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First published online 3 July 2008
doi: 10.1242/dev.021493

Development 135, 2593-2602 (2008)
Published by The Company of Biologists 2008
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Targeted disruption of β-catenin in Sf1-expressing cells impairs development and maintenance of the adrenal cortex

Alex C. Kim1,*, Anne L. Reuter2,*, Mohamad Zubair2, Tobias Else1, Kerri Serecky1, Nathan C. Bingham2, Gareth G. Lavery2, Keith L. Parker2 and Gary D. Hammer1,{dagger}

1 Department of Medicine, University of Michigan Medical School, Ann Arbor, MI 48109-0678. USA.
2 Departments of Internal Medicine and Pharmacology, UT Southwestern Medical Center, Dallas, TX 75390-8857, USA.


Figure 1
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  		Fig. 1. Developmental profile of β-catenin and canonical Wnt signaling in the embryonic and adult adrenal glands. Immunohistochemical analyses of Sf1 (using antiserum A), β-catenin and tyrosine hydroxylase (Th) were performed as described in the Materials and methods. Colorometric determinations of lacZ activity, as a surrogate for active canonical Wnt signaling, in adrenal glands from Wnt-Gal mice at E12.5, E18.5, P0 and P21 were performed as described in the Materials and methods. The black arrowheads highlight lacZ staining in the E12.5 section. Scale bars: 100 µm. The inset in the E12.5 section shows a 40x magnification of the area stained for lacZ. c, adrenal cortex; x, adrenal fetal/X-zone; m, adrenal medulla.

 

Figure 2
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  		Fig. 2. Cre expression in the adult adrenal gland. (A) Southern blot analysis of isolated genomic DNA from wild-type, Sf1/Crelow, and Sf1/Crehigh mice. (B) LacZ activity staining of adrenal glands from 6-week-old male Z/AP, Sf1/Crelow x Z/AP and Sf1/Crehigh x Z/AP, as described in the Materials and methods. Scale bars: 100 µm. (C) Quantitative-PCR analysis of hAP expression in adrenal glands from 6-week-old male wild-type, Z/AP, Sf1/Crelow x Z/AP and Sf1/Crehigh x Z/AP mice.

 

Figure 3
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  		Fig. 3. Comparison of wild type and Sf1/Crehigh-mediated β-catenin KO embryos at different developmental stages. Embryos were harvested from timed-pregnant dams at the indicated stages and sagittal sections were processed and analyzed as described in the Materials and methods. (A) Immunohistochemical analysis of β-catenin expression. (B) Hematoxylin and Eosin (H&E) staining of sections. (C) Immunohistochemical analysis of SF1 expression using antiserum B against Sf1 to identify adrenocortical progenitors. The offset shows a section from an E16.5 embryo stained with an antiserum against tyrosine hydroxylase, which detects chromaffin cell precursors. DA, dorsal aorta; G, gonad. Scale bars: 100 µm.

 

Figure 4
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  		Fig. 4. Effect of Sf1/Crehigh-mediated β-catenin KO on adrenal expression of steroidogenic enzymes. Embryos from mice of the indicated genotypes were harvested at E12.5 or E13.5, processed as described in the Materials and methods, and used for in situ hybridization analyses. Probes included cholesterol side-chain cleavage enzyme (Cyp11a1), 3β-hydroxysteroid dehydrogenase (3β-HSD) and 21-hydroxylase (Cyp21). Scale bars: 100 µm.

 

Figure 5
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  		Fig. 5. Loss of β-catenin mediated by the Sf1/Crehigh transgene results in a decreased number of adrenocortical cells at least partly due to decreased proliferation. BrdU incorporation into wild-type and Sf1/Crehigh-mediated KO mice was determined at the indicated stages as described in the Materials and methods. Mice with Sf1/Crehigh-mediated β-catenin KO appeared to have decreased BrdU staining in the region of the adrenal primordium. The graphs show quantitation of Sf1-positive cells (top) and Sf1-positive cells that were also positive for BrdU (bottom) in E12.5 and E13.5 embryos. The number of Sf1-positive cells was decreased significantly at both E12.5 (*P<0.01 versus wild type) and E13.5 (**P<0.01 versus wild type), whereas the number of doubly labeled cells was only decreased significantly at E13.5 (**P<0.01 versus wild type). Scale bars: 50 µm.

 

Figure 6
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  		Fig. 6. Sf1/Crelow-driven loss of β-catenin permits adrenal survival owing to incomplete inactivation of β-catenin. (A) Photograph of the urogenital region of newborn (P0) WT, Sf1/Crelow- and Sf1/Crehigh-β-catenin KO mice. H&E staining of sagittal sections in the region of the adrenal gland from P0 wild-type mice or from mice with β-catenin KO mediated by the indicated Sf1/Cre transgenes. (B) LacZ stained sections of Wnt-Gal and Wnt-Gal/β-catenin KOlow, as described in the Materials and methods. Scale bars: 200 µm. (C) Quantitation of active canonical Wnt signaling by comparing the area of lacZ staining versus the total adrenal area (P<0.05 versus Cre-negative littermates). Quantitative-PCR analysis of Axin2 expression in adrenal glands from wild-type and Sf1/Crelow-β-catenin KO mice (P<0.05 versus Cre-negative littermates). Ad, adrenal gland; K, kidney; l, liver. (D) Demonstration of recombination of the β-catenin gene in DNA samples from wild-type mice and those with β-catenin inactivation mediated by the Sf1/Crelow transgene.

 

Figure 7
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  		Fig. 7. Progressive decline of adrenocortical volume in Sf1/Crelow-mediated β-catenin KO mice. (A) Histological analysis of the adrenal glands from wild-type and Sf1/Crelow-mediated β-catenin KO mice at different ages. Adrenal glands were isolated from mice of the indicated genotypes as described in Materials and methods, and processed for immunohistochemical detection of Sf1 (antiserum A), β-catenin and tyrosine hydroxylase (Th). TUNEL assay was conducted on the section as described in the Materials and methods. (B) H&E staining and Sf1 immunohistochemistry (antiserum A) of adrenal from wild type and Sf1/Crelow-mediated β-catenin KO mice at 45 weeks of age. The black bar highlights the adrenal cortex. Scale bars: 100 µm. C, adrenal cortex; M, adrenal medulla.

 

Figure 8
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  		Fig. 8. Analysis of differentiated adrenocortical markers. The quantitative PCR was performed on adrenal cDNA from the 30-week-old wild-type and Sf1/Crelow-β-catenin KO-histological failure mice as described in the Materials and methods.

 

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