Page 36 - 《水产学报》2026年第01期

1 期 水 产 学 报 50 卷

Functional differentiation of pcolcea and pcolceb genes in
bone development in Danio rerio

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WANG Wenxiu , CHEN Yulong , XIAO Zhengyu , GAO Zexia 1,2,3 , NIE Chunhong 1,2*
(1. Key Lab of Freshwater Animal Breeding, Ministry of Agriculture and Rural Affairs,
Engineering Research Center of Green Development for Conventional Aquatic Biological Industry in
the Yangtze River Economic Belt, Ministry of Education,
College of Fisheries, Huazhong Agricultural University, Wuhan 430070, China;
2. Hubei Hongshan Laboratory, Wuhan 430070, China;
3. Hubei Province Famous Fish Breeding and
Healthy Aquaculture Engineering Technology Research Center, Wuhan 430070, China)

Abstract: Procollagen C-proteinase enhancer protein (PCOLCE) is a secreted protein that enhances the activity of procollagen
C-proteinase, thereby facilitating the maturation of collagen and influencing the process of bone formation. Down-regulation of
pcolce expression compromises the maturation of collagen, ultimately leading to a reduction in bone mass. In teleost fish, the
occurrence of genomic duplication events has led to the emergence of two paralogs of pcolce, designated pcolcea and pcolceb,
whose functional roles in skeletal development remain incompletely characterized. To delineate the functional divergence of
pcolcea and pcolceb in zebrafish (Danio rerio) osteogenesis, this study employed a multi-faceted approach integrating compu-
tational analysis of sequence features, spatiotemporal expression profiling, and phenotypic characterization of targeted mutants.
The results indicated that pcolcea and pcolceb comprise 8 and 9 exons, respectively, with pcolceb being significantly longer;
both genes were predominantly expressed in the vertebrae, head, and pectoral fins. Through targeted gene knockout, we gener-
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ated pcolcea and pcolceb homozygous mutants. The pcolcea mutant carried a 229-bp deletion in exon 1, while the pcolceb -/-
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mutant carried a 7-bp deletion in exon 3. Compared to wild-type D. rerio, pcolcea and pcolcea ; pcolceb double mutant D.
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rerio exhibited a marked reduction in vertebral tissue mineral density, whereas pcolceb D. rerio showed no significant differ-
ence. Subsequently, the expression of genes related to bone development (runx2b, entpd5a, alpl, bglap, sp7), collagen develop-
ment (col1a1a, col1a1b, col1a2), and downstream effector genes (bmp1a, bmp1b) was quantified by RT-qPCR in the caudal
vertebrae of the mutants. RT-qPCR results showed that expression of bmp1b, col1a1a, col1a2, runx2b, alpl, and sp7 was signi-
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ficantly downregulated in pcolcea mutants. In pcolcea ; pcolceb double mutants, the expression of bmp1b, col1a1a, col1a1b,
col1a2, runx2b, entpd5a, bglap, and sp7 was significantly lower than in wild-type D. rerio. Collectively, these results demon-
strated that loss of pcolcea impaired skeletal tissue mineralization in D. rerio, and this phenotype was significantly exacerbated
by the concurrent deletion of pcolceb. This study elucidates the distinct roles of pcolcea and pcolceb in D. rerio bone develop-
ment and provides a foundation for understanding the molecular mechanisms governing skeletal formation in fish.
Key words: Danio rerio; pcolcea; pcolceb; bone development; CRISPR/Cas9; gene expression
Corresponding author: NIE Chunhong. E-mail: chunhongnie@mail.hzau.edu.cn
Funding projects: Young Scientists Fund of the National Natural Science Foundation of China (32202917); Key Program of
the National Natural Science Foundation of China (32330108); Fundamental Research Funds for the Central Universities
(2662023SCQD002); Hubei Provincial Natural Science Foundation of China (2022CFB819)

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