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doi: 10.1242/10.1242/dev.00536

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Six1 is required for the early organogenesis of mammalian kidney

¹ McLaughlin Research Institute, 1520 23rd Street South, Great Falls, MT 59405, USA
² INSERM 129, ICGM, F-75014 Paris, France

View larger version (77K): [in a new window]   Fig. 1. Expression of Six1 during kidney development analyzed by in situ and X-gal staining of heterozygous Six1lacZ embryos for the Six1lacZ allele. (A) In situ hybridization showing Six1 expression in metanephric mesenchyme (mm) at E10.5. (B) X-gal staining of Six1lacZ heterozygous embryos showing strong Six1lacZ expression at E10.5, similar to that observed by in situ hybridization (A). (C) At E11.5, Six1 is expressed in the induced mesenchyme (mm) around the ureteric bud epithelium (ur). (D) X-gal staining of E17.5 Six1lacZ heterozygous kidneys showing Six1lacZ expression in collecting tubules (arrow). Six1lacZ is also expressed in the muscles surrounding the ureter (ur). (E,F) Transverse sections of X-gal-stained Six1lacZ heterozygous kidneys at E17.5 revealed that the Six1lacZ-expressing cells are localized in the collecting tubules (arrows). F is higher magnification of the boxed area in E. p, renal pelvis; wd, Wolffian duct.

View larger version (104K): [in a new window]   Fig. 2. Kidney development in Six1-deficient mice. (A-D) Metanephros (mt) in Six1+/- (A,C) and Six1-/- (B,D) mice at E12.5. Kidneys are absent in Six1-/- mice (*), but ureter (ur) was observed in the left primordium (D). The genital tracts appeared to be normal both in males and females. (E) In Six1+/- embryos of E11.5, ureteric bud (ub) grows out from Wolffian duct and invades the mesenchyme and the metanephric mesenchyme (mm) are condensed around the bulging ureteric bud. (F) In Six1-/- embryos, although the ureteric buds grow out from Wolffian ducts, they fail to invade the mesenchyme (arrows) completely. (G-J) TUNEL analysis of metanephros in Six1+/- and Six1-/- embryos at E11.5. Note the apoptotic cells were markedly increased in Six1-/- metanephric mesenchyme (arrows). g, gonad; wd, Wolffian duct.

View larger version (139K): [in a new window]   Fig. 3. Six1 is required for the expression of Pax2 and Six2 but not Eya1 in the metanephric mesenchyme (mm) at E10.5. (A-D) Pax2 is normally expressed in the intermediate mesoderm (A,B), the metanephric mesenchyme before and after induction and in the ureteric epithelium (ub). In Six1-/- embryos, however, its expression in the metanephric mesenchyme at E10.5 is undetectable (arrows in D). (E,F) Eya1 is expressed in the metanephric mesenchyme before and after induction and its expression is not affected in Six1-/- mesenchyme at E10.5. (G,H) Six2 is also expressed in the metanephric mesenchyme before and after induction and its expression is undetectable in Six1-/- mesenchyme at E10.5 (arrows).

View larger version (114K): [in a new window]   Fig. 4. Six1 is required for the expression of Sall1 in the metanephric mesenchyme (mm). (A,B) Bmp4 is normally expressed in the mesenchyme around the ureteric stalk (ub) and its expression was not affected in Six1-/- embryos at E10.5. (C,D) Bmp7 is normally expressed in the ureteric bud (ub) and metanephric mesenchyme at E10.5 and its expression levels are normal in both structures in Six1-/- embryos at E10.5. However, its expression domain is reduced in size in Six1-/- metanephric mesenchyme (D). (E,F) Wt1 is widely expressed in the mesenchyme and urogenital ridge region during kidney development and its expression level is not affected in Six1-/- embryos at E10.5. However, its expression domain in the metanephric mesenchyme also appears to be reduced in size in E10.5 Six1-/- embryos. (G,H) Sall1 is expressed in the metanephric mesenchyme before and after induction, however its expression was undetectable in the mesenchyme in Six1-/- embryos at E10.5 (arrows). (I,J) Gdnf is expressed in the metanephric mesenchyme and its expression levels are normal in Six1-/- embryos at E10.5. However, its expression domain appears to be reduced in size in Six1-/- embryos at E10.5 (arrows).

View larger version (116K): [in a new window]   Fig. 5. Sall1, Eya1 and Six1 expression is not affected in Pax2-/- metanephric mesenchyme (mm). (A,B) Sall1 is expressed in the metanephric mesenchyme and its expression is not affected in Pax2-/- mesenchyme at E10.5. (C,D) Eya1 is expressed in the metanephric mesenchyme and its expression level is unaffected in Pax2-/- embryos at E10.5. (E,F) Six1 is expressed in the metanephric mesenchyme and its expression level is also unaffected in Pax2-/- embryos at E10.5. (G,H) However, Six1 expression in Eya1-/- mesenchyme is significantly reduced when compared to that in wild-type embryos at E10.5. Its expression in the limb bud and somites is also significantly reduced. Six homozygous embryos were used for each probe.

View larger version (94K): [in a new window]   Fig. 6. Renal hypoplasia or agenesis in Eya1/Six1 double heterozygous animals. (A-D) E17.5 wild type, Six1+/- or Eya1+/-/Six1+/- kidneys. Samples shown in A, C and D were X-gal stained for Six1lacZ and it is expressed in the testis (t) and muscles surrounding the ureters and kidneys (D). Eya1+/-/Six1+/- animals show either smaller kidneys bilaterally (A), a small kidney on one side and no kidney on the other side (B), or complete absence of kidneys (C,D). In some Eya1+/-/Six1+/- animals that show renal agenesis, ureters that end blindly are observed (arrow in B,C). Adrenal glands and the genital tracts appeared to be normal in all compound heterozygous animals analyzed so far. (E,F) Histological analysis of kidneys of E17.5 Six1+/- and Eya1+/-/Six1+/- animals. The number of nephrons was markedly reduced in the double heterozygous kidneys, but normal developing structures are present. The lower panels are higher magnification of the boxed areas. In Six1+/- animals, the differentiating metanephric cap tissue (vesicles) in the peripheral nephrogenic zone, in which ureteric bud branching and induction of new nephrons takes place, are morphologically apparent (arrow in E). However, in the hypoplastic Eya1+/-/Six1+/- kidney, the differentiating metanephric vesicles in the peripheral nephrogenic zone were largely reduced in number (arrow in F). (G,H) A reduced number of ureteric bud branches is detected in the compound heterozygous animals at E13.5. ct, convoluting tubule; g, glomerulus; ur, ureter.

View larger version (104K): [in a new window]   Fig. 7. Six1 mutant mesenchyme is unresponsive to induction. (A) E11.0 Six1+/- kidney rudiments cultured for 5 days and stained with Pax2 in situ probe. After 5 days of culture, they developed into a fully branched kidney structure (k) showing Pax2 expression in the collecting tubules and in the nephrons. (B) E11.0 Six1-/- metanephric rudiments cultured for 5 days and stained with Pax2 in situ probe. No kidney formation was observed (arrow). (C) E11.0 Six1+/- metanephric mesenchyme cultured with spinal cord (sc) for 5 days and stained with Pax2 in situ probe. The Six1+/- mesenchymal cultures exhibited characteristic tubules (k) showing Pax2 expression. (D) E11.0 Six1-/- metanephric mesenchyme cultured with hetorozygous spinal cord for 5 days and stained with Pax2 in situ probe. None of the Six1-/- mesenchymes exhibited any sign of tubule formation. Note the disappearance of Six1 mutant mesenchyme (arrow), which shows no Pax2 expression.