Hossain Waheeda A, D'Sa Chrystal, Morest D Kent
Department of Neuroscience, University of Connecticut Health Center, Farmington, 06030, USA.
J Neurobiol. 2006 Aug;66(9):897-915. doi: 10.1002/neu.20264.
Neurotrophins and FGF2 contribute to formation of the cochlea, but their roles in cochlear nucleus development are unknown. The effects of these factors may differ in the cochlea and cochlear nucleus, which may influence each other's development. It is important to analyze the effects of these factors on cellular structures at well-defined steps in the normal morphogenetic sequence. The present study used immunohistochemistry to localize factors in situ and to test hypotheses about their roles in an in vitro model. Specific antibody staining revealed that TrkC, the NT3 receptor, is present in neural precursors prior to embryonic day E11 until after birth. NT3 appeared in precursor cells during migration (E13-E15) and disappeared at birth. TrkC and NT3 occurred in the same structures, including growing axons, terminals, and their synaptic targets. Thus, NT3 tracks the migration routes and the morphogenetic sequences within a window defined by TrkC. In vitro, the cochlear nucleus anlage was explanted from E11 embryos. Cultures were divided into groups fed with defined medium, with or without FGF2, BDNF, and NT3 supplements, alone or in combinations, for 7 days. When neuroblasts migrated and differentiated, immunostaining was used for locating NT3 and TrkC in the morphogenetic sequence, bromodeoxyuridine for proliferation, and synaptic vesicle protein for synaptogenesis. By time-lapse imaging and quantitative measures, the results support the hypothesis that FGF2 promotes proliferation and migration. NT3 interacts with FGF2 and BDNF to promote neurite outgrowth, fasciculation, and synapse formation. Factors and receptors localize to the structural sites undergoing critical changes.
神经营养因子和FGF2有助于耳蜗的形成，但其在蜗神经核发育中的作用尚不清楚。这些因子在耳蜗和蜗神经核中的作用可能不同，而这可能会相互影响彼此的发育。分析这些因子在正常形态发生序列中明确阶段对细胞结构的影响很重要。本研究采用免疫组织化学方法在原位定位这些因子，并在体外模型中验证关于它们作用的假设。特异性抗体染色显示，神经营养因子3（NT3）的受体TrkC在胚胎第11天（E11）之前直至出生后的神经前体细胞中均有表达。NT3在迁移期（E13 - E15）出现在前体细胞中，并在出生时消失。TrkC和NT3出现在相同的结构中，包括生长中的轴突、终末及其突触靶点。因此，NT3追踪由TrkC界定的窗口内的迁移路径和形态发生序列。在体外，从E11胚胎中取出蜗神经核原基。将培养物分为添加或不添加FGF2、脑源性神经营养因子（BDNF）和NT3补充剂的不同组，单独或组合添加，培养7天。当神经母细胞迁移和分化时，使用免疫染色在形态发生序列中定位NT3和TrkC，使用溴脱氧尿苷检测增殖情况，使用突触小泡蛋白检测突触形成情况。通过延时成像和定量测量，结果支持了FGF2促进增殖和迁移的假设。NT3与FGF2和BDNF相互作用以促进神经突生长、束状化和突触形成。因子和受体定位于正在发生关键变化的结构部位。
