PMID- 22065447 OWN - NLM STAT- MEDLINE VI - 772 TI - Whole-mount in situ hybridization of sectioned tissues of species hybrids to detect cis-regulatory changes in gene expression pattern. PG - 319-28 LA - eng PT - Journal Article PL - United States TA - Methods Mol Biol JT - Methods in molecular biology (Clifton, N.J.) JID - 9214969 IS - 1940-6029 (Electronic) LID - 10.1007/978-1-61779-228-1_19 [doi] FAU - Futahashi, Ryo AU - Futahashi R AD - Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan. ryo-futahashi@aist.go.jp IS - 1064-3745 (Linking) RN - 0 (RNA Probes) RN - 0 (RNA, Messenger) SB - IM MH - Animals MH - Base Sequence MH - Butterflies/genetics MH - Crosses, Genetic MH - Female MH - *Gene Expression Regulation MH - *Hybridization, Genetic MH - In Situ Hybridization/*methods MH - Larva/genetics MH - Male MH - Molecular Sequence Data MH - *Organ Specificity MH - RNA Probes/metabolism MH - RNA, Messenger/genetics/metabolism MH - Regulatory Sequences, Nucleic Acid/*genetics MH - Species Specificity DCOM- 20120222 LR - 20111108 DP - 2011 AB - To distinguish whether differences in gene expression between species or between individuals of the same species are caused by cis-regulatory changes or by distribution differences in trans-regulatory proteins, comparison of species-specific mRNA expression in an F1 hybrid by whole-mount in situ hybridization is a rarely used yet very powerful tool. If asymmetric expression pattern is observed for the two alleles, this implies a cis-regulatory divergence of this gene. Alternatively, if symmetric expression pattern is observed for both alleles, the change in expression of this gene is probably caused by changes in the distribution of trans-regulatory proteins. In this chapter, I describe how to prepare RNA probes, tissue samples and how to detect mRNA expression pattern using in situ hybridization. Although I choose to present here the detection of yellow-related gene (YRG) expression pattern in the larval epidermis of swallowtail butterflies, this protocol can be adapted to other species and tissues. YRG mRNA expression is correlated with interspecific differences of yellow and green larval color pattern such as V-shaped markings in swallowtail butterflies. F1 hybrids show an intermediate color pattern between parental species. In this case, both species-specific YRG mRNA showed a similar expression pattern in F1 hybrids, suggesting that the change in expression of YRG is mainly caused by changes in the distribution of trans-regulatory proteins.