A neural crest-specific overexpression mice model reveals the transcriptional regulatory effects of Dlx2 during maxillary process development

Craniofacial morphogenesis is an intricate process that requires precise regulation of cell proliferation, migration, and differentiation. Perturbations of this process cause a series of craniofacial deformities. Dlx2 is a critical transcription factor that regulates the development of the first branchial arch. However, the transcriptional regulatory functions of Dlx2 during craniofacial development have been poorly understood due to the lack of animal models in that the levels of Dlx2 can be precisely modulated. In this study, we constructed a Rosa26 site-directed Dlx2 gene knock-in mouse model Rosa26CAG-LSL-Dlx2-3xFlag for conditionally overexpressing Dlx2. By breeding with Wnt1cre mice, we obtained Wnt1cre; Rosa26Dlx2/- mice in which Dlx2 is overexpressed in neural crest lineage at about three times the endogenous level. The Wnt1cre; Rosa26Dlx2/- mice exhibited consistent phenotypes that include cleft palate across generations and individual animals. Using this model, we demonstrated that Dlx2 caused cleft palate by affecting maxillary growth in the early-stage development of maxillary prominences. By performing bulk RNA-seq, we demonstrated that the overexpression of Dlx2 induced significant changes of many genes related to critical developmental pathways. In summary, our novel mouse model provides a reliable and consistent system for investigating the functions of Dlx2 during development and for dissecting the gene regulatory networks underlying craniofacial development. Bulk RNA sequencing of E12.5 WT and Wnt1cre; Rosa26Dlx2/- mouse embryonic maxillary prominence tissue. Three biological replicates per group颅面形态发生是一项精密复杂的生物学过程，需对细胞增殖、迁移与分化进行精准调控。该过程的异常扰动会引发一系列颅面畸形。Dlx2是调控第一鳃弓（first branchial arch）发育的关键转录因子（transcription factor）。然而，由于缺乏可精准调控Dlx2表达水平的动物模型，学界对Dlx2在颅面发育过程中的转录调控功能始终缺乏深入认知。本研究构建了一种靶向Rosa26位点的Dlx2基因敲入小鼠模型Rosa26^{CAG-LSL-Dlx2-3×Flag}，该模型可实现Dlx2的条件性过表达。通过与Wnt1^Cre小鼠交配，我们获得了Wnt1^Cre; Rosa26^Dlx2/-小鼠，该模型可在神经嵴谱系（neural crest lineage）中以约为内源基因表达水平3倍的剂量过表达Dlx2。Wnt1^Cre; Rosa26^Dlx2/-小鼠呈现出稳定一致的表型，不同世代及个体均表现出腭裂（cleft palate）。利用该模型，我们证实Dlx2可通过影响上颌突早期发育阶段的上颌生长，进而引发腭裂。通过批量RNA测序（bulk RNA-seq）分析，我们发现Dlx2过表达会诱导诸多与关键发育通路相关的基因发生显著表达变化。综上，本研究构建的新型小鼠模型为探究Dlx2在发育过程中的功能，以及解析颅面发育背后的基因调控网络提供了一套可靠且稳定的研究体系。本研究对胚胎第12.5天（E12.5）野生型（Wild Type, WT）与Wnt1^Cre; Rosa26^Dlx2/-小鼠的胚胎上颌突组织进行了批量RNA测序，每组设置3次生物学重复。