Visualization and quantification of mouse prostate development by in situ hybridization.

Area of Science:

• Developmental biology
• Genetics
• Molecular biology

Background:

• Prostate development studies require accurate methods for visualizing and quantifying prostatic buds.
• Refining animal usage is crucial for ethical and efficient research.

Purpose of the Study:

• To validate a combined in situ hybridization (ISH)/immunohistochemistry (IHC) staining method for mouse prostatic buds.
• To determine if male and female mouse urogenital sinus (UGS) explants yield comparable prostatic bud numbers in vitro.
• To identify optimal riboprobes for marking prostatic buds.

Main Methods:

• Utilized IHC to label UGS epithelium and ISH to detect prostatic buds using Nkx3-1, Wnt10b, and Edar riboprobes.
• Quantified total prostatic buds per UGS and the proportion marked by each mRNA.
• Compared bud formation in male UGS in vivo and male/female UGS in vitro.

Main Results:

• Nkx3-1 marked the prostate field early in vivo; all three mRNAs marked buds later.
• mRNAs showed distinct localization: Nkx3-1 along bud length, Edar/Wnt10b at distal tips.
• In vitro, no single mRNA marked all buds; marking was stage- and gender-dependent.
• Nkx3-1 marked the highest proportion of buds during in vitro UGS development.

Conclusions:

• ISH staining of mouse UGS effectively quantifies prostatic bud number.
• Nkx3-1 is the most suitable riboprobe for this ISH-based quantification method.
• Female UGS explants are not interchangeable with male UGS explants in in vitro prostate development studies.
• Nkx3-1, Edar, and Wnt10b mark distinct prostatic bud regions, aiding future gene expression studies.