RNAseq comparison of gene expression profiles in Hand2-Cre driven Msx1/Msx2 double conditional mutant and control mouse embryonic mandible. RNAseq comparison of gene expression profiles in Hand2-Cre driven Msx1/Msx2 double conditional mutant and control mouse embryonic mandible

The mandible of the jawed vertebrate is derived from the mandibular process of the first pharyngeal arch of the early embryo. The first pharyngeal arch consists of cells from all three germ layers, with the neural crest giving rise to all the skeletal elements of the mandible. The correct patterning of the neural crest cells by spatially and temporally controlled expression of various transcription factors during mandible development is crucial for the proper morphogenesis of the lower jaw. Msx family genes encode transcription factors which contain the conserved homeodomain. Among the three members of Msx genes, Msx1 and Msx2 are expressed in the neural crest and epithelium of distal mandibular process during early embryonic development with partially overlapped expression patterns. Msx1-/- mouse embryos develop multiple craniofacial developmental defects including tooth agenesis, cleft palate, and hypoplastic mandible. Although no jaw defects were observed in Msx2-/- mouse embryos, Msx1-/-Msx2-/- mouse embryos exhibit significantly severer mandibular defects compared to Msx1-/- mouse embryos, suggesting a partial functional redundancy between the two genes in mandible development. Besides the direct roles of Msx1 and Msx2 in the development of the mandibular neural crest, the phenotypes of Msx1-/-Msx2-/- may also contributed by secondary impacts from defective pre-migrative neural crest and non-neural crest tissues. Here we performed the gene expression profiling by RNA-seq in the distal mandibular processes of Msx1f/f;Msx2f/f;Hand2-Cre and Msx1f/+;Msx2f/f;Hand2-Cre embryos at E10.75, the former developed distally truncated mandible at later stages while the latter served as morphologically normal littermate control. Comparing the gene expression profiles of the two will give us insight into the functions of Msx1 and Msx2 expressed in mandibular neural crest cells in the development of the mandible. Overall design: Msx1f/f;Msx2f/f;Rosa26mTmG/mTmG female mice were crossed with Msx1f/+;Msx2f/f;Hand2-Cre males. The embryos were collected at E10.75. The GFP+ embryos were collected and the GFP+ distal mandibles were dissected in cold PBS under fluorescent microscope and snap frozen. A total of 3 litters of embryos were dissected. From each litter, 1 embryo of Msx1f/f;Msx2f/f;Rosa26mTmG/+;Hand2-Cre genotype and 1 embryo of Msx1f/+;Msx2f/f;Rosa26mTmG/+;Hand2-Cre genotype were selected for total RNA extraction, sequencing library preparation, and paired-ended sequencing.

有颌脊椎动物的下颌骨（mandible）起源于早期胚胎第一鳃弓（pharyngeal arch）的下颌突。第一鳃弓包含来自三个胚层的细胞，其中神经嵴（neural crest）可分化为下颌骨的所有骨骼组分。在下颌发育过程中，各类转录因子（transcription factor）的时空特异性表达对神经嵴细胞的正确模式化至关重要，这直接影响下颌的正常形态发生。Msx家族基因编码含有保守同源结构域（homeodomain）的转录因子。在Msx基因的三个家族成员中，Msx1与Msx2可在早期胚胎发育阶段的下颌突远端神经嵴与上皮组织中表达，且二者的表达模式存在部分重叠。Msx1纯合敲除（Msx1-/-）的小鼠胚胎会出现多种颅面发育缺陷，包括牙齿缺失、腭裂及下颌发育不全。尽管Msx2纯合敲除（Msx2-/-）的小鼠胚胎未观察到下颌发育异常，但Msx1-/-Msx2-/-双敲除小鼠胚胎的下颌缺陷程度相较于Msx1-/-小鼠胚胎显著更严重，提示这两个基因在下颌发育过程中存在部分功能冗余。除Msx1与Msx2在下颌神经嵴发育中的直接作用外，Msx1-/-Msx2-/-小鼠的表型可能还受到迁移前神经嵴及非神经嵴组织缺陷带来的次级影响。本研究通过RNA测序（RNA-seq）对E10.75时期的Msx1f/f;Msx2f/f;Hand2-Cre与Msx1f/+;Msx2f/f;Hand2-Cre胚胎的下颌突远端组织进行了基因表达谱分析。前者在后续发育阶段会出现远端截断的下颌骨，而后者作为形态正常的同窝对照。对二者的基因表达谱进行比较，将有助于我们深入解析在下颌神经嵴细胞中表达的Msx1与Msx2在下颌发育中的功能。实验整体设计：将Msx1f/f;Msx2f/f;Rosa26mTmG/mTmG雌性小鼠与Msx1f/+;Msx2f/f;Hand2-Cre雄性小鼠进行交配。于胚胎发育第10.75天（E10.75）收集胚胎。收集GFP阳性胚胎后，在荧光显微镜下于预冷的PBS中解剖其下颌突远端组织并快速冷冻。本研究共解剖了3窝胚胎，从每窝中分别选取1只Msx1f/f;Msx2f/f;Rosa26mTmG/+;Hand2-Cre基因型胚胎与1只Msx1f/+;Msx2f/f;Rosa26mTmG/+;Hand2-Cre基因型胚胎，进行总RNA提取、测序文库构建及双端测序。