A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Summary

Here, we describe an efficient high throughput in situ hybridization (ISH) method for visualizing patterns of mRNA expression in developing fetal mouse prostate tissue sections. The method can be easily adapted to visualize mRNA expression patterns in other mouse tissues or in tissues from other species.

Abstract

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male urethra, which relies on androgenic signals and epithelial-mesenchymal interactions^1,2. Understanding the molecular mechanisms responsible for prostate development may reveal growth mechanisms that are inappropriately reawakened later in life to give rise to prostate diseases such as benign prostatic hyperplasia and prostate cancer.

The developing LUT is anatomically complex. By the time prostatic budding begins on 16.5 days post conception (dpc), numerous cell types are present. Vasculature, nerves and smooth muscle reside within the mesenchymal stroma³. This stroma surrounds a multilayered epithelium and gives rise to the fetal prostate through androgen receptor-dependent paracrine signals⁴. The identity of the stromal androgen receptor-responsive genes required for prostate development and the mechanism by which prostate ductal epithelium forms in response to these genes is not fully understood. The ability to precisely identify cell types and localize expression of specific factors within them is imperative to further understand prostate development. In situ hybridization (ISH) allows for localization of mRNAs within a tissue. Thus, this method can be used to identify pattern and timing of expression of signaling molecules and their receptors, thereby elucidating potential prostate developmental regulators.

Here, we describe a high throughput ISH technique to identify mRNA expression patterns in the fetal mouse LUT using vibrating microtome-cut sections. This method offers several advantages over other ISH protocols. Performing ISH on thin sections adhered to a slide is technically difficult; cryosections frequently have poor structural quality while both cryosections and paraffin sections often result in weak signal resolution. Performing ISH on whole mount tissues can result in probe trapping. In contrast, our high throughput technique utilizes thick-cut sections that reveal detailed tissue architecture. Modified microfuge tubes allow easy handling of sections during the ISH procedure. A maximum of 4 mRNA transcripts can be screened from a single 17.5dpc LUT with up to 24 mRNA transcripts detected in a single run, thereby reducing cost and maximizing efficiency. This method allows multiple treatment groups to be processed identically and as a single unit, thereby removing any bias for interpreting data. Most pertinently for prostate researchers, this method provides a spatial and temporal location of low and high abundance mRNA transcripts in the fetal mouse urethra that gives rise to the prostate ductal network.

Protocol

1. Synthesis of a Digoxigenin-11-UTP-Labeled Riboprobe from a PCR-Generated Template

1. To synthesize a gene-specific riboprobe, use Entrez Gene (http://www.ncbi.nlm.nih.gov/sites/entrez) to obtain the gene cDNA reference sequence (RefSeq). Use the Primer3 program (http://frodo.wi.mit.edu/primer3/)⁵ to design gene-specific PCR primers against the 3’-region of the cDNA sequence. Recommended parameters for PCR primer selection are described elsewhere (http://www.gudmap.org/Research/Protocols/Vezina/Riboprobe_Syn.html).
2. Use the MegaBLAST Program (http://www.ncbi.nlm.nih.gov/genome/seq/BlastGen/BlastGen.cgi?taxid=10090)⁶ to ....

Discussion

Using the method described here, it is possible to detect mRNAs in all of the major cell types and tissue compartments of the fetal male and female mouse LUTs including the mesenchymal pads, urothelium, smooth muscle, prostatic buds, ejaculatory duct, and vagina. The 50μm sections used in this protocol have the advantage of being thick enough to resolve tissue architecture (such as blood vessels) but are thin enough to avoid probe trapping, which is a methodological problem commonly encountered during whole-mount ISH........

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anti-Digoxigenin antibody, Fab fragments	Roche Group	11214667001
Blocking reagent	Roche Group	11096176001
BM Purple AP substrate, precipitating	Roche Group	11442074001
Bovine Serum Albumin	Fisher Scientific	BP1600-100
Cell culture plate, 24 well	Corning	3524
Digoxigenin 11-UTP	Roche Group	1277073910
dNTPs	Roche Group	11969064001
Double-edged razor blade	Wilkinson Sword	Classic Model
Eliminase RNase remover	Decon Laboratories	1102
Formamide	Sigma-Aldrich	F5786-1L
Gel extraction kit	Qiagen	28704
Glutaraldehyde, 25% solution in H2O	Sigma-Aldrich	G6257-100ML
Heparin, sodium salt	Sigma-Aldrich	H3393
Hydrogen peroxide, 30% solution in H2O	Fisher Scientific	BP2633-500
Levamisole	Sigma-Aldrich	L9756
Loctite 404 quick set instant adhesive	Henkel Corp	46551
Magnesium chloride	Fisher Scientific	M33-500
Maleic acid	Sigma-Aldrich	M0375-500G
Microcentrifuge tubes, 1.5mL	Biologix Research Company	BP337-100
Millicell culture plate insert	EMD Millipore	PICM01250
Molecular grinding resin	G-Biosciences	786-138PR
Paraformaldehyde, 4% solution in phosphate buffered saline	Affymetrix	19943
Phosphate buffered saline, without Ca & Mg	MP Biomedicals	ICN1760420
Polyester mesh, 33 micron, 12" x 24"	Small Parts, Inc.	CMY-0033-D
Proteinase K solution, 20mg/ml	Amresco	E195-5ML
QIAshredder Columns	Qiagen	79654
Q solution	Qiagen	Provided with Taq DNA polymerase
RNase	Sigma-Aldrich	R6513
RNase inhibitor	Roche Group	03335399001
RNeasy mini kit	Qiagen	74104
RNEASY mini kit	Qiagen	74104
RQ1 RNase-free DNase	Promega Corp.	M6101
SeaPlaque low-melt agarose	Lonza Inc.	50101
Sheep serum	Sigma-Aldrich	S2263-500mL
Stericup filter unit, 0.22μm, polyethersulfone, 500mL	EMD Millipore	SCGPU05RE
Sodium azide, granular	Fisher Scientific	S227I-100
Sodium chloride	Fisher Scientific	BP358-212
Sodium dodecyl sulfate	Fisher Scientific	S529-500
SSC, 20X solution	Research Products International Corp.	S24022-4000.0
SuperScript III first-strand synthesis system	Invitrogen	18080-051
T7 RNA polymerase	Roche Group	10881767001
Taq DNA polymerase	Qiagen	201203
Tris-HCl	Fisher Scientific	BP153-1
Tween 20	Fisher Scientific	BP337-100
Vibrating microtome with deluxe specimen bath	Leica Microsystems	VT1000A
Yeast tRNA	Roche Group	109495