Sun G et al. (2010), Spatio-temporal expression profile of stem cell...

XB-ART-42224

PLoS One 2010 Oct 22;510:e13605. doi: 10.1371/journal.pone.0013605.

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Spatio-temporal expression profile of stem cell-associated gene LGR5 in the intestine during thyroid hormone-dependent metamorphosis in Xenopus laevis.

Sun G , Hasebe T , Fujimoto K , Lu R , Fu L , Matsuda H , Kajita M , Ishizuya-Oka A , Shi YB .

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The intestinal epithelium undergoes constant self-renewal throughout adult life across vertebrates. This is accomplished through the proliferation and subsequent differentiation of the adult stem cells. This self-renewal system is established in the so-called postembryonic developmental period in mammals when endogenous thyroid hormone (T3) levels are high. The T3-dependent metamorphosis in anurans like Xenopus laevis resembles the mammalian postembryonic development and offers a unique opportunity to study how the adult stem cells are developed. The tadpole intestine is predominantly a monolayer of larval epithelial cells. During metamorphosis, the larval epithelial cells undergo apoptosis and, concurrently, adult epithelial stem/progenitor cells develop de novo, rapidly proliferate, and then differentiate to establish a trough-crest axis of the epithelial fold, resembling the crypt-villus axis in the adult mammalian intestine. The leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is a well-established stem cell marker in the adult mouse intestinal crypt. Here we have cloned and analyzed the spatiotemporal expression profile of LGR5 gene during frog metamorphosis. We show that the two duplicated LGR5 genes in Xenopus laevis and the LGR5 gene in Xenopus tropicalis are highly homologous to the LGR5 in other vertebrates. The expression of LGR5 is induced in the limb, tail, and intestine by T3 during metamorphosis. More importantly, LGR5 mRNA is localized to the developing adult epithelial stem cells of the intestine. These results suggest that LGR5-expressing cells are the stem/progenitor cells of the adult intestine and that LGR5 plays a role in the development and/or maintenance of the adult intestinal stem cells during postembryonic development in vertebrates.

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Species referenced: Xenopus tropicalis Xenopus laevis
Genes referenced: cd44 fshr ins lgr4 lgr5 rpl8 tshr

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	Figure 1. The X. laevis (Xl) LGR5 shares a high degree of homology with X. tropicalis (Xt), human (Hu) and mouse (Mm) LGR5.There are two duplicated LGR5 genes in X. laevis and only one (Xl-LGR5a) was used for comparison. The deduced amino acid (aa) sequences were aligned and gaps were introduced to achieve best alignments among species. Dots indicate the aa are the same as that of Xl-LGR5a and conserved changes among species are marked with stars under the Mm-LGR5 sequences. Number (1) to (17) indicate the highly conserved leucine-rich repeats. The conserved transmembrane domains are indicated with TM1-7. The N-terminal signal peptide, N-, C-flanking cysteine-rich sequences, and C-terminal tail are not well conserved.
	Figure 2. A phylogenetic tree of LGR proteins.Amino acid sequences of indicated LGR proteins were analyzed with Multiple Sequence Alignment by CLUSTALW (http://align.genome.jp/). The proteins form three subgroups: one containing Hu-LHR, Hu-FSHR, and Hu-TSHR; another containing LGR7 and LGR8, whose ligands are small heterodimeric peptides with homology to insulin, including the pregnancy hormone relaxin and insulin-like 3 [33], [34], [35]; and the third containing LGR4, LGR5, and LGR6, including the X. laevis LGR5a, b as well as X. tropicalis LGR5, and LGR6. Hu, human, Ck, chicken, Tg, Taeniopygia guttata, Md: Monodelphis domestica.
	Figure 3. Stage- and tissue-dependent regulation of LGR5 expression during development.(A) Zygotic transcription of LGR5 begins in late embryogenesis during development in X. laevis. Total RNA was isolated from whole animals from stage 1 (fertilized egg) to stage 66 (the end of metamorphosis) and analyzed by qRT-PCR. The LGR5 expression shown in (C) was normalized against rpl8 expression determined at the same time. (B, C) Organ-specific temporal regulation of LGR5 during natural metamorphosis in X. laevis. Total RNA was isolated from tail, limb, and intestine of tadpoles at the indicated developmental stages and used for regular RT-PCR (B) or qRT-PCR (C) analysis of the LGR5 expression. In each RT-PCR, a primer set for the ribosomal gene rpl8 was also included in the same PCR reaction as an internal control, although the rpl8 result was shown only for the intestine in (B). The LGR5 expression shown in (C) was normalized against rpl8 expression determined at the same time.
	Figure 4. T3 up-regulates LGR5 expression in premetamorphic tadpoles.Tadpoles at stage 54 were exposed to 5 nM T3 for 0–5 days before tissue isolation, RNA extraction, and qRT-PCR analyses as in Fig. 3C.
	Figure 5. Spatiotemporal expression of LGR5 mRNA in the small intestine during natural metamorphosis.Cross sections of the intestine at premetamorphic stage 54 (A, Aâ€²), prometamorphic stage 56/57 (B, Bâ€²), metamorphic climax stages 61 (C, Câ€², F) and 62 (D, Dâ€², G), and the end of metamorphosis (stage 66) (E, Eâ€²) were hybridized with LGR5 antisense (Aâ€“Eâ€²) or sense probe (G). To compare the localization of LGR5 mRNA (C, Câ€²) with that of adult epithelial progenitor cells, the serial sections at stage 60/61 were stained with methyl green-pyronin Y (MG-PY) (F). Arrows indicate the cells expressing LGR5 (Aâ€²â€“Eâ€²), while arrowheads indicate adult epithelial progenitor cells (F). Higher magnification of boxed areas in (A)â€“(E) are shown in (Aâ€²)â€“(Eâ€²). Sense probe did not produce any signal (G). Note that at metamorphic climax stage 61, LGR5 mRNA was localized in the islets between the larval epithelial cells and the connective tissue (C, Câ€²). These islet cells were identified as the adult epithelial progenitor cells strongly stained red with pyronin Y (F) [29]. AE: adult epithelial cell including progenitor/stem cell, CT: connective tissue, LE: larval epithelial cell, Lu: lumen, M: muscle layer, Ty: typhlosole. Scale bars are 100 Âµm (Aâ€“E, G) and 20 Âµm (Aâ€²â€“Eâ€², F), respectively.
	Figure 6. T3-dependent expression of LGR5 mRNA in the small intestine.Cross sections of the intestine from premetamorphic tadpoles (stage 54) treated with 10 nM T3 for 1 (A, Aâ€²), 3 (B, Bâ€²) and 5 days (C, Câ€²) were hybridized with LGR5 antisense probe. AE: adult epithelial cell including the progenitor/stem cell, CT: connective tissue, LE: larval epithelial cell, Lu: lumen, M: muscle layer. Scale bars are 100 Âµm (Aâ€“C) and 20 Âµm (Aâ€²â€“Câ€²), respectively.
	lgr5 (leucine-rich repeat containing G protein-coupled receptor 5) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, prometamorphic, NF stage 56/57, cross section of small intestine.
	lgr5 (leucine-rich repeat containing G protein-coupled receptor 5) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, metamorphic climax, NF stage 60/61, cross section of small intestine.
	lgr5 (leucine-rich repeat containing G protein-coupled receptor 5) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, metamorphic climax, NF stage 626, cross section of small intestine.
	lgr5 (leucine-rich repeat containing G protein-coupled receptor 5) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, at end of metamorphosis, NF stage 66, cross section of small intestine.

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