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Thyroid hormone (TH) is required for limb development in Xenopus laevis. Specific cell types in the growing limb were targeted for expression of a dominant negative form of the TH receptor by sperm-mediated transgenesis. Limb muscle development, the innervation of muscle from the spinal cord, and cartilage growth can be inhibited without affecting patterning of the limb or differentiation of other cell types. Remodeling of the skin occurs late in metamorphosis after the limb has formed. The coordination of these independent programs is affected in part by the control that TH exerts over DNA replication in all cell types of the limb.
Fig. 1. Hind limb development in X. laevis tadpoles. The NF numbers shown are those assigned by Nieuwkoop and Faber (6) to normal hind limb development. An arrested hind limb bud of a tadpole grown continuously in the presence of 1 mM methimazole (a goitrogen) for 16 months is shown. The inhibitor was added at the end of embryogenesis (1 week after fertilization, NF 46), when the tadpole begins to feed. This tadpole developed a large goiter (arrow). (Scale bar: 0.5 mm.)
Fig. 2. TH controls bone and cartilage formation (A and D), muscle development (B and E), and DNA replication (C and F) in the limb. Premetamorphic NF 55 tadpoles were grown for 1 week in 1 mM methimazole. Then 10 nM T3 was added to the rearing water of one group of tadpoles (D–F) but not others (A–C) for 3 days. The hind limbs were fixed, and frontal sections were prepared for in situ hybridization (15). sox-9 (CF285736; A and D), alpha 1 skeletal muscle actin (BC046739; B and E), and cyclin D2 (CA792959; C and F) were used. (Scale bar: 200 μm.)
Fig. 3. Transgenic NF 63 tadpoles at the climax of metamorphosis showing the typical limb phenotypes that occur when these three promoters drive a TRDN transgene. (A) Wild type. (B) Constitutive NβT/TRDN paralyzed phenotype (ventral view). (C) Constitutive collagen/TRDN “pig†phenotype. (D) Inducible pCar/TRDN paralyzed phenotype. This F1 progeny was induced with 50 μg/ml doxycycline from NF 52 to NF 63. (Scale bar: 2 mm.)
Fig. 4. Tadpoleskin covering the limb changes to adult skin at the climax of metamorphosis. Shown are in situ hybridizations of limb cross sections (14) with larval keratin (X04807; A–C) and adult keratin (X02895; D–F). Stages were as follows: NF 53 (A and D), NF 59 (B and E), and NF 62 (C and F). (Scale bar: 200 μm.)
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