RNAseq comparison of gene expression profiles in Osr1 mutant and control mouse embryonic tongue

The tongue is a muscular organ in the vertebrate oral cavity that performs complex functions in daily life, including feeding and phonetic articulation. The tongue consists of mesenchyme cells of two distinct origins: the muscle cells are derived from occipital somites whereas the tendons and other connective tissues derived from the cranial neural crest. Cranial neural crest cells are important for the initiation of tongue swelling and proper patterning of intrinsic and extrinsic tongue muscle groups. However, little is known regarding the molecular and cellular mechanisms of tongue morphogenesis. We show that the odd-skipped related 1 (Osr1) transcription factor exhibits dynamic expression in the tongue mesenchyme during early tongue development. Tissue-specific inactivation of Osr1 in the early neural crest cells resulted in ectopic cartilage formation in the mouse tongue. We show that Sox9, the master regulator of chondrocyte differentiation, is initially widely expressed in the neural crest derived mesenchyme in the tongue and subsequently down-regulated concomitant by up-regulation of Osr1 expression. Osr1 mutant embryos exhibit persistent expression of Sox9 and chondrocyte differentiation from the neural crest derived tongue mesenchyme. Further biochemical analyses indicate that Osr1 may directly suppresses Sox9 gene expression in the tongue mesenchyme. These data reveal a novel mechanism in suppression of chondrogenic fate during tongue development. Remarkably, the ectopic cartilage in the Osr1 mutant mice resembles the entoglossal cartilage naturally develops in the avian tongue. These results suggest that modulation of expression of Osr1 may underline the evolutionary divergence in tongue cartilage formation. RNAs were isolated from microdissected E12 embryonic mouse tongue of Osr1f/-;Wnt1cre and control littermates and characterized by RNAseq E12 mouse embryonic tongues were micro-dissceted, 3 pairs of control and mutant samples were pooled for the RNA extraction舌是脊椎动物口腔内的肌肉性器官，在日常生命活动中承担摄食与语音发音等复杂功能。舌的间充质（mesenchyme）细胞存在两种截然不同的起源：肌肉细胞源自枕体节（occipital somites），而肌腱及其他结缔组织则来源于颅神经嵴（cranial neural crest）。颅神经嵴细胞对于舌肿胀的启动以及舌内、外肌群的正常模式构建至关重要。然而，目前学界对舌形态发生的分子与细胞机制仍知之甚少。 本研究发现，奇数跳过相关1（odd-skipped related 1, Osr1）转录因子在舌发育早期的舌间充质中呈现动态表达模式。在早期神经嵴细胞中对Osr1进行组织特异性失活，会导致小鼠舌内出现异位软骨形成。研究还表明，作为软骨细胞分化核心调控因子的Sox9，最初在舌内源自神经嵴的间充质中广泛表达，随后随着Osr1表达上调而下调。Osr1突变胚胎中，源自神经嵴的舌间充质会持续表达Sox9并发生软骨细胞分化。进一步的生化分析显示，Osr1可能直接抑制舌间充质中Sox9基因的表达。上述结果揭示了舌发育过程中软骨细胞命运抑制的全新机制。 值得注意的是，Osr1突变小鼠体内的异位软骨，与鸟类舌内天然发育的舌内软骨（entoglossal cartilage）形态一致。本研究结果提示，对Osr1表达的调控可能是舌软骨形成过程中进化分化的分子基础。 研究人员从经显微解剖的Osr1f/-;Wnt1-Cre基因型小鼠胚胎期12天（embryonic day 12, E12）舌组织及其同窝对照样本中分离RNA，并通过RNA测序（RNA-seq）进行表征。本次实验中共显微解剖E12小鼠胚胎舌组织，将3对对照与突变样本混合后开展RNA提取。