College of Ecology, Lishui University, Lishui 323000, China.
Department of Biological Science, National University of Singapore, Singapore 117558, Singapore.
Comp Biochem Physiol Part D Genomics Proteomics. 2022 Dec;44:101030. doi: 10.1016/j.cbd.2022.101030. Epub 2022 Nov 2.
Intermuscular bones (IBs), which are little, bony spicules in muscle, are embedded in lower teleosts' myosepta. Despite the importance of studying IB development in freshwater aquaculture species, the genes associated with IB development need to be further explored. In the present study, we identified four stages of IB development in barbel steed (Hemibarbus labeo), namely stage 1: IBs have not emerged, stage 2: a few small IBs have emerged in the tail, stage 3: longer IBs gradually emerged in the tail and stage 4: all of the IBs in the tail are mature and long, via Alizarin red staining. Subsequently, we used the HiseqXTen platform to sequence and de novo assemble the transcriptome of epaxial muscle (between 35th and 40th myomere) of barbel steed at 29 days (stage 1) and 42 days (stage 3) after hatching. A total of 190,814 unigenes were obtained with an average length and N50 of 648 bp and 1027 bp, respectively. We found 2174 differentially expressed genes (DEGs) between stages 1 and 3, of which 378 and 1796 were up- and down-regulated, respectively. Functional enrichment analysis showed that several DEGs functioned in ossification, positive regulation of osteoblast differentiation, osteoblast differentiation, and BMP signaling pathway, and were further enriched in signal pathway, including osteoclast differentiation, TGF-β signaling pathway, cytokine-cytokine receptor interaction, Jak-STAT signaling pathway, and other KEEG pathways. In conclusion, we identified genes that may be related to IB development, such as kazal type serine peptidase inhibitor domain 1 (KAZALD1), extracellular matrix protein 1 (ECM1), tetranectin, bone morphogenetic protein 1 (bmp1), acid phosphatase 5 (ACP5), collagen type XI alpha 1 chain (COL11A1), matrix metallopeptidase 9 (MMP9), pannexin-3 (PANX3), sp7 transcription factor (Sp7), and c-x-c motif chemokine ligand 8 (CXCL8), by comparing the transcriptomes of epaxial muscle before and after IB ossification. This study provided a theoretical basis for identifying the molecular mechanisms underlying IB development in fish.
肌间骨（IBs）是肌肉中的小骨棘，嵌入低等硬骨鱼的肌膜中。尽管研究淡水水产养殖物种中 IB 发育的重要性，但与 IB 发育相关的基因仍需要进一步探索。在本研究中，我们在黄河雅罗鱼（Hemibarbus labeo）中鉴定出 IB 发育的四个阶段，即阶段 1：IBs 尚未出现；阶段 2：尾部出现少量小 IBs；阶段 3：尾部逐渐出现较长的 IBs；阶段 4：尾部所有的 IBs 成熟且较长，通过茜素红染色。随后，我们使用 HiseqXTen 平台对孵化后 29 天（阶段 1）和 42 天（阶段 3）黄河雅罗鱼背肌（第 35 到 40 肌节之间）的转录组进行测序和从头组装。共获得 190814 个 unigenes，平均长度和 N50 分别为 648bp 和 1027bp。我们在阶段 1 和 3 之间发现了 2174 个差异表达基因（DEGs），其中 378 个上调，1796 个下调。功能富集分析表明，一些 DEGs 参与了骨化、成骨细胞分化的正调控、成骨细胞分化和 BMP 信号通路，并且进一步富集在信号通路中，包括破骨细胞分化、TGF-β 信号通路、细胞因子-细胞因子受体相互作用、Jak-STAT 信号通路和其他 KEEG 通路。总之，我们通过比较 IB 骨化前后背肌的转录组，鉴定了可能与 IB 发育相关的基因，如 kazal 型丝氨酸蛋白酶抑制剂域 1（KAZALD1）、细胞外基质蛋白 1（ECM1）、四连接蛋白、骨形态发生蛋白 1（bmp1）、酸性磷酸酶 5（ACP5）、胶原 XI 型α1 链（COL11A1）、基质金属蛋白酶 9（MMP9）、pannexin-3（PANX3）、sp7 转录因子（Sp7）和 C-X-C 基序趋化因子配体 8（CXCL8）。本研究为鉴定鱼类 IB 发育的分子机制提供了理论依据。
