Characterization of zebrafish mutants with defects in bone calcification during development.
Characterization of zebrafish mutants with defects in bone calcification during development.
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DOI：
10.1016/j.bbrc.2013.09.043
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发表时间：
2013-10
影响因子：
3.1
通讯作者：
Yang Xi;Dong-Yang Chen;Lei Sun;Yuhao Li;Lei Li
中科院分区：
文献类型：
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作者：
Yang Xi;Dong-Yang Chen;Lei Sun;Yuhao Li;Lei Li
利用荧光染料钙黄绿素和阿辛蓝，我们染色的F3代化学（ENU）诱变的斑马鱼胚胎和幼虫，并筛选突变体的骨发育缺陷。我们发现了一个突变株，骨钙化缓慢（bcs），在发育过程中表现出延迟的轴向椎骨钙化。在受精后4-5天（dpf）之前，bcsembryos在骨骼发育方面没有表现出明显的异常（即，软骨和椎骨的正常数量、大小和形状）。在5-6 dpf，当椎骨开始钙化时，bcsembryos开始显示缺陷。例如，在7 dpf，在大多数检查的胚胎中，钙黄绿素染色显示没有椎骨矿化的信号，而在相同的发育阶段，在野生型动物中观察到2-14个矿化的椎骨。分别在9和11 dpf检查时，在bcsmutants中也观察到钙化椎骨数量的减少。有趣的是，到13 dpf，bcsmutants中的缺陷不再明显。野生型和突变型动物之间的钙化椎骨数量没有显着差异。我们检测了骨发育标记基因的表达（例如，Sox 9 b、Bmp 2b和Cyp 26 b1，它们在骨形成和钙化中起重要作用）。在突变鱼中，我们观察到Sox 9 b表达略有增加，Bmp 2b表达无变化，但Cyp 26 b1表达显著增加。总之，这些数据表明bcs延缓了轴向骨骼钙化，但不影响骨形成和成熟。
Using the fluorescent dyes calcein and alcian blue, we stained the F3 generation of chemically (ENU) mutagenized zebrafish embryos and larvae, and screened for mutants with defects in bone development. We identified a mutant line,bone calcification slow(bcs), which showed delayed axial vertebra calcification during development. Before 4–5 days post-fertilization (dpf), thebcsembryos did not display obvious abnormalities in bone development (i.e., normal number, size and shape of cartilage and vertebrae). At 5–6 dpf, when vertebrae calcification starts,bcsembryos began to show defects. At 7 dpf, for example, in most of thebcsembryos examined, calcein staining revealed no signals of vertebrae mineralization, whereas during the same developmental stages, 2–14 mineralized vertebrae were observed in wild-type animals. Decreases in the number of calcified vertebrae were also observed inbcsmutants when examined at 9 and 11 dpf, respectively. Interestingly, by 13 dpf the defects inbcsmutants were no longer evident. There were no significant differences in the number of calcified vertebrae between wild-type and mutant animals. We examined the expression of bone development marker genes (e.g.,Sox9b,Bmp2b, andCyp26b1, which play important roles in bone formation and calcification). In mutant fish, we observed slight increases inSox9bexpression, no alterations inBmp2bexpression, but significant increases inCyp26b1expression. Together, the data suggest thatbcsdelays axial skeletal calcification, but does not affect bone formation and maturation.