Doodnath Reshma, Dervan Adrian, Wride Michael A, Puri Prem
National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin 12, Ireland.
Pediatr Surg Int. 2010 Dec;26(12):1217-21. doi: 10.1007/s00383-010-2746-7. Epub 2010 Oct 24.
Recently, the zebrafish (Danio rerio) has been shown to be an excellent model for human paediatric research. Advantages over other models include its small size, externally visually accessible development and ease of experimental manipulation. The enteric nervous system (ENS) consists of neurons and enteric glia. Glial cells permit cell bodies and processes of neurons to be arranged and maintained in a proper spatial arrangement, and are essential in the maintenance of basic physiological functions of neurons. Glial fibrillary acidic protein (GFAP) is expressed in astrocytes, but also expressed outside of the central nervous system. The aim of this study was to investigate the spatio-temporal pattern of GFAP expression in developing zebrafish ENS from 24 h post-fertilization (hpf), using transgenic fish that express green fluorescent protein (GFP).
Zebrafish embryos were collected from transgenic GFP Tg(GFAP:GFP)(mi2001) adult zebrafish from 24 to 120 hpf, fixed and processed for whole mount immunohistochemistry. Antibodies to Phox2b were used to identify enteric neurons. Specimens were mounted on slides and imaging was performed using a fluorescent laser confocal microscope.
GFAP:GFP labelling outside the spinal cord was identified in embryos from 48 hpf. The patterning was intracellular and consisted of elongated profiles that appeared to migrate away from the spinal cord into the periphery. At 72 and 96 hpf, GFAP:GFP was expressed dorsally and ventrally to the intestinal tract. At 120 hpf, GFAP:GFP was expressed throughout the intestinal wall, and clusters of enteric neurons were identified using Phox2b immunofluorescence along the pathway of GFAP:GFP positive processes, indicative of a migratory pathway of ENS precursors from the spinal cord into the intestine.
The pattern of migration of GFAP:GFP expressing cells outside the spinal cord suggests an organized, early developing migratory pathway to the ENS. This shows for the first time that Tg(GFAP:GFP)(mi2001) zebrafish model is an ideal one to study spatio-temporal patterning of early ENS development.
最近，斑马鱼（Danio rerio）已被证明是人类儿科研究的优秀模型。与其他模型相比，其优势包括体型小、发育过程在外部可见且易于进行实验操作。肠神经系统（ENS）由神经元和肠神经胶质细胞组成。神经胶质细胞使神经元的细胞体和突起能够以适当的空间排列方式进行排列和维持，并且对于维持神经元的基本生理功能至关重要。胶质纤维酸性蛋白（GFAP）在星形胶质细胞中表达，但也在中枢神经系统之外表达。本研究的目的是利用表达绿色荧光蛋白（GFP）的转基因鱼，研究受精后24小时（hpf）起发育中的斑马鱼肠神经系统中GFAP表达的时空模式。
从转基因GFP Tg(GFAP:GFP)(mi2001)成年斑马鱼收集24至120 hpf的斑马鱼胚胎，固定并进行整体免疫组织化学处理。使用抗Phox2b抗体来识别肠神经元。将标本固定在载玻片上，并用荧光激光共聚焦显微镜进行成像。
在48 hpf的胚胎中，在脊髓外发现了GFAP:GFP标记。其模式为细胞内标记，由细长的形态组成，这些形态似乎从脊髓迁移到外周。在72和96 hpf时，GFAP:GFP在肠道的背侧和腹侧表达。在120 hpf时，GFAP:GFP在整个肠壁表达，并且沿着GFAP:GFP阳性突起的路径使用Phox2b免疫荧光鉴定出肠神经元簇，这表明肠神经系统前体从脊髓迁移到肠道的迁移途径。
脊髓外表达GFAP:GFP的细胞的迁移模式表明存在一条有组织的、早期发育的向肠神经系统的迁移途径。这首次表明Tg(GFAP:GFP)(mi2001)斑马鱼模型是研究早期肠神经系统发育时空模式的理想模型。
