Rabelo EM et al. (1994), Interplay between thyroid hormone and estrogen ...

XB-ART-21729

Mech Dev 1994 Jan 01;451:49-57.

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Interplay between thyroid hormone and estrogen in modulating expression of their receptor and vitellogenin genes during Xenopus metamorphosis.

Rabelo EM , Baker BS , Tata JR .

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Many postembryonic developmental processes are regulated by an intricate interplay among hormones and growth factors. Thyroid hormone (TH) and estrogen are well known to be individually and obligatorily required for the initiation and progression of amphibian metamorphosis and vitellogenesis. However, whether or not a possible interplay between these two hormones would affect these two developmental processes is not known. Here we report on how triiodothyronine (T3) enhances the precocious activation of vitellogenin (Vit) genes by estradiol (E2) in Xenopus tadpoles during metamorphosis. Using a combination of filter hybridization, RNase protection assay and in situ hybridization, we first show that very low doses (10(-9) M) of exogenous T3 will autoinduce thyroid hormone receptor (TR) mRNA in several tissues of premetamorphic tadpoles. The same treatment enhances and accelerates the precocious activation of the silent vitellogenin genes by E2 at metamorphic climax (stages 60-64) but not before mid-metamorphosis (stages 56-58). This developmental stage dependency may be explained by our finding that, under the same experimental conditions, T3 fails to alter the autoinduction of ER mRNA at mid-metamorphosis but strongly potentiates it at metamorphic climax. Thus a developmental stage specific interplay between thyroid hormone and estrogen determines the kinetics and extent of activation of vitellogenin and estrogen receptor genes during Xenopus postembryonic development.

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Species referenced: Xenopus laevis
Genes referenced: mt-tr thra tra trna vit vtga2 vtgb1

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	Fig. 1. Upregulation by T 3 of TR mRNA in different tissues of premetamorphic Xenopus tadpoles. The figure shows dark-field imaging of localization by in situ hybridization of 35S-labelled antisense Xenoptts TRa cRNA probe (Kawahara et al., 1991) in saggital sections of brain (a,b), intestine (c,d) and liver (e,f) of stage 52 tadpoles. (a,c,e) Control (untreated) tadpoles. (b,d,f) Tadpoles treated for 4 days with 10-9M T 3. Sense probe gave virtually no hybridization signal and the autoradiographs are thus not shown. Arrows in liver sections (e,f) indicate an artefact produced in black-and-white photography by pigmentation (Pi) surrounding the bulk of the parenchymal cells (Pa) of the liver. The sections were exposed for one week for autoradiography. Other details as in Kawahara et al. (1991). Bars represent 100 um.
	Fig. 2. Autoinduction by T 3 of TR mRNA in newly emerging hind limb of stage 53 Xenopus tadpole. The figure shows (a,c) bright- and (b,d) dark-field imaging of a saggital section of a region comprising the limb bud or growing hind limb (indicated by arrowheads) of a stage 53 tadpole after 5 days of immersion in 10-9M T 3. (a,b) Untreated control; (c,d) Ta-treated. The section of untreated control shows that the limb bud is small and poorly developed in tadpoles at this stage. Note that 5 days of T 3 treatment at stage 53 has accelerated hind limb bud to reach a stage equivalent to 54/55 of untreated tadpoles. Other details as in Fig. 1. Bars represent 100um.
	Fig. 3. Developmental stage-dependent enhancement by T 3 of activation by E 2 of Vit gene expression in Xenopus tadpole liver. Batches of 6-14 tadpoles at stage 56 (slots 1-3) or stage 60 (slots 4-6) at the commencement of the experiment were treated or not with E 2 alone or with T 3 and E 2 together. After 5 days total RNA was extracted from liver pooled from each batch and analyzed by slot-blot hybridization (Baker and Tata, 1990). The RNA samples were hybridized either to: A) a 1.53 kb PstI-HindlII fragment of Xenopus cytoskeletal actin cDNA (Mohun and Garret, 1987) or (B) a 1.40 kb HindlII-EcoRI cDNA probe derived from the Xenopus vitellogenin B1 cDNA containing plasmid pXlvcl0 (Wahli et al., 1979). 2 tzg of total liver RNA along with 2 /zg of yeast tRNA were loaded on each slot before hybridization with the 32p-labelled Vit or actin cDNAs. RNA samples: slots 1,4: control (no treatment); 2,5: 10-TM E 2 alone for 5 days; 3,6: tadpoles immersed in 10-9M T 3 for 1 day, then in 10-gM T 3 and 10-TM E 2 together for 4 days; 7: tRNA alone.
	Fig. 4. T 3 strongly potentiates the induction of Vit mRNA by E 2 in livers of Xenopus tadpoles at late stages of metamorphic climax. Vit mRNA accumulation was visualized by in situ hybridization in saggital sections of livers of stage 63/64 tadpoles treated or not with 10-7M E 2 alone or pre-treated with T 3 for different periods of time. Where animals were pre-treated with T3, it was maintained in the water throughout the period of exposure to E 2. Hybridization was performed with 35S-labelled sense and antisense cRNA probes derived from pXlvc 10 Xenopus vitellogenin B1 cDNA (Wahli et al., 1979). Only the dark-field imaging with the antisense probe (a,c-f) is shown since the sense probe gave a negative signal after 1 week of exposure for autoradiography. (a) Dark-field image of control (untreated) tadpole liver. (b) Bright-field image of liver of control tadpole. (c) Liver of tadpole after 1 day treatment with E 2 alone. (d) 1 day T 3 followed by l day E 2. (e) 2 days E 2 alone. (f) 1 day T 3 followed by 2 days E 2. (g) 4 days E 2 alone. (h) 2 days T 3 followed by 2 days E 2. Arrows indicate pigmentation artefact surrounding the liver (see also Fig. le,f). Bars represent 100 um.
	Fig. 5. T 3 enhances the autoinduction of ER mRNA by E 2 in stage 60 (lanes 4-6) but not 56 (lanes 1-3) Xenopus tadpole liver. ER mRNA was determined by RNase protection assay of 20 gg of the same total RNA samples used for slot-blot determination of Vit mRNA, as shown in Fig. 3. The hybridization probe was 32p-labelled 167 bp BglII-HindIII fragment from Xenopus ER cDNA (Weiler et al., 1987) while a 32p-labelled 58 RNA probe (Xu and Tata, 1992) was used as a RNA loading control. The arrows show the 197 bp free probe, the 167 bp protected band for Xenopus ER and the smaller 5S RNA bands at the bottom of the gel. Lanes: 1,4: control (untreated) tadpoles; 2,5: treated with 10-7M E 2 alone; 3,6: pre-treatment with 10-9M T 3 for 1 day, followed by T 3 and E 2 together for 4 days; 7: tRNA alone.