Noggin is required for normal lobe patterning and ductal budding in the mouse prostate

Abstract

Mesenchymal expression of the BMP antagonist NOGGIN during prostate development plays a critical role in pre-natal ventral prostate development and opposes BMP4-mediated inhibition of cell proliferation during postnatal ductal development. Morphologic examination of newborn Noggin-/- male fetuses revealed genitourinary anomalies including cryptorchidism, incomplete separation of the hindgut from the urogenital sinus (UGS), absence of the ventral mesenchymal pad, and a complete loss of ventral prostate (VP) budding. Examination of lobe-specific marker expression in the E14 Noggin-/- UGS rescued by transplantation under the renal capsule of a male nude mouse confirmed a complete loss of VP determination. More modest effects were observed in the other lobes, including decreased number of ductal buds in the dorsal and lateral prostates of newborn Noggin-/- males. BMP4 and BMP7 have been shown to inhibit ductal budding and outgrowth by negatively regulating epithelial cell proliferation. We show here that NOGGIN can neutralize budding inhibition by BMP4 and rescues branching morphogenesis of BMP4-exposed UGS in organ culture and show that the effects of BMP4 and NOGGIN activities converge on P63+ epithelial cells located at nascent duct tips. Together, these studies show that the BMP-NOGGIN axis regulates patterning of the ventral prostate, regulates ductal budding, and controls proliferation of P63+ epithelial cells in the nascent ducts of developing mouse prostate.

Figure 1. Temporal and spatial expression of Noggin in the developing UGS and prostate of male mice

(A) To assess the temporal expression of Noggin during prostate development, whole UGS from embryonic male mice and isolated ventral prostate (VP), dorsolateral prostate (DLP) and coagulating gland (GC) of postnatal male mice were collected at regular intervals during development (E14-P10). RNA was isolated from pooled UGS tissues (n = 6-9 E14-18 male fetuses from multiple litters) or individual prostate tissues from P1-P10 male neonates. Noggin mRNA abundance was determined by real-time PCR and normalized to glyceraldehyde phosphate dehydrogenase gene expression. Results are the mean (± SEM) of two RNA pools for E14-18 UGS, or three separate prostates (P1-P10. (B) Temporal and spatial expression of the Noggin^tm1(Lacz)Am transgene in developing UGS and prostate from Noggin^+/- transgenic mice. UGS or prostate tissues were removed from male mice at different days during gestation and postnatal development and probed for Noggin^tm1(Lacz)Am-mediated β-galactosidase activity or Noggin mRNA expression by whole-mount ISH. Photographs are representative of n > 10 UGS specimens. To visualize the global expression of Noggin as well as its expression in specific prostate structures, β-galactosidase activity in UGS specimens from P3 mice was visualized by whole mount imaging (left, center row), imaging of sagittal sections cut along the caudal-cranial urethral axis (center, center row), and imaging of transverse sections cut along the lateral-medial urethral axis (right, center row). Noggin expression was observed in mesenchyme surrounding seminal vesicles and prostatic buds of the VP, DLP, and CG in P1-10 mouse prostate. Noggin expression was most concentrated at distal tips of individual nascent DLP buds and decreased proximally towards the base of buds (right, center row).

Figure 2. Regulation of Noggin expression in the UGS

(A) To assess the regulatory relationship between NOGGIN, SHH, and BMP4, UGSs from E14 WT male mice were exposed in organ culture to recombinant octylated SHH (5 nM), BMP4 (300 ng/ml), NOGGIN (1 μg/ml), the hedgehog inhibitor cyclopamine (10 μM) or vehicle (0.1% BSA, control) for 3 days and the relative abundance of Noggin mRNA was determined for individual cultured UGS tissues by real-time RT-PCR. Results are mean (± s.e.m.), n ≥ 5 UGS specimens. (B) To determine whether BMP4-mediated induction of Noggin mRNA was caused by these agents acting directly on UGS mesenchyme, the UGSM-2 cell line (Shaw et al., 2006) was exposed to vehicle (0.01% BSA) or recombinant BMP4 for 24 hours and the expression of Noggin mRNA mean (± s.e.m.) was evaluated by real-time RT-PCR from three independent experiments. Significant differences between groups were determined by Analysis of Variance (ANOVA), followed by the Fisher Least Significant Difference post-hoc test. “*” Indicates significantly different from vehicle-exposed control UGS (p < 0.01).

Figure 3. Phenotype of the developing prostate from Noggin^-/- male mouse fetuses

(A) UGS/hindgut complexes from WT and Noggin^-/- male mice were removed from representative E17 male mouse fetuses (n = 3 per genotype) and visualized by light microscopy. The urogenital division (UD) that separates the hindgut (HG) from the UGS in the WT specimen was absent (denoted by “*”) and the membranous portion of the urethra (MU) failed to elongate in the Noggin^-/- mouse specimen. (B) To better visualize the overall architecture of the Noggin^+/+ and Noggin^-/- UGS, UGS epithelium (UGE) was separated from mesenchyme and visualized by scanning electron microscopy (SEM). Isolated UGE of a representative WT E17 male fetus (n = 3) revealed seminal vesicles (SV) and ductus deferens (DD) that drained into the ejaculatory duct located within the dorsal sulcus (white arrowhead), or groove, created by two prominent dorsal ridges (DR) on the lateral surfaces of the UGS. Emerging VP (blue), dorsal (green), lateral (yellow) CG (red) buds, and the prostatic utricle (PU) were also evident. On the other hand, VP buds and the PU were missing, DLP and CG budding was reduced, and the ejaculatory ducts were laterally displaced and drained into the DR instead of the dorsal sulcus (*) in the representative Noggin^-/- UGS. Also, the angle between the bladder neck and urethra of WT UGS approximated 90°, whereas this angle was closer to 160° in the Noggin^-/- UGS. (C) SEM images of Gremlin^-/- and Chordin^-/- male mouse prostate at P1 reveal budding patterns and prostate architecture that is very similar to WT littermates.

Figure 4. Noggin is required for formation of the ventral mesenchymal pad and for ventral UGS epithelial cell proliferation

(A, left column) Representative H&E stained prostate tissue sections (n = 5 per genotype) of P1 mouse prostate indicating thinning or absence of the ventral mesenchymal pad (VMP) in the P1 Noggin^-/- prostate. (A, right column) E14 WT and Noggin^-/- UGS were labeled in utero with BrdU 2 hr prior to euthanasia, sectioned and stained to evaluate epithelial cell proliferation. Representative photomicrographs (n = 3 UGS per genotype) from WT and Noggin^-/- UGS reveals decreased ventral UGS mesenchymal cell (UGM) density (outlined in pink) and fewer proliferating ventral UGS epithelial cells (UGE; white arrowheads), while cell proliferation in the Wolffian-derived epithelium (WE) was comparable between groups. (B) Proliferating ventral UGS epithelial cells (% of total) were quantified from a fixed area within 3-6 sections per UGS (3 UGS per genotype). Results are expressed as mean (± s.e.m.), n = 3. Significant differences between groups were determined by Student’s T test. “*” Indicates significantly different from WT (p < 0.05). Scale bar = 100 μm.

Figure 5. Ventral prostate development is inhibited in Noggin^-/- male mice

To determine if UGSs from Noggin^-/- male mice are capable of supporting development and differentiation into mature prostate tissue, prostates from WT, Noggin^+/-, and Noggin^-/- P1 newborn mice were grafted under the kidney capsule of nude mice for 21 days. (A) Hematoxylin and eosin stained graft sections of WT and Noggin^-/- mice revealed a ductal network characteristic of mature prostate containing prostatic secretions. There were no qualitative differences in the dorsolateral prostate ductal structure of WT and Noggin^-/- UGS renal grafts. Scale bar = 100 μm. (B) Prostate lobe-specific marker abundance was compared between the WT and Noggin^-/- prostate renal grafts. Results are expressed as mean (± s.e.m.), n = 3. Significant differences between groups were determined by Student’s T test. “*” Indicates significantly different from WT (p < 0.05). Specificity of Sbp, Ren1, and Pbsn expression for VP, CG, and DLP, respectively, was established by comparing the relative mRNA abundance of each message in prostate tissue from P35 WT mice. Results are expressed as mean (± s.e.m.), n = 3. Significant differences between groups were determined by ANOVA followed by the Fisher Least Significant Difference post-hoc test. †” Indicates significantly different from VP, CG, and DLP, for Sbp, Ren1, and Pbsn, respectively (p < 0.05). Significant differences between groups were determined by Student’s T test. ((C) To determine whether Noggin^+/- mice exhibited a haploinsufficient phenotype, P35 WT and Noggin^+/- prostate lobe weights for were measured and normalized to total body weight. Results are mean (± s.e.m.), n ≥ 18 for each genotype. “*” Indicates significantly different from WT (p < 0.05).

Figure 6. Effect of NOGGIN on prostatic budding, branching, and cell proliferation in UGS or neonatal prostate organ culture

To determine whether exposure to recombinant NOGGIN and/or recombinant BMP4 alters prostatic budding or E14 WT UGS was incubated for 7 days in organ culture containing vehicle (0.01% BSA; control), recombinant BMP4 (300 ng/ml), recombinant NOGGIN (1 μg/ml) or a combination of NOGGIN and BMP4. (A) The number of bud tips (□) and main ducts (●) per UGS were determined. Results are mean (± s.e.m.), n ≥ 10 samples per group. Significant differences between groups were determined by ANOVA, followed by the Fisher Least Significant Difference test. “*” Indicates significantly different from control (p < 0.001). Representative UGS samples after 7 days in organ culture are shown for each treatment group.

Figure 7. Ontogeny of P63 expresssion in developing WT UGS and early postnatal prostate

CD-1 mouse UGS and prostate tissue specimens were analyzed for P63 expression by immunohistochemistry. (A) Multiple layers of P63-positive staining basal epithelial cells accumulated beneath the basal lamina (partially contoured in white) of the UGS and urethra at E16 in WT male mice. (B-E) During postnatal development from P1-P10, P63 expression was gradually restricted to a single P63+ epithelial cell layer beneath the basal lamina of developing prostate ducts. (E) Immunohistochemical analysis of P63 and Ki67 expression in prostate sections from P1 WT male newborn mice revealed numerous P63+ basal epithelial cells (red) throughout prostatic epithelium, including prostate buds. Ki67-positive cells (green) were observed in prostatic mesenchyme and prostate epithelium. P63+, proliferating cells (yellow) in prostate epithelium were restricted to the tips of emerging prostate buds (arrowheads). Scale bar = 100 μm.

Figure 8. NOGGIN exposure induces proliferation of BMP4-exposed prostate in organ culture

P1 prostates were incubated for 3 days in organ culture with vehicle (0.1% BSA; control) or recombinant BMP4 (300 ng/ml). The organ culture media was then replaced with media containing vehicle, recombinant NOGGIN (1 μg/ml), or BMP4 and cultures were incubated for an additional day. Four hours before harvesting the tissue, BrdU was added to the media to stain proliferating cells. Tissue sections were then assayed for BrdU (green) and P63 (pink) expression by IHC. Prostatic buds are indicated by arrowheads and the basal lamina for one bud in panels A-D is traced in white. (A) Vehicle-exposed prostates showed proliferation in prostate mesenchyme and P63+ and P63- epithelial cells. (B) NOGGIN (NOG) exposure for 1 day does not change the proliferation of P63- epithelial cells within the core of prostatic buds compared to control prostates. (C) BMP4 exposure for 4 days decreased proliferation of P63+ but not P63- epithelial cells compared to control. (D) BMP4 exposure for 3 days followed by NOGGIN exposure for 1 day caused a burst of proliferation in P63+ but not P63- epithelial cells at the leading edge of ducts. Scale bar = 100 μm. (E) Mitotic indices for P63+ cells were calculated by counting the number of proliferating, BrdU and P63 positive cells as a fraction of the total P63+ cell population for each treatment group. Results are the mean (± s.e.m) of 2 to 6 sections from each of four UGS specimens per treatment group. Differences between groups were determined by Fisher’s protected least significant difference test. “*” indicates significantly different from control (p < 0.05). Analysis of mitotic indices for P63- cells revealed no differences between any of the treatment groups (not shown).