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. Author manuscript; available in PMC: 2015 Jun 15.
Published in final edited form as: Dev Biol. 2014 Mar 31;390(2):160–169. doi: 10.1016/j.ydbio.2014.03.014

DLG1 influences distal ureter maturation via a non-epithelial cell autonomous mechanism involving reduced retinoic acid signaling, Ret expression, and apoptosis

Sung Tae Kim a, Sun-Young Ahn b,c, Wojciech Swat d, Jeffrey H Miner a
PMCID: PMC4038003  NIHMSID: NIHMS581572  PMID: 24699546

Summary

The absence of Discs-large 1 (DLG1), the mouse ortholog of the Drosophila discs-large tumor suppressor, results in congenital hydronephrosis characterized by urinary tract abnormalities, reduced ureteric bud branching, and delayed disconnection of the ureter from the common nephric duct (CND). To define the specific cellular requirements for Dlg1 expression during urogenital development, we used a floxed Dlg1 allele and Pax2-Cre, Pax3-Cre, Six2-Cre, and HoxB7-Cre transgenes to generate cell type-restricted Dlg1 mutants. In addition, we used RetGFP knockin and retinoic acid response element-lacZ transgenic mice to determine the effects of Dlg1 mutation on the respective morphogenetic signaling pathways. Mutation of Dlg1 in urothelium and collecting ducts (via HoxB7-Cre or Pax2-Cre) and in nephron precursors (via Pax2-Cre and Six2-Cre) resulted in no apparent abnormalities in ureteric bud branching or in distal ureter maturation, and no hydronephrosis. Mutation in nephrons, ureteric smooth muscle, and mesenchyme surrounding the lower urinary tract (via the Pax3-Cre transgene) resulted in congenital hydronephrosis accompanied by reduced branching, abnormal distal ureter maturation and insertion, and smooth muscle orientation defects, phenotypes very similar to those in Dlg1 null mice. Dlg1 null mice showed reduced Ret expression and apoptosis during ureter maturation and evidence of reduced retinoic acid signaling in the kidney. Taken together, these results suggest that Dlg1 expression in ureter and CND-associated mesenchymal cells is essential for ensuring distal ureter maturation by facilitating retinoic acid signaling, Ret expression, and apoptosis of the urothelium.

Keywords: Urogenital system, PDZ domain, hydronephrosis

Introduction

Discs-large homolog 1 (DLG1, also known as synapse-associated protein 97/SAP97), is a founding member of the membrane-associated guanylate kinase (MAGUK) protein family. MAGUKs are scaffolding proteins characterized by a core structure containing one or more postsynaptic density-95/discs-large/zonula occludens-1 (PDZ) domains, a Src homology 3 (SH3) domain, and a guanylate kinase (GK) domain (te Velthuis et al., 2007). DLG1’s functions include establishing apico-basal polarity in epithelial cells through its participation in forming the Scribble complex, clustering neurotransmitter receptors and ion channels at the post-synaptic membrane, and mediating cell-cell adhesion (Humbert et al., 2006; Kim and Sheng, 2004; Laprise et al., 2004).

Drosophila discs-large mutants exhibit loss of imaginal disc apicobasal cell polarity and neoplastic tissue overgrowth (Woods et al., 1996). In mammals, DLG1 and its paralogs (DLG2, DLG3, and DLG4) have been widely studied in neurons due to their roles in clustering neurotransmitter receptors (reviewed in Oliva et al., 2012; Zheng et al., 2011), but Dlg1 is also broadly expressed in the kidney and ureter (Ahn et al., 2013; Naim et al., 2005). Dlg1 mutant mice exhibit congenital hydronephrosis, reduced ureteric bud branching, and various urogenital abnormalities. In the ureter, these mice display an absence of the stromal cell layer, misorientation of smooth muscle cells, abnormal peristalsis, and delayed disconnection of the ureter from the common nephric duct (CND) (Iizuka-Kogo et al., 2013; Iizuka-Kogo et al., 2007; Mahoney et al., 2006; Naim et al., 2005).

Congenital anomalies of the kidney and urinary tract (CAKUT) are the most common cause of chronic kidney disease in children (Kerecuk et al., 2008). Normal urinary tract morphogenesis is highly dependent upon Wolffian duct (also called nephric or mesonephric duct) formation, ureteric bud induction, distal ureter maturation, and the ureter-bladder connection. In mice, the Wolffian duct begins to form from the intermediate mesoderm at E8.5. At E10.5, the ureteric bud arises from the caudal Wolffian duct and invades the metanephric mesenchyme, where it branches and induces nephrogenesis; it eventually forms the ureter and the collecting system of the kidney. At E11.5, the ureter is joined to the CND, which in turn is joined to the cloaca (the precursor of the bladder and urethra). The CND subsequently disappears by apoptosis at approximately E13, thereby allowing the ureter to both separate from the Wolffian duct and come into direct contact with the cloaca to form a connection that will become the ureteral orifice (Mendelsohn, 2009).

Distal ureter maturation is emerging as a crucial process in urogenital development. The CND is eliminated by apoptosis induced by vitamin A and Ret signaling, which is crucial for separation of the ureter and for transposition of the ureter orifice to the urogenital sinus (Batourina et al., 2002; Batourina et al., 2005; Uetani et al., 2009). A failure or delay in either elimination of the CND or distal ureter maturation can lead to vesicoureteral junction obstruction and hydronephrosis. A recent report shows that a Gata3-Raldh2-Ret molecular network plays a crucial role in normal CND insertion into the cloaca (Chia et al., 2011). However, there is still much to discover about the molecular mechanisms of distal ureter maturation. Here, we report our investigations into the cellular requirements for Dlg1 expression during urogenital development. Our results indicate a novel link between mesenchymal expression of Dlg1 and retinoic acid signaling and Ret expression in the epithelium.

MATERIALS AND METHODS

Genetically altered mice

All animal studies were approved by the Washington University Animal Studies Committee. For timed matings, the morning a vaginal plug was found was considered embryonic day (E) 0.5. The mutant alleles and transgenes used in these studies have been previously described, as follows: Dlg1− and Dlg1fl mice (Mahoney et al., 2006; Stephenson et al., 2007) generated by the Swat laboratory; Pax3-Cre transgenic (P3Pro-Cre) mice (Li et al., 2000), obtained from Dr. Jonathan Epstein (University of Pennsylvania, Philadelphia, PA, USA); Pax2-Cre transgenic mice (Ohyama and Groves, 2004), obtained from Dr. Andy Groves (Baylor College of Medicine, Houston, TX, USA); Hoxb7-Cre-EGFP transgenic mice (Zhao et al., 2004), obtained from Dr. Carlton Bates (University of Pittsburgh, Pittsburgh, PA, USA); Six2-EGFP-Cre transgenic mice (Kobayashi et al., 2008), obtained from Dr. Andy McMahon (University of Southern California, Los Angeles, CA, USA); Hoxb7-EGFP mice (Srinivas et al., 1999), obtained from Dr. Frank Costantini (Columbia University, New York City, NY, USA); Ret-EGFP knockin (Ret haploinsufficient) mice (Jain et al., 2006b), obtained from Dr. Sanjay Jain (Washington University, St. Louis, MO, USA); and RAREhsplacZ (RARElacZ) transgenic mice (Rossant et al., 1991), obtained from Dr. Janet Rossant (The Hospital for Sick Children, Toronto, ON, Canada). Mice were maintained on a primarily C57BL/6J-CBA/J mixed strain background. Littermates with at least one wild-type allele or with one or two floxed alleles but lacking Cre were used as controls.

Antibodies

The antibodies or reagents used were as follows: rabbit or mouse anti-GFP (Invitrogen, Carlsbad, CA, USA); rabbit anti-active Caspase-3 (Promega, Madison, WI, USA); rat anti-cytokeratin 8 (TROMA-1, Developmental Studies Hybridoma Bank, Iowa City, IA, USA); and Alexa Fluor 488- or 594-conjugated secondary antibodies (Molecular Probes, Eugene, OR, USA).

Immunofluorescence and whole mount immunofluorescence

Frozen sections (8 μm) were fixed in 4% paraformaldehyde in PBS for 10 minutes and washed three times with PBS. After blocking with 5% normal goat serum in 1% BSA-PBS for 1 hour, the sections were incubated with the primary antibody overnight at 4°C, washed three times for 10 minutes with PBS, and incubated with secondary antibody for 30 minutes. Images were viewed with a Nikon Eclipse E800 microscope (Nikon Instruments Corp., Melville, NY, USA) and captured with an Olympus DP2 digital camera using Olympus DP2-BSW software (Olympus America, Center Valley, PA, USA).

For whole-mount immunofluorescence, isolated tissues were fixed in 4% paraformaldehyde in PBS overnight at 4°C. After washing 4 times in PBS/0.3% Triton X-100 for 30 minutes and incubating with 5% normal goat serum overnight at 4°C for blocking, tissue was incubated with primary antibodies overnight at 4°C. After washing 4 times in PBS/0.3% Triton X-100 for 60 minutes each, the tissue was incubated with secondary antibodies overnight at 4°C, washed, and mounted with 90% glycerol. Images of stained tissue were captured using a Nikon fluorescence microscope or confocal microscope system and analyzed using NIS-Elements BR 3.2 software.

RNA extraction and quantitative real-time PCR

Total RNA was isolated using RNeasy Mini or Micro Kit (Qiagen, Chatsworth, CA, USA). Reverse transcription with oligo (dT) priming was performed using Superscript III (Invitrogen, Carlsbad, CA, USA). The relative expression of each transcript was determined by quantitative real-time PCR in the fast mode (annealing and extending at 60°C) with a 7900 HT Fast Real-Time PCR System (Applied Biosystems, Forrest City, CA, USA). Each well of the 96-well reaction plate contained a total volume of 20 μL with Fast Power SYBR Green PCR Master Mix (Applied Biosystems). The abundance of mRNA transcript was measured and normalized to glyceraldehyde 3-phosphate dehydrogenase (Gapdh). The primer sequences were: for Dlg1 (forward: gtg gat cat tca aag cag tgt ga, reverse: agg ctg gca atc tcc caa gt), Ret (forward: tgc ccc cag gac tgt ctc cg, reverse: tca cac agt ggg ccc tgg ct), Rara (forward: cgc caa ggg agc tga acg gg, reverse: ctg gga ctg agg ctg ggg ct), Rarb (forward: tca gcg cga aag gtg ccg aa, reverse: ggg aca cgc tgg gac tgt gc), Raldh2 (forward: aac ccg ctg agc aga cac cg, reverse: ttg ctg ccc ctg ctg ttg gc), Raldh3 (forward: aga cca tgg aca ccg gca agc, reverse: acc ccg atg ggc tca tgc ct), and Gapdh (forward: agg tcg gtg tga acg gat ttg, reverse: tgt aga cca tgt agt tga ggt ca).

Trypan blue solution injections

To reveal possible ureteric obstruction, 0.4% trypan blue solution (Sigma, St. Louis, MO, USA) was injected into the renal pelvis of embryonic kidneys using a pulled Pasteur glass pipette. Hydrostatic pressure was then applied to push the solution through the ureter towards the bladder.

Embryonic organ culture and time-lapse photography

The common nephric duct was dissected under sterile conditions from E11.5 embryos and grown at the air-medium interface on 0.4 μm pore size PET track-etched membranes (Falcon, Franklin Lakes, NJ, USA) in 6-well plates with Dulbecco’s modified Eagle’s medium (DMEM)/F12 with 15 mM Hepes (Gibco, Grand Island, NY, USA), 0.01 nM PGE1 (Prostaglandin E1, Sigma, St. Louis, MO, USA), 5 μg/ml ion-saturated transferrin (Sigma), 10 nM sodium selenate (Sigma), and 1% penicillin/streptomycin in a 37°C incubator for 7 days. A maximum of four explants were grown on each membrane. For time-lapse photography, we incubated the organs in a chamber attached to a Nikon Eclipse TE300 microscope in the Washington University Center for Kidney Disease Research Organogenesis Core. We acquired live images every 60 minutes. The medium was changed every 48 hours.

Statistical analysis

Two-tailed, unpaired Student’s t-tests were used to determine statistical difference. Differences were considered significant when the P value was < 0.05.

Results

Impaired descending movement of the ureter in Dlg1−/− embryos

To easily visualize and analyze the urogenital system in Dlg1 mutant embryos, we crossed in the Hoxb7-EGFP transgene (Srinivas et al., 1999), which is expressed in the Wolffian duct and ureteric bud epithelium and in their derivatives (Fig. 1). In our Dlg1−/− embryos, the common nephric duct (CND) was still retained at E14.5 or E15.5 (Fig. 1L-N; compare with control, J–K), as also found in the analysis of an independently generated Dlg1 knockout (Iizuka-Kogo et al., 2013; Iizuka-Kogo et al., 2007). At E11.5, the ureteric bud’s location on the Wolffian duct and the length of the CND were similar in controls and mutants (Fig. 1A–B). However, at E12.5 the CND’s length differed significantly between the two genotypes, being longer in the mutant (Fig. 1C–D). By E13.5, control ureters were already separated from the CND (Fig. 1E–G), whereas Dlg1−/− ureters were still present on the CND (Fig. 1H–I). At E14.5 and E15.5, the mutant CND was much shorter than at earlier stages (Fig. 1L–N; compare to H). This suggests that Dlg1−/− ureters have an impaired descending movement on the CND; this was confirmed by time-lapse video of CND organ cultures (Supplemental Fig. 1 and 2), which showed better separation of the ureter from the CND in the control as compared to the mutant.

Fig. 1. Impaired descending movement of the ureter from the common nephric duct (CND) to the cloaca.

Fig. 1

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The HoxB7-EGFP transgene was used to visualize the urogenital system. At E11.5, the length of the CND was similar between control (A) and Dlg1 KO (B). At E12.5, the Dlg1 KO CND (D) was longer than the control CND (C). By E13.5 control ureters were already separated from the CND (E, F, J, K), though at E13.5 some remnant CND cells expressing HoxB7-EGFP (arrow in G) had not yet been eliminated by apoptosis. Dlg1 KO ureters were still on the CND at E13.5 (H, I), E14.5 (L, M) and E15.5 (N). Dlg1 KO CNDs at E14.5 and 15.5 (L–N) were shorter than that at E12.5 (D) or E13.5 (H). When Dlg1 was mutated in epithelial cells by HoxB7-Cre, in nephron progenitors by Six2-Cre, or in both by Pax2-Cre, ureters were separated normally from the CND at E14.5 (O, P, Q), as in control (J). However, when Dlg1 was mutated in surrounding mesenchyme using the Pax3-Cre transgene, ureters were still on the CND (arrows in R, S), as in the Dlg1 KO (L–N). This suggests that Dlg1 expression in the mesenchyme is essential for proper distal ureter maturation. CND, common nephric duct; UB, ureteric bud; WD, Wolffian duct. Scale bars, 0.5 mm (E, H, J–S), 25 μm (F, G, I).

To identify the cells responsible for this defect in eliminating the CND, we used a floxed allele of Dlg1 (Dlg1fl) and different Cre transgenes to mutate Dlg1 in different cell populations associated with urogenital development. When DLG1 was deleted in nephron progenitors and/or Wolffian duct/ureteric bud epithelial cells using Six2Cre, HoxB7-Cre, or Pax2-Cre, ureters separated normally from the CND at E14.5 (Fig. 1O–Q), similar to controls. However, when DLG1 was deleted in the metanephric mesenchyme and in the mesenchyme surrounding the Wolffian duct, ureter, and cloaca using the Pax3-Cre transgene, ureters were still present on the CND (Fig. 1R–S), as also observed in the complete Dlg1 knockout (Fig. 1L–N). Together, these results suggest that Dlg1 expression in the surrounding mesenchyme is essential for elimination of the CND and for proper distal ureter maturation, whereas expression in the epithelium is dispensable.

Reduced Ret expression, retinoic acid signaling, and apoptosis in Dlg1−/− embryos

Ret is a receptor tyrosine kinase activated in the urinary tract by glial cell-derived neurotrophic factor (GDNF) and the co-receptor GFRα1 (reviewed in Jain, 2009). Ret signaling is involved in ureter formation and maturation, and its expression in the Wolffian duct and ureteric bud is governed in part by retinoic acid signaling (Batourina et al., 2005). We therefore used RetGFP knock-in mice (which are also Ret haploinsufficient) to explore the potential relationship between these signaling pathways and defects in elimination of the CND. In Dlg1−/−; RetGFP mice, Ret expression, as indicated by GFP fluorescence, was significantly reduced in the CND at E12.5 as compared to the WT CND (Fig. 2A).

Fig. 2. Reduced Ret expression and retinoic acid signaling in the Dlg1 KO.

Fig. 2

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(A) Dlg1 KO; RetGFP embryos indirectly show Ret expression in the CND (arrows) via GFP fluorescence intensity. Ret expression was reduced in the Dlg1 KO CND at E12.5 (right panels) compared to the WT CND (left panels). (B) Ret expression in the kidney was also analyzed by RetGFP fluorescence. Ret was highly expressed in the WT but reduced in the Dlg1 KO ureteric bud tips. Image capture conditions were rigorously controlled for in both A and B so that valid comparisons could be made. (C) Quantitative real-time RT-PCR revealed reductions in mRNAs for Dlg1, Ret, retinoic acid receptors (Rara, Rarb), and retinaldehyde dehydrogenases 2 and 3 (Raldh2 and Raldh3) in the Dlg1 KO kidney. *, P < 0.01 compared with wild type.

Given that ureteric bud branching was reported to be reduced in the Dlg1 mutant kidney (Naim et al., 2005), and RET signaling is critical for branching morphogenesis (Costantini, 2010), we also analyzed Ret expression within the kidney. By GFP fluorescence, Ret was highly expressed in the ureteric bud tips of the wild-type embryo, but it was reduced in the Dlg1 KO (Fig. 2B). Similarly, by real-time RT-PCR, the Dlg1 KO kidneys exhibited reduced levels of Ret mRNA, as well as reductions in mRNAs for retinoic acid receptors (Rara and Rarb) and enzymes involved in retinoic acid synthesis (Raldh2 and Raldh3) (Fig. 2C). To further explore the importance of these findings, we used mice carrying the retinoic acid response element (RARE)-lacZ transgene and Xgal staining of urogenital tissues to assay the level of retinoic acid signaling. In wild-type mice, RARE-lacZ activity was most robust in ureteric bud lineage cells (Fig. 3A–E). In the Dlg1 KO, lacZ activity was significantly decreased, and some cells in the ureter were negative (Fig. 3A′–E′). This suggests that retinoic acid signaling is reduced in the Dlg1 KO. This is likely mechanistically related to the observed reduction in Ret expression.

Fig. 3. Reduced retinoic acid response element (RARE) activity in Dlg1 KO CND and kidney.

Fig. 3

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Expression of the RARE-lacZ transgene (revealed by Xgal staining) was used to assay retinoic acid signaling in WT (A–E) and Dlg1 KO (A′-E′) tissues. LacZ was expressed in cells of the CND (A, A′) and ureteric bud lineage (B–E, B′-E′). In the Dlg1 KO, lacZ activity was significantly decreased, and some cells in the ureter were negative (A′-E′). Insets in B and B′ show high power views of ureteric bud tips. Scale bars, 100 μm (A,A′), 200 μm (B, B′, D, D′), 50 μm (C, C′, E, E′).

Apoptosis is one of the key processes for elimination of the CND and for distal ureter maturation (Batourina et al., 2005). We therefore assayed for apoptosis by whole mount staining for active caspase-3, a marker of apoptosis. By confocal microscopy, we found that apoptosis was significantly reduced in the Dlg1 KO CND at E11.5 to 14.5 (Fig. 4). Together, these results suggest that the absence of DLG1 leads to defects in retinoic acid signaling, Ret expression, and the induction of apoptosis that they normally promote in the CND.

Fig. 4. Decreased apoptosis in the Dlg1 KO CND.

Fig. 4

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Antibody staining for activated caspase-3 (red) was used to identify apoptotic cells. By confocal microscopy, apoptosis was reduced in the Dlg1 KO CND at E11.5 to 14.5 as compared to the control littermate. Insets show higher magnification views of the boxed structures. The HoxB7-EGFP transgene allowed visualization of the urothelium.

Congenital hydronephrosis in Dlg1flox/ko;Pax3-Cre mice

Because Dlg1 KO mice die shortly after birth, we analyzed floxed Dlg1 mice carrying different Cre genes such that DLG1 was absent only from specific cell types. With HoxB7-Cre/EGFP (which deletes in Wolffian duct and ureteric epithelia), the urogenital system, including the collecting ducts and papilla (Fig. 5A, B), was normal, and the mice were viable and fertile, indicating that DLG1 is not required in the epithelial cells. Similarly, with Pax2-Cre (which deletes in Wolffian duct and ureteric epithelia as well as in metanephric mesenchyme), the urogenital system, including the collecting ducts and papilla, was normal (Fig. 5C, D), but these mice died within one week after birth for unknown reasons; this lethality could be related to the expression of Pax2-Cre in the nervous system (reviewed in Oliva et al., 2012; Zheng et al., 2011), where DLG1 likely has important functions (reviewed in Zheng et al., 2011). With Six2-Cre/EGFP (which deletes only in the nephron progenitors of the metanephric mesenchyme), the urogenital system was normal, indicating that DLG1 is dispensable for the function of nephron epithelial cells, including podocytes, parietal epithelial cells, and tubular epithelial cells. However, when DLG1 was deleted by the Pax3-Cre transgene in ureteric mesenchyme, metanephric mesenchyme, and much of the remaining urogenital mesenchyme, congenital hydronephrosis occurred. At E18.5, the renal pelvis was dilated, and more severe hydronephrosis occurred within a month. Dlg1flox/ko;Pax3-Cre mice usually died at approximately 4–5 months of age due to the effects of hydronephrosis, including loss of renal parenchyma and consequent impaired kidney function (Fig. 5).

Fig. 5. Utilization of different Cre transgenic mice reveals congenital hydronephrosis in Dlg1flox/ko;Pax3-Cre mice.

Fig. 5

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Using different Cre transgenic mice, we generated cell type-restricted Dlg1 KOs. The collecting duct systems and papillae of Dlg1flox/ko;HoxB7-Cre and Dlg1flox/ko;Pax2-Cre mice appeared similar to controls (Fig. 5A–D). However, when Dlg1 was mutated in metanephric, ureteric, and Wolffian duct mesenchyme by Pax3-Cre (Fig. 5 E–J, bottom panels, except where noted as Control), hydronephrosis occurred. At E18.5, the renal pelvis was dilated, and severe hydronephrosis was apparent at various adult ages (P41, P108, and P420 are shown). Scale bars, 300 μm (A–E), 1.2 mm (J).

To investigate the potential causes of hydronephrosis in Dlg1 KO mice, we injected Trypan blue solution into the renal pelvis to determine whether it could reach the bladder. As shown in Fig. 6, the Dlg1 KO did not have a physical urinary tract obstruction, as the injected solution was detected in the ureter and bladder, similar to the control. This suggests that hydronephrosis in Dlg1 KO is not caused by a physical urinary obstruction, but due to failure of functional urinary propulsion from the kidney to the bladder.

Fig. 6. Dlg1 KO mice do not have a physical urinary tract obstruction.

Fig. 6

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To assay for a physical obstruction, a blue solution was injected into the renal pelvis. The solution successfully traveled down the ureter (yellow arrows) to the bladder in both control and Dlg1 KO mice at E18.5.

We previously reported a smooth muscle cell (SMC) orientation defect in the Dlg1 KO ureter associated with a defect in peristalsis (Mahoney et al., 2006). By electron microscopy, Dlg1flox/ko;Pax3-Cre mice exhibited a similar defect in ureteric SMC orientation, primarily in the proximal portion of the ureter (Fig. 7E). In cross sections of the normal ureter, the SMCs closest to the urothelium show a spindle shape, indicating the presence of circular smooth muscle (Fig. 7A, F). However, SMCs in Dlg1flox/ko;Pax3-Cre mice showed a more rounded shape (Fig. 7E), similar to the Dlg1 KO (Fig. 7D), indicating the presence of longitudinal smooth muscle. In contrast, the orientation of SMCs in Dlg1flox/ko;Pax2-Cre (Fig. 7B) and Dlg1flox/ko;HoxB7-Cre (Fig. 7C) mice was normal and similar to that of control. This suggests that Dlg1 expression in the ureteric mesenchyme (where the Pax3-Cre transgene but not Pax2-Cre or HoxB7-Cre is expressed) is essential for proper smooth muscle orientation.

Fig. 7. Smooth muscle orientation defects are present in Dlg1flox/ko; Pax3-Cre ureter.

Fig. 7

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By viewing cross sections of the ureter by electron microscopy, we found that the inner ring of normally circular smooth muscle cells (SMCs) showed the normal spindle shape in Dlg1flox/ko;Pax2-Cre (B) and in Dlg1flox/ko;HoxB7-Cre (C) mice, similar to controls (A, F). But note the rounded, less spindle shape of SMCs in Dlg1flox/ko;Pax3-Cre mice (E; arrow), which was similar to the Dlg1 KO (D; arrow). Ep, epithelial cells; S, stromal cells; SM, smooth muscle cells. Scale bars, 10 μm.

Abnormal distal ureter insertion in Dlg1 KO and Dlg1flox/ko;Pax3-Cre mice

Next, we analyzed ureter insertion in the conditional as well as constitutive mutants, again using expression of the HoxB7-EGFP transgene to facilitate the analysis. Control ureters were inserted normally into the bladder; however, Dlg1 KO ureters were inserted into the urethra, and at an abnormal angle (Fig. 8). This abnormal insertion was also found in Dlg1flox/ko;Pax3-Cre mice (Fig. 8). In serial sections, control ureters were connected normally to the bladder epithelium through the bladder wall (Supplementary Fig. 3); however, ureters of Dlg1 KO mice were abnormally connected to the urethra epithelium and to the seminal vesicle instead of to the bladder epithelium. In addition, the angle of insertion was abnormal (Supplementary Fig. 4). In adult male Dlg1flox/ko;Pax3-Cre mice the seminal vesicle lumen was connected to the bladder lumen (data not shown), which could explain the observed lack of male fertility.

Fig. 8. Abnormal distal ureter insertion into the bladder was observed in Dlg1 KO and Dlg1flox/ko; Pax3-Cre mice at E18.5.

Fig. 8

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The HoxB7-EGFP transgene was included to facilitate the analysis. (A, A′) Whole mount staining of anti-GFP (in green) and cytokeratin 8 (in red) was performed. In the control (A), ureters were inserted normally into the bladder; however, in the Dlg1 KO (A′), ureters were inserted into the urethra, and at an abnormal entry angle. Similarly, by direct EGFP fluorescence, abnormal insertion of the ureters was observed in Dlg1flox/ko;Pax3-Cre mice (B′-D′) compared to control (B–D), demonstrating the importance of Dlg1 expression in mesenchymal cells surrounding the lower urinary tract epithelial structures.

Discussion

We have provided novel insights into the role of Dlg1 in regulating the development of the lower urinary tract. Although there is much research focused on the functions of DLG1 in the nervous system, where it is commonly referred to as synapse-associated protein 97, DLG1 is also considered to be an important epithelial cell polarity protein because it is present in the Scribble complex, which is composed of Scribble, DLG1, and lethal giant larvae (Bryant and Mostov, 2008; Lee and Vasioukhin, 2008; Pieczynski and Margolis, 2011). This complex has been shown to localize to the basolateral aspect of epithelial cells and to promote basolateral membrane domain identity. However, our studies did not uncover an obligatory role for DLG1 in epithelial cells of the urogenital system, as selective mutation of Dlg1 either in precursors of epithelial cells of the nephron (with Six2-Cre) or in the ureter and collecting duct system (with HoxB7-Cre) did not have serious consequences, either for cell polarity or for kidney function. Moreover, mutation of Dlg1 in both the nephron and the ureteric bud derivatives (with Pax2-Cre) resulted in apparently normal kidneys and ureters, but the mutation was lethal shortly after birth for reasons that are likely related to the importance of DLG1 in the nervous system (Oliva et al., 2012; Zheng et al., 2011), where Pax2-Cre is also expressed during development (Ohyama and Groves, 2004).

In contrast to the apparent lack of a cell autonomous role for DLG1 in epithelial cells, mutating Dlg1 with the Pax3-Cre transgene in metanephric mesenchyme and in mesenchyme surrounding the ureteric bud, Wolffian duct, and cloaca resulted in defects in maturation of the distal ureter. These defects are very similar to those that we (Figs. 1 and 8) and others (Iizuka-Kogo et al., 2013; Iizuka-Kogo et al., 2007) observed in constitutive Dlg1−/− embryos. Taken together with the results discussed above, these data suggest that DLG1 has an important role in the mesenchyme surrounding the ureteric bud and/or Wolffian duct epithelia for promoting proper distal ureter maturation, independent of any cell autonomous role in the epithelial cells themselves. Similarly, the lack of DLG1 in metanephric mesenchyme could cause the renal hypoplasia observed in Dlg1−/− mice (Iizuka-Kogo et al., 2007; Mahoney et al., 2006; Naim et al., 2005) in an analogous non-cell autonomous fashion.

The notion that DLG1 could have a non-cell autonomous role is supported by studies of the Drosophila neuromuscular junction. In the total absence of discs-large, there are defects in both pre- and postsynaptic aspects of the larval neuromuscular junction. Targeted expression of discs-large solely to the presynaptic cell is sufficient to rescue defects in both the pre- and postsynaptic cells; this is suggestive of a cell autonomous role in the presynaptic cell in promoting proper neurotransmitter release and a non-cell autonomous role in the responding postsynaptic cell (Budnik et al., 1996).

Retinoic acid signaling is crucial for both upper and lower urinary tract development and maturation, in part via its activation of Ret gene expression (Batourina et al., 2002; Batourina et al., 2005). RET gene mutations in humans and mice have been associated with urinary tract defects that include renal hypoplasia, hydronephrosis, vesicoureteral reflux, bilateral megaureters, distal ureteral stricture, and ectopic ureter insertion (Jain et al., 2006a; Pini Prato et al., 2009). Indeed, similar to our Dlg1 KO phenotype, the ectopically terminating ureters in Rara/Rarb double mutants open into the urethra instead of the base of the urinary bladder (Mendelsohn et al., 1994). We therefore investigated the hypothesis that the absence of DLG1 affects the retinoic acid signaling pathway. We found a dampening of retinoic acid signaling in Dlg1−/− embryos using the RARE-lacZ reporter, which is expressed primarily in epithelial cells, suggesting that DLG1 has a cell autonomous role in regulating production and/or secretion of retinoic acid by the surrounding mesenchymal cells. In addition, the significant reductions in both Raldh2 and Raldh3 mRNA in Dlg1−/− kidney is consistent with this hypothesis, as these genes encode enzymes involved in retinoic acid synthesis. Similarly, retinoic acid produced by kidney stromal cells (which derive from a subset of the metanephric mesenchyme that expresses the Pax3-Cre transgene) has been shown to be important to induce retinoic acid signaling and Ret expression in the ureteric bud, thereby promoting ureteric bud branching (Rosselot et al., 2010). By extension, we speculate that retinoic acid production and/or secretion by Dlg1-expressing mesenchymal cells associated with the developing lower urinary tract promotes CND maturation via direct effects on retinoic acid receptor signaling in the constituent epithelial cells.

Other mouse models have been described with aberrant distal ureter maturation. Hains et al. described longer CNDs and cranially shifted ureteric buds in Fgfr2Mes−/ − mice (Hains et al., 2010). The ureters of these mutant mice had a low insertion into the bladder near the bladder neck compared to controls, which resulted in a high rate of vesicoureteral reflux postnatally (Hains et al., 2010). Similarly, the CND was found to be longer in Bmp4+/− mice, with the ureteral orifices observed to be abnormally close to the urethra at E16.5. In severe Bmp4+/− mutants, the ureter terminated in the seminal vesicle or the vas deferens (Miyazaki et al., 2000). In our Dlg1 KO mice, the CND was also found to be longer in the mutants compared to controls at E12.5, and there was impaired descending movement of the ureter on the CND. A unique feature, however, of the Dlg1 KO mice is that the ureters maintained a patent connection with the bladder, whereas in other mutants such as the Ret or Rar double mutants, the distal ureter would occasionally be stenotic or fail to join the lower urogenital tract (Jain et al., 2006a; Mendelsohn et al., 1994). This difference in phenotype may be due to a difference in the process of separation of the ureter from the CND observed in the Dlg1 KO mice.

In our previous studies of Dlg1−/− mice, we observed congenital hydronephrosis that we ascribed to defects in ureteric smooth muscle alignment that impaired the peristaltic movements that propel urine from the kidney to the bladder (Mahoney et al., 2006). Here we showed that these defects are cell autonomous, as mesenchymal but not epithelial mutation of Dlg1 caused a similar smooth muscle cell defect. But in addition, we found defects in distal ureter maturation that likely also contribute to hydronephrosis. These results show that DLG1 has dual roles in the lower urinary tract, making it a potentially important target for involvement in human CAKUT. Whether mutations that affect human DLG1 or associated proteins cause hydronephrosis or other urinary tract defects is currently under investigation.

Supplementary Material

01
Download video file (2.7MB, mov)
02
Download video file (2.6MB, mov)
03

Supplementary Fig. 1. Time-lapse fluorescence video of in vitro organ culture of control; HoxB7-EGFP embryonic CND. The CND and surrounding tissues were collected at E12.5 and cultured for 7 days. Note the almost complete separation of the ureter from the CND.

Supplementary Fig. 2. Time-lapse fluorescence video of in vitro organ culture of Dlg1 KO; HoxB7-EGFP embryonic CND. The CND and surrounding tissues were collected at E12.5 and cultured for 7 days. Note that the ureter did not separate from the CND; failure is most obvious in the lower ureter.

Supplementary Fig. 3. Serial sections of E18.5 male control; HoxB7-EGFP ureters and bladder. After whole mount staining with anti-GFP in green and cytokeratin 8 in red, serial sectioning was performed to visualize the insertion angles and connections of the ureters with the bladder. Ureters and seminal vesicles are yellow (GFP+, cytokeratin 8+); urethra and bladder epithelial cells are red (GFP−, cytokeratin 8+). The ureters are connected to the bladder epithelium through the bladder wall.

Supplementary Fig. 4. Serial sections of E18.5 male Dlg1 KO; HoxB7-EGFP ureters and bladder. After whole mount staining with anti-GFP in green and cytokeratin 8 in red, serial sectioning was performed to visualize the insertion angle and connections of the ureters with the bladder. Ureters and seminal vesicle are yellow (GFP+, cytokeratin 8+); urethra and bladder epithelial cells are red (GFP−, cytokeratin 8+). The ureters were not connected to the bladder epithelium, but instead were abnormally connected to the urethra epithelium and to the seminal vesicles. The seminal vesicle lumen was also observed to be connected to the bladder lumen (middle panel, row 2).

Highlights.

  • Dlg1flox/ko;Pax3-Cre mice demonstrate congenital hydronephrosis and reduced branching

  • Dlg1flox/ko;Pax3-Cre mice manifest distal ureter insertion and smooth muscle orientation defects

  • Dlg1 null mice show reduced Ret expression during ureter maturation

  • Dlg1 null mice showed reduced retinoic acid signaling in the kidney

  • Dlg1 in ureter and CND-associated mesenchymal cells is essential for distal ureter maturation

Acknowledgments

We thank Jeanette Cunningham for performing electron microscopy, Jennifer Richardson for genotyping the mice, the WU Mouse Genetics Core for care of mice, and Drs. Epstein, Groves, Bates, Costantini, McMahon, Jain, and Rossant for providing the genetically altered mice that made this study possible.

Funding

This work was funded by NIH grant R01DK081156 to JHM and WS, and in part by the Children’s Discovery Institute. Microscopy and Mouse Genetics Core services were supported in part by the WU Center for Kidney Disease Research (NIH P30DK079333) and by the WU Dept. of Otolaryngology, Research Center for Auditory and Visual Studies (NIH P30DC004665).

Footnotes

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References

  1. Ahn SY, Kim Y, Kim ST, Swat W, Miner JH. Scaffolding Proteins DLG1 and CASK Cooperate to Maintain the Nephron Progenitor Population during Kidney Development. J Am Soc Nephrol. 2013;24:1127–1138. doi: 10.1681/ASN.2012111074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Batourina E, Choi C, Paragas N, Bello N, Hensle T, Costantini FD, Schuchardt A, Bacallao RL, Mendelsohn CL. Distal ureter morphogenesis depends on epithelial cell remodeling mediated by vitamin A and Ret. Nat Genet. 2002;32:109–115. doi: 10.1038/ng952. [DOI] [PubMed] [Google Scholar]
  3. Batourina E, Tsai S, Lambert S, Sprenkle P, Viana R, Dutta S, Hensle T, Wang F, Niederreither K, McMahon AP, Carroll TJ, Mendelsohn CL. Apoptosis induced by vitamin A signaling is crucial for connecting the ureters to the bladder. Nat Genet. 2005;37:1082–1089. doi: 10.1038/ng1645. [DOI] [PubMed] [Google Scholar]
  4. Bryant DM, Mostov KE. From cells to organs: building polarized tissue. Nat Rev Mol Cell Biol. 2008;9:887–901. doi: 10.1038/nrm2523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Budnik V, Koh YH, Guan B, Hartmann B, Hough C, Woods D, Gorczyca M. Regulation of synapse structure and function by the Drosophila tumor suppressor gene dlg. Neuron. 1996;17:627–640. doi: 10.1016/s0896-6273(00)80196-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chia I, Grote D, Marcotte M, Batourina E, Mendelsohn C, Bouchard M. Nephric duct insertion is a crucial step in urinary tract maturation that is regulated by a Gata3-Raldh2-Ret molecular network in mice. Development. 2011;138:2089–2097. doi: 10.1242/dev.056838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Costantini F. GDNF/Ret signaling and renal branching morphogenesis: From mesenchymal signals to epithelial cell behaviors. Organogenesis. 2010;6:252–262. doi: 10.4161/org.6.4.12680. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hains DS, Sims-Lucas S, Carpenter A, Saha M, Murawski I, Kish K, Gupta I, McHugh K, Bates CM. High incidence of vesicoureteral reflux in mice with Fgfr2 deletion in kidney mesenchyma. The Journal of urology. 2010;183:2077–2084. doi: 10.1016/j.juro.2009.12.095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Humbert PO, Dow LE, Russell SM. The Scribble and Par complexes in polarity and migration: friends or foes? Trends Cell Biol. 2006;16:622–630. doi: 10.1016/j.tcb.2006.10.005. [DOI] [PubMed] [Google Scholar]
  10. Iizuka-Kogo A, Akiyama T, Senda T. Decreased apoptosis and persistence of the common nephric duct during the development of an aberrant vesicoureteral junction in Dlg1 gene-targeted mice. Anat Rec (Hoboken) 2013;296:1936–1942. doi: 10.1002/ar.22814. [DOI] [PubMed] [Google Scholar]
  11. Iizuka-Kogo A, Ishidao T, Akiyama T, Senda T. Abnormal development of urogenital organs in Dlgh1-deficient mice. Development. 2007;134:1799–1807. doi: 10.1242/dev.02830. [DOI] [PubMed] [Google Scholar]
  12. Jain S. The many faces of RET dysfunction in kidney. Organogenesis. 2009;5:177–190. doi: 10.4161/org.5.4.10048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jain S, Encinas M, Johnson EM, Jr, Milbrandt J. Critical and distinct roles for key RET tyrosine docking sites in renal development. Genes Dev. 2006a;20:321–333. doi: 10.1101/gad.1387206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jain S, Golden JP, Wozniak D, Pehek E, Johnson EM, Jr, Milbrandt J. RET is dispensable for maintenance of midbrain dopaminergic neurons in adult mice. J Neurosci. 2006b;26:11230–11238. doi: 10.1523/JNEUROSCI.1876-06.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kerecuk L, Schreuder MF, Woolf AS. Renal tract malformations: perspectives for nephrologists. Nat Clin Pract Nephrol. 2008;4:312–325. doi: 10.1038/ncpneph0807. [DOI] [PubMed] [Google Scholar]
  16. Kim E, Sheng M. PDZ domain proteins of synapses. Nat Rev Neurosci. 2004;5:771–781. doi: 10.1038/nrn1517. [DOI] [PubMed] [Google Scholar]
  17. Kobayashi A, Valerius MT, Mugford JW, Carroll TJ, Self M, Oliver G, McMahon AP. Six2 defines and regulates a multipotent self-renewing nephron progenitor population throughout mammalian kidney development. Cell Stem Cell. 2008;3:169–181. doi: 10.1016/j.stem.2008.05.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Laprise P, Viel A, Rivard N. Human homolog of disc-large is required for adherens junction assembly and differentiation of human intestinal epithelial cells. J Biol Chem. 2004;279:10157–10166. doi: 10.1074/jbc.M309843200. [DOI] [PubMed] [Google Scholar]
  19. Lee M, Vasioukhin V. Cell polarity and cancer--cell and tissue polarity as a non- canonical tumor suppressor. J Cell Sci. 2008;121:1141–1150. doi: 10.1242/jcs.016634. [DOI] [PubMed] [Google Scholar]
  20. Li J, Chen F, Epstein JA. Neural crest expression of Cre recombinase directed by the proximal Pax3 promoter in transgenic mice. Genesis. 2000;26:162–164. doi: 10.1002/(sici)1526-968x(200002)26:2<162::aid-gene21>3.0.co;2-r. [DOI] [PubMed] [Google Scholar]
  21. Mahoney ZX, Sammut B, Xavier RJ, Cunningham J, Go G, Brim KL, Stappenbeck TS, Miner JH, Swat W. Discs-large homolog 1 regulates smooth muscle orientation in the mouse ureter. Proc Natl Acad Sci U S A. 2006;103:19872–19877. doi: 10.1073/pnas.0609326103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mendelsohn C. Using mouse models to understand normal and abnormal urogenital tract development. Organogenesis. 2009;5:306–314. doi: 10.4161/org.8173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mendelsohn C, Lohnes D, Decimo D, Lufkin T, LeMeur M, Chambon P, Mark M. Function of the retinoic acid receptors (RARs) during development (II). Multiple abnormalities at various stages of organogenesis in RAR double mutants. Development. 1994;120:2749–2771. doi: 10.1242/dev.120.10.2749. [DOI] [PubMed] [Google Scholar]
  24. Miyazaki Y, Oshima K, Fogo A, Hogan BL, Ichikawa I. Bone morphogenetic protein 4 regulates the budding site and elongation of the mouse ureter. The Journal of clinical investigation. 2000;105:863–873. doi: 10.1172/JCI8256. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Naim E, Bernstein A, Bertram JF, Caruana G. Mutagenesis of the epithelial polarity gene, discs large 1, perturbs nephrogenesis in the developing mouse kidney. Kidney Int. 2005;68:955–965. doi: 10.1111/j.1523-1755.2005.00489.x. [DOI] [PubMed] [Google Scholar]
  26. Ohyama T, Groves AK. Generation of Pax2-Cre mice by modification of a Pax2 bacterial artificial chromosome. Genesis. 2004;38:195–199. doi: 10.1002/gene.20017. [DOI] [PubMed] [Google Scholar]
  27. Oliva C, Escobedo P, Astorga C, Molina C, Sierralta J. Role of the MAGUK protein family in synapse formation and function. Dev Neurobiol. 2012;72:57–72. doi: 10.1002/dneu.20949. [DOI] [PubMed] [Google Scholar]
  28. Pieczynski J, Margolis B. Protein complexes that control renal epithelial polarity. Am J Physiol Renal Physiol. 2011;300:F589–601. doi: 10.1152/ajprenal.00615.2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pini Prato A, Musso M, Ceccherini I, Mattioli G, Giunta C, Ghiggeri GM, Jasonni V. Hirschsprung disease and congenital anomalies of the kidney and urinary tract (CAKUT): a novel syndromic association. Medicine (Baltimore) 2009;88:83–90. doi: 10.1097/MD.0b013e31819cf5da. [DOI] [PubMed] [Google Scholar]
  30. Rossant J, Zirngibl R, Cado D, Shago M, Giguere V. Expression of a retinoic acid response element-hsplacZ transgene defines specific domains of transcriptional activity during mouse embryogenesis. Genes Dev. 1991;5:1333–1344. doi: 10.1101/gad.5.8.1333. [DOI] [PubMed] [Google Scholar]
  31. Rosselot C, Spraggon L, Chia I, Batourina E, Riccio P, Lu B, Niederreither K, Dolle P, Duester G, Chambon P, Costantini F, Gilbert T, Molotkov A, Mendelsohn C. Non-cell-autonomous retinoid signaling is crucial for renal development. Development. 2010;137:283–292. doi: 10.1242/dev.040287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Srinivas S, Goldberg MR, Watanabe T, D’Agati V, al-Awqati Q, Costantini F. Expression of green fluorescent protein in the ureteric bud of transgenic mice: a new tool for the analysis of ureteric bud morphogenesis. Dev Genet. 1999;24:241–251. doi: 10.1002/(SICI)1520-6408(1999)24:3/4<241::AID-DVG7>3.0.CO;2-R. [DOI] [PubMed] [Google Scholar]
  33. Stephenson LM, Sammut B, Graham DB, Chan-Wang J, Brim KL, Huett AS, Miletic AV, Kloeppel T, Landry A, Xavier R, Swat W. DLGH1 is a negative regulator of T-lymphocyte proliferation. Mol Cell Biol. 2007;27:7574–7581. doi: 10.1128/MCB.00439-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. te Velthuis AJ, Admiraal JF, Bagowski CP. Molecular evolution of the MAGUK family in metazoan genomes. BMC Evol Biol. 2007;7:129. doi: 10.1186/1471-2148-7-129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Uetani N, Bertozzi K, Chagnon MJ, Hendriks W, Tremblay ML, Bouchard M. Maturation of ureter-bladder connection in mice is controlled by LAR family receptor protein tyrosine phosphatases. J Clin Invest. 2009;119:924–935. doi: 10.1172/JCI37196. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Woods DF, Hough C, Peel D, Callaini G, Bryant PJ. Dlg protein is required for junction structure, cell polarity, and proliferation control in Drosophila epithelia. J Cell Biol. 1996;134:1469–1482. doi: 10.1083/jcb.134.6.1469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Zhao H, Kegg H, Grady S, Truong HT, Robinson ML, Baum M, Bates CM. Role of fibroblast growth factor receptors 1 and 2 in the ureteric bud. Dev Biol. 2004;276:403–415. doi: 10.1016/j.ydbio.2004.09.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Zheng CY, Seabold GK, Horak M, Petralia RS. MAGUKs, synaptic development, and synaptic plasticity. Neuroscientist. 2011;17:493–512. doi: 10.1177/1073858410386384. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

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Supplementary Materials

01
Download video file (2.7MB, mov)
02
Download video file (2.6MB, mov)
03

Supplementary Fig. 1. Time-lapse fluorescence video of in vitro organ culture of control; HoxB7-EGFP embryonic CND. The CND and surrounding tissues were collected at E12.5 and cultured for 7 days. Note the almost complete separation of the ureter from the CND.

Supplementary Fig. 2. Time-lapse fluorescence video of in vitro organ culture of Dlg1 KO; HoxB7-EGFP embryonic CND. The CND and surrounding tissues were collected at E12.5 and cultured for 7 days. Note that the ureter did not separate from the CND; failure is most obvious in the lower ureter.

Supplementary Fig. 3. Serial sections of E18.5 male control; HoxB7-EGFP ureters and bladder. After whole mount staining with anti-GFP in green and cytokeratin 8 in red, serial sectioning was performed to visualize the insertion angles and connections of the ureters with the bladder. Ureters and seminal vesicles are yellow (GFP+, cytokeratin 8+); urethra and bladder epithelial cells are red (GFP−, cytokeratin 8+). The ureters are connected to the bladder epithelium through the bladder wall.

Supplementary Fig. 4. Serial sections of E18.5 male Dlg1 KO; HoxB7-EGFP ureters and bladder. After whole mount staining with anti-GFP in green and cytokeratin 8 in red, serial sectioning was performed to visualize the insertion angle and connections of the ureters with the bladder. Ureters and seminal vesicle are yellow (GFP+, cytokeratin 8+); urethra and bladder epithelial cells are red (GFP−, cytokeratin 8+). The ureters were not connected to the bladder epithelium, but instead were abnormally connected to the urethra epithelium and to the seminal vesicles. The seminal vesicle lumen was also observed to be connected to the bladder lumen (middle panel, row 2).

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