A zebrafish sox9 gene required for cartilage morphogenesis.
A zebrafish sox9 gene required for cartilage morphogenesis.
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发表时间：
2002-11
期刊：
影响因子：
4.6
通讯作者：
Yi-Lin Yan;Craig T. Miller;R. Nissen;A. Singer;Dong Liu;A. Kirn;B. Draper;J. Willoughby;P. Morcos;A. Amsterdam;B. Chung;M. Westerfield;P. Haffter;N. Hopkins;C. Kimmel;J. Postlethwait
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作者：
Yi-Lin Yan;Craig T. Miller;R. Nissen;A. Singer;Dong Liu;A. Kirn;B. Draper;J. Willoughby;P. Morcos;A. Amsterdam;B. Chung;M. Westerfield;P. Haffter;N. Hopkins;C. Kimmel;J. Postlethwait
软骨构建的分子遗传机制还不完全清楚。水母（jef）突变纯合子的斑马鱼胚胎表现出颅面缺陷，缺乏脑颅、咽弓和肩带的软骨成分，类似于人类的肢端发育不良。我们发现，两个等位基因的jef包含突变的sox 9a，两个斑马鱼直系同源的人类转录因子SOX 9。乙基亚硝基脲诱导的突变改变了剪接点的保守核苷酸，严重减少了sox 9a转录本的剪接。插入sox 9a的逆转录病毒破坏了其DNA结合域。用剪接位点定向的吗啉代反义寡核苷酸抑制野生型胚胎中sox 9a转录本的剪接，产生了类似jef突变幼虫的表型，并导致sox 9a转录本在细胞核中积累;这种积累可以作为一种测定吗啉代的功效而不依赖于表型。RNA酶保护试验表明，在注射吗啉的动物中，剪接抑制的百分比从受精后28小时的80%下降到4天的45%。纯合突变胚胎的col 2a 1信息（软骨的主要胶原蛋白）的数量大大减少。dlx 2的表达分析表明，jef（sox 9a）胚胎神经嵴的特化和迁移是正常的。用BODIPY-神经酰胺染色的活胚胎的共聚焦图像在单细胞分辨率下显示突变胚胎中形成前胶原缩合。除了缺乏明显的软骨分化外，jef（sox 9a）突变体中的咽弓浓缩缺乏三种特定的形态发生行为：软骨细胞有序排列的堆叠，咽软骨器官的个体化和个体软骨的正确成形。尽管软骨严重减少，肌联蛋白表达的分析显示，在jef（sox 9a）突变体正常的肌肉图案。同样，钙黄绿素标记显示，jef（sox 9a）突变体的早期骨形成在很大程度上不受影响。这些研究表明，jef（sox 9a）对于冷凝的形态发生和明显的软骨分化都至关重要。
The molecular genetic mechanisms of cartilage construction are incompletely understood. Zebrafish embryos homozygous for jellyfish (jef) mutations show craniofacial defects and lack cartilage elements of the neurocranium, pharyngeal arches, and pectoral girdle similar to humans with campomelic dysplasia. We show that two alleles of jef contain mutations in sox9a, one of two zebrafish orthologs of the human transcription factor SOX9. A mutation induced by ethyl nitrosourea changed a conserved nucleotide at a splice junction and severely reduced splicing of sox9a transcript. A retrovirus insertion into sox9a disrupted its DNA-binding domain. Inhibiting splicing of the sox9a transcript in wild-type embryos with splice site-directed morpholino antisense oligonucleotides produced a phenotype like jef mutant larvae, and caused sox9a transcript to accumulate in the nucleus; this accumulation can serve as an assay for the efficacy of a morpholino independent of phenotype. RNase-protection assays showed that in morpholino-injected animals, the percent of splicing inhibition decreased from 80% at 28 hours post fertilization to 45% by 4 days. Homozygous mutant embryos had greatly reduced quantities of col2a1 message, the major collagen of cartilage. Analysis of dlx2 expression showed that neural crest specification and migration was normal in jef (sox9a) embryos. Confocal images of living embryos stained with BODIPY-ceramide revealed at single-cell resolution the formation of precartilage condensations in mutant embryos. Besides the lack of overt cartilage differentiation, pharyngeal arch condensations in jef (sox9a) mutants lacked three specific morphogenetic behaviors: the stacking of chondrocytes into orderly arrays, the individuation of pharyngeal cartilage organs and the proper shaping of individual cartilages. Despite the severe reduction of cartilages, analysis of titin expression showed normal muscle patterning in jef (sox9a) mutants. Likewise, calcein labeling revealed that early bone formation was largely unaffected in jef (sox9a) mutants. These studies show that jef (sox9a) is essential for both morphogenesis of condensations and overt cartilage differentiation.