QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 29 June 2005
doi: 10.1242/dev.01917

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gao, N. Articles by Matusik, R. J. Search for Related Content PubMed PubMed Citation Articles by Gao, N. Articles by Matusik, R. J.

Forkhead box A1 regulates prostate ductal morphogenesis and promotes epithelial cell maturation

Nan Gao^1,*, Kenichiro Ishii^2,*, Janni Mirosevich², Satoru Kuwajima⁴, Stacey R. Oppenheimer^3,5, Richard L. Roberts⁶, Ming Jiang², Xiuping Yu², Scott B. Shappell⁶, Richard M. Caprioli^3,5, Markus Stoffel⁴, Simon W. Hayward^2,3 and Robert J. Matusik^1,2,3,

¹ Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN 37232, USA
² Department of Urologic Surgery, Vanderbilt University, Nashville, TN 37232, USA
³ Department of Cancer Biology and Vanderbilt-Ingram Cancer Center, Vanderbilt University, Nashville, TN 37232, USA
⁴ Laboratory of Metabolic Diseases, The Rockefeller University, New York, NY 10021, USA
⁵ Mass Spectrometry Research Center, Vanderbilt University, Nashville, TN 37232, USA
⁶ Department of Pathology, Vanderbilt University, Nashville, TN 37232, USA

View larger version (96K): [in a new window]   Fig. 1. Distribution of Foxa1 and Foxa2 in mouse UGS. (A) Foxa1 (green) is expressed in E18 mouse UGE (asterisk). (B) Ck14 staining (red) reveals peripheral basal epithelial layers. (C) Triple immunofluorescence merges from A and B, plus DAPI. (D) Nuclear Foxa1 staining (inset) is seen in 6-week-old mature prostate epithelium. (E) E18 UGE expresses Foxa2 (green nuclear staining) with strongest level in peripheral basal epithelium. Inset shows entire UGS counterstained with DAPI. (F) Same basal epithelial cells express Shh (red signal on cell-membrane). (G) Merges of E and F. (H) In E21 UGS, Ptch1 (green) is expressed in both UGE and mesenchymal cells (arrow). Arrowhead indicates Ptch1-expressing nascent prostatic buds. (I) Shh is co-expressed in E21 UGE and nascent epithelial buds (arrowhead). (J) Merges of H and I. (K) E21 UGS double stained for AR (green) and SMA (red). SMA-expressing cells are the same population expressing Ptch1 (arrow). (L) E21 UGS double-stained for AR (green) and Ck14 (red). (M) E21 UGS double-stained for Ck8 (red) and p63 (green). (N) ß-Catenin (red) is expressed in both epithelium and surrounding mesenchyme in E18 UGS, with strongest signal detected in the peripheral epithelium (arrowhead). (O) E18 UGS stained for p63. (P) Magnified merges of N and O. (Q,R) In situ hybridization. Foxa1 and Foxa2 are expressed in P1 prostate rudiments (arrows, parasagittal sections). Insets show strongly stained epithelial buds (arrowheads in R). (S,T) Foxa1, but not Foxa2, is expressed in mature glands. BL, Bladder.

View larger version (102K): [in a new window]   Fig. 2. Foxa1 regulates prostate ductal morphogenesis. (A) Upper panels: urogenital organs (lateral, left, and dorsal, right, views) dissected from P1 pups. The bladder was removed as indicated by broken lines. Lower panels: prostate rudiments (asterisks) and seminal vesicle (SV) were grafted as renal rescue tissue. (B-D) Upper panels: 8-week-old rescued tissues, with indicated genotypes, developed in the host renal capsules. SVs are circled with broken lines. Rescued Foxa1–/– prostate (asterisk in D) is smaller than controls upon comparison after fine dissection (lower panels). (E-G) ß-Galactosidase staining on 8-week-old rescued prostates. (H) Twelve-week-old tissue recombinants derived from wild-type (left) or Foxa1–/– (right) epithelium that was recombined with E18 rUGM. (I) The Foxa1–/– recombinants have significantly lower weights than controls (n=3, P<0.01). (J-K) Hematoxylin and Eosin staining of 4-week-old rescued Foxa1–/– and Foxa1+/+ VPs. (L,M) AR staining. (N,O) High magnification views of regions framed in L and M (arrowheads). (P,Q) Toluidine Blue staining on 1 µm thin section of 12-week-old rescued Foxa1–/– and wild-type VPs. (R,S) E-cadherin staining (red) on 12-week-old rescued Foxa1–/– and wild-type VPs. Foxa1-null epithelial cell polarity is disrupted (arrowheads). (T,U) Hematoxylin and Eosin staining of 12-week-old tissue recombinants from Foxa1–/– and control epithelium.

View larger version (145K): [in a new window]   Fig. 3. Foxa1-deficient prostate has altered epithelial cell population and stromal pattern. (A) Ck5 staining on 4-week-old rescued Foxa1–/– VP. (B) Ck5-positive basal epithelial cells (arrow) expanded within the null epithelial cords. (C) Ck5 staining on rescued wild-type VP. (D,E) Dual-staining of p63 (green) and Ck8 (red) on 4-week-old rescued Foxa1–/– and Foxa1+/+ DLPs. (F,G) Some Foxa1–/– cells co-expressed of both markers (arrowhead in F), while control cells expressed these markers separately (G). (H-J) SMA staining on 4-week-old rescued VPs. Expansion in smooth muscle layer (arrowheads) was seen in null prostate (H,I), but not in control (J).

View larger version (34K): [in a new window]   Fig. 4. Altered gene expression profiles in Foxa1–/– prostates. RT-PCR was performed using gene specific primers, on rescued Foxa1+/+ and Foxa1–/– prostates, with Gapdh gene as an internal standard. Cycle numbers of amplification are indicated on the top.

View larger version (73K): [in a new window]   Fig. 5. No mature luminal cells in Foxa1–/– prostate. (A-D) EM analysis on 12-week-old rescued Foxa1+/+ and Foxa1–/– VPs. Scale bars: 2 µm in A,B; 500 nm in C,D. Asterisks indicate the lumen. Wild-type cells (A,C) contain secretory materials seen in apical vesicles (arrow) and at luminal surface (arrowhead). Secretory material was absent in Foxa1–/– cells (B,D). (E) MALDI-MS protein profiles of m/z range 3000-16,000 obtained from 4-week-old rescued Foxa1+/+, Foxa1+/– and Foxa1–/– prostates. Continuous profiles were zoomed in at m/z range 16,100-26,000. Arrows indicate peaks that were absent in Foxa1–/– but present in control prostates. (F) RT-PCR for Pbsn and Sbp. Bs, basement membrane.

View larger version (101K): [in a new window]   Fig. 6. Elevated Shh and Foxa2 but reduced Nkx3.1 expression in Foxa1–/– epithelium. (A) Shh expression in 4-week-old rescued Foxa1–/– VP epithelium, with strong and focused activity evident in the epithelial buds (B). (C) Ptch1 is detected in the same null epithelial buds. (D,E) Triple-immunofluorescence of ß-catenin (red), p63 (green) and DAPI (blue) on rescued Foxa1–/– and Foxa1+/+ prostates. (F-I) Foxa1–/– epithelium is negatively or faintly stained for nuclear Nkx3.1 (F,H). Arrowheads indicate the same epithelial buds illustrated in B. Foxa1+/+ luminal epithelium show strong nuclear Nkx3.1 immunoreactivity (G,I). (J-L) Foxa2 is expressed in Foxa1–/– epithelium (J,K), but not in Foxa1+/+ epithelium (L).

View larger version (53K): [in a new window]   Fig. 7. Foxa1 regulates early ductal pattern formation and promotes epithelial cell maturation. The embryonic urogenital sinus is composed of undifferentiated epithelial and stromal cells. During early prostate morphogenesis, systemic androgens and elevated epithelial cell Foxa1 and Nkx3.1 proteins modulate cell growth and differentiation. The Foxa1-null prevents cytodifferentiation and results in a population of intermediate epithelial and basal-like cells that individually express markers representative of both cell types (i.e. Ck5, Ck8 and Ck14). The mesenchymal cells differentiate into an atypically thick layer of smooth muscle. Prostatic embryonic signaling pathways remain active as reflected by the elevation of Shh, Bmp, Fgf and Notch. Foxa2, which is normally expressed only in prostatic buds in the embryo, remains elevated while Nkx3.1 is downregulated. The Foxa1-null prostate produces limited secretory proteins. An Nkx3.1-null prostate shows an epithelial cell hyperplasia by four weeks of age with limited differentiation as reflected by dramatically reduced levels of secretory proteins. By 40 weeks of age, the Nkx3.1-null prostate contains both epithelial cells hyperplasia and prostatic intraepithelial neoplasia – a precursor lesion for prostate cancer. A prostate that has AR-null epithelium but retains AR in the stromal cells results in normal development of the ductal structural, including both epithelial and basal cells. However, full differentiation does not occur as secretory proteins are not expressed. The normal adult prostate exhibits functional cytodifferentiation with fully differentiated basal and luminal cells exhibiting a full profile of secretory activity.