Combinatorial analysis of mRNA expression patterns in mouse embryos using hybridization chain reaction.
Multiplexed fluorescent hybridization chain reaction (HCR) and advanced imaging techniques can be used to evaluate combinatorial gene expression patterns in whole mouse embryos with unprecedented spatial resolution. Using HCR, DNA probes complementary to mRNA targets trigger chain reactions in which metastable fluorophore-labeled DNA HCR hairpins self-assemble into tethered fluorescent amplification polymers. Each target mRNA is detected by a probe set containing one or more DNA probes, with each probe carrying two HCR initiators. For multiplexed experiments, probe sets for different target mRNAs carry orthogonal initiators that trigger orthogonal DNA HCR amplification cascades labeled by spectrally distinct fluorophores. As a result, in situ amplification is performed for all targets simultaneously, and the duration of the experiment is independent of the number of target mRNAs. We have used multiplexed fluorescent in situ HCR and advanced imaging technologies to address questions of cell heterogeneity and tissue complexity in craniofacial patterning and anterior neural development. In the sample protocol presented here, we detect three different mRNA targets: Tg(egfp), encoding the enhanced green fluorescent protein (GFP) transgene (typically used as a control); Twist1, encoding a transcription factor involved in cell lineage determination and differentiation; and Pax2, encoding a transcription factor expressed in the mid-hindbrain region of the mouse embryo.
Keck School of Medicine, University of Southern California, Children's Hospital Los Angeles, California 90027;
David
D
Huss
Division of Biology & Biological Engineering, California Institute of Technology, Pasadena, California 91125;
Harry M T
HM
Choi
Translational Imaging Center, University of Southern California, Los Angeles, California 90089; Division of Biology & Biological Engineering, California Institute of Technology, Pasadena, California 91125;
Carol
C
Readhead
Translational Imaging Center, University of Southern California, Los Angeles, California 90089;
Scott E
SE
Fraser
Division of Biology & Biological Engineering, California Institute of Technology, Pasadena, California 91125; Division of Engineering & Applied Science, California Institute of Technology, Pasadena, California 91125.
Niles A
NA
Pierce
Keck School of Medicine, University of Southern California, Children's Hospital Los Angeles, California 90027;
Rusty
R
Lansford
D000818:Animals; D001921:Brain / Q000196:embryology; D004622:Embryo, Mammalian; D020869:Gene Expression Profiling / Q000379:methods*; D018507:Gene Expression Regulation, Developmental; D051379:Mice; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009693:Nucleic Acid Hybridization / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis*
D012333:RNA, Messenger