Proc Natl Acad Sci U S A
February 2, 1999;
Metamorphosis is inhibited in transgenic Xenopus laevis tadpoles that overexpress type III deiodinase.
One of the genes that is up-regulated by thyroid
hormone (TH) during Xenopus laevis metamorphosis encodes a type III deiodinase (D3) that inactivates TH. Transgenic X. laevis tadpoles overexpressing a GFP-D3 fusion protein were produced. These transgenic tadpoles had high levels of deiodinase activity and were resistant to exogenous TH added 1 week after fertilization. They developed normally throughout embryogenesis and premetamorphic stages but became retarded in their development late in prometamorphosis
when endogenous TH reaches its highest level. Gill
resorption were delayed and most of the animals arrested and died. One tadpole
completed its metamorphosis without resorbing its tail
. These results demonstrate that D3 can modulate the action of TH in vivo, and document the value of the new transgenic method for functional analysis of genes involved in metamorphosis.
Proc Natl Acad Sci U S A
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References [+] :
The subcellular localization of GFP and GFP-D3 proteins in X. laevis XTC cells and the expression pattern of transgenic animals. (A) GFP fluorescence in transfected XTC cell. (B) ER-Tracker fluorescence of the same cell shown in A. (C) GFP-D3 fluorescence in transfected cell next to untransfected cell. (D) ER-Tracker fluorescence of the same cells shown in C. (Bar = 20 μm, A–D.) (E) GFP fluorescence of a 2-wk-old pCS2+GFP transgenic animal. (F) GFP fluorescence of a 2-wk-old pCS2+GFP-D3 transgenic animal expressing high levels of GFP-D3. (Bar = 2 mm, E and F.)
The D3 enzymatic activities in extracts of 1-wk-old normal fertilized, pCS2+GFP-D3 nontransgenic siblings and pCS2+GFP-D3 transgenic tadpoles. Transgenic tadpoles were screened by GFP fluorescence. Values shown are the average of five individual animals in each group. The error bar represents SD.
Tadpoles overexpressing GFP-D3 are resistant to exogenous T3. One-week-old tadpoles were screened by using GFP fluorescence and treated with 10 nM T3 for 1 wk. The reshaping of their heads and the protrusion of the lower jaw are the most obvious TH-induced changes. (A) Normal fertilized animals, no T3. (B) Normal fertilized animals treated with T3. (C) pCS2+GFP-D3 transgenic animals, no T3. (D) pCS2+GFP-D3 transgenic animals treated with T3. The arrowhead points to an animal showing partial response as evidenced by slight protrusion of the lower jaw. (E) Nontransgenic siblings, no T3. (F) Nontransgenic siblings treated with T3. (Bar = 4 mm.)
Cartilage staining of tadpoles after the T3 induction assay. (A) Normal fertilized animal. (B) Normal fertilized animal treated with T3. (C) pCS2+GFP-D3 transgenic animal treated with T3. (D) Nontransgenic sibling treated with T3. M, Meckel’s cartilage; BA, branchial arch cartilages. (Bar = 1 mm.)
Arrest of pCS2+GFP-D3 transgenic animals at stage 60–61. (A) Control animal, stage 60. (B) Control animal, stage 62. (C, D, and E) Three arrested transgenic animals showing retardation of gill and tail resorption. The brackets indicate the gills. (Bar = 1 cm.)
Tailed transgenic frog overexpressing GFP-D3. The animal having just completed gill resorption (A) and the same animal 2 months later (B). (Bar = 1 cm.)
Auf dem Brinke, Subcellular localization of thyroxine-5-deiodinase in rat liver. 1980, Pubmed