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In the intestine during metamorphosis of the frog Xenopus laevis, most of the larval epithelial cells are induced to undergo apoptosis by thyroid hormone (TH), and under continued TH action, the remaining epithelial cells dedifferentiate into stem cells (SCs), which then newly generate an adult epithelium analogous to the mammalian intestinal epithelium. Previously, we have shown that the precursors of the SCs that exist in the larval epithelium as differentiated absorptive cells specifically express receptor tyrosine kinase-like orphan receptor 2 (Ror2). By using Ror2 as a marker, we have immunohistochemically shown here that these SC precursors, but not the larval epithelial cells destined to die by apoptosis, express TH receptor α (TRα). Upon initiation of TH-dependent remodeling, TRα expression remains restricted to the SCs as well as proliferating adult epithelial primordia derived from them. As intestinal folds form, TRα expression becomes localized in the trough of the folds where the SCs reside. In contrast, TRβ expression is transiently up-regulated in the entire intestine concomitantly with the increase of endogenous TH levels and is most highly expressed in the developing adult epithelial primordia. Moreover, we have shown here that global histone H4 acetylation is enhanced in the SC precursors and adult primordia including the SCs, while tri-methylation of histone H3 lysine 27 is lacking in those cells during metamorphosis. Our results strongly suggest distinct roles of TRα and TRβ in the intestinal larval-to-adult remodeling, involving distinctive epigenetic modifications in the SC lineage., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In Xenopus laevis intestine during metamorphosis, the larval epithelial cells are removed by apoptosis, and the adult epithelial stem (AE) cells appear concomitantly. They proliferate and differentiate to form the adult epithelium (Ep). Thyroid hormone (TH) is well established to trigger this remodeling by regulating the expression of various genes including Notch receptor. To study the role of Notch signaling, we have analyzed the expression of its components, including the ligands (DLL and Jag), receptor (Notch), and targets (Hairy), in the metamorphosing intestine by real-time reverse transcription-polymerase chain reaction and in situ hybridization or immunohistochemistry. We show that they are up-regulated during both natural and TH-induced metamorphosis in a tissue-specific manner. Particularly, Hairy1 is specifically expressed in the AE cells. Moreover, up-regulation of Hairy1 and Hairy2b by TH was prevented by treating tadpoles with a γ-secretase inhibitor (GSI), which inhibits Notch signaling. More importantly, TH-induced up-regulation of LGR5, an adult intestinal stem cell marker, was suppressed by GSI treatment. Our results suggest that Notch signaling plays a role in stem cell development by regulating the expression of Hairy genes during intestinal remodeling. Furthermore, we show with organ culture experiments that prolonged exposure of tadpole intestine to TH plus GSI leads to hyperplasia of secretory cells and reduction of absorptive cells. Our findings here thus provide evidence for evolutionarily conserved role of Notch signaling in intestinal cell fate determination but more importantly reveal, for the first time, an important role of Notch pathway in the formation of adult intestinal stem cells during vertebrate development. Stem Cells 2017;35:1028-1039., (© 2016 The Authors STEM CELLS published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

In the amphibian intestine during metamorphosis, most of the larval epithelial cells undergo apoptosis, whereas a small number of them survive. These cells dedifferentiate into stem cells through interactions with the microenvironment referred to as "stem cell niche" and generate the adult epithelium analogous to the mammalian counterpart. Since all processes of the larval-to-adult intestinal remodeling can be experimentally induced by thyroid hormone (TH) both in vivo and in vitro, the amphibian intestine provides us a valuable opportunity to study how adult stem cells and their niche are formed during postembryonic development. To address this issue, a number of expression and functional analyses of TH response genes have been intensely performed in the Xenopus laevis over the past two decades, by using organ culture and transgenic techniques. We here review recent progress in this field, focusing on key signaling pathways involved in establishment of the stem cell niche and discuss their evolutionarily conserved roles in the vertebrate intestine., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: Intestinal remodeling during amphibian metamorphosis resembles the maturation of the adult intestine during mammalian postembryonic development when the adult epithelial self-renewing system is established under the influence of high concentrations of plasma thyroid hormone (T3). This process involves de novo formation and subsequent proliferation and differentiation of the adult stem cells., Methodology/principal Findings: The T3-dependence of the formation of adult intestinal stem cell during Xenopus laevis metamorphosis offers a unique opportunity to identify genes likely important for adult organ-specific stem cell development. We have cloned and characterized the ectopic viral integration site 1 (EVI) and its variant myelodysplastic syndrome 1 (MDS)/EVI generated via transcription from the upstream MDS promoter and alternative splicing. EVI and MDS/EVI have been implicated in a number of cancers including breast, leukemia, ovarian, and intestinal cancers. We show that EVI and MDS/EVI transcripts are upregulated by T3 in the epithelium but not the rest of the intestine in Xenopus laevis when adult stem cells are forming in the epithelium., Conclusions/significance: Our results suggest that EVI and MDS/EVI are likely involved in the development and/or proliferation of newly forming adult intestinal epithelial cells.

Background: During Xenopus laevis metamorphosis, Sonic hedgehog (Shh) is directly induced by thyroid hormone (TH) at the transcription level as one of the earliest events in intestinal remodeling. However, the regulation of other components of this signaling pathway remains to be analyzed. Here, we analyzed the spatiotemporal expression of Patched (Ptc)-1, Smoothened (Smo), Gli1, Gli2, and Gli3 during natural and TH-induced intestinal remodeling., Results: We show that all of the genes examined are transiently up-regulated in the mesenchymal tissues during intestinal metamorphosis., Conclusions: Interestingly, in the presence of protein synthesis inhibitors, Gli2 but not the others was induced by TH, suggesting that Gli2 is a direct TH response gene, while the others are likely indirect ones. Furthermore, we demonstrate by the organ culture experiment that overexpression of Shh enhances the expression of Ptc-1, Smo, and Glis even in the absence of TH, indicating that Shh regulates its own pathway components during intestinal remodeling., (Copyright © 2011 Wiley Periodicals, Inc.)

A Xenopus laevis homolog of nucleophosmin/nucleoplasmin3 (NPM3), no29, has been previously identified as a thyroid hormone (TH)-response gene during TH-induced metamorphosis. X. laevis has another NPM3 homolog (npm3) in the pseudo-tetraploid genome, whereas X. tropicalis possesses one ortholog in the diploid genome. To assess the possible roles of these NPM3 homologs in amphibian metamorphosis, we have analyzed their expression profiles in X. laevis tadpoles. Levels of no29 and npm3 mRNA are rapidly up-regulated by exogenous TH in various organs of the premetamorphic tadpoles. Notably, in the small intestine, no29 and npm3 mRNA levels are transiently up-regulated during metamorphic climax, when progenitor/stem cells of the adult epithelium appear and actively proliferate. In situ hybridization analysis has revealed that the no29 transcript is specifically localized in adult epithelial progenitor/stem cells of the intestine during natural and TH-induced metamorphosis. Double-staining for in situ hybridization and immunohistochemistry has shown co-expression of no29 mRNA and no38 protein (an ortholog of NPM1), which is known to interact with NPM3 and to regulate cell proliferation in mammals. Thus, no29/npm3 might serve as a stem cell marker in the intestine during metamorphosis.

During amphibian metamorphosis, the animal body dramatically remodels to adapt from the aquatic to the terrestrial life. Cell death of larval organs/tissues occurs massively in balance with proliferation of adult organs/tissues, to ensure survival of the individuals. Thus, amphibian metamorphosis provides a unique and valuable opportunity to study regulatory mechanisms of cell death. The advantage of this animal model is the absolute dependence of amphibian metamorphosis on thyroid hormone (TH). Since the 1990s, a number of TH response genes have been identified in several organs of Xenopus laevis tadpoles such as the tail and the intestine by subtractive hybridization and more recently by cDNA microarrays. Their expression and functional analyses, which are still ongoing, have shed light on molecular mechanisms of TH-induced cell death during amphibian metamorphosis. In this review, I survey the recent progress of research in this field, focusing on the X. laevis intestine where apoptotic process is well characterized at the cellular level and can be easily manipulated in vitro. A growing body of evidence indicates that apoptosis during the intestinal remodeling occurs not only via a cell-autonomous pathway but also via cell-cell and/or cell-extracellular matrix (ECM) interactions. Especially, stromelysin-3, a matrix metalloproteinase, has been shown to alter cell-ECM interactions by cleaving a laminin receptor and induce apoptosis in the larval intestinal epithelium. Here, I emphasize the importance of TH-induced multiple apoptotic pathways for massive and well-organized apoptosis in the amphibian organs and discuss their conservation in the mammalian organs., (© 2011 The Author. Journal compilation © 2011 Japanese Society of Developmental Biologists.)

Background: 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., Methodology/principal Findings: 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., Conclusions/significance: 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.

Matrix metalloproteinases (MMPs) are a superfamily of Zn(2+)-dependent proteases that are capable of cleaving the proteinaceous component of the extracellular matrix (ECM). The ECM is a critical medium for cell-cell interactions and can also directly signal cells through cell surface ECM receptors, such as integrins. In addition, many growth factors and signaling molecules are stored in the ECM. Thus, ECM remodeling and/or degradation by MMPs are expected to affect cell fate and behavior during many developmental and pathological processes. Numerous studies have shown that the expression of MMP mRNAs and proteins associates tightly with diverse developmental and pathological processes, such as tumor metastasis and mammary gland involution. In vivo evidence to support the roles of MMPs in these processes has been much harder to get. Here, we will review some of our studies on MMP11, or stromelysin-3, during the thyroid hormone-dependent amphibian metamorphosis, a process that resembles the so-called postembryonic development in mammals (from a few months before to several months after birth in humans when organ growth and maturation take place). Our investigations demonstrate that stromelysin-3 controls apoptosis in different tissues via at least two distinct mechanisms., ((c) 2010 Wiley-Liss, Inc.)

During amphibian metamorphosis, the larval tissues/organs rapidly degenerate to adapt from the aquatic to the terrestrial life. At the cellular level, a large quantity of apoptosis occurs in a spatiotemporally-regulated fashion in different organs to ensure timely removal of larval organs/tissues and the development of adult ones for the survival of the individuals. Thus, amphibian metamorphosis provides us a good opportunity to understand the mechanisms regulating apoptosis. To investigate this process at the molecular level, a number of thyroid hormone (TH) response genes have been isolated from several organs of Xenopus laevis tadpoles and their expression and functional analyses are now in progress using modern molecular and genetic technologies. In this review, we will first summarize when and where apoptosis occurs in typical larva-specific and larval-to-adult remodeling amphibian organs to highlight that the timing of apoptosis is different in different tissues/organs, even though all are induced by the same circulating TH. Next, to discuss how TH spatiotemporally regulates the apoptosis, we will focus on apoptosis of the X. laevis small intestine, one of the best characterized remodeling organs. Functional studies of TH response genes using transgenic frogs and culture techniques have shown that apoptosis of larval epithelial cells can be induced by TH either cell-autonomously or indirectly through interactions with extracellular matrix (ECM) components of the underlying basal lamina. Here, we propose that multiple intra- and extracellular apoptotic pathways are coordinately controlled by TH to ensure massive but well-organized apoptosis, which is essential for the proper progression of amphibian metamorphosis.

Interactions between cells and extracellular matrix (ECM), in particular the basement membrane (BM), are fundamentally important for the regulation of a wide variety of physiological and pathological processes. Matrix metalloproteinases (MMP) play critical roles in ECM remodeling and/or regulation of cell-ECM interactions because of their ability to cleave protein components of the ECM. Of particular interest among MMP is stromelysin-3 (ST3), which was first isolated from a human breast cancer and also shown to be correlated with apoptosis during development and invasion of tumor cells in mammals. We have been using intestinal remodeling during thyroid hormone (TH)-dependent amphibian metamorphosis as a model to study the role of ST3 during post-embryonic tissue remodeling and organ development in vertebrates. This process involves complete degeneration of the tadpole or larval epithelium through apoptosis and de novo development of the adult epithelium. Here, we will first summarize expression studies by us and others showing a tight spatial and temporal correlation of the expression of ST3 mRNA and protein with larval cell death and adult tissue development. We will then review in vitro and in vivo data supporting a critical role of ST3 in TH-induced larval epithelial cell death and ECM remodeling. We will further discuss the potential mechanisms of ST3 function during metamorphosis and its broader implications.

Matrix metalloproteinases (MMPs) play a pivotal role in development and/or pathogenesis through degrading extracellular matrix (ECM) components. We have previously shown that Xenopus MMP-9 gene is duplicated. To assess possible roles of MMP-9 and MMP-9TH in X. laevis intestinal remodeling, we here analyzed their expression profiles by in situ hybridization and show that their expression is transiently up-regulated during thyroid hormone-dependent metamorphosis. Of interest, MMP-9TH mRNA is strictly localized in the connective tissue and most highly expressed just beneath the larval epithelium that begins to undergo apoptosis. On the other hand, cells expressing MMP-9 mRNA become first detectable in the connective tissue and then, after the start of epithelial apoptosis, also in the larval epithelium. These results strongly suggest that MMP-9TH is responsible in the larval epithelial apoptosis through degrading ECM components in the basal lamina, whereas MMP-9 is involved in the removal of dying epithelial cells during amphibian intestinal remodeling., ((c) 2007 Wiley-Liss, Inc.)

To clarify connective-tissue-specific genes involved in adult epithelial development during amphibian intestinal remodeling, we have isolated 16 cDNA clones derived from the anterior part of Xenopus laevis intestine cultured in vitro by using subtractive suppression hybridization. Among four genes identified, the expression of Xtld, a Xenopus homolog of Drosophila Tolloid closely related to bone morphogenic protein-1 (BMP-1), was most remarkably up-regulated during metamorphosis. To further explore the roles of Xtld in intestinal remodeling, we examined its developmental expression in the X. laevis intestine by in situ hybridization and northern blot analysis. Xtld mRNA first became detectable in the connective tissue just before the appearance of adult epithelial primordia. Subsequently, the level of Xtld mRNA reached a high in the connective tissue, concomitantly with adult epithelial development along the anteroposterior axis of the intestine. Thereafter, towards the completion of metamorphosis, the expression of Xtld mRNA was down-regulated. Thus, the expression profile of Xtld mRNA spatiotemporally correlates well with adult epithelial development in vivo. Furthermore, the present culture study has shown that thyroid hormone (TH) up-regulates the expression of Xtld mRNA organ-autonomously in the anterior part of the intestine, but not in its posterior part, and that TH up-regulation of Xtld expression is not mediated by the epithelium. These results suggest that TH directly up-regulates Xtld expression in the connective tissue along the anteroposterior axis, which in turn plays important roles in adult epithelial development during amphibian intestinal remodeling.

We have identified one of the genes that are up-regulated by thyroid hormone (TH) in Xenopus laevis small intestine as the Xenopus homolog of bone morphogenetic protein-4 (BMP-4). To clarify possible roles of BMP-4 in intestinal remodeling during metamorphosis, we have examined its expression in X. laevis intestine by using in situ hybridization and organ culture techniques. At the beginning of metamorphic climax, BMP-4 mRNA first becomes detectable in the connective tissue, concurrently with the appearance of adult epithelial primordia. Subsequently, when the adult epithelial primordia are actively proliferating, BMP-4 mRNA becomes more abundant only in the connective tissue with a gradient toward the epithelium. Thereafter, as the adult primordia differentiate, the level of BMP-4 mRNA gradually decreases. Thus, BMP-4 expression correlates well with cell proliferation and/or initial differentiation of the adult epithelium, but not with apoptosis of the larval epithelium. Furthermore, the present culture study indicates that (1) TH-induced expression of BMP-4 mRNA is higher in the anterior part of the intestine than in the posterior part, which agrees with the better development of the adult epithelium in the more anterior part, and that (2) the expression of BMP-4 mRNA is up-regulated by TH in the presence of epithelium, but not in its absence. Therefore, BMP-4, which is indirectly induced by TH through some epithelial factor(s), probably plays important roles in adult epithelial development during amphibian intestinal remodeling.

Organogenesis and tissue remodeling are critical processes during postembryonic animal development. Anuran metamorphosis has for nearly a century served as an excellent model to study these processes in vertebrates. Frogs not only have essentially the same organs with the same functions as higher vertebrates such as humans, but also employ similar organogenic processes involving highly conserved genes. Development of frog organs takes place during metamorphosis, which is free of any maternal influences but absolutely dependent on the presence of thyroid hormone. Furthermore, this process can be easily manipulated both in intact tadpoles and in organ cultures by controlling the availability of thyroid hormone. These interesting properties have led to extensive morphological, cellular, and biochemical studies on amphibian metamorphosis. More recently, the cloning of thyroid hormone receptors and the demonstration that they are transcription factors have encouraged enormous interest in the molecular pathways controlling tissue remodeling induced by thyroid hormone during metamorphosis. This article summarizes some of the recent studies on the mechanisms of gene regulation by thyroid hormone receptors and isolation and functional characterization of genes induced by thyroid hormone during Xenopus metamorphosis. Particular focus is placed on the remodeling of the animal intestine, which involves both apoptosis (programmed cell death) of larval cells and de novo development of adult tissues, and the roles of thyroid hormone-induced genes that encode matrix metalloproteinases during this process.

Thyroid hormone (TH) plays a causative role in anuran metamorphosis. This effect is presumed to be manifested through the regulation of gene expression by TH receptors (TRs). TRs can act as both activators and repressors of a TH-inducible gene depending upon the presence and absence of TH, respectively. We have been investigating the roles of TRs during Xenopus laevis development, including premetamorphic and metamorphosing stages. In this review, we summarize some of the studies on the TRs by others and us. These studies reveal that TRs have dual functions in frog development as reflected in the following two aspects. First, TRs function initially as repressors of TH-inducible genes in premetamorphic tadpoles to prevent precocious metamorphosis, thus ensuring a proper period of tadpole growth, and later as activators of these genes to activate the metamorphic process. Second, TRs can promote both cell proliferation and apoptosis during metamorphosis, depending upon the cell type in which they are expressed.

Programmed cell death or apoptosis is an important aspect in organogenesis and tissue remodeling. It is precisely controlled both temporally and spatially during development. Amphibian metamorphosis is an excellent model to study developmental control of apoptosis in vertebrates. This process involves the transformation of essentially every organ/tissue as tadpoles change to frogs, yet is controlled by a single hormone, thyroid hormone (TH). Although different organs and tissues undergo vastly different developmental changes, including de novo development and total resorption, most require apoptotic elimination of at least some cell types. Such properties and the dependence on TH make frog metamorphosis a unique model to isolate and functionally characterize genes participating in the regulation of tissue specific cell death during organ development in vertebrates. Indeed, molecular studies of the TH-dependent gene regulation cascade have led to the discovery of a group of genes encoding matrix metalloproteinases (MMPs) participating in metamorphosis. In vivo and in vitro studies have provided strong evidence to support a role of MMP-mediated remodeling of the extracellular matrix in regulating apoptotic tissue remodeling during metamorphosis.

Matrix metalloproteinases (MMPs) are a family of extracellular proteases capable of degrading various proteinaceous components of the extracellular matrix (ECM). They have been implicated to play important roles in a number of developmental and pathological processes, such as tumor metastasis and inflammation. Relatively few studies have been carried out to investigate the function of MMPs during postembryonic organ-development. Using Xenopus laevis development as a model system, we demonstrate here that three MMPs, stromelysin-3 (ST3), collagenases-3 (Col3), and Col4, have distinct spatial and temporal expression profiles during metamorphosis as the tadpole transforms into a frog. In situ hybridizations reveal a tight, but distinct, association of individual MMPs with tissue remodeling in the tail and intestine during metamorphosis. In particular, ST3 expression is strongly correlated with apoptosis in both organs as demonstrated by analyses of serial sections with in situ hybridization for ST3 mRNA and TUNEL (terminal deoxyribonucleotidyl transferase-mediated dUTP-biotin nick end labeling) for apoptosis, respectively. On the other hand, Col3 and Col4 are present in regions where extensive connective tissue remodeling take place. These results indicate that ST3 is likely to play a role in ECM-remodeling that facilitate apoptotic tissue remodeling or resorption while Col3 and Col4 appear to participate in connective tissue degradation during development.

The regulatory effects of the thyroid hormone on amphibian metamorphosis is mediated by thyroid hormone receptors. Using Xenopus laevis as a model system, we and others have shown that the mRNA levels of thyroid hormone receptors and 9-cis retinoic acid receptors, which form the functional heterodimers with thyroid hormone receptors, are regulated temporally in a tissue-dependent manner so that high levels of their mRNAs are present in an organ when metamorphosis is occurring. By overexpressing thyroid hormone receptors, 9-cis retinoic acid receptors, or both into developing Xenopus embryos, we have shown that both thyroid hormone receptors and 9-cis retinoic acid receptors are required for mediating the effects of thyroid hormone on embryo development and precocious but specific regulation of the genes, which are normally regulated by thyroid hormone during metamorphosis. Analyses of the developmental expression of one class of thyroid hormone response genes, which encode extracellular matrix-degrading metalloproteinases, suggest that extra cellular remodeling plays an important role during tissue remodeling, including cell death (apoptosis) and cell proliferation and differentiation. This effect of extracellular matrix on cell behavior has been supported directly by in vitro primary cell culture experiments, in which intestinal epithelial cells undergo thyroid hormone-induced apoptosis, just like that during natural metamorphosis.

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.

In the amphibian small intestine, the epithelial transformation from the larval to adult type is mainly the result of degeneration of the larval epithelium and development of the new (adult) epithelium. In this analysis at the cellular level, we chronologically examined apoptosis and cell proliferation in the Xenopus intestine by using in situ nick end-labeling of genomic DNA (TUNEL) and bromodeoxyuridine (BrdU) immunohistochemistry. During pre- and prometamorphosis, few apoptotic cells were detected by TUNEL, and a small number of proliferating cells randomly distributed in the larval epithelium were labeled by BrdU. At the beginning of the metamorphic climax, when primordia of the adult epithelium were first detected, numbers of apoptotic cells suddenly increased in the larval epithelium, whereas numbers of proliferating cells increased only in the adult epithelium. Subsequently, a dramatic cell loss of the larval epithelium and a rapid growth of the adult epithelium occurred. Following complete epithelial replacement, the adult epithelium became differentiated into a simple columnar epithelium possessing a cell renewal system similar to that of mammalian intestinal epithelium. These results indicate that larval epithelial apoptosis progresses simultaneously with active proliferation of the adult epithelium during the early period of metamorphic climax, which coincides with the modification of the basement membrane lining both types of epithelia.

Programmed cell death occurs in a variety of organs during amphibian metamorphosis and is usually identified by electron microscopy as apoptosis or its modifications. Because of the massive cell death that occurs during a short period, amphibian organs serve as an ideal model system for the study of mechanisms underlying programmed cell death. In this article, a series of morphological changes in apoptosis from their nuclear changes to removal by phagocytic macrophages is reviewed, mainly in the small intestine of metamorphosing Xenopus laevis tadpoles. It is well known that cell death during amphibian metamorphosis is under the control of thyroid hormone (TH), and changes in gene expression induced by TH have been recently analyzed in a few Xenopus organs. On the other hand, there is a growing body of evidence that cell death is regulated by various kinds of local factors. For example, roles of interactions with other tissue cells and/or participation of immunocompetent cells in cell death have been experimentally shown. Therefore, to clarify the mechanisms of this complicated process, it is important at present that TH-induced changes in gene expression of each cell type comprising the organ are chronologically examined by combining morphological and molecular biological techniques.

The action of the epithelium on differentiation of connective tissue cells of Xenopus small intestine during metamorphosis was investigated by using culture and morphological techniques. Connective tissue fragments isolated from the small intestine at stage 57 were cultivated in the presence or absence of homologous epithelium. In the presence of the epithelium, metamorphic changes in the connective tissue were fully induced by hormones including thyroid hormone (T3), as during spontaneous metamorphosis, whereas they were partially induced in the absence of the epithelium. Macrophage-like cells showing non-specific esterase activity in the connective tissue were much fewer in the absence of the epithelium than in the presence of it, and aggregates of fibroblasts possessing well-developed rough endoplasmic reticulum developed only in the presence of the epithelium. Just before the aggregation of the fibroblasts, the connective tissue close to the epithelium became intensely stained with concanavalin A (ConA) and wheat germ agglutinin (WGA). The present results indicate that the epithelium plays important roles in the differentiation of intestinal connective tissue cells, which in turn affect the epithelial transformation from larval to adult form during anuran metamorphosis. Thus, the tissue interaction between the epithelium and the connective tissue in the anuran small intestine is truly bidirectional.

The degenerative processes in the larval small intestine of Xenopus laevis tadpoles during spontaneous metamorphosis and during thyroid hormone-induced metamorphosis in vitro were examined by electron microscopy. Around the beginning of spontaneous metamorphic climax (stages 59-61), both apoptotic bodies derived from larval epithelial cells and intraepithelial macrophage-like cells suddenly increase in number. The macrophage-like cells become rounded and enlarged because of numerous vacuoles containing the apoptotic bodies. Mitotic profiles of the macrophage-like cells, however, are localized in the connective tissue where different developmental stages of macrophage-like cells are present. After stage 62, the intraepithelial macrophage-like cells decrease in number, while large macrophage-like cells which include the apoptotic bodies and retain intact cell membranes and nuclei appear in the lumen. Degenerative changes similar to those during spontaneous metamorphosis described above could be reproduced in vitro. In tissue fragments isolated from the small intestine of stage 57 tadpoles and cultured in the presence of thyroid hormone, the number of intraepithelial macrophage-like cells reaches its maximum around the 3rd day of cultivation when the larval epithelial cells most rapidly decrease in number. These results suggest that the rapid degeneration of larval epithelial cells occurs not only because of apoptosis of the epithelial cells themselves but also from heterolysis by macrophages. The macrophages probably originate in the connective tissue, actively proliferate, migrate into the larval epithelium around the beginning of metamorphic climax, and are finally extruded into the lumen.

We have developed an organ culture system of the anuran small intestine to reproduce in vitro the transition from larval to adult epithelial form which occurs during spontaneous metamorphosis. Tubular fragments isolated from the small intestine of Xenopus laevis tadpoles were slit open and placed on membrane filters in culture dishes. In 60% Leibovitz 15 medium supplemented with 10% charcoal-treated serum, the explants were maintained in good condition for at least 10 days without any morphologic changes. Addition of triiodothyronine (T3) at a concentration higher than 10(-9) M to the medium could induce cell death of larval epithelial cells, but T3 alone was not sufficient for proliferation and differentiation of adult epithelial cells. When insulin (5 micrograms/ml) and cortisol (0.5 microgram/ml) besides T3 were added, the adult cells proliferated and differentiated just as during spontaneous metamorphosis. On Day 5 of cultivation, the adult cells rapidly proliferated to form typical islets, whereas the larval ones rapidly degenerated. At the same time, the connective tissue beneath the epithelium suddenly increased in cell density. These changes correspond to those occurring at the onset of metamorphic climax. By Day 10, the adult cells differentiated into a simple columnar epithelium which possessed the brush border and showed the adult-type lectin-binding pattern. Therefore, the larval epithelium of the small intestine responded to the hormones and transformed into the adult one. This organ culture system may be useful for clarifying the mechanism of the epithelial transition from larval to adult type during metamorphosis.

Ultrastructural changes in the intestinal connective tissue of Xenopus laevis during metamorphosis have been studied. Throughout the larval period to stage 60, the connective tissue consists of a few immature fibroblasts surrounded by a sparse extracellular matrix: few collagen fibrils are visible except close to the thin basal lamina. At the beginning of the transition from larval to adult epithelial form around stage 60, extensive changes are observed in connective tissue. The cells become more numerous and different types appear as the collagen fibrils increase in number and density. Through gaps in the thickened and extensively folded basal lamina, frequent contacts between epithelial and connective tissue cells are established. Thereafter, with the progression of fold formation, the connective tissue cells become oriented according to their position relative to the fold structure. The basal lamina beneath the adult epithelium becomes thin after stage 62, while that beneath the larval epithelium remains thick. Upon the completion of metamorphosis, the connective tissue consists mainly of typical fibroblasts with definite orientation and numerous collagen fibrils. These observations indicate that developmental changes in the connective tissue, especially in the region close to the epithelium, are closely related spatiotemporarily to the transition from the larval to the adult epithelial form. This suggests that tissue interactions between the connective tissue and the epithelium play important roles in controlling the epithelial degeneration, proliferation, and differentiation during metamorphic climax.

Developmental changes, until the completion of metamorphosis, of the connective tissue of the digestive tract in the South African clawed frog Xenopus laevis were examined by light and scanning electron microscopy. During the pre- and prometamorphosis (stages 50 through 59) the layer of connective tissue was prominent in the typhlosole, while that in the rest of the intestine and stomach remained very thin. Most of the connective tissue cells were fibroblasts and were concentrated mainly in the typhlosole, the boundaries of which were distinct. In the anterior part of the typhlosole just behind the stomach, the layers of connective tissue and muscle were the thickest and the boundary between these 2 layers was obscure. Although the mitotic cells of the connective tissue were rare, if any, and no migrating cells were observed on the surface of the connective tissue, the connective tissue of the intestine gradually increased in cell number during the pre- and prometamorphosis. In the stomach, on the other hand, there was no such increase. At the beginning of the metamorphic climax (stages 60 and/or 61), the layer of connective tissue suddenly became thick in both the stomach and the intestine. The boundaries of the typhlosole became less distinct. Mitotic index of the connective tissue became high, and its cells dramatically increased in number and contained many undifferentiated mesenchyme-like cells. In the stomach, these connective tissue cells invaded the glands of the larval epithelium into just below the surface epithelium and mitotic index of the entire stomach epithelium increased. In the intestine, in contrast, mitotic epithelial cells were localized in the islets which invaginated into the connective tissue and later formed the adult epithelium. With the growth of islets, the intestinal folds were formed as straight rows and, after stage 63, were modified into a zigzag pattern and had mitotic cells localized in the troughs of folds. The present results indicate that dramatic changes of the connective tissue of both the stomach and the intestine occur at the beginning of the metamorphic climax, concomitantly with those of the epithelium. Moreover, the results also show that, before the climax, even when there were no indications of metamorphic changes in the epithelium, cells of the connective tissue gradually increased in number in the intestine (but not in the stomach). It is also suggested that the muscle cells of the typhlosole just behind the stomach may participate in this gradual increase of connective tissue cells.