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XB-ART-50072
J Vis Exp January 12, 2015; (95): e51526.

Understanding early organogenesis using a simplified in situ hybridization protocol in Xenopus.

Deimling SJ , Halabi RR , Grover SA , Wang JH , Drysdale TA .


Abstract
Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

PubMed ID: 25651461
PMC ID: PMC4354522
Article link: J Vis Exp
Grant support: [+]

Species referenced: Xenopus
Genes referenced: astl2c astl3a.1 etv2 hand1 hba1 mmp15 pax2 pax8 tnni3


Article Images: [+] show captions
References [+] :
Carroll, Molecular regulation of pronephric development. 1999, Pubmed, Xenbase