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First published online 24 September 2003
doi: 10.1242/dev.00795

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Novel roles of Fgfr2 in AER differentiation and positioning of the dorsoventral limb interface

Marat Gorivodsky* and Peter Lonai{dagger}

Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot, Israel



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  		Fig. 1. Defective limb morphogenesis in perinatal chimeras. Fixed whole-mount preparations, with the exception of C, which is labeled for ß-galactosidase. Arrows indicate digits or mini-limbs with reversed polarity. A-D are forelimbs, E and F are hindlimbs.

 


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  		Fig. 2. Skeletal defects in perinatal chimeras. (A-C,E,F) Alcian Blue and Alizarin staining. (D) Alizarin staining only. A and E show control fore- and hind limbs, respectively. B-D show chimeric forelimbs; F shows a chimeric hindlimb.

 


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  		Fig. 3. Mid-gestation chimeric embryos. (A) Control Fgfr2+/+{leftrightarrow}Fgfr2+/+ chimera; (B) moderate and (C) high-level Fgfr2{Delta}2/{Delta}2{leftrightarrow}Fgfr2+/+ aggregation chimeras. All were recovered at E10.5 and stained for ß-galactosidase. Limb development is absent in the high-grade mutant chimera (C), and is impaired in the medium-grade mutant chimera (B; arrows).

 


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  		Fig. 4. Mutant cells do not colonize the AER. Localization of Fgfr2{Delta}2/{Delta}2 mutant cells in chimeric limb buds, as shown by (A,C) whole-mount ß-galactosidase staining and (B,D,E,F) histological sections of ß-galactosidase-stained limb buds. (A,B,E) Control chimera; (C,D,F) mutant chimera. Arrowheads indicate the AER, arrows point to ß-galactosidase-positive cells in the surface ectoderm. Note that in D and F there is little contribution of mutant cells to the surface ectoderm, and none to the AER.

 


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  		Fig. 5. Interruption and ectopic displacement of the AER, and low-level cell death in Fgfr2{Delta}2/{Delta}2{leftrightarrow}Fgfr2+/+ chimeras. (A,B) ß-galactosidase staining of and (C-L) whole-mount in situ hybridization in control (C,G,I,K) and Fgfr2{Delta}2/{Delta}2{leftrightarrow}Fgfr2+/+ mutant chimeric (A,B,D,F,H,J,L) mice. (M,N) TUNEL assay for the detection of apoptotic cells in control (M) and Fgfr2{Delta}2/{Delta}2{leftrightarrow}Fgfr2+/+ mutant chimeric (N) mice. Staining and probes are shown on the left side of the images. Note the disruption and displacement of AER markers in mutant chimeras (arrowheads in E,F and L).

 


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  		Fig. 6. Reduced expression of mesenchymal markers in the chimeric limb bud. Whole-mount in situ hybridization. (A,B) Msx1 signals in the wild-type and chimeric progress zone mesenchyme. (C,D) Shh expression in the zone of polarizing activity (ZPA). (A,C) Wild type; (B,D) Chimera. Arrowhead indicates a faint Shh signal in the ZPA of the mutant chimeric forelimb bud.

 


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  		Fig. 7. Downregulation of En1 and Wnt7a expression, and the ectopic localization of En1-positive cells in the dorsal ectoderm. (A-C) En1 and (D,E) Wnt7a hybridization in wild type (A,D) and chimeras (B,C,E). B shows a ventral view, and C the dorsal view of En1 hybridization in the chimeric limb bud. Arrows indicate patches formed by En1 or Wnt7a in the dorsal ectoderm.

 


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  		Fig. 8. En1 and Wnt7a expression in the limb fields of (non-chimeric) Fgfr2{Delta}2/{Delta}2 E9.5 mutant embryos. A and B show En1 (arrows), and C and D show Wnt7a (arrowheads) in situ hybridization. (A,C) Wild type. (B,D) Mutant embryos. Note that neither marker is expressed in the limb fields of the mutant.

 


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  		Fig. 9. The mutually exclusive localization of (A) Fgf8- or (B) En1-positive wild-type cells (purple) and ß-galactosidase-positive mutant cells (pink) is shown by double staining. Note, Fgf8- and En1-positive cells detected by in situ hybridization are observed in the AER; ß-galactosidase staining with the pinkish Salmon-gal chromophore indicates mutant cells. Arrow indicates dorsally shifted ectopic AER fragment.

 

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© The Company of Biologists Ltd 2003