肌间刺缺失对斑马鱼骨骼发育的影响

COMPARATIVE ANALYSIS OF SKELETAL DEVELOPMENT BETWEEN WILDTYPE ZEBRAFISH AND INTERMUSCULAR BONE-DEFICIENT MUTANTS

  • 摘要: 利用斑马鱼(Danio rerio)野生型与肌间刺完全缺失突变型个体, 从骨骼染色和骨骼发育相关基因表达两方面, 初步评价了肌间刺缺失对斑马鱼骨骼发育的影响。通过骨骼染色对比观察了两种肌间刺表型个体受精后8dpf(days post fertilization, dpf)到56dpf的骨骼发育情况, 结果显示, 两种肌间刺表型除肌间刺外, 其他骨骼发育基本同步。此外, 通过qRT-PCR实验检测分析了6个骨骼发育相关基因(bmp2abmp4、smad1、smad4arunx2asp7)在不同肌间刺表型5个胚胎发育时期(3hpf囊胚期、6hpf原肠胚期、12hpf体节期、24hpf咽囊期和72hpf孵化期)和5个胚后生长阶段(15、30、45、60和75dpf)的表达情况。结果显示:在胚胎发育时期, 野生型和突变型个体中bmp2abmp4、smad1、smad4a基因和突变型个体中sp7基因的表达均呈现先升后降的变化趋势, 且在体节期达到最高表达水平;野生型和突变型个体中runx2a基因和野生型个体中sp7基因则表现为逐渐上升的趋势。6个基因在囊胚期和原肠胚期表达量无显著差异, bmp2a的表达水平在体节期、咽囊期和孵化期无显著差异, 野生型个体bmp4、smad1、smad4arunx2a基因在体节期、咽囊期和孵化期的表达水平明显高于突变型, 而sp7基因则表现为突变型明显高于野生型。胚后发育阶段 6个基因在5个生长阶段均呈现逐渐下降的趋势, 且在两种肌间刺表型间其表达仅在个别时期差异显著。综上所述, 肌间刺的缺失对斑马鱼骨骼发育表现型无显著影响, 只在胚胎发育时期影响骨骼相关基因表达水平的变化;结合骨骼染色结果, 推测肌间刺缺失对斑马鱼骨骼发育无显著影响。

     

    Abstract: This study verified the impact of Imb (intermuscular bone) deficiency on skeletal development in two ways: analyses of skeletal staining and the expression of skeletal development-related genes between wildtype and Imb mutant zebrafish. First, this study revealed the skeletal development of two Imb phenotypes from 8 dpf to 56 dpf. The results indicated that the development of most of the skeleton was synchronous except for the intermuscular bones. Furthermore, we detected the expression of 6 skeletal development-related genes (bmp2a, bmp4, smad1, smad4a, runx2a and sp7) in 5 embryonic stages (blastula, 3 hpf; gastrula, 6 hpf; segmentation, 12 hpf; pharyngula, 24 hpf; and the hatching period, 72 hpf) and 5 postembryonic stages (15 dpf, 30 dpf, 45 dpf, 60 dpf and 75 dpf). The results for the different embryonic stages showed that the expression of 4 genes (bmp2a, bmp4, smad1 and smad4a) in both wildtype and mutant individuals and the expression of sp7 in mutant individuals was increased in the blastula, gastrula and segmentation stages, then declined in the pharyngula and hatching period. However, the expression of runx2a in both wildtype and mutant individuals and the expression of sp7 in wildtype zebrafish continued to rise during embryonic development. In addition, there was no difference in the expression of the 6 genes between wildtype and mutant zebrafish in the blastula and gastrula stages, whereas a significant difference in the expression of the genes existed in the other 3 stages. The expression of bmp4, smad1, smad4a and runx2a in wildtype zebrafish was higher than that in mutant zebrafish, and the expression of sp7 in wildtype zebrafish was lower than that in mutant individuals, whereas there was no difference in bmp2a expression between wildtype and mutant individuals. Moreover, the results during postembryonic development indicated that the expression of the 6 genes declined significantly in 5 periods and that there was no difference between wildtype and mutant individuals. In summary, considering the results of the skeletal staining and gene expression analyses together, we speculate that the deletion of intermuscular bones does not impact the skeletal development of zebrafish.

     

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