Laser microdissection coupled with RNA-seq profiling of the E11.5 mouse mandibular epithelium along the dorsoventral axis

How the dorsal-ventral axis of the vertebrate jaw, particularly the position of tooth initiation site, is established remains a critical and unresolved question. Tooth development starts with the formation of the dental lamina, a localized thickened strip within the maxillary and mandibular epithelium. To identify transcriptional regulatory networks (TRN) controlling the specification of dental lamina from the naïve mandibular epithelium, we utilized Laser Microdissection coupled low-input RNA-seq (LMD-RNA-seq) to profile gene expression of different domains of the mandibular epithelium along the dorsal-ventral axis in wild type mouse and two loss-of-function mouse models of domain specific transcription factors. To identify genes that are critical for mandibular epithelial patterning, we applied Laser Microdissection (LMD) coupled Smart-Seq2 RNA-seq to generate transcriptome profiles of four domains along the dorsal-ventral axis of the E11.5 mouse mandibular epithelium (also known as the oral-aboral axis). The four domains, from dorsal to ventral, included: 1) a posterior/dorsal domain; 2) a Dental Lamina (DL) domain; 3) an anterior domain; and, 4) an aboral/ventral domain. To determine the role of domain specific TFs in mandibular epithelium patterning and DL formation, we also generated transcriptome profiles of two loss-of-function mouse models: 1) DL specific transcription factor: Pitx2; 2) Aboral domain specific transcription factor: Tfap2a and Tfap2b.脊椎动物颌骨的背腹轴，尤其是牙齿起始位点的建立机制，仍是一个关键且尚未解决的科学问题。牙齿发育起始于牙板的形成——上颌与下颌上皮内局部增厚的条带结构。为了鉴定调控未成熟下颌上皮向牙板特化的转录调控网络 (transcriptional regulatory networks, TRN)，我们采用激光显微切割 (Laser Microdissection) 偶联低起始量RNA测序（LMD-RNA-seq）技术，对野生型小鼠以及两种结构域特异性转录因子 (transcription factors) 功能缺失小鼠模型中，下颌上皮沿背腹轴的不同区域进行基因表达谱分析。 为了鉴定对下颌上皮模式建成至关重要的基因，我们还应用激光显微切割 (LMD) 偶联Smart-Seq2 RNA测序技术，对胚胎期11.5天（E11.5）小鼠下颌上皮（又称口-非口轴）沿背腹轴的四个区域进行转录组谱分析。从背侧到腹侧的四个区域依次为：1）后/背侧区域；2）牙板 (DL) 区域；3）前侧区域；4）非口/腹侧区域。 为了明确结构域特异性转录因子在下颌上皮模式建成与牙板形成中的作用，我们还构建了两种功能缺失小鼠模型并获取其转录组谱：1）牙板特异性转录因子Pitx2；2）非口区域特异性转录因子Tfap2a与Tfap2b。