Studies into the molecular basis of morphogenesis frequently begin with investigations into gene expression across time and cell type in that organ. One of the most anatomically informative approaches to such studies is the use of in situ hybridization, either of intact or histologically sectioned tissues. Here, we describe the optimization of this approach for use in the temporal and spatial analysis of gene expression in the urogenital system, from embryonic development to the postnatal period. The methods described are applicable for high throughput analysis of large gene sets. As such, ISH has become a powerful technique for gene expression profiling and is valuable for the validation of profiling analyses performed using other approaches such as microarrays.
In situ hybridization Metanephros Kidney Urogenital system Genitourinary system Gene expression mRNA expression
This is a preview of subscription content, log in to check access.
Springer Nature is developing a new tool to find and evaluate Protocols. Learn more
This work was supported by NIH NIDDK grant to M.H.L. (DK070136). M.H.L. is a Principal Research Fellow of the National Health and Medical Research Council of Australia.
Thiagarajan RD, Georgas KM, Rumballe BA et al (2011) Identification of anchor genes during kidney development defines ontological relationships, molecular subcompartments and regulatory pathways. PLoS One 6(2):e17286PubMedCentralPubMedCrossRefGoogle Scholar
Chiu HS, Szucsik JC, Georgas KM et al (2010) Comparative gene expression analysis of genital tubercle development reveals a putative appendicular Wnt7 network for the epidermal differentiation. Dev Biol 344(2):1071–1087PubMedCentralPubMedCrossRefGoogle Scholar
Georgas KM, Chiu HS, Lesieur E, Rumballe BA, Little MH (2011) Expression of metanephric nephron-patterning genes in differentiating mesonephric tubules. Dev Dyn 240(6):1600–1612PubMedCrossRefGoogle Scholar
Georgas K, Rumballe B, Valerius MT et al (2009) Analysis of early nephron patterning reveals a role for distal RV proliferation in fusion to the ureteric tip via a cap mesenchyme-derived connecting segment. Dev Biol 332(2):273–286PubMedCrossRefGoogle Scholar
Brunskill EW, Aronow BJ, Georgas K et al (2008) Atlas of gene expression in the developing kidney at microanatomic resolution. Dev Cell 5:781–791CrossRefGoogle Scholar
Rumballe BA, Georgas KM, Combes A et al (2011) Nephron formation adopts a novel spatial topology at cessation of nephrogenesis. Dev Biol 360(1):110–122Google Scholar
Wilkinson DG, Nieto MA (1993) Detection of messenger RNA by in situ hybridization to tissue sections and whole mounts. Methods Enzymol 225:361–373PubMedCrossRefGoogle Scholar
Challen G, Gardiner B, Caruana G et al (2005) Temporal and spatial transcriptional programs in murine kidney development. Physiol Genomics 23(2):159–171PubMedCrossRefGoogle Scholar
Wilhelm D, Hiramatsu R, Mizusaki H et al (2007) SOX9 regulates prostaglandin D synthase gene transcription in vivo to ensure testis development. J Biol Chem 282:10553–10560PubMedCrossRefGoogle Scholar
Rumballe B, Georgas K, Little MH (2008) High-throughput paraffin section in situ hybridization and dual immunohistochemistry on mouse tissues. CSH Protoc 5030:1. doi:10.1101/pdb.prot5030Google Scholar
Georgas K, Rumballe B, Wilkinson L et al (2008) Use of dual section mRNA in situ hybridisation/immunohistochemistry to clarify gene expression patterns during the early stages of nephron development in the embryo and in the mature nephron of the adult mouse kidney. Histochem Cell Biol 130(5):927–942PubMedCrossRefGoogle Scholar