Msx2 function in tooth crown morphogenesis

描述(申请人提供)：牙齿是上皮性附属物，其形态发生受保守的信号级联调节。牙冠的花纹和形状由牙尖的位置、方向、形状和高度以及邻近上皮的增殖和折叠决定。在分子水平上，牙冠的图案和形状是由信号及其转录介质控制的，这些信号来自釉质结，釉质结是一种被认为是牙齿形态发生的组织中心的上皮结构。有10多种信号蛋白在釉质结节中受到限制表达，包括成纤维细胞生长因子(FGFs)、HH、WNTs和转化生长因子b(TGFb)超家族以及p21、Edar、Lef1和MSX2等信号调节因子。缺乏同源异型盒基因MSX2的小鼠表现出冠状形态发生缺陷。我们的研究表明，在MSX2基因敲除小鼠中，(I)牙尖形成但畸形，(Ii)磨牙上皮折叠异常，表现为局部的细胞增殖增加，(Iii)牙釉质结中的Bmp4信号依赖于MSX2，这表明Bmp4和MSX2基因在同一遗传途径中发挥作用。这些观察结果为我们提出的假设提供了基础，即依赖于MSX2的信号BMP4是通过控制牙尖形状和牙冠正常形态发生而在釉质结中发挥MSX2功能的潜在主要效应者。在此背景下，为了进一步研究MSX2在牙齿发育中的作用，我们将(I)通过研究Bmp4等与MSX2在釉质结中共表达的基因的表达模式，确定MSX2在釉质结中的调控基因；(Ii)通过检测BMP4在体外和体内修复牙尖形状和牙冠形态发生缺陷的充分性，确定MSX2下游基因BMP4是否是MSX2功能的主要效应者。总之，这些实验应该从遗传学和分子水平上确定MSX2及其下游基因Bmp4在牙齿发育后期的调控层次，并为理解牙尖形状和牙冠形态发生提供一个分子框架。 叙述：牙齿是上皮性附属物，其形态发生受复杂的遗传途径调节。几种颅面部和外胚层发育不良疾病通过影响牙齿的早期发育(牙齿发育不全)或晚期发育(牙冠发育不良、釉质发育不良)来影响牙齿的发育。MSX2突变的牙齿发育不良，表现为牙冠形状异常。虽然MSX2基因突变时对牙冠形态发生起关键作用，但其功能仍不清楚。本项目旨在了解MSX2在牙齿变形症发病机制中的作用。为了实现这一目标，将利用强大的分子生物学、转基因和器官培养方法，寻找受MSX2调控的基因。预计这些结果将对MSX2基因的功能、牙齿发育的发病机制提供有价值的见解，也可能为牙齿变形的治疗提供有价值的信息。4.

DESCRIPTION (provided by applicant): Teeth are epithelial appendages whose morphogenesis is regulated by conserved signaling cascades. The patterning and shape of tooth crowns is determined by the location, orientation, shape and height of tooth cusps, and by the proliferation and folding of the adjacent epithelium. At the molecular level, the patterning and shape of tooth crowns is controlled by signals and their transcriptional mediators derived from the enamel knot, an epithelial structure considered the organizing center for tooth morphogenesis. More than 10 signaling proteins have restricted expression in the enamel knots, including fibroblast growth factors (FGFs), hedgehog (HH), WNTs and transforming growth factor b (TGFb) superfamilies as well as mediators of signaling such as p21, Edar, Lef1 and Msx2. Mice lacking the homeobox gene Msx2 exhibit defects in crown morphogenesis. Our studies show that in the Msx2 knock out mice (i) the cusps are formed but they are misshapen, (ii) the folding of the dental epithelium is aberrant in the molars exhibiting localized, increased cell proliferation and that (iii) Bmp4 signaling in the enamel knot is Msx2-dependent suggesting that Bmp4 and Msx2 genes function within the same genetic pathway. These observations serve as the basis for our proposed hypothesis that an Msx2-dependent signal BMP4 is a potential major effector of the Msx2 function in the enamel knot by controlling the shape of cusps and crown normal morphogenesis. In this context and to further investigate the role of Msx2 during tooth development, we will (i) identify genes regulated by Msx2 in the enamel knot, by investigating the expression patterns of genes that, like Bmp4, are co-expressed with Msx2 in the enamel knot and (ii) determine whether the Msx2 downstream gene, BMP4, is a major effector of the Msx2 function in the enamel knot, by testing the sufficiency of BMP4 to rescue the cusp shape and crown morphogenesis defect in vitro and in vivo. Collectively these experiments should define a regulatory hierarchy between Msx2 and its downstream gene Bmp4 in both genetic and molecular terms during late tooth development and provide a molecular framework for understanding cusp shape and crown morphogenesis. Narrative: Teeth are epithelial appendages whose morphogenesis is regulated by complex genetic pathways. Several craniofacial and ectodermal dysplasia disorders affect tooth development by affecting their early development (tooth agenesis) or late development (dysplastic crown, enamel hypoplasia). Msx2 mutant teeth are dysplastic, exhibiting crown shape abnormalities. Although, Msx2 is critical for tooth crown morphogenesis when mutated, its function remains unknown. This project aims at understanding the role of Msx2 in the pathogenesis of tooth dysmorphias. To accomplish this aim, a search for genes regulated by Msx2 will be conducted, using powerful molecular biology, transgenic and organ culture approaches. It is anticipated that these results will provide valuable insight on the function of Msx2 gene, on the pathogenetic mechanism of tooth development and may also provide valuable information towards treatment of tooth dysmorphias. 4