title: A continuum of transcriptional identities visualized by combinatorial fluorescent in situ hybridization.
abstract: Oligonucleotide-based fluorescent in situ hybridization (FISH) coupled with high-resolution high-sensitivity microscopy allows the visualization of single RNA molecules within fixed cells and tissues as distinct foci. We show here that combinatorial labeling of RNA molecules with several fluorescent dyes extends the number of genes that can be targeted simultaneously beyond the number of fluorophores used. This approach also inherently validates the identification of transcripts reducing false positive counts. We have used combinatorial FISH and image analysis to measure the transcript densities of six genes using three fluorophores. This has allowed us to visualize the endothelial maturation of lateral mesoderm in an in vitro ES differentiation assay from a single snapshot of molecular identities. Our observations show that, under these specific conditions, endothelial maturation follows a homogeneous course with a gradual increase in expression of Cdh5 and a concomitant loss of early transcription factors, arguing that maturation is governed in a generally deterministic manner. This methodology is limited by the number of fluorophores that can be used and by the available microscopic resolution, but currently available equipment should allow the visualization of transcripts from 10 or more genes simultaneously.
authors.affiliation: Stem Cell Biology Group, Riken Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Japan. mjakt@cdb.riken.jp
authors.forename: Lars Martin
authors.initials: LM
authors.lastname: Jakt
authors.forename: Satoko
authors.initials: S
authors.lastname: Moriwaki
authors.forename: Shinichi
authors.initials: S
authors.lastname: Nishikawa
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D015820:Cadherins / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics; D002478:Cells, Cultured; D002642:Chick Embryo; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D015342:DNA Probes / Q000138:chemical synthesis; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D005456:Fluorescent Dyes; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D051379:Mice; D014158:Transcription, Genetic; D000099152:Cadherin 5
chemical_list: D015703:Antigens, CD; D015820:Cadherins; D015342:DNA Probes; D005456:Fluorescent Dyes; D000099152:Cadherin 5