Amano T et al. (1998), Metamorphosis-associated and region-specific ex...

XB-ART-15087

Dev Growth Differ 1998 Apr 01;402:177-88. doi: 10.1046/j.1440-169x.1998.00007.x.

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Metamorphosis-associated and region-specific expression of calbindin gene in the posterior intestinal epithelium of Xenopus laevis larva.

Amano T , Noro N , Kawabata H , Kobayashi Y , Yoshizato K .

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The present study used a molecular approach toward understanding the mechanism of hormone- and region-dependent remodeling of the small intestine during metamorphosis of Xenopus laevis. A protein spot was noticed on a two-dimensional polyacrylamide gel as a protein whose expression was metamorphic stage- and region-dependent. The protein was identified as the Xenopus homolog (Xcalbindin) of chick calbindin D28k. Xcalbindin expression in the intestine was restricted to absorptive cells in the posterior part, being detectable at stages 49-61, not detectable at stages 62-63, detectable again at stages 64-66, and finally becoming undetectable in the adult. During spontaneous metamorphosis, the level of Xcalbindin mRNA was significantly increased between stages 57 and 58, dramatically reduced at stage 59, and the mRNA was undetectable from stages 60-63, after which it was weakly re-expressed until the end of metamorphosis. Such up- and down-regulation of Xcalbindin mRNA was induced precociously by exogenous thyroid hormone. These results indicated that Xcalbindin is a specific marker of the differentiated absorptive cells of the intestine. Immunohistochemistry with specific antibodies against Xcalbindin demonstrated that precursor cells of adult intestinal epithelial cells expressed Xcalbindin. Considering these results, the origin of adult intestinal epithelial cells was discussed.

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Species referenced: Xenopus laevis
Genes referenced: calb1 ednra eef1a2 gopc mtor uqcc6

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	Fig. 1. Two-dimensional polyacrylamide gel electrophoresis of proteins of the small intestine. The small intestine was removed from tadpoles at stage 57 (A,B,D) or at stage 63 (C). The stage 57 small intestine was amputated at the posterior end of the typhlosole to obtain the anterior (A) and posterior (B,D) part of the organ. The stage 63 organ was analyzed as a whole. Small fragments of intestine were homogenized and 25 iJQ of protein per gel was subjected to 2-D PAGE. Gels were stained by silver (A.B,C), or blotted onto a nitrocellulose filter and stained with anti-XPI-28 antibody (D). Arrowheads (B,D) indicate the XPI-28 spot, which is specifically expressed in the posterior part. Values of pi and molecular weights are indicated at the top and left side of (A), respectively.
	Fig. 2. Nucleotide and its deduced amino acid sequences of the full-length eDNA encoding XPI-28. The clone has 3597 bases comprising 41 bases in a 5'-untranslated region, the translation start site initiating an open reading frame that encodes 260 amino acids, the stop codon marked by an asterisk, and 2676 bases in a 3'-untranslated region. Five polyadenylation signals (AATAAA) are present, the last of which is boxed. The repeat sequence of ATTT or TTTA in the 3'-untranslated region is underlined, and is found in the calcium binding protein gene family (Minghetti eta/. 1988).
	Fig. 3. Comparison of Xenopus and chick cal bind in D2sk. The whole sequence of Xcalbindin and chick (chk) calbindin D2sk (Hunziker 1986) is shown in a 6-lolded manner with theN-terminal methionine at the top lett and the C-terminal asparagine at the bottom right. Calbindin has six putative calcium binding EF hand domains, each of which consists of the helix-loop-helix structure. Five oxygen atom-containing amino acid residues in each domain are important lor calcium binding proteins to co-ordinate with calcium (Kretsinger 1976). The glycine residue located at the center of loop structures and the glutamic acidleucine sequence at the end of the loops are invariable among calcium binding proteins (Kretsinger 1976). These were all found in Xcalbindin. Xcalbindin and chick calbindin D28k share the amino acid sequence at a rate of 80%. Amino acid residues different between the two are indicated tor the chick. Dashes in the chk cal bind in and a double line ( =) inserted between arginine and glutamine residues in the calcium binding domain II of Xcalbindin indicate common amino acids and a gap, respectively. Calcium is shown to bind the EF-hand structure (Kretsinger 1976). Invariant residues of glycine, glutamic acid, and leucine are boxed. Oxygen atom-containing amino acids marked by asterisks (')are conserved well among calcium binding proteins. Arabic numerals, 260 and 262, shown at the bottom right indicate the sequence number of carboxyl end residues, asparagine lor Xcalbindin and chk calbindin, respectively.
	Fig. 4. Western blot analysis of Xcalbindin. (A) Tissue distribution of Xcalbindin. Proteins were extracted from the indicated tissues of stage 58 tadpoles and 10 J-lg was subjected to SDS-PAGE, blotted, and stained with anti-Xcalbindin antibody. ai, anterior part of small intestine; br, brain; co, colon; he, heart; ki, kidney; li, liver; mu, limb muscle; pi, posterior part of small intestine; sk, skin; st, stomach. Molecular weights are indicated at the lett side. An arrow to the right side of the gel points to the position of Xcalbindin. (B) Developmental changes in Xcalbindin expression. Proteins were extracted from the whole embryo at stage 47 and from the whole tissue of small intestine at stages from stage 49 through to the adult (lett _ panel). Proteins were extracted from the colon at stage 62 through to the adult (right panel). Ten or 20 J..lg (stage 47) of proteins was run on SDS-PAGE, blotted, and stained with anti-Xcalbindin antibody. ad, adult. Arrows at the right side of gels point to the position of Xcalbindin
	Fig. 5. Immunohistochemistry of Xcalbindin. (A) Whole-mount immunohistochemistry of the small intestine at stage 57. a, anterior side of the organ; p, posterior side; ty, typhlosole. Arrowheads and a small arrow point to the typhlosole and the posterior end of it, respectively. A curved arrow at the right side of the organ points to the region that started to be weakly stained by the antibody. Bar, 5 mm. (B) Whole-mount immunohistochemistry of the digestive tract at stage 66 (froglet). The upper organ was stained with antiXcalbindin antibody and the lower was a negative control. The size of digestive tract was varied among individual froglets. Positively stained areas are indicated by arrowheads. An arrow shows the anterior end of the positive region. The anterior and posterior ends of the small intestine are marked by asterisks. Stomach tissues are thick and folded and, as a result, look dark. The black spot between the intestine and colon is caused by pigmentation. a, anterior side of the organ; p, posterior side; st, stomach; int, small intestine; co, colon. Bar, 5 mm (C-K) Cross-section immunostaining. C,F,I, anterior part; D,E,G,H,J,K, posterior part; C-E, stage 57; F-H, stage 60; 1-K, stage 66. E,H are an enlargement of a part of D,G, respectively. J,K are the middle and terminal region of the Xcalbindinexpressing posterior intestine, respectively. Arrows in G,H indicate Xcalbindin positive primary epithelial cells. Secondary epithelium cells (H) are surrounded by a dotted line. ab, absorptive cells; ct, connective tissue; ep, epithelium; go, goblet cells; lu, lumen; se, secondary epithelium. Bars, 50 um.
	Fig. 6. Expression of Xcalbindin in the islet cells. Cross-sections (4 [Jm) were prepared for various regions of the posterior part of the small intestine at stage 60 (A,B), or 61 (C-H). Serial sections were stained with methylgreen pyronin Y (A,C,E,G) or anti-Xcalbindin antibody (B,D,F,H). The vision field of B,D,F,H corresponds to that of A,C,E,G, respectively. ct, connective tissue; pe, primary epithelium. Arrowheads indicate the islets. Each arrowhead in B,D,F,H points to the islet indicated by each corresponding arrowhead in A,C,E,G, respectively. Bar, 100 um.
	Fig. 7. Northern blot analysis of the Xcalbindin gene during spontaneous metamorphosis. Total RNA was extracted from the whole small intestine at stages indicated and was subjected to northern hybridization ( 10 ~g RNA/lane) using 32P-Iabeled Xcalbindin or EF1a as a probe.
	Fig. 8. Changes in the small intestine during TH-induced metamorphosis. Tadpoles at stage 53 were treated with 1 o-s moi/L T3 up to 72 h. Identical results were obtained for tadpoles at stage 55. (A) The length of small intestine was determined and its change is shown in relative length making the length at day 0 ofT 3-treatment 1.0. 0, control animals; 0. T3·treated animals. Each point represents the average of five determinations. Vertical bars represent the standard deviation of the mean. (B) Northern hybridization of Xcal bindin gene. Ten micrograms of RNA at indicated time points of T3-treatment was electrophoresed and subjected to northern hybridization as in Fig . 7. (C) Immunohistochemistry of Xcalbindin was performed for a posterior intestinal cross-section from a tadpole treated with T 3 for 3 days. ct. connective tissue; lu, lumen; m, muscle; pe, primary epithelium . Bar, 50 um.