The spatio-temporal patterning of Hoxa9 and Hoxa13 in the developing zebrafish enteric nervous system
The spatio-temporal patterning of Hoxa9 and Hoxa13 in the developing zebrafish enteric nervous system
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DOI：
10.1007/s00383-011-2992-3
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发表时间：
2012-02-01
影响因子：
1.8
通讯作者：
Puri, P.
中科院分区：
文献类型：
--
作者：
Doodnath, R.;Wride, M.;Puri, P.
背景先天性巨结肠症的特点是远端肠道中没有神经节细胞，这一过程由复杂的遗传途径控制。同源框基因在肠道发育中起着重要作用，肠道HOX编码描述了HOX基因在时间和空间上的不同表达。在先天性巨结肠(HD)患者中发现了Hoxa9和Hoxa13突变。本研究旨在利用斑马鱼模型确定Hoxa9和Hoxa13在肠道神经系统(ENS)发育中的时空模式。方法获得含有目的基因的纯化质粒并接种到培养基中，以指数级增加含有该基因的细菌数量。然后用离心法收集细胞，提取质粒DNA，线性化后沉淀。通过体外转录反应制备地高辛标记的RNA探针。用这些探针对受精后24~120h的斑马鱼胚胎进行了原位杂交，此时斑马鱼的肠道已经完全发育。然后将胚胎置于甘油中，用奥林巴斯B40显微镜成像，用奥林巴斯Super F1.8数码相机拍摄图像。结果在24 HPF时，Hoxa9在前脑和后脑以及非常远端的肌节都有表达，而Hoxa13只在前脑和后脑表达。在48HPF，可以看到Hoxa9和Hoxa13标记的细胞从前脑向远端迁移到脊索和脊髓。在72HPF，Hoxa9标记的细胞遍布脊髓，而Hoxa13阳性细胞则从脊髓和近端肠道向下迁移。到96HPF时，Hoxa9和Hoxa13标记的细胞已经沿着脊髓全长和近端和中肠迁移。120HPF可见Hoxa9和Hoxa13阳性细胞分布于斑马鱼肠道全长。结论进一步证实Hoxa9和Hoxa13参与斑马鱼ENS早期有组织的发育。
Background Hirschsprung's disease is characterised by the absence of ganglion cells in the distal bowel, a process which is controlled by complex genetic pathways. Homeobox genes have a major role in gut development and this is depicted by the enteric Hox code which describes the different spatial and temporal expression of Hox genes. Hoxa9 and Hoxa13 mutations have been discovered in patients with Hirschsprung's disease (HD). The aim of this study was to determine the spatio-temporal pattern of Hoxa9 and Hoxa13 in enteric nervous system (ENS) development using the zebrafish model.Methods Purified plasmids that contained the gene of interest were obtained and inoculated into culture medium to exponentially increase the number of bacteria containing the plasmid. Cells were then harvested by centrifugation and plasmid DNA was extracted, which was then linearised and precipitated. RNA digoxigenin-labelled probes were made by in vitro transcription reaction. In situ hybridisation was carried out using these probes on zebrafish embryos which were collected from 24 to 120 h post fertilisation (hpf), by which time the zebrafish intestine is fully developed. Embryos were then mounted in glycerol and imaged using an Olympus B40 microscope and images were taken using an Olympus Super F1.8 digital camera.Results At 24 hpf, Hoxa9 expression is seen in the forebrain and hindbrain and also in the very distal myotome whereas Hoxa13 expression, however, is seen only at the forebrain and hindbrain. At 48 hpf, Hoxa9- and Hoxa13-labelled cells are seen migrating distally from the forebrain into the notochord and spinal cord. At 72 hpf, Hoxa9-labelled cells can be seen throughout the spinal cord whereas Hoxa13 positive cells are seen migrating down from the spinal cord and in the proximal gut. By 96 hpf, Hoxa9-and Hoxa13-labelled cells have migrated down the full length of the spinal cord and along the proximal and mid intestine. By 120 hpf, Hoxa9 and Hoxa13 positive cells can be seen along the entire length of the zebrafish intestine.Conclusions These results show further evidence that Hoxa9 and Hoxa13 are involved in the early and organised patterning of ENS development in the zebrafish model.