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First published online May 30, 2007
doi: 10.1242/10.1242/dev.004390

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Adrenal development is initiated by Cited2 and Wt1 through modulation of Sf-1 dosage

¹ Section of Gene Function and Regulation, Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK.
² Neural Development Unit, Institute of Child Health, University College London, London, UK.

View larger version (91K): [in this window] [in a new window]   Fig. 1. Cited2 acts in the adrenogonadal primordium (AGP) to promote adrenal cortex development. (A) Adrenal cortex differentiation is markedly impaired in Cited2 mutant mouse embryos. Urogenital regions from wild-type (WT) or mutant embryos (Mut) were subjected to whole-mount in situ hybridisation (WISH) for Sf-1 or Hoxb9 expression and sectioned. Arrowhead, position of the adrenal anlagen; arrow, Sf-1 expression in the developing gonad; DA, dorsal aorta. (B) Neural crest migration to the adrenal anlagen occurs at E13. Neural crest migration to the adrenal anlagen (arrowhead) was evaluated by double WISH for Cyp11a1 (orange) and Mash1 (purple). The lower left panel shows a section (taken at the line in the top panel) through the adrenal anlagen. (C) Neural crest migration is only mildly affected in Cited2 mutant embryos. WISH for Mash1 or tyrosine hydroxylase (Th) at E13.5 shows that neural crest cells still migrate to the presumptive adrenal area (arrowhead) of mutant embryos, albeit in reduced numbers. (D) Cited2 is expressed in the AGP and the adrenal cortex anlagen. Cited2 mRNA is expressed in the genital ridge (arrow) and the presumptive adrenal area (arrowhead) at E10.5. At E12 (left panel shows WISH; right panel shows section of WISH sample at the white line), expression in the gonad (G) is barely detectable but Cited2 is present in the adrenal anlagen (arrowhead). At E13.5, Cited2 is faintly expressed in the gonads of both sexes. At E14.5, Cited2 is strongly expressed in the cells of the adrenal cortex (C) and weakly expressed in the medulla (M).

View larger version (54K): [in this window] [in a new window]   Fig. 2. Early gonad development is affected in Cited2 mutant mouse embryos. (A) Gonad development was analysed in wild-type (WT, left panels) and mutant (Mut, right panels) embryos by WISH for Sox9, Cyp11a1 and Sf-1 mRNAs at E11.5 and E13.5. Arrow, gonad; arrowhead, adrenal primordium. (B) Expression of Sox9, Cyp11a1 and Sf-1 was analysed by quantitative real-time RT-PCR on E11.5 microdissected XY gonads from wild-type (black bars) and Cited2 mutant (grey bars) embryos. Results are shown as a percentage of mRNA accumulation in wild-type embryos and represent the mean values obtained with at least four urogenital regions for each genotype (±s.d.); *, P<0.05 in Student's t-test.

View larger version (76K): [in this window] [in a new window]   Fig. 3. Apoptosis and proliferation in Cited2 mutant mouse embryos. (A) Analysis of apoptosis in E10.5 and E12.5 Cited2 mutant embryos. Apoptosis was analysed by incubation of E10.5 and E12.5 wild-type (WT) and mutant (Mut) embryos with Lysotracker Red (red staining). Urogenital regions were then sectioned and incubated with anti-laminin antibody (green) and stained with DAPI (blue). Arrowhead, mesonephric tubules; arrow, coelomic epithelium; dashed line, adrenal anlage at E12.5. (B) Proliferation is not decreased in E10.5 Cited2 mutant embryos. Proliferation was detected by BrdU incorporation at E10.5. Sections of the urogenital region were stained with anti-BrdU (red) and anti-laminin or anti-Sf-1 (green) antibodies. Arrow, coelomic epithelium. Note the decrease in Sf-1 protein levels in the Cited2 mutant.

View larger version (89K): [in this window] [in a new window]   Fig. 4. Similar mesonephric tubule phenotype in Cited2 and Wt1 mutant mice. (A) Cited2 mutant mouse embryos lack caudal mesonephric tubules. Development of mesonephric tubules was analysed by WISH for Lim1 at E10 (upper panel) and by immunohistochemistry for laminin at E11.5 (lower panel). Caudal mesonephric tubules (arrowheads) are absent from Cited2 mutant (Mut) embryos. (B) Cited2, Wt1 and Sf-1 are coexpressed in the early AGP. Expression of Cited2, Wt1 and Sf-1 was analysed by WISH during urogenital development. The three factors are colocalised in the coelomic epithelium (arrow) and underlying cell layers at E10. Cited2 and Sf-1, but not Wt1, are coexpressed in the adrenal primordium (arrowhead) at E12.5. G, gonad.

View larger version (29K): [in this window] [in a new window]   Fig. 5. Cited2 and Wt1 are in the same genetic pathway. Genetic interaction between Cited2 and Wt1 was analysed by interbreeding Cited2+/- and Wt1+/- mice. Urogenital regions from embryos of the four resulting genotypes were analysed by WISH for Sf-1 at E11.5 (left panel). Adrenal surface was measured as the non-gonadal area staining positive for Sf-1 (arrowhead) and is expressed as a percentage of wild-type surface (right panel). Results are the mean of three independent experiments; *, P<0.05 in Student's t-test.

View larger version (27K): [in this window] [in a new window]   Fig. 6. Mouse Cited2 and Wt1 interact physically. (A) Wt1 and Cited2 endogenous proteins interact physically in M15 cells. Nuclear proteins from M15 cells were immunoprecipitated with antibodies for Wt1 (IP Wt1) or ß-galactosidase (IP ßGal, specificity control). Cited2 (C) was only detected in the immunoprecipitate formed with Wt1 antibody. `5%' means 5% of the amount of protein used for immunoprecipitation. (B) Wt1 and Cited2 interact physically in transfected cells. 293A cells were transfected with expression vectors for Wt1(-/-) or Wt1(+/+) and Cited2. Proteins were immunoprecipitated with antibodies raised against Wt1 (left panel) or Cited2 (right panel). Non-immune rabbit IgGs or rabbit anti-Sf-1 antibody did not immunoprecipitate Cited2 in control experiments. EV, empty vector; C, western blot with Cited2 antibody; W, western blot with Wt1 antibody. (C) Cited2 interacts directly with the Wt1 DNA-binding domain. In vitro translated 35S-labelled Cited2 protein (IVT Cited) was incubated with GST alone (GST, negative control) or immobilised GST-tagged full-length (FL), N-terminal (N-ter) or zinc-finger DNA-binding domain (ZF) fragments of Wt1(-/-) protein. After washes, bound proteins were eluted and separated on a SDS-PAGE gel and detected by autoradiography (upper panel). A Coomassie staining of the gel is provided as a loading control for the amounts of the GST fusion proteins (lower panel). Arrowheads indicate the GST-fused Wt1 proteins.

View larger version (45K): [in this window] [in a new window]   Fig. 7. Cited2 and Wt1 coordinately control Sf-1 mRNA accumulation in the AGP. (A) Sf-1 accumulation is decreased in the urogenital region of Cited2-/- mouse embryos. In the upper panel, Sf-1 expression was analysed by WISH in Cited2+/+ and Cited2-/- embryos at E10.5. In the lower panels, urogenital regions were sectioned at the line shown in the upper panels. (B) Quantitative real-time RT-PCR analysis of Sf-1 and Wt1 mRNA accumulation at E10.5. Urogenital regions from Cited2+/+ and Cited2-/- embryos were microdissected and relative Sf-1 and Wt1 mRNA accumulation was analysed by quantitative real-time RT-PCR. Results are shown as a percentage of mRNA accumulation in wild-type embryos and represent the mean values obtained with at least four urogenital regions for each genotype (±s.d.). (C) Sf-1 mRNA accumulation in the offspring of Cited2+/-xWt1+/- matings at E10.5. Experiments were performed as in B (*, P<0.05 in Student's t-test). (D) Cited2 and Sf-1 are in the same genetic pathway. Genetic interaction between Cited2 and Sf-1 was analysed by interbreeding Cited2+/- and Sf1+/- mice. E13.5 adrenals and kidneys were then subjected to WISH for Hoxb9. The adrenal gland was identified as the area outside the kidney staining positive for Hoxb9 (arrowhead). Its surface was measured using ImageJ software and is expressed as a percentage of the wild-type surface (values included in the appropriate panels). Results are the mean of four independent experiments (±s.d.). (E) Cited2 stimulates Wt1 transcriptional activity at the Sf-1 promoter. C2C12 cells were transfected with luciferase reporter genes driven by either the Sf-1 (Sf1P, Sf1Pm, left panel) or amphiregulin (labelled as pAmphiregulin, right panel) promoters. The effect of 50 ng RcCMV-Wt1(-/-), 50 ng pCMV5-Cited2, or both, was evaluated in co-transfection experiments as indicated. The version of the Sf-1 promoter harbouring four mutated Wt1-responsive elements (Sf1Pm) was included as a control. Results are expressed as fold induction over the activity of Sf1P or amphiregulin in the absence of Wt1 and Cited2, and represent the mean of at least three experiments performed in triplicate (±s.d.).