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

First published online 3 January 2007
doi: 10.1242/dev.02741

This Article Summary Full Text Full Text (PDF) 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 Kuslak, S. L. Articles by Marker, P. C. Search for Related Content PubMed PubMed Citation Articles by Kuslak, S. L. Articles by Marker, P. C.

The mouse seminal vesicle shape mutation is allelic with Fgfr2

Department of Genetics, Cell Biology and Development, University of Minnesota Comprehensive Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA.

View larger version (30K): [in this window] [in a new window]   Fig. 1. Identification of a candidate svs mutation in a non-recombinant interval. (A) Block diagram showing the number of each chromosome type isolated in crosses between svs M. mus musculus and wild-type M. mus castaneus mice. Meiotic chromosomes typed for phenotypic and molecular loci are depicted as columns of blocks. White and black blocks indicate the presence of the M. mus musculus allele (svs strain) or the M. mus castaneus allele, respectively. The number of chromosomes observed with each genotype is shown at the top of the column (from a total of 222 informative chromosomes analyzed). Mapped loci are indicated on the left. (B) Genetic and physical maps of the svs candidate interval. The svs mutation maps between D7Mit134 and D7Mit43 on mouse chromosome 7 (top). In the NCBI m34 mouse genome sequence assembly (Waterston et al., 2002), this genetic interval corresponds to a 410.3 kb physical interval. Annotation of the genome sequence reveals the presence of two known and six predicted genes in this candidate interval (bottom) (Birney et al., 2006).

View larger version (57K): [in this window] [in a new window]   Fig. 2. Identification of a candidate svs mutation. (A) Representative Southern blot of genomic DNA from svs, C57BL/6By (B6), and Balb/cBy (Balb) mice digested with BamHI and probed with a fragment from Fgfr2. Note the shift of a major band only in svs mutant mice (arrowhead). (B) PCR of svs, C57BL/6By (B6), and Balb/cBy (Balb) mouse genomic DNA using primers flanking the candidate mutation. The parental control strains Balb/cBy and C57BL/6By have a 399 bp band, whereas svs mutant mice have an 890 bp band. (C) Genomic structure of Fgfr2. The horizontal arrow indicates the direction of transcription; the vertical lines indicate exons; and the vertical arrow indicates the intron which harbors the svs insertion (top). Sequencing of PCR products from svs mutant mice revealed a 491 bp insertion flanked by intronic Fgfr2 sequences (bottom). Abbreviations: mChr7, mouse chromosome 7.

View larger version (33K): [in this window] [in a new window]   Fig. 3. FGFR2 protein levels and message localization are unchanged in svs mutant mice. (A) Western blot of FGFR2 expression using two antibodies that bind to different regions in the protein in ventral prostates (VP) and seminal vesicles (SV) from svs mutant or heterozygous mice. No difference in protein expression is seen. Top, anti-FGFR2 carboxy-terminus; middle, anti-FGFR2 extracellular domain; bottom, actin was used as a loading control. (B-D) In situ hybridization using an antisense probe to Fgfr2 shows positive staining only in the epithelium of P5 seminal vesicles from wild-type (B) or svs mutant (D) mice. No staining was observed using the sense control probe (C). Abbreviations: epi, epithelium; mes, mesenchyme.

View larger version (51K): [in this window] [in a new window]   Fig. 4. Dramatic alterations in alternative splicing of Fgfr2 in svs mutant mice. (A) FGFR2 is represented by a schematic of its protein domains with the corresponding exons numbered below each domain. The bar below the diagram indicates the location of the in situ hybridization probe used in Fig. 3. (B) The transcript structure of the 19 different isoforms of Fgfr2 identified in svs mutant and wild-type seminal vesicles, along with the number of times each transcript was found and the percentage of all isoforms identified in parentheses. The complete sequence of each transcript is available from GenBank with accession numbers EF143322-EF143340. Abbreviations: Ig, Immunoglobulin-like; AB, acid box; TMD, transmembrane domain; TK, tyrosine kinase domain; 8b/c, exon 8IIIb or 8IIIc.

View larger version (77K): [in this window] [in a new window]   Fig. 5. The svs mutation is allelic with Fgfr2. A genetic complementation test to determine if svs is an allele of Fgfr2. (A) P5 seminal vesicles from Fgfr2flox/svs mice initiated normal branching morphogenesis indicating that a functional Fgfr2 gene complements the svs mutation. Arrows indicate branched tips of the seminal vesicle. (B) Conversely, p5 seminal vesicles from Fgfr2/svs mice failed to initiate branching morphogenesis, indicating that a null allele of Fgfr2 fails to complement the svs mutation. (C) A p5 svs homozygous mutant seminal vesicle is shown for comparison. (D,E) Frozen sections of seminal vesicles from fully developed Fgfr2flox/svs and Fgfr2/svs adult mice were stained with Hematoxylin and Eosin. (D) Upper image depicts a cross-section of seminal vesicles from Fgfr2flox/svs mice with a complex branched structure. The bottom image shows the same seminal vesicle at increased magnification showing the presence of macroscopic clefts (arrowhead) that result from developmental branching morphogenesis. (E) Upper image depicts seminal vesicles from Fgfr2/svs mice that lack all branching. The bottom image shows the same seminal vesicle at increased magnification showing the lack of macroscopic clefts.

View larger version (39K): [in this window] [in a new window]   Fig. 6. Signal transduction through the MEK/ERK pathway is defective in svs mutant mice. (A) Western blots of P5 seminal vesicles from svs mutant and heterozygous control mice showing the loss of activated ERK1/2 in seminal vesicles from the svs mutant mice. (B) Western blot of seminal vesicles from P1 wild-type mice stimulated with recombinant FGF10 protein for 0, 20, 40 or 120 minutes, revealing a 2-fold activation of ERK1/2 by 20 minutes of stimulation, which recedes to near basal levels by 2 hours. Graph below shows quantification of the western blot results. (C) P1 wild-type seminal vesicles were cultured in serum-free medium (data not shown), serum-free medium plus testosterone (T), or serum-free medium with testosterone and UO126 (T+UO126). Testosterone stimulates lateral branching during a 4-day culture period (arrowhead). UO126 completely abrogates all testosterone-induced branching (bottom panels). (D) Western blot of cultured seminal vesicles confirms that testosterone does not stimulate activation of ERK1/2, and that UO126 inhibits ERK1/2 phosphorylation.

View larger version (20K): [in this window] [in a new window]   Fig. 7. Gene expression defects in svs mutant mice. (A-G) Real time RT-PCR comparison of mRNA levels in littermate control and svs homozygous mutant P5 seminal vesicles for genes implicated as branching morphogenesis regulators in the prostate and/or seminal vesicles. Bar graphs show the ratio of gene expression relative to 18S RNA expression; error bars show s.d. for three replicates of each measurement. Expression of Fgf10 was the same in wild-type and svs mutant mice (A). Expression of Shh (B), Gli1 (C), Gli2 (D), Ptch1 (E), Bmp4 (F) and Bmp7 (G) were reduced in svs mutant mice as compared with wild-type animals. Differences observed in B-G were statistically significant (ANOVA test with least significant difference post-hoc analysis; P value shown above each graph).