Gersch Robert P, Kirmizitas Arif, Sobkow Lidia, Sorrentino Gina, Thomsen Gerald H, Hadjiargyrou Michael
Department of Biomedical Engineering, Stony Brook University, Stony Brook, NY 11794-5281, United States.
Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794-8575, United States.
Gene Expr Patterns. 2012 Mar-Apr;12(3-4):145-53. doi: 10.1016/j.gep.2012.01.002. Epub 2012 Jan 18.
Mustn1 is a vertebrate-specific protein that, in vitro, was showed to be essential for prechondrocyte function and thus it has the potential to regulate chondrogenesis during embryonic development. We use Xenopus laevis as a model to examine Mustn1 involvement in chondrogenesis. Previous work suggests that Mustn1 is necessary but not sufficient for chondrogenic proliferation and differentiation, as well as myogenic differentiation in vitro. Mustn1 was quantified and localized in developing Xenopus embryos using RT-PCR and whole mount in situ hybridization. Xenopus embryos were injected with either control morpholinos (Co-MO) or one designed against Mustn1 (Mustn1-MO) at the four cell stage. Embryos were scored for morphological defects and Sox9 was visualized via in situ hybridization. Finally, Mustn1-MO-injected embryos were co-injected with Mustn1-MO resistant mRNA to confirm the specificity of the observed phenotype. Mustn1 is expressed from the mid-neurula stage to the swimming tadpole stages, predominantly in anterior structures including the pharyngeal arches and associated craniofacial tissues, and the developing somites. Targeted knockdown of Mustn1 in craniofacial and dorsal axial tissues resulted in phenotypes characterized by small or absent eye(s), a shortened body axis, and tail kinks. Further, Mustn1 knockdown reduced cranial Sox9 mRNA expression and resulted in the loss of differentiated cartilaginous head structures (e.g. ceratohyal and pharyngeal arches). Reintroduction of MO-resistant Mustn1 mRNA rescued these effects. We conclude that Mustn1 is necessary for normal craniofacial cartilage development in vivo, although the exact molecular mechanism remains unknown.
Mustn1是一种脊椎动物特有的蛋白质，在体外实验中，它被证明对前软骨细胞功能至关重要，因此有潜力在胚胎发育过程中调节软骨形成。我们以非洲爪蟾作为模型来研究Mustn1在软骨形成中的作用。先前的研究表明，Mustn1对于软骨生成增殖和分化以及体外成肌分化是必要的，但并不充分。我们使用逆转录聚合酶链反应（RT-PCR）和全胚胎原位杂交技术对发育中的非洲爪蟾胚胎中的Mustn1进行定量和定位。在四细胞期，将非洲爪蟾胚胎注射对照吗啉代寡核苷酸（Co-MO）或针对Mustn1设计的吗啉代寡核苷酸（Mustn1-MO）。对胚胎的形态缺陷进行评分，并通过原位杂交观察Sox9的表达情况。最后，将Mustn1-MO注射的胚胎与抗Mustn1-MO的mRNA共注射，以确认所观察到的表型的特异性。Mustn1从中神经胚期到游动蝌蚪期均有表达，主要在前部结构中表达，包括咽弓及相关的颅面部组织，以及发育中的体节。在颅面部和背轴组织中靶向敲低Mustn1会导致出现小眼或无眼、体轴缩短和尾部扭结等表型。此外，Mustn1敲低会降低颅部Sox9 mRNA的表达，并导致分化的软骨头部结构（如角舌骨和咽弓）缺失。重新引入抗MO的Mustn1 mRNA可挽救这些效应。我们得出结论，Mustn1在体内对于正常的颅面部软骨发育是必要的，尽管确切的分子机制仍不清楚。
