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First published online 10 January 2007
doi: 10.1242/dev.02765

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Fibroblast growth factor receptor 2 tyrosine kinase is required for prostatic morphogenesis and the acquisition of strict androgen dependency for adult tissue homeostasis

¹ Center for Cancer Biology and Nutrition, Institute of Biosciences and Technology, Texas A&M Health Science Center, 2121 W. Holcombe Blvd, Houston, TX 77030-3303, USA.
² State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100094, P.R. China.
³ Center for Advanced Biotechnology and Medicine, UMDNJ-Robert Wood Johnson Medical School, 679 Hoes Lane, Piscataway, NJ 08854, USA.
⁴ Department of Molecular Biology and Pharmacology, Washington University, School of Medicine, 660 South Euclid Avenue, St Louis, MO, 63110, USA.
⁵ Department of Surgery, University of Western Ontario, London, ON, N6A 4G5, Canada.
⁶ Clinical Research Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue, Seattle, WA 98109-1024, USA.

View larger version (71K): [in this window] [in a new window]   Fig. 1. Disruption of the Fgfr2 alleles in prostate epithelium. (A) Schematic of the floxed Fgfr2 alleles for conditional disruption. The genomic DNA containing exons 6-10 and the adjacent introns is shown. The primers for PCR genotyping and FGFR2 expression analyses are indicated. (B) The Fgfr2cn alleles only encode a truncated ectodomain. (C) PCR genotyping. Genomic DNAs extracted from different lobes of Fgfr2cn and control prostates of 3-week-old mice were PCR analyzed with the indicated primers. Primers f1 and f2 amplify a fragment of 207 bp from floxed Fgfr2 alleles. Primers f1 and f3 amplify a fragment of 471 bp from Fgfr2-null alleles, and give no amplification for wild-type Fgfr2 or Fgfr2flox alleles. (D,E) Diminished FGFR2 expression in the epithelium of Fgfr2cn prostates. The expression of FGFR2 was assessed with RT-PCR (D) and in situ hybridization (E). Primers R2f and R2r amplify both FGFR2IIIb (IIIb) and FGFR2IIIc (IIIc) isoforms; primers b and t only amplify FGFR2IIIb isoform, primers c and t only amplify FGFR2IIIc isoform. -, negative control without cDNA templates. Panel E shows strong expression of FGFR2 in the epithelial compartment of control prostates, which was diminished in Fgfr2cn prostates. ap, anterior prostate; dlp, dorsolateral prostate; vp, ventral prostate; S, signal peptide; I/II/III, immunoglobulin loop I, II and III, respectively; TM, transmembrane domain; F/F, homozygous Fgfr2flox mice; CN, Fgfr2cn mice.

View larger version (100K): [in this window] [in a new window]   Fig. 2. Disruption of Fgfr2 alleles leads to perturbed prostate morphogenesis. (A) The urogenital sinuses were dissected from embryos or postnatal pups carrying ROSA26/NKX3.1-Cre, and Fgfr2flox or wild-type Fgfr2 alleles at the indicated days. The tissues were lightly fixed and stained with X-Gal, as described. The stained tissues representing each prostatic lobe are indicated. Notice no significant difference exists in prostatic buds at E17.5 between Fgfr2cn and wild-type controls. Insert: section from the same tissue showing that NKX3.1-Cre efficiently excised the silencing cassette of the ROSA26 allele. (B) The prostate and urethra were dissected from mice at the indicated ages (left panels). Right panels: dorsolateral prostate lobes dissected from tissues shown in left panels. (B',B'') Epithelial ducts were dissected from dorsolateral prostates of 6-week-old mice with the indicated genotypes. (C) The prostate tissues were collected from Fgfr2cn and control mice at the indicated ages, and proliferating cells were identified immunohistochemically by expression of PCNA. Inserts: high-magnification views from the same sections. ap, anterior prostate; dlp, dorsolateral prostate; vp, ventral prostate; u, urethra; b, bladder; s, seminal vesicle; F/F, homozygous Fgfr2flox mice; CN, Fgfr2cn mice. Scar bars: 2 mm.

View larger version (146K): [in this window] [in a new window]   Fig. 3. Fgfr2cn prostates exhibited basic prostate characteristics. (A) Prostate tissues from 6-week-old mice were sectioned and stained with HE for histological analyses. Inserts: high-magnification views from the same tissues. (B) Total RNAs were extracted from dorsolateral prostates of 3-week-old mice and reverse translated with random hexanucleotide primers. RT-PCR was performed as indicated, with ß-actin and Gapdh as internal standards. Cycle numbers of amplification are indicated at the top. (C) Real-time RT-PCR analyses of the same panel of molecules as in B. Data were normalized with ß-actin loading control and were expressed as folds of difference from the control prostates. Data were means of triplicate samples. *P<0.001. F/F, homozygous Fgfr2flox mice; CN, Fgfr2cn mice; ap, anterior prostate; dp, dorsal prostate; lp, lateral prostate; vp, ventral prostate.

View larger version (103K): [in this window] [in a new window]   Fig. 4. Immunohistochemical characterization of the Fgfr2cn prostate. (A) The prostate sections from 4-week-old mice were immunostained with anti--actin or anti-cytokeratin 8, as indicated. (B) Prostate sections from Fgfr2cn and control mice at the indicated ages were immunohistochemically stained with anti-p63 antibodies. Inserts: high-magnification views from the same section. (C) Ratios of p63-positive cells in the epithelial compartment were calculated from three samples. Data representing means and s.d. of triplicate samples. F/F, homozygous Fgfr2flox mice; CN, Fgfr2cn mice.

View larger version (146K): [in this window] [in a new window]   Fig. 5. Diminished androgen dependency in Fgfr2cn prostates with respect to tissue homeostasis. (A) Fgfr2cn and control mice were orchiectomized to eliminate testisderived androgens. At the indicated day after the operation, the prostate tissues were harvested and apoptotic cells were detected with TUNEL assay. (B) HE staining of the same tissues showing that castration failed to induce tissue atrophy in Fgfr2cn prostates. F/F, homozygous Fgfr2flox mice; CN, Fgfr2cn mice.

View larger version (113K): [in this window] [in a new window]   Fig. 6. Expression of the androgen receptor in Fgfr2cn prostates. Prostates were harvested from 6-week-old mice before (0 day) and at the indicated days after the castration. Expression and cellular localization of the AR were assessed with immunostaining with anti-AR antibodies. Notice that a considerable amount of AR in Fgfr2cn prostates remained in the nuclei at day 14 after the castration. ap, anterior prostate; dp, dorsal prostate; lp, lateral prostate; vp, ventral prostate; F/F, homozygous Fgfr2flox mice; CN, Fgfr2cn mice.

View larger version (124K): [in this window] [in a new window]   Fig. 7. Fgfr2cn prostates responded only weakly to androgen replenishment. (A) Gross tissue appearance of dorsolateral prostates from Fgfr2cn and control mice 2 weeks after castration or uncastrated mice at the same age. (B) HE staining of dlp dissected from the mice 2 weeks after castration (0 day) and at the indicated days after the androgen replenishment. (C) Sections were immunostained with anti-PCNA antibodies to reveal the proliferating cells. Inserts: high-magnification views of the same tissue. (D) The mean percentage of PCNA-positive cells in regenerating prostates was calculated from three samples. Data represents means of triplicate samples. CN, Fgfr2cn mice; F/F, Fgfr2flox homozygous mice.

View larger version (73K): [in this window] [in a new window]   Fig. 8. Prostate development in Fgfr2cn and control mice was partially inhibited by neonatal castration. Left panels: gross tissue appearance of prostates from Fgfr2cn and control mice at the indicated ages with or without neonatal castration. Right panels: the same tissues were micro-dissected to reveal detailed ductal structures. CN, Fgfr2cn mice; F/F, Fgfr2flox homozygous mice. Scale bars: 1 mm.

View larger version (51K): [in this window] [in a new window]   Fig. 9. Production of secretory proteins in Fgfr2cn prostates remained androgen dependent. (A) Profiles of secretory proteins. The PBS-extracted proteins from the dlp of 2-month-old mice were separated on 5-20% gradient SDS-PAGE and stained with Coomassie Brilliant Blue G250. (B) Expression of probasin and PSP94 in Fgfr2cn prostates. The PBS-extracted proteins from castrated or uncastrated mice were separated on SDS-PAGE and were western blotted with anti-probasin and anti-PSP94 antibodies, as indicated. (C) Total RNA was extracted from adult Fgfr2cn and control prostates, and expression of probasin and PSP94 was determined by real-time RT-PCR. Expression levels were normalized to ß-actin loading controls. The expression of each gene in control prostates was set to 1. Data are mean±s.d. of three independent experiments. F/F, homozygous Fgfr2flox mice; CN, Fgfr2cn mice.