Molecular Mechanisms of Trigeminal Development

描述(由申请人提供)： 在当前的资金周期中，我们揭示了神经营养因子对发育中的三叉神经(V)初级传入神经元的营养和趋化作用，并表明神经营养因子的树枝和延伸效应是由Rho家族GTP酶介导的。拟议的实验旨在通过定向删除感觉神经元生存所必需的特定基因来解决V神经节细胞的分化及其在小鼠中的投射。凋亡前基因(Bax)缺失的小鼠将与NGF-、NT3-、TrkA-或TrkC-基因敲除小鼠杂交，揭示神经营养因子对V神经节细胞的轴突分化作用，而不依赖于它们的细胞存活效应。为了研究NGF和NTS对胚胎V传入投射的趋化作用，将进行功能丧失和功能获得的研究。将通过超微结构和免疫组织化学研究分析NGF和NTS在这些不同制剂中的突触营养作用。1.在Bax/NGF或Bax/NTS双基因敲除小鼠中，V神经节中所有类型的初级感觉神经元都将存活，但它们在轴突分化方面存在缺陷，从而阻止了胡须相关脑干模式的形成。细胞死亡标记物、细胞数量的体视学估计(核心A)和细胞类别特异性标记物将用于评估胚胎V神经节细胞的存活。轴突标记和投射的量化将揭示轴突分化相对于野生型对照的变化(核心B)。代谢染色将揭示对中枢神经系统模式形成的影响(核心A)。2.神经营养因子促进V类初级传入神经元的突触发生。在外源性神经营养素强化方案的作用下，V通路的大量培养将被用来研究V脑干核团(核心A)突触蛋白表达和突触轮廓的发展。在缺乏NGF和NTS高亲和力受体的情况下，将在体内对TrkA和TrkC基因敲除胚胎进行补充分析，以研究突触的发育。这两个实验将揭示NGF家族在V系统发育中的作用。我们进一步的假设是：3.GDNF家族配体(GDNF、Neurturin、Artemin)参与了围产期V神经节细胞的存活和轴突的发育。V初级传入细胞的存活、分化、表型、形态和模式将在可比的基因靶向和因子增强整体范例中进行评估，因为它们与GDNF家族的配体和受体(核心A)有关。也将通过使用嵌入GDNF家族配体的局部来源的分离的V神经节来测试趋化效应。将分析靠近或远离此GDNF来源的V轴突生长(核心B)。

Description (provided by applicant): During the current funding cycle, we revealed trophic and tropic actions of neurotrophins on specific classes of developing trigeminal (V) primary afferent neurons, and showed that arborization and elongation effects of neurotrophins are mediated by the Rho family GTPases. Proposed experiments address the differentiation of V ganglion cells and their projections in mice with targeted deletion of specific genes known to be necessary for sensory neuron survival. Mice with deletion of a preapoptotic gene (bax) will be crossed with NGF-, NT3-,TrkA-, or TrkC-knockout mice to reveal axonal differentiation effects of neurotrophins on V ganglion cells, independent of their cell survival effects. To address chemotropic actions of NGF and NTS on embryonic V afferent projections, loss-of-function and gain-of-function studies will be performed. Synaptotrophic actions of NGF and NTS will be analyzed in these various preparations by ultrastructural and immunohistochemical studies. Pilot data prompt the following 3 hypotheses and experiments: 1. In Bax/NGF or Bax/NTS double knockout mice, all classes of primary sensory neurons in the V ganglion will survive, but they display deficits in axon differentiation, therein preventing whisker related brainstem pattern formation. Cell death markers, stereologic estimates of cell numbers (Core A), and cell class-specific markers will be used to assess embryonic V ganglion cell survival. Axonal labeling and quantification of projections will reveal changes in axonal differentiation relative to wildtype controls (Core B). Metabolic staining will reveal effects on CNS pattern formation (Core A). 2. Neurotrophins facilitate synaptogenesis in developing V primary afferents. Wholemount cultures of the V pathway subjected to exogenous neurotrophin augmentation regimens will be used to study development of synaptic protein expression and synaptic profiles in V brainstem nuclei (Core A). Complementary analyses will be performed in vivo in TrkA and TrkC knockout embryos to study synaptic development in the absence of high-affinity receptors for NGF and NTS. These 2 experiments will reveal NGF family actions in V system development. We further hypothesize: 3. The GDNF family ligands (GDNF, neurturin, artemin) contribute to perinatal V ganglion cell survival and axon development. V primary afferent survival, differentiation, phenotypes, morphology and patterning will be assessed in comparable gene targeting and factor augmentation wholemount paradigms as they pertain to the GDNF family ligands and receptors (Core A). Chemotropic effects will also be tested by using isolated V ganglia embedded with a localized source of GDNF family ligand. V axon growth towards or away from this GDNF source will be analyzed (Core B).