Antenatal Hydronephrosis and VUR in Robo2 Mice Figure 3. Natural history of antenatal hydronephrosis in Robo2 mosaic mice. (A) Upper figure panels: ultrasound noninvasive monitoring of hydronephrosis (asterisks) progression in a Robo2 mosaic mutant mouse from embryonic E18.5 to postnatal 6-week old (W6) showing no spontaneous resolution; white dot line representing the anteroposterior renal pelvic diameter. Lower figure panels: ultrasound noninvasive monitoring of a wild-type littermate control showing absence of hydronephrosis at any stages. The outline of the kidneys depicted in ultrasound images have been marked by red dot line. P1: postnatal day-1; W1 to W6: week-1 to week-6; b: bladder; k: kidney. (B) Survival curve of Robo2 mice with antenatal hydronephrosis (red line) compared with the survival curve of Robo2 mice without antenatal hydronephrosis (blue line). (C) Small body size of a Robo2 mosaic mouse with hydronephrosis at one week compared with its age matched wild-type littermate. (D) H&E histology of nephropathy and renal scar in a Robo2 mosaic mouse showing atrophic tubules, substantial inflammatory cellular infiltrate, degeneration of remnant glomeruli and proteinaceous casts (asterisk); there was a reflux nephropathy characteristic abrupt transition from scar tissue to relatively normal parenchyma (arrow in D). (E) The severity of hydronephrosis measured by the anteroposterior renal pelvic diameter at different stages from prenatal E18.5 to postnatal week-6 showing continuous progression of hydronephrosis in Robo2 mice without spontaneous resolution. doi:10.1371/journal.pone.0024763.g003 Fetal origin of antenatal hydronephrosis and VUR phenotype in Robo2 mutant mice To determine if an enlarged ureterovesical junction was the primary congenital defect at an early developmental stage in Robo2 mutant embryos and later caused reflux and hydronephrosis, we examined the structure of the ureterovesical junction and entire upper urinary tract by micro-ultrasonography in 30 Robo2 newborn mice with antenatal hydronephrosis (Video S7 and Video S8). Interestingly, 25 (83%) of 30 Robo2 newborn mutant mice with hydronephrosis displayed wide open golf-hole like ureterovesical junctions (Fig. 6A, Video S7 and Video S8) and were confirmed by histology (Fig. 6B). The ureterovesical junction structure was barely detectable by ultrasound in a cohort of 30 (30/30, 100%) control newborn mice without hydronephrosis (Fig. 6C,D). To determine if Robo2 and its ligand Slit2 have intrinsic roles and are expressed in the ureterovesical junction region during development, we performed in situ hybridization and found both Robo2 and Slit2 mRNA in the distal ureter inside the bladder wall of developing mouse embryos (Fig. 6E and Figure S7). These results suggest that Robo2 is required for normal ureterovesical junction formation and that loss of Robo2 can lead to early congenital ureterovesical junction dilatation, which disrupts the anti-reflux mechanism in the bladder trigone. To determine if ureterovesical junction dilatation is an early developmental defect in Robo2-deficient mice, we utilized the expression of the Hoxb7-GFP transgene, an excellent marker of the ureteric bud and its derivatives from E10.5 on [32]. Since the Hoxb7-GFP transgene is allelic to Robo2flox or Robo2del5 allele, this precluded its use in Robo2del5/del5 homozygotes and Robo2del5/del5«Robo2del5/flox mosaic mice. However, we found three newborn Robo2del5/+ heterozygous mice with unilateral dilatation of ureterovesical junction (Fig. 6F,G), two of which had a dilated ureter on the same side (Fig. 6F). This unilateral ureterovesical junction dilatation was confirmed by histology (Fig. 6H) and was not found in 20 wild-type newborn controls (Fig. 6I,J). Since formation of the normal ureterovesical junction during early embryonic development is critically dependent on the primordial ureteric budding site from the Wolffian duct [33], we examined the ureteric bud outgrowth in early Robo2del5/del5 homozygous embryos. By pan-cytokeratin staining (another marker for ureteric bud and its derivatives) of five cultured kidney explants, we found supernumerary ureteric buds in all PLoS ONE | www.plosone.org 5 September 2011 | Volume 6 | Issue 9 | e24763