Novel roles ofFgfr2in AER differentiation and positioning of the dorsoventral limb interface

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Abstract
The epithelial b variant of Fgfr2 is active in the entire surface ectoderm of the early embryo, and later in the limb ectoderm and AER, where it is required for limb outgrowth. As limb buds do not form in the absence of Fgfr2, we used chimera analysis to investigate the mechanism of action of this receptor in limb development. ES cells homozygous for a loss-of-function mutation of Fgfr2 that carry aβ -galactosidase reporter were aggregated with normal pre-implantation embryos. Chimeras with a high proportion of mutant cells did not form limbs, whereas those with a moderate proportion formed limb buds with a lobular structure and a discontinuous AER. Where present, the AER did not contain mutant cells, although mutant cells did localize to the adjacent surface ectoderm and limb mesenchyme. In the underlying mesenchyme of AER-free areas, cell proliferation was reduced, and transcription of Shh and Msx1 was diminished. En1 expression in the ventral ectoderm was discontinuous and exhibited ectopic dorsal localization, whereas Wnt7a expression was diminished in the dorsal ectoderm but remained confined to that site. En1 and Wnt7a were not expressed in non-chimeric Fgfr2-null mutant embryos, revealing that they are downstream of Fgfr2. In late gestation chimeras, defects presented in all three limb segments as bone duplications, bone loss or ectopic outgrowths. We suggest that Fgfr2 is required for AER differentiation, as well as for En1 and Wnt7a expression. This receptor also mediates signals from the limb mesenchyme to the limb ectoderm throughout limb development, affecting the position and morphogenesis of precursor cells in the dorsal and ventral limb ectoderm, and AER.