转录因子Isl1通过Wnt及Shh信号通路调控舌早期发育的机理研究

Craniofacial abnormalities account for most of congenital malformations. The normal development of tongue is critical for the development of palate. There are limited reports on the molecular mechanism of tongue development. Transcription factor Isl1 as an LIM family member plays an important role during pancreas maturation and heart development. Whereas, there are no reports about its function in the development of tongue. The defect of tongue development was found in Isl1 conditional knockout mice (Isl1 (loxp/loxp);Shh-cre). The lingual swellings were not detected at E11.5 in Isl1 mutant mice. The results of experiments showed that the activity of Wnt/β-catenin and shh signaling pathway was significantly inhibited in the lower jaw of Isl1 mutant mice. The results highly suggest that transcription factor Isl1 is important for tongue development. We are going to study the regulation mechanism of Wnt/β-catenin and shh signaling by Isl1. Conditinal knockout mice, Dual-Luciferase Reporter Assay and Chip assay will be used to address the function of Isl1 during the development of tongue. The project will contribute to understanding of the mechanism of tongue development and supply a new target for the gene therapy.

颌面部发育畸形发病率位于新生儿先天性发育畸形的首位。舌发育与腭发生密切相关，而目前对舌发育机理的研究少有报道。Isl1是属于LIM-HD家族的一个转录因子，在心脏发育及胰岛成熟等过程中起重要作用，然而尚无Isl1 基因参与舌发育的报道。我们前期首次构建了Isl1在下颌上皮细胞条件性敲除小鼠，首次发现Isl1突变型小鼠在胚胎第11.5天舌隆突消失，舌几乎完全未发育。并且突变型小鼠下颌舌隆突中Wnt和Shh信号通路受到显著抑制。我们的前期工作提示Isl1对启动和调控舌发育起关键作用。本项目拟通过基因条件敲除小鼠、体外双荧光报告基因、染色质免疫沉淀分析等方法，进一步深入研究Isl1在下颌中对Wnt和Shh信号通路的调节机制，解析Isl1调控舌发育过程的分子机制。本项目的开展有助于深入了解胚胎早期下颌上皮细胞中的信号分子对于舌发育的关键启动作用和调控机制，为治疗颌面部发育畸形疾病提供新药靶点。

颌面部发育畸形发病率位于先天性发育畸形的首位。一直以来，大多数的颌面部发育畸形病因学研究集中在腭突的发育异常。然而，舌的发育分子调控机制少见报道。舌是参与发音、咀嚼、感知味觉等功能的重要器官，舌的正常发育对口腔内相关结构的正常发育十分重要。Isl1 是属于LIM-Homeodomain (LIM-HD)家族的一个转录因子，在胰岛的成熟、运动神经元特化及心脏发育过程中都起着重要作用。我们用Shh-Cre 小鼠与Isl1（loxp/loxp）小鼠交配，得到Isl1 在下颌上皮细胞条件性敲除小鼠。我们的研究结果首次发现，Isl1 突变型小鼠表现出舌发育异常，在胚胎第11.5 天舌隆突消失，舌几乎完全未发育，显示下颌上皮细胞中转录因子Isl1 对启动和调控舌发育起着不可或缺的关键作用。实验结果显示Hedgehog信号通路Shh、Ptch1及Gli1等基因的表达明显下降，相反Wnt信号通路抑制因子Dkk1、Dkk2、Dkk3及Wif1等基因的表达都明显上升。TOPGAL转基因小鼠染色结果显示Wnt信号通路在下颌上皮细胞中的活性明显下降，Chip assay结果进一步证实Isl1蛋白可以结合到Dkk1、Dkk2及Wif1基因的启动子区域。实验结果显示Isl1通过调控Wnt信号通路调节Shh信号通路，进而影响下颌及舌的发育。进一步研究发现，过表达β-catenin或Ihh可以部分挽救Isl1基因敲除导致的下颌及舌发育缺陷。本项目的开展有助于深入了解舌发育的分子机制，尤其是胚胎早期下颌上皮细胞对于舌发育的关键启动作用和调控机制，为预防舌发育缺陷疾病、发现治疗颌面部发育畸形疾病新药靶点和有效途径提供理论依据。