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.