Cellular mechanisms underlying Fgf8-mediated asymmetry of the pharyngeal endoderm

项目摘要 咽囊发育成胸腺、甲状腺和甲状旁腺， 耳朵和扁桃腺它们还提供了对颅面形态发生至关重要的信号 骷髅在后一种作用中，囊袋相对于间充质的特定形态和位置， 咽弓对于指导骨骼的形成至关重要。尽管这些重要的作用， 脊椎动物头部的发育，哺乳动物咽囊的发育仍然知之甚少。 咽囊的形成由两个独立的形态发生过程组成， 近端-远端延伸。本提案中概述的工作重点是了解Fgf 8在细胞凋亡中的作用。 后一个过程。使用Fgf 8突变小鼠的等位基因系列，可以将不同Fgf 8剂量的胚胎移植到小鼠中。 产生，包括轻度和重度突变。与斑马鱼相反，在两种小鼠Fgf 8突变体中， 基因型，第一咽囊（pp 1）外袋接触外胚层，但未能沿沿着 近端-远端轴。在轻度突变体中，pp 1的大小减小，但确实延伸。在严重突变体中，pp 1 外胚叶裂隙发育不全，组织紊乱，导致失败 使第一和第二弓形在远端分开。严重的突变体有小而圆的囊袋， 看起来是互相叠加的总之，这些数据表明Fgf 8在咽部炎症中具有额外的作用。 袋形成超出了引导侧向外袋化，其可以包括两个袋的近侧-远侧延伸， 咽内胚层和外胚层。先前的研究表明，Fgf 8调节细胞极性， 肌动蛋白细胞骨架的极性对于咽囊的延伸是必不可少的。具体的假设是 Fgf 8具有调节细胞增殖和细胞极性的旁分泌功能， 咽囊上皮细胞这一假设将通过两个具体目标进行检验。具体目标1、 在Fgf 8已经减少的胚胎中，将定量囊上皮细胞的增殖和极性 在全球在特定目标2中，将在胚胎中评价囊形状，其中Fgf 8在胚胎中特异性消融。 咽中胚层或外胚层。这项工作的未来研究目标是调查遗传和 在颅面形态发生的变化，特别是在变化的发展相互作用， 颅面疾病的严重程度和发病率。Fgf 8等位基因系列表现出大范围的 形态变异，包括两种突变基因型中存在的双侧变异。Fgf 8突变体 基因型表现出下颌的方向不对称性，例如Fgf 8 Neo/Neo小鼠，其中单侧融合 33%的新生儿仅观察到左侧颌骨。虽然还没有直接展示， 方向不对称可能是由于在颅中胚层中Fgf 8表达的双侧不对称， 心脏发育的结果。面部不对称和心脏缺陷与几种综合征有关， 特别是CHARGE和DiGeorge（22 q11缺失）综合征，其具有与Fgf 8突变体相似的表型。

PROJECT SUMMARY The pharyngeal pouches develop into the thymus, thyroid and parathyroid glands and contribute to formation of the ear and tonsils. They also provide signals that are essential to the morphogenesis of the craniofacial skeleton. In this latter role, the specific morphology and position of the pouches relative to the mesenchyme of the pharyngeal arches is critical to instruct skeletal formation. Despite these essential roles for proper development of the vertebrate head, pharyngeal pouch development in mammals remains poorly understood. Pharyngeal pouch formation consists of two separate morphogenetic processes, lateral out-pocketing and proximal-distal extension. The work outlined in this proposal focuses on understanding the role of Fgf8 in the latter process. Using an allelic series of Fgf8 mutant mice, embryos of different Fgf8 dosages can be generated, including both a mild and severe mutant. In contrast to zebrafish, in both mouse Fgf8 mutant genotypes, the first pharyngeal pouch (pp1) out-pockets to contact the ectoderm but fails to extend along the proximal-distal axis. In mild mutants, pp1 is reduced in size, but does extend. In the severe mutant, pp1 does not extend proximo-distally and the ectodermal cleft is also hypoplastic and disorganized, contributing to failure of the first and second arches to separate distally. Severe mutants have small, round pouches in which cells appear to stack upon each other. Together, these data suggest that Fgf8 has additional roles in pharyngeal pouch formation beyond directing lateral out-pocketing, which may include proximal-distal extension of both the pharyngeal endoderm and ectoderm. Previous research has suggested that Fgf8 regulates cell polarity, and that polarity of the actin cytoskeleton is essential to pharyngeal pouch extension. The specific hypothesis to be tested by the proposed research is that Fgf8 has a paracrine function regulating proliferation and polarity of pharyngeal pouch epithelial cells. This hypothesis will be tested through two specific aims. In Specific Aim 1, proliferation and polarity in pouch epithelial cells will be quantified in embryos in which Fgf8 has been reduced globally. In Specific Aim 2, pouch shape will be evaluated in embryos in which Fgf8 is specifically ablated in the pharyngeal mesoderm or ectoderm. The future research goals of this work are to investigate genetic and developmental interactions underlying variation in craniofacial morphogenesis, particularly variation in the severity and penetrance of craniofacial disorders. The Fgf8 allelic series exhibits a large range of morphological variation, including the bilateral variation present in both mutant genotypes. Both Fgf8 mutant genotypes exhibit directional asymmetry of the jaw, exemplified by Fgf8Neo/Neo mice in which unilateral fusion of the jaw on the left side only is observed in 33% of neonates. Although it has yet to be shown directly, directional asymmetry is likely due bilateral asymmetry in Fgf8 expression in the cranial mesoderm as a consequence of heart development. Facial asymmetry and heart defects are associated in several syndromes, notably CHARGE and DiGeorge (22q11 deletion) syndrome, which have similar phenotypes to Fgf8 mutants.