Cellular mechanisms underlying Fgf8-mediated asymmetry of the pharyngeal endoderm

项目总结 咽囊发育成胸腺、甲状腺和甲状旁腺，并有助于形成。 耳朵和扁桃体。它们还提供对颅面形态发生至关重要的信号。 骷髅。在后一种作用中，囊相对于间充质的特定形态和位置 咽弓是指导骨骼形成的关键。尽管这些角色是正确的 脊椎动物头部、咽囊的发育在哺乳动物中的发育仍然知之甚少。 咽袋的形成由两个独立的形态发生过程组成，即侧向外袋形成和 近-远端伸展。本提案中概述的工作重点是了解Fgf8在 后一个过程。利用一系列Fgf8突变小鼠的等位基因，不同Fgf8剂量的胚胎可以 产生的，包括轻度和重度突变。与斑马鱼相反，在小鼠Fgf8突变体中 第一个咽袋(PP1)外袋接触外胚层，但不能沿着外胚层延伸 近端-远端轴线。在温和的突变体中，PP1的大小减少，但确实延长。在严重的突变体中，PP1 没有向近端延伸，外胚层裂隙也发育不良，组织紊乱，导致失败 第一拱门和第二拱门向远端分开。严重的突变株有小而圆的囊袋，其中的细胞 看起来彼此堆叠在一起。综上所述，这些数据表明Fgf8在咽部还有其他作用 眼袋形成超过引导外侧外袋，这可能包括近端-远端延伸的两个 咽内胚层和外胚层。先前的研究表明，Fgf8调节细胞的极性，并且 肌动蛋白细胞骨架的这种极性对于咽袋的伸展是必不可少的。具体的假设是 被提出的研究验证的是Fgf8具有旁分泌功能，调节细胞的增殖和极性 咽袋上皮细胞。这一假设将通过两个具体目标进行检验。在具体目标1中， 在Fgf8减少的胚胎中，囊袋上皮细胞的增殖和极性将被量化 全球范围内。在特定目标2中，将评估在FGF8被特定消融的胚胎中的眼袋形状 咽中胚层或外胚层。这项工作的未来研究目标是研究遗传和 颅面形态发生变异背后的发育相互作用，特别是 颅面部疾病的严重程度和外显率。Fgf8等位基因序列表现出很大范围的 形态变异，包括两个突变型中存在的双侧变异。两个Fgf8突变体 Fgf8Neo/Neo小鼠表现出颌骨的方向性不对称，在Fgf8Neo/Neo小鼠中，一侧融合 只有33%的新生儿可以观察到左侧的颌骨。虽然它还没有直接展示出来， 定向不对称可能是由于Fgf8在颅骨中胚层中表达的两侧不对称。 心脏发育的后果。面部不对称和心脏缺陷与几种综合征有关， 值得注意的是，Charge和DiGeorge(22q11缺失)综合征与Fgf8突变体具有相似的表型。

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.