Endo T et al. (1997), Shh expression in developing and regenerating l...

XB-ART-16445

Dev Dyn 1997 Jun 01;2092:227-32. doi: 10.1002/(SICI)1097-0177(199706)209:2<227::AID-AJA8>3.0.CO;2-K.

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Shh expression in developing and regenerating limb buds of Xenopus laevis.

Endo T , Yokoyama H , Tamura K , Ide H .

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The zone of polarizing activity (ZPA) is a specialized region involved in the antero-posterior (A-P) axis formation in chick and mouse limb buds. The existence of ZPA in the posterior margin is suggested in Xenopus hindlimb buds because 180 degree rotation of the distal limb tip induces the supernumerary limb. In this study, we investigated the expression of Sonic hedgehog (shh), a molecular marker for ZPA, in Xenopus developing limb buds and regenerating blastemas by whole-mount in situ hybridization. Although shh was expressed in the posterior margin of the limb bud like in chicks, its expression domain did not correspond to the ZPA map of Xenopus hindlimb buds. shh expression was distant from the ZPA at stage 53 in particular. To clarify the difference between the shh expression domain and ZPA, we examined shh expression in 180 degree rotated limb buds. As a result, ectopic shh expression was newly induced in the proximal region to its original expression domain. These results suggest that ZPA is accompanied by shh expression as in chick limb buds. Furthermore we examined shh expression in regenerating blastemas. shh was reexpressed in the posterior margin of the blastema. This result supports the possibility that ZPA also exists in the regenerating blastema.

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Species referenced: Xenopus laevis
Genes referenced: shh zp2

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	Fig. 2. shh expression in developing hindlimb buds at stage 50 (a, b), stage 51 (c), stage 52 (d), stage 53 (e), and stage 55 (f). a,c: Hybridization was done at 60. b: Hybridization was done at 50. Arrowheads indicate the expression domains. Scale bars 5 200 μm for a, b, c, d, and e, and 250 μm for f. A: Anterior; P: posterior.
	Fig. 3. shh expression in 180degree rotated limb buds. a: The stage 53 limb bud before amputation. Arrows indicate the amputation level. b,c: A 180rotated limb bud viewed from the ventral side (b) and the ventro-posterior side (c) 6 days after rotation. d: Control limb bud. The amputated distal tip was returned to the stump in the proper orientation and observed 6 days after amputation. e: Diagram showing shh expressions in the stage 53 limb bud before rotation (left) and 6 days after rotation (right). Arrowheads and the shaded area indicate shh expression domains. The stippled area indicates high polarizing activity. Vertical solid lines indicate the amputation planes (we determined the amputation planes by the distribution of melanophores). Scale bar 5 200 μm. A: Anterior; P: posterior.
	Fig. 4. shh expression in regenerating blastemas. The blastemas were formed from the limb buds amputated at stages 53 (a), 55 (b), and 57 (c). Arrowheads indicate the expression domains. Vertical solid lines indicate the amputation planes (prospective ankle level). Scale bars 5 200 μm for a and b, and 400 μm for c. A: Anterior; P: posterior.