Supporting Information Figure S1 Mouse fetal kidneys can be detected by transabdominal ultrasonography at E15.5. (PDF) Figure S2 Breeding scheme to generate Robo2del5/del5 homozygotes, Robo2del5/del5«Robo2del5/flox mosaic mu- tant mice, and Robo2flox/+ controls. (PDF) Figure S3 Antenatal congenital urinary tract defects in the same Robo2 mutant mice can be monitored and followed from prenatal to postnatal stages. (PDF) Figure S4 Spontaneous regression of hydronephrosis in a Robo2 mosaic mouse with duplex kidney. (PDF) Figure S5 Urethral catheter inside a mouse bladder can be detected by urosonography. (PDF) Figure S6 Microbubble ultrasound contrast agents (UCA) were washed out from the renal pelvis shortly after reflux in a Robo2 mosaic mouse. (PDF) Figure S7 Slit2 is expressed in the developing mouse UVJ region. (PDF) Figure S8 Ureteral smooth muscle defect in a Robo2 homozygous embryo at lower magnification. (PDF) Table S1 Sensitivity and specificity of antenatal hydro- nephrosis detection in E18.5 Robo2 mutant mouse embryos by micro-ultrasonography. (PDF) Table S2 Sensitivity and specificity of hydronephrosis detection in Robo2 mutant newborn mice by micro- ultrasonography. (PDF) Table S3 Survival of 29 Robo2 mutant mice with antenatal hydronephrosis (25 bilateral; 4 unilateral) in the first 6 weeks after birth. (PDF) Table S4 Largest anteroposterior renal pelvic diameter (mm) measured at different stages from prenatal E18.5 to postnatal week-6 in seven survived Robo2 mosaic mice with antenatal hydronephrosis (2 bilateral; 5 unilateral – total 9 kidney units). (PDF) Video S1 Infusion of microbubble ultrasound contrast agents into the bladder of a 6-week old Robo2 mouse through a urethra catheter in gray-scale. (WMV) References Antenatal Hydronephrosis and VUR in Robo2 Mice Video S2 Infusion of microbubble ultrasound contrast agents in green-scale into the bladder of a 6-week old Robo2 mouse under contrast imaging mode. (WMV) Video S3 Retrograde flow of microbubble ultrasound contrast agents in green-scale refluxing into dilated renal pelvis from the bladder of a Robo2 mosaic mouse under contrast imaging mode. (WMV) Video S4 Microbubble ultrasound contrast agents (UCA) washout from renal pelvis shortly after reflux in a Robo2 mosaic mouse under B-mode ultrasound in gray-scale. (WMV) Video S5 Ultrasound-guided percutaneous needle in- sertion into the renal pelvis of a Robo2 mosaic mouse with hydronephrosis. (WMV) Video S6 Ultrasound-guided percutaneous needle aspi- ration of urine from dilated renal pelvis of a Robo2 mosaic mouse with hydronephrosis. Noting the collapse of the renal pelvis after ultrasound-guided needle aspiration. (WMV) Video S7 Two-dimensional B-mode ultrasound video of the entire urinary tract structure in a Robo2 newborn mosaic mouse in the caudal-cephalic scanning direction (i.e. ultrasound imaging began with the bladder, then golf-hole like enlarged ureterovesical junction, moving upper to the dilated ureter, and finally showing the renal pelvis region with hydronephrosis). (WMV) Video S8 Three-dimensional reassembly of the entire urinary tract structure using 2D images captured in video S7 from the same Robo2 newborn mosaic mouse with golf-hole like enlarged ureterovesical junction, megaureter and hydronephrosis. (WMV) Acknowledgments We thank Victoria Herrera and Julius Decano and Boston University Medical Center Micro-ultrasound Imaging Core for technical assistance and support. Jizeng Qiao for help with early mouse embryonic kidney organ culture. Frank Costantini for the Hoxb7-GFP transgenic mouse. Ramon Bonegio for help with survival curve analysis. Xueping Fan, Anna Pisarek-Horowitz and Hila Milo Rasooly for helpful discussion. These data were presented in part as an abstract at the annual meeting of the American Society of Nephrology, Renal Week 2009. Author Contributions Conceived and designed the experiments: HW WL. Performed the experiments: HW QL JL CM WL. Analyzed the data: HW QL JL CM DJS WL. Contributed reagents/materials/analysis tools: CM DJS WL. Wrote the paper: HW DJS WL. 1. Pope JCt, Brock JW, 3rd, Adams MC, Stephens FD, Ichikawa I (1999) How they begin and how they end: classic and new theories for the development and deterioration of congenital anomalies of the kidney and urinary tract, CAKUT. J Am Soc Nephrol 10: 2018–2028. 2. Sanyanusin P, McNoe LA, Sullivan MJ, Weaver RG, Eccles MR (1995) Mutation of PAX2 in two siblings with renal-coloboma syndrome. Hum Mol Genet 4: 2183–2184. 3. Ulinski T, Lescure S, Beaufils S, Guigonis V, Decramer S, et al. (2006) Renal phenotypes related to hepatocyte nuclear factor-1beta (TCF2) mutations in a pediatric cohort. J Am Soc Nephrol 17: 497–503. 4. Lu W, van Eerde AM, Fan X, Quintero-Rivera F, Kulkarni S, et al. (2007) Disruption of ROBO2 is associated with urinary tract anomalies and confers risk of vesicoureteral reflux. Am J Hum Genet 80: 616–632. PLoS ONE | www.plosone.org 12 September 2011 | Volume 6 | Issue 9 | e24763