Molecular and cytological analyses reveal distinct transformations of intestinal epithelial cells during Xenopus metamorphosis
© Okada et al. 2015
Received: 24 November 2015
Accepted: 22 December 2015
Published: 29 December 2015
The thyroid hormone (T3)-induced formation of adult intestine during amphibian metamorphosis resembles the maturation of the mammalian intestine during postembryonic development, the period around birth when plasma T3 level peaks. This process involves de novo formation of adult intestinal stem cells as well as the removal of the larval epithelial cells through apoptosis. Earlier studies have revealed a number of cytological and molecular markers for the epithelial cells undergoing different changes during metamorphosis. However, the lack of established double labeling has made it difficult to ascertain the identities of the metamorphosing epithelial cells.
Here, we carried out different double-staining with a number of cytological and molecular markers during T3-induced and natural metamorphosis in Xenopus laevis. Our studies demonstrated conclusively that the clusters of proliferating cells in the epithelium at the climax of metamorphosis are undifferentiated epithelial cells and express the well-known adult intestinal stem cell marker gene Lgr5. We further show that the adult stem cells and apoptotic larval epithelial cells are distinct epithelial cells during metamorphosis.
Our findings suggest that morphologically identical larval epithelial cells choose two alternative paths: programmed cell death or dedifferentiation to form adult stem cells, in response to T3 during metamorphosis with apoptosis occurring prior to the formation of the proliferating adult stem cell clusters (islets).
KeywordsThyroid hormone Metamorphosis Xenopus laevis Thyroid hormone receptor Stem cells Intestine
Intestinal remodeling during Xenopus metamorphosis serves as an excellent model to study the development of vertebrate adult organ-specific adult stem cells, which are essential for physiological tissue renewal and regeneration. This transformation of the larval intestine to the adult form during amphibian metamorphosis involves the removal of larval epithelium and de novo formation of the adult epithelium with concurrent maturation of the other intestinal tissues in a process similar to the maturation of the mammalian intestine around birth [1–5]. The tadpole intestine consists of largely a monolayer of larval epithelial cells surrounded by thin layers of connective tissue and muscles. During metamorphosis, the larval epithelial cells undergo apoptosis and clusters of proliferating adult epithelial cells are formed de novo, which subsequently proliferate and differentiation to form a multiply folded adult epithelium surrounded by thick layers of connective tissue and muscles [1, 6–12]. Gene expression analyses of known adult stem cell markers of mammalian intestine, such as Lgr5 , suggest that the clusters of proliferating cells are adult stem cells.
Like all other processes during amphibian metamorphosis, intestinal remodeling is under the control of thyroid hormone (T3) [14, 15]. This process can be easily induced by adding physiological concentrations of T3 to premetamorphic tadpole rearing water or prevented by blocking the synthesis of endogenous T3. In addition, it is organ autonomous and can be induced with T3 even in intestinal organ cultures of premetamorphic tadpoles. Such properties makes intestinal remodeling a superior model to study the development of adult organ-specific stem cells as compared to the mammalian models, where it is difficult to manipulate the uterus-enclosed late stage embryos for such studies.
Earlier work in Xenopus laevis has shown that T3 induces the vast majority of the larval epithelial cells to undergo programmed cell death or apoptosis and that the proliferating adult epithelial cells are formed de novo, apparently from the dedifferentiation of a small number of larval epithelial cells, via a yet-unknown mechanism [1, 7–12, 16–19]. These proliferating adult epithelial cells can be easily identified as clusters of cells or islets that can be labeled with DNA synthesis markers, such as 3H and 5-bromo-2′-deoxyuridine, or strongly stained with methyl green-pyronin Y (MPGY) at the climax of metamorphosis [16–18, 20]. In addition, in situ hybridization analyses have shown that well-known markers of the adult mammalian intestinal stem cells, such as leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5) and Musashi-1 (Msi-1), are expressed in clusters of intestinal epithelial cells at the climax of metamorphosis, suggesting that the clusters or islets are proliferating adult stem cells. However, there has been no report of using double labeling to ascertain the identities and property of these cell clusters. Here by using a combination of different staining methods, we successfully carried out different double labeling that allowed us to conclusively demonstrate that the clusters of epithelial cells induced by T3 at the climax of intestinal metamorphosis are proliferating, Lgr5+ adult stem cells. We further show that these cells can be strongly stained with MPGY and lack intestinal fatty acid binding protein (IFABP), which is expressed in the differentiated epithelial cells. Finally, we demonstrated that apoptotic and the proliferating cells are distinct populations of epithelial cells at the climax of metamorphosis.
Results and discussions
Proliferating adult intestinal epithelial cells exist as cell clusters and lack the differentiation marker IFABP
When we carried out similar analyses on intestinal cross-sections during natural metamorphosis, we also observed that at the climax of metamorphosis (stage 62), the EdU labeled cells were present as clusters between the luminal, larval epithelial cells that were positive for IFABP and the connective tissue (Fig. 4B, b″), while before (stage 54) or after (stage 66) metamorphosis (Fig. 4A, C, respectively), the EdU positive cells had IFABP, although at the troughs of the epithelial folds of post-metamorphic intestine (stage 66), the EdU positive cells had little or lower levels of IFABP (Fig. 4c″). These findings suggest that T3 induces the formation of clusters of proliferating cells that are dedifferentiated or undifferentiated. The same conclusion was reached when double-labeling was carried out with immunohistochemistry against IFABP and PCNA (proliferating cell nuclear antigen) (data not shown).
The proliferating epithelial cell clusters express adult intestinal stem cell marker Lgr5
Apoptotic and proliferating cells represent distinct populations of epithelial cells at the climax of metamorphosis
Adult organ-specific stem cells are critical for organ homeostasis, repair, and regeneration and mis-regulation of such stem cells often leads to diseases such as cancer. Thus, extensive studies have been carried out to understand the regulation of organ-specific stem cells as well as cancer stem cells [28–36]. Intestinal remodeling during amphibian metamorphosis resembles the maturation of mammalian intestine around birth and thus has served as a model to study the development of adult organ-specific stem cells in vertebrates [2, 4, 5, 9, 37–43]. The advancements in genetic approaches for gene function studies in vivo, such as the knockout and knockin in Xenopus [44–46], undoubtedly further enhance the value of this unique model system for studying adult organ-specific stem cells. While earlier single labeling studies have provided valuable information for analyzing cell transformations in the epithelium, the lack of double-labeling has hindered analyses and/or interpretations regarding adult stem cells. Here, we have adapted different protocols that allowed us to double label different epithelial cells with several combinations of different labeling methods, including chemical labeling with EdU, staining with MGPY, in situ hybridization, and immunohistochemistry. These have allowed us to directly demonstrate experimentally for the first time that adult intestinal stem cells formed during metamorphosis are the proliferating cell clusters formed at the climax of metamorphosis. Considering our earlier findings that the adult stem cells are derived from larval epithelium [12, 19], our double-labeling studies of proliferating and apoptotic cells indicate that in response to T3, the epithelial cells take two mutually exclusive pathways, apoptosis or dedifferentiation followed by proliferation, which leads to the formation of the adult intestinal epithelium. Finally, our findings here pave the way to use any one of the labeling methods in this study to analyze stem cell development during metamorphosis.
Animals and treatments
Wild-type X. laevis tadpoles were reared in the laboratory or purchased from Nasco or Xenopus 1. The tadpoles were staged based on Nieuwkoop and Faber . Premetamorphic X. laevis tadpoles at stage 54 were treated with 10 nM T3 for 0–6 days at 18 °C. At least 3 tadpoles were analyzed for each stage or day of T3 treatment. All animal studies were done in accordance with the guidelines established by the National Institute of Child Health and Human Development Animal Use and Care Committee.
In situ hybridization
The in situ probe for Lgr5 was made as described previously . Intestinal fragments were isolated from the anterior part of the small intestine of tadpoles at indicated stages, fixed in 4 % MOPS/EGTA/magnesium sulfate/formaldehyde buffer (MEMFA), followed by cryosectioning. Tissue sections cut at 7 µm were subjected to in situ hybridization by using the antisense probe as previously described . For double staining with EdU staining, the sections were first processed for digoxygenin in situ hybridization, and then the slides were washed in 1× phosphate buffered saline plus 0.05 % Tween-20 for 5 min, followed by EdU staining.
5-Ethynyl-2′-deoxyuridine (EdU) labeling
EdU staining was performed as described . Briefly, 6.7, 40 and 40 µL of 2.5-mg/mL EdU were injected into stage 54, 62, and 66 tadpoles, respectively. One hour after injection, the tadpoles were sacrificed, and the intestine was fixed in 4 % MEMFA and processed for cryosectioning. EdU was detected by using the Click-iT Plus EdU Alexa Fluor 594 Imaging kit (Life Technologies) according to supplier’s instructions.
To identify differentiated intestinal absorptive cells, the sections were incubated with the rabbit anti-IFABP (intestinal fatty acid binding protein) antibody (diluted 1:500; ) overnight at 4 °C. Samples were washed several times with 1× phosphate buffered saline and primary antibodies were detected by using Alexa Fluor 568 Goat Anti-Rabbit IgG (H+L) Antibody (diluted 1:100; molecular probes). For double labeling with EdU staining, the sections were first processed for immunostaining, and then the slides were washed in 1× phosphate buffered saline plus 0.05 % Tween-20 for 5 min, followed by EdU staining.
Methyl green-pyronin Y (MPGY) staining
Sections were stained with MPGY (Muto), a mixture of methyl green, which binds strongly to DNA, and pyronin Y, which binds strongly to RNA, for 5 min at room temperature . Adult epithelial stem/progenitor cells were intensely stained red because of their RNA-rich cytoplasm . For double staining with EdU labeling, the sections were first processed for EdU staining. After photographing the EdU labeling, the slides were washed in 1X Phosphate Buffered Saline plus 0.05 % Tween-20 for 5 min, followed by MPGY staining. The image of the MPGY staining was taken. The images from MPGY and EdU staining from the same slide were merged by using Adobe Photoshop CS5.1 to determine whether MPGY and EdU labeled the same cells.
TUNEL (terminal deoxyribonucleotidyl transferase-mediated dUTP-biotin nick end labeling) assays were performed by using DeadEnd™ Colorimetric TUNEL System (Promega) as described . For double staining, EdU staining was performed after the TUNEL assays.
Thyroid hormone receptor
Intestinal fatty acid binding protein
Terminal deoxyribonucleotidyl transferase-mediated dUTP-biotin nick end labeling
Methyl green pyronin Y
MO, LW, TCM, and DS designed and carried out experiments and interpreted the findings. MO and YBS prepared the manuscript. All authors read and approved the final manuscript.
This work was supported by the intramural Research Program of NICHD, NIH. MO was supported in part by Japan Society for the Promotion of Science (NIH) Fellowship.
The authors declare that they have no competing interests.
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