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Abstract
Transgenic Xenopus laevis tadpoles that express a dominant negative form of the thyroid hormone receptor (TRDN) controlled by the cardiac actinmuscle promoter (pCar) develop with very little limbmuscle. Under the control of the tetracycline system the transgene can be induced at will by adding doxycycline to the rearing water. Pre-existing limbmuscle fibers begins to disintegrate within 2 days after up-regulation of the TRDN transgene. The muscle cells do not die even after weeks of transgene exposure when the myofibrils have degenerated completely and the tadpole is nearing death. A microarray analysis after 2 weeks of exposure to the transgene identified 24 muscle genes whose expression was altered in such a way that they might cause the muscle phenotype. These candidate genes are normally activated in growing limbmuscle but they are repressed by the TRDN transgene. Several of these genes have been implicated in mammalian myopathies. However, the expression of only one of these genes, calsequestrin, is down-regulated in 1 day and therefore might initiate the degeneration. Calsequestrin is one of several affected genes that encode proteins involved in calcium sequestration, transport and utilization in muscle suggesting that uncontrolled calcium influx into the growing limbmuscle fibers causes rhabdomyolysis. Many of the same genes that are down-regulated in the tail at the peak of metamorphic climax just before it is resorbed are suppressed in the transgenic limbmuscle in effect turning the limb growth program into a tail resorption program.
Fig. 1. The transgene is expressed exclusively in muscle. A transgenic limb (NF55) frontal section simultaneously reacted with (A) an antibody to GFP (red) and DAPI (blue); (B) an antibody to myosin (My32, green) and DAPI (blue); (C) merge of panels A and B without the DAPI. The scale bar=200 μm.
Fig. 2. Electron micrographs of the limbmuscle phenotype at NF61. (A, C) control, and (B, D) pCar/TRDN limb. Panels A and B are longitudinal sections and panels C and D are cross sections. The sibling control and pCar/TRDN tadpoles were raised for 6 weeks in Dox. The scale bar=5 μm.
Fig. 3. Design of the limb microarray experiment. Hind limbs were removed from the circled stages for RNA extraction. In the text the expression results always
compare the transgenic limbs and the frog limbs with the GFP negative NF56 controls.
Fig. 5. In situ hybridization with panelsA�F, alpha actin (BC046739) and panelsA′�F′, LIM (BC041221). PanelsA andA′, cross sections of control NF55 tail; panels B
and B′, cross sections of NF 62 tail; panels C and C′, are frontal sections of control NF56 limb. Panels D and D′, are frontal sections of NF56 limb treated for 3 days with
10 nM T3; panels E, E′, frontal section of control NF61 limb; panels F, F′, pCar transgenic limb that was induced for 6 weeks. with Dox. Scale bars=200 μm.
Fig. 6. Kinetics of limbmuscle fiber degradation after inducing the transgene by the addition of the inducer, Dox. Whole mount My32 (myosin) immunostain. Transgenic tadpoles at NF 55 were induced with Dox for 2 days, 4 days, and 1 week. Scale bar=200 μm.
Fig. 7. Early disappearance of calsequestrin mRNA in hind limbmuscle after the up-regulation of the TRDN transgene with Dox. Panels A, B, E�H are in situ
hybridization; panels C and D are the same sections as panels A and B immunostained with My32 (green). Panels A, C, E, and G are uninduced NF 55 transgenic
tadpole limb sections; panels B, D, F, and H are limb sections of NF 55 transgenic tadpoles induced for 1 day with Dox. (A, B) calsequestrin; (E, F) actin; (G, H)
SERCA Ca(2+)-ATPase. Scale bar=200 μm.
pdlim7 (PDZ and LIM domain 7 (enigma)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 55, coronal histological section, dorsal up.
casq1 (calsequestrin 1 (fast-twitch, skeletal muscle) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 55, transverse histological section of limb.
Supplementary Figure S1.
Muscle progenitor cells are present in the pCar/TRDN limb even at the climax of metamorphosis (NF61), and they incorporate Brdu. (A) Brdu positive cells (green); (B) satellite muscle cells labeled with an antibody to Pax 7 (red); C, a merged picture of A and B. Scale bar = 200 μm.
Supplementary Figure S2.
In situ hybridization shows that titin (A, B) and desmin (A′, B′) gene expression are not affected by the transgene. (A, A′) NF61 limb without Dox; (B, B′) pCar/TRDN NF61 limb after 6 weeks of Dox treatment; (C, C′) same sections as B, B′ but immunostained with My32 (myosin). Scale bar = 200 μm.
Supplementary Figure S3.
Kinetics of gene expression after addition of Dox to pCar/TRDN tadpoles at NF56. (A1–D1) calsequestrin 1; (A2–D2) actin alpha 1; (A3–D3) phosphoglycerate mutase 2. (A1–A3) control; (B1–B3) 2 days of Dox; (C1–C3) 4 days of Dox; (D1–D3) 1 week of Dox. Sections of bone can be seen in some panels (black arrow) at background levels. Scale bar = 200 μm.
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