title: mRNA fluorescence in situ hybridization to determine overlapping gene expression in whole-mount mouse embryos.
abstract: BACKGROUND
  Whole-mount in situ hybridization (ISH) is a prevalent tool to examine the spatial distribution of gene transcripts in intact embryos. Chromogenic-based methods of signal development are commonly used in mouse embryos because of their high sensitivity. Fluorescence techniques, however, offer several advantages over chromogenic methods including the ability to visualize multiple signals in a specimen at once.
  
  
  RESULTS
  We describe a procedure for fluorescence in situ hybridization (FISH) for whole mouse embryos up to embryonic day 13.5. We show that this approach successfully produces a bright expression signal for several genes, validating the procedure in multiple tissues. Further, we show that double FISH can be used to visualize the expression of two genes in a single embryo by determining that Hoxd13 and Shh are co-expressed in both the limb bud and the hindgut. Finally, we demonstrate that FISH can be paired with confocal microscopy to take optical sections of interior regions of the embryo.
  
  
  CONCLUSIONS
  FISH is a valid alternative to chromogenic-based ISH for visualizing gene expression in whole mouse embryos. This work provides a framework to add additional fluorescence signals in the mouse such as visualizing both mRNA and protein by pairing the procedure with immunofluorescence.
authors.affiliation: Department of Biological Sciences, University of Calgary, Calgary, Canada.
authors.forename: Stanley J
authors.initials: SJ
authors.lastname: Neufeld
authors.forename: Xiaolan
authors.initials: X
authors.lastname: Zhou
authors.forename: Peter D
authors.initials: PD
authors.lastname: Vize
authors.forename: John
authors.initials: J
authors.lastname: Cobb
mesh_terms: D000818:Animals; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental / Q000502:physiology*; D053823:Hedgehog Proteins / Q000096:biosynthesis; D006614:Hindlimb / Q000166:cytology / Q000196:embryology; D018398:Homeodomain Proteins / Q000096:biosynthesis; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D007422:Intestines / Q000166:cytology / Q000196:embryology; D051379:Mice; D012333:RNA, Messenger / Q000096:biosynthesis*; D014157:Transcription Factors / Q000096:biosynthesis
chemical_list: D053823:Hedgehog Proteins; D018398:Homeodomain Proteins; C084118:Hoxd13 protein, mouse; D012333:RNA, Messenger; C465570:Shh protein, mouse; D014157:Transcription Factors
keywords: Hoxd13; Shh; co-expression; fluorescence in situ hybridization; limb development; mouse; whole-mount