Shi YB , Hasebe T , Fu L , Fujimoto K , Ishizuya-Oka A .

	Figure 1. T3-dependent intestinal remodeling during Xenopus laevis metamorphosis serves as a model to study adult organ-specific stem cell development in vertebrates. Top: In premetamorphic tadpoles, there is little or no T3 and the intestine has a simple structure with only a single fold, the typhlosole. This structure is similar to the mammalian embryonic intestine. At the metamorphic climax when T3 level is high, the larval epithelial cells begin to undergo apoptosis, as indicated by the circles. Concurrently, the proliferating adult progenitor/stem cells develop de novo from larval epithelial cells through dedifferentiation, as indicated by black dots. By the end of metamorphosis, the newly differentiated adult epithelial cells form a multiply folded epithelium, similar to mammalian adult intestines. This process is entirely controlled by T3 and can be induced even in organ cultures of tadpole intestine with T3 treatment. In mammals, the intestine undergoes postembryonic maturation into the adult form around or shortly after birth when T3 levels are also high. Thus, intestinal metamorphosis offers a unique opportunity to study the development of adult intestinal stem cells. Bottom: T3 functions by regulating gene transcription through TRs. In the absence of T3 (as in premetamorphic tadpole), TR forms heterodimers with RXR and the heterodimer binds to target gene promoters to repress their expression by recruiting corepressor complexes containing the related proteins N-CoR or SMRT and histone deacetylases. When T3 is present, the corepressor complexes are released upon T3 binding to TR, and simultaneously coactivator complexes such as those containing SRC, p300, and PRMT1, are recruited to activate target gene expression. SRC and p300 are histone acetyltransferases and PRMT1 is a histone methyltransferase. The coactivator complexes will modify histones and activate gene expression to induce metamorphosis.
	Figure 2. Recombinant intestinal organ culture studies using transgenic tadpole. A. Schematic diagram for tissue recombination and organ culture of the Xenopus laevis intestine. Transgenic (Tg) frogs are generated using a double promoter construct [61] where one GFP is expressed in the lens of the eye under the control of the γ-crystallin gene promoter for identification of the transgenic animals and another GFP is expressed ubiquitously under the CMV promoter or a dominant positive thyroid hormone receptor (dpTR, which resembles T3-bound TR, or activated TR, but does not need T3) is expressed under the control of a heat shock inducible promoter. The adult Tg frogs are used to produce Tg and wild type (Wt) sibling tadpoles. Tubular fragments are isolated from the small intestine just behind the bile duct junction (bd) of premetamorphic (when there is little endogenous T3 present) Tg and Wt tadpoles, slit open lengthwise, and separated into epithelium (Ep) and non-epithelial tissues (non-Ep), which consists of mainly the connective tissue underlying the epithelium. Each Ep is then recombined with homologous or heterologous non-Ep. The four kinds of recombinant intestines are placed on membrane filters on grids and cultured in vitro. Heat shock treatment, when using dpTR tadpoles, or T3 treatment, when using GFP animals, is applied to induce intestinal remodeling. * indicated type of Ep/type of non-Ep used in the recombinant organ culture: e.g., Tg/Wt indicating Tg epithelium recombined with Wt non-epithelial tissues. B. Recombinant organ culture studies using dpTR-transgenic and Wt tadpoles indicate that only when both the Ep and non-Ep were derived from Tg animals, i.e., Tg/Tg, were true stem cells and adult epithelium formed after heat shock treatment. Recombinants made of Tg EP and Tg non-EP (Tg/Tg) (A-D) and Tg Ep and Wt non-EP (Tg/Wt) (E-H) of the intestines were cultured with heat shock treatment for 5 (A-C, E-G) or 7 (D, H) days in vitro. Cross sections were double-immuno-stained with anti-Shh (green, sonic hedgehog, an adult progenitor cell marker) and anti-CK19 (red, cytokeratin-19, which is expressed in epithelial cells. (A, E)), anti-Msi-1 (green, Musashi-1, a stem cell marker of the vertebrate adult intestine) and anti-CK19 (red; (B, F)), or anti-Akt (green, a stem cell marker of the vertebrate adult intestine) and anti-CK19 (red) antibodies (C, G), or immunostained with anti-IFABP antibody (green; D, H). In both Tg/Tg (arrowheads; (A)) and Tg/Wt intestines (E), cells positive for Shh become detectable on day 5 among cells expressing CK19, indicating the adult progenitor cells can be induced by cell-autonomous action of activated TR in the epithelium. Cells positive for Msi1 and Akt are also detected among CK19- immunoreactive cells in Tg/Tg intestine (arrowheads; (B, C)) but not in Tg/Wt intestine (F, G). In addition, differentiated epithelial cells expressing IFABP were present after extended culturing only in Tg/Tg organ cultures (D). Thus, activation of TR in the non-epithelial tissues is also required for the stem cell formation, likely by contributing to the formation of the stem cell niche. Not shown here is that most of the epithelial cells undergo apoptosis when dpTR is expressed in either the EP or non-EP or both, just like that during metamorphosis when T3 binds to TR. See [47] for details.
	Figure 3. PRMT1 is required for the development and/or proliferation of adult intestinal progenitor/stem cells. A. In vivo knock-down of PRMT1 expression. Xenopus laevis tadpoles at stage 53/54 were injected with (MO, Co) or without (Un) PRMT1 (MO) or control (Co) anti-sense Vivo-morpholino oligos for 4 consecutive days. Intestines were dissected for western blot analysis of the expression of the endogenous PRMT1 or histone H4 (as a control). B. Knocking-down the expression of endogenous PRMT1 results in the reduction in the number of proliferating adult cells in the intestine. Stage 53/54 premetamorphic tadpoles were injected without (untreated) or with PRMT1 (MO) or control Vivo-morpholino oligos for four days. All tadpoles were then treated with 5 nM of thyroid hormone for 4 days. The tadpoles were sacrificed and the intestine was analyzed for the number of proliferating adult cells. See [53] for details.
	Figure 4. Conserved spatiotemporal expression patterns of PRMT1 in the postembryonic intestines of fish and mouse suggest a conserved role of PRMT1 in adult intestinal stem cell development. PRMT1 mRNA was analyzed by in situ hybridization in the intestines of zebrafish at 15 days post fertilization (dpf) (A), 20 dpf (B) and 1 year post fertilization (ypf) (C) and mouse at postnatal day 7 (D, P7), P21 (E), and postnatal week 14 (F, P14w). Arrows indicate PRMT1 positive cells in the intestinal epithelium. Note that high levels of PRMT1 expression is detected only in the proliferating/stem cells located in the crypts in both species, resembling that in Xenopus laevis. In addition, the transition period in both species when PRMT1 is upregulated is characterized by high levels of T3 in the serum, just like that during Xenopus metamorphosis. ep, epithelium. l, lumen. Bars, 50 μm. See [53] for details.

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