mechanistic insights into the role of Robo2 in the development of ureter smooth muscle cells and ureterovesical junction. We have applied a micro-ultrasound system for noninvasive and longitudinal investigations of antenatal hydronephrosis and VUR in a genetically engineered mouse model of a recently identified human primary VUR disorder (VUR2, OMIM #610878). With this noninvasive imaging modality, we were able to study the natural history and disease progression of complex CAKUT birth defects in the same individual mouse from embryonic stage to adulthood. Our results showed that loss of Robo2 gene could cause progressive congenital hydronephrosis and high-grade dilating VUR with little chance of spontaneous resolution after birth. Perinatal genetic analysis for ROBO2 mutations might serve as an alternative to medical surveillance and watchful waiting, and may be used in the future as an early diagnostic tool to identify a subset of affected fetuses and newborns with progressive hydronephrosis and VUR. With this genetic testing, early ureterovesical junction surgery such as ureteral reimplantation might be planned for newborns with ROBO2 mutations to minimize further renal injury caused by progressive reflux and preserve remaining renal function. In addition, our mouse model also informs us that we may uncover more deleterious mutations in ROBO2 gene if we focus on DNA analysis of patients with both progressive antenatal hydronephrosis and congenital high-grade dilating VUR. Finally, our study also provides a reproducible genetic mouse model of congenital progressive antenatal hydronephrosis and primary dilating VUR, which will improve our understanding of the pathophysiology and disease process of these challenging condi- tions so that we may treat patients more efficiently [51]. Materials and Methods Animals Ethics Statement. Mouse protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at Boston University Medical Center (#14388). Preparation of Robo2 mutant mice for analysis The generation and genotyping of Robo2flox conditional allele, Robo2del5 germline mutant allele, Robo2+ wild-type allele, and Robo2 homozygous, heterozygous and mosaic mice were described previously [4]. Briefly, the Robo2 heterozygous breeding scheme in Figure S2 was followed to generate F1 pregnant mice carrying embryos of Robo2del5/del5 homozygotes, Robo2del5/flox;TgElla-Cre+ (Robo2del5/del5«Robo2del5/flox) mosaics, Robo2del5/+ heterozygous, and Robo2flox/+ controls as well as newborn and adult mice in a mixed C57BL/6-129/Sv background. In Robo2del5/flox;TgElla-Cre+ mosaic mice, EIIa-Cre recombinase deletes only the Robo2flox allele to facilitate the penetrance of CAKUT phenotype when the Robo2 gene dosage is reduced below haploinsufficiency (the other allele, Robo2del5, has been deleted ubiquitously from germline expression). The Robo2 mosaicism originates from the incomplete, stochastic action of the EIIa-Cre transgene on the Robo2flox alleles in the early embryo before implantation [4,20]. This particular union of Robo2del5;TgElla-Cre and Robo2flox gametes results in Robo2del5/del5 «Robo2del5/flox mosaic mice. These mosaics are genotypically denoted Robo2del5/del5«Robo2del5/flox because they contain both Robo2del5/del5 and Robo2del5/flox cells (i.e. a mixture of homozygous and heterozygous cells). Cells carrying the Robo2del5/del5 genotype in these Robo2del5/del5«Robo2del5/flox mosaics derive from the action of EIIa-Cre in cells that commence embryogenesis with the Robo2del5/flox genotype. Robo2del5/flox cells remain when Ella-Cre activity in those cells is incomplete or insufficient to cause Cre-loxP mediated recombination. The authenticity of Robo2del5/flox; Antenatal Hydronephrosis and VUR in Robo2 Mice TgElla-Cre+ (Robo2del5/del5«Robo2del5/flox) mosaic mice was deter- mined by tail DNA genotyping for the presence of Robo2del5 and Robo2flox alleles as well as TgElla-Cre transgene. F1 timed pregnant female mice (after positive vaginal plug) were examined by micro- ultrasound for prenatal CAKUT phenotype in mouse embryos. F2 Robo2del5/del5 homozygous and Robo2del5/del5«Robo2del5/flox mo- saic mice were examined by micro-ultrasound for postnatal CAKUT phenotype. F2 littermates Robo2flox/+ mice without CAKUT phenotype were used as controls. To examine the ureterovesical junction defects in Robo2del5/+ newborn mice, Hoxb7-GFP transgenic mice (a gift from Dr. Frank Costantini, Columbia University) were bred with Robo2del5/+ mutants. GFP fluorescence was monitored and photographed using an Olympus SZX16 epifluorescence stereomicroscope. Micro-ultrasound analysis of prenatal CAKUT phenotype in mouse embryos The Vevo 770 micro-ultrasound imaging system from Visual- Sonics (www.visualsonics.com) was used for all noninvasive ultrasound imaging analyses. Vevo 770 (VisualSonics, Toronto, Canada; #VS-11392) is a high frequency (25–55 MHz) micro- ultrasound system specially designed to assess anatomical struc- tures and flow dynamics in small animals such as mice. It provides spatial resolution down to 30 microns and enables noninvasive, in vivo visualization of embryonic (E5.5) through to adult mice in real- time, and has been used successfully in detecting mouse embryos [52]. To examine antenatal CAKUT phenotype in Robo2 embryos, F1 timed pregnant Robo2del5/flox female mice (after positive vaginal plug with Robo2del5/+;TgElla-Cre+ male mice) were anesthetized using inhalant anesthesia of mixed oxygen and anesthetic gas isoflurane (2.5% for anesthesia induction and 1.5% for anesthesia maintenance) through a mouse nose mask tube on the VisualSonics Vevo Compact Anesthesia System (VisualSonics, #SA-11201). Pregnant mice were placed in a supine position on the mouse handling platform with controlled heating, ECG electrodes and temperature monitoring system (VisualSonics, #SA-11436). The mouse heart rate was maintained at 450–500 beats per minute and core body temperature was monitored with a rectal temperature probe coupled to a digital thermometer and was maintained at 37.0uC. The fur of abdominal area was removed using Depilatory cream (Nair, Church & Dwight Co.) and ultrasound transmission gel (Aquasonic 100, Parker Labora- tories, NJ) was used as a coupling agent between the skin and the ultrasound transducer. Real-time Micro Visualization (RMV) transducer RMV-708 scanhead (55 MHz center frequency with frequency band between 27.5 MHz to 82.5 MHz, 4.5 mm focal length and axial resolution of 30 mm) was used and mounted on a motorized Vevo integrated rail system III (VisualSonics, #SA- 11433) for hand-free serial imaging. Two-dimensional images of individual embryos were obtained in B-mode at a frequency of 30 MHz with an imaging thickness of 40 mm. Grayscale values of ultrasound imaging were calculated by Vevo 770 analytic software for defined areas as quantitative tissue echogenicities to identify and distinguish anatomical structures (e.g. renal pelvis, renal parenchyma, urinary bladder, stomach, spine, etc). The position of most embryos inside the pregnant female body is lateral decubitus. Because the in vivo embryo position could not be adjusted inside the uterus, we used easily visible fluid-filled embryonic stomach as a hallmark structure to identify the left side of the body. The enlarged echoless region of renal pelvis (i.e. hydronephrosis) or the ureter (i.e. ureterectasis) could be detected inside Robo2 mouse embryos by B-mode ultrasound imaging after E17.5 days. Duplex kidney was usually identified under B-mode ultrasound imaging as a combined structure with an echoless hydronephrotic upper pole PLoS ONE | www.plosone.org 9 September 2011 | Volume 6 | Issue 9 | e24763