条件性敲除颅神经嵴源性间充质细胞Jagged1基因致小鼠腭发育不足机制的研究

It has been found that Jagged1-Notch signaling pathway takes part in the modulation of palate development; however, little is known about its cellular and molecular mechanisms. In the previous work, through conditional knockout(CKO) of the Jagged1 (JAG1) gene in the cranial neural crest(CNC),we created a JAG1-CKO mouse model, which showed delayed palate fusion in embryo and progessive palate hypoplasia after birth. Intrigued by these findings，in this project, we are going to make full use of this novel mouse model and clarify the cellular and molecular mechanisms behind the phenotype of palate hypoplasia in the JAG1-CKO mouse. Briefly, 1)the differentially expressed genes in the palate of JAG1-CKO mice compared to that of wild type(WT) mice are to be investigated, using Whole Mouse Genome Oligo Microarray, on embryonic day 13.5(E13.5) and post-natal day 1(P1) respectively. Among the differentially expressed genes, one or several down-stream genes are to be hypothesized as potential pivotal genes, which may play an essential role in the modulation of palate development. 2)The expression of the potential pivotal gene is to be up- or down-regulated in the in vitro cultured palate process on E13.5, or in the mouse palate in vivo on P1 respectively, with the expectation that the phenotype of palate hypoplasia could be recapitulated in the WT mice, or partly rescued in the JAG1-CKO mice. 3)Furthermore, the cellular and histological mechanisms of formation and rescue of the palate hypoplasia are to be investigated using PHH3,TUNEL and hyaluronic acid staining, for cell proliferation, apoptosis and extracellular matrix respectively. In summary, the molecular and cellular mechanisms of the mouse palate hypoplasia induced by Jagged1 gene deletion in CNC are to be expatiated,which will enlarge our understanding in the role of Notch signaling pathway in palate development modulation, and enlighten the potential of rescuing human palate dysplasia through gene or molecular approaches.

Jagged1-Notch信号通路参与了腭发育的调控，但目前对其调控机制还知之甚少。申请人在前期研究中建立了条件性敲除颅神经嵴源性间充质细胞Jagged1基因的小鼠模型（JAG1-CKO），发现其具有进行性腭发育不足表现型。利用该模型，本项目拟： 1）在胚胎第13.5天（E13.5）和出生后第1天(P1)两个时间点，分别使用基因芯片检测JAG1-CKO小鼠腭部相对于野生型（WT）的差异表达基因，从中推测可能对腭发育不足起关键调控作用的下游基因。2）通过上调或下调关键下游基因的表达，分别对体外培养的腭胚突（E13.5）和活体小鼠（P1）的腭发育进行干预,观察能否调控腭发育不足表现型。3）利用PHH3、TUNEL及透明质酸染色等方法，明确其细胞组织学机制。本研究有望获得对Jagged1-Notch信号通路调控腭发育机制的进一步认识，并为通过基因分子手段干预人类腭发育不足畸形提供借鉴和参考。

Jagged1-Notch 信号通路参与了腭发育的调控，但目前对其调控机制还知之甚少。申请人在前期研究中建立了条件性敲除颅神经嵴源性间充质细胞Jagged1 基因的小鼠模型（JAG1-CKO），发现其具有进行性腭发育不足表现型。利用该模型，本项目通过体外腭胚突的培养及相应的免疫荧光染色对比观察JMKO及同窝wt小鼠胚胎在腭部生长发育过程中，其腭胚突细胞增殖、凋亡等生物学行为以及胞外基质的变化。 使用Jagged1、PECAM-1双重免疫荧光染色及缺氧探针技术，观察并比较JMKO小鼠及同窝wt小鼠胚胎腭胚突的血管发育及缺氧状况。 利用实时定量PCR实验技术，检测并比较Jagged1-Notch信号通路中各配体、受体在JMKO小鼠及同窝wt小鼠胚胎腭胚突的表达。 发现 E14.5~E15.5，JMKO小鼠胚胎腭胚突间充质可见明显不规则血管腔形成，血管分支减少，其血管分布密度与wt相比显著下降，缺氧探针显示此阶段JMKO小鼠腭胚突间充质缺氧区域比wt明显增大，Jagged1表达下降部位与血管分布密度下降及缺氧探针阳性部位一致。 E13.5~E14.5，JMKO小鼠胚胎腭胚突组织中Notch2及Notch3 mRNA的表达与同窝wt比较均显著上调，Notch1 mRNA的表达与同窝wt比较无显著差异。 由此我们可以认为在Jagged1-Notch信号通路中，Jagged1、Notch2及Notch3可能在腭胚突生长发育过程中发挥了重要作用。选择性敲除颅神经嵴源性间充质细胞Jagged1基因导致JMKO小鼠腭胚突血管发育异常及缺氧区域面积增大，腭胚突的血供及供氧不足使其间充质细胞的正常增殖凋亡及胞外基质的正常分泌被干扰，这可能是导致JMKO小鼠硬腭短小伴腭皱襞数量减少、上颌发育不足及反牙合等畸形产生的主要原因。