Shu-Ming Zou

Although fibroblast growth factor 8 (FGF8) is a critical regulator of skeletal morphogenesis in vertebrates, its specific role in the formation and development of intermuscular bones (IBs) in teleost fish remains insufficiently characterized. In this study, we generated double mutants (fgf8a^+/- + fgf8b^+/-) in diploid Chongming crucian carp (Carassius auratus) using CRISPR/Cas9-mediated gene editing. Compared to wild-type fish, the double mutants exhibited significantly reduced IB number (p < 0.01), demonstrating a synergistic role of fgf8a and fgf8b in IB formation. Notably, reduced number of IBs did not compromise overall growth, muscle architecture, or reproductive performance. Integrated transcriptomic and metabolomic analyses revealed that the reduced IB phenotype was linked to modifications in relevant signaling pathways and a concomitant upregulation of metabolites beneficial for muscle quality and health. Our findings highlight the crucial role of fgf8 in regulating IB formation and development in crucian carp, providing insights into the genetic mechanisms underlying this process in teleosts.

In the present study, vertebral bone tissues derived from Chongming crucian carp (Carassius carassius), a dominant species during the summer and autumn seasons on Chongming Island in the lower Yangtze River, were used to establish and characterize a Carassius carassius osteoblast cell line (COBC). The established COBCs were assessed using chromosome analysis, osteocalcin enzyme-linked immunosorbent assay (ELISA), and osteogenesis-related gene expression analysis. Additionally, cellular responses to environmental stress were assessed. The results showed that COBC exhibited optimal proliferation in L-15 medium supplemented with 20% fetal bovine serum at 28 °C. The histochemical staining assay results were all positive, thereby confirming that the isolated cells display typical osteoblast characteristics. Quantitative PCR analysis revealed that osteogenic marker genes, including runx2a and runx2b, were expressed at significantly higher levels in COBCs than in fish tissues. Under hypoxic stress, COBCs exhibited morphological changes, an increase in cell death, significant alterations in gene expression, and variations in antioxidant enzyme activity. These responses facilitate adaptation to hypoxic stress. This study established the first osteoblast cell line of the Chongming crucian carp and characterized its biological properties and response to hypoxic stress. These findings offer a valuable in vitro cell model and technical support for research on fish bone tissue biology and the assessment of environmental stress effects.

A reliable and reproducible method for the isolation, culture, and identification of an osteoblast cell line from crucian carp (Carassius auratus) was established in this study using vertebral bone tissue from Chongming crucian carp, a locally important aquaculture strain from the lower Yangtze River region. O steoblast cells were isolated using a tissue explant culture method, and optimal in vitro culture conditions were systematically evaluated. The established osteoblast cell line, designated Chongming Carassius auratus osteoblast cells (COBC), was characterized through chromosomal karyotype analysis, osteocalcin enzyme-linked immunosorbent assay (ELISA), and osteogenesis-related gene expression analysis. Additionally, cellular responses to environmental stress were assessed. The results showed that COBC exhibited optimal proliferation in L-15 medium supplemented with 20% fetal bovine serum at 28 ℃ under 5% CO₂. Alkaline phosphatase staining, Alizarin Red staining, and von Kossa staining all yielded positive results, thereby confirming that the isolated cells possessed typical and stable osteoblastic properties, with the osteocalcin content of 36,884 ng / L. Quantitative PCR analysis revealed that osteogenic marker genes, including runx2a and runx2b, were expressed at significantly higher levels in COBC s than in muscle tissue. Under hypoxia - reoxygenation stress, COBC exhibited enhanced apoptotic responses, marked alterations in related gene expression, and modulation of antioxidant enzyme activities, suggesting a certain degree of adaptive capacity to oxygen fluctuations. This study provides the first systematic description of the establishment and biological characterization of COBC, as well as its responses to hypoxic stress. These findings offer a valuable in vitro cell model and technical support for studies on fish bone tissue biology and the assessment of environmental stress effects.

Megalobrama amblycephala is a key freshwater fish species in Chinese aquaculture, valued for its high artificial propagation success and relative resistance to common aquaculture diseases. However, it is sensitive to hypoxia, and its molecular responses to hypoxic stress in the heart remain relatively unknown. Herein, we investigated the effects of hypoxia on myocardial enzyme activities (creatine kinase, creatine kinase myocardial band isoenzyme, lactate dehydrogenase, and α-hydroxybutyrate dehydrogenase), histological structure, and transcriptome changes in the heart of M. amblycephala. The fish were divided into five groups, including normoxia control (N0), hypoxia for 6, 12, and 24 h (H6, H12, and H24, respectively), and reoxygenation for 24 h (R24) groups. The results showed that hypoxia significantly increased myocardial enzyme activities. Histological analysis revealed that hypoxia induced slight swelling and disorganization of myocardial fibers, while reoxygenation partially restored their structural integrity. Transcriptome sequencing identified 4,269 DEGs, with 964, 1,159, 812, and 1,334 identified in the H6, H12, H24, and R24 groups, respectively, compared to the N0 group. KEGG pathway analysis identified circadian rhythm, cardiac muscle contraction, fatty acid degradation, hypertrophic cardiomyopathy, and fluid shear stress and atherosclerosis pathways as candidate molecular pathways involved in hypoxia tolerance. Notably, several key genes, including cry5, LOC125248347, cacng5b, atp2a1l, LOC125252961, aldh3a1, gcdha, edn1, atp2a1, gpc1a, and hsp90aa1.2, were significantly up-regulated in the H24 group, highlighting their potential roles in the adaptive responses to hypoxic stress. These findings provide new insights into the physiological, histological, and molecular responses underlying hypoxia adaptation in M. amblycephala, laying the groundwork for future studies on hypoxia-related molecular mechanisms in other economically important fish species.