Molecular Mechanisms of Trigeminal Development

描述（申请人提供）： 在当前的资助周期中，我们揭示了神经营养因子对特定类别的发育中三叉神经（V）初级传入神经元的营养和向性作用，并表明神经营养因子的树枝化和延伸作用是由Rho家族GTP酶介导的。提出的实验解决了V神经节细胞的分化和它们在小鼠中的投射，已知特定基因的靶向缺失是感觉神经元存活所必需的。将缺失凋亡前基因（bax）的小鼠与NGF-、NT 3-、TrkA-或TrkC-敲除小鼠杂交，以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作用。为了说明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研究。通过超微结构和免疫组织化学研究，将在这些不同的制剂中分析NGF和NTS的突触营养作用。初步数据提示以下3个假设和实验：1。在Bax/NGF或Bax/NTS双敲除小鼠中，V神经节中的所有类别的初级感觉神经元都将存活，但它们显示轴突分化的缺陷，从而阻止了须相关的脑干模式形成。将使用细胞死亡标志物、细胞数量的体视学估计（核心A）和细胞类别特异性标志物评估胚胎V神经节细胞存活。轴突标记和投射的定量将揭示轴突分化相对于野生型对照（核心B）的变化。代谢染色将揭示对CNS模式形成的影响（核心A）。2.神经营养因子促进V初级传入神经的突触发生。接受外源性神经营养因子增强方案的V通路的全细胞培养物将用于研究V脑干核（核心A）中突触蛋白表达和突触特征的发展。将在TrkA和TrkC敲除胚胎中进行体内补充分析，以研究在缺乏NGF和NTS的高亲和力受体的情况下的突触发育。这两个实验将揭示V系统发育中NGF家族的作用。我们进一步假设：3。GDNF家族配体（GDNF、neurturin、artemin）有助于围产期V神经节细胞存活和轴突发育。将在与GDNF家族配体和受体（核心A）相关的可比基因靶向和因子增强整体装载范例中评估V初级传入存活、分化、表型、形态和模式。还将通过使用包埋有GDNF家族配体的局部来源的分离的V神经节来测试趋化作用。将分析朝向或远离该GDNF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).