Antenatal Hydronephrosis and VUR in Robo2 Mice Figure 6. Fetal origin of antenatal hydronephrosis and VUR phenotype in Robo2 mutant mice. (A) Noninvasive in vivo ultrasound detection of bilateral golf-hole like ureterovesical junction (UVJ) dilatation (arrows) in a newborn Robo2 mosaic mouse (also see Video S7); Scale bar, 1.0 mm; R-UVJ: right UVJ; L-UVJ: left UVJ; b: bladder. (B) H&E histology of the same newborn mice in (A) confirmed the bilateral golf-hole like UVJ dilatation (arrows); Magnification: 1006. (C) An un-dilated normal UVJ (arrow) was barely detectable under ultrasound scan in a wild-type control newborn mouse; Scale bar, 1.0 mm. (D) H&E histology of the same control newborn mice in (C) showing un-dilated right and left UVJs outlined by red dot line; Magnification: 1006. (E) In situ hybridization analysis showing Robo2 transcripts expressed around the developing UVJ: in the distal ureter (u, arrow) inside the bladder (b) wall of an E17.5 mouse embryo; Magnification: 506. (F–H) Unilateral UVJ dilatation in Robo2 heterozygous newborn mice: (F,G) Hoxb7-GFP reporter gene positive Robo2 newborn heterozygotes showing unilateral right UVJ dilatation (red dot line in panel F,G), associated with right hydroureter (hy-u in panel F) or undilated ureter (u in panel G); the left UVJ in Robo2 newborn heterozygotes (L-UVJ in panel F and G) appeared normal; Magnification: 256. (H) H&E histology confirmed right UVJ dilatation (arrow) in a Robo2 newborn heterozygote; Magnification: 1006. (I,J) Both right and left UVJ (arrows) of a wild-type newborn control appeared normal by Hoxb7-GFP (panel I) or H&E histology (panel J) analyses; Magnification: 256 for panel I, 1006 for panel J. (K) Early multiple ureteric buds (UB) outgrowth from the Wolffian duct (WD) in a Robo2del5/del5 homozygous embryo (E11.5 kidney cultured for 1-day and stained with pan-cytokeratin); magnification: 606. A wild-type embryo in (L) showing a single UB with one budding site (asterisk) from the Wolffian duct (WD); magnification: 606. (M,N) Anti-smooth muscle a actin antibody (SMAa, red) showing reduced and irregular ureteral smooth muscle staining (arrow in M) in an E14.5 Robo2del5/del5 homozygous embryo compared with its wild-type littermate control (arrow in N). The urothelium (stained by anti-E-cadherin antibody, green) appeared normal in both Robo2del5/del5 homozygous and wild-type controls; DAPI (blue) marked cell nuclei; Magnification: 4006. doi:10.1371/journal.pone.0024763.g006 [34]. Similarly, the muscular wall of the distal ureter in VUR children is decreased and replaced with dysplastic and atrophic smooth muscle cells [38,39,48]. The inner layer of ureteral smooth muscle cells are derived from the early mesenchymal cells that surround the mesonephric duct [34,49]. Since Robo2 is specifically expressed in the early mesenchymal cells surrounding the mesonephric duct before ureteric bud outgrowth [18], it is not unlikely that loss of Robo2 in early mesenchymal progenitor cells may lead to abnormal ureteral smooth muscle cell development, ureterovesical junction dilata- tion, trigonal weakness and VUR [50]. Because an early ectopic ureteric bud could not acquire adequate surrounding mesenchy- mal tissue to form proper trigonal musculature and ureterovesical junction, it is more prone to become a refluxing ureter later. Thus, multiple ureteric buds in Robo2 mutant embryos could also cause weak ureteral musculature and gaping ureteral orifices with incompetent anti-reflux mechanism [33,50]. Future studies are required to test these hypotheses and provide additional PLoS ONE | www.plosone.org 8 September 2011 | Volume 6 | Issue 9 | e24763