【作者】 白仲添；

【导师】 王建林；

【作者基本信息】 兰州大学 ， 动物学， 2011， 博士

【摘要】 分节是脊椎动物早期脑部发育中最普遍和重要的特征。神经上皮在发育中分节形成一系列菱形区(rhombomere1-8, r1-8),这种分节过程参与协调脑运动神经的发育。尤其在后脑,鳃运动神经的定位和各自的特性,与这些菱形区的形成密不可分。鳃运动神经轴突起自神经板并向背侧延伸到达特定轴突出口(exit point,EP),穿过EP在神经上皮内向外侧投射进入周围组织。到达EP之前,鳃运动神经轴突的投射受一系列排斥、吸引和黏附性扩散因子调控,并受诸如轴突出口、胶质细胞等一系列“路标”的指引。但是,当神经轴突通过EP后,是什么因素决定鳃运动神经轴突的寻路特征,目前尚未见报道。副神经(即第11对脑神经)是运动神经,其寻路特征较其它脑神经而言具有独特性：副神经神经元胞体起自神经板,向背侧延伸并停留在神经管背外侧,形成一系列头尾方向区域性分布的神经核,每一个神经元发出的轴突对应一个轴突出口,轴突出口由迁移的神经嵴界帽细胞组成,它可阻止神经元胞体通过EP而只允许其轴突通过。穿过EP以后,副神经轴突并不像其它脑神经运动轴突一样直接进入周围组织,而是先穿过神经上皮,转向头侧延伸,随着神经管的发育延伸一段距离,然后汇聚所有轴突形成副神经,沿垂直于纵轴的方向迁移出神经管进入周围组织。由于其寻路方式的特异性,本研究以副神经轴突作为研究对象,来揭示鳃运动神经轴突导向的共性。由于副神经轴突延伸受周围环境因素的影响,因此,我们着重探究第八菱形区(r8)的伸长、r8区体节、神经嵴细胞,神经上皮及神经元胞体本身是否参与鳃运动神经轴突的寻路导向等问题。首先,解答副神经核的特异性分布是否受r8的伸长所影响,也就是说,副神经神经元胞体沿神经管向背侧的迁移特性是否由于起源于发育中r8的伸长所致。第二,解答副神经根的寻路轨迹是受来源于内胚层的环境因子所影响,还是受神经管内在因子所调控。实验对r8区进行异种同位移植(鹌鹑r8一鸡r8),并没有发现在副神经轴突延伸区域发现r8的延伸。对r8区进行从头部到胸部的异位移植后,可以在受体移植区观察到副神经根的典型特征。反过来,胸部神经管异位移植到r8区时,并不能在r8区产生副神经根。并且,对r8区体节的切除也不能影响手术区副神经根的导向。以上结果说明,头部神经管本身具有调控r8区副神经神经元轴向分布的特性。副神经根的寻路特征受神经管内在因子的决定。基于以上的结论,本实验进一步研究运动神经轴突寻路是否受神经上皮和神经嵴的调控。Sox10基因对神经嵴前体细胞的迁移起至关重要的作用,Wntl基因则在神经嵴细胞的形成和迁移中起决定性的作用。因此,本实验设计用SoxlO/acZ/lacZ基因突变鼠和Wntl基因敲除小鼠,来研究神经嵴的形成和迁移受阻后副神经轴突的导向变化。实验发现,在基因突变鼠和敲除鼠,副神经根的寻路特征并没有受到这两种基因突变和敲除的影响。对副神经区域进行冰冻切片后用HNK-1进行特异性免疫组织化学染色发现,在r8区,有残留的神经嵴细胞存在。因此,对Sox10和Wntl基因进行突变和敲除,并不能完全去除神经嵴细胞,也许少量的神经嵴细胞足以介导对轴突寻路的调控。由于突变鼠和敲除鼠在本实验中的局限性,在接下来的实验中,我们以鸡胚和鹌鹑作为研究对象,利用显微切除和种植携带Noggin念珠的方法来局部切除和影响神经嵴。结果显示,穿过EP以后,副神经轴突寻路特征发生改变,与其它鳃运动神经轴突一样,垂直于神经管纵轴向背腹侧延伸到周围组织。但是对神经嵴从r8区到躯干段的异位移植并不能使副神经的轴突寻路发生改变。接着,对含有神经上皮的背侧神经管在r8区、躯干段和r5区(分布面神经运动神经核)之间进行异位移植,来研究神经上皮在鳃运动神经轴突导向中是否起引导和调节作用。结果显示,移植到躯干段和r5区的r8神经上皮排斥宿主脊神经和面神经轴突直接进入周围组织；移植到r8区的躯干段和r5区的神经上皮则允许宿主副神经轴突垂直于纵轴方向进入到周围组织。实验证实,神经上皮含有引导鳃运动神经轴突延伸的导向因子,而且神经嵴细胞可能传递指令给神经上皮,来参与神经管内鳃运动神经轴突导向的决定。更多还原

【Abstract】 Segmentation is a widely employed strategy in development. In the head, the segmentation of the neuroepithelium of the hindbrain coordinates the development of the cranial motor nerves. Particularly in hindbrain, the branchiomotor nerves reflect this rhombomeric organization. The branchiomotor axons grow dorsally away from the floor plate, project laterally within the neuroepithelium to exit to the periphery. During extending in the neural tube, the projecting branchiomotor axons are guided by diffusible cues including repulsive, attractive and adhesive properties, and aided by guideposts, such as nerve exit points, glial cells etc. However, after exiting to the exit point, nothing is known what determine the pathfinding pattern of branchiomotor axons. The accessory nerve, XI cranial nerve, is a pure motor nerve. It displays a unique organization in that its somata located in an extended region of the neural tube, forming a large rostro-caudal extending nucleus, and its axons turn immediately after exiting the neural epithelium cranially, and ascendant along the developing neural tube before they extend ventrally into the periphery. Due to the unique pathfinding pattern, the cranial accessory axons were used as a model to explore the pathfinding pattern determination of the branchiomotor axons. In our research, the roles of elongation of rhomobomere 8, neural crest cells, neuroepithelium were revealed in guiding pathfinding pattern of branchiomotor axons. Firstly, we investigated whether the extended nucleus domain arises by an elongation of rhomobomere 8 or whether this was due to a migration of neurons along the neural tube which had originated from rhombomere 8. Secondly we determined whether accessory nerve root patterning was influenced by environmental cues derived from the mesoderm or whether it represented an intrinsic property of the neural tube. After the homotopic transplantation of a rhombomere 8 segment, we did not observe an elongation of the grafted tissue. Furthermore migration of cells within the neural tube was not detected. After the heterotopic tranplantation of a rhombomere 8 segment from occipital to cervical and thoracic level, we observed the typical accessory nerve root pattern. In contrast a cervical neural tube graft was not able to give rise to the typical accessory nerve root pattern when transplanted to hindbrain level. The absence of somites did not alter the accessory nerve roots in the operated region. Our results reveal that neurons within rhombomere 8 maintain their axial position within the neural tube in the occipital region. The formation of the accessory nerve root pattern is an intrinsic property of the neural tube. Based on these results, we detect whether the motor axon pattern was determined by neuroepithelium and (or) neural crest. Since Sox 10 signal is essential for the migration of nascent neural crest cells, the mutant mouse SoxlOlacZ/lacZ experiment suggests that the accessory pattern extends at the same pattern as wild-type. Then, the mutant mouse WntlCre-/- was used expectantly to delete all the neural crest cells. We found the same result displayed as mutant mouse SoxlOlacZ/lacZ. Using neural crest cells marker HNK-1, we found some neural crest left in the rhombomere 8 (r8). The data suggest the SoxlOlacZ/lacZ and WntlCre-/- mutant mouse can not inhibit the migration of all the neural crest cells. May be the remanent crest cells is sufficient to carry out the hypothetic axon guidance role of neural crest. Elimination of neural crest source by surgical or Noggin Beads implantation results in accessory axons pathfinding vertically extend dorsally and ventrally. Furthermore, the neural crest transplantation experiment between trunk and r8 was used to validate the result. We found that the accessory and spinal axon patterns were not affected by graft. Further, the heterotopic graft of dorsal neural tube between trunk and r8, which concludes neural crest and neuroepithelium was used to reveal whether the neuroepithelium owns a role of axon guidance. The data tell us that in the trunk level, the grafted r8 neuroepithelium repelled the spinal axons extend directly to periphery. Coincidentally, the grafted trunk neuroepithelium allows accessory axons vertically extending. The neuroepithelium therefore contains guidance cues for axon extending. Together, we conclude that the neuraoepithelium may drive neural crest cells to instruct accessory axons running on the neural tube.更多还原