Development of Avian Extraocular Muscles and Nerves

描述(申请人提供)：我们的长期目标是了解指导眼外肌的起始和形态形成的机制，并引导适当的运动轴突到达它们，其缺陷导致先天性动眼神经疾病，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，Fgf8)在内的正负向肌源性信号联合作用于相邻的轴旁中胚层细胞。我们的初步数据显示，通过异位表达Fgf8来改变鸟类中脑的信号特性，扰乱了背斜肌(DO)的发育。我们将在禽类胚胎中筛选假定的肌源性信号，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(用于侧直肌，LR诱导)来测试候选信号。这将通过电穿孔和感染RCAS逆转录病毒载体来完成。为了检测肌肉原基的反应能力，感染了反应阻断结构的躯干中胚层，例如Sprouty1，将被移植到抗感染的鸡胚胎品系中，以取代正常的LR原基。对胚胎进行眼外肌分化的几个标志的检测，基因表达和基因表达的变化 大脑中的神经发生，以及轴突生长的模式。其中，IV和VI中的脑神经分别出现在不同的位置，每一种都显示出独特的早期发育模式。我们的假设是，超过生长的外展神经和动眼神经轴突使用不同的指导系统组合，包括生长因子-受体对和缝隙-ROBO、信号素-神经匹林和ePhin-Eph家族的成员。在筛选候选引导分子后，我们将通过在神经III、VI和XIII形成部位之间移植运动神经元前体来测试运动轴突预指定特性，然后通过电穿孔产生运动神经元候选基因表达的变化来评估识别的引导分子的作用。目的3利用伴随Duane综合征的动眼神经轴突交叉支配下丘脑肌的禽类模型。这是由外展肌原基的简单早期损伤造成的。我们的假设是，在没有外展神经的情况下，在LR内存在神经驱避剂的上调和/或神经诱导剂的下调，从而允许n.III内生。我们将筛选候选分子，特别是HGF和GDNF，然后通过向LR原基过度表达神经营养候选来测试它们的作用。

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.