-6339860/v1/e270fb8f335d1c5d7d268d8e.png"},{"id":88814238,"identity":"280a3aea-d0ba-457f-bbbd-b84a7124bd70","added_by":"auto","created_at":"2025-08-11 16:08:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":16738699,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6339860/v1/c0892066-7a8c-4060-bf94-86e84b47ede3.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exploring in vivo pectoral muscle differentiation in Ross chickens: sustained expression of MMP-2 and MT1-MMP throughout all stages and absence of MMP-9","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMost of our understanding of muscle development derives from experiments conducted on various vertebrate models, including fish, amphibians, mammals, and birds (Dali et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Yabe and Takada, 2016; Sabillo et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Garcia et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, due to its advantages in handling and developmental timeframe, the chicken embryo is a widely used model for studies investigating muscle development biology, meat production, human and animal pathologies, and bioengineering approaches (Hirst and Marcelle, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Possidonio et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Costa et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Wachholz et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Fern\u0026aacute;ndez-Costa et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Malila et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Sola and Caillot, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Wang et al., 2024).\u003c/p\u003e \u003cp\u003eMuscle development begins in the pre-embryo phase with the gastrulation process, during which several epiblast cells migrate to different regions, preceding tissue differentiation in vertebrates. During gastrulation, the inward movement of epiblast cells through the primitive streak results in the formation of the ectoderm, mesoderm, and endoderm (Acloque et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Notably, the mesoderm is the tissue responsible for the development of connective and muscle tissues. The mesoderm is divided into paraxial, intermediate, and lateral regions, which are situated alongside the notochord and the neural tube (Maroto et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn chickens, the paraxial mesoderm self-organizes, forming somites every 90 minutes in a rostrocaudal direction. Between days 1 and 5 of development, approximately 52 pairs of somites are formed. They consist of a ventral region (sclerotome), responsible for forming most of the skeletal bones, and a dorsal region (dermomyotome), which can differentiate into dermal tissue or skeletal muscles as well (Pourqui\u0026eacute;, 2004; 2018) in a process referred to as myogenesis. During the differentiation of dermomyotome cells, various signaling pathways play crucial roles, including Notch, Wnt, FGF, BMP, and Sonic Hedgehog (Chal and Pourqui\u0026eacute;, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Sieiro et al., 2016; Scall and Marcelle, 2018).\u003c/p\u003e \u003cp\u003eMyogenesis in chickens begins with progenitor cells located in the dermomyotome, marking the primary phase of myogenesis, which is identified between days 3 and 7 of development (the first phase of myogenesis). This phase begins with the expression of specific myogenic determinant genes such as Pax3\u0026thinsp;+\u0026thinsp;and Pax7+, and these cells are referred to as myocytes. The myocytes migrate from the dorsal region of the somite to the body wall, and through a fusion process, they lead to the formation of the first myotubes. These myotubes develop into primary myofibers that express Myogenin (Myog), MyoD, slow myosin heavy chain (MyHC), and myosin light chain 1 (Chal and Pourqui\u0026eacute;, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The next phase of myogenesis, denominated as the fetal phase, begins on day E8 and is characterized by the preferential expression of Pax7\u0026thinsp;+\u0026thinsp;in myocytes. Using primary fibers as a scaffold, these myocytes differentiate into secondary or fetal fibers, expressing specific markers such as β-enolase and Nfix. Muscle growth during this phase occurs primarily through the continuous fusion of progenitor myoblasts, which is distinct from adult myogenesis, involving the proliferation of satellite cells (also Pax7) that augment an already differentiated cell population (Chal and Pourqui\u0026eacute;, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Additionally, during development, cells may produce various extracellular components, including enzymes such as matrix metalloproteinases (MMPs). MMPs are zinc-dependent enzymes essential for remodeling extracellular components, playing vital roles in differentiation, proliferation, apoptosis, angiogenesis, tissue repair, and the maintenance of homeostasis (Cui et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Duong and Erickson, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). The widely studied MMPs are MMP-2, MMP-9, and MT1-MMP, and some of them have well-established roles in different experimental conditions (Gomes et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; dos Reis and Gomes, 2019; Christensen and Purslow, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Chan et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Moracho et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHowever, concerning to stages of chicken muscle differentiation, there is no reports about the expression of MMPs, however those that exist used precursor cells obtained from rats such as the C2C12 cell lineage which is a subclone from a myoblast line established from normal adult C3H mouse leg muscle (Blau et al, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1985\u003c/span\u003e). The original cell line, C2, was established in primary cultures from the thigh muscle of 2-month-old normal mice, 70 hours after crush injury cell lineage (Yaffe and Saxel, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1977\u003c/span\u003e). Although it is not a chicken myocyte lineage but cells derived from adult mammalian muscle, the expression of matrix metalloproteinases (MMPs) such as MMP-2, MMP-9 and MT1-MMP has been described during the in vitro differentiation of these cells into muscle cells (Couch and Strittmatter, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1983\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1984\u003c/span\u003e; Gu\u0026eacute;rin and Holland, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Lewis et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Chen and Li, 2009; Smith et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In contrast, in vivo approaches have investigated the processes of muscle morphology and the expression of MMPs in rodent muscles in the context of diseases or following muscle injuries (Fukushima et al., 2007; Zimowska et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Kumar et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Chen et al., 2023). Although the importance of this results already reported they were obtained in several experimental conditions that are different from those found during the embryonic stages in vivo, which need to be clarified yet.\u003c/p\u003e \u003cp\u003eThus, the aim of this study was to investigate in vivo the expression of MMP-2, MT1-MMP, and MMP-9 in the pectoral muscle of Ross lineage chickens during the secondary or fetal stages of development. These findings will contribute to a more comprehensive understanding of myogenesis and the roles of MMPs in muscle development during in vivo chicken embryo-fetal development stages.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1 Design of Experiments and Ethical Approval\u003c/strong\u003e: The calculation of sample size was based on a pilot experiment in which the height of 30 villi from the intestines of two chicken embryos, at 16 and 17 days, was measured (Paiva et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). The linear tool of the ImageJ software, which was downloaded from the public domain (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://imagej.nih.gov\u003c/span\u003e\u003c/span\u003e), was calibrated using a 20-micrometer bar that was present in the images of semi-serial sections. Following that, the measurements were loaded into the Bioestat 5.3 software, which can be downloaded from this link: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.mamiraua.org.br/downloads/programas/\u003c/span\u003e\u003c/span\u003e. Before the measurements were transformed to square roots, the Shapiro-Wilk test was performed to check the normality of the data. Subsequently, descriptive statistics were calculated and tested using the two-sample Z test to provide sample mean data and standard deviation. This data was then subjected to independent sample testing to determine the sample size for each time point studied. Based on the means and standard deviations of villus height, with a 1:1 relationship between samples, a test power of 80%, and an alpha level of 0.05 for reliability, a sample size of 3 embryos was obtained for each time evaluated (n = 9). All experiments in this work were authorized by the University Committee for Ethics in Animal Research, under protocol number 23.000028800-9.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Experimental Procedures\u003c/strong\u003e: Granja Econômica Avícola LTDA (Carambeí-PR, Brazil) kindly provided nine fertilized eggs (ROSS lineage). They were incubated at 37.8°C and 60% relative humidity until E11, E15, and E19 days. To prevent contamination, each embryo was decapitated, and its chest was removed while still in a sterilized state and placed into the laminar flow at each evaluation point. According to subsequent analyses, detailed below, the chest was divided into three pieces and submerged in each of three solutions. \u003cspan\u003e\u003cstrong\u003e2.3 Morphological Analysis\u003c/strong\u003e: The chest fragments from each embryo were immersed for 48 hours in a 2% paraformaldehyde/0.1 M phosphate buffer at pH 7.2. They were then dehydrated in alcohol and embedded in paraplast to create three serial sections of 7 µm. Sections were dewaxed and rehydrated before staining with Masson's trichromic solution. Regions of samples exhibiting longitudinal or cross sections areas were selected for posterior analysis according to methods used.\u003cbr\u003e\u003c/span\u003e\u003cspan\u003e\u003cstrong\u003e2.4 Immunohistochemistry for MMP-2, MMP-9, MT1-MMP, and actin\u003c/strong\u003e: For each embryo/time evaluated, slides were prepared with three serial sections of 7 µm. After dewaxing and rehydrating procedures, the sections were subjected to antigen retrieval using a citrate buffer solution at pH 6.0 for 20 minutes at 98°C. Afterward, they were quenched three times with 2% hydrogen peroxide for 10 minutes each to inhibit endogenous peroxidase activity. Slides were washed in water and PBS (pH 7.4) and incubated in PBS containing 3% BSA to block non-specific antigen binding for 1 hour. After washing in PBS again, they were covered with monoclonal primary antibodies (2 µg/ml, purchased from Chemicon, USA) as follows: MMP-9 (1:200), MMP-2 (1:200), and MT1-MMP (1:200) in PBS containing 1% BSA, and incubated overnight. Additionally, sections of cerebellum and incisor tooth, in which the presence of MMP-9 had been previously demonstrated (Gomes et al.,\u0026nbsp;\u003cspan class=\"CitationRef\"\u003e2011\u003c/span\u003e; Yasmin and Gomes, 2023), were used as positive controls for MMP-9.\u003cbr\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eSections were then washed in PBS and incubated with secondary antibodies using the DAKO (Universal LSAB kit) for 30 minutes at 37°C in each solution. After washing in PBS, sections were incubated with the DAB reagent (SIGMA) prepared in PBS in the presence of hydrogen peroxide. Negative control staining was performed for each molecule by either omitting the primary antiserum or substituting the primary antiserum with non-immune serum.\u003c/p\u003e\n\u003cp\u003eActin detection was performed by immunofluorescence as follows: After passing through a 3% BSA solution for 1 hour, the sections were incubated with a primary monoclonal beta-actin antibody (1:500) (ThermoFisher Scientific, USA, MA5-15739) in PBS containing 1% BSA and 0.1% Triton X-100 overnight. After washing in PBS (pH 7.4), they were incubated with a secondary antibody, Alexa Fluor 488 (1:1000) (Life Technologies, Brazil, Lot No. 1832425), in PBS containing 1% BSA for 1 hour, then washed again at room temperature. After this and PBS washing, the sections were incubated with DAPI (2 µg/mL, Sigma-Aldrich, USA) for 30 minutes, washed, and covered with Fluorescence Mounting Medium (DAKO, USA) under coverslips. The Masson's trichromic staining images were taken using an Olympus DP72 microscope and epifluorescence microscopy (Leica, China) for photos of immunofluorescence sections.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Procedures for the Zymography Assay\u003c/strong\u003e: Small pieces of chest muscle were taken under sterile conditions for each evaluation, and then washed in PBS, cut into smaller pieces with a surgical blade, and distributed into three wells of a 96-well plate. The plate was then washed once with Dulbecco's Modified Eagle Medium (purchased from SIGMA), which contained 100 µg/ml of the antibiotic gentamicin, without the addition of fetal bovine serum. The fragments were incubated for 24 hours at 37°C to produce MMP-conditioned media (Gomes et al., \u003cspan class=\"CitationRef\"\u003e2011\u003c/span\u003e). Following that, the material was collected into a microtube and centrifuged at 2000 rpm for one minute.\u003c/p\u003e\n\u003cp\u003eFor the zymography and protease detection tests, 0.5 µg of each sample recovered from the conditioned medium was resuspended, briefly sonicated in Laemmli buffer under non-reducing conditions, and then subjected to SDS-PAGE in a 10% polyacrylamide gel containing 5% gelatin. A positive sample for MMP-9 previously identified (Dos Reis et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e) and a sample of medium free of fragments were used as negative controls for the zymography test. Following electrophoresis, the gels were shaken and rinsed twice for thirty minutes in 2.5% Triton X-100. They were then submerged in 1 M CaCl₂ and 1 M Tris-HCl incubation buffer for sixteen hours at 37°C to activate the proteases in the samples. Gels were then stained with Coomassie Blue until the degraded bands were visible, and the molecular weight of the bands was compared to a molecular weight standard obtained from Fermentas Life Science (Burlington, Ontario, Canada). To characterize the protease family, various inhibitors were added to the incubation buffer of other gels. These inhibitors included 0.5 mM N-Ethylmaleimide (NEM) (cysteine inhibitor), 1,10-phenanthroline (PHE), and EDTA (MMP inhibitors); 2 mM phenylmethylsulfonyl fluoride (PMSF) (serine inhibitor); 2 mM benzamidine (trypsin serine inhibitor); and 0.5 mM N-Ethylmaleimide (NEM) (cysteine inhibitor) at 37°C for 16 hours. Gels were then stained with Coomassie Blue until the degraded bands were visible.\u003c/p\u003e\n\n\n\n\n\n\n\n\n\n"},{"header":"Results","content":"\u003cp\u003e\u003cspan type=\"BoldSmallCaps\" class=\"BoldSmallCaps\" name=\"Emphasis\"\u003e3.1 The stage of myocyte fusion begins on day 11, while the primary and secondary stages of myotube formation occur on days 15 and 19 during the development of the chicken pectoral muscle.\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eThe morphological phases of muscle cell differentiation are shown in detail in\u003c/span\u003e Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eInitially, on day E11 of development, myocytes with spherical nuclei are observed in a clustered organization (oval circle) with thin cell membrane extensions (arrows) touching the membranes of adjacent myocytes. In\u003c/span\u003e Figs. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eand 1b1, the fusion process among the myocytes is clearly visible on day E15, producing a syncytium structure (indicated by the oval circle) to generate primary myotubes of varying thicknesses, as shown in\u003c/span\u003e Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eb1. The nuclei of the myotubes assume an elliptical morphology (arrow) with active fusion of myoblasts, while other myocytes are outside the myotubes (tip of arrow). Some transverse striations are visible (dashed lines). This day, E15, is considered the beginning of the secondary phase of pectoral muscle cell differentiation.\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eAs seen in\u003c/span\u003e Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eby day E19 of development, the myotubes are already very thick, with elongated regions in the cytoplasm showing stronger red staining that we identified as nascent myofibrils (arrows). Figure\u0026nbsp;1c1 shows transverse striations (dashed lines) running parallel to the cytoplasm of muscle cells, as well as thin blue-stained structures emerging from surrounding tissue and branching out over the muscle cells (arrows), which could be nerve fiber ramifications.\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003e3.2 The distribution of actin in the cytoplasm changes depending on the differentiation stages of muscle cells.\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e shows the immunostaining for actin during the phases of muscle cell differentiation. On day E11, actin is distributed throughout the myoblast cytoplasm during their fusion process (Fig. 2a1 and a2). However, on day E15, although actin can be detected in the cytoplasm of the myotubes, it appears to begin concentrating in specific regions within the cytoplasm of the myotubes, as observed in the images (Fig. 2b1,b3) in a longitudinal section (indicated by white arrows), as well as in the transversal sections (Fig. 2b2,b4), where the strong actin staining appears in the shape of a ring delimiting a core region with less staining (b4 detailed). This core can be interpreted as a long, tubular-shaped structure in a cross-section. On day E19, actin staining is detected in the same longitudinal stick-shaped sections along the cytoplasm of muscle cells, as demonstrated in Fig. 2c1, indicated by white arrows. It is also clear that the actin staining is not continuous in this structure, which is interrupted by small dark gaps between regions of actin staining, as represented by the white arrows in Fig. 2c2, suggesting its association with the myofibril organization.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003e3.3 MMP-2 and MT1-MMP were expressed at all stages of muscle cell differentiation; however, MMP-9 was not expressed.\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA shows immunostaining for MMP-2 and MT1-MMP on days E11, E15, and E19 in myocytes (a and d), myotubes (b and e), and muscle cells (c and f), respectively. However, MMP-9 was not detected at any time evaluated, as shown in images g, h, and i, when compared to positive controls in sections of the cerebellum (J) and tooth (k and l), respectively. When the optical density of immunostaining of MMP-2 and MT1-MMP was measured and compared using ANOVA with Tukey's post-hoc test, MMP-2 was significantly (p \u0026lt; 0.05) more expressed on day 15 when compared to MT1-MMP on day 11 and with MMP-2 on day 19 of development, as demonstrated in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB.\u003c/p\u003e\u003cp\u003eThe zymogram obtained from the conditioned medium of muscle cell fragments from the pectoral muscle samples is shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA, where gelatin band degradations with molecular weights below 72 kDa were detected on all days (11, 15, and 19), with the absence of gelatin band degradation at the molecular weight for MMP-9 when compared to that obtained from the control samples. The bands corresponding to pre-, pro-, and active forms of MMP-2 were confirmed after the inhibitory test, the results of which are shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB. It was found that EDTA and PMSF completely inhibited the activation of proteases in the conditioned medium. On the other hand, PHE completely inhibited the activity on day 15 and partially on days 11 and 19. Benzamidine and NEM, however, were unable to inhibit the enzymes. These results confirm the expression of MMP-2, but also the presence of serine proteases by the muscle cells during their differentiation process. The band area of gelatin degradation produced by MMP-2 was not significantly different among the times evaluated when tested using ANOVA with Tukey's post-hoc test, as demonstrated in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn vivo, the investigation into muscle differentiation is crucial not only for understanding myopathies and muscle regeneration but also for improving meat production in the poultry industry (Gaglianone et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e; Wachholz et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e; Orlowski et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e; Gus et al., \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e). The literature reports that the morphology of myogenesis in chicken development is divided into an embryonic period that spans from days E4 to E7, during which myocytes from the myotome migrate to different body parts and fuse to form primary myotubes. The next stage, termed fetal, begins on day E8, characterized by the formation of a secondary wave of myotubes, where new myocytes fuse to the previously formed myotubes. During this stage, cells actively produce various isoforms of muscle proteins organized into myofibrils (Stockdale, \u003cspan class=\"CitationRef\"\u003e1992\u003c/span\u003e., 2001; Chal and Pourquié, \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOn the other hand, findings related to morphological events of muscle differentiation have been derived from in vitro studies using muscle cell precursors from adult muscle lesioned or from the C2C12 cell lineage, which was isolated from the muscle of adult rodents. The C2C12 cell lineage may not be considered an embryonic cell but rather a lineage that already has determination and the potential to differentiate into several types of cells, such as adipocytes, osteocytes, fibroblasts, and also muscle cells. Thus, the differentiation pathway of C2C12 cells depends on specific components added to the culture medium to trigger the differentiation process (Strittmatter, \u003cspan class=\"CitationRef\"\u003e1984\u003c/span\u003e; Fahime et al., \u003cspan class=\"CitationRef\"\u003e2000\u003c/span\u003e; C). Concurrently, some studies have demonstrated that several MMPs are expressed during muscle differentiation in response to the components of the culture medium (Lluri and Jaworski, \u003cspan class=\"CitationRef\"\u003e2005\u003c/span\u003e; Ohtake et al., \u003cspan class=\"CitationRef\"\u003e2006\u003c/span\u003e; Cha and Purslow, \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHowever, in vitro studies do not replicate the real conditions present in the embryo during its development. Due to the limited information on the relationship between the morphological phases of muscle differentiation and the expression of MMPs related to that, we present here the in vivo results regarding the morphology and expression of MMPs in the differentiation of chicken pectoral muscle during days E11, E15, and E19 of development. These time points were chosen based on previous reports that used primary culture to study different aspects of muscle differentiation from development day E11 (Mermelstein et al., \u003cspan class=\"CitationRef\"\u003e2006\u003c/span\u003e; Bagri et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOur results, while aligning in some aspects with previous reports related to the cellular morphological phases of muscle differentiation, such as fusion of myocytes to form myotubes, differ in other respects when compared to in vitro cell lineages or in vivo studies using chicken. The first difference found relates to the timing of myocyte fusion and the formation of primary myotubes in our study when compared to described previously. While the literature describes that primary myotubes occur around day E7, termed the embryonic phase (Stockdale, \u003cspan class=\"CitationRef\"\u003e1992\u003c/span\u003e; Chal et al., 2016), in our in vivo study using the eggs of Ross chicken lineage, we observed that the onset of myocyte fusion, forming the primary wave of myotube differentiation, occurs on day E11, which, according to the same reports, corresponds to the fetal phase where the second wave of myotubes should be in progress. However, in our study, the second wave was observed only on day E15, while on day E19, the myotubes were in an advanced stage of differentiation. Thus, the timing observed within embryonic and fetal periods differs from what has been reported previously (Stockdale, \u003cspan class=\"CitationRef\"\u003e1992\u003c/span\u003e; Chal et al., 2016).\u003c/p\u003e\u003cp\u003eTaken together, our results on days E11, E15, and E19 are more similar to the myogenesis phases described in mice, as previously reported (Biressi et al., \u003cspan class=\"CitationRef\"\u003e2007\u003c/span\u003e), than when compared to those reported in chickens (Stockdale, \u003cspan class=\"CitationRef\"\u003e1992\u003c/span\u003e; Chal et al., 2016). This indicates that in the pectoral muscle of Ross chickens, muscle cell differentiation may be more advanced than previously recognized, suggesting that variations in the timing of differentiation processes among different muscles or even between species should not be dismissed.\u003c/p\u003e\u003cp\u003eAnother relevant aspect discussed in this study is the expression of matrix metalloproteinases (MMPs). It has been reported that MMP-9 activity is detectable at all stages of myoblast differentiation isolated from rats, whereas MMP-2 activity reaches its highest level during myoblast fusion (Zimowska et al., \u003cspan class=\"CitationRef\"\u003e2008\u003c/span\u003e). In satellite cells isolated from adult mouse muscle, maintained in vitro with silenced expression of MMP-9, some disruption in their differentiation was observed (Nowak et al., 2028). However, during muscle regeneration in adult animals, it was demonstrated that the expression of MMP-2 and MMP-9 depends on the type of muscle evaluated (slow or fast) as well as the phase of regeneration. Additionally, when C2C12 cells are under normoxic conditions, MMP-9 is required in the earlier phases of myoblast differentiation (Chellini et al., \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, other studies show that the expression of MMP-9 in C2C12 cells may respond differently to substrate coating and cyclic mechanical stretching (Cha and Purslow, \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e). Therefore, the results for MMP-9 expression obtained from studies using muscle cell precursors isolated from adult rats, such as satellite cells or for the C2C12 lineage, are still controversial and dependent on several differing experimental conditions, thus opposing our findings in the real developmental context of chickens, where MMP-9 was not detected by immunohistochemistry or zymography. This absence raises questions about the plasticity and regulatory mechanisms present during in vivo muscle development that may not exist when cells are studied under in vitro conditions.\u003c/p\u003e\u003cp\u003eNonetheless, our in vivo results confirmed the continuous expression of MMP-2 and MT1-MMP from day E11 up to E19 of the chicken Roos lineage, aligning with previous reports in vitro from precursor cells obtained from adult muscle of mice (Nowak et al., 2028). Although they do not represent the real conditions of muscle differentiation as demonstrated in our in vivo study, the results indicate that MMP-2 and MT1-MMP are important during the differentiation of muscle in vivo as well.\u003c/p\u003e\u003cp\u003eFinally, the chicken model proves promising for elucidating the underlying mechanisms of muscle differentiation and MMPs for further investigations that not only expand our understanding of myogenesis but also explore the practical application of this knowledge in improving production conditions to understand muscle health in poultry (Christensen and Purslow, \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e) and myopathies (Kumar et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eIn conclusion, during in vivo pectoral muscle development in Ross chickens, MMP-9 is not expressed at any stage of muscle differentiation, underscoring its non-essential role, whereas the expression of MMP-2 and MT1-MMP is crucial, with primary myotubes emerging on day E11 and secondary muscle cells appearing on days E15 and E19, reflecting a distinct timeline for muscle differentiation in this species.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eC\u003c/strong\u003e\u003cstrong\u003er\u003c/strong\u003e\u003cstrong\u003eediT\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003cstrong\u003euthorship contribution statement\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eAlisson Rodrigo de Oliveira: experimental methodology, investigation and formal analysis execution. Maria Albertina de Miranda Soares: formal analysis, data curation and original write revision. Jose Rosa Gomes: conceptualization, final writing and edition, formal analysis, data curation, conceptualization, general supervision.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003eData will be made available on request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eThe authors would like to acknowledge for Coordination for the improvement of higher education personnel (CAPES) by the student scholarship provided and to Granja Economica Avicola LTDA (Carambe\u0026iacute;-PR-Brazil) that kindly provided the fertilized eggs (ROSS lineage).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAcloque H, Oca\u0026ntilde;a OH, Matheu A, Rizzoti K, Wise C, Lovell-Badge R, Nieto MA (2011). Reciprocal repression between Sox3 and snail transcription factors defines embryonic territories at gastrulation. Dev Cell 21(3): 546\u0026ndash;558. https://doi.org/10.1016/j.devcel.2011.07.005\u003c/li\u003e\n\u003cli\u003eBagri KM, Oliveira LF, Pereira MG, Abreu JG, Mermelstein C (2022). The Wnt/Beta-Catenin Pathway and Cytoskeletal Filaments Are Involved in the Positioning, Size, and Function of Lysosomes during Chick Myogenesis. Cells 11(21): 3402. https://doi.org/10.3390/cells11213402\u003c/li\u003e\n\u003cli\u003eBlau HM, Pavlath GK, Hardeman EC, Chiu CP, Silberstein L, Webster SG, Miller SC, Webster C (1985). Plasticity of the differentiated state. Science 230(4727): 758\u0026ndash;766. https://doi.org/10.1126/science.2414846\u003c/li\u003e\n\u003cli\u003eCha MC, Purslow PP (2010). The activities of MMP-9 and total gelatinase respond differently to substrate coating and cyclic mechanical stretching in fibroblasts and myoblasts. Cell Biol Int 34(6): 587\u0026ndash;591. https://doi.org/10.1042/CBI20090096\u003c/li\u003e\n\u003cli\u003eChal J, Pourqui\u0026eacute; O (2017). Making muscle: skeletal myogenesis in vivo and in vitro. Development. https://doi.org/10.1242/dev.151035\u003c/li\u003e\n\u003cli\u003eBiressi S, Molinaro M, Cossu G (2007). Cellular heterogeneity during vertebrate skeletal muscle development. Dev Biol 308(2): 281\u0026ndash;293. https://doi.org/10.1016/j.ydbio.2007.06.00\u003c/li\u003e\n\u003cli\u003eChan ZC, Oentaryo MJ, Lee CW (2020). MMP-mediated modulation of ECM environment during axonal growth and NMJ development. Neurosci Lett 724: 134822. https://doi.org/10.1016/j.neulet.2020.134822\u003c/li\u003e\n\u003cli\u003eChellini F, Tani A, Parigi M, Palmieri F, Garella R, Zecchi-Orlandini S, Squecco R, Sassoli C (2023). HIF-1\u0026alpha;/MMP-9 Axis Is Required in the Early Phases of Skeletal Myoblast Differentiation under Normoxia Condition In Vitro. Cells 12(24): 2851. https://doi.org/10.3390/cells12242851\u003c/li\u003e\n\u003cli\u003eChristensen S, Purslow PP (2016). The role of matrix metalloproteinases in muscle and adipose tissue development and meat quality: A review. Meat Sci 119: 138\u0026ndash;146. https://doi.org/10.1016/j.meatsci.2016.04.025\u003c/li\u003e\n\u003cli\u003eCouch CB, Strittmatter WJ (1983). Rat myoblast fusion requires metalloendoprotease activity. Cell https://doi.org/10.1016/0092-8674(83)90516-0\u003c/li\u003e\n\u003cli\u003eCouch CB, Strittmatter WJ (1984). Specific blockers of myoblast fusion inhibit a soluble and not the membrane-associated metalloendoprotease in myoblasts. J Biol Chem 259(9): 5396\u0026ndash;5399.\u003c/li\u003e\n\u003cli\u003eCosta ML, Jurberg AD, Mermelstein C (2021). The Role of Embryonic Chick Muscle Cell Culture in the Study of Skeletal Myogenesis. Front Physiol 12: 668600. https://doi.org/10.3389/fphys.2021.668600\u003c/li\u003e\n\u003cli\u003eCui N, Hu M, Khalil RA (2017). Biochemical and Biological Attributes of Matrix Metalloproteinases. Prog Mol Biol Transl Sci. https://doi.org/10.1016/bs.pmbts.2017.02.005\u003c/li\u003e\n\u003cli\u003eDali L, Gustin J, Perry K, Domingo CR (2002). Signals that instruct somite and myotome formation persist in Xenopus laevis early tailbud stage embryos. Cells Tissues Organs https://doi.org/10.1159/000064387\u003c/li\u003e\n\u003cli\u003eDos Reis CA, de Miranda Soares MA, Gomes JR (2020). Expression of the matrix metalloproteinases 2 and 9 in the rat small intestine during intrauterine and postnatal life. Anat Rec (Hoboken) https://doi.org/10.1002/ar.24314\u003c/li\u003e\n\u003cli\u003eDuong TD, Erickson CA (2004). MMP-2 plays an essential role in producing epithelial-mesenchymal transformations in the avian embryo. Dev Dyn https://doi.org/10.1002/dvdy.10465\u003c/li\u003e\n\u003cli\u003eEl Fahime E, Torrente Y, Caron NJ, Bresolin MD, Tremblay JP (2000). In vivo migration of transplanted myoblasts requires matrix metalloproteinase activity. Exp Cell Res https://doi.org/10.1006/excr.2000.4962\u003c/li\u003e\n\u003cli\u003eFern\u0026aacute;ndez-Costa JM, Fern\u0026aacute;ndez-Garibay X, Velasco-Mallorqu\u0026iacute; F, Ram\u0026oacute;n-Azc\u0026oacute;n J (2021). Bioengineered in vitro skeletal muscles as new tools for muscular dystrophies preclinical studies. J Tissue Eng https://doi.org/10.1177/2041731420981339\u003c/li\u003e\n\u003cli\u003eGaglianone RB, Bloise FF, Ortiga-Carvalho TM, Quirico-Santos T, Costa ML, Mermelstein C (2020). Comparative study of calcium and calcium-related enzymes with differentiation markers in different ages and muscle types in mdx mice. Histol Histopathol 35(2): 203\u0026ndash;216. https://doi.org/10.14670/HH-18-145\u003c/li\u003e\n\u003cli\u003eGarcia P, Wang Y, Viallet J, Macek J, Jilkova Z (2021). The Chicken Embryo Model: A Novel and Relevant Model for Immune-Based Studies. Front Immunol https://doi.org/10.3389/fimmu.2021.791081\u003c/li\u003e\n\u003cli\u003eGomes JR, Omar NF, dos Santos Neves J, Narvaes EA, Novaes PD (2011). Immunolocalization and activity of the MMP-9 and MMP-2 in odontogenic region of the rat incisor tooth after post shortening procedure. J Mol Histol https://doi.org/10.1007/s10735-011-9318-6\u003c/li\u003e\n\u003cli\u003eGu\u0026eacute;rin CW, Holland PC (1995). Synthesis and secretion of matrix-degrading metalloproteases by human skeletal muscle satellite cells. Dev Dyn https://doi.org/10.1002/aja.1002020109\u003c/li\u003e\n\u003cli\u003eGus S, Huang Q, Sun C, Wen C, Yang N (2024). Transcriptomic and epigenomic insights into pectoral muscle fiber formation at the late embryonic development in pure chicken lines. Poultry Sci 103(8): 103882. https://doi.org/10.1016/j.psj.2024.103882\u003c/li\u003e\n\u003cli\u003eHirst CE, Marcelle C (2015). The avian embryo as a model system for skeletal myogenesis. Results Probl Cell Differ. https://doi.org/10.1007/978-3-662-44608-9_5\u003c/li\u003e\n\u003cli\u003eKumar L, Bisen M, Khan A, Kumar P, Patel SKS (2022). Role of Matrix Metalloproteinases in Musculoskeletal Diseases. Biomedicines https://doi.org/10.3390/biomedicines10102477\u003c/li\u003e\n\u003cli\u003eLewis MP, Tippett HL, Sinanan AC, Morgan MJ, Hunt NP (2000). Gelatinase-B (matrix metalloproteinase-9; MMP-9) secretion is involved in the migratory phase of human and murine muscle cell cultures. J Muscle Res Cell Motil https://doi.org/10.1023/a:1005670507906\u003c/li\u003e\n\u003cli\u003eLluri G, Jaworski DM (2005). Regulation of TIMP-2, MT1-MMP, and MMP-2 expression during C2C12 differentiation. Muscle Nerve https://doi.org/10.1002/mus.20383\u003c/li\u003e\n\u003cli\u003eMalila Y, Thanatsang KV, Sanpinit P, Arayamethakorn S, Soglia F, Zappaterra M, Bordini M, Sirri F, Rungrassamee W, Davoli R, Petracci M (2022). Differential expression patterns of genes associated with metabolisms, muscle growth and repair in Pectoralis major muscles of fast- and medium-growing chickens. PLOS ONE 17(10): e0275160. https://doi.org/10.1371/journal.pone.0275160\u003c/li\u003e\n\u003cli\u003eMaroto M, Bone RA, Dale JK (2012). Somitogenesis. Development (Cambridge, England) https://doi.org/10.1242/dev.069310\u003c/li\u003e\n\u003cli\u003eMermelstein CS, Andrade LR, Portilho DM, Costa ML (2006). Desmin filaments are stably associated with the outer nuclear surface in chick myoblasts. Cell Tissue Res 323(2): 351\u0026ndash;357. https://doi.org/10.1007/s00441-005-0063-6\u003c/li\u003e\n\u003cli\u003eMoracho N, Learte AI, Mu\u0026ntilde;oz-S\u0026aacute;ez E, Marchena MA, Cid MA, Arroyo AG, S\u0026aacute;nchez-Camacho C (2022). Emerging roles of MT-MMPs in embryonic development. Dev Dyn 251(2): 240\u0026ndash;275. https://doi.org/10.1002/dvdy.398\u003c/li\u003e\n\u003cli\u003eNowak E, Gawor M, Ciemerych MA, Zimowska M (2018). Silencing of gelatinase expression delays myoblast differentiation in vitro. Cell Biol Int 42(3): 373\u0026ndash;382. https://doi.org/10.1002/cbin.10914\u003c/li\u003e\n\u003cli\u003eOhtake Y, Tojo H, Seiki M (2006). Multifunctional roles of MT1-MMP in myofiber formation and morphostatic maintenance of skeletal muscle. J Cell Sci https://doi.org/10.1242/jcs.03158\u003c/li\u003e\n\u003cli\u003eOrlowski SK, Dridi S, Greene ES, Coy CS, Velleman SG, Anthony NB (2021). Histological Analysis and Gene Expression of Satellite Cell Markers in the Pectoralis Major Muscle in Broiler Lines Divergently Selected for Percent 4-Day Breast Yield. Front Physiol 12: 712095. https://doi.org/10.3389/fphys.2021.712095\u003c/li\u003e\n\u003cli\u003ePaiva NH, Gomes JR, Loddi MM, Dos Reis CA, de Miranda Soares MA (2019). Spatial and temporal changes in cell proliferation in the chick jejunum during the folding of the ridges into zigzags. Acta Histochem https://doi.org/10.1016/j.acthis.2019.02.005\u003c/li\u003e\n\u003cli\u003ePossidonio AC, Soares CP, Portilho DM, Midlej V, Benchimol M, Butler-Browne G, Costa ML, Mermelstein C (2014). Differences in the expression and distribution of flotillin-2 in chick, mice and human muscle cells. PLOS ONE 9(8): e103990. https://doi.org/10.1371/journal.pone.0103990\u003c/li\u003e\n\u003cli\u003eSabillo A, Ramirez J, Domingo CR (2016). Making muscle: Morphogenetic movements and molecular mechanisms of myogenesis in Xenopus laevis. Semin Cell Dev Biol https://doi.org/10.1016/j.semcdb.2016.02.006\u003c/li\u003e\n\u003cli\u003eScaal M, Marcelle C (2018). Chick muscle development. Int J Dev Biol https://doi.org/10.1387/ijdb.170312cm\u003c/li\u003e\n\u003cli\u003eSmith LR, Kok HJ, Zhang B, Chung D, Spradlin RA, Rakoczy KD, Lei H, Boesze-Battaglia K, Barton ER (2020). Matrix Metalloproteinase 13 from Satellite Cells is Required for Efficient Muscle Growth and Regeneration. Cell Physiol Biochem 54(3): 333\u0026ndash;353. https://doi.org/10.33594/000000223\u003c/li\u003e\n\u003cli\u003eStockdale FE (1992). Myogenic cell lineages. Dev Biol 154(2): 284\u0026ndash;298. https://doi.org/10.1016/0012-1606(92)90068-r\u003c/li\u003e\n\u003cli\u003eSola B, Caillot M (2022). L\u003cspan dir=\"RTL\"\u003e\u0026rsquo;\u003c/span\u003eembryon de poule - Un mod\u0026egrave;le pr\u0026eacute;clinique alternatif en canc\u0026eacute;rologie [The hen embryo: An alternative preclinical model in cancer]. M\u0026eacute;decine et Sciences https://doi.org/10.1051/medsci/2022123\u003c/li\u003e\n\u003cli\u003eStrittmatter WJ (1984). Specific blockers of myoblast fusion inhibit a soluble and not the membrane-associated metalloendoprotease in myoblasts. J Biol Chem 259(9): 5396\u0026ndash;5399.\u003c/li\u003e\n\u003cli\u003eWachholz GE, Rengel BD, Vargesson N, Fraga LR (2021). From the Farm to the Lab: How Chicken Embryos Contribute to the Field of Teratology. Front Genet https://doi.org/10.3389/fgene.2021.666726\u003c/li\u003e\n\u003cli\u003eYaffe D, Saxel O (1977). Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle. Nature 270(5639): 725\u0026ndash;727. https://doi.org/10.1038/270725a0\u003c/li\u003e\n\u003cli\u003eFidler Y, Gomes JR (2023). Effects of a Single Dose of X-Ray Irradiation on MMP-9 Expression and Morphology of the Cerebellum Cortex of Adult Rats. Cerebellum 22(2): 240\u0026ndash;248. https://doi.org/10.1007/s12311-022-01386-4\u003c/li\u003e\n\u003cli\u003eZimowska M, Brzoska E, Swierczynska M, Streminska W, Moraczewski J (2008). Distinct patterns of MMP-9 and MMP-2 activity in slow and fast twitch skeletal muscle regeneration in vivo. Int J Dev Biol https://doi.org/10.1387/ijdb.072331mz\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"histochemistry-and-cell-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"hacb","sideBox":"Learn more about [Histochemistry and Cell Biology](http://link.springer.com/journal/418)","snPcode":"418","submissionUrl":"https://submission.nature.com/new-submission/418/3","title":"Histochemistry and Cell Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"chicken, muscle cell, differentiation, MMPs","lastPublishedDoi":"10.21203/rs.3.rs-6339860/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6339860/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThere are pertinent studies on the relationship between MMPs and muscle cell differentiation in vitro, but there are currently few studies in vivo exploring MMP expressions in muscle chicken during different stages of differentiation. Therefore, we aimed to investigate in vivo, whether MMP-2, MT1-MMP, and MMP-9 are expressed in pectoral muscle during the stages of chicken development on days E11, E15, and E19. Our results demonstrated that, in contrast to earlier reports in vitro, that primary myotubes are formed on E11 while on E15 and E19 is occurring the secondary muscle waves. MMP-2, on the other hand, appears to be more expressed than MT1-MMP throughout the differentiation process, whereas MMP-9 is not expressed at any point. Additionally, serine was discovered as an unexpected finding in the zymogram analysis. We conclude that during in vivo pectoral muscle development in Ross chickens, MMP-9 is not expressed at any stage of muscle differentiation, underscoring its non-essential role, whereas the expression of MMP-2 and MT1-MMP is crucial, with primary myotubes emerging on day E11 and secondary muscle cells appearing on days E15 and E19, reflecting a distinct timeline for this muscle differentiation in this chicken lineage.\u003c/p\u003e","manuscriptTitle":"Exploring in vivo pectoral muscle differentiation in Ross chickens: sustained expression of MMP-2 and MT1-MMP throughout all stages and absence of MMP-9","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-22 13:36:31","doi":"10.21203/rs.3.rs-6339860/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-07T17:10:17+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-23T14:01:30+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-15T15:38:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"144817257377561152194073007094253290725","date":"2025-04-09T08:09:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"268214363822453562051935625374694302068","date":"2025-04-02T07:55:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"301740504939280188961336014237514498652","date":"2025-04-01T20:53:47+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-01T15:52:27+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-01T14:20:11+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-31T09:14:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"Histochemistry and Cell Biology","date":"2025-03-30T18:15:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"histochemistry-and-cell-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"hacb","sideBox":"Learn more about [Histochemistry and Cell Biology](http://link.springer.com/journal/418)","snPcode":"418","submissionUrl":"https://submission.nature.com/new-submission/418/3","title":"Histochemistry and Cell Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f506c0a0-935b-49d2-9b63-c5301d5080bf","owner":[],"postedDate":"April 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-08-11T16:04:47+00:00","versionOfRecord":{"articleIdentity":"rs-6339860","link":"https://doi.org/10.1007/s00418-025-02405-1","journal":{"identity":"histochemistry-and-cell-biology","isVorOnly":false,"title":"Histochemistry and Cell Biology"},"publishedOn":"2025-08-05 15:57:21","publishedOnDateReadable":"August 5th, 2025"},"versionCreatedAt":"2025-04-22 13:36:31","video":"","vorDoi":"10.1007/s00418-025-02405-1","vorDoiUrl":"https://doi.org/10.1007/s00418-025-02405-1","workflowStages":[]},"version":"v1","identity":"rs-6339860","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6339860","identity":"rs-6339860","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}