Molecular Characterization of the Genital Organizer: Gene Expression Profile of the Mouse Urethral Plate Epithelium

Abstract

Purpose: Lower urinary tract malformations are among the most common congenital anomalies in humans. Molecular genetic studies of mouse external genital development have begun to identify mechanisms that pattern the genital tubercle and orchestrate urethral tubulogenesis. The urethral plate epithelium is an endodermal signaling region that has an essential role in external genital development. However, little is known about the molecular identity of this cell population or the genes that regulate its activity.

Materials and methods: We used microarray analysis to characterize differences in gene expression between urethral plate epithelium and surrounding tissue in mouse genital tubercles. In situ hybridizations were performed to map gene expression patterns and ToppCluster (https://toppcluster.cchmc.org/) was used to analyze gene associations.

Results: A total of 84 genes were enriched at least 20-fold in urethral plate epithelium relative to surrounding tissue. The majority of these genes were expressed throughout the urethral plate in males and females at embryonic day 12.5 when the urethral plate is known to signal. Functional analysis using ToppCluster revealed genetic pathways with known functions in other organ systems but unknown roles in external genital development. Additionally, a 3-dimensional molecular atlas of genes enriched in urethral plate epithelium was generated and deposited at the GUDMAP (GenitoUrinary Development Molecular Anatomy Project) website (http://gudmap.org/).

Conclusions: We identified dozens of genes previously unknown to be expressed in urethral plate epithelium at a crucial developmental period. It provides a novel panel of genes for analysis in animal models and in humans with external genital anomalies.

Keywords: congenital abnormalities; gene expression; genitalia; microarray analysis; urethra.

Figure 1

A. Whole mount Shh^GFPCre;R26R^eYFP mouse at E12.5. Insert shows high magnification image of the genital tubercle. B. Schematic of a sagittal section through an E12.5 mouse genital tubercle showing the Shh-expressing epithelial tissue (blue) and the plane of dissection (dashed red line). C. Cell distribution according to amount of fluorescence emitted during FACS sorting. GT- Genital tubercle, cm - Cloacal membrane, UPE- Urethral plate epithelium, UGS- Urogenital sinus, URS- Urorectal Septum.

Figure 2

In situ hybridizations of selected genes. Note that patterns show little spatial regionalization along the urethral plate and there are no obvious differences between male and female expression patterns.

Figure 3

In situ hybridization showing expression patterns of genes not found in the urethral plate. A. Gene expressed in the mesenchyme surrounding the distal urethral plate. B. Genes expressed in the mesenchyme surrounding the midurethral plate. C. Genes expressed throughout the mesenchyme.

Figure 4

ToppCluster analysis of genes enriched in the urethral epithelium associated with molecular function (light blue), pathways (green), and biological process (teal).