Disruption of Gen1 Causes Congenital Anomalies of the Kidney and Urinary Tract in Mice

Abstract

Congenital anomalies of the kidney and urinary tract (CAKUT) are among the most common developmental defects in humans. Despite of several known CAKUT-related loci (HNF1B, PAX2, EYA1, etc.), the genetic etiology of CAKUT remains to be elucidated for most patients. In this study, we report that disruption of the Holliday Junction resolvase gene Gen1 leads to renal agenesis, duplex kidney, hydronephrosis, and vesicoureteral reflux (VUR) in mice. GEN1 interacts with SIX1 and enhances the transcriptional activity of SIX1/EYA1, a key regulatory complex of the GDNF morphogen. Gen1 mutation impairs Grem1 and Gdnf expression, resulting in excessive ureteric bud formation and defective ureteric bud branching during early kidney development. These results revealed an unidentified role of GEN1 in kidney development and suggested its contribution to CAKUT.

Figure 1

Kidney and urinary tract defects in Gen1 mutants. (A) Genomic structure of the Gen1^PB allele. A PB[Act-RFP] transposon was inserted in the second intron (solid lines) of Gen1. Open boxes, exons; black arrow, transcription direction; green arrows, PB termini; red arrow, RFP expression cassette. (B) Real-time PCR analysis of Gen1 expression in E12.5 embryos. Data is shown as the mean ± s.e.m. *** P < 0.001 determined by t test. (C) Percentages of newborn mice with normal number of kidneys (NK), unilateral renal agenesis (URA), or duplex kidneys (DK). Percentages of mice having accompanying hydronephrosis (HN) are indicated at the top of each column. (D-G) Representative images of the urinary system in newborn mice. URA (E), unilateral DK (F,G), severe HN and hydroureter (G) are presented. (H-O) Hematoxylin-eosin (H&E) staining of the paraffin sections of kidneys in newborn mice. A wild type kidney (H), URA (I), DK (J), and severe HN (K) are presented. Details within the boxes are enlarged in (L-O), respectively. DK is defined by double renal pelvises (arrowheads). Well differentiated glomeruli (arrows) could be observed in the wild type, URA, and DK. In contrast, tubular elements and glomeruli are almost missing in severe HN (O). Scale bars, 2 mm in D-G; 250 μm in H-K; 50 μm in L-O.

Figure 2

Gen1 mutation leads to ectopic UBs accompanied by altered Grem1 and Gdnf expression. (A-C) Ectopic UBs detected in E11 embryos by c-Ret RNA in situ hybridization. (D-F) Gdnf RNA in situ hybridization in E10.5 embryos. Homozygous embryos (E,F) express less Gdnf than the wild type (D). (G) Real-time RT-PCR of Grem1 from E10.5 wild type (n=4) and homozygous mutant (n=6) kidney primordia. Dashed lines indicate UB. Scale bars, 0.2 mm. * P<0.05.

Figure 3

Ureteric branching defects in Gen1 mutants. (A) c-Ret RNA in situ hybridization at E11.5 showed normal branch in wild type embryos. (B) Both UBs at the same side failed to branch in some E11.5 Gen1^PB/PB embryos. (C) Gdnf RNA in situ hybridization showed Gdnf expression around the UB branches in E11.5 wild type embryos. (D) No Gdnf expression was detected around the un-branched UBs in E11.5 Gen1^PB/PB embryos. (E) Quantitative RT-PCR showed decreased Gdnf expression in MM of E11.5 Gen1^PB/PB embryos. Dashed lines indicate UB. Scale bars, 0.2 mm.

Figure 4

GEN1 binds with SIX1 and enhances transcriptional activity of the SIX1/EYA1 complex. (A) HA-tagged SIX1 can be pulled down by FLAG-tagged GEN1. (B) GFP-tagged GEN1 can be pulled down by FLAG-tagged SIX1. (C) Luciferase activity of a SIX1/EYA1 reporter. Supplemented by SIX1, EYA1, and GEN1, the luciferase became more active than under any other environment. * P < 0.05, ** P < 0.01, *** P < 0.001 determined by t test. (D,E) In situ hybridization at E10.5 revealed lower Six2 expression in homozygous (E) than in wild type embryos (D). Dashed lines indicate UB. Full-length blots are presented in Supplementary Figure 6. Scale bar, 0.1 mm.

Figure 5

Impaired interaction between mutant GEN1 and SIX1. (A) Schematic view of mutant GEN1 proteins. (B) Luciferase assay showed potent activity of GEN1-2A in enhancing the transcriptional activity of SIX1/EYA1 complex. (C) Luciferase assay showed impaired activity of truncated GEN1s in enhancing the transcriptional activity of SIX1/EYA1 complex. (D) Co-IP experiments showed impaired binding activity between truncated GEN1s and SIX1. Full-length blots are presented in Supplementary Figure 7. XPG-N, N-terminal nuclease domain; XPG-I, internal nuclease domain; H3TH, helix-hairpin-helix DNA binding domain. FL, full length; 2A, E134A/E136A.