Development of Avian Extraocular Muscles and Nerves

描述（由申请人提供）：我们的长期目标是了解指导眼外肌起始和形态发生并引导适当运动轴突到达眼外肌的机制，其缺陷导致先天性眼病，如Duane综合征。我们的假设是，包括来自大脑的高度局部化信号（例如，FGF 8）作用于相邻的近轴中胚层细胞。我们的初步数据表明，通过异位表达fgf 8改变鸟类中脑的信号传导特性会破坏背斜肌（DO）的发育。我们将在禽类胚胎中筛选推定的肌源性信号，然后通过将信号激动剂或拮抗剂引入中脑（用于DO诱导）或后脑（用于外直肌，LR，诱导）来测试候选物。这将使用电穿孔和用RCAS逆转录病毒构建体感染来完成。为了检查肌肉原基的反应能力，将用反应阻断构建体（例如sprouty 1）感染的躯干中胚层移植到鸡胚的抗感染品系中，以代替正常的LR原基。测定胚胎的眼外肌分化的几种标志物，基因表达的变化， 大脑中的神经发生，以及轴突生长的模式。颅神经In、IV和VI出现在不同的位置，并且每个都显示出独特的早期生长模式。我们的假设是，外展神经和眼神经轴突使用不同的组合的指导系统，包括生长因子受体对和成员的狭缝机器人，脑信号蛋白neuropilin，和ephrin的eph家庭。筛选候选的指导分子后，我们将通过在神经III，VI和XIII形成的位点之间移植运动神经元前体来测试运动轴突特性的预先指定，然后通过使用电穿孔在运动神经元中产生候选基因表达的变化来评估所识别的指导分子的作用。目的3利用鸟类模型的交叉神经支配的LR肌肉的眼神经轴突，伴随杜安综合征。这是由一个简单的早期病变外展神经原基。我们的假设是，在外展神经的情况下，有一个上调的神经排斥剂和/或下调的LR内的神经引诱剂，允许n。III向内生长。我们将筛选候选分子，特别是HGF和GDNF，然后通过将神经营养候选物过表达到LR原基中来测试它们的作用。

DESCRIPTION (provided by applicant): Our long-term goal is to understand mechanisms directing the initiation and morphogenesis of the extra-ocular muscles and guiding appropriate motor axons to them, defects of which contribute to congenital oculomotor diseases such as Duane syndrome. Our hypothesis is that a consortium of positive and negative myogenic signals including highly localized signals from the brain (e.g., fgf8) act upon adjacent paraxial mesoderm cells. Our preliminary data show that altering signaling properties of the avian midbrain by ectopic expression of fgf8 disrupts development of the dorsal oblique (DO). We will screen for putative myogenic signals in the avian embryo, then test candidates by introducing signal agonists or antagonists into the midbrain (for DO induction) or hindbrain (for lateral rectus, LR, induction). This will be done using electroporation and infection with RCAS retroviral constructs. To examine the response capabilities of muscle primordia, trunk mesoderm infected with response blocking constructs, e.g. sprouty1, will be transplanted into infection-resistant strains of chick embryos in place of the normal LR primordium. Embryos are assayed for several markers of extra-ocular muscle differentiation, for changes in gene expression and neurogenesis in the brain, and for patterns of axon outgrowth. Cranial nerves In, IV, and VI arise in separate locations and each shows patterns of early outgrowth that are unique. Our hypothesis is that outgrowing abducens and oculomotor axons use different combinations of guidance systems, including growth factor-receptor pairs and members of the slit-robo, semaphorin-neuropilin, and ephrin-eph families. After screening for candidate guidance molecules, we will test for prespecification of motor axon properties by transplanting motor neuron precursors between sites of nerve III, VI, and XIII formation, then evaluate the roles of identified guidance molecules by creating changes in candidate gene expression in motor neurons using electroporation. Aim 3 exploits an avian model of the cross-innervation of the LR muscle by oculomotor axons that accompanies Duane syndrome. This is produced by a simple early lesion to the abducens primordium. Our hypothesis is that in the absence of the abducens nerve, there is an upregulation of neurorepellants and/or downregulation of neuroattractants within the LR that allows n. III ingrowth. We will screen for candidate molecules, especially HGF and GDNF, then test for their roles by overexpressing neurotrophic candidates into the LR primordium.