Yan Yi-Lin, Miller Craig T, Nissen Robert M, Singer Amy, Liu Dong, Kirn Anette, Draper Bruce, Willoughby John, Morcos Paul A, Amsterdam Adam, Chung Bon-Chu, Westerfield Monte, Haffter Pascal, Hopkins Nancy, Kimmel Charles, Postlethwait John H
Institute of Neuroscience, University of Oregon, Eugene 97403, USA.
Development. 2002 Nov;129(21):5065-79. doi: 10.1242/dev.129.21.5065.
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.
软骨构建的分子遗传机制尚未完全明确。水母（jef）突变纯合的斑马鱼胚胎表现出颅面缺陷，并且缺乏神经颅、咽弓和肩带的软骨成分，这与患有弯肢侏儒症的人类相似。我们发现jef的两个等位基因在sox9a中存在突变，sox9a是人类转录因子SOX9在斑马鱼中的两个直系同源基因之一。亚硝酸乙酯诱导的突变改变了剪接连接处的一个保守核苷酸，并严重减少了sox9a转录本的剪接。逆转录病毒插入sox9a破坏了其DNA结合结构域。用剪接位点定向吗啉代反义寡核苷酸抑制野生型胚胎中sox9a转录本的剪接会产生类似jef突变幼虫的表型，并导致sox9a转录本在细胞核中积累；这种积累可作为一种独立于表型的吗啉代功效检测方法。核糖核酸酶保护分析表明，在注射吗啉代的动物中，剪接抑制百分比从受精后28小时的80%下降到4天后的45%。纯合突变胚胎中软骨的主要胶原蛋白col2a1信息的数量大幅减少。对dlx2表达的分析表明，jef（sox9a）胚胎中的神经嵴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以单细胞分辨率揭示了突变胚胎中软骨前凝聚物的形成。除了缺乏明显的软骨分化外，jef（sox9a）突变体中的咽弓凝聚物缺乏三种特定的形态发生行为：软骨细胞堆叠成有序阵列、咽软骨器官个体化以及单个软骨的正确塑形。尽管软骨严重减少，但对肌联蛋白表达的分析表明jef（sox9a）突变体中的肌肉模式正常。同样，钙黄绿素标记显示jef（sox9a）突变体中的早期骨形成在很大程度上未受影响。这些研究表明，jef（sox9a）对于凝聚物的形态发生和明显的软骨分化都至关重要。
