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doi: 10.1242/10.1242/dev.00432

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Hoxb13 is required for normal differentiation and secretory function of the ventral prostate

Kyriakos D. Economides* and Mario R. Capecchi{dagger}

Howard Hughes Medical Institute, Department of Human Genetics, University of Utah, Salt Lake City, Utah 84112, USA
* Present address: Center for Advanced Biotechnology and Medicine, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA



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  		Fig. 1. Gross morphology of mutant ventral prostates. Ventral prostate showing the opaque appearance of control ducts (A) and the transparent appearance of Hoxb13 homozygous mutant ducts (B) (yellow arrows). (C) X-gal staining of 5-week-old Hoxb13lacZ heterozygote reproductive organs show preferential expression of Hoxb13 within the ventral prostate; Hoxb13 continues to be strongly expressed in the ventral prostate of 1-year-old animals. (D-F) There is a dose-dependent X-gal staining intensity between wild-type (D), Hoxb13 heterozygote (E) and Hoxb13 homozygous mutant (F) ventral prostates (white arrowheads). Homozygous mutant prostates frequently exhibit swelling in individual ducts (E; red arrowhead). (G-I) Reductions in ventral prostate duct tips are observed in Hoxb13/Hoxd13 double homozygous mutants. Dark-field micrographs of ventral prostate microdissections of wild type (G), Hoxb13 homozygous mutants (H) and Hoxb13/Hoxd13 double homozygous mutants (I). Significant reductions in number of duct tips and outgrowth of branches are only seen in double Hoxb13/Hoxd13 mutants (averages and standard deviations for number of duct tips from 3 prostates for each genotype are shown below representative photo). (J-L) Postnatal X-gal staining of developing prostates showing Hoxb13 expression during the time of extensive ductile morphogenesis. At P10, X-gal staining is barely detectable (J), but is very strong by P12 in the ventral prostate (K). (L) During development, all prostatic lobes show strong X-gal staining (P16 shown). B, bladder; VP,ventral prostate; AP, anterior prostate; dlp, dorsolateral prostate; SV, seminal vesicle. Scale bars (A-I) 2 mm, (J-L) 1 mm.

 


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  		Fig. 2. Sections of Hoxb13 mutant ventral prostates. (A,B) Hematoxylin and Eosin stained sections, and (C,D) high magnification X-gal and eosin stained sections of heterozygous (A,C) and mutant (B,D) ventral prostates. There is a lack of secretory proteins within the ducts of the homozygous mutant ventral prostates (B) when compared to heterozygotes (A, black arrows). X-gal staining reveals strong expression of the Hoxb13lacZ reporter in the luminal epithelium (C,D) Note the tall columnar epithelium in heterozygotes compared with the simple cuboidal epithelium in homozygous mutants (compare C and D, white arrowheads). Scale bars: 25 µm (in B for A,B; in D, for C,D).

 


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  		Fig. 3. Hoxb13 is required for secretion of major ventral prostate proteins. The lack of ventral secretory protein expression is specific to Hoxb13 homozygous mutants. (A) SDS-PAGE of secretory proteins. Hoxb13 homozygous mutants do not express p25 a 25-40 kDa glycoprotein or p12 a 6 kDa serine protease inhibitor (lane 3). Hoxd13 mutants do not exhibit defects in their ventral prostatic secretory protein profile. In the absence of Hoxd13, one copy of Hoxb13 is sufficient for expression of both p12 and p25 (lane 4). (B) The Hoxb13 mutation appears to affect secretory function of only the ventral prostate. SDS-PAGE of secretory proteins from anterior prostates (AP) ventral prostates (VP). (C) Hoxb13 homozygous mutant ventral prostates do not produce secretory protein mRNA. Semi-quantitative RT-PCR using primers for the two major ventral secretory proteins p12 and p25, reveals absence of these mRNAs in ventral prostates from Hoxb13 homozygous mutants. Heart and liver RNAs are used as negative controls, GAPDH is an internal loading control. Molecular mass markers indicated by bars on the left of A and B are 188, 98, 62, 49, 38, 28, 17, 14, and 6 kDa.

 


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  		Fig. 4. Immunofluorescence of basal and epithelial markers. Hoxb13 homozygous mutant luminal cells display loss of polarity. (A,B) Hoxb13 expression and localization was determined by using an antibody to the ß-galactosidase reporter. (C,D) Androgen Receptor (AR) expression is intact in homozygous mutant epithelium. Basal cell marker CD44 is mis-expressed on apical surface of mutant epithelium (B,D yellow arrowheads). The tall-columnar morphology of heterozygous luminal cells (A,C) is evident when compared to the simple-cuboidal morphology of homozygous mutant luminal cells (B,D). St, stroma; Lu, lumen. Scale bar: 10 µm.

 


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  		Fig. 5. Nkx3.1 expression is not reduced in Hoxb13 homozygous mutants. (A) Western blots using an anti-Nkx3.1 antibody and an anti-ventral prostate protein antibody were performed on samples of protein extracts from ventral duct cells where secreted proteins were first removed. Nkx3.1 levels are not reduced in mutant ventral prostates, while antibodies to the ventral prostate proteins show complete absence of ventral prostate-specific proteins in the mutant VP. (B) Western blots using an anti-Nkx3.1 antibody and an anti-ventral prostate protein antibody on total protein samples show that Seminal vesicles but not anterior prostates secrete p12. Furthermore, expression of p12 in the seminal vesicle is not affected in Hoxb13 homozygous mutants. (C) Coomassie Blue stained gel of total proteins shows that homozygous mutant ventral prostates share many common bands with control anterior prostates (black arrowheads), but also display some prominent ventral-specific bands (asterisks). SV, seminal vesicle; AP, anterior prostate; VP, ventral prostate, B, bladder.

 

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