[18]. Therefore, we have studied a mouse model with a conditional Robo2 floxed allele, Robo2flox. The Robo2flox/flox homozygotes are viable, fertile and lack urinary tract abnormalities [4]. Using an EIIa-Cre transgene that has variable Cre activity in the early pre-implantation embryo [20], we generated a Robo2del5/del5« Robo2del5/flox mosaic mouse line with Robo2 gene dosage below haploinsufficiency [4]. Most Robo2 mosaic mutant mice develop antenatal hydronephrosis and abnormally located ureterovesical junctions (UVJs). However, it is not clear if these abnormally located UVJs lead to urinary obstruction or reflux, both of which elevate the pressure in the upper urinary tract and are the common causes of antenatal hydronephrosis. Although many die in the postnatal period due to severe bilateral CAKUT, the variable deletion of the floxed Robo2 allele in these mosaic mice allows some of them to survive into adulthood because the disease affects only one side [4]. This model has enabled us to answer several questions regarding the natural history, causal relationship and developmental origins of antenatal hydronephrosis and VUR, and to distinguish experimentally between urinary obstruction and reflux as a cause for antenatal hydrone- phrosis in Robo2-deficient mice. In the study reported here, we used noninvasive micro- ultrasonography and pathological analysis to determine the natural history of antenatal hydronephrosis in individual Robo2- deficient mice from embryo to adulthood. With the use of a microbubble ultrasound contrast agent and dye tracing, we observed that Robo2-deficient mice developed high-grade dilating VUR accompanied by wide-open incompetent ureterovesical junctions without obstruction. By ultrasound-guided percutaneous aspiration, we determined that unilateral hydronephrosis in Robo2- deficient mice was exacerbated by the retrograde flow of urine from the contralateral functional kidney through an enlarged ureterovesical junction. Further developmental studies revealed early congenital dilatation of the ureteral orifice in Robo2-deficient mice with antenatal hydronephrosis and VUR. Our data provide strong evidence that Robo2 is critical for maintaining both active and passive anti-reflux mechanisms and loss of Robo2 gene could cause progressive congenital hydronephrosis and high-grade dilating VUR with little chance of spontaneous resolution after birth. This study also establishes that complex renal tract birth defects like antenatal hydronephrosis and high-grade VUR can be effectively studied noninvasively in vivo in mice. Results Noninvasive evaluation of perinatal hydronephrosis in Robo2 mutant mice In order to study the natural history of antenatal hydronephrosis in Robo2-deficient mice, we first established a noninvasive method to evaluate renal collecting system in mouse embryos. The human metanephric kidney begins to form at 4 weeks of gestation and is functional at approximately 11 gestational weeks, which corre- sponds to embryonic stages E15.5–E16.5 in mice. Using the transvaginal route [21] or Doppler imaging [22], a detailed evaluation of human embryonic kidney and renal pelvis by ultrasound is possible as early as 12 weeks [23]. To determine if ultrasonography can visualize mouse embryonic kidney at a comparable gestational age, we used a Visualsonic Vevo 770 high- frequency micro-ultrasound system to examine timed pregnant female mice (Fig. 1A and Figure S1A). The mouse fetal kidney could be detected by transabdominal ultrasonography as early as E15.5 when it started to produce urine (Figure S1), however the pelvis and ureters of normal wild-type and Robo2 mutant embryos were indistinguishable at this stage (Figure S1B and S1C). Antenatal Hydronephrosis and VUR in Robo2 Mice With rapid maturation of mouse embryonic kidney and urinary tract from E16.5 to birth, bilateral and unilateral antenatal hydronephrosis and duplex kidneys in Robo2 mutant embryos could be diagnosed starting at E17.5–18.5 (Fig. 1B–J). We analyzed four E18.5 pregnant mice from Robo2 heterozygous matings (Figure S2). Twenty-seven embryos were detected using Vevo 770 micro-ultrasound. Among these 27 embryos, 13 were diagnosed with antenatal hydronephrosis based on renal pelvic dilatation (Fig. 1D,F). After dissection, we recovered 29 E18.5 embryos and confirmed the presence of hydronephrosis in 13 embryos by histology (Fig. 1C,E,J). There were two false positives and two false negatives for the diagnosis of antenatal hydrone- phrosis with this micro-ultrasound method, which corresponds to 84.6% sensitivity and 87.5% specificity (Table S1). We then analyzed newborn mice from six Robo2 heterozygous matings when matched individual dissection and histological examination became possible. All 13 Robo2del5/del5 homozygous newborn mice had hydronephrosis on micro-ultrasonography, which was confirmed by histology. Of 12 Robo2del5/del5« Robo2del5/flox mosaic newborn mice, eight (67%) had hydrone- phrosis detected by micro-ultrasonography and confirmed histo- logically (Fig. 2). Thus, micro-ultrasonography is 100% sensitive and 100% specific for detecting hydronephrosis in neonatal mice (Table S2). Interestingly, the majority (5/8, 63%) of Robo2del5/del5«Robo2del5/flox mosaic newborn mice with hydrone- phrosis displayed similar severity of hydronephrosis and cystic renal dysplasia phenotype as Robo2del5/del5 homozygotes (Fig. 2F– H). This finding suggests that Robo2del5/del5«Robo2del5/flox mosaic mice can develop severe Robo2-null like hydronephrosis defects. Since Robo2del5/del5«Robo2del5/flox mosaic mice have variable ratios of the Robo2flox and the Robo2del5 alleles due to the incomplete, stochastic action of the EIIa-Cre transgene on the Robo2flox alleles in the early embryo before implantation [4,20], some mosaic mice also survive to adulthood with relatively mild hydronephrosis [4]. Therefore, the mosaic Robo2 genotype alone cannot predict the severity of hydronephrosis, and variable Robo2 gene dosage may be responsible for the broad-spectrum and variable severity of the CAKUT phenotype in Robo2del5/del5 «Robo2del5/flox mosaic mice. Of note, a similarly broad-spectrum and variable severity of urinary tract abnormalities has also been found in human CAKUT patients [24,25]. Thus, these studies establish that micro-ultrasonography is a sensitive and reliable method for noninvasive in vivo investigation of complex renal and urological birth defects like CAKUT in embryonic and newborn mice. Antenatal hydronephrosis in Robo2 knockout mice progresses continuously without spontaneous resolution after birth Compared with the prenatal diagnosis of hydronephrosis in humans, a challenge in mouse fetal ultrasound examination is the large number of mouse embryos in each pregnancy with an average litter size of eight fetuses. To study the natural history of antenatal hydronephrosis, it is important to identify individual embryos with hydronephrosis prenatally and follow its disease progression after birth. Although it is difficult to distinguish individual normal embryos because their anatomies are similar, by monitoring hydronephrosis severity, symmetry and position of the urinary tract, we have successfully identified and followed E18.5 mutant embryos from eight pregnant mice of Robo2 heterozygous mice matings (Figure S2). Using Vevo 770 micro-ultrasound, we matched individual embryos and newborn mice with the same characteristic urinary tract structural abnormalities such as hydronephrosis, hydroureter and duplex kidney (Figure S3). This PLoS ONE | www.plosone.org 2 September 2011 | Volume 6 | Issue 9 | e24763