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. 2013 Apr 15;376(2):125-35.
doi: 10.1016/j.ydbio.2013.01.034. Epub 2013 Feb 8.

Beta-catenin (CTNNB1) Induces Bmp Expression in Urogenital Sinus Epithelium and Participates in Prostatic Bud Initiation and Patterning

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Beta-catenin (CTNNB1) Induces Bmp Expression in Urogenital Sinus Epithelium and Participates in Prostatic Bud Initiation and Patterning

Vatsal Mehta et al. Dev Biol. .
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Abstract

Fetal prostate development is initiated by androgens and patterned by androgen dependent and independent signals. How these signals integrate to control epithelial cell differentiation and prostatic bud patterning is not fully understood. To test the role of beta-catenin (Ctnnb1) in this process, we used a genetic approach to conditionally delete or stabilize Ctnnb1 in urogenital sinus (UGS) epithelium from which the prostate derives. Two opposing mechanisms of action were revealed. By deleting Ctnnb1, we found it is required for separation of UGS from cloaca, emergence or maintenance of differentiated UGS basal epithelium and formation of prostatic buds. By genetically inducing a patchy subset of UGS epithelial cells to express excess CTNNB1, we found its excess abundance increases Bmp expression and leads to a global impairment of prostatic bud formation. Addition of NOGGIN partially restores prostatic budding in UGS explants with excess Ctnnb1. These results indicate a requirement for Ctnnb1 in UGS basal epithelial cell differentiation, prostatic bud initiation and bud spacing and suggest some of these actions are mediated in part through activation of BMP signaling.

Figures

Fig. 1
Fig. 1. Ctnnb1 deletion from early-stage mouse UGS epithelium (UGE) impairs cloacal division, prostate field specification and formation of prostatic buds and KRT14+ basal epithelium
(A–B) Electron micrographs of E17.5 Shhcre/+;Ctnnb1tm2Kem/+ (control) and Shhcre/+; Ctnnb1tm2Kem/tm2Kem (Ctnnb1cLOF) UGE after removal of overlying UGS stroma. Prostatic buds are pseudocolored red. Arrow indicates an inappropriate hindgut-UGE connection (rectourethral fistula, rf) in Ctnnb1cLOF mice. (C–D) E17.5 male UGSs were stained by ISH to visualize the prostatic bud marker Nkx3-1 (purple) and counterstained by IHC to visualize UGE marked by cadherin 1 (CDH1). (E–H) E17.5 UGS sections were immunofluorescently labeled to detect CTNNB1, CDH1 and the UGS basal epithelial cell marker KRT14. Nuclei were stained with DAPI. Staining patterns in each panel represent three males. Abbreviations are bl: bladder, bn: bladder neck, hg: hindgut, sv: seminal vesicle, wd: Wolffian duct. Arrowheads indicate prostatic buds.
Fig. 2
Fig. 2. Ctnnb1 deletion from late-stage mouse UGE impairs prostatic bud formation but not KRT14+ mature UGS basal epithelium formation
Male Shh+/+;Ctnnb1tm2Kem/tm2Kem (control) and ShhcreERT2/+;Ctnnb1tm2Kem/tm2Kem (Ctnnb1iLOF) embryos were exposed to tamoxifen and progesterone as described. (A–B) E18.5 male UGSs were stained by ISH to visualize Nkx3-1 (purple) and counterstained by IHC to visualize UGE marked by CDH1. Blue arrowheads indicate ventral prostatic buds, red arrowheads are anterior prostatic buds and green arrowheads are dorsolateral prostatic buds. (C) Nkx3-1+ bud numbers are mean ± SE of five independent samples per group from at least three litters. Asterisks indicate significant differences from controls (p < 0.05). (D–G) Near mid-sagittal sections from three UGSs per genotype UGS sections were immunofluorescently labeled to visualize CTNNB1, CDH1 and KRT14. Cell nuclei are stained with DAPI (blue). Arrowheads indicate prostatic buds. The arrow in panel E (inset) indicates a representative CTNNB1 immunonegative cell. Abbreviations are bl: bladder, sv: seminal vesicle.
Fig. 3
Fig. 3. Ctnnb1-positive cells are more likely than Ctnnb1-negative cells to become UGS basal epithelium
Male ShhcreERT2/+;Ctnnb1tm2Kem/+;R26R+ (control;R26R) and ShhcreERT2/+;Ctnnb1tm2Kem/tm2Kem;R26R+ (Ctnnb1iLOF;R26R) embryos were exposed to tamoxifen and progesterone as described. (A–B) E18.5 UGSs were stained to visualize LacZ activity, embedded in paraffin, sectioned and immunofluorescently labeled to detect the cell proliferation marker Ki67 and KRT14. Brightfield LacZ staining images were captured, inverted, pseudocolored yellow and merged with fluorescent images. Nuclei were stained with DAPI. Staining patterns represent at least three UGSs per genotype. Arrowheads indicate prostatic buds. (C) The LacZ+ cell percentage of KRT14+ cells and (D) the Ki67+ cell percentage of LacZ+;KRT14+ cells are mean ± SE of three independent samples per group from at least three litters. Asterisks indicate significant differences from controls (p < 0.05).
Fig. 4
Fig. 4. Excess CTNNB1 in UGE impairs prostatic bud formation and causes an inappropriate pattern of CTNNB1-responsive mRNA expression
Male Shh+/+; Ctnnb1tm1Mmt/tm1Mmt (control) and ShhcreERT2/+;Ctnnb1tm1Mmt/tm1Mmt (Ctnnb1iGOF) embryos were exposed to tamoxifen and progesterone as described. (A–B) E18.5 male UGSs were stained by ISH to visualize Nkx3-1 (purple) and counterstained by IHC to visualize UGE marked by CDH1. Blue arrowheads indicate ventral prostatic buds, red arrowheads are anterior prostatic buds and green arrowheads are dorsolateral prostatic buds. (C) Nkx3-1+ bud numbers are mean ± SE of five independent samples per group from at least three litters. Asterisks indicate significant differences from controls (p < 0.05). (D–M) Sections from three UGSs per genotype were immunofluorescently labeled to visualize CTNNB1 and CDH1 proteins or stained by ISH to visualize mRNA expression (purple) of the urethra lamina propria mesenchyme Foxf1a or the CTNNB1 responsive genes Axin2, Lef1 and Wif1. Sections were immunofluorescently counterstained to visualize UGE marked by CDH1. Abbreviations are bl: bladder, sv: seminal vesicle. Arrowheads indicate prostatic buds.
Fig. 5
Fig. 5. Excess CTNNB1 in UGE is not sufficient for prostate identity but is sufficient for basal epithelial cell differentiation
Male Shh+/+; Ctnnb1tm1Mmt/tm1Mmt (control) and ShhcreERT2/+; Ctnnb1tm1Mmt/tm1Mmt (Ctnnb1iGOF) embryos were exposed to tamoxifen and progesterone as described. (A-B, E-H) At E18.5, UGS sections were stained by ISH to visualize mRNAs (purple) for the prostatic bud marker Nkx3-1, the basal epithelial marker Krt5 and the intermediate and superficial urothelial marker Upk1b. Sections were immunofluorescently counterstained to visualize UGE marked by CDH1. (C–D) E18.5 male UGS sections were immunofluorescently labeled to visualize CTNNB1 and the basal epithelial marker TRP63. Staining patterns in each panel represent three males. Abbreviations are bl: bladder, sv: seminal vesicle. Arrowheads indicate prostatic buds.
Fig. 6
Fig. 6. Excess CTNNB1 in UGE induces BMP expression and increases BMP signaling
E18.5 Male Shh+/+;Ctnnb1tm1Mmt/tm1Mmt (control) and ShhcreERT2/+;Ctnnb1tm1Mmt/tm1Mmt (Ctnnb1iGOF) embryos were exposed to tamoxifen and progesterone as described. (A–F) Sections stained by ISH to visualize mRNA expression (purple) of Bmp 2, 4 and 7 Black arrowheads indicate prostatic bud tips. (G–H) Sections immunofluorescently labeled to visualize CTNNB1 (green), phospho-SMAD (pSMAD) 1/5/8 (red) and nuclei stained with DAPI (blue). White lines demarcate the interface between UGE and UGM. Yellow arrows indicate cells in which CTNNB1 is known to be high (prostatic bud tips in G and CTNNB1 cell islands in H). White arrows indicate cells surrounding those with high CTNNB1. Abbreviations are bl: bladder, sv: seminal vesicle.
Fig. 7
Fig. 7. BMP signaling inhibition in cultured UGS with excess CTNNB1 partially restores prostatic bud formation
E14.5 male Shh+/+;Ctnnb1tm1Mmt/tm1Mmt (control) and ShhcreERT2/+;Ctnnb1tm1Mmt/tm1Mmt (Ctnnb1iGOF) UGSs were incubated for 4 d in organ culture medium containing 1 µm 4-hydroxytamoxifen, 10nM 5α dihydrotestosterone and either vehicle or recombinant NOGGIN protein (rNOGGIN, 1 µg/ml). Media and supplements were replenished after the second day. (A–D). Whole UGSs were stained by ISH to visualize Nkx3-1 mRNA (purple). Abbreviations are bl: bladder, sv: seminal vesicle. (E) Nkx3-1+ bud numbers are mean ± SE of five independent samples per group from at least three litters. Asterisks indicate a significant difference (p < 0.05) in prostatic bud number compared to Ctnnb1iGOF UGS grown in the absence of rNOGGIN.
Fig. 8
Fig. 8. Proposed dichotomous CTNNB1 activity during mouse prostate development
(A) During early prostate development, functional CTNNB1 in primitive UGS epithelium supports formation or maintenance of mature KRT14+ basal epithelium, which is needed for prostatic bud formation. (B) During prostatic bud formation and elongation, CTNNB1 is activated in prostatic bud tips where it initiates BMP 2, 4 and 7 synthesis, which act upon UGS epithelium to prevent additional buds from forming. We propose that these CTNNB1 mechanisms in part establish prostatic bud spacing intervals.

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