A high-resolution molecular atlas of the fetal mouse lower urogenital tract

Abstract

Epithelial-stromal interactions in the lower urogenital tract (LUT) are integral to prostatic and seminal vesicle development in males, vaginal and uterine development in females, and urethral development in both sexes. Gene expression profiling of isolated LUT stroma and epithelium has unraveled mechanisms of LUT development, but such studies are confounded by heterogeneous and ill-defined cell sub-populations contained within each tissue compartment. We used in situ hybridization to synthesize a high-resolution molecular atlas of 17-day post-coitus fetal mouse LUT. We identified mRNAs that mark selective cell populations of the seminal vesicle, ejaculatory duct, prostate, urethra, and vagina, subdividing these tissues into 16 stromal and 8 epithelial sub-compartments. These results provide a powerful tool for mapping LUT gene expression patterns and also reveal previously uncharacterized sub-compartments that may play mechanistic roles in LUT development of which we were previously unaware.

Fig. 1

Selective mRNA markers of basal, intermediate, and superficial urothelial cell compartments in the 17 dpc mouse urethra. Near mid-sagittal sections (50 μm) of 17 dpc male and female LUT were stained by ISH to visualize mRNA expression (purple) of (A-B) the basal urothelial marker keratin 14 (Krt14), (C-D) the intermediate and superficial urothelial marker uroplakin 1b (Upk1b), and (E-F) the basal and intermediate urothelial marker kringle containing transmembrane protein 1 (Kremen1). Sections were then stained by immunofluorescence with an anti-smooth muscle actin alpha 2 (ACTA2) antibody that recognizes muscularis mucosa and muscularis propria (green) and an anti-cadherin 1 (CDH1) antibody that recognizes all urothelium (red). Inset images are magnified sections of urothelium that reveal the basal urothelium (bu), intermediate urothelium (iu), and superficial urothelium (su) sub-compartments. Results in each panel are representative of three males and three females. Abbreviations used are BL: bladder, ED: ejaculatory duct, LV: lower vagina, SV: seminal vesicle, UV: upper vagina. Arrowheads indicate prostatic buds. All images are of the same magnification.

Fig. 2

KRT14 protein is more selective than TRP63 protein for marking basal urothelial cells in the 17 dpc mouse urethra. (A, a’) 17 dpc male and (B, b’) female mouse LUT near mid-sagittal sections (5 μm) were stained by immunofluorescence to detect keratin 14 (KRT14, red) and transformation related protein 63 (TRP63, green). Cell nuclei were stained with DAPI (blue). Staining patterns in each panel are representative of three males and three females. Abbreviations used are BL: bladder, ED: ejaculatory duct, LV: lower vagina, SV: seminal vesicle, UV: upper vagina. White arrowheads indicate prostatic buds. Yellow arrowheads indicate KRT14⁻;TRP63⁺ intermediate urothelial cells. All images are of the same magnification.

Fig. 3

Wnt10b is a selective and early stage marker of mouse prostate identity. ISH was used to visualize expression of wingless-related MMTV integration site 10B (Wnt10b) mRNA (purple) in near mid-sagittal sections (50 μm) of 17 dpc (A) male and (B) female mouse LUT. ISH was also used to compare, after formation of prostatic buds and urethral gland buds in P0 male LUT, (C) Wnt10b and (D) NK-3 transcription factor, locus 1 (Nkx3-1) mRNA expression. Sections were then stained by immunofluorescence with an anti-smooth muscle actin alpha 2 (ACTA2) antibody that recognizes muscularis mucosa and muscularis propria (green) and an anti-cadherin 1 (CDH1) antibody that recognizes all urothelium (red). Staining patterns are representative of three separate LUTs for each transcript and embryonic stage. Abbreviations used are BL: bladder, ED: ejaculatory duct, LV: lower vagina, SV: seminal vesicle, UV: upper vagina. Black arrowheads indicate representative prostatic buds. White arrowheads indicate representative urethral buds. All images are of the same magnification.

Fig. 4

Selective mRNA markers of vagina and ejaculatory duct in 17 dpc mice. Near mid-sagittal sections (50 μm) of 17 dpc male and female LUT were stained by ISH to visualize mRNA expression (purple) of (A-B) the ejaculatory duct mesenchyme marker anti-Müllerian hormone receptor, type II (Amhr2), (C-D) the upper vagina epithelium marker wingless-related MMTV integration site 7a (Wnt7a), (E-F) the seminal vesicle epithelium marker Wnt9b, and (G-H) the seminal vesicle mesenchyme marker cellular retinoic acid binding protein I (Crabp1). Sections were then stained by immunofluorescence with an anti-smooth muscle actin alpha 2 (ACTA2) antibody that recognizes muscularis mucosa and muscularis propria (green) and an anti-cadherin 1 (CDH1) antibody that recognizes all urothelium (red). Staining patterns are representative of three males and three females. Abbreviations used are BL: bladder, ED: ejaculatory duct, LV: lower vagina, SV: seminal vesicle, UV: upper vagina. Arrowheads indicate prostatic buds. All images are of the same magnification.

Fig. 5

Selective mRNA markers of 17 dpc mouse pelvic urethra lamina propria, muscularis mucosa, submucosa, and muscularis propria. Near mid-sagittal sections (50 μm) of 17 dpc male and female LUT were stained by ISH to visualize mRNA expression (purple) of (A-B) the lamina propria marker snail homolog 1 (Snai1), (C-D) the lamina propria and submucosa marker forkhead box f1a (Foxf1a), and (E-F) the muscularis mucosa and muscularis propria marker actin alpha 2 (Acta2). Sections were then stained by immunofluorescence with an anti-smooth muscle actin alpha 2 (ACTA2) antibody that recognizes muscularis mucosa and muscularis propria (green) and an anti-cadherin 1 (CDH1) antibody that recognizes all urothelium (red). Inset images are magnified sections of mesenchyme that reveal the lamina propria (lp), muscularis mucosa (mm), submucosa (sm), and muscularis propria (mp) compartments. Staining patterns are representative of three males and three females. Abbreviations used are BL: bladder, ED: ejaculatory duct, LV: lower vagina, SV: seminal vesicle, UV: upper vagina. All images are of the same magnification.

Fig. 6

Selective mRNA markers of mesenchymal pads of 17 dpc mouse pelvic urethra mesenchyme. (A-B) Near mid-sagittal sections (5 μm) of 17 dpc male and female LUT were stained with hematoxylin and eosin to reveal mesenchymal cell condensates that comprise the three mesenchymal pads of the pelvic urethra. Mesenchymal condensates are outlined with dashed lines. (C-F) Near mid-sagittal sections (50 μm) of 17 dpc male and female LUT were stained by ISH to visualize mRNA expression (purple) of (C-D) fibroblast growth factor 10 (Fgf10) that marks all mesenchymal pads and (E-F) sex comb on midleg homolog 1 (Scmh1) that marks only the ventral mesenchymal pad. Sections were then stained by immunofluorescence with an anti-smooth muscle actin alpha 2 (ACTA2) antibody that recognizes muscularis mucosa and muscularis propria (green) and an anti-cadherin 1 (CDH1) antibody that recognizes all urothelium (red). Results in each panel are representative of three males and three females. Abbreviations used are AMP: anterior mesenchymal pad, BL: bladder, DMP: dorsal mesenchymal pad, ED: ejaculatory duct, LV: lower vagina, MP: muscularis propria, SV: seminal vesicle, UV: upper vagina, VMP: ventral mesenchymal pad. All images are of the same magnification.

Fig. 7

Selective mRNA markers of vascular endothelium and pericytes in 17 dpc mouse pelvic urethra. Near mid-sagittal sections (50 μm) of 17 dpc male and female LUT were stained by ISH to visualize mRNA expression (purple) of (A-B) the vascular endothelium marker E26 avian leukemia oncogene 1, 5′ domain (Ets1) and (C-D) the presumptive pericyte marker regulator of G-protein signaling 5 (Rgs5). Sections were then stained by immunofluorescence with an anti-smooth muscle actin alpha 2 (ACTA2) antibody that recognizes muscularis mucosa and muscularis propria (green) and an anti-cadherin 1 (CDH1) antibody that recognizes all urothelium (red). Results in each panel are representative of three males and three females. Abbreviations used are BL: bladder, ED: ejaculatory duct, LV: lower vagina, MM: muscularis mucosa, MP: muscularis propria, UV: upper vagina. Arrowheads indicate prostatic buds. All images are of the same magnification.

Fig. 8

Selective mRNA markers of presumptive neural crest cells, macrophages, and interstitial cells of Cajal in 17 dpc mouse pelvic urethra. Near mid-sagittal sections (50 μm) of 17 dpc male and female LUT were stained by ISH to visualize mRNA expression (purple) of (A-B) the presumptive macrophage marker egf-like module containing, mucin-like, hormone receptor-like 1 (Emr1), (C-D) the presumptive neural crest derived cell marker SRY (sex determining region Y)-box 10 (Sox10), and (E-F) the presumptive interstitial cell of Cajal (ICC) marker kit oncogene (Kit). Sections were then stained by immunofluorescence with an anti-smooth muscle actin alpha 2 (ACTA2) antibody that recognizes muscularis mucosa and muscularis propria (green) and an anti-cadherin 1 (CDH1) antibody that recognizes all urothelium (red). Results in each panel are representative of three males and three females. Abbreviations used are BL: bladder, ED: ejaculatory duct, LV: lower vagina, MM: muscularis mucosa, MP: muscularis propria, UV: upper vagina. Black arrowheads indicate prostatic buds and white arrowheads indicate presumptive ICC. All images are of the same magnification.

Fig. 9

Srd5a2 mRNA expression marks a sub-population of 17 dpc male LUT stroma where nuclear androgen receptor protein expression is abundant. Near mid-sagittal sections (50 μm) of 17 dpc male and female LUT were stained by ISH to visualize mRNA expression (purple) of (A-B) steroid 5 alpha reductase type 2 (Srd5a2). Sections were then stained by immunofluorescence with an anti-smooth muscle actin alpha 2 (ACTA2) antibody that recognizes muscularis mucosa and muscularis propria (green) and an anti-cadherin 1 (CDH1) antibody that recognizes all urothelium (red). In panels C-D, IHC was used to visualize the distribution of nuclear androgen receptor (AR) protein (green). CDH1 (red) was used to visualize urothelium and DAPI (blue) was used to visualize cell nuclei. Results in each panel are representative of three males and three females. Abbreviations used are BL: bladder, ED: ejaculatory duct, LV: lower vagina, MM: muscularis mucosa, MP: muscularis propria, UV: upper vagina. Arrowheads indicate prostatic buds. All images are of the same magnification.

Fig. 10

Selective mRNA marker of dorsal LUT in 17 dpc mice. Near mid-sagittal sections (50 μm) of 17 dpc male and female LUT were stained by ISH to visualize mRNA expression (purple) of (A-B) the dorsal urethral marker GATA binding protein 2 (Gata2) and (C-D) the ventral urethral marker frizzled-related protein (Frzb). Sections were then stained by immunofluorescence with an anti-smooth muscle actin alpha 2 (ACTA2) antibody that recognizes muscularis mucosa and muscularis propria (green) and an anti-cadherin 1 (CDH1) antibody that recognizes all urothelium (red). Inset images are transverse sections (50 μm) of 17 dpc male and female LUT. Results in each panel are representative of three males and three females. Abbreviations used are BL: bladder, D: dorsal, ED: ejaculatory duct, LV: lower vagina, UV: upper vagina, V: ventral. Arrowheads indicate prostatic buds. All images are of the same magnification.

Fig. 11

Schematic representation of stromal and epithelial sub-compartments in 17 dpc mouse LUT. A representation of a near mid-sagittal 17 dpc mouse LUT is shown and tissue sub-compartments identified in the current study are color-coded according to the key. Supplemental Fig. 1. Functional androgen receptor signaling is required for Srd5a2 mRNA expression in the mouse UGS. (A) E14.5 male C57BL/6J fetal mouse UGS tissues were incubated for 24 hr in organ culture media containing 10 μm hydoxyflutamide (OHF, androgen receptor [AR] antagonist) or 10 nM 5α dihydrotestosterone (DHT, AR agonist). (B) UGS tissues were collected from male E18.5 wild type fetal mice that harbor a functional Ar and from testicular feminization (Tfm) fetal mice that harbor a spontaneous Ar mutation that renders it less sensitive to androgens. Srd5a2 mRNA abundance was assessed by real-time RT-PCR and normalized to peptidyl prolyl isomerase mRNA abundance. Results are mean ± SE of n = 3 litter-independent samples per group. Significant differences from the control group (p < 0.05) are indicated by an asterisk and were identified by Student’s T-Test.