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First published online April 13, 2007
doi: 10.1242/10.1242/dev.02830

Development 134, 1799-1807 (2007)
Published by The Company of Biologists 2007
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Abnormal development of urogenital organs in Dlgh1-deficient mice

Akiko Iizuka-Kogo1,*, Takefumi Ishidao2,*,{dagger}, Tetsu Akiyama2,{ddagger} and Takao Senda1,§

1 Department of Anatomy I, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan.
2 Laboratory of Molecular and Genetic Information, Institute for Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan.


Figure 1
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  		Fig. 1. Targeted disruption of the mouse Dlgh1 gene. (A) The design of the Dlgh1 targeting construct and the structure of the mutant allele. The neo gene is inserted into a ClaI site, and is flanked by 0.85 kb and 8.5 kb homologous sequences at the 5' and 3' sides (thick lines), respectively. The thin lines indicate sequences derived from the pBluescript vector. The locations of the DNA probe used for Southern blot analysis and the PCR primers used for genotyping are indicated. Black boxes represent exons. Bam, BamHI; Cla, ClaI; Eco, EcoRI; Xho, XhoI. (B) Southern blot analysis of tail DNA from F3 progeny using EcoRI-digested genomic DNA. (C) PCR genotyping of Dlgh1-/- mice. Competitive PCR was performed on genomic tail DNA with three primers (see A): one common 3' primer (R), a 5' Dlgh1-specific primer (F) and a 5' neo-specific primer (N). DNA fragments of 503 bp and 273 bp are amplified from the wild-type and mutant alleles, respectively. (D) Western blot analysis of brain and kidney extracts from Dlgh1+/+, Dlgh1+/- and Dlgh1-/- mice at E15.5. Dlgh1 is not detected in the homozygous mutant mice, and the expression levels of G3PDH are similar among the genotypes.

 

Figure 2
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  		Fig. 2. Abnormal urogenital organs in prenatal Dlgh1-/- mice at E18.5. (A,D-F) Urinary organs of male Dlgh1+/+ (A), and male (D) or female (E,F) Dlgh1-/- mice. (D) Hypoplastic kidney. (E) Bilateral megaureter. (F) Unilateral duplicated ureter. The two ureters are marked with asterisks. (B,G) Hematoxylin and Eosin staining of kidney sections from Dlgh1+/+ (B) and Dlgh1-/- (G) mice showing hydronephrosis. (C,H) Lateral view of the vagina and urethra of female mice. A vaginal lumen can be seen on the hindside of the urethra in Dlgh1+/+ mice (C), whereas no vagina is formed in Dlgh1-/- mice (H). (I) Comparison of the longitudinal kidney length relative to the body length. (J) Comparison of the ureteric length (from the hilum of the kidney to the vesicoureteral junction) relative to the body length. The values in each column represent the mean±s.d. and the relative ratio to the mean value for Dlgh1+/+ mice. The red points in each graph show the average of each group. The red lines indicate the mean value for Dlgh1+/+ mice±2 s.d. P values are shown for significant differences between two groups. n, the number of kidneys used for the analyses. ad, adrenal gland; bl, urinary bladder; k, kidney; t, testis; u, urethra; ur, ureter; v, vagina. Scale bars: 1 mm in A,D-F,C,H; 200 µm in B,G.

 

Figure 3
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  		Fig. 3. Development of the embryonic metanephros and ureter in Dlgh1-/- mice. Immunofluorescence staining of urinary organs of Dlgh1+/+ (upper panels) and Dlgh1-/- (lower panels) mice with an anti-Pax2 antibody (A,D) or anti-Pax2 (green) and anti-pan-cytokeratin (red) antibodies (B,C). Note that the metanephric Pax2-positive area is contracted in Dlgh1-/- mice. The insets in B and C show staining with an anti-pan-cytokeratin antibody. The pairs of arrowheads delineate the region of the common nephric duct. Md, Müllerian duct; ur, ureteric bud; Wd, Wolffian duct.

 

Figure 4
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  		Fig. 4. Heterogeneous mesenchymal cells around the common nephric duct in Dlgh1-/- mice. Hematoxylin and Eosin-stained images of the ureter (A) and Wolffian duct (B) in Dlgh1+/+ mice and the common nephric duct in Dlgh1-/- mice (C) at E13.5. The mesenchymal tissue around the common nephric duct in Dlgh1-/- mice is heterogeneous in different regions. Dorsal is up. cnd, common nephric duct; ur, ureter; Wd, Wolffian duct. Scale bars: 50 µm.

 

Figure 5
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  		Fig. 5. Abnormal development of the Wolffian and Müllerian ducts. Whole-mount immunostaining of urogenital tracts from Dlgh1+/+ (A-F) and Dlgh1-/- (G-J) mice at E14.5 (A,B,G), E15.5 (C,D,H) and E16.5 (E,F,I,J) with an anti-Pax2 antibody. (A,C-J) Dorsal view of the urethra and bladder. (B) Diagonally oriented dorsal view of the basal part of the right ureter. The Müllerian ducts do not fuse laterally in Dlgh1-/- mice and the ureter and Wolffian duct are often connected to the common nephric duct, which is indicated by arrowheads (G,H). bl, urinary bladder; Md, Müllerian duct; sv, primordium for the seminal vesicle; ur, ureter; Wd, Wolffian duct. Scale bars: 200 µm.

 

Figure 6
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  		Fig. 6. Failure of Müllerian duct fusion in Dlgh1-/- mice at E18.5. Cross-sections at the vesicoureteral junction of Dlgh1+/+ (A) and Dlgh1-/- (B) female mice at E18.5. The right and left uteri are joined to form the cervical lumen in Dlgh1+/+ mice, but are separated in Dlgh1-/- mice. The uteri in Dlgh1-/- mice ended blindly in a more-caudal serial section. bl, urinary bladder; cx, cervix; ur, ureter; ut, uterus.

 

Figure 7
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  		Fig. 7. Expression patterns of Dlgh1 and Ecadherin in embryos. (A,B) Expression patterns of Dlgh1 (A) and Pax2 (B) in Dlgh1+/+ mice at E11.5. Dlgh1 is expressed not only in the Wolffian duct and ureter, which are Pax2-positive, but also in the epithelial cells of the hindgut, which are Pax2-negative. In addition, mesenchymal cells within the metanephric blastema are also weakly Dlgh1-positive. (C-J) Subcellular distributions of Dlgh1 (C,G), E-cadherin (D,H, red in E,F,I,J), occludin (green in E,I) and ZO-1 (green in F,J) in the ureteric epithelium of Dlgh1+/+ (C-F) and Dlgh1-/- (G-J) mice at E14.5 (C,D,G,H) and E15.5 (E,F,I,J). C,D and G,H represent each color of the same sections, respectively. In Dlgh1+/+ mice, Dlgh1 is accumulated at the basolateral region of the ureteric epithelium (C), whereas no significant Dlgh1-positive signals are detected in Dlgh1-/- mice (G). The subcellular distributions of E-cadherin, occludin and ZO-1 in Dlgh1-/- mice are similar to those in Dlgh1+/+ mice; staining of E-cadherin in the basement membrane of frozen sections (E,F,I,J) tended to be more intense than that in paraffin sections (C,D,G,H). hg, hindgut; mn, metanephros; ur, ureter; Wd, Wolffian duct. Scale bars: 100 µm in A,B; 10 µm in C-J.

 

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© The Company of Biologists Ltd 2007