To assess the temporal expression of retinoid signaling during prostatic budding initiation and elongation, the UGS from fetal male mice that express a LacZ transgene under control of a RARE-containing promoter were collected at regular intervals during development from embryonic day (E)14.5 to postnatal day (P)0.5 and probed for β-galactosidase activity. Photographs are representative of n = 5 UGS specimens per developmental time point. To evaluate global retinoid activity and activity within specific UGS structures, β-galactosidase activity in UGS specimens was visualized by whole mount imaging (A-D), imaging of sections cut near the mid-sagittal plane (a’-c’) or along the sagittal plane near the lateral surface (d’), and imaging of sections taken along the transverse plane (b”). To enhance visualization, a dotted black line was added to illustrate the boundary between the inner UGS epithelial cell layer and outer mesenchymal cell layer and a solid black line was added to indicate the ventral mesenchymal pad (VMP). Prostatic buds in tissue sections are indicated by asterisks and by colored dotted lines in whole-mount images (blue = ventral buds, green = dorsolateral buds, and red = anterior buds). SV: seminal vesicle; ED: ejactulatory duct; U: ureter.

The expression of RARs and RXRs was evaluated at E16.5, during the initiation of prostatic buds from the UGS. Since retinoid activity was most abundant in UGS mesenchyme in Fig. 1, cDNA was synthesized from isolated E16.5 UGS mesenchyme (pooled from n = 5 separate fetuses) and purity of this mesenchymal cDNA preparation was demonstrated by showing that it was deficient in the epithelial-specific gene keratin 14 (Krt14). All six of the Rar and Rxr mRNAs were detected in UGS mesenchyme. Immunoblotting was used to determine which RAR and RXR isoforms were present.

The spatial expression pattern of retinaldehyde dehydrogenase (Aldh) 1a1, 1a2 and 1a3 mRNAs was determined in representative E15.5 - 17.5 UGS specimens by ISH on sagittal UGS sections (80 μm) and whole mount UGS and prostate specimens (n = 5 UGS and prostate specimens per group). A dotted black line was added to indicate the boundary between the inner layer of UGS epithelium (UGE) and the outer layer of UGS mesenchyme (UGM). Ur: urethra; WE: Wolffian-derived epithelium; BN: bladder neck.

The spatial expression pattern of cytochrome P450 (Cyp) 26a1 and 26b1 mRNA was determined in representative E15.5-17 UGS specimens by ISH on 80 μm sagittal UGS sections (n = 3 UGS specimens per group). A black line was added to indicate the boundary between the inner layer of UGE and the outer layer of UGM. At all stages examined between E15.5 - 17.5, there was a gap in Cyp26b1 expression (indicated by a diamond) proximal to the VMP.

UGS from E14.5 male C57BL/6J mouse fetuses were incubated for 24 h in organ culture media containing vehicle or RA (0-10 μM). Abundance of Cyp26b1 and Nr2f2 was determined by real-time RT-PCR and normalized to the mRNA abundance of the housekeeping gene peptidyl prolyl isomerase a (Ppia). Results are mean ± SEM of n ≥ 4 independent samples per group from at least 3 separate litters. Significant differences among groups were determined by ANOVA followed by Fisher’s Least Significant Difference test. The presence of an asterisk indicates significantly different from vehicle (open bars, p ≤ 0.05). The presence of a dagger indicates significantly different from 10 μM RA (p ≤ 0.05).

UGS from E14.5 male C57BL/6J mouse fetuses were incubated for 3 d in organ culture media containing vehicle or graded concentrations of RA (0-10 μM). Media and RA were replenished after each day. At the end of the incubation, UGE was separated from UGM as described in methods and visualized by SEM. A micrograph representing a single UGE from each exposure group is shown (results are n ≥ 6 independent samples per group from at least 3 separate litters). Prostatic buds are pseudocolored green.

UGS from E14.5 male C57BL/6J mouse fetuses were incubated for 24 h (for RT-PCR) or 3 d (for prostatic bud counting) in organ culture media containing vehicle or graded concentrations of RA (0-10 μM). Media and RA were replenished after each day. At the end of the 3d incubation, UGE was separated from UGM and visualized by SEM. Prostatic buds were counted using micrographs of each UGS taken from four separate angles in order to observe the entire UGS and ensure that all prostatic buds on the entire UGS epithelial surface were counted. Results for the total number of prostatic buds per UGS are mean ± SEM of n ≥ 6 independent samples per group from at least 3 separate litters. Abundance of Shh and Bmp4 was determined by real-time RT-PCR and normalized to the mRNA abundance of the housekeeping gene peptidyl prolyl isomerase a (Ppia). Results are mean ± SEM of n ≥ 4 independent samples per group from at least 3 separate litters. Significant differences among means were determined using one-way ANOVA, followed by Fisher’s LSD post-hoc test. The presence of an asterisk indicates a mean that is significantly different from the vehicle control (open bars, p ≤ 0.05). The presence of a dagger indicates it is significantly different from 1 μM RA (p ≤ 0.05).

UGS from E14.5 male C57BL/6J mouse fetuses were incubated for 3 d in organ culture media containing vehicle, recombinant SHH protein (5 μM), recombinant NOGGIN protein (1 ug/ml)or a combination of SHH + NOGGIN. Media and recombinant proteins were replenished daily. At the end of the incubation, UGE was separated from UGM as described in Methods and visualized by SEM. Prostatic buds were counted using micrographs of each UGS that were taken from four separate angles in order to observe the entire UGS epithelial surface and ensure that all prostatic buds present were counted. Results for the total number of prostatic buds per UGS are expressed as the mean ± SEM of n ≥ 6 independent samples per group for at least 3 separate litters. Significant differences among means were determined using one-way ANOVA, followed by Fisher’s LSD post-hoc test. The presence of an asterisk indicates a mean that is significantly different from the vehicle (control) mean (p ≤ 0.05).