Ishizuya-Oka A et al. (1997), Temporal and spatial expression of an intestina...

XB-ART-17163

Dev Genet 1997 Jan 01;201:53-66. doi: 10.1002/(SICI)1520-6408(1997)20:1<53::AID-DVG7>3.0.CO;2-8.

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Temporal and spatial expression of an intestinal Na+/PO4 3- cotransporter correlates with epithelial transformation during thyroid hormone-dependent frog metamorphosis.

Ishizuya-Oka A , Stolow MA , Ueda S , Shi YB .

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The amphibian intestine has two morphologically distinct structures during development. Early embryogenesis generates a simple, tube-like intestine in the tadpole whereas after thyroid hormone (T3)-dependent metamorphosis a newly remodeled adult intestine is formed similar to that of higher vertebrates. This change requires a drastic transformation of the epithelial layer We have isolated a Na+/PO4 3- cotransporter gene that may contribute to this transformation. The deduced amino acid sequence of this gene shows a high degree of homology to the mammalian renal Na+/PO4 3- cotransporters, which have little or no expression in organs other than the kidney. The frog gene is highly expressed and regulated by T3 in the intestine with little expression and/or regulation by T3 in most other organs. Its mRNA is restricted to the differentiated epithelial cells both in tadpoles and postmetamorphic frogs. Interestingly, its expression is low in premetamorphic tadpoles, but up-regulated when metamorphosis is initiated by endogenous T3. As the larval epithelium undergoes programmed cell death (apoptosis), the mRNa level drops to a minimum. Subsequently, the gene is reactivated at the tip region of the newly formed adult intestinal folds and a crest-trough polarity of expression is established by the end of metamorphosis. This temporal regulation profile is also reproduced when premetamorphic tadpoles are treated with T3 to induce precocious intestinal remodeling. These results suggest a possible role of the Na+/PO 4 3- cotransporter during metamorphosis and demonstrate that the adult epithelial cell differentiation pattern is established in the direction of crest-to-trough of the intestinal fold, concurrent with the epithelial morphogenic process.

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Species referenced: Xenopus laevis
Genes referenced: rpl8 slc34a2

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	Fig. 1. A: Organization of two cDNA clones of Xenopus intestinal Na1/PO4 32 cotransporter. The xNaPi-B clone is longer than the xNaPi-A clone by 178 bp at 58-end and 2.5 Kb at 38-end. The initiation codon (AUG), termination codon (TAG), and the putative polyadenylation signal (arrow) for each clone are indicated. B: Sequence comparison among the Xenopus Na1/PO4 32 cotransporter cDNAs and the flounder Na1/PO4 32 cotransport cDNA [fNaPi, Werner et al., 1994]. Only sequences differ from xNaPi-A are shown for xNaPi-B and fNaPi and gaps (indicated by dots) were introduced for better alignment. The initiation and stop codons are underlined. The two Xenopus cDNA clones are over 99% identical. Both Xenopus clones are highly homologous to fNaPi throughout the coding region, sharing about 67% identity over 1750 bp. It should be pointed out that the exact 58-ends of the xNaPi-A and xNaPi-B mRNA are unknown. However, it is interesting to note that xNaPi-B has a relatively long 58-untranslated region, which may suggest the existence of possible translational regulation for the NaPi expression. Each Xenopus cDNAencodes a 674 amino acid polypeptide that is also over 99% identical to the other. The high degrees of homologies at cDNA and amino acid sequence levels suggest that the two Xenopus cDNA clones are derived from a single gene but with alternative polyadenylations. However, it can not be completely ruled out that they are from two highly conserved genes as Xenopus laevis is a pseudotetraploid animal with many of its genes duplicated.
	Fig. 2. Amino acid sequence comparison among Xenopus intestinal Na1/PO4 32 cotransporter (NaPi), flounder NaPi [Werner et al., 1994], human NaPi [Magagnin et al., 1993], and opossum NaPi [Sorribas et al., 1994]. Only sequences that differ from the XenopusNaPi sequences are shown for the rest of the NaPis. Gaps (indicated by dots) were introduced for better alignment. Note that all NaPis were predicted to have eight transmembrane domains (M1 to M8) (either M3a or M3b may serve as the third transmembrane domain for opossum NaPi) [Sorribas et al., 1994]. The Xenopus NaPi shares 66%, 58%, and 57% identity with the flounder, human, and opossum NaPi, respectively.
	Fig. 3. Organ-specific expression of Xenopus Na1/PO4 32 cotransporter gene (xNaPi). A: Tadpoles at stage 54 were treated in the presence (1) or absence (2) of T3 for up to 24 hours. After treatment, poly (A)1 RNA was isolated from brain, hindlimb (limb), tail, and intestine. RNA was copied into cDNA by reverse transcription, restricted to small fragments, ligated to a PCR-linker, and amplified by PCR. Southern blot hybridization was performed on the cDNA, using a xNaPi or rpL8 probe, which served as a control for loading and PCR. Note that the smeary signals were due to the procedure used to generate the PCR-cDNAs, which resulted in the restriction of fulllength cDNAs into multiple fragments (see references in Materials and Methods). B: The xNaPi is expressed in the small intestine of juvenile frog. The small intestine, large intestine (codon), stomach, and pancreas were dissected from juvenile frog for RNA isolation. Ten μg total RNA was used per lane for Northern blot analysis. C: The xNaPi is also highly expressed in frog lung. A mature frog was dissected in order to isolate RNA from different visceral organs. Approximately 5 μg RNA/lane was used for Northern blot analysis. Although the loading was not quite equal for RNA from different organs (see rpL8 control), the results clearly demonstrated high levels of xNaPi expression in the intestine and lung but not the kidney. For both (B) and (C), the smeary signals migrated faster that the largest mRNA band were most likely due to in part partial degradation of the large mRNAs and in part the alternatively processed mRNA forms. The bars indicate the positions of the 28S and 18S rRNA.
	Fig. 4. Developmental regulation of Xenopus Na1/PO4 32 cotransporter (xNaPi). Each lane had 10 μg RNAexcept the lane for the tail at stage 64, which had only 5 μg. Note the drastic regulation during intestinal remodeling between stages 58–66 and the absence of expression in the tail throughout development. The hybridization with rpL8 served as a control.
	Fig. 5. The expression profiles of xNaPi during T3-induced metamorphosis mimic those during natural development (Fig. 4). Each lane had 10 μg RNA. Note the up-regulation followed by down-regulation and then reactivation of the xNaPi gene in the intestine during T3 treatment and the absence of expression in the tail throughout the treatment.
	Fig. 6. In situ hybridization localizes the expression of Na1/PO4 32 cotransporter to the differentiated epithelial cells of the small intestine. A: Anterior small intestine of a stage 58 tadpole hybridized with the antisense Na1/PO4 32 cotransporter probe. The connective tissue (CT) was mostly concentrated in the typhlosole (Ty). The muscle layers (M) were very thin. Signals were localized in the simple columnar epithelium (E). B: Anterior intestine of a postmetamprhic frog (stage 66) hybridized with the antisense probe. Note the presence of multiple adult intestinal folds (Fo). Almost all of the epithelial cells were strongly positive except for the trough region of the folds (arrowheads). C,D: Control sections of the small intestine at stage 58 and stage 66 (frog), respectively, hybridized with the sense probe. No specific signals were detected. E: Higher magnification of the tadpole small intestine at stage 59 hybridized with the antisense probe. Signals were detected in absorptive epithelial cells (Ab) possessing the brush border (bb) but not in goblet cells (Go). The connective tissue and the muscular layer were negative. F: Higher magnification of the frog small intestine at stage 66 hybridized with the antisense probe. Signals were also localized in absorptive epithelial cells. L, lumen. Scale bars 5 20 μm.
	Fig. 7. Na1/PO4 32 expression was first up- and then downregulated in the intestine between stage 55 and 61. In situ hybridization was performed on the intestinal cross-sections with the antisense probe. A: Anterior part of the small intestine at stage 55. Signals in the epithelium (E) were very weak. The connective tissue (CT) and the muscles (M) were negative. B: Anterior part of the small intestine at stage 57. Signals in the epithelium were stronger than those at stage 55 in (A). The other tissues remained negative. C: Anterior part of the small intestine at stage 59. Signals in the epithelium attained their maxima by stages 58–59. D: Distal part of the small intestine at stage 59. Signals in the epithelium appeared to be similar to those in the anterior part of the intestine in (C). E: Anterior part of the small intestine at stage 61. The epithelium consisted of the degenerating larval (LE) and adult (AE) epithelial cells. No signals could be detected in either type of cells. L, lumen. Scale bars 5 20 μm.
	Fig. 8. The anterior-posterior and the crest-trough gradients of Na1/PO4 32 cotransporter expression were established in the small intestine between stages 63–66 as adult epithelial cells differentiated. A: Cross section of the anterior part of the small intestine at stage 63, hybridized with the antisense probe. Intestinal folds (Fo) had begun to form. Signals in the epithelium (E) increased in intensity towards the crest of the folds. B: Higher magnification of an intestinal fold of the anterior part of the small intestine at stage 63, hybridized with the antisense probe. Strong signals were detected only at the crest of the fold (arrowhead). C: Anterior part of the small intestine at stage 64, hybridized with the antisense probe. There were regional differences in the intensities of the signals along the crest-trough axis of a fold. Signals were strong at the crest of a fold, but decreased in intensity towards the trough region (arrowheads). D: Anterior part of the small intestine at stage 66, hybridized with the antisense probe. Signals were generally much stronger than those at stage 64 (C). However, signals in the trough region of a fold (arrowheads) remained very weak or absent. E: Distal part of the small intestine at stage 66, hybridized with the antisense probe. Intestinal folds were smaller in number and size than those in the anterior part (D). Signals were detected in the entire epthelium lining a fold except for the trough region (arrowheads). F: Control section of the distal part of the small intestine at stage 66, hybridized with the sense probe. No specific signals were present. CT, connective tissue; M, muscular layer; L, lumen. Scale bars 5 20 μm.