Antenatal Hydronephrosis and VUR in Robo2 Mice Figure 4. Hydronephrosis in Robo2 mosaic mice is caused by high-grade VUR. (A–D) Ultrasonographic images (green-scale under contrast- mode) depicted the urinary tract system and hydronephrosis (asterisk in C) in a Robo2 mosaic mouse (A–C) and the kidney pelvis region of a Robo2flox/+ control (D) before infusion of microbubble ultrasound contrast agent into the mouse bladder. Some background green signals were also seen in the green-scale setting. (E–G) Ultrasonographic images (green-scale under contrast-mode) after microbubble contrast agent infusion showing the flow of microbubble (strong and fresh green signals) inside the same urinary tract system of the Robo2 mosaic mouse after infusion of microbubble into the bladder. The green color enhanced microbubble was refluxed into the renal pelvis (asterisks in G; also see Video S3) through the ureter (F). Only background green signals were seen in the Robo2flox/+ control (H) without reflux. (I–K) Methylene blue cystogram was used to confirm reflux phenotype in an one week old Robo2 mosaic mouse (I,J) and a six weeks old Robo2 mosaic mutant mouse (K) showing retrograde flow of methylene blue into the renal pelvis (asterisks) through the ureter (white arrows in J,K). (L) A six weeks old wild-type control with methylene blue inside the bladder without reflux. Scale bars, 1.0 mm. Abbreviation: b, urinary bladder; k, kidney; u, ureter. doi:10.1371/journal.pone.0024763.g004 Robo2del5/del5 homozygous embryos (Fig. 6K), but not in wild-type controls (Fig. 6L). Because normal ureteral smooth muscle development is critical for ureterovesical junction formation and bladder trigone structure [34], we next examined the morphology of ureteral smooth muscle in early Robo2del5/del5 homozygous embryos before ureter dilatation by triple and dual immunohis- tochemistry staining. Using anti-smooth muscle a actin antibody (a ureter smooth muscle marker), E-cadherin (a marker for ureter urothelium) and lectin Dolichos Biflorus (DBA, another marker for ureter urothelium), we found normal looking urothelium but disarrangement and reduced abundance of ureteral smooth muscle in three E14.5 Robo2del5/del5 homozygous embryos (Fig. 6M,N and Figure S8) before any ureteral dilatation or hydronephrosis. These results suggest that Robo2 signaling may be necessary for the intercalation of ureteric smooth muscle into the bladder trigone to form a normal ureterovesical junction [34]. Discussion The ureter normally enters the bladder in an oblique direction, perforating the bladder muscle (detrusor) laterally and proceeding between the bladder mucosa and detrusor muscle to form the ureterovesical junction (UVJ) also called ureteral orifice. The intravesical portion of the ureter collapses during bladder voiding and creates a ‘‘passive’’ so-called flap-valve mechanism that prevents reflux [35,36]. However, recent studies show that the ratio of the intravesical ureter length to the ureter diameter is smaller than previously thought [37], and suggest that there is also an ‘‘active’’ ostial-valve anti-reflux mechanism controlled by the contraction of the muscle coat around the ureterovesical junction [38,39]. The muscle fiber of the ureterovesical junction functions as an ‘‘ureterovesical sphincter’’ that contracts in response to bladder contraction during voiding and subsequently relaxes PLoS ONE | www.plosone.org 6 September 2011 | Volume 6 | Issue 9 | e24763