Characterization, expression profiling, and functional analysis of col10a1 in cobia (Rachycentron canadum)

Characterization, expression profiling, and functional analysis of col10a1 in cobia (Rachycentron canadum) | 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 Characterization, expression profiling, and functional analysis of col10a1 in cobia (Rachycentron canadum) Xiaolong Zhao, Yunsheng Yang, Qian Ma, Zhe Zheng, Yuwei Wu, Weijie Guo, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9410040/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Background Collagens are key extracellular matrix components in vertebrates, with type X collagen (COL10) crucial for skeletal development. In mammals, expression of col10a1 is limited to hypertrophic chondrocytes, yet in teleosts, it is also in osteoblasts, hinting at its role in both cartilage and bone mineralization. However, most prior research focused on model fish, little is known about col10a1 in the other teleost taxa, particularly in Perciformes species. Results We cloned cobia’s col10a1 gene with its 2000 bp upstream region, revealing it spans 4585 bp, encodes 659 amino acids, and shares a close evolutionary relationship with that of Echeneis naucrates. Through the analysis of luciferase activities of various promoter constructs, a potential promoter region (spanning from − 300 to + 200 bp) and a potential region (located between − 1800 and − 1300 bp) harboring an enhancer element were identified. We used quantitative real time PCR and fluorescence in situ hybridization to study its expression, finding high levels in skeletal tissues such as scales and cartilage. During early development, col10a1 expression starts at the multi-cell stage, peaks at organogenesis, increases again from 1 day post-hatching, reaching a maximum at 9 days before declining. In cobia larvae, it's mainly expressed in cartilage cells, notochord sheath cells, and muscle fibers. Comparing deformed and normal skeletal tissues, col10a1 expression is suppressed in deformed Meckel’s cartilage, but no significant change in deformed vertebrae. We then constructed a prokaryotic expression vector for cobia COL10A1, expressed, and purified the protein. This protein boosted alkaline phosphatase activity and Alizarin red staining, and upregulated mineralization-related genes in mouse osteoblastic cells. Conclusions Our study confirms col10a1 's key role in cartilage and bone development of cobia, consistent with Cypriniformes and Beloniformes species. The cobia COL10A1 protein was shown to regulate mineralization in mouse osteoblastic cells. This further substantiates that col10a1 and its protein are vital for skeletal development and mineralization across vertebrates, highlighting their conserved and crucial functions in skeletal biology. Rachycentron canadum col10a1 gene expression function skeleton Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Background Collagens are the most abundant extracellular matrix components in vertebrate species, playing pivotal roles in various physiological functions and providing mechanical properties to bones, cartilage, tendons, and other tissues [1–3]. Among the 28 members of the collagen superfamily, type X collagen (COL10) is particularly crucial for skeletal development. Originally discovered in chicken long bones in 1985 [4], its encoded gene, col10a1 , is shown to be expressed in hypertrophic chondrocytes during endochondral ossification in mammals [5]. Unlike fibrillar collagens, COL10 forms a non-fibrillar network and is vital for initiating calcium precipitation within the cartilaginous extracellular matrix [6]. In mammals, col10a1 expression is restricted to hypertrophic chondrocytes [7]. However, Renn and Winkler [8] reported that col10a1 is absent in chondrocytes but is expressed early in regions of intramembranous and perichondral bone formation within the axial skeleton during medaka ( Oryzias latipes ) embryogenesis. This suggests that col10a1 marks pre-osteoblasts in O. latipes . Furthermore, osteoblasts in both the spotted gar ( Lepisosteus oculatus ) and zebrafish ( Danio rerio ) were shown to express col10a1 expression [9], highlighting significant differences in col10a1 expression patterns between teleosts and mammalian species. In addition, vertebrate skeleton formation involves two distinct modes: chondral and intramembranous ossification, and teleosts are no exception [10]. Chondrocytes are crucial for cartilage formation, while osteoblasts are responsible for both chondral and intramembranous ossification. The expression of col10a1 in both chondrocytes and osteoblasts suggests a potential role for type X collagen in the mineralization of both cartilage and bone in fish [11]. Further research on vertebral body formation in O. latipes using a transgenic line expressing nlGFP under the control of the col10a1 promoter revealed that col10a1 : nlGFP cells emerge in a segmental pattern within the axial skeleton before the mineralization of the notochordal sheath. These cells persist on the outer surface of the chordal centra during mineralization and subsequent perichordal ossification of the vertebral bodies [12]. Recently, Raman [13] demonstrated that a mutant D. rerio line for col10a1 exhibited reduced chondrocranium size and decreased bone mineralization in larvae, as well as reduced vertebral thickness and tissue mineral density in adults. Although these findings have enhanced our understanding of col10a1 ’s role in regulating skeleton development, most of the aforementioned research was conducted on model organisms in fish (zebrafish in Cypriniformes and medaka in Beloniformes). However, there is still limited knowledge regarding the genetic characteristics and functions of col10a1 in other teleost taxa, particularly the Perciformes, which represent a taxonomically distinct group both morphologically and phylogenetically. The cobia ( Rachycentron canadum ), a marine teleost belonging to Rachycentridae family within Perciformes order, is widely distributed across tropical, subtropical, and warm temperate seas globally [14]. In this study, we cloned the sequence of cobia col10a1 gene and utilized it for gene structure and phylogenetic analysis. Furthermore, the promoter activity of the 5′-flanking region of col10a1 gene was assessed using Luciferase reporter assays to investigate its transcriptional regulatory mechanisms. Tissue distribution of this gene was explored through quantitative real-time PCR (qRT-PCR) and fluorescence in situ hybridization (FISH). Additionally, the expression pattern of col10a1 across developmental stages, from embryos to juveniles, has also been investigated. Finally, we evaluated the functions of cobia col10a1 by expressing and purifying the protein in a prokaryotic system, followed by analyzing its effect on calcification and the expression of mineralization-associated genes in murine osteoblastic cells. Materials and methods Sample collection Cobia samples used in this study were obtained from the marine biology research base of Guangdong Ocean University (Zhanjiang, China). They are categorized as follows: (1) Embryos: a total of 30 embryos were collected at each of the following developmental stages: one-cell, two-cell, four-cell, eight-cell, 16-cell, multi-cell, blastula, gastrula, neurula, organogenesis, and hatching. These stages were determined based on the criteria established by Kuang [15]. (2) Larvae and juveniles: six individuals were sampled at every other day from 1 to 15 days post hatching (dph). Additional samples were collected at 18, 20, 25, and 30 dph. (3) Adults: three females and three males, with an average body weight of 4410.7 ± 409.9 g and an average body length of 64.8 ± 2.2 cm, were selected for tissue sampling. Nineteen tissues were sampled, including brain, branchial arch, eye, fin rays, gill, head kidney, heart, intestine, liver, Meckel’s cartilage, muscle, ovary, scales, skin, spleen, stomach, testis, trunk kidney, and vertebrae. In addition, as reported by Ma [16], samples of Mickel's cartilage and vertebrae were collected from three fish displaying jaw deformities or spinal curvatures. Prior to sampling, all fish were anesthetized and euthanized using an overdose eugenol (Shanghai Macklin Biochemical Technology Co., Ltd, China). Subsequently, the whole fish or the dissected tissues were immediately frozen in liquid nitrogen and stored in a refrigerator at − 80°C. To elucidate the tissue distribution of col10a1 during early developmental stages, cobia larvae were preserved in 4% paraformaldehyde (PFA) at 4°C for 24 h, followed by storage in 75% ethanol until further use for paraffin section and in situ hybridization. Total RNA extraction and first-strand cDNA synthesis Total RNA was extracted using TRIzol reagent (Invitrogen, USA) following the manufacturer’s protocol. The quality and concentration of the extracted RNA were assessed using the Bioanalyzer 2100 (Agilent Technologies, USA) and the NanoDrop 2000 (Thermo Fisher Scientific, USA). Subsequently, 1 µg of the total RNA was used to synthesize cDNA with the EasyScript® First-Strand cDNA Synthesis SuperMix (TransGen Biotech, China), according to the manufacturer's instructions. DNA extraction Muscle samples were used for DNA extraction using a marine animal genomic DNA extraction kit (TransGen Biotech, China) according to the manufacturer's instructions. Cloning of cobia col10a1 gene and sequence analysis The entire gene sequence of the cobia col10a1 along with its upstream 2000 bp was extracted from the cobia genome dataset (PRJNA634421). To ensure sequence accuracy, primers for amplifying various length fragments of the 5’-flanking region and the CDS were designed using Primer premier 6.0 software (Supplementary Table 1, Table S1 ). PCR amplification was then conducted using these primers following the protocol of Accurate Taq Master Mix (Accurate Biology, China). The PCR amplification products were subsequently analyzed using 1.2% (mass fraction) agarose gel electrophoresis, and then sequenced by Shanghai Sangon Biotech Co., Ltd. (Shanghai, China). To determine the chromosomal location of the cobia col10a1 gene, both the genome annotation data and the col10a1 gene sequence were imported into TBtools software 2.027 [17]. The exon/intron structures were visualized utilizing the Gene Structure Display Server (GSDS, http://gsds.cbi.pku.edu.cn ) by aligning the coding sequence with the corresponding genomic DNA sequences. Using DNAMAN software, the deduced amino acid sequence of cobia col10a1 was obtained and employed for homology comparison and identity analysis. Corresponding COL10A1 amino acid sequences from other vertebrate species were retrieved from the NCBI GenBank ( http://www.ncbi.nlm.nih.gov/genbank/ ). Multiple sequence alignment of COL10A1 was conducted using DNAMAN software. Subsequently, phylogenetic analysis was performed using the neighbor-joining (NJ) method in MEGA11 software. The resulting phylogenetic tree was visualized using the Chiplot website ( https://www.chiplot.online/tvbot.html ). The whole-genome sequences and their corresponding gene annotation files for D. rerio , O. latipes , Echeneis naucrates , and Scyliorhinus canicula were obtained from the NCBI database. Subsequently, an interspecies collinearity analysis of the col10a1 genes was carried out among cobia and these teleost species, utilizing the MCScanX plugin integrated within the TBtools software. Furthermore, the physicochemical properties of cobia COL10A1, including its molecular weight and isoelectric point, were analyzed using the ExPASy tool ( http://web.expasy.org/protparam/ ). The secondary and tertiary structures of COL10A1 were predicted using SOPMA ( https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html ) and SWISS ( http://swissmodel.expasy.org/ ). To predict potential transcriptional start sites of the cobia col10a1 gene, the online analysis software BDGP ( https://www.fruitfly.org/seq_tools/other.html ) and Promoter 2.0 Prediction Server ( http://www.cbs.dtu.dk/services/Promoter/ ) were utilized. Furthermore, AliBaba2.1 software ( http://gene-regulation.com/pub/programs/alibaba2/index.html ) was used to identify potential transcription factor binding sites for the col10a1 gene. Quantitative real-time PCR (qRT-PCR) The specific primer sequences for the col10a1 gene and the internal control β-actin used in qRT-PCR are listed in Table S1 . The qRT-PCR reactions were carried out in a final volume of 20 µL using the PerfectStart™ Green qPCR SuperMix Kit (TransGen Biotech, China) on the LightCycler 96 system (Roche, Switzerland). Subsequently, a dissociation protocol was executed to further confirm target specificity. PCR efficiency was determined based on the slopes of standard curves generated from serial 10-fold dilutions of cDNA samples, with an acceptable efficiency range of 90% to 110%. Correlation coefficients (R 2 ) were calculated using linear regression analysis in SPSS 26.0. The mRNA levels of each sample were normalized to the β-actin levels. The relative changes in mRNA expression were calculated using the 2 −ΔΔCT method. All qRT-PCR reactions were performed in triplicate to ensure accuracy and reproducibility. Paraffin section, Masson's trichome, and fluorescence in situ hybridization (FISH) of cobia col10a1 Fixed larval and juvenile samples were dehydrated in an ethanol series, and embedding in paraffin. Longitudinal 4 µm sections were cut and stained by Masson's trichome (Servicebio, China) to identify collagen. FISH probe was synthesized using a multi-sequence hybrid probe by Servicebio Inc. (Servicebio, China). Fluorescence in situ hybridization was performed with a FISH kit (Servicebio, Wuhan, China) according to the manufacturer’s protocol. Images were acquired using ortho-fluorescent microscope (Nikon Eclipse ci, Japan) and the Nikon DS-U3 imaging system. The probe sequences were shown in Table S1 . Recombinant vector construction of fragments in 5’-flanking region of cobia col10a1 To construct recombinant vectors for fragments in the 5'-flanking region of cobia col10a1 , all primers were designed with tailing sequences specific for either the NheI site (for forward primers) or the HindIII site (for reverse primers), as detailed in Table S1 . Following amplification, the DNA fragments were digested using NheI and HindIII restriction enzymes (Takara Bio, China). These fragments were then inserted into the pGL3-basic vector (Promega, United States) and subsequently sequenced for verification. Once the sequences were confirmed, the plasmids, designated as pGL3-col10a1-P1 through pGL3-col10a1-P4, were extracted utilizing an endotoxin-free plasmid bulk extraction kit (Sangon Biotech, China). Cell Culture, Transfection, and Dual Luciferase Assay HEK 293T cells were cultivated and subsequently transfected following the method described by Ma [18]. In brief, the transfection process involved co-introducing 100 ng of the promoter reporter plasmid along with the internal control vector, pGL3-Basic, into the cells. Luciferase activity was quantified using the Dual Luciferase Reporter Assay System E2940 (Promega, USA) according to the manufacturer's instructions. The activity of each promoter construct was determined by calculating the luminescence ratio of firefly luciferase to renilla luciferase. Each experimental group underwent three replicates to ensure accuracy [8]. Prokaryotic Expression and Protein Purification of cobia COL10A1 The primers used for amplification of cobia COL10A1, which included NdeI and HindIII restriction sites along with protective bases, are listed in Table S1 . Following sequencing and verification, the recombinant cloning vector pMD19-T-col10a1 was constructed. Both the pET-32a vector and the pMD19-T-col10a1 were subsequently subjected to double digestion using NdeI and HindIII enzymes (Takara Bio, China). The recovered fragments were then ligated overnight at 16°C using T4 DNA ligase. The resulting expression vector, named pET-30a-COL10A1, was transformed into the DH5α cloning strain (TransGen Biotech, China). Positive clones were identified by colony PCR, and the correctness of the construct was confirmed by sequencing (Sangon biotech, China). Subsequently, the recombinant plasmid pET-30a-COL10A1 was transformed into the BL21 (DE3) strain (Sangon biotech, China) for expression. The procedures for transformation, expression, and purification of the recombinant fusion proteins were conducted following the methodology described by Li [19].The concentration of the purified protein was determined using the Bradford protein assay, with BSA serving as the standard (Sangon biotech, China). The purity and molecular weight of the protein were assessed by standard sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis for confirmation. Cultivation of MC3T3-E1 cells and Treatment with cobia COL10A1 Murine osteoblastic cell MC3T3-E1 were acquired from Pricella Biotechnology Co., Ltd., (Wuhan, China) and cultivated in α-MEM medium supplemented with 10% fetal bovine serum (Pricella Biotechnology Co., Ltd, Wuhan, China). The cells were incubated at 37 ℃ in an atmosphere containing 5% CO 2 . For the experiment, passage 3 MC3T3-E1 cells were seeded into a 48-well plate at a density of 1 × 10 4 cells per well and cultured for 24 h. Subsequently, the medium was replaced with osteogenic differentiation induction medium (Pricella Biotechnology Co., Ltd, Wuhan, China) containing cobia COL10A1 protein with a purity of ≥ 90%. The cobia COL10A1 protein was added at concentrations of 0 µg/mL, 1 µg/mL, and 10 µg/mL, respectively. Four replicate wells were established for each concentration, and the medium was changed every 3 days. Determination of Alkaline phosphatase (ALP) Activity and Calcification After 7 days of treatment, alkaline phosphatase (ALP) staining was performed using an ALP staining kit (Puhe Biological Co., Ltd., Wuxi, China). Furthermore, alizarin red staining was conducted using alizarin red S staining solution from the same company, following 14 days of treatment. The procedures for both staining experiments were carried out in accordance with the manufacturer's instructions. Images were captured using a stereoscopic microscope (M205 FCA, Leica, Germany). Image J software was utilized to determine the average optical density (AOD) value, which indicates the concentration of blue-purple precipitate per unit area in the ALP staining results. Similarly, AOD value of red calcified nodules per unit area in the Alizarin red staining results were analyzed to detect the calcification of cells. Assessment of Gene Expression in MC3T3-E1 cells MC3T3-E1 cells were treated with cobia COL10A1 for 7 days, following which they were collected for RNA extraction. Subsequently, cDNA synthesis and qRT-PCR were carried out as previously described. The primers utilized for detectin g of mouse skeleton formation-related genes, including Runt-related transcription factor 2 ( runx2 ), osterix , Osteocalcin ( ocn ), Alkaline phosphatase ( alp ) with glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) serving as the internal reference, are listed in Table S1 . Statistical analysis The statistical analysis was performed using one-way ANOVA in SPSS 20.0. Data were expressed as the mean ± SD, P < 0.05 was considered to indicate a significant difference. Results Characteristics of cobia COL10A1 The CDS of cobia col10a1 is 1977 base pairs (bp) in length and encodes a protein comprising 659 amino acids (aa). Analysis of the predicted physical and chemical properties of this protein revealed a relative molecular mass of 63.34 kDa and an isoelectric point (pI) of 9.22. As illustrated in Supplementary Fig. 1A (Fig. S1 A), the predicted secondary structure of cobia COL10A1 reveals that it is composed of 6.69% α-helix, 6.23% β-turn, 77.05% random coil, and 10.03% extended strand. The tertiary structure of the COL10A1 protein, as presented in Fig. S1 D, is similar with the secondary structure prediction, with the random coil conformation being the most dominant. In addition, the COL10A1 proteins of mouse ( Mus musculus ), D. rerio and cobia revealed similar structure. Gene structure, chromosome localization and Phylogenetic analysis of cobia col10a1 As shown in Fig. S2 , cobia col10a1 gene consists of two exons (120 and 1857 bp) and one intron (2608 bp). A comparative analysis of the col10a1 gene sequences across several species showed that the exon-intron organization of the cobia col10a1 gene is similar to that of the col10a1 genes in Centropristis striata and Lampris incognitus . However, the col10a1 gene in six other teleost species, such as Echeneis naucrates , Seriola aureovittata , and Paralichthys olivaceus , comprises three exons and two introns (Fig. 1 ). R. canadum (Gene id: PQ246021), C. striata (Gene id: 131990873), D. rerio (Gene id: 555804), E. naucrates (Gene id: 115038291), L. incognitus (Gene id: 130125025), O. latipes (Gene id: 110017749), P. olivaceus (Gene id: 109632608), S. aureovittata (Gene id: 130187688), T. jaculatrix (Gene id: 121199749). The collinearity analysis presented in Fig. 2 reveals that cobia col10a1 gene is situated on chromosome 18. This gene demonstrates collinear relationships with chromosome 24 in both E. naucrates and O. latipes , chromosome 6 in Scyliorhinus canicula , as well as with two chromosomes (Chr17 and Chr20) in D. rerio . A phylogenetic tree based on the amino acid sequences of COL10A1 demonstrated that cobia COL10A1 firstly clusters with E. naucrates , and then groups with S. aureovittata , P. olivaceus , and Thunnus albacares , forming a distinct clade. This clade subsequently clusters with other fish species, Amphibia ( Xenopus laevis ), Aves ( Gallus gallus ), and Mammalian ( Homo sapiens and M. musculus ) (Fig. 3 ). The bootstrap consensus tree inferred from 1000 replicates was taken to represent the evolutionary history of the taxa analyzed. Cloning and bioinformatics analysis of the promoter sequence of the cobia col10a1 gene A 2000 bp sequence upstream from the 5′-flanking of the col10a1 gene in cobia was used as the promoter region, ranging from − 1800 bp upstream to + 200 bp downstream of the transcription start site (TSS), was amplified by PCR. Bioinformatics analysis identified four core promoters within this promoter region using BDGP, and determined one transcription start site using Promoter 2.0, which are listed in Fig. 4 . Four core promoters (marked in red) were identified in this promoter region by BDGP, and one transcription start site (marked in blue) was identified by Promoter 2.0. All scores exceeded their respective prediction thresholds (0.8 for BDGP and 1 for Promoter 2.0), indicating a high degree of confidence in these identifications. Predictive analysis of potential transcriptional binding sites in the col10a1 gene The potential transcription factor binding sites within the cobia col10a1 gene were predicted, and the results revealed a total of 14 transcription factor binding sites in the promoter region of the cobia col10a1 gene (Fig. 4 ). Notably, three primary transcription factors, namely Sp1, AP1, and Oct-1, were found to be predominantly represented among these binding sites. Analysis of the promoter activity of cobia col10a1 gene As shown in Fig. 5 , the dual luciferase reporter gene system revealed that among four constructed recombinant plasmids with cobia col10a1 promoter fragments, they all exhibited significantly higher activities than the control pGL3-Basic. In addition, a significantly higher expression in pGL3-col10a1-P1 was identified compared to the other three plasmids, which located at -1800 to -1300 bp (relative to the transcription start site). Different lowercase letters in the superscript indicate significant differences ( P < 0.05). Expression pattern of cobia col10a1 gene As shown in Fig. 6 A, the col10a1 gene was expressed in all 19 tissues examined in cobia, with the highest expression levels observed in skeletal tissues, namely scales, Meckel's cartilage, and fin rays. The expression of col10a1 in the muscle and heart were significantly lower than that in the bony tissues. Furthermore, these expression levels were significantly higher compared to those in the other 14 tissues ( P < 0.05). During the initial stages of cobia embryonic development, ranging from the 2-cell stage to the 16-cell stage, the expression of col10a1 was low and showed no significant variations across the developmental time points. However, a significant up-regulation of col10a1 was observed beginning at the multi-cell stage, with the highest level detected at the organogenesis stage (Fig. 6 B). The expression of col10a1 in cobia larvae constantly increased from 1 dph and peaked at 9 dph (Fig. 6 C). Subsequently, a significant decrease in col10a1 expression was observed at 11 dph, and this level of expression remained relatively stable until 30 dph. As illustrated in Fig. 6 D, the expression level of col10a1 in the deformed Meckel’s cartilage of cobia was significantly lower than that in the normal tissue ( P 0.05). A. Tissue expression distribution of cobia col10a 1 gene. Ba: branchial arch; Br: brain; Ey: eye; Fr: fin rays; Gi: gill; He: heart; Hk: head kidney; In: intestine; Li: liver; Mec: Meckel’s cartilage; Mu: muscle; Ov: ovary; Sc: scales; Sk: skin; Sp: spleen; St: stomach; Te: testis; Tk: trunk kidney; Ve: vertebrae. B . Expression pattern of col10a1 gene during cobia embryonic development. C . Relative expression levels of col10a1 gene in cobia at different developmental stages. D . Expression of cobia col10a1 gene in deformed and normal skeletal tissues. Different lowercase letters in the superscript indicate significant differences ( P < 0.05); ** indicate statistically significant differences with P values less than 0.05. Localization of expression of col10a1 in cobia larvae As shown in Fig. 7 A, the expression of col10a1 in the vertebrae of cobia larvae (7 dph) was mainly distributed in notochord sheath cells, but absent in vacuolar cells. In the head skeleton, the col10a1 mRNA can be detected in both the immature and mature chondrocytes. The expression patterns of col10a1 in the vertebrae of cobia juveniles (21dph) was similar to that observed at 7dph, with hybridization signals primarily localized in the notochord sheath cells during spinal development (Fig. 7 B). In the head skeleton, col10a1 mRNA was also detectable in both immature and mature chondrocytes. Moreover, the hybridization signal intensity in mature chondrocytes at 7 dph and 21 dph was significantly lower than that in immature chondrocytes. (a, c, e, g): Vertebrae tissue. (b, d, f, h): Head tissue. The col10a 1 gene is labelled in red and nuclei are blue (DAPI). Ey: Eyes; IMM: Immature chondrocytes; IVL: Intervertebral ligament; MAT: Mature chondrocytes; Mf: Muscle fibers; Nsc: Notochord sheath cells; V: Vacuole. Scale bar = 100 um. ALP and Alizarin red staining of MC3T3-E1 cells treated with cobia COL10A1 The ALP activity of MC3T3-E1 cells cultured in a differentiation medium containing varying concentrations of COL10A1 for 7 days was shown in Fig. 8 . The results indicated that the ALP activity in the COL10A1 treatment group at 10 µg/mL was significantly higher compared to the control group ( P 0.05). A . the control group (CG); B . osteoblasts treated with 1 µg/mL of COL10A1; C . osteoblasts treated with 10 µg/mL of COL10A1; D . Average optical density (AOD) of ALP staining. Different lowercase letters in the superscript indicate significant differences ( P < 0.05). The results of Alizarin red staining demonstrated that after 14 days of treatment with cobia COL10A1, the number of calcified nodules was significantly increased in the 10 µg/mL treatment group ( P < 0.05). However, no significant difference was observed in the 1 µg/mL treatment group compared to the control (Fig. 9 ). A . the control group (CG); B . osteoblasts treated with 1 µg/mL of COL10A1; C . osteoblasts treated with 10 µg/mL of COL10A1; D . Average optical density (AOD) of Alizarin red staining. Different letters indicate a statistically significant difference with P values less than 0.05. Effect of COL10A1 on the expression of mineralization-associated genes in MC3T3-E1 cells As shown in Fig. 10 , the highest expression level of runx2 , osterix , and alp were identified in the 10 µg/mL COL10A1 treatment group. However, the highest expression of ocn was identified in the 1 µg/mL group. For runx2 , osterix , and alp , no significant differences were detected between the gene expression in 1 µg/mL COL10A1 treatment group and the control group ( P > 0.05). CG, the control group; 1 µg/mL, osteoblasts treated with 1 µg/mL of COL10A1; 10 µg/mL, osteoblasts treated with 10 µg/mL of COL10A1. Different lowercase letters in the superscript indicate significant differences ( P < 0.05). Discussion Phylogenetic analysis showed that the cobia COL10A1 clade first clusters with those of E. nauerates , S. aureovittata , and P. olivaceus , and subsequently groups with other teleosts and higher vertebrates. This is consistent with traditional taxonomy and evolutionary principles governing vertebrates (Fig. 3 ). However, the exon-intron organization of col10a1 in cobia exhibits a different structure of two exons and one intron, contrasting with the three-exon/two-intron configuration observed in the three aforementioned species (which cluster with cobia). This arrangement is also found in mammalian col10a (e.g., M. musculus ) [20]. A similar exon-intron organization was identified in C. striata and L. incognitus , which form a distinct group from cobia in the NJ tree (Fig. 3 ). These findings imply that phylogenetic relationships and gene structures may evolve under different selective pressures. In higher vertebrates, such as avians, tetrapods and mammals, COL10 is mainly synthesized by hypertrophic chondrocytes during endochondral ossification, and is absent in osteoblasts and other chondrocyte maturation stages, making it a marker for maturing chondrocytes [21, 22]. It also plays a crucial role in the normal distribution of matrix vesicles and proteoglycans in the calcifying zone of the cartilage growth plate [23]. In contrast, the major expression sites of COL10 expand in amphibians and fish. In Xenopus tropicalis , col10a1 is expressed in osteoblasts besides cartilage mineralization [24]. In D. rerio , col10a1 is expressed in both osteoblasts and chondrocytes [9]. In O. latipes , col10a1 transcription occurs before osterix expression and the bone matrix mineralization, enabling the identification of pre-osteoblasts. Furthermore, the expression of col10a1 in cartilaginous fish ameloblasts and/or odontoblasts implies its involvement in odontode skeleton (teeth and scales) formation [11]. In this study, tissue distribution of cobia col10a1 reveals that it exhibits significantly higher expression in the scales, Meckel’s cartilage, and fin rays, with relatively lower expression in vertebrae. Similarly, col10a1 was also found to be expressed in the vertebrae of flounder, P. olivaceus [25]. A comparable distribution pattern is reported in O. latipes , where the gene is expressed in early osteoblasts across most of the skeleton, encompassing head elements, axial skeleton (centrae and neural arches), and fins. Additionally, the possibility that osteoblast-like cells mineralize the sheath before ossifying the perichordal centrum has been proposed[26, 27]. The time-point concurrent of col10a1 : nlGFP positive cells with the onset of mineralization of the notochordal sheath in O. latipes make these cells promising candidates for being involved in mineralization of the notochordal sheath [8, 12]. FISH confirms high col10a1 expression in the notochord sheath cells of cobia larvae, indicating its role in perichondral bone formation. Thus, col10a1 is expressed in both osteoblasts and chondrocytes across teleosts. Besides, it has been shown that COL10A1 mutations can cause skeletal disorders like Schmid type metaphyseal chondrodysplasia [28]. The chick COL10 transgene product is linked to craniofacial skeletal abnormalities [29]. These suggests that COL10A1 may play a role in regulating abnormal skeletal development. In cobia with jaw deformities or spinal curvatures, col10a1 is up-regulated in deformed cartilage but unchanged in vertebrae, indicating its intricate involvement in abnormal cartilage development regulation in teleosts. In O. latipes , col10a1 transcript commences earlier in developing intramembranous and perichondral bones of the head and trunk before ossification [8]. Thus it has been concluded that col10a1 marks pre-osteoblasts and osteoblasts. In cobia, col10a1 has higher expression during late embryonic and early larval periods (from neurula stage to 9 dph), aligning with the onset of skeletal development reported by Mao [30]. FISH analysis of cobia larvae at 7 dph and 21 dph revealed that col10a1 is present in both mature and immature cartilage, a pattern also observed in other teleosts like zebrafish [9]. It has been reported that osteoblasts in X. tropicalis , O. latipes , D. rerio and L. oculatus express col10a1 [8, 9, 24]. Col10a1a −/− mutant D. rerio larvae display reduced bone mineralization, while adult fish exhibit decreased vertebral thickness and tissue mineral density [13]. These are similar to observations in mice and humans. An accumulation of col10a1 : nlGFP positive cells is found in areas of increased mineralization during notochordal sheath repair [12]. In this study, we obtain cobia COL10A1 proteins through prokaryotic expression and purification. Treating murine osteoblastic cells with cobia COL10A1 enhanced ALP activity and alizarin red staining intensity, indicating its role in promoting active mineralization and creating a suitable environment for mineralization and bone remodeling [31]. The impact of cobia COL10A1 on murine cells supports that both the triple-helical domain and the C-terminal domain are highly conserved across all species, thereby implying the preservation of COL10A1's protein functions [8]. Our study shows cobia COL10A1 significantly elevates runx2 and osterix mRNA levels in murine osteoblastic cells. Runx2 is crucial for the development of osteogenic lineage in mammals, birds, and teleosts such as D. rerio [32, 33]. Sp7 / osterix is involved in multiple aspects of bone development, including the differentiation and maturation of osteoblasts, as well as the formation of osteocytes. Notably, col10a1 is the target gene of osterix in osteoblasts [34], with a positive correlation between them. The up-regulation of runx2 and osterix in MC3T3-E1 cells treated with COL10A1 supports a positive regulatory relationship and highlights its impact on osteoblast differentiation [13]. Comparative sequence analysis reveals high promoter conservation among mammalian species near the transcription start site (ranging from − 120 to + 1 bp), likely serving as the basal promoter [35]. Using the luciferase reporter gene system, we identified a potential promoter region in cobia col10a1 (-300 to + 200 bp) with multiple potential regulatory elements like AP1, Sp1, c-Jun, and c-Fos [36]. AP1, composed of Jun and Fos proteins [37], is crucial for bone formation and cartilage development [22]. In addition, c-Jun is highly expressed in hypertrophic chondrocytes, and c-Fos may suppress col10a1 expression in a tissue-specific manner [38]. Collectively, these findings suggest that AP1 elements possess a silencing activity on col10a1 [39, 40]. The regulation of type X collagen expression is crucial for its role in endochondral ossification, particularly in hypertrophic chondrocytes. Enhancer elements play a significant role in this regulation, ensuring tissue-specific expression [22]. In studies of human enhancer activity, multiple luciferase reporter constructs were prepared using different fragments of the COL10A1 promoter and transfected into hypertrophic chondrocytes. The results indicate that enhancer activity was localized to the region upstream of the transcription start site, spanning − 2407 bp to -1870 bp [38]. In the chicken col10a1 gene, an additional enhancer element resides within the region spanning − 2.9 kb to -2.4 kb upstream of the transcription start site [41]. In the bovine col10a1 gene, the enhancer element is located within the region extending from − 3381 bp to -2875 bp upstream of the transcription start site. The mouse enhancer element resides within the Col10a1 distal promoter region (-4.4 kb to -3.8 kb) and mediates high levels of hypertrophic chondrocyte-specific expression both in vitro and in vivo [39]. What's more, the enhancer element within the mouse Col10a1 distal promoter exhibits high homology with enhancers from human and bovine sources. Further experiments confirmed that transgenic constructs containing the 600 bp enhancer element mediated high reporter activity, whereas vector controls without the enhancer produced only low reporter activity in hypertrophic chondrocytes [40]. In this study, we identified a construct, designated as pGL3-col10a1-P4, which encompasses a potential promoter region. Concurrently, another distinct region (represented by the construct pGL3-col10a1-P1), spanning from − 1800 to -1300 bp, exhibits a significantly higher luciferase activity than pGL3-col10a1-P4, suggesting the existence of an enhancer element within this specific region. Conclusion We present here a comprehensive report on the cobia col10a1 gene, encompassing its full-length sequence, chromosome localization, gene structure, phylogenetic relationship, and potential transcriptional region. Moreover, we have elucidated the expression pattern of this gene, examining its tissue distribution, expression across various developmental stages, and its association with skeletal deformities. Our findings confirm that this gene plays a pivotal role in both cartilage and bone development, aligning with recently published data on freshwater species. Additionally, the cobia COL10A1 protein has been demonstrated to regulate the mineralization process of murine osteoblastic cells. This evidence further supports the notion that the col10a1 gene and its protein product are crucial for skeletal development and mineralization across vertebrates. Abbreviations AA Amino Acids ALP Alkaline Phosphatase AOD Average Optical Density BP Base Pairs CDS The Coding Sequence COL10 Collagen Type X DPH Days Post Hatching GAPDH Glyceraldehyde-3-Phosphate Dehydrogenase NJ Neighbor-Joining OCN Osteocalcin PFA Paraformaldehyde PI Point QRT-PCR Quantitative Real-Time PCR FISH Fluorescence in Situ Hybridization RUNX2 Runt-Related Transcription Factor 2 SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis TSS Transcription Start Site Declarations Ethics approval and consent to participate All animal experiments in this study were handled in strict accordance with the ARRIVE guidelines and China legislation on scientific procedures on living animals. The protocol was approved by the Animal Research and Ethics Committees of Guangdong Ocean University. Consent for publication Not applicable. Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding This research was funded by Guangdong Basic and Applied Basic Research Foundation (grant number 2022A1515012244) and the National Natural Science Foundation of China (grant number 32373153). Author Contribution Q. Ma performed project conception and experiment design; X.L. Zhao, Y.S. Yang, Y.W. Wu, Z. Zheng, W.J. Guo and D.H. Huang conducted the experiments and analyzed the data; X.L. Zhao and Q. Ma wrote the original manuscript and thoroughly revised and finalized the manuscript. All authors read and approved the final manuscript. Acknowledgements Not applicable. Data Availability The sequence data that support the findings of this study are openly available in GenBank of NCBI at under accession PQ246021. The dataset supporting the conclusions of this article is available in the NCBI Short Read Archive (SRA) repository, as BioProject PRJNA634421. References Bretaud S, Nauroy P, Malbouyres M, Ruggiero F. Fishing for collagen function: About development, regeneration and disease. Semin Cell Dev Biol. 2019;89:100–8. https://doi.org/10.1016/j.semcdb.2018.10.002. Gordon MK, Hahn RA. Collagens. Cell Tissue Res. 2010;339:247–57. https://doi.org/10.1007/s00441-009-0844-4. Ikoma T, Kobayashi H, Tanaka J, Walsh D, Mann S. Microstructure, mechanical, and biomimetic properties of fish scales from Pagrus major. J Struct Biol. 2003;142:327–33. https://doi.org/10.1016/S1047-8477(03)00053-4. Schmid TM, Linsenmayer TF. Developmental acquisition of type X collagen in the embryonic chick tibiotarsus. Dev Biol. 1985;107:373–81. https://doi.org/10.1016/0012-1606(85)90319-7. Kwan KM, Pang MK, Zhou S, Cowan SK, Kong RY, Pfordte T, et al. Abnormal compartmentalization of cartilage matrix components in mice lacking collagen X: implications for function. J Membr Biol. 1997;136:459–71. https://doi.org/10.1083/jcb.136.2.459. Kirsch T, Wuthier RE. Stimulation of calcification of growth plate cartilage matrix vesicles by binding to type II and X collagens. J Biol Chem. 1994; 269:11462–9. https://doi.org/10.1016/S0021-9258(19)78146-0. Zheng Q, Zhou G, Morello R, Chen Y, Garcia-Rojas X, Lee B. Type X collagen gene regulation by Runx2 contributes directly to its hypertrophic chondrocyte–specific expression in vivo. J Cell Biol. 2003; 162(5): 833-842. https://doi.org/10.1083/jcb.200211089. Renn J, Winkler C. Characterization of collagen type 10a1 and osteocalcin in early and mature osteoblasts during skeleton formation in medaka. J Appl Ichthyol. 2010;26:196–201. https://doi.org/10.1111/j.1439-0426.2010.01404.x. Eames BF, Amores A, Yan Y-L, Postlethwait JH. Evolution of the osteoblast: skeletogenesis in gar and zebrafish. BMC Evol Biol. 2012;12:27. https://doi.org/10.1186/1471-2148-12-27. Renn J, Winkler C. Osterix-mCherry Transgenic Medaka for In ViVo Imaging of Bone Formation. Dev Dyn. 2009;238:241–8. https://doi.org/10.1002/dvdy.21836. Debiais-Thibaud M, Simion P, Venteo S, Munoz D, Marcellini S, Mazan S, et al. Skeletal Mineralization in Association with Type X Collagen Expression Is an Ancestral Feature for Jawed Vertebrates. Mol Biol Evol. 2019;36:2265–76. https://doi.org/10.1093/molbev/msz145. Renn J, Buettner A, Thuy TT, Chan SJH, Winkler C. A col10a1:nlGFP transgenic line displays putative osteoblast precursors at the medaka notochordal sheath prior to mineralization. Dev Biol. 2013;381:134–43. https://doi.org/10.1016/j.ydbio.2013.05.030. Raman R, Antony M, Nivelle R, Lavergne A, Zappia J, Guerrero-Limon G, et al. The Osteoblast Transcriptome in Developing Zebrafish Reveals Key Roles for Extracellular Matrix Proteins Col10a1a and Fbln1 in Skeletal Development and Homeostasis. Biomolecules. 2024;14:139. https://doi.org/10.3390/biom14020139. Schaffer RV, Nakamura EL. Synopsis of biological data on the cobia Rachycentron canadum (Pisces: Rachycentridae). laguna. 1989. J.H K, G C, Ma Q, Huang J.S, Zhang J.D, Shi G, et al. Embryonic development and morphological characteristics of larvae and juveniles of cobia ( Rachycentron canadum ). scxb. 2021;45:1814–24. https://doi.org/10.11964/jfc.20200812389. Ma Q, Yang Y, Mao F, Zhou Q, Wang L, Chen G. Genome-wide identification, phylogeny and expression analysis of the bmp gene family associated with development and skeleton deformity in cobia ( Rachycentron canadum ). Aquac Rep. 2023;31:101644. https://doi.org/10.1016/j.aqrep.2023.101644. Chen C, Wu Y, Li J, Wang X, Zeng Z, Xu J, et al. TBtools-II: A “one for all, all for one”bioinformatics platform for biological big-data mining. Mol Plant. 2023;16:1733–42. https://doi.org/10.1016/j.molp.2023.09.010. Ma Q, Fan Y, Zhuang Z, Liu S. Cloning, expression profiling and promoter functional analysis of bone morphogenetic protein 2 in the tongue sole ( Cynoglossus semilaevis ). Acta Oceanol Sin. 2018;37:76–84. https://doi.org/10.1007/s13131-018-1164-x. Li G, Wubshet AK, Ding Y, Li Q, Dai J, Wang Y, et al. Antigenicity and Immunogenicity Analysis of the E. coli Expressed FMDV Structural Proteins; VP1, VP0, VP3 of the South African Territories Type 2 Virus. Viruses. 2021; 13 (6): 1005. https://doi.org/10.3390/v13061005. Kong RYC, Kwan KM, Lau ET, Thomas JT, Boot‐Handford RP, Grant ME, et al. Intron‐exon structure, alternative use of promoter and expression of the mouse collagen X gene, Col10a‐1 . Eur J Biochem. 1993;213:99–111. https://doi.org/10.1111/j.1432-1033.1993.tb17739.x. Gomez-Picos P, Ovens K, Eames BF. Limb Mesoderm and Head Ectomesenchyme Both Express a Core Transcriptional Program During Chondrocyte Differentiation. Front Cell Dev Biol. 2022; 10: 876825. https://doi.org/10.3389/fcell.2022.876825. Lu Y, Qiao L, Lei G, Mira RR, Gu J, Zheng Q. Col10a1 gene expression and chondrocyte hypertrophy during skeletal development and disease. Front Biol. 2014;9:195–204. https://doi.org/10.1007/s11515-014-1310-6. Gudmann NS, Karsdal, M.A. Chapter 10-Type X Collagen Karsdal MA. Biochemistry of Collagens, Laminins and Elastin. 2016:73-76. https://doi.org/10.1016/B978-0-443-15617-5.00028-7. Aldea D, Hanna P, Munoz D, Espinoza J, Torrejon M, Sachs L, et al. Evolution of the vertebrate bone matrix: An expression analysis of the network forming collagen paralogues in amphibian osteoblasts. J Exp Zool B Mol Dev Evol. 2013;320:375–84. https://doi.org/10.1002/jez.b.22511. Ibaraki H, Wu X, Uji S, Yokoi H, Sakai Y, Suzuki T. Transcriptome analysis of vertebral bone in the flounder, Paralichthys olivaceus (Teleostei, Pleuronectiformes), using Illumina sequencing. Mar Genom. 2015;24:269–76. https://doi.org/10.1016/j.margen.2015.09.009. Inohaya K, Takano Y, Kudo A. The teleost intervertebral region acts as a growth center of the centrum: In vivo visualization of osteoblasts and their progenitors in transgenic fish. Dev Dyn. 2007;236:3031–46. https://doi.org/10.1002/dvdy.21329. Spoorendonk KM, Peterson-Maduro J, Renn J, Trowe T, Kranenbarg S, Winkler C, et al. Retinoic acid and Cyp26b1 are critical regulators of osteogenesis in the axial skeleton. Development. 2008;135:3765–74. https://doi.org/10.1242/dev.024034. Chan D, Jacenko O. Phenotypic and biochemical consequences of collagen X mutations in mice and humans. Matrix Biol. 1998;17:169–84. https://doi.org/10.1016/S0945-053X(98)90056-7. Chung KS, Jacenko O, Boyle P, Olsen BR, Nishimura I. Craniofacial abnormalities in mice carrying a dominant interference mutation in type X collagen. Dev Dyn. 1997; 208:544–52. https://doi.org/10.1002/(SICI)1097-0177(199704)208:43.0.CO;2-X. Mao FF, Chen G, Ma Q, ZHOU QL, Shi G, Huang JS, et al. Development characteristics of the vertebral column and the appendicular skeleton in larval and juvenile cobia ( Rachycentron canadum ). scxb. 2023;47:089105–9. https://doi.org/10.11964/jfc.20210712985. Kirsch T, Ishikawa Y, Mwale F, Wuthier RE. Roles of the nucleational core complex and collagens (types II and X) in calcification of growth plate cartilage matrix vesicles. J Biol Chem. 1994; 269:20103–9. https://doi.org/10.1016/S0021-9258(17)32133-6. Komori T. Regulation of bone development and extracellular matrix protein genes by RUNX2. Cell Tissue Res. 2010;339:189–95. https://doi.org/10.1007/s00441-009-0832-8. Simoes B, Conceicao N, Viegas CSB, Pinto JP, Gavaia PJ, Hurst LD, et al. Identification of a promoter element within the zebrafish colXα1 gene responsive to Runx2 isoforms Osf2/Cbfa1 and til-1 but not to pebp2αA2. Calcif Tissue Int. 2006;79:230–44. https://doi.org/10.1007/s00223-006-0111-6. Niu P, Zhong Z, Wang M, Huang G, Xu S, Hou Y, et al. Zinc finger transcription factor Sp7/Osterix acts on bone formation and regulates col10a1a expression in zebrafish. Sci Bull. 2017;62:174–84. https://doi.org/10.1016/j.scib.2017.01.009. Beier F, Lammi MJ, Bertling W, Von Der Mark K. Transcriptional Regulation of the Human Type X Collagen Gene Expression. Ann N Y Acad Sci. 1996;785:209–11. https://doi.org/10.1111/j.1749-6632.1996.tb56263.x. Gu J, Lu Y, Li F, Qiao L, Wang Q, Li N, et al. Identification and characterization of the novel Col10a1 regulatory mechanism during chondrocyte hypertrophic differentiation. Cell Death Dis. 2014;5:e1469–e1469. https://doi.org/10.1038/cddis.2014.444. Eferl R, Hoebertz A, Schilling AF, Rath M, Karreth F, Kenner L, et al. The Fos-related antigen Fra-1 is an activator of bone matrix formation. EMBO J. 2004;23:2789–99. https://doi.org/10.1038/sj.emboj.7600282. Riemer S, Gebhard S, Beier F, Pöschl E, Von Der Mark K. Role of c‐fos in the regulation of type X collagen gene expression by PTH and PTHrP: Localization of a PTH/PTHrP‐responsive region in the human COL10A1 enhancer. J Cell Biochem. 2002;86:688–99. https://doi.org/10.1002/jcb.10260. Gebhard S, Pöschl E, Riemer S, Bauer E, Hattori T, Eberspaecher H, et al. A highly conserved enhancer in mammalian type X collagen genes drives high levels of tissue-specific expression in hypertrophic cartilage in vitro and in vivo. Matrix Biol. 2004;23:309–22. https://doi.org/10.1016/j.matbio.2004.05.010. Zheng Q, Keller B, Zhou G, Napierala D, Chen Y, Zabel B, et al. Localization of the Cis -Enhancer Element for Mouse Type X Collagen Expression in Hypertrophic Chondrocytes In Vivo. J Bone Miner Res. 2009;24:1022–32. https://doi.org/10.1359/jbmr.081249. Volk SW, Luvalle P, Leask T, Leboy PS. A BMP responsive transcriptional region in the chicken type X collagen gene. Journal of Bone and Mineral Research. 1998;13:1521–9. https://doi.org/10.1359/jbmr.1998.13.10.1521. Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterial1.TableS1.docx SupplementaryMaterial2.FigureS1S2.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 08 May, 2026 Reviewers agreed at journal 05 May, 2026 Reviewers agreed at journal 02 May, 2026 Reviewers agreed at journal 24 Apr, 2026 Reviewers invited by journal 18 Apr, 2026 Editor invited by journal 17 Apr, 2026 Editor assigned by journal 14 Apr, 2026 Submission checks completed at journal 14 Apr, 2026 First submitted to journal 13 Apr, 2026 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. 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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-9410040","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":629663834,"identity":"d60ffd04-1c34-4412-a29d-03722b08d307","order_by":0,"name":"Xiaolong Zhao","email":"","orcid":"","institution":"Guangdong Ocean University","correspondingAuthor":false,"prefix":"","firstName":"Xiaolong","middleName":"","lastName":"Zhao","suffix":""},{"id":629663835,"identity":"61ea33ec-3eb2-4571-bd40-2373889d6b83","order_by":1,"name":"Yunsheng Yang","email":"","orcid":"","institution":"Guangdong Ocean 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03:38:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9410040/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9410040/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107871348,"identity":"86b75d11-0be1-4f55-8861-762ad94d1f42","added_by":"auto","created_at":"2026-04-27 07:48:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":55404,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of genome structure of \u003cem\u003ecol10a1\u003c/em\u003e genes in cobia and eight other teleost species\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eR. canadum\u003c/em\u003e (Gene id: PQ246021), \u003cem\u003eC. striata\u003c/em\u003e (Gene id: 131990873), \u003cem\u003eD. rerio\u003c/em\u003e (Gene id: 555804), \u003cem\u003eE. naucrates\u003c/em\u003e (Gene id: 115038291), \u003cem\u003eL. incognitus\u003c/em\u003e (Gene id: 130125025), \u003cem\u003eO. latipes\u003c/em\u003e (Gene id: 110017749), \u003cem\u003eP. olivaceus\u003c/em\u003e (Gene id: 109632608), \u003cem\u003eS. aureovittata\u003c/em\u003e (Gene id: 130187688),\u003cem\u003e T. jaculatrix\u003c/em\u003e (Gene id: 121199749).\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-9410040/v1/b4c1b571f499284546c411d8.png"},{"id":107871353,"identity":"3080c16a-a197-4759-b34b-c0355ae90bd6","added_by":"auto","created_at":"2026-04-27 07:48:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":14260869,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of collinearity of \u003cem\u003ecol10a1\u003c/em\u003e gene between genomes of cobia and other vertebrate species\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-9410040/v1/1b439eeead4b3c5d33d55e61.png"},{"id":107873029,"identity":"0b257b40-7ef9-4f88-b9a4-e36639c4f1be","added_by":"auto","created_at":"2026-04-27 08:01:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":558620,"visible":true,"origin":"","legend":"\u003cp\u003eNJ-based phylogenetic tree of amino acid sequences of COL10A1 across various vertebrate species\u003c/p\u003e\n\u003cp\u003eThe bootstrap consensus tree inferred from 1000 replicates was taken to represent the evolutionary history of the taxa analyzed.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-9410040/v1/a45dfc90136bd71485f91516.png"},{"id":107871346,"identity":"483dc39c-59f8-4180-a20e-037721f8a3a9","added_by":"auto","created_at":"2026-04-27 07:48:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":12876346,"visible":true,"origin":"","legend":"\u003cp\u003ePrediction of the cobia \u003cem\u003ecol10a1\u003c/em\u003e core promoter and transcription factor binding site in the 2000 bp upstream 5’-flanking region\u003c/p\u003e\n\u003cp\u003eFour core promoters (marked in red) were identified in this promoter region by BDGP, and one transcription start site (marked in blue) was identified by Promoter 2.0. All scores exceeded their respective prediction thresholds (0.8 for BDGP and 1 for Promoter 2.0), indicating a high degree of confidence in these identifications.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-9410040/v1/01fe6e21a14ada0b19c4217f.png"},{"id":108006141,"identity":"0def371b-53be-4066-ba47-904ed807c329","added_by":"auto","created_at":"2026-04-28 12:53:58","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":48430,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of cobia \u003cem\u003ecol10a1\u003c/em\u003e gene promoter activity by the dual luciferase reporter gene system\u003c/p\u003e\n\u003cp\u003eDifferent lowercase letters in the superscript indicate significant differences (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-9410040/v1/ac7011337cc7c8b20c06777d.png"},{"id":107871349,"identity":"0d0e35e3-0ddf-4f56-997b-c2ec9e8f7104","added_by":"auto","created_at":"2026-04-27 07:48:33","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":70311,"visible":true,"origin":"","legend":"\u003cp\u003eExpression pattern of cobia \u003cem\u003ecol10a1\u003c/em\u003e gene\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Tissue expression distribution of cobia \u003cem\u003ecol10a\u003c/em\u003e1 gene. Ba: branchial arch; Br: brain; Ey: eye; Fr: fin rays; Gi: gill; He: heart; Hk: head kidney; In: intestine; Li: liver; Mec: Meckel’s cartilage; Mu: muscle; Ov: ovary; Sc: scales; Sk: skin; Sp: spleen; St: stomach; Te: testis; Tk: trunk kidney; Ve: vertebrae. \u003cstrong\u003eB\u003c/strong\u003e. Expression pattern of \u003cem\u003ecol10a1\u003c/em\u003e gene during cobia embryonic development. \u003cstrong\u003eC\u003c/strong\u003e. Relative expression levels of \u003cem\u003ecol10a1\u003c/em\u003e gene in cobia at different developmental stages. \u003cstrong\u003eD\u003c/strong\u003e. Expression of cobia \u003cem\u003ecol10a1\u003c/em\u003e gene in deformed and normal skeletal tissues. Different lowercase letters in the superscript indicate significant differences (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05); ** indicate statistically significant differences with P values less than 0.05.\u003c/p\u003e","description":"","filename":"Fig.6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9410040/v1/bc48d955ed7606f1a8c668d1.jpg"},{"id":107872235,"identity":"c310e47e-bd25-431c-a2ae-ac8f9b284b12","added_by":"auto","created_at":"2026-04-27 07:56:20","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":785850,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence in situ hybridization of \u003cem\u003ecol10a1\u003c/em\u003emRNA at 7 dph (\u003cstrong\u003eA\u003c/strong\u003e) and 21 dph (\u003cstrong\u003eB\u003c/strong\u003e) in cobia\u003c/p\u003e\n\u003cp\u003e(a, c, e, g): Vertebrae tissue. (b, d, f, h): Head tissue. The \u003cem\u003ecol10a\u003c/em\u003e1 gene is labelled in red and nuclei are blue (DAPI). Ey: Eyes; IMM: Immature chondrocytes; IVL: Intervertebral ligament; MAT: Mature chondrocytes; Mf: Muscle fibers; Nsc: Notochord sheath cells; V: Vacuole. Scale bar = 100 um.\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-9410040/v1/3ed085e470e26c401303d95e.png"},{"id":107871355,"identity":"5eca991d-42c4-4da2-80c8-37f3eed9c018","added_by":"auto","created_at":"2026-04-27 07:48:34","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1060424,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAlkaline phosphatase (ALP) staining of MC3T3-E1 cells following treatment with cobia COL10A1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e. the control group (CG); \u003cstrong\u003eB\u003c/strong\u003e. osteoblasts treated with 1 μg/mL of COL10A1; \u003cstrong\u003eC\u003c/strong\u003e. osteoblasts treated with 10 μg/mL of COL10A1; \u003cstrong\u003eD\u003c/strong\u003e. Average optical density (AOD) of ALP staining. Different lowercase letters in the superscript indicate significant differences (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Fig.8.png","url":"https://assets-eu.researchsquare.com/files/rs-9410040/v1/d6c7b96532388ce0c946a882.png"},{"id":107871278,"identity":"0c42a7a2-9915-43ac-81ce-f209d6ac36eb","added_by":"auto","created_at":"2026-04-27 07:48:01","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":945386,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAlizarin red staining of MC3T3-E1 cells following treatment with cobia COL10A1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e. the control group (CG); \u003cstrong\u003eB\u003c/strong\u003e. osteoblasts treated with 1 μg/mL of COL10A1; \u003cstrong\u003eC\u003c/strong\u003e. osteoblasts treated with 10 μg/mL of COL10A1; \u003cstrong\u003eD\u003c/strong\u003e. Average optical density (AOD) of Alizarin red staining. Different letters indicate a statistically significant difference with \u003cem\u003eP\u003c/em\u003e values less than 0.05.\u003c/p\u003e","description":"","filename":"Fig.9.png","url":"https://assets-eu.researchsquare.com/files/rs-9410040/v1/b1de274143f93949f1be4c5b.png"},{"id":107872165,"identity":"6dc01c15-25a0-41f6-9d21-6df9d205a9e9","added_by":"auto","created_at":"2026-04-27 07:55:47","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":52129,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRelative expression of mineralization-associated genes in MC3T3-E1 cells following treatments with cobia COL10A1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCG, the control group; 1 μg/mL, osteoblasts treated with 1 μg/mL of COL10A1; 10 μg/mL, osteoblasts treated with 10 μg/mL of COL10A1. Different lowercase letters in the superscript indicate significant differences (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Fig.10.png","url":"https://assets-eu.researchsquare.com/files/rs-9410040/v1/b27ab27a45321fb957322eb5.png"},{"id":108009371,"identity":"a7d73f92-7e18-46d1-8eb7-dc8139e7e5fe","added_by":"auto","created_at":"2026-04-28 13:10:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":27223637,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9410040/v1/d06a6cdd-595f-449f-bbfd-e69d90213b27.pdf"},{"id":107872287,"identity":"934695bf-0cde-4767-8bb6-d02277bc4e88","added_by":"auto","created_at":"2026-04-27 07:56:29","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":21080,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial1.TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-9410040/v1/dd6d080ea7903b5228e36a02.docx"},{"id":107871225,"identity":"4947724b-1e08-46f2-8622-18fe5fcaa476","added_by":"auto","created_at":"2026-04-27 07:47:39","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1264676,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial2.FigureS1S2.docx","url":"https://assets-eu.researchsquare.com/files/rs-9410040/v1/0ec14e8253e5a5bf49671c9b.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Characterization, expression profiling, and functional analysis of col10a1 in cobia (Rachycentron canadum)","fulltext":[{"header":"Background","content":"\u003cp\u003eCollagens are the most abundant extracellular matrix components in vertebrate species, playing pivotal roles in various physiological functions and providing mechanical properties to bones, cartilage, tendons, and other tissues [1\u0026ndash;3]. Among the 28 members of the collagen superfamily, type X collagen (COL10) is particularly crucial for skeletal development. Originally discovered in chicken long bones in 1985 [4], its encoded gene, \u003cem\u003ecol10a1\u003c/em\u003e, is shown to be expressed in hypertrophic chondrocytes during endochondral ossification in mammals [5]. Unlike fibrillar collagens, COL10 forms a non-fibrillar network and is vital for initiating calcium precipitation within the cartilaginous extracellular matrix [6].\u003c/p\u003e \u003cp\u003eIn mammals, \u003cem\u003ecol10a1\u003c/em\u003e expression is restricted to hypertrophic chondrocytes [7]. However, Renn and Winkler [8] reported that \u003cem\u003ecol10a1\u003c/em\u003e is absent in chondrocytes but is expressed early in regions of intramembranous and perichondral bone formation within the axial skeleton during medaka (\u003cem\u003eOryzias latipes\u003c/em\u003e) embryogenesis. This suggests that \u003cem\u003ecol10a1\u003c/em\u003e marks pre-osteoblasts in \u003cem\u003eO. latipes\u003c/em\u003e. Furthermore, osteoblasts in both the spotted gar (\u003cem\u003eLepisosteus oculatus\u003c/em\u003e) and zebrafish (\u003cem\u003eDanio rerio\u003c/em\u003e) were shown to express \u003cem\u003ecol10a1\u003c/em\u003e expression [9], highlighting significant differences in \u003cem\u003ecol10a1\u003c/em\u003e expression patterns between teleosts and mammalian species. In addition, vertebrate skeleton formation involves two distinct modes: chondral and intramembranous ossification, and teleosts are no exception [10]. Chondrocytes are crucial for cartilage formation, while osteoblasts are responsible for both chondral and intramembranous ossification. The expression of \u003cem\u003ecol10a1\u003c/em\u003e in both chondrocytes and osteoblasts suggests a potential role for type X collagen in the mineralization of both cartilage and bone in fish [11].\u003c/p\u003e \u003cp\u003eFurther research on vertebral body formation in \u003cem\u003eO. latipes\u003c/em\u003e using a transgenic line expressing \u003cem\u003enlGFP\u003c/em\u003e under the control of the \u003cem\u003ecol10a1\u003c/em\u003e promoter revealed that \u003cem\u003ecol10a1\u003c/em\u003e: \u003cem\u003enlGFP\u003c/em\u003e cells emerge in a segmental pattern within the axial skeleton before the mineralization of the notochordal sheath. These cells persist on the outer surface of the chordal centra during mineralization and subsequent perichordal ossification of the vertebral bodies [12]. Recently, Raman [13] demonstrated that a mutant \u003cem\u003eD. rerio\u003c/em\u003e line for \u003cem\u003ecol10a1\u003c/em\u003e exhibited reduced chondrocranium size and decreased bone mineralization in larvae, as well as reduced vertebral thickness and tissue mineral density in adults. Although these findings have enhanced our understanding of \u003cem\u003ecol10a1\u003c/em\u003e\u0026rsquo;s role in regulating skeleton development, most of the aforementioned research was conducted on model organisms in fish (zebrafish in Cypriniformes and medaka in Beloniformes). However, there is still limited knowledge regarding the genetic characteristics and functions of \u003cem\u003ecol10a1\u003c/em\u003e in other teleost taxa, particularly the Perciformes, which represent a taxonomically distinct group both morphologically and phylogenetically.\u003c/p\u003e \u003cp\u003eThe cobia (\u003cem\u003eRachycentron canadum\u003c/em\u003e), a marine teleost belonging to Rachycentridae family within Perciformes order, is widely distributed across tropical, subtropical, and warm temperate seas globally [14]. In this study, we cloned the sequence of cobia \u003cem\u003ecol10a1\u003c/em\u003e gene and utilized it for gene structure and phylogenetic analysis. Furthermore, the promoter activity of the 5\u0026prime;-flanking region of \u003cem\u003ecol10a1\u003c/em\u003e gene was assessed using Luciferase reporter assays to investigate its transcriptional regulatory mechanisms. Tissue distribution of this gene was explored through quantitative real-time PCR (qRT-PCR) and fluorescence in situ hybridization (FISH). Additionally, the expression pattern of \u003cem\u003ecol10a1\u003c/em\u003e across developmental stages, from embryos to juveniles, has also been investigated. Finally, we evaluated the functions of cobia \u003cem\u003ecol10a1\u003c/em\u003e by expressing and purifying the protein in a prokaryotic system, followed by analyzing its effect on calcification and the expression of mineralization-associated genes in murine osteoblastic cells.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSample collection\u003c/h2\u003e \u003cp\u003eCobia samples used in this study were obtained from the marine biology research base of Guangdong Ocean University (Zhanjiang, China). They are categorized as follows: (1) Embryos: a total of 30 embryos were collected at each of the following developmental stages: one-cell, two-cell, four-cell, eight-cell, 16-cell, multi-cell, blastula, gastrula, neurula, organogenesis, and hatching. These stages were determined based on the criteria established by Kuang [15]. (2) Larvae and juveniles: six individuals were sampled at every other day from 1 to 15 days post hatching (dph). Additional samples were collected at 18, 20, 25, and 30 dph. (3) Adults: three females and three males, with an average body weight of 4410.7\u0026thinsp;\u0026plusmn;\u0026thinsp;409.9 g and an average body length of 64.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2 cm, were selected for tissue sampling. Nineteen tissues were sampled, including brain, branchial arch, eye, fin rays, gill, head kidney, heart, intestine, liver, Meckel\u0026rsquo;s cartilage, muscle, ovary, scales, skin, spleen, stomach, testis, trunk kidney, and vertebrae. In addition, as reported by Ma [16], samples of Mickel's cartilage and vertebrae were collected from three fish displaying jaw deformities or spinal curvatures. Prior to sampling, all fish were anesthetized and euthanized using an overdose eugenol (Shanghai Macklin Biochemical Technology Co., Ltd, China). Subsequently, the whole fish or the dissected tissues were immediately frozen in liquid nitrogen and stored in a refrigerator at \u0026minus;\u0026thinsp;80\u0026deg;C. To elucidate the tissue distribution of \u003cem\u003ecol10a1\u003c/em\u003e during early developmental stages, cobia larvae were preserved in 4% paraformaldehyde (PFA) at 4\u0026deg;C for 24 h, followed by storage in 75% ethanol until further use for paraffin section and in situ hybridization.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTotal RNA extraction and first-strand cDNA synthesis\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted using TRIzol reagent (Invitrogen, USA) following the manufacturer\u0026rsquo;s protocol. The quality and concentration of the extracted RNA were assessed using the Bioanalyzer 2100 (Agilent Technologies, USA) and the NanoDrop 2000 (Thermo Fisher Scientific, USA). Subsequently, 1 \u0026micro;g of the total RNA was used to synthesize cDNA with the EasyScript\u0026reg; First-Strand cDNA Synthesis SuperMix (TransGen Biotech, China), according to the manufacturer's instructions.\u003c/p\u003e\n\u003ch3\u003eDNA extraction\u003c/h3\u003e\n\u003cp\u003eMuscle samples were used for DNA extraction using a marine animal genomic DNA extraction kit (TransGen Biotech, China) according to the manufacturer's instructions.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCloning of cobia\u003c/b\u003e \u003cb\u003ecol10a1\u003c/b\u003e \u003cb\u003egene and sequence analysis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe entire gene sequence of the cobia \u003cem\u003ecol10a1\u003c/em\u003e along with its upstream 2000 bp was extracted from the cobia genome dataset (PRJNA634421). To ensure sequence accuracy, primers for amplifying various length fragments of the 5\u0026rsquo;-flanking region and the CDS were designed using Primer premier 6.0 software (Supplementary Table\u0026nbsp;1, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). PCR amplification was then conducted using these primers following the protocol of Accurate Taq Master Mix (Accurate Biology, China). The PCR amplification products were subsequently analyzed using 1.2% (mass fraction) agarose gel electrophoresis, and then sequenced by Shanghai Sangon Biotech Co., Ltd. (Shanghai, China).\u003c/p\u003e \u003cp\u003eTo determine the chromosomal location of the cobia \u003cem\u003ecol10a1\u003c/em\u003e gene, both the genome annotation data and the \u003cem\u003ecol10a1\u003c/em\u003e gene sequence were imported into TBtools software 2.027 [17]. The exon/intron structures were visualized utilizing the Gene Structure Display Server (GSDS, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gsds.cbi.pku.edu.cn\u003c/span\u003e\u003cspan address=\"http://gsds.cbi.pku.edu.cn\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) by aligning the coding sequence with the corresponding genomic DNA sequences. Using DNAMAN software, the deduced amino acid sequence of cobia \u003cem\u003ecol10a1\u003c/em\u003e was obtained and employed for homology comparison and identity analysis. Corresponding COL10A1 amino acid sequences from other vertebrate species were retrieved from the NCBI GenBank (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/genbank/\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/genbank/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Multiple sequence alignment of COL10A1 was conducted using DNAMAN software. Subsequently, phylogenetic analysis was performed using the neighbor-joining (NJ) method in MEGA11 software. The resulting phylogenetic tree was visualized using the Chiplot website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.chiplot.online/tvbot.html\u003c/span\u003e\u003cspan address=\"https://www.chiplot.online/tvbot.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The whole-genome sequences and their corresponding gene annotation files for \u003cem\u003eD. rerio\u003c/em\u003e, \u003cem\u003eO. latipes\u003c/em\u003e, \u003cem\u003eEcheneis naucrates\u003c/em\u003e, and \u003cem\u003eScyliorhinus canicula\u003c/em\u003e were obtained from the NCBI database. Subsequently, an interspecies collinearity analysis of the \u003cem\u003ecol10a1\u003c/em\u003e genes was carried out among cobia and these teleost species, utilizing the MCScanX plugin integrated within the TBtools software.\u003c/p\u003e \u003cp\u003eFurthermore, the physicochemical properties of cobia COL10A1, including its molecular weight and isoelectric point, were analyzed using the ExPASy tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://web.expasy.org/protparam/\u003c/span\u003e\u003cspan address=\"http://web.expasy.org/protparam/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The secondary and tertiary structures of COL10A1 were predicted using SOPMA (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html\u003c/span\u003e\u003cspan address=\"https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and SWISS (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://swissmodel.expasy.org/\u003c/span\u003e\u003cspan address=\"http://swissmodel.expasy.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo predict potential transcriptional start sites of the cobia \u003cem\u003ecol10a1\u003c/em\u003e gene, the online analysis software BDGP (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.fruitfly.org/seq_tools/other.html\u003c/span\u003e\u003cspan address=\"https://www.fruitfly.org/seq_tools/other.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and Promoter 2.0 Prediction Server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.cbs.dtu.dk/services/Promoter/\u003c/span\u003e\u003cspan address=\"http://www.cbs.dtu.dk/services/Promoter/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) were utilized. Furthermore, AliBaba2.1 software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gene-regulation.com/pub/programs/alibaba2/index.html\u003c/span\u003e\u003cspan address=\"http://gene-regulation.com/pub/programs/alibaba2/index.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to identify potential transcription factor binding sites for the \u003cem\u003ecol10a1\u003c/em\u003e gene.\u003c/p\u003e\n\u003ch3\u003eQuantitative real-time PCR (qRT-PCR)\u003c/h3\u003e\n\u003cp\u003eThe specific primer sequences for the \u003cem\u003ecol10a1\u003c/em\u003e gene and the internal control \u003cem\u003eβ-actin\u003c/em\u003e used in qRT-PCR are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The qRT-PCR reactions were carried out in a final volume of 20 \u0026micro;L using the PerfectStart\u0026trade; Green qPCR SuperMix Kit (TransGen Biotech, China) on the LightCycler 96 system (Roche, Switzerland). Subsequently, a dissociation protocol was executed to further confirm target specificity. PCR efficiency was determined based on the slopes of standard curves generated from serial 10-fold dilutions of cDNA samples, with an acceptable efficiency range of 90% to 110%. Correlation coefficients (R\u003csup\u003e2\u003c/sup\u003e) were calculated using linear regression analysis in SPSS 26.0. The mRNA levels of each sample were normalized to the \u003cem\u003eβ-actin\u003c/em\u003e levels. The relative changes in mRNA expression were calculated using the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003emethod. All qRT-PCR reactions were performed in triplicate to ensure accuracy and reproducibility.\u003c/p\u003e \u003cp\u003e \u003cb\u003eParaffin section, Masson's trichome, and fluorescence in situ hybridization (FISH) of cobia\u003c/b\u003e \u003cb\u003ecol10a1\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFixed larval and juvenile samples were dehydrated in an ethanol series, and embedding in paraffin. Longitudinal 4 \u0026micro;m sections were cut and stained by Masson's trichome (Servicebio, China) to identify collagen. FISH probe was synthesized using a multi-sequence hybrid probe by Servicebio Inc. (Servicebio, China). Fluorescence in situ hybridization was performed with a FISH kit (Servicebio, Wuhan, China) according to the manufacturer\u0026rsquo;s protocol. Images were acquired using ortho-fluorescent microscope (Nikon Eclipse ci, Japan) and the Nikon DS-U3 imaging system. The probe sequences were shown in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRecombinant vector construction of fragments in 5\u0026rsquo;-flanking region of cobia\u003c/b\u003e \u003cb\u003ecol10a1\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo construct recombinant vectors for fragments in the 5'-flanking region of cobia \u003cem\u003ecol10a1\u003c/em\u003e, all primers were designed with tailing sequences specific for either the NheI site (for forward primers) or the HindIII site (for reverse primers), as detailed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Following amplification, the DNA fragments were digested using NheI and HindIII restriction enzymes (Takara Bio, China). These fragments were then inserted into the pGL3-basic vector (Promega, United States) and subsequently sequenced for verification. Once the sequences were confirmed, the plasmids, designated as pGL3-col10a1-P1 through pGL3-col10a1-P4, were extracted utilizing an endotoxin-free plasmid bulk extraction kit (Sangon Biotech, China).\u003c/p\u003e\n\u003ch3\u003eCell Culture, Transfection, and Dual Luciferase Assay\u003c/h3\u003e\n\u003cp\u003eHEK 293T cells were cultivated and subsequently transfected following the method described by Ma [18]. In brief, the transfection process involved co-introducing 100 ng of the promoter reporter plasmid along with the internal control vector, pGL3-Basic, into the cells. Luciferase activity was quantified using the Dual Luciferase Reporter Assay System E2940 (Promega, USA) according to the manufacturer's instructions. The activity of each promoter construct was determined by calculating the luminescence ratio of firefly luciferase to renilla luciferase. Each experimental group underwent three replicates to ensure accuracy [8].\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eProkaryotic Expression and Protein Purification of cobia COL10A1\u003c/h2\u003e \u003cp\u003eThe primers used for amplification of cobia COL10A1, which included NdeI and HindIII restriction sites along with protective bases, are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Following sequencing and verification, the recombinant cloning vector pMD19-T-col10a1 was constructed. Both the pET-32a vector and the pMD19-T-col10a1 were subsequently subjected to double digestion using NdeI and HindIII enzymes (Takara Bio, China). The recovered fragments were then ligated overnight at 16\u0026deg;C using T4 DNA ligase. The resulting expression vector, named pET-30a-COL10A1, was transformed into the DH5α cloning strain (TransGen Biotech, China). Positive clones were identified by colony PCR, and the correctness of the construct was confirmed by sequencing (Sangon biotech, China).\u003c/p\u003e \u003cp\u003eSubsequently, the recombinant plasmid pET-30a-COL10A1 was transformed into the BL21 (DE3) strain (Sangon biotech, China) for expression. The procedures for transformation, expression, and purification of the recombinant fusion proteins were conducted following the methodology described by Li [19].The concentration of the purified protein was determined using the Bradford protein assay, with BSA serving as the standard (Sangon biotech, China). The purity and molecular weight of the protein were assessed by standard sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis for confirmation.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCultivation of MC3T3-E1 cells and Treatment with cobia COL10A1\u003c/h3\u003e\n\u003cp\u003eMurine osteoblastic cell MC3T3-E1 were acquired from Pricella Biotechnology Co., Ltd., (Wuhan, China) and cultivated in α-MEM medium supplemented with 10% fetal bovine serum (Pricella Biotechnology Co., Ltd, Wuhan, China). The cells were incubated at 37 ℃ in an atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eFor the experiment, passage 3 MC3T3-E1 cells were seeded into a 48-well plate at a density of 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well and cultured for 24 h. Subsequently, the medium was replaced with osteogenic differentiation induction medium (Pricella Biotechnology Co., Ltd, Wuhan, China) containing cobia COL10A1 protein with a purity of \u0026ge;\u0026thinsp;90%. The cobia COL10A1 protein was added at concentrations of 0 \u0026micro;g/mL, 1 \u0026micro;g/mL, and 10 \u0026micro;g/mL, respectively. Four replicate wells were established for each concentration, and the medium was changed every 3 days.\u003c/p\u003e\n\u003ch3\u003eDetermination of Alkaline phosphatase (ALP) Activity and Calcification\u003c/h3\u003e\n\u003cp\u003eAfter 7 days of treatment, alkaline phosphatase (ALP) staining was performed using an ALP staining kit (Puhe Biological Co., Ltd., Wuxi, China). Furthermore, alizarin red staining was conducted using alizarin red S staining solution from the same company, following 14 days of treatment. The procedures for both staining experiments were carried out in accordance with the manufacturer's instructions. Images were captured using a stereoscopic microscope (M205 FCA, Leica, Germany). Image J software was utilized to determine the average optical density (AOD) value, which indicates the concentration of blue-purple precipitate per unit area in the ALP staining results. Similarly, AOD value of red calcified nodules per unit area in the Alizarin red staining results were analyzed to detect the calcification of cells.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of Gene Expression in MC3T3-E1 cells\u003c/h2\u003e \u003cp\u003eMC3T3-E1 cells were treated with cobia COL10A1 for 7 days, following which they were collected for RNA extraction. Subsequently, cDNA synthesis and qRT-PCR were carried out as previously described. The primers utilized for detectin\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eg\u003c/span\u003e of mouse skeleton formation-related genes, including Runt-related transcription factor 2 (\u003cem\u003erunx2\u003c/em\u003e), \u003cem\u003eosterix\u003c/em\u003e, Osteocalcin (\u003cem\u003eocn\u003c/em\u003e), Alkaline phosphatase (\u003cem\u003ealp\u003c/em\u003e) with glyceraldehyde-3-phosphate dehydrogenase (\u003cem\u003egapdh\u003c/em\u003e) serving as the internal reference, are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe statistical analysis was performed using one-way ANOVA in SPSS 20.0. Data were expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05 was considered to indicate a significant difference.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCharacteristics of cobia COL10A1\u003c/h2\u003e \u003cp\u003eThe CDS of cobia \u003cem\u003ecol10a1\u003c/em\u003e is 1977 base pairs (bp) in length and encodes a protein comprising 659 amino acids (aa). Analysis of the predicted physical and chemical properties of this protein revealed a relative molecular mass of 63.34 kDa and an isoelectric point (pI) of 9.22. As illustrated in Supplementary Fig.\u0026nbsp;1A (Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA), the predicted secondary structure of cobia COL10A1 reveals that it is composed of 6.69% α-helix, 6.23% β-turn, 77.05% random coil, and 10.03% extended strand. The tertiary structure of the COL10A1 protein, as presented in Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD, is similar with the secondary structure prediction, with the random coil conformation being the most dominant. In addition, the COL10A1 proteins of mouse (\u003cem\u003eMus musculus\u003c/em\u003e), \u003cem\u003eD. rerio\u003c/em\u003e and cobia revealed similar structure.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGene structure, chromosome localization and Phylogenetic analysis of cobia\u003c/b\u003e \u003cb\u003ecol10a1\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e, cobia \u003cem\u003ecol10a1\u003c/em\u003e gene consists of two exons (120 and 1857 bp) and one intron (2608 bp). A comparative analysis of the \u003cem\u003ecol10a1\u003c/em\u003e gene sequences across several species showed that the exon-intron organization of the cobia \u003cem\u003ecol10a1\u003c/em\u003e gene is similar to that of the \u003cem\u003ecol10a1\u003c/em\u003e genes in \u003cem\u003eCentropristis striata\u003c/em\u003e and \u003cem\u003eLampris incognitus\u003c/em\u003e. However, the \u003cem\u003ecol10a1\u003c/em\u003e gene in six other teleost species, such as \u003cem\u003eEcheneis naucrates\u003c/em\u003e, \u003cem\u003eSeriola aureovittata\u003c/em\u003e, and \u003cem\u003eParalichthys olivaceus\u003c/em\u003e, comprises three exons and two introns (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eR. canadum\u003c/em\u003e (Gene id: PQ246021), \u003cem\u003eC. striata\u003c/em\u003e (Gene id: 131990873), \u003cem\u003eD. rerio\u003c/em\u003e (Gene id: 555804), \u003cem\u003eE. naucrates\u003c/em\u003e (Gene id: 115038291), \u003cem\u003eL. incognitus\u003c/em\u003e (Gene id: 130125025), \u003cem\u003eO. latipes\u003c/em\u003e (Gene id: 110017749), \u003cem\u003eP. olivaceus\u003c/em\u003e (Gene id: 109632608), \u003cem\u003eS. aureovittata\u003c/em\u003e (Gene id: 130187688), \u003cem\u003eT. jaculatrix\u003c/em\u003e (Gene id: 121199749).\u003c/p\u003e \u003cp\u003eThe collinearity analysis presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e reveals that cobia \u003cem\u003ecol10a1\u003c/em\u003e gene is situated on chromosome 18. This gene demonstrates collinear relationships with chromosome 24 in both \u003cem\u003eE. naucrates\u003c/em\u003e and \u003cem\u003eO. latipes\u003c/em\u003e, chromosome 6 in \u003cem\u003eScyliorhinus canicula\u003c/em\u003e, as well as with two chromosomes (Chr17 and Chr20) in \u003cem\u003eD. rerio\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA phylogenetic tree based on the amino acid sequences of COL10A1 demonstrated that cobia COL10A1 firstly clusters with \u003cem\u003eE. naucrates\u003c/em\u003e, and then groups with \u003cem\u003eS. aureovittata\u003c/em\u003e, \u003cem\u003eP. olivaceus\u003c/em\u003e, and \u003cem\u003eThunnus albacares\u003c/em\u003e, forming a distinct clade. This clade subsequently clusters with other fish species, Amphibia (\u003cem\u003eXenopus laevis\u003c/em\u003e), Aves (\u003cem\u003eGallus gallus\u003c/em\u003e), and Mammalian (\u003cem\u003eHomo sapiens\u003c/em\u003e and \u003cem\u003eM. musculus\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe bootstrap consensus tree inferred from 1000 replicates was taken to represent the evolutionary history of the taxa analyzed.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCloning and bioinformatics analysis of the promoter sequence of the cobia\u003c/b\u003e \u003cb\u003ecol10a1\u003c/b\u003e \u003cb\u003egene\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA 2000 bp sequence upstream from the 5\u0026prime;-flanking of the \u003cem\u003ecol10a1\u003c/em\u003e gene in cobia was used as the promoter region, ranging from \u0026minus;\u0026thinsp;1800 bp upstream to +\u0026thinsp;200 bp downstream of the transcription start site (TSS), was amplified by PCR. Bioinformatics analysis identified four core promoters within this promoter region using BDGP, and determined one transcription start site using Promoter 2.0, which are listed in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFour core promoters (marked in red) were identified in this promoter region by BDGP, and one transcription start site (marked in blue) was identified by Promoter 2.0. All scores exceeded their respective prediction thresholds (0.8 for BDGP and 1 for Promoter 2.0), indicating a high degree of confidence in these identifications.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePredictive analysis of potential transcriptional binding sites in the\u003c/b\u003e \u003cb\u003ecol10a1\u003c/b\u003e \u003cb\u003egene\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe potential transcription factor binding sites within the cobia \u003cem\u003ecol10a1\u003c/em\u003e gene were predicted, and the results revealed a total of 14 transcription factor binding sites in the promoter region of the cobia \u003cem\u003ecol10a1\u003c/em\u003e gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Notably, three primary transcription factors, namely Sp1, AP1, and Oct-1, were found to be predominantly represented among these binding sites.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of the promoter activity of cobia\u003c/b\u003e \u003cb\u003ecol10a1\u003c/b\u003e \u003cb\u003egene\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, the dual luciferase reporter gene system revealed that among four constructed recombinant plasmids with cobia \u003cem\u003ecol10a1\u003c/em\u003e promoter fragments, they all exhibited significantly higher activities than the control pGL3-Basic. In addition, a significantly higher expression in pGL3-col10a1-P1 was identified compared to the other three plasmids, which located at -1800 to -1300 bp (relative to the transcription start site).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDifferent lowercase letters in the superscript indicate significant differences (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003cb\u003eExpression pattern of cobia\u003c/b\u003e \u003cb\u003ecol10a1\u003c/b\u003e \u003cb\u003egene\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, the \u003cem\u003ecol10a1\u003c/em\u003e gene was expressed in all 19 tissues examined in cobia, with the highest expression levels observed in skeletal tissues, namely scales, Meckel's cartilage, and fin rays. The expression of \u003cem\u003ecol10a1\u003c/em\u003e in the muscle and heart were significantly lower than that in the bony tissues. Furthermore, these expression levels were significantly higher compared to those in the other 14 tissues (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eDuring the initial stages of cobia embryonic development, ranging from the 2-cell stage to the 16-cell stage, the expression of \u003cem\u003ecol10a1\u003c/em\u003e was low and showed no significant variations across the developmental time points. However, a significant up-regulation of \u003cem\u003ecol10a1\u003c/em\u003e was observed beginning at the multi-cell stage, with the highest level detected at the organogenesis stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eThe expression of \u003cem\u003ecol10a1\u003c/em\u003e in cobia larvae constantly increased from 1 dph and peaked at 9 dph (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Subsequently, a significant decrease in \u003cem\u003ecol10a1\u003c/em\u003e expression was observed at 11 dph, and this level of expression remained relatively stable until 30 dph.\u003c/p\u003e \u003cp\u003eAs illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, the expression level of \u003cem\u003ecol10a1\u003c/em\u003e in the deformed Meckel\u0026rsquo;s cartilage of cobia was significantly lower than that in the normal tissue (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, no significant difference was observed between the deformed and normal vertebrae tissues (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eA.\u003c/b\u003e Tissue expression distribution of cobia \u003cem\u003ecol10a\u003c/em\u003e1 gene. Ba: branchial arch; Br: brain; Ey: eye; Fr: fin rays; Gi: gill; He: heart; Hk: head kidney; In: intestine; Li: liver; Mec: Meckel\u0026rsquo;s cartilage; Mu: muscle; Ov: ovary; Sc: scales; Sk: skin; Sp: spleen; St: stomach; Te: testis; Tk: trunk kidney; Ve: vertebrae. \u003cb\u003eB\u003c/b\u003e. Expression pattern of \u003cem\u003ecol10a1\u003c/em\u003e gene during cobia embryonic development. \u003cb\u003eC\u003c/b\u003e. Relative expression levels of \u003cem\u003ecol10a1\u003c/em\u003e gene in cobia at different developmental stages. \u003cb\u003eD\u003c/b\u003e. Expression of cobia \u003cem\u003ecol10a1\u003c/em\u003e gene in deformed and normal skeletal tissues. Different lowercase letters in the superscript indicate significant differences (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05); ** indicate statistically significant differences with P values less than 0.05.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLocalization of expression of\u003c/b\u003e \u003cb\u003ecol10a1\u003c/b\u003e \u003cb\u003ein cobia larvae\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, the expression of \u003cem\u003ecol10a1\u003c/em\u003e in the vertebrae of cobia larvae (7 dph) was mainly distributed in notochord sheath cells, but absent in vacuolar cells. In the head skeleton, the \u003cem\u003ecol10a1\u003c/em\u003e mRNA can be detected in both the immature and mature chondrocytes. The expression patterns of \u003cem\u003ecol10a1\u003c/em\u003e in the vertebrae of cobia juveniles (21dph) was similar to that observed at 7dph, with hybridization signals primarily localized in the notochord sheath cells during spinal development (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). In the head skeleton, \u003cem\u003ecol10a1\u003c/em\u003emRNA was also detectable in both immature and mature chondrocytes. Moreover, the hybridization signal intensity in mature chondrocytes at 7 dph and 21 dph was significantly lower than that in immature chondrocytes.\u003c/p\u003e \u003cp\u003e(a, c, e, g): Vertebrae tissue. (b, d, f, h): Head tissue. The \u003cem\u003ecol10a\u003c/em\u003e1 gene is labelled in red and nuclei are blue (DAPI). Ey: Eyes; IMM: Immature chondrocytes; IVL: Intervertebral ligament; MAT: Mature chondrocytes; Mf: Muscle fibers; Nsc: Notochord sheath cells; V: Vacuole. Scale bar =\u0026thinsp;100 um.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eALP and Alizarin red staining of MC3T3-E1 cells treated with cobia COL10A1\u003c/h2\u003e \u003cp\u003eThe ALP activity of MC3T3-E1 cells cultured in a differentiation medium containing varying concentrations of COL10A1 for 7 days was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e. The results indicated that the ALP activity in the COL10A1 treatment group at 10 \u0026micro;g/mL was significantly higher compared to the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, no significant difference was observed between the COL10A1 treatment group at 1 \u0026micro;g/mL and the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eA\u003c/b\u003e. the control group (CG); \u003cb\u003eB\u003c/b\u003e. osteoblasts treated with 1 \u0026micro;g/mL of COL10A1; \u003cb\u003eC\u003c/b\u003e. osteoblasts treated with 10 \u0026micro;g/mL of COL10A1; \u003cb\u003eD\u003c/b\u003e. Average optical density (AOD) of ALP staining. Different lowercase letters in the superscript indicate significant differences (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eThe results of Alizarin red staining demonstrated that after 14 days of treatment with cobia COL10A1, the number of calcified nodules was significantly increased in the 10 \u0026micro;g/mL treatment group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, no significant difference was observed in the 1 \u0026micro;g/mL treatment group compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eA\u003c/b\u003e. the control group (CG); \u003cb\u003eB\u003c/b\u003e. osteoblasts treated with 1 \u0026micro;g/mL of COL10A1; \u003cb\u003eC\u003c/b\u003e. osteoblasts treated with 10 \u0026micro;g/mL of COL10A1; \u003cb\u003eD\u003c/b\u003e. Average optical density (AOD) of Alizarin red staining. Different letters indicate a statistically significant difference with \u003cem\u003eP\u003c/em\u003e values less than 0.05.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eEffect of COL10A1 on the expression of mineralization-associated genes in MC3T3-E1 cells\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e, the highest expression level of \u003cem\u003erunx2\u003c/em\u003e, \u003cem\u003eosterix\u003c/em\u003e, and \u003cem\u003ealp\u003c/em\u003e were identified in the 10 \u0026micro;g/mL COL10A1 treatment group. However, the highest expression of \u003cem\u003eocn\u003c/em\u003e was identified in the 1 \u0026micro;g/mL group. For \u003cem\u003erunx2\u003c/em\u003e, \u003cem\u003eosterix\u003c/em\u003e, and \u003cem\u003ealp\u003c/em\u003e, no significant differences were detected between the gene expression in 1 \u0026micro;g/mL COL10A1 treatment group and the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCG, the control group; 1 \u0026micro;g/mL, osteoblasts treated with 1 \u0026micro;g/mL of COL10A1; 10 \u0026micro;g/mL, osteoblasts treated with 10 \u0026micro;g/mL of COL10A1. Different lowercase letters in the superscript indicate significant differences (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePhylogenetic analysis showed that the cobia COL10A1 clade first clusters with those of \u003cem\u003eE. nauerates\u003c/em\u003e, \u003cem\u003eS. aureovittata\u003c/em\u003e, and \u003cem\u003eP. olivaceus\u003c/em\u003e, and subsequently groups with other teleosts and higher vertebrates. This is consistent with traditional taxonomy and evolutionary principles governing vertebrates (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). However, the exon-intron organization of \u003cem\u003ecol10a1\u003c/em\u003e in cobia exhibits a different structure of two exons and one intron, contrasting with the three-exon/two-intron configuration observed in the three aforementioned species (which cluster with cobia). This arrangement is also found in mammalian \u003cem\u003ecol10a\u003c/em\u003e (e.g., \u003cem\u003eM. musculus\u003c/em\u003e) [20]. A similar exon-intron organization was identified in \u003cem\u003eC. striata\u003c/em\u003e and \u003cem\u003eL. incognitus\u003c/em\u003e, which form a distinct group from cobia in the NJ tree (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These findings imply that phylogenetic relationships and gene structures may evolve under different selective pressures.\u003c/p\u003e \u003cp\u003eIn higher vertebrates, such as avians, tetrapods and mammals, COL10 is mainly synthesized by hypertrophic chondrocytes during endochondral ossification, and is absent in osteoblasts and other chondrocyte maturation stages, making it a marker for maturing chondrocytes [21, 22]. It also plays a crucial role in the normal distribution of matrix vesicles and proteoglycans in the calcifying zone of the cartilage growth plate [23].\u003c/p\u003e \u003cp\u003eIn contrast, the major expression sites of COL10 expand in amphibians and fish. In \u003cem\u003eXenopus tropicalis\u003c/em\u003e, \u003cem\u003ecol10a1\u003c/em\u003e is expressed in osteoblasts besides cartilage mineralization [24]. In \u003cem\u003eD. rerio\u003c/em\u003e, \u003cem\u003ecol10a1\u003c/em\u003e is expressed in both osteoblasts and chondrocytes [9]. In \u003cem\u003eO. latipes\u003c/em\u003e, \u003cem\u003ecol10a1\u003c/em\u003e transcription occurs before osterix expression and the bone matrix mineralization, enabling the identification of pre-osteoblasts. Furthermore, the expression of \u003cem\u003ecol10a1\u003c/em\u003e in cartilaginous fish ameloblasts and/or odontoblasts implies its involvement in odontode skeleton (teeth and scales) formation [11].\u003c/p\u003e \u003cp\u003eIn this study, tissue distribution of cobia \u003cem\u003ecol10a1\u003c/em\u003e reveals that it exhibits significantly higher expression in the scales, Meckel\u0026rsquo;s cartilage, and fin rays, with relatively lower expression in vertebrae. Similarly, \u003cem\u003ecol10a1\u003c/em\u003e was also found to be expressed in the vertebrae of flounder, \u003cem\u003eP. olivaceus\u003c/em\u003e [25]. A comparable distribution pattern is reported in \u003cem\u003eO. latipes\u003c/em\u003e, where the gene is expressed in early osteoblasts across most of the skeleton, encompassing head elements, axial skeleton (centrae and neural arches), and fins. Additionally, the possibility that osteoblast-like cells mineralize the sheath before ossifying the perichordal centrum has been proposed[26, 27]. The time-point concurrent of \u003cem\u003ecol10a1\u003c/em\u003e: \u003cem\u003enlGFP\u003c/em\u003e positive cells with the onset of mineralization of the notochordal sheath in \u003cem\u003eO. latipes\u003c/em\u003e make these cells promising candidates for being involved in mineralization of the notochordal sheath [8, 12]. FISH confirms high \u003cem\u003ecol10a1\u003c/em\u003e expression in the notochord sheath cells of cobia larvae, indicating its role in perichondral bone formation. Thus, \u003cem\u003ecol10a1\u003c/em\u003e is expressed in both osteoblasts and chondrocytes across teleosts.\u003c/p\u003e \u003cp\u003eBesides, it has been shown that COL10A1 mutations can cause skeletal disorders like Schmid type metaphyseal chondrodysplasia [28]. The chick COL10 transgene product is linked to craniofacial skeletal abnormalities [29]. These suggests that COL10A1 may play a role in regulating abnormal skeletal development. In cobia with jaw deformities or spinal curvatures, \u003cem\u003ecol10a1\u003c/em\u003e is up-regulated in deformed cartilage but unchanged in vertebrae, indicating its intricate involvement in abnormal cartilage development regulation in teleosts.\u003c/p\u003e \u003cp\u003eIn \u003cem\u003eO. latipes\u003c/em\u003e, \u003cem\u003ecol10a1\u003c/em\u003e transcript commences earlier in developing intramembranous and perichondral bones of the head and trunk before ossification [8]. Thus it has been concluded that \u003cem\u003ecol10a1\u003c/em\u003e marks pre-osteoblasts and osteoblasts. In cobia, \u003cem\u003ecol10a1\u003c/em\u003e has higher expression during late embryonic and early larval periods (from neurula stage to 9 dph), aligning with the onset of skeletal development reported by Mao [30]. FISH analysis of cobia larvae at 7 dph and 21 dph revealed that \u003cem\u003ecol10a1\u003c/em\u003e is present in both mature and immature cartilage, a pattern also observed in other teleosts like zebrafish [9].\u003c/p\u003e \u003cp\u003eIt has been reported that osteoblasts in \u003cem\u003eX. tropicalis\u003c/em\u003e, \u003cem\u003eO. latipes\u003c/em\u003e, \u003cem\u003eD. rerio\u003c/em\u003e and \u003cem\u003eL. oculatus\u003c/em\u003e express \u003cem\u003ecol10a1\u003c/em\u003e [8, 9, 24]. \u003cem\u003eCol10a1a\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mutant \u003cem\u003eD. rerio\u003c/em\u003e larvae display reduced bone mineralization, while adult fish exhibit decreased vertebral thickness and tissue mineral density [13]. These are similar to observations in mice and humans. An accumulation of \u003cem\u003ecol10a1\u003c/em\u003e: \u003cem\u003enlGFP\u003c/em\u003e positive cells is found in areas of increased mineralization during notochordal sheath repair [12]. In this study, we obtain cobia COL10A1 proteins through prokaryotic expression and purification. Treating murine osteoblastic cells with cobia COL10A1 enhanced ALP activity and alizarin red staining intensity, indicating its role in promoting active mineralization and creating a suitable environment for mineralization and bone remodeling [31]. The impact of cobia COL10A1 on murine cells supports that both the triple-helical domain and the C-terminal domain are highly conserved across all species, thereby implying the preservation of COL10A1's protein functions [8].\u003c/p\u003e \u003cp\u003eOur study shows cobia COL10A1 significantly elevates \u003cem\u003erunx2\u003c/em\u003e and \u003cem\u003eosterix\u003c/em\u003e mRNA levels in murine osteoblastic cells. \u003cem\u003eRunx2\u003c/em\u003e is crucial for the development of osteogenic lineage in mammals, birds, and teleosts such as \u003cem\u003eD. rerio\u003c/em\u003e [32, 33]. \u003cem\u003eSp7\u003c/em\u003e/\u003cem\u003eosterix\u003c/em\u003e is involved in multiple aspects of bone development, including the differentiation and maturation of osteoblasts, as well as the formation of osteocytes. Notably, \u003cem\u003ecol10a1\u003c/em\u003e is the target gene of \u003cem\u003eosterix\u003c/em\u003e in osteoblasts [34], with a positive correlation between them. The up-regulation of \u003cem\u003erunx2\u003c/em\u003e and \u003cem\u003eosterix\u003c/em\u003e in MC3T3-E1 cells treated with COL10A1 supports a positive regulatory relationship and highlights its impact on osteoblast differentiation [13].\u003c/p\u003e \u003cp\u003eComparative sequence analysis reveals high promoter conservation among mammalian species near the transcription start site (ranging from \u0026minus;\u0026thinsp;120 to +\u0026thinsp;1 bp), likely serving as the basal promoter [35]. Using the luciferase reporter gene system, we identified a potential promoter region in cobia \u003cem\u003ecol10a1\u003c/em\u003e (-300 to +\u0026thinsp;200 bp) with multiple potential regulatory elements like AP1, Sp1, c-Jun, and c-Fos [36]. AP1, composed of Jun and Fos proteins [37], is crucial for bone formation and cartilage development [22]. In addition, c-Jun is highly expressed in hypertrophic chondrocytes, and c-Fos may suppress \u003cem\u003ecol10a1\u003c/em\u003e expression in a tissue-specific manner [38]. Collectively, these findings suggest that AP1 elements possess a silencing activity on \u003cem\u003ecol10a1\u003c/em\u003e [39, 40].\u003c/p\u003e \u003cp\u003eThe regulation of type X collagen expression is crucial for its role in endochondral ossification, particularly in hypertrophic chondrocytes. Enhancer elements play a significant role in this regulation, ensuring tissue-specific expression [22]. In studies of human enhancer activity, multiple luciferase reporter constructs were prepared using different fragments of the COL10A1 promoter and transfected into hypertrophic chondrocytes. The results indicate that enhancer activity was localized to the region upstream of the transcription start site, spanning\u0026thinsp;\u0026minus;\u0026thinsp;2407 bp to -1870 bp [38]. In the chicken \u003cem\u003ecol10a1\u003c/em\u003e gene, an additional enhancer element resides within the region spanning\u0026thinsp;\u0026minus;\u0026thinsp;2.9 kb to -2.4 kb upstream of the transcription start site [41]. In the bovine \u003cem\u003ecol10a1\u003c/em\u003e gene, the enhancer element is located within the region extending from \u0026minus;\u0026thinsp;3381 bp to -2875 bp upstream of the transcription start site. The mouse enhancer element resides within the Col10a1 distal promoter region (-4.4 kb to -3.8 kb) and mediates high levels of hypertrophic chondrocyte-specific expression both in vitro and in vivo [39].\u003c/p\u003e \u003cp\u003eWhat's more, the enhancer element within the mouse Col10a1 distal promoter exhibits high homology with enhancers from human and bovine sources. Further experiments confirmed that transgenic constructs containing the 600 bp enhancer element mediated high reporter activity, whereas vector controls without the enhancer produced only low reporter activity in hypertrophic chondrocytes [40]. In this study, we identified a construct, designated as pGL3-col10a1-P4, which encompasses a potential promoter region. Concurrently, another distinct region (represented by the construct pGL3-col10a1-P1), spanning from \u0026minus;\u0026thinsp;1800 to -1300 bp, exhibits a significantly higher luciferase activity than pGL3-col10a1-P4, suggesting the existence of an enhancer element within this specific region.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eWe present here a comprehensive report on the cobia \u003cem\u003ecol10a1\u003c/em\u003e gene, encompassing its full-length sequence, chromosome localization, gene structure, phylogenetic relationship, and potential transcriptional region. Moreover, we have elucidated the expression pattern of this gene, examining its tissue distribution, expression across various developmental stages, and its association with skeletal deformities. Our findings confirm that this gene plays a pivotal role in both cartilage and bone development, aligning with recently published data on freshwater species. Additionally, the cobia COL10A1 protein has been demonstrated to regulate the mineralization process of murine osteoblastic cells. This evidence further supports the notion that the \u003cem\u003ecol10a1\u003c/em\u003e gene and its protein product are crucial for skeletal development and mineralization across vertebrates.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAmino Acids\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eALP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAlkaline Phosphatase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAOD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAverage Optical Density\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBase Pairs\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCDS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eThe Coding Sequence\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCOL10\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCollagen Type X\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDPH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDays Post Hatching\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGAPDH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGlyceraldehyde-3-Phosphate Dehydrogenase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNJ\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNeighbor-Joining\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOCN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOsteocalcin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePFA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eParaformaldehyde\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePoint\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eQRT-PCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eQuantitative Real-Time PCR\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFISH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFluorescence in Situ Hybridization\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRUNX2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRunt-Related Transcription Factor 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSDS-PAGE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTSS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTranscription Start Site\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003e All animal experiments in this study were handled in strict accordance with the ARRIVE guidelines and China legislation on scientific procedures on living animals. The protocol was approved by the Animal Research and Ethics Committees of Guangdong Ocean University.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was funded by Guangdong Basic and Applied Basic Research Foundation (grant number 2022A1515012244) and the National Natural Science Foundation of China (grant number 32373153).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eQ. Ma performed project conception and experiment design; X.L. Zhao, Y.S. Yang, Y.W. Wu, Z. Zheng, W.J. Guo and D.H. Huang conducted the experiments and analyzed the data; X.L. Zhao and Q. Ma wrote the original manuscript and thoroughly revised and finalized the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe sequence data that support the findings of this study are openly available in GenBank of NCBI at under accession PQ246021. The dataset supporting the conclusions of this article is available in the NCBI Short Read Archive (SRA) repository, as BioProject PRJNA634421.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBretaud S, Nauroy P, Malbouyres M, Ruggiero F. Fishing for collagen function: About development, regeneration and disease. Semin Cell Dev Biol. 2019;89:100\u0026ndash;8. https://doi.org/10.1016/j.semcdb.2018.10.002.\u003c/li\u003e\n\u003cli\u003eGordon MK, Hahn RA. Collagens. Cell Tissue Res. 2010;339:247\u0026ndash;57. https://doi.org/10.1007/s00441-009-0844-4.\u003c/li\u003e\n\u003cli\u003eIkoma T, Kobayashi H, Tanaka J, Walsh D, Mann S. Microstructure, mechanical, and biomimetic properties of fish scales from Pagrus major. J Struct Biol. 2003;142:327\u0026ndash;33. https://doi.org/10.1016/S1047-8477(03)00053-4.\u003c/li\u003e\n\u003cli\u003eSchmid TM, Linsenmayer TF. Developmental acquisition of type X collagen in the embryonic chick tibiotarsus. Dev Biol. 1985;107:373\u0026ndash;81. https://doi.org/10.1016/0012-1606(85)90319-7.\u003c/li\u003e\n\u003cli\u003eKwan KM, Pang MK, Zhou S, Cowan SK, Kong RY, Pfordte T, et al. Abnormal compartmentalization of cartilage matrix components in mice lacking collagen X: implications for function. J Membr Biol. 1997;136:459\u0026ndash;71. https://doi.org/10.1083/jcb.136.2.459.\u003c/li\u003e\n\u003cli\u003eKirsch T, Wuthier RE. Stimulation of calcification of growth plate cartilage matrix vesicles by binding to type II and X collagens. J Biol Chem. 1994; 269:11462\u0026ndash;9. https://doi.org/10.1016/S0021-9258(19)78146-0.\u003c/li\u003e\n\u003cli\u003eZheng Q, Zhou G, Morello R, Chen Y, Garcia-Rojas X, Lee B. Type X collagen gene regulation by Runx2 contributes directly to its hypertrophic chondrocyte\u0026ndash;specific expression in vivo. J Cell Biol. 2003; 162(5): 833-842. https://doi.org/10.1083/jcb.200211089.\u003c/li\u003e\n\u003cli\u003eRenn J, Winkler C. Characterization of collagen type 10a1 and osteocalcin in early and mature osteoblasts during skeleton formation in medaka. J Appl Ichthyol. 2010;26:196\u0026ndash;201. https://doi.org/10.1111/j.1439-0426.2010.01404.x.\u003c/li\u003e\n\u003cli\u003eEames BF, Amores A, Yan Y-L, Postlethwait JH. Evolution of the osteoblast: skeletogenesis in gar and zebrafish. BMC Evol Biol. 2012;12:27. https://doi.org/10.1186/1471-2148-12-27.\u003c/li\u003e\n\u003cli\u003eRenn J, Winkler C. Osterix-mCherry Transgenic Medaka for In ViVo Imaging of Bone Formation. Dev Dyn. 2009;238:241\u0026ndash;8. https://doi.org/10.1002/dvdy.21836.\u003c/li\u003e\n\u003cli\u003eDebiais-Thibaud M, Simion P, Venteo S, Munoz D, Marcellini S, Mazan S, et al. Skeletal Mineralization in Association with Type X Collagen Expression Is an Ancestral Feature for Jawed Vertebrates. Mol Biol Evol. 2019;36:2265\u0026ndash;76. https://doi.org/10.1093/molbev/msz145.\u003c/li\u003e\n\u003cli\u003eRenn J, Buettner A, Thuy TT, Chan SJH, Winkler C. A col10a1:nlGFP transgenic line displays putative osteoblast precursors at the medaka notochordal sheath prior to mineralization. Dev Biol. 2013;381:134\u0026ndash;43. https://doi.org/10.1016/j.ydbio.2013.05.030.\u003c/li\u003e\n\u003cli\u003eRaman R, Antony M, Nivelle R, Lavergne A, Zappia J, Guerrero-Limon G, et al. The Osteoblast Transcriptome in Developing Zebrafish Reveals Key Roles for Extracellular Matrix Proteins Col10a1a and Fbln1 in Skeletal Development and Homeostasis. Biomolecules. 2024;14:139. https://doi.org/10.3390/biom14020139.\u003c/li\u003e\n\u003cli\u003eSchaffer RV, Nakamura EL. Synopsis of biological data on the cobia \u003cem\u003eRachycentron canadum\u003c/em\u003e (Pisces: Rachycentridae). laguna. 1989.\u003c/li\u003e\n\u003cli\u003eJ.H K, G C, Ma Q, Huang J.S, Zhang J.D, Shi G, et al. Embryonic development and morphological characteristics of larvae and juveniles of cobia (\u003cem\u003eRachycentron canadum\u003c/em\u003e). scxb. 2021;45:1814\u0026ndash;24. https://doi.org/10.11964/jfc.20200812389.\u003c/li\u003e\n\u003cli\u003eMa Q, Yang Y, Mao F, Zhou Q, Wang L, Chen G. Genome-wide identification, phylogeny and expression analysis of the \u003cem\u003ebmp\u003c/em\u003e gene family associated with development and skeleton deformity in cobia (\u003cem\u003eRachycentron canadum\u003c/em\u003e). Aquac Rep. 2023;31:101644. https://doi.org/10.1016/j.aqrep.2023.101644.\u003c/li\u003e\n\u003cli\u003eChen C, Wu Y, Li J, Wang X, Zeng Z, Xu J, et al. TBtools-II: A \u0026ldquo;one for all, all for one\u0026rdquo;bioinformatics platform for biological big-data mining. Mol Plant. 2023;16:1733\u0026ndash;42. https://doi.org/10.1016/j.molp.2023.09.010.\u003c/li\u003e\n\u003cli\u003eMa Q, Fan Y, Zhuang Z, Liu S. Cloning, expression profiling and promoter functional analysis of bone morphogenetic protein 2 in the tongue sole (\u003cem\u003eCynoglossus semilaevis\u003c/em\u003e). Acta Oceanol Sin. 2018;37:76\u0026ndash;84. https://doi.org/10.1007/s13131-018-1164-x.\u003c/li\u003e\n\u003cli\u003eLi G, Wubshet AK, Ding Y, Li Q, Dai J, Wang Y, et al. Antigenicity and Immunogenicity Analysis of the E. coli Expressed FMDV Structural Proteins; VP1, VP0, VP3 of the South African Territories Type 2 Virus. Viruses. 2021; 13 (6): 1005. https://doi.org/10.3390/v13061005.\u003c/li\u003e\n\u003cli\u003eKong RYC, Kwan KM, Lau ET, Thomas JT, Boot‐Handford RP, Grant ME, et al. Intron‐exon structure, alternative use of promoter and expression of the mouse collagen X gene, \u003cem\u003eCol10a‐1\u003c/em\u003e. Eur J Biochem. 1993;213:99\u0026ndash;111. https://doi.org/10.1111/j.1432-1033.1993.tb17739.x.\u003c/li\u003e\n\u003cli\u003eGomez-Picos P, Ovens K, Eames BF. Limb Mesoderm and Head Ectomesenchyme Both Express a Core Transcriptional Program During Chondrocyte Differentiation. Front Cell Dev Biol. 2022; 10: 876825. https://doi.org/10.3389/fcell.2022.876825.\u003c/li\u003e\n\u003cli\u003eLu Y, Qiao L, Lei G, Mira RR, Gu J, Zheng Q. \u003cem\u003eCol10a1\u003c/em\u003e gene expression and chondrocyte hypertrophy during skeletal development and disease. Front Biol. 2014;9:195\u0026ndash;204. https://doi.org/10.1007/s11515-014-1310-6.\u003c/li\u003e\n\u003cli\u003eGudmann NS, Karsdal, M.A. Chapter 10-Type X Collagen Karsdal MA. Biochemistry of Collagens, Laminins and Elastin. 2016:73-76. https://doi.org/10.1016/B978-0-443-15617-5.00028-7.\u003c/li\u003e\n\u003cli\u003eAldea D, Hanna P, Munoz D, Espinoza J, Torrejon M, Sachs L, et al. Evolution of the vertebrate bone matrix: An expression analysis of the network forming collagen paralogues in amphibian osteoblasts. J Exp Zool B Mol Dev Evol. 2013;320:375\u0026ndash;84. https://doi.org/10.1002/jez.b.22511.\u003c/li\u003e\n\u003cli\u003eIbaraki H, Wu X, Uji S, Yokoi H, Sakai Y, Suzuki T. Transcriptome analysis of vertebral bone in the flounder, \u003cem\u003eParalichthys olivaceus\u003c/em\u003e (Teleostei, Pleuronectiformes), using Illumina sequencing. Mar Genom. 2015;24:269\u0026ndash;76. https://doi.org/10.1016/j.margen.2015.09.009.\u003c/li\u003e\n\u003cli\u003eInohaya K, Takano Y, Kudo A. The teleost intervertebral region acts as a growth center of the centrum: In vivo visualization of osteoblasts and their progenitors in transgenic fish. Dev Dyn. 2007;236:3031\u0026ndash;46. https://doi.org/10.1002/dvdy.21329.\u003c/li\u003e\n\u003cli\u003eSpoorendonk KM, Peterson-Maduro J, Renn J, Trowe T, Kranenbarg S, Winkler C, et al. Retinoic acid and Cyp26b1 are critical regulators of osteogenesis in the axial skeleton. Development. 2008;135:3765\u0026ndash;74. https://doi.org/10.1242/dev.024034.\u003c/li\u003e\n\u003cli\u003eChan D, Jacenko O. Phenotypic and biochemical consequences of collagen X mutations in mice and humans. Matrix Biol. 1998;17:169\u0026ndash;84. https://doi.org/10.1016/S0945-053X(98)90056-7.\u003c/li\u003e\n\u003cli\u003eChung KS, Jacenko O, Boyle P, Olsen BR, Nishimura I. Craniofacial abnormalities in mice carrying a dominant interference mutation in type X collagen. Dev Dyn. 1997; 208:544\u0026ndash;52. https://doi.org/10.1002/(SICI)1097-0177(199704)208:4\u0026lt;544::AID-AJA10\u0026gt;3.0.CO;2-X.\u003c/li\u003e\n\u003cli\u003eMao FF, Chen G, Ma Q, ZHOU QL, Shi G, Huang JS, et al. Development characteristics of the vertebral column and the appendicular skeleton in larval and juvenile cobia (\u003cem\u003eRachycentron canadum\u003c/em\u003e). scxb. 2023;47:089105\u0026ndash;9. https://doi.org/10.11964/jfc.20210712985.\u003c/li\u003e\n\u003cli\u003eKirsch T, Ishikawa Y, Mwale F, Wuthier RE. Roles of the nucleational core complex and collagens (types II and X) in calcification of growth plate cartilage matrix vesicles. J Biol Chem. 1994; 269:20103\u0026ndash;9. https://doi.org/10.1016/S0021-9258(17)32133-6.\u003c/li\u003e\n\u003cli\u003eKomori T. Regulation of bone development and extracellular matrix protein genes by RUNX2. Cell Tissue Res. 2010;339:189\u0026ndash;95. https://doi.org/10.1007/s00441-009-0832-8.\u003c/li\u003e\n\u003cli\u003eSimoes B, Conceicao N, Viegas CSB, Pinto JP, Gavaia PJ, Hurst LD, et al. Identification of a promoter element within the zebrafish \u003cem\u003ecolX\u0026alpha;1\u003c/em\u003e gene responsive to Runx2 isoforms Osf2/Cbfa1 and til-1 but not to pebp2\u0026alpha;A2. Calcif Tissue Int. 2006;79:230\u0026ndash;44. https://doi.org/10.1007/s00223-006-0111-6.\u003c/li\u003e\n\u003cli\u003eNiu P, Zhong Z, Wang M, Huang G, Xu S, Hou Y, et al. Zinc finger transcription factor Sp7/Osterix acts on bone formation and regulates col10a1a expression in zebrafish. Sci Bull. 2017;62:174\u0026ndash;84. https://doi.org/10.1016/j.scib.2017.01.009.\u003c/li\u003e\n\u003cli\u003eBeier F, Lammi MJ, Bertling W, Von Der Mark K. Transcriptional Regulation of the Human Type X Collagen Gene Expression. Ann N Y Acad Sci. 1996;785:209\u0026ndash;11. https://doi.org/10.1111/j.1749-6632.1996.tb56263.x.\u003c/li\u003e\n\u003cli\u003eGu J, Lu Y, Li F, Qiao L, Wang Q, Li N, et al. Identification and characterization of the novel Col10a1 regulatory mechanism during chondrocyte hypertrophic differentiation. Cell Death Dis. 2014;5:e1469\u0026ndash;e1469. https://doi.org/10.1038/cddis.2014.444.\u003c/li\u003e\n\u003cli\u003eEferl R, Hoebertz A, Schilling AF, Rath M, Karreth F, Kenner L, et al. The Fos-related antigen Fra-1 is an activator of bone matrix formation. EMBO J. 2004;23:2789\u0026ndash;99. https://doi.org/10.1038/sj.emboj.7600282.\u003c/li\u003e\n\u003cli\u003eRiemer S, Gebhard S, Beier F, P\u0026ouml;schl E, Von Der Mark K. Role of c‐fos in the regulation of type X collagen gene expression by PTH and PTHrP: Localization of a PTH/PTHrP‐responsive region in the human COL10A1 enhancer. J Cell Biochem. 2002;86:688\u0026ndash;99. https://doi.org/10.1002/jcb.10260.\u003c/li\u003e\n\u003cli\u003eGebhard S, P\u0026ouml;schl E, Riemer S, Bauer E, Hattori T, Eberspaecher H, et al. A highly conserved enhancer in mammalian type X collagen genes drives high levels of tissue-specific expression in hypertrophic cartilage in vitro and in vivo. Matrix Biol. 2004;23:309\u0026ndash;22. https://doi.org/10.1016/j.matbio.2004.05.010.\u003c/li\u003e\n\u003cli\u003eZheng Q, Keller B, Zhou G, Napierala D, Chen Y, Zabel B, et al. Localization of the \u003cem\u003eCis\u003c/em\u003e -Enhancer Element for Mouse Type X Collagen Expression in Hypertrophic Chondrocytes In Vivo. J Bone Miner Res. 2009;24:1022\u0026ndash;32. https://doi.org/10.1359/jbmr.081249.\u003c/li\u003e\n\u003cli\u003eVolk SW, Luvalle P, Leask T, Leboy PS. A BMP responsive transcriptional region in the chicken type X collagen gene. Journal of Bone and Mineral Research. 1998;13:1521\u0026ndash;9. https://doi.org/10.1359/jbmr.1998.13.10.1521.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Rachycentron canadum, col10a1, gene expression, function, skeleton","lastPublishedDoi":"10.21203/rs.3.rs-9410040/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9410040/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eCollagens are key extracellular matrix components in vertebrates, with type X collagen (COL10) crucial for skeletal development. In mammals, expression of \u003cem\u003ecol10a1\u003c/em\u003e is limited to hypertrophic chondrocytes, yet in teleosts, it is also in osteoblasts, hinting at its role in both cartilage and bone mineralization. However, most prior research focused on model fish, little is known about \u003cem\u003ecol10a1\u003c/em\u003e in the other teleost taxa, particularly in Perciformes species.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe cloned cobia\u0026rsquo;s \u003cem\u003ecol10a1\u003c/em\u003e gene with its 2000 bp upstream region, revealing it spans 4585 bp, encodes 659 amino acids, and shares a close evolutionary relationship with that of \u003cem\u003eEcheneis naucrates.\u003c/em\u003e Through the analysis of luciferase activities of various promoter constructs, a potential promoter region (spanning from \u0026minus;\u0026thinsp;300 to +\u0026thinsp;200 bp) and a potential region (located between \u0026minus;\u0026thinsp;1800 and \u0026minus;\u0026thinsp;1300 bp) harboring an enhancer element were identified. We used quantitative real time PCR and fluorescence in situ hybridization to study its expression, finding high levels in skeletal tissues such as scales and cartilage. During early development, \u003cem\u003ecol10a1\u003c/em\u003e expression starts at the multi-cell stage, peaks at organogenesis, increases again from 1 day post-hatching, reaching a maximum at 9 days before declining. In cobia larvae, it's mainly expressed in cartilage cells, notochord sheath cells, and muscle fibers. Comparing deformed and normal skeletal tissues, \u003cem\u003ecol10a1\u003c/em\u003e expression is suppressed in deformed Meckel\u0026rsquo;s cartilage, but no significant change in deformed vertebrae. We then constructed a prokaryotic expression vector for cobia COL10A1, expressed, and purified the protein. This protein boosted alkaline phosphatase activity and Alizarin red staining, and upregulated mineralization-related genes in mouse osteoblastic cells.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur study confirms \u003cem\u003ecol10a1\u003c/em\u003e's key role in cartilage and bone development of cobia, consistent with Cypriniformes and Beloniformes species. The cobia COL10A1 protein was shown to regulate mineralization in mouse osteoblastic cells. This further substantiates that \u003cem\u003ecol10a1\u003c/em\u003e and its protein are vital for skeletal development and mineralization across vertebrates, highlighting their conserved and crucial functions in skeletal biology.\u003c/p\u003e","manuscriptTitle":"Characterization, expression profiling, and functional analysis of col10a1 in cobia (Rachycentron canadum)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-27 06:11:43","doi":"10.21203/rs.3.rs-9410040/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-08T14:26:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"175765811265139617015606735564562444566","date":"2026-05-05T07:04:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"42159644742607827337464334321592594498","date":"2026-05-03T02:13:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"81369181678281735852280563668027992800","date":"2026-04-24T14:30:55+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-18T12:28:04+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-17T18:12:02+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-15T01:37:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-15T01:36:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Genomics","date":"2026-04-14T03:20:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"dbf761f3-3ec8-4c56-9b9a-2dff2b8c7251","owner":[],"postedDate":"April 27th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-08T14:26:57+00:00","index":36,"fulltext":""},{"type":"reviewerAgreed","content":"175765811265139617015606735564562444566","date":"2026-05-05T07:04:23+00:00","index":35,"fulltext":""},{"type":"reviewerAgreed","content":"42159644742607827337464334321592594498","date":"2026-05-03T02:13:33+00:00","index":33,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-27T06:11:43+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-27 06:11:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9410040","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9410040","identity":"rs-9410040","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}