Kawahara A et al. (1991), Developmental and regional expression of thyroi...

XB-ART-24637

Development 1991 Aug 01;1124:933-43.

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Developmental and regional expression of thyroid hormone receptor genes during Xenopus metamorphosis.

Kawahara A , Baker BS , Tata JR .

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A characteristic feature of the obligatory control of amphibian metamorphosis by thyroid hormones is the early acquisition of response of tadpole tissues to these hormones well before the latter are secreted, with 'exponentially' increasing hormonal sensitivity upon the onset of metamorphosis. We have therefore analyzed the expression of the two thyroid hormone receptor genes (TR alpha and beta) before, during and after metamorphosis in Xenopus tadpoles and froglets. Using non-cross-hybridizing cRNA probes for 5' and 3' sequences of Xenopus TR alpha and beta transcripts for RNAase protection assays, the two mRNAs can be detected in tadpoles as early as stage 39. Their concentration increases abruptly at stage 44 and continues to increase differentially at the onset of metamorphosis (stage 55) and through metamorphic climax at stages 58-62, after which they decline upon completion of metamorphosis at stage 66. Quantitative densitometric scanning of autoradiograms showed that, although the concentration of TR beta transcripts is about 1/30th of that of TR alpha mRNA at stages 44-48, depending on the region, it accumulates 3-10 times more rapidly than does the alpha isoform during further development. A substantial proportion of the increase in TR beta mRNA is localized to the head region of tadpoles. Using the hormone-binding domain (HBD) and 3' end of Xenopus TR alpha cRNA as probe for in situ hybridization, the highest concentration of TR transcripts in stage 44 tadpoles is seen in the brain and spinal cord. High concentrations of mRNA are also present in the intestinal epithelium and tail tip, tissues programmed for regression. At later stages (55 onwards), strong hybridization signals are also exhibited by hindlimb buds. This pattern persists through metamorphic climax, after which TR mRNAs decline in all tissues to low levels in froglets at stage 66. In developing froglets, TR transcripts were detected in large amounts in the cytoplasm of stage 1 and 2 oocytes but the rate of their accumulation did not increase with further oocyte growth. This observation raises the possibility that the response to thyroid hormones at early stages of tadpoles (42-44) may be due to TR synthesized on maternally derived mRNA. Exposure of tadpoles at premetamorphic stages (48-52) to exogenous thyroid hormone (T3) substantially enhanced the accumulation of TR mRNA, especially that of TR beta message, which could explain the accelerated increase in sensitivity of tadpoles to thyroid hormones at the onset of natural metamorphosis.(ABSTRACT TRUNCATED AT 400 WORDS)

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Species referenced: Xenopus laevis
Genes referenced: actc1 actl6a hbd mt-tr tbx2 thra thrb tra trna uqcc6

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	Fig. 1. Diagrammatic representation of fragments of cloned cDNAs to Xenopus TRa and P, and ER mRNAs to generate cRNA probes for RNAase protection assay (fragments I and IV) and in situ hybridization (fragments II, III, V). HBD: hormone-binding domain (•); DBD: DNAbinding domain (S); The length of the fragments and their position is given by nucleotide numbers and restriction sites. Cloning sites ( • ) are also indicated. Details of these and other probes are given in Materials and methods.
	Fig. 2. Detection by RNAase protection assay of TRa- and fi transcripts in different regions and tissues of Xenopus tadpoles and froglets at different developmental stages before, during and after metamorphosis. 10 (C) or 20 (A,B) ng of total RNA from whole tadpoles (stages 28, 39, 44, 48), the head (h), middle (m) and tail (t) portions of tadpoles, hindlimbs (li) of froglets and liver (1) of adult Xenopus were hybridized to lxlCPctsmin"1 of 32P-labelled cRNA probes (Pr) for (A) TRa- cRNA, (B) TR/3 or (C) cardiac actin cRNA. Unhybridized RNA and probe were digested with RNAase A and Tl and the protected fragments resolved by electrophoresis on sequencing gels with appropriate size markers (Zinn et at. 1985). tRNA was used as a negative control (not shown) and the complete digestion of the unprotected probe was verified for each run. Autoradiograms were exposed for 16 h for A and B and for 30min for C. Arrow indicates the cRNA probe lengths and the expected size of the protected probes (260, 261 nt, respectively) for TRa-, TR0 CRN As (see Fig. 1). The multiple protected bands of actin cRNA probe are indicated by small arrows in C.
	Fig. 3. Dark-field imaging of localization by in situ hybridization of TR mRNA in different tissues of stage 44 (premetamorphosis) and 55 (prometamorphosis) Xenopus tadpoles. Sagittal sections were prepared from different regions of tadpoles hybridized to a mixture of antisense 35S-labelled cRNA probe 3 (1x10^5 cts min-1 uL-1), as shown in Fig. 1, unhybridized probe digested with RNAase and the distribution of transcripts visualized by autoradiography. Sense probes gave virtually no hybridization and the autoradiographs are not shown here. (A) Head region of stage 44 tadpole; (B) middle or trunk region, stage 44; (C) tail, stage 44; (D) trunk and tail with hindlimb bud in stage 55 (prometamorphosis). Left and right sides of each image are the ventral and dorsal sides of the tadpoles, respectively. Bright-field micrographs were also prepared (not shown) and used for identifying the different organs and tissues for which the abbreviations are as follows: Br, brain; Ct, connective tissue; Gi, gill; He, heart; Hlb, hindlimb bud; In, intestinal epithelium; Ki, kidney; Li, liver; Mu, muscle; No, notochord; Pf, pharyngeal floor epithelium; Ph, pharyngeal cavity; Sc, spinal chord; Sk, skinj.Tf, tail fin; Tm, tail muscle. Note that pigmented layers (Pi) and food particles (Fp) appear as bright zones, also seen with sense probe controls. Autoradiograms exposed for 9 days.
	Fig. 4. Composite assembly of sagittal sections of (A) bright- and (B) dark-field imaging of Xenopus tadpoles at different developmental stages to show the localization of TR mRNA using the labelled TRa cRNA as probe. (1) Stage 44; (2) stage 56 (prometamorphosis); (3) stage 62 (late-metamorphic climax). The three stages shown are to the same scale. Abbreviations: Ne, nasal epithelium; Oe, oesophagus; Gb, gall bladder; Go, gonad; Hlc, hindlimb connection. Other abbreviations as in Fig. 3.
	Fig. 5. Localization of TR mRNA in stages 1 and 2 oocytes visualized in sections through Xenopus froglet ovary hybridized to Xenopus TRa cRNA. (A) Antisense probe; (B) sense probe. Autoradiograms exposed for 3 and 9 days for antisense and sense probes, respectively.
	Fig. 6. Preferential autoinduction of TRb mRNA by T3 in different regions of Xenopus tadpoles, as revealed by RNAase protection assays. 20 fig of RNA from head, middle and tail regions of stage 52 tadpoles, treated or not with 2X10~9M T3 or 10~8M oestrogen (E2) were hybridized to 32P-labelled TRo- or TR/3 cRNA probes and analyzed as described in Fig. 2. Autoradiograms of TRa and TRb probes were exposed for 16 and 40 h, respectively. AH abbreviations and sizes of probes and protected fragments also as in Fig. 2.