非洲爪狼是研究脊椎动物发育的重要模型系统之一。然而,这个系统的一个缺点是,因为非洲爪狼胚胎的不透明,3D成像分析仅限于表面结构,外植体培养物,和胚胎后的t。为了开发一种3D组织/器官成像技术,在整个非洲爪的胚胎,我们确定了使用胎盘碱性磷酸酶(PLAP)作为转基因报道分子的最佳条件,并将其应用于相关光学显微镜和块面成像(CoMBI)方法,以可视化表达PLAP的组织/器官.在内源性碱性磷酸酶活性被热灭活的胚胎中,PLAP染色以与绿色荧光蛋白(GFP)荧光一致的方式显现各种组织特异性增强子/启动子活性。此外,PLAP染色似乎比GFP荧光更敏感,由此产生的表达模式不是马赛克,与通过相同转基因方法引入的lacZ基因表达的β-半乳糖苷酶的镶嵌染色模式形成鲜明对比。由于碱性磷酸酶底物的有效渗透,在深部组织中检测到PLAP活性,比如发育中的大脑,脊髓,心,和躯体,通过全装染色。通过CoMBI方法分析染色的胚胎,导致PLAP表达组织的3D图像的数字重建。这些结果证明了PLAP报告系统用于检测驱动深层组织表达的增强子/启动子活性的功效及其与CoMBI方法的组合作为用于非洲爪狼胚胎中特定组织/器官结构的3D数字成像分析的强大方法。
Xenopus is one of the essential model systems for studying vertebrate development. However, one drawback of this system is that, because of the opacity of Xenopus embryos, 3D imaging analysis is limited to surface structures, explant cultures, and post-embryonic tadpoles. To develop a technique for 3D tissue/organ imaging in whole Xenopus embryos, we identified optimal conditions for using placental alkaline phosphatase (PLAP) as a transgenic
reporter and applied it to the correlative light microscopy and block-face imaging (CoMBI) method for visualization of PLAP-expressing tissues/organs. In embryos whose endogenous alkaline phosphatase activities were heat-inactivated, PLAP staining visualized various tissue-specific enhancer/promoter activities in a manner consistent with green fluorescent protein (GFP) fluorescence. Furthermore, PLAP staining appeared to be more sensitive than GFP fluorescence as a
reporter, and the resulting expression patterns were not mosaic, in striking contrast to the mosaic staining pattern of β-galactosidase expressed from the lacZ gene that was introduced by the same transgenesis method. Owing to efficient penetration of alkaline phosphatase substrates, PLAP activity was detected in deep tissues, such as the developing brain, spinal cord, heart, and somites, by whole-mount staining. The stained embryos were analyzed by the CoMBI method, resulting in the digital reconstruction of 3D images of the PLAP-expressing tissues. These results demonstrate the efficacy of the PLAP
reporter system for detecting enhancer/promoter activities driving deep tissue expression and its combination with the CoMBI method as a powerful approach for 3D digital imaging analysis of specific tissue/organ structures in Xenopus embryos.