title: Whole-mount fluorescent in situ hybridization staining of the colonial tunicate Botryllus schlosseri.
abstract: Botryllus schlosseri is a colonial ascidian with characteristics that make it an attractive model for studying immunology, stem cell biology, evolutionary biology, and regeneration. Transcriptome sequencing and the recent completion of a draft genome sequence for B. schlosseri have revealed a large number of genes, both with and without vertebrate homologs, but analyzing the spatial and temporal expression of these genes in situ has remained a challenge. Here, we report a robust protocol for in situ hybridization that enables the simultaneous detection of multiple transcripts in whole adult B. schlosseri using Tyramide Signal Amplification in conjunction with digoxigenin- and dinitrophenol-labeled RNA probes. Using this protocol, we have identified a number of genes that can serve as markers for developing and mature structures in B. schlosseri, permitting analysis of phenotypes induced in loss-of-function experiments.
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, University of California Santa Barbara, Santa Barbara, California.
authors.forename: Adam D
authors.initials: AD
authors.lastname: Langenbacher
authors.forename: Delany
authors.initials: D
authors.lastname: Rodriguez
authors.forename: Alessandro
authors.initials: A
authors.lastname: Di Maio
authors.forename: Anthony W
authors.initials: AW
authors.lastname: De Tomaso
mesh_terms: D000818:Animals; D005786:Gene Expression Regulation; D005819:Genetic Markers; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D015347:RNA Probes / Q000235:genetics; D016372:RNA, Antisense / Q000235:genetics; D013194:Staining and Labeling; D014561:Urochordata / Q000235:genetics*
chemical_list: D005819:Genetic Markers; D015347:RNA Probes; D016372:RNA, Antisense
keywords: allorecognition; ascidian; gene expression; stem cells; tyramide signal amplification