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First published online 30 November 2005
doi: 10.1242/dev.02184

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Pax2/8-regulated Gata3 expression is necessary for morphogenesis and guidance of the nephric duct in the developing kidney

¹ McGill Cancer Centre and Biochemistry Department, McGill University, 3655 Promenade Sir-William-Osler, Montreal, Quebec H3G 1Y6, Canada.
² Research Institute of Molecular Pathology, Vienna Biocenter, Dr Bohr-Gasse 7, 1030 Vienna, Austria.

View larger version (101K): [in a new window]   Fig. 1. Gata3 expression in wild-type nephric tissues. A Gata3 cRNA probe was used for in situ hybridization on whole-mount embryos (A,B) or metanephros sections (C,D) at the indicated stages. Gata3 expression is detected in the pronephros anlage (pa; A), in the nephric duct (nd) of the mesonephros (B), and in the collecting duct (cd) and ureter tip (ut) of the metanephros (C,D).

View larger version (136K): [in a new window]   Fig. 2. Regulation of Gata3 by Pax transcription factors in the pro/mesonephros. Wild-type (A,C) and Pax2-/-Pax8+/- (B,D) embryos were analyzed at the 15-somite stage by in situ hybridization of Gata3 (A,B) and Pax8 (C,D) cRNA probes on adjacent sections. Gata3 expression is specifically lost in the Pax2-/-Pax8+/- nephric duct. nc, nephric cord; nd, nephric duct.

View larger version (51K): [in a new window]   Fig. 3. Inactivation of the Gata3 gene. (A) The Gata3 gene was targeted by insertion of loxP sites (grey triangles) flanking exon 4 (encoding the first zinc finger), followed by a cassette, flanked by frt sites (grey rectangles), containing a translation stop codon, an internal ribosome entry site (Ires) driving the GFP gene, and the neomycin selection gene (neo). The GFP and neo genes were followed by SV40 polyadenylation signals. The targeting construct additionally contained the thymidine kinase (tk) and dyphteria toxin A (DT-A) genes at the 5' and 3' ends, respectively, for selection against random genomic integration. Mice with the original targeted allele (Gata3ex4GFP) were mated with the More-cre germline deleter strain (Tallquist and Soriano, 2000) to excise exon 4 and generate the Gata3GFP allele (referred to as Gata3- allele). The different alleles were detected by Southern blot analysis of BamHI-digested genomic DNA with the indicated probe. The exons are numbered according to Pandolfi et al. (Pandolfi et al., 1995). (B) Southern blot analysis of wild-type (+/+), Gata3ex4GFP (ex4GFP/+) and Gata3GFP (GFP/+) tail DNA digested with BamHI. (C) GFP expression of the targeted Gata3 gene in Gata3GFP/+ embryo at E9.5. Broken line indicates the contour of the embryo. ba, branchial arches; op, otic placode; nd, nephric duct; va, vitelline artery.

View larger version (102K): [in a new window]   Fig. 4. Mesonephric defects in Gata3-/- embryos. Pax2 expression was detected in wild-type (A,C,E) or Gata3-/- (B,D,F) embryos at 17 somites (A,B), 21 somites (C,D) and 27 somites (E,F). (A-D) A delay is observed in the caudal extension of Pax2+ mesonephric cells (broken red lines in B,D,F) of Gata3-/- embryos. (E,F). The distinct Pax2 expression domains in the nephric cord and duct of wild-type embryos (insert in E) were replaced by a single stream of Pax2+ cells (black arrowheads, insert in F) with regions of discontinuity (white arrowheads) in Gata3-/- embryos. ms, mesonephros; mst, mesonephric tubules; nc, nephric cord; nd, nephric duct.

View larger version (81K): [in a new window]   Fig. 5. Nephric cord extension in Gata3-/- embryos. Control Gata3+/+ or Gata3+/- embryos (A,C,E,G,I,K) and Gata3-/- embryos (B,D,F,H,J,L) were analyzed for marker expression on sections of caudal E9.75 mesonephros (A-F) and E10.5 metanephros (G-L). (A,B) Pax2 (red) and Wt1 (green) antibody staining reveals the presence of Pax2+ Wt1+ nephric cord cells (nc; yellow), but an absence of Pax2+ Wt1- nephric duct cells in Gata3-/- embryos. (C,D) Immunostaining of the epithelial marker E-cadherin (green) further reveals the absence of a nephric duct in Gata3-/- embryos (asterisk). Some non-specific signal caused by blood cell autofluorescence is apparent and reflects Gata3-dependent hemorrhages (white arrowhead) (Pandolfi et al., 1995). (E,F) TUNEL signals (red) indicated an increase in apoptosis in Gata3-/- nephric cord cells. Sections C-F were counterstained with DAPI (blue). (G,H) In situ hybridization with a Pax2 cRNA probe showed the presence of reduced levels of Pax2 expression in the metanephric mesenchyme (mm) in the absence of a ureteric bud (ub). (I,J) The lack of a ureteric bud at the hindlimb level of Gata3-/- embryos was confirmed by the absence of Emx2 mRNA expression. Emx2 expression in surrounding tissues was unaffected. (K,L) Wt1 mRNA expression in the metanephric mesenchyme is still detected in Gata3-/- embryos.

View larger version (91K): [in a new window]   Fig. 6. Ectopic tubulogenesis and guidance defects in the Gata3-/- mesonephros. Control Gata3+/+ (+/+) or Gata3+/- (+/-) embryos (A,C,E,G,I,M,O) and Gata3-/- (-/-) (B,D,F,H,J,K,L,N,P) embryos were analyzed for nephric duct defects at E9.25 (A,B,K), E9.5 (C-J,L-N) and E10.5 (O,P). (A,B) Confocal analysis of GFP expression from the Pax2GFP BAC transgene shows the initiation of aberrant nephric duct morphogenesis. (C,D) Extreme case of mesonephros hypercellularity revealed by immunohistochemistry with an anti-Pax2 antibody. (E,F) Gata3-/- ectopic ducts expressed the nephric duct marker E-cadherin. (G,H) Same section as in E,F, showing the colocalization of E-cadherin with GFP expressed from the targeted Gata3 locus. (I,J) Whole-mount in situ hybridization with a Pax2 cRNA probe reveals the presence of multiple nephric ducts (nd) growing towards the ectoderm at the level of the mesonephric tubules (mst) in Gata3-/- embryos. (K,L) Gata3-/- embryos in which a nephric duct grew caudally showed a guidance defect, as revealed by whole-mount in situ hybridization with a Pax2 cRNA probe. (M,N) Immunohistochemistry with an anti-Pax2 antibody showed an abnormally large distance between the nephric duct and nephric cord in Gata3-/- embryos. (O,P) Brn1 cRNA in situ staining of a representative E10.5 embryo in which the duct turned abruptly in the direction of the surface ectoderm (broken line) and fused with it. da, dorsal aorta; ec, ectoderm; ms, mesonephros; mst, mesonephric tubules; nc, nephric cord; nd, nephric duct.

View larger version (128K): [in a new window]   Fig. 7. Regulation of cell proliferation and gene expression by Gata3. Control wild-type or Gata3+/- embryos (A,C,E) and Gata3-/- embryos (B,D,F) were analyzed by immunohistochemistry with anti-phosphorylated-histone H3 and anti-E-cadherin antibodies (A,B) and by in situ hybridization with Ret (C,D) and Wnt11 (E,F) cRNA probes. (A,B) The number of mitotic cells (white arrows) and the cellularity (broken lines) of the nephric duct were significantly higher in Gata3-/- compared with control embryos. Sections A,B were counterstained with DAPI (blue) (C,D) Ret expression was absent from Gata3-/- hypercellular nephric ducts. Strong Ret expression is still present in the Gata3-/- spinal cord (sc). (E,F) Expression of the Ret-regulated Wnt11 gene was completely lost in Gata3-/- nephric ducts in contrast to control embryos. Broken lines in A,B,D,F demarcate the epithelial nephric duct, as defined by E-cadherin staining. da, dorsal aorta; ec, ectoderm; nd, nephric duct; sc, spinal cord.