Ishizuya-Oka A et al. (1997), Temporal and spatial regulation of a putative t...

XB-ART-17128

Dev Genet 1997 Jan 01;204:329-37. doi: 10.1002/(SICI)1520-6408(1997)20:4<329::AID-DVG4>3.0.CO;2-9.

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Temporal and spatial regulation of a putative transcriptional repressor implicates it as playing a role in thyroid hormone-dependent organ transformation.

Ishizuya-Oka A , Ueda S , Shi YB .

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Thyroid hormone (T3) induces both larval cell death and adult cell proliferation and differentiation during amphibian metamorphosis. We have previously isolated a bZip transcription factor (TH/bZip) as a T3 response gene in the metamorphosing Xenopus intestine. We demonstrate that the Xenopus TH/bZip gene is a direct T3-response gene and ubiquitously regulated by T3 in tadpoles. Developmental in situ hybridization analyses have shown that TH/bZip gene is regulated in a cell-type-specific manner that correlates with tissue transformation. In particular, it is found to be expressed in the larval intestinal epithelial cells prior to their apoptotic degeneration and in the proliferating adult cell types. However, the gene is repressed again upon adult cell differentiation. This regulation pattern mimics that of the thyroid hormone receptor (TR)beta genes. Since the TH/bZip gene is a direct T3-response gene, such a correlation suggests that TR beta may be involved in the regulation of the TH/bZip gene. More importantly, in situ hybridization reveals a strong spatiotemporal correlation of TH/bZip expression with the tissue-specific remodeling in the intestine, suggesting that TH/bZip gene may participate, depending on the cell types, in both inducing apoptosis and stimulating cell proliferation. A similar role has been reported for the proto-oncogene c-myc, another leucine-zipper-containing transcription factor, in tissue culture cell systems.

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Species referenced: Xenopus laevis
Genes referenced: myc rpl8 thibz

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	Fig. 1. Schematics of the intestinal development in Xenopus laevis. The tadpole small intestine has a single epithelial fold (Ty), the typhlosole, where the undeveloped immature connective tissue (CT) is concentrated. The simple columnar epithelium (E) and the connective tissue are surrounded by a thin layer of immature muscles (M). By contrast, a frog has multiple intestinal folds (Fo) with elaborate connective tissue. The folds are surrounded by a well-developed, thick layer of muscles. Filled circles proliferating adult epithelial cells; open circles: apoptotic primary epithelial cells. The developmental stages are based on Nieuwkoop and Faber (1956).
	Fig. 2. The TH/bZip gene is a direct T3 response gene. (A) Kinetics of T3 induction. Stage 54 tadpoles were treated with 5 nM T3 and RNA was isolated from the intestine. Northern blot hybridization showed that the TH/bZip mRNA could be detected as early as 8 hr after T3 addition. The positions of 28S and 18S rRNAs were indicated by the bars on the side. Because of the weak signal, the autoradiogram was scanned using a phosphorimager in order to reveal the low level of TH/bZip mRNAafter 8 hr of T3 treatment. (B) T3-induction of TH/bZip gene is independent of new protein synthesis. Stage 54 tadpoles were treated with 5 nM T3 in the presence or absence of protein synthesis inhibitors (CHX) for 12 hr. The RNA was isolated and analyzed by Northern blot hybridization. Ten μg RNA was used per lane. The smeary signals migrated faster than the full-length mRNA band were most likely due to partial degradations of the mRNA. The hybridization of the duplicate filters with rpL8 served as a loading control.
	Fig. 3. The Xenopus TH/bZip gene is highly expressed during both natural and T3-induced metamorphosis. (A) Developmental expression of TH/bZip mRNA. Each lane had 10 μg RNA except the lane for the tail at stage 64, which had only 5 μg. (B) TH/bZip expression during T3 treatment. Stage 54 tadpoles were treated with 5 nM T3 for 0–5 days, and RNAwas isolated from the intestine. 5 μg RNAwas used per lane. The hybridization with rpL8 served as a loading control. Bars indicate the position of the 18S and 28S rRNA. It should be pointed that the plasma T3 concentration peaked at stage 62 (Leloup and Buscaglia, 1977), when the TH/bZip mRNA was at the maximal level, and gradually decreased toward the end of metamorphosis as the TH/bZip mRNA was down-regulated. By contrast, a constant level of T3 was maintained during in vitro treatment. This decrease in plasma T3 is probably not critical for the down-regulation of the TH/bZip gene as the mRNAlevel was repressed by stage 64 when the plasma T3 was still at 45% of the peak level (Leloup and Buscaglia, 1977).
	Fig. 4. In situ hybridization analysis of TH/bZip gene in the small intestine before stage 60. (A) Small intestine at stage 55, hybridized with the antisense TH/bZip probe. The intestinal wall consists of the simple columnar epithelium (E) facing the lumen (L), the immature connective tissue (CT), and a thin layer of muscles (M). No specific signals are detected in any tissues. 3570. (B) Control section of the small intestine at stage 55, hybridized with the sense probe. No specific signals are detected. 3570. (C) Small intestine at stage 59, hybridized with the antisense probe. Signals are detected in the epithelium (arrows), but not in the connective tissue and the muscles. 3570. (D) Control section of the small intestine at stage 59, hybridized with the sense probe. No specific signals are detected. 3570. Bars: 20 μm.
	Fig. 5. In situ hybridization analysis of TH/bZip gene in the small intestine during stages 60–61. (A) Cross section of the small intestine at stage 60. The connective tissue begins to increase in thickness outside the typhlosole (Ty). Signals are detected not only in the epithelium (E) facing the lumen (L), but also in the connective tissue (CT) and the muscles (M). 3160. (B) Control section of the small intestine at stage 60, hybridizaed with the sense probe. No specific signals are detected. 3160. (C) Lower region of the typhlosole at stage 60, hybridized with the antisense probe. Positive cells in the connective tissue cells are localized in the bottom of the typhlosole (arrows). 3525. (D) Intestinal wall at stage 60, hybridized with the antisense probe. Islets of adult epithelial cells (arrowheads) appear between the larval epithelium (LE) and the connective tissue. They are intensely positive. 3525. (E) Intestinal epithelium at stage 61, hybridized with the antisense probe. The adult epithelium (AE) remains positive, but the degenerating larval epithelium is negative. 3525. Bars: 20 μm.
	Fig. 6. In situ hybridization analysis of TH/bZip gene in the small intestine during stages 62–66. (A) Cross section of the small intestine at the early phase of stage 62, hybridized with the antisense probe. The typhlosole (Ty) becomes shorter, while the adult intestinal folds (Fo) are developing. The larval epithelium is almost completely replaced by the adult epithelium (AE), except for the crest region of the typhlosole and the folds (arrows). Signals in the muscles (M) and the connective tissue (CT), especially in the typhlosole, are stronger than those at stage 60 in Fig. 5A, while signals in the epithelium are weaker. 3130. (B) Control section of the small intestine at the early phase of stage 62, hybridized with the sense probe. No specific signals are detected. 3130. (C) Developing intestinal fold of the small intestine at stage 62, hybridized with the antisense probe. Signals are intense in the muscles and the connective tissue but not in the adult epithelium. 3525. (D) Cross section of the small intestine at stage 63, hybridized with the antisense probe. The connective tissue (CT) remains positive, but signals are weaker than those at stage 62 in Fig. 6A and C. The muscles are almost negative. 3190. (E) Cross section of the small intestine at stage 66, hybridized with the antisense probe. No specific signals are definitively detected in any tissues. 3175. (F) Control section of the small intestine at stage 66, hybridized with the sense probe. No specific signals are detected. 3175. L, lumen. Bars: 20 μm.