Fairclough L and Tata JR (1997), An immunocytochemical analysis of the expressio...

XB-ART-16399

Dev Growth Differ 1997 Jun 01;393:273-83. doi: 10.1046/j.1440-169x.1997.t01-2-00003.x.

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An immunocytochemical analysis of the expression of thyroid hormone receptor alpha and beta proteins during natural and thyroid hormone-induced metamorphosis in Xenopus.

Fairclough L , Tata JR .

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Amphibian metamorphosis is characterized by the upregulation of thyroid hormone receptor (TR) mRNA in all tissues of tadpole during both the natural and thyroid hormone (TH)-induced development. The two TR genes, termed alpha and beta, are members of a large multigene family of nuclear receptors related to the cellular homolog of the oncogene c-erbA. The phenomenon of upregulation is more marked for the beta than the alpha isoform. To determine whether or not the auto-induction of the transcripts is paralleled by that of TR proteins, non-cross-reacting monoclonal antibodies were prepared against Xenopus laevis TR alpha and beta (xTR alpha, beta) in order to analyze immunocytochemically their expression and localization. Three tadpole tissues that exemplify three major consequences of gene re-programing during natural and TH-induced metamorphosis were studied: (i) Liver that undergoes extensive functional switching; (ii) small intestinal epithelium that exhibits substantial cell death prior to major structural and biochemical modifications; and (iii) hind limb-bud as an example of de novo morphogenesis. It was shown that xTR alpha protein is generally more abundant in these tissues, and its expression is developmentally and hormonally less regulated, than is xTR beta. The auto-induction of xTR beta was particularly intense at 5 days after administration of triiodo-thyronine (T3) to both pre-metamorphic (stage 52) tadpoles and at the onset of natural metamorphosis (stage 55). In the developing hind limb-bud at both stages the upregulation of TR beta is topologically restricted, being particularly intense in dense pockets of cells, presumably rich in chondrocytes. It was concluded that the distribution and expression of xTR alpha and beta proteins match partially, but not fully, those of their transcripts during natural and hormone-induced metamorphosis.

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Species referenced: Xenopus laevis
Genes referenced: ednra tdrd6 thra thrb

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	Fig.1. Antigens used for raising and characterizing antibodies against xTRa and (3. A, full-length xTRa produced as a fusion protein (Machuca eta!. 1995; Ulisse eta!. 1996). B, full-length xTR(3 produced as a fusion protein. C, synthetic 25-mer peptide representing a sequence in the hinge region that is unique to xTR(3 (Yaoita eta/. 1990). A/8, N-terminal domain; DBD, DNA-binding domain; HR, hinge region; LBO, ligand-binding domain.
	Fig. 2. Western blot characterizing antibodies (Ab) against xTRa and ~ pGEX fusion proteins. Antibodies: Lanes 1, polyclonal Ab against recombinant full-length xTRa fusion protein; 2 and 3, MAb against xTRa; 4, polyclonal Ab against xTR~-specific peptide; 5 and 6, MAb against xTR~ peptide; 7 and 8, control MAb against ~-galactosidase. xTR antigens: Lanes 1 ,2,5,7: xTRa fusion protein; 3,4,6,8: xTR~ fusion protein. The two antigens are as described in Fig. 1. Bold arrowheads denote the positions of xTRa and ~ pGEX fusion proteins. Small arrows marked 1, 2, 3 and 4 give the positions of molecular weight marker proteins of 220, 97, 66 and 46 kDa, respectively. Note that a value of 27 kDa, which is the size of the pGEX protein, has to be deducted from the values for fusion proteins to derive the molecular weights of xTRa and~ alone.
	Fig. 3. Immunocytochemical localization of xTRa and xTRB in stage 55 (onset of metamorphosis) Xenopus tadpole liver (a,c,e,g) and small intestine (b,d,f,h). Panels a-d show the reaction with monoclonal antibodies against xTRa (a, b) and B (c,d); (e,f) controls for liver and small intestine, respectively, using a Mab against b-galactosidase; (g,h) histological staining of liver and small intestine, respectively, in which the dense dark bodies are nuclei. Arrowheads point to a few representative nuclei of liver parenchymal and intestinal epithelial cells that are positive for xTRa and 13. Bar indicates magnification, which is the same for all figures, except Fig. 6.
	Fig. 4. Strong upregulation by T3 of xTRB protein in liver (a,b,e,f,) and intestinal epithelium (c,d,g,h). Stage 52 (a-d) and 55 (e-h) Xenopus tadpoles were either untreated (a,c,e,g) or exposed to 2.5 nM T3 for 5 days before the tissues were sectioned for immunocytochemical analysis. Arrowheads indicate a few sites of xTRB localization. Note the marked morphological change in intestinal structure induced by T3 (h), which is similar to that seen at natural metamorphic climax (stages 57-60).
	Fig. 5. Spatially preferential localization of xTRB in hind limb-buds whose growth is accelerated by T3 administered for 5 days to stages 52 and 55 Xenopus tadpoles. (a,b,e,f) stage 52 tadpoles, (c,d,g,h) stage 55 tadpoles. a-d, control; e-h, T3-treated. (a,c,e,g) immunocytochemical localization of xTRB; (b,d,f,h) histochemical staining. Arrowheads indicate spatially restricted dense pockets of cells particularly enriched in xTRB. Note that the limb-bud in (d) is sectioned through a different plane from that of other sections, also that the magnification of (h) is different from that of other sections (x 100 instead of x 400).