Figure 1.
Upk3b expression in embryonic development.
In situ hybridization analysis of Upk3b expression in whole wildtype embryos (A, B), on sagittal embryo sections (C–J and M–O) and on transverse embryo sections (K, L). (A–E) Overview images of embryos; anterior is up, dorsal is to the right. (F–O) Higher magnification images of the regions marked by open rectangles (in A–E). Stages are as indicated. Arrows point to the epicardium. a, atrium; at, epicardial covering of the dorsal wall of the great arterial trunks; bl, urinary bladder; bw, body wall; da, dorsal aorta; fl, fore limb bud; g, gut; h, heart; ki, kidney; li, liver; lu, lung; nd, nephric duct; nt, neural tube; ur, urogenital ridge; pe, pericardium of the dorsal wall of the pericardial coelom; peo, proepicardium; ppm, pleuropericardial membrane; v, ventricle.
Figure 2.
Upk3b expression in adult tissues.
In situ hybridization analysis of Upk3b expression on sections of the adult heart (A, D), the urinary bladder (B, E) and the kidney (C, F). (A–C) Overview images of whole organ sections, (D–F) higher magnification images of the regions marked by open rectangles (in A–C). The arrow points to the epicardium. bl, urinary bladder; cv, coronary vessel; la, left atrium; lu, urinary bladder lumen; lv, left ventricle; m, urinary bladder mesothelium; p, renal pelvis; pa, renal papilla; ra, right atrium; rv, right ventricle; sm, smooth muscle layer, u, urothelium.
Figure 3.
Generation and confirmation of a creERT2 knock-in allele of Upk3b.
(A) Scheme of the targeted insertion of a creERT2 recombinase gene/loxP-flanked neomycin selection cassette in the Upk3b locus. Exons are shown in black, regions for homologous recombination in red. Screening for clones with correct integration of the creERT2/neo cassette was performed using a PCR for the 3′-region, primers are indicated in pink. (B) A BamHI restriction fragment length polymorphism (RFLP) with the indicated 5′-probe was used to check for correct 5′-integration, wildtype (wt) and mutant (mut) bands are shown on the Southern blot. (C) A long-range PCR was used to verify the correct 3′-integration of the targeting vector by detection of a 3.5 kbp fragment in the mutant allele. ATG, transcriptional start codon; B, BamHI; creERT2, cre recombinase fused to a triple mutant form of the human estrogen receptor expression cassette; kbp, kilo base pairs; loxP, locus of X-over P1; Neo, neomycin resistance gene; pA, polyadenylation signal; pgk, phosphoglycerate kinase I promoter.
Figure 4.
Upk3bcreERT2/+ mice neither express cre nor mediate recombination of loxP-flanked sequences in the ureter and in the epicardium.
Pregnant mothers were injected with 2 mg tamoxifen at E9.5 and Upk3bcreERT2/+;Rosa26mTmG/+ embryos were analyzed at E15.5 by in situ hybridization for expression of Upk3b and cre on transverse sections of the ureter (A, B) and the heart (D, E). Immunofluorescence analysis of the lineage marker GFP was performed on transverse sections of the ureter (C) and the heart (F). Arrows (in A) point to the urothelium, arrows (in D) point to the epicardium. lv, left ventricle; ppm, pleuropericardial membrane; rv, right ventricle; u, ureter. Nuclei are counter-stained with 4′,6-diamidino-2-phenylindole.
Figure 5.
Upk3bcreERT2/creERT2 mice are Upk3b null mutants.
In situ hybridization analysis for expression of Upk3b and cre on transverse sections of an E18.5 heart (A, C, E) and ureter (B, D, F) in wildtype and homozygous knock-out embryos. (G) Semiquantitative RT-PCR analysis of E12.5 isolated hearts derived from wildtype, heterozygous and knock-out (Upk3bcreERT2/creERT2) animals. lu, lung; ppm, pleuropericardial membrane; ra, right atrium; rv, right ventricle; u, urothelium.
Figure 6.
Upk3b is dispensable for normal heart formation.
Hematoxylin and eosin staining (H&E) and immunofluorescence analysis of markers of capillary endothelia (IB4), cardiomyocytes (MF20), fibroblasts (Postn) and coronary smooth muscle cells (Tagln) on sections of 6-month old hearts of wildtype and homozygous knock-out mice. Magnified areas are indicated with rectangles. Arrows point to the epicardium. cv, coronary vessel; lv, left ventricle; rv, right ventricle. Nuclei are counter-stained with 4′,6-diamidino-2-phenylindole.
Figure 7.
Upk3b is dispensable for normal urinary bladder formation.
(A) Hematoxylin and eosin staining (H&E) and immunofluorescence analysis for markers of the urothelium (Upk1b), vessel endothelium (Emcn), fibroblasts (Postn) and smooth muscle cells (Tagln) on sections of the bladder of 6-month old wildtype and homozygous knock-out animals. (B) Scanning electron micrograph of the urothelial apical surface shows large polygonal superficial cells covered with microridges (arrows) in wildtype and homozygous knock-out animals. Magnified areas are indicated with rectangles. Bars represent 5-µm and 2-µm, respectively. d, detrusor; u, urothelium. Nuclei are counter-stained with 4′,6-diamidino-2-phenylindole.
Figure 8.
Upk3b is dispensable for normal kidney and ureter formation.
(A) Hematoxylin and eosin staining (H&E) and immunofluorescence analysis for markers of the urothelium (Upk1b), collecting ducts (Aqp2) and proximal tubules (LTA) on sagittal sections of the kidney of 12-month old wildtype and homozygous knock-out animals. (B) Hematoxylin and eosin staining (H&E) and immunofluorescence analysis for markers of the urothelium (Upk1b) and smooth muscle cells (Acta2) and transmission electron micrographs on sections of the ureter of 12-month old wildtype and homozygous knock-out animals. TEM of mouse ureter umbrella cells showing fusiform cytoplasmic vesicles (asterisk) and apical plaques (arrowheads). Magnified areas are indicated with rectangles. Bars represent 1 µm and 100 nm, respectively. c, renal cortex; md, renal medulla; pa, renal papilla. Nuclei are counter-stained with 4′,6-diamidino-2-phenylindole.
Figure 9.
Upk3a expression in embryonic development and in adult tissues.
In situ hybridization analysis of Upk3a expression in whole wildtype embryos (A, B), on sagittal embryo sections (C, D and G, H), on transverse embryo sections (E, F) and on sections of the adult heart (I, L), the urinary bladder (J, M) and the kidney (K, N). (A–D) Overview images of embryos; anterior is up, dorsal is to the right. (I–K) Overview images of whole organ sections; (E–H and L–N) higher magnification images of the regions marked by open rectangles (in A–D and I–K). Stages are as indicated. Arrows point to the epicardium. bl, urinary bladder; da, dorsal aorta; fl, fore limb bud; g, gut; h, heart; ki, kidney; la, left atrium; li, liver; lu, lung; lv, left ventricle; nd, nephric duct; nt, neural tube; p, renal pelvis; pa, renal papilla; ra, right atrium; rv, right ventricle; sm, smooth muscle layer; u, urothelium; ur, urogenital ridge; v, ventricle.