Nephric duct insertion is a crucial step in urinary tract maturation that is regulated by a Gata3-Raldh2-Ret molecular network in mice

Abstract

Urinary tract development depends on a complex series of events in which the ureter moves from its initial branch point on the nephric duct (ND) to its final insertion site in the cloaca (the primitive bladder and urethra). Defects in this maturation process can result in malpositioned ureters and hydronephrosis, a common cause of renal disease in children. Here, we report that insertion of the ND into the cloaca is an unrecognized but crucial step that is required for proper positioning of the ureter and that depends on Ret signaling. Analysis of Ret mutant mice at birth reveals hydronephrosis and defective ureter maturation, abnormalities that our results suggest are caused, at least in part, by delayed insertion of the ND. We find a similar set of malformations in mutants lacking either Gata3 or Raldh2. We show that these factors act in parallel to regulate ND insertion via Ret. Morphological analysis of ND extension in wild-type embryos reveals elaborate cellular protrusions at ND tips that are not detected in Ret, Gata3 or Raldh2 mutant embryos, suggesting that these protrusions may normally be important for fusion with the cloaca. Together, our studies reveal a novel Ret-dependent event, ND insertion, that, when abnormal, can cause obstruction and hydronephrosis at birth; whether ND defects underlie similar types of urinary tract abnormalities in humans is an interesting possibility.

Fig. 1.

Abnormal ureter insertion in Ret^−/− mouse embryos. (A,B) Whole-mount urinary tract from wild-type (A) and Ret mutant (B) embryos at E18.5. The black arrow denotes the abnormal connection between the ureter and vas deferens, which are not separated and are dilated owing to obstruction (ur/vd*). (C,D) Whole-mount urinary tract from E17.5 control (C) and Ret mutant (D) embryos labeled with Hoxb7-Gfp transgene. The white arrow denotes the trajectory of the ureter as it passes through the bladder wall. Note the abnormal path of the intravesicular ureter as it crosses the bladder at a 90° angle (D), rather than a 45° angle as in controls (C). (E) Hematoxylin and Eosin (H&E) stained frontal section of the E17.5 wild-type embryo shown in C. Note the smooth trajectory of the intravesicular ureter as it passes through the bladder wall at a 45° angle and joins the lumen (black arrow). (F) H&E stained section from the sample shown in D. The black arrow traces the path of the mutant ureter that, in addition to its abnormal angle of entry, also appears to be kinked. (G,H) Sagittal section from wild-type (G) and Ret mutant (H) embryos labeled with Hoxb7-Gfp transgene. Note the separation between the common nephric duct and urogenital sinus in the Ret mutant embryo (white arrowhead). GFP is shown in green, DAPI staining in blue. (I,J) H&E stained sections through E9.5 wild-type (U) and Ret mutant (J) embryos. Note the distance between the nephric duct (ND) tips and cloaca (white arrowhead) in the Ret mutant embryo, whereas control NDs reach the cloaca at this stage. cl, cloaca; cnd, common nephric duct; ki, kidney; nd, nephric duct; te, testis; ugs, urogenital sinus; uo, ureter orifice; ur, ureter; vd, vas deferens. Magnifications: 2.5× in A,B; 10× in C-F; 20× in G,H.

Fig. 2.

Hydronephrosis and ectopic ureter termination in Gata3 and Raldh2 mutant mouse embryos resemble those present in Ret mutants. (A) Whole-mount urinary tract from a wild-type control E18.5 embryo showing the kidney and ureter. (B) Whole-mount E18.5 Gata3^ND−/− embryo showing hydronephrosis and megaureter. The asterisk denotes the dilated mutant ureter. (C) Hematoxylin and Eosin (H&E) stained section through an E18.5 wild-type embryo at the position where the ureter enters the bladder. (D) Gata3^ND−/− embryo showing an abnormal ureter that terminates bluntly rather than connecting to the bladder. The asterisk denotes the dilated mutant ureter. (E,F) Whole-mount urinary tracts from E18.5 wild-type (E) and Raldh2^−/− (F) embryos. Note the hydronephrotic kidney and dilated ureter in F (asterisk). (G,H) H&E-stained section through E18.5 wild-type (G) and Raldh2^−/− (H) kidneys. Note that the renal pelvis is not dilated in G, in contrast to the hydronephrosis and hydroureter observed in H (asterisks). (I) H&E-stained section through the bladder of an E18.5 wild-type embryo showing normal entry of the ureter. (J) Section through an E18.5 Raldh2^−/− embryo at the level of the prostatic urethra, showing the ectopic ureteral connection in the vas deferens. The asterisk denotes the dilated distal ureter. ki, kidney; sv, seminal vesicle; ur, ureter; vd, vas deferens. Magnifications: 10× in A,B,E-H; 20× in C,D,I,J.

Fig. 3.

Abnormal nephric duct (ND) insertion leads to disrupted ureter maturation in Gata3^ND−/−, Raldh2^−/− and Ret^−/− mouse embryos. (A-D) Sagittal Hematoxylin and Eosin (H&E)-stained sections through the urogenital sinus of E14.5 wild-type (A), Gata3^ND−/− (B), Raldh2^−/− (C) and Ret^−/− (D) embryos. Note that in the three mutants, the NDs are not joined to the urogenital sinus (white arrowheads). Black arrowhead in A indicates ureter entry point. (E-H) E-cadherin-stained sagittal vibratome sections through E12.5 wild-type (E), Gata3^ND−/− (F), Raldh2^−/− (G) and Ret^−/− (H) embryos. Unlike controls, the NDs in the three mutants are intact and have not inserted in the urogenital sinus (white arrowheads). (I-L) Whole-mount in situ hybridization showing Pax2 expression in the cloaca and nephric ducts of E9.5 wild-type (I), Gata3^ND−/− (J), Raldh2^−/− (K) and Ret^−/− (L) embryos. NDs in the control embryo terminate in the cloaca (arrow in I); however, mutant nephric ducts terminate prematurely and fail to join the cloaca (arrows in J-L). cl, cloaca; cnd, common nephric duct; nd, nephric duct; ugs, urogenital sinus. Magnifications: 20× in A-H, 10× in I-L.

Fig. 4.

Retinoids and Gata3 act in parallel to regulate Ret expression in the mouse nephric duct (ND). (A) Ret expression in the nephric ducts of a whole-mount E9.5 wild-type embryo. (B) In situ hybridization analysis of an E9.5 Raldh2^−/− mutant embryo showing downregulation of Ret in the ND, which has terminated prematurely. (C) Whole-mount in situ hybridization analysis of an E9.5 wild-type embryo hybridized with Ret probe. (D) Downregulation of Ret in an E9.5 whole-mount Gata3^ND−/− mutant. (E) Gata3 expression in a wild-type E9.5 embryo. (F) Gata3 expression in an E9.5 Raldh2^−/− embryo. (G,H) Raldh2 expression in control (G) and in a Gata3^ND−/− mutant embryo (H). cl, cloaca; nd, nephric duct. Magnifications: ×20 in all panels.

Fig. 5.

Wild-type embryos display prominent extensions that are not visible in Ret, Gata3 or Raldh2 mutant embryos. (A) A whole-mount wild-type Hoxb7-Gfp embryo at E9.5 showing nephric ducts (NDs) that have extended and migrated to the cloaca (dashed oval). Cellular extensions are visible in the more posterior segment (arrowheads). (B) Higher magnification image showing posterior cellular extensions in a control Hoxb7-Gfp embryo (arrowheads). (C) High magnification image showing cellular extensions (arrowheads) at the tip of a E9.5 Hoxb7-Gfp control nephric duct. (D) Absence of visible ND extensions in Hoxb7-Gfp;Ret mutant at E9.5. (E) Absence of visible GFP-positive ND extensions in Gata3^ND−/− mutant embryo at E9.5. (F) Absence of ND extensions in E9.5 Raldh2 mutant embryo. White arrows in B-F denote the direction of growth. nd, nephric ducts. Magnifications: 10× in A; 12× in B; 40× in D-F.

Fig. 6.

Model showing how delayed or failed nephric duct (ND) insertion can influence the final position of the ureter orifice. (A) Nephric duct insertion and ureter maturation in wild-type embryos. NDs extend to the caudal region of the embryo and insert in the cloaca at E9.5. Ureteric bud formation begins at E10. By 11.5, ureters have elongated and reached the kidney blastema, but are still attached to the caudal ND segment (the CND). Between E12.5 and E13.5, the CND merges with the urogenital sinus and undergoes apoptosis, forming a patent connection between the kidneys and the bladder. Between E13.5 and E14.5, the ureter aligns with and fuses with the urogenital sinus epithelium; as this occurs, the caudal-most ureter segment undergoes apoptosis, generating a new ureter orifice in the bladder primordium. (B) Delayed ND insertion at E9.5 affects CND apoptosis and results in defects in alignment and insertion of the ureter into the bladder. As a consequence, distal ureters end up closer to the ND than in controls, which can cause obstruction once renal function begins (dilated ureters in right-most panel). (C) Complete failure in ND insertion at E9.5 results in ectopic vas deferens, where the ureter and vas deferens remain fused and fail to connect with the bladder and urethra, respectively, causing obstruction (dilated ureter in right-most panel). Bl, bladder; CND, common nephric duct; epi, cloacal epithelium; Ki, kidney; Mes, mesenchyme; ND, nephric duct; Ur, ureter.