Essential roles of YAP-TEAD complex in adult stem cell development during thyroid hormone-induced intestinal remodeling of Xenopus laevis.

During amphibian metamorphosis which is triggered by thyroid hormone (TH), the small intestine is extensively remodeled from the larval to adult form. In the Xenopus laevis intestine, some of the larval epithelial cells dedifferentiate into adult stem cells, which newly form the adult epithelium similar to the mammalian one. We have previously shown that TH-activated Shh, Wnt and Notch signaling pathways play important roles in adult epithelial development. Here we focus on the Hippo signaling pathway, which is known to interact with these pathways in the mammalian intestine. Our quantitative RT-PCR analysis indicates that the expression of genes involved in this pathway including YAP1, TAZ, TEAD1 and core kinases is differently regulated by TH in the metamorphosing intestine. Additionally, we show by in situ hybridization and immunohistochemistry that the transcriptional co-activator YAP1, a major effector of the Hippo signaling, is expressed in the adult stem cells and connective tissue cells surrounding them and that YAP1 protein is localized in either nucleus or cytoplasm of the stem cells. We further show that YAP1 binds its binding partner TEAD1 (transcription factor) in vivo and that their interaction is inhibited by verteporfin (VP). More importantly, by using VP in organ culture of the tadpole intestine, we experimentally demonstrate that the inhibition of YAP1-TEAD1 interaction decreases both TH-induced stem cells expressing LGR5 and nearby connective tissue cells in number and proliferation, leading to the failure of adult epithelial development. Our results indicate that YAP-TEAD complex is required for stem cell development during intestinal remodeling.

Stem cell development involves divergent thyroid hormone receptor subtype expression and epigenetic modifications in the Xenopus metamorphosing intestine.

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, TRß 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 TRß in the intestinal larval-to-adult remodeling, involving distinctive epigenetic modifications in the SC lineage.

Expression of hyaluronan synthases upregulated by thyroid hormone is involved in intestinal stem cell development during Xenopus laevis metamorphosis.

During amphibian intestinal remodeling, thyroid hormone (TH) induces adult stem cells, which newly generate the absorptive epithelium analogous to the mammalian one. We have previously shown that hyaluronan (HA) is newly synthesized and plays an essential role in the development of the stem cells via its major receptor CD44 in the Xenopus laevis intestine. We here focused on HA synthase (HAS) and examined how the expression of HAS family genes is regulated during natural and TH-induced metamorphosis. Our quantitative RT-PCR analysis indicated that the mRNA expression of HAS2 and HAS3, but not that of HAS1 and HAS-rs, a unique Xenopus HAS-related sequence, is upregulated concomitantly with the development of adult epithelial primordia consisting of the stem/progenitor cells during the metamorphic climax. In addition, our in situ hybridization analysis indicated that the HAS3 mRNA is specifically expressed in the adult epithelial primordia, whereas HAS2 mRNA is expressed in both the adult epithelial primordia and nearby connective tissue cells during this period. Furthermore, by treating X. laevis tadpoles with 4-methylumbelliferone, a HA synthesis inhibitor, we have experimentally shown that inhibition of HA synthesis leads to suppression of TH-upregulated expression of leucine-rich repeat-containing G protein-coupled 5 (LGR5), an intestinal stem cell marker, CD44, HAS2, HAS3, and gelatinase A in vivo. These findings suggest that HA newly synthesized by HAS2 and/or HAS3 is required for intestinal stem cell development through a positive feedback loop and is involved in the formation of the stem cell niche during metamorphosis.

Essential Roles of Thyroid Hormone-Regulated Hyaluronan/CD44 Signaling in Adult Stem Cell Development During Xenopus laevis Intestinal Remodeling.

In the amphibian intestine during metamorphosis, thyroid hormone (TH) induces some larval epithelial cells to dedifferentiate into stem cells, which generate the adult epithelium analogous to the mammalian intestinal epithelium. We have previously shown that the canonical Wnt signaling pathway is involved in adult epithelial development in the Xenopus laevis intestine. To understand the function of this pathway more precisely, we here focused on CD44, a major Wnt target, which has been identified as a TH response gene in the X. laevis intestine. Our in situ hybridization analysis indicated that CD44 mRNA is detectable in adult epithelial primordia consisting of the adult stem/progenitor cells and is strongly expressed in the connective tissue (CT) cells surrounding them. Interestingly, when the expression of CD44 mRNA is the highest, hyaluronan (HA), a principle ligand of CD44, is newly synthesized and becomes most abundantly distributed in the CT just beneath the adult epithelial primordia that are actively proliferating. Thereafter, as the adult primordia differentiate into the simple columnar epithelium, the expression of CD44 mRNA is gradually downregulated. More importantly, using organ cultures of the X. laevis tadpole intestine in the presence of TH, we have experimentally shown that inhibition of HA synthesis by 4-methylumbelliferone suppresses development of not only the CT but also the epithelial stem cells, resulting in failure to generate the AE. Our findings strongly suggest that TH-upregulated HA/CD44 signaling plays an essential role in formation of the intestinal stem cell niche during vertebrate postembryonic development. Stem Cells 2017;35:2175-2183.

Thyroid Hormone-Induced Activation of Notch Signaling is Required for Adult Intestinal Stem Cell Development During Xenopus Laevis Metamorphosis.

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.

Thyroid hormone activates Wnt/ß-catenin signaling involved in adult epithelial development during intestinal remodeling in Xenopus laevis.

During amphibian intestinal remodeling, thyroid hormone (TH) induces some larval epithelial cells to dedifferentiate into adult stem cells, which newly generate the absorptive epithelium analogous to the mammalian epithelium. To clarify molecular mechanisms underlying adult epithelial development, we here focus on TH response genes that are associated with the canonical Wnt pathway. Our quantitative reverse transcription plus polymerase chain reaction and immunohistochemical analyses indicate that all of the genes examined, including ß-catenin, c-Myc and secreted frizzle-related protein 2 (SFRP2), are up-regulated in Xenopus laevis intestine during both natural and TH-induced metamorphosis. Moreover, immunoreactivity for nuclear ß-catenin becomes detectable in adult stem cells from the start of their appearance and then increases in intensity in adult epithelial primordia derived from the stem cells, which actively proliferate and coexpress Wnt target genes c-Myc and LGR5. These expression profiles strongly suggest the involvement of the canonical Wnt pathway in the maintenance and/or proliferation of adult stem/progenitor cells. More importantly, by using organ cultures of the tadpole intestine, we have experimentally shown that the addition of exogenous SFRP2 protein to the culture medium promotes cell proliferation of the adult epithelial primordia, whereas inhibition of endogenous SFRP2 by its antibody suppresses their proliferation. The inhibition of SFRP2 suppresses larval epithelial changes in shape from simple columnar to stem-cell-like roundish cells, resulting in the failure of epithelial dedifferentiation. Thus, TH-up-regulated SFRP2 in the postembryonic intestine promotes adult stem cell development, possibly by acting as an agonist of both canonical and non-canonical Wnt signaling.

Thyroid hormone-induced sonic hedgehog signal up-regulates its own pathway in a paracrine manner in the Xenopus laevis intestine during metamorphosis.

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.

Stem cell development involves divergent thyroid hormone receptor subtype expression and epigenetic modifications in the amphibian intestine during metamorphosis.

In the amphibian intestine during metamorphosis, most of the larval epithelial cells undergo apoptosis, while a small number of the epithelial cells dedifferentiate into stem cells (SCs). The SCs actively proliferate and then newly generate the adult epithelium analogous to the mammalian counterpart, which is continuously renewed from the SCs throughout adulthood. This larval-to-adult intestinal remodeling can be experimentally induced by thyroid hormone (TH) through interacting with the surrounding connective tissue that develops as the stem cell niche. Thus, the amphibian intestine provides us a valuable opportunity to study how the SCs and their niche are formed during development. To clarify the TH-induced and evolutionally conserved mechanism of SC development at the molecular level, numerous TH response genes have been identified in the Xenopus laevis intestine over the last three decades and extensively analyzed for their expression and function by using wild-type and transgenic Xenopus tadpoles. Interestingly, accumulating evidence indicates that thyroid hormone receptor (TR) epigenetically regulates the expression of TH response genes involved in the remodeling. In this review, we highlight recent progress in the understanding of SC development, focusing on epigenetic gene regulation by TH/TR signaling in the X. laevis intestine. We here propose that two subtypes of TRs, TRα and TRß, play distinct roles in the intestinal SC development via different histone modifications in different cell types.

Epithelial-connective tissue interactions induced by thyroid hormone receptor are essential for adult stem cell development in the Xenopus laevis intestine.

In the amphibian intestine during metamorphosis, stem cells appear and generate the adult absorptive epithelium, analogous to the mammalian one, under the control of thyroid hormone (TH). We have previously shown that the adult stem cells originate from differentiated larval epithelial cells in the Xenopus laevis intestine. To clarify whether TH signaling in the epithelium alone is sufficient for inducing the stem cells, we have now performed tissue recombinant culture experiments using transgenic X. laevis tadpoles that express a dominant-positive TH receptor (dpTR) under a control of heat shock promoter. Wild-type (Wt) or dpTR transgenic (Tg) larval epithelium (Ep) was isolated from the tadpole intestine, recombined with homologous or heterologous nonepithelial tissues (non-Ep), and then cultivated in the absence of TH with daily heat shocks to induce transgenic dpTR expression. Adult epithelial progenitor cells expressing sonic hedgehog became detectable on day 5 in both the recombinant intestine of Tg Ep and Tg non-Ep (Tg/Tg) and that of Tg Ep and Wt non-Ep (Tg/Wt). However, in Tg/Wt intestine, they did not express other stem cell markers such as Musashi-1 and never generated the adult epithelium expressing a marker for absorptive epithelial cells. Our results indicate that, while it is unclear why some larval epithelial cells dedifferentiate into adult progenitor/stem cells, TR-mediated gene expression in the surrounding tissues other than the epithelium is required for them to develop into adult stem cells, suggesting the importance of TH-inducible epithelial-connective tissue interactions in establishment of the stem cell niche in the amphibian intestine.

Thyroid hormone-regulated expression of nuclear lamins correlates with dedifferentiation of intestinal epithelial cells during Xenopus laevis metamorphosis.

In the Xenopus laevis intestine during metamorphosis, which is triggered by thyroid hormone (TH), the adult epithelium develops and replaces the larval one undergoing apoptosis. We have previously shown that progenitor/stem cells of the adult epithelium originate from some differentiated larval epithelial cells. To investigate molecular mechanisms underlying larval epithelial dedifferentiation into the adult progenitor/stem cells, we here focused on nuclear lamin A (LA) and lamin LIII (LIII), whose expression is generally known to be correlated with the state of cell differentiation. We analyzed the spatiotemporal expression of LA and LIII during X. laevis intestinal remodeling by reverse transcription PCR, Western blotting, and immunohistochemistry. At the onset of natural metamorphosis, when the adult epithelial progenitor cells appear as small islets, the expression of LA is down-regulated, but that of LIII is up-regulated only in the islets. Then, as the adult progenitor cells differentiate, the expression of LA is up-regulated, whereas that of LIII is down-regulated in the adult cells. As multiple intestinal folds form, adult epithelial cells positive for LIII become restricted only to the troughs of the folds. In addition, we have shown that TH up- or down-regulates the expression of these lamins in the premetamorphic intestine as during natural metamorphosis. These results indicate that TH-regulated expression of LA and LIII closely correlates with dedifferentiation of the epithelial cells in the X. laevis intestine, suggesting the involvement of the lamins in the process of dedifferentiation during amphibian metamorphosis.

Spatiotemporal expression profile of no29/nucleophosmin3 in the intestine of Xenopus laevis during metamorphosis.

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.

RCAN1 regulates vascular branching during Xenopus laevis angiogenesis.

BACKGROUND/AIMS: The mechanisms that regulate the size-related morphologies of various blood vessels from the aorta to capillary vessels are still poorly understood. In this study, we evaluate the involvement of regulator of calcineurin 1 (RCAN1), a regulatory protein in the calcineurin/NFAT signal transduction pathway, in vascular morphology to gain further insight into these mechanisms. METHODS AND RESULTS: We first generated 2 types of vasculature in vitro from the same source of human umbilical vein endothelial cells by fibrin gel assay. We found that RCAN1 was significantly upregulated in large vessels with low branching frequencies when compared with small vessels with high branching frequencies. Next, to clarify whether RCAN1 regulates the branching of blood vessels in vivo, we injected RCAN1 mRNA into fertilized Xenopus laevis eggs. Overexpression of RCAN1 decreased the number of branching points that sprouted from intersomitic vessels during X. laevis angiogenesis. In addition, coexpression of calcineurin A, a target of RCAN1, could rescue RCAN1-suppressed vascular branching. CONCLUSIONS: These results provide in vivo evidence of RCAN1-regulated vascular branching which may play a role in the patterning of morphologically different vasculature.

Amphibian organ remodeling during metamorphosis: insight into thyroid hormone-induced apoptosis.

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.

Tissue-dependent induction of apoptosis by matrix metalloproteinase stromelysin-3 during amphibian metamorphosis.

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.

Apoptosis in amphibian organs during metamorphosis.

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.

Origin of the adult intestinal stem cells induced by thyroid hormone in Xenopus laevis.

In the amphibian intestine during metamorphosis, de novo stem cells generate the adult epithelium analogous to the mammalian counterpart. Interestingly, to date the exact origin of these stem cells remains to be determined, making intestinal metamorphosis a unique model to study development of adult organ-specific stem cells. Here, to determine their origin, we made use of transgenic Xenopus tadpoles expressing green fluorescent protein (GFP) for recombinant organ cultures. The larval epithelium separated from the wild-type (Wt) or GFP transgenic (Tg) intestine before metamorphic climax was recombined with homologous and heterologous nonepithelial tissues and was cultivated in the presence of thyroid hormone, the causative agent of metamorphosis. In all kinds of recombinant intestine, adult progenitor cells expressing markers for intestinal stem cells such as sonic hedgehog became detectable and then differentiated into the adult epithelium expressing intestinal fatty acid binding-protein, a marker for absorptive cells. Notably, whenever the epithelium was derived from Tg intestine, both the adult progenitor/stem cells and their differentiated cells expressed GFP, whereas neither of them expressed GFP in the Wt-derived epithelium. Our results provide direct evidence that stem cells that generate the adult intestinal epithelium originate from the larval epithelium, through thyroid hormone-induced dedifferentiation.

Thyroid hormone-induced expression of Foxl1 in subepithelial fibroblasts correlates with adult stem cell development during Xenopus intestinal remodeling.

In the Xenopus laevis intestine during metamorphosis, stem cells appear and generate the adult epithelium analogous to the mammalian one. We have previously shown that connective tissue cells surrounding the epithelium are essential for the stem cell development. To clarify whether such cells correspond to mammalian Foxl1-expressing mesenchymal cells, which have recently been shown to be a critical component of intestinal stem cell niche, we here examined the expression profile of Foxl1 in the X. laevis intestine by using RT-PCR and immunohistochemistry. Foxl1 expression was transiently upregulated only in connective tissue cells during the early period of metamorphic climax and was the highest just beneath the proliferating stem/progenitor cells. In addition, electron microscopic analysis showed that these subepithelial cells are ultrastructurally identified as telocytes like the mammalian Foxl1-expressing cells. Furthermore, we experimentally showed that Foxl1 expression is indirectly upregulated by thyroid hormone (TH) through Shh signaling and that TH organ-autonomously induces the Foxl1-expressing cells concomitantly with appearance of the stem cells in the tadpole intestine in vitro. The present results suggest that intestinal niche cells expressing Foxl1 are evolutionally conserved among terrestrial vertebrates and can be induced by TH/Shh signaling during amphibian metamorphosis for stem cell development.

Regulation of extracellular matrix remodeling and cell fate determination by matrix metalloproteinase stromelysin-3 during thyroid hormone-dependent post-embryonic development.

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.

Thyroid hormone regulation of stem cell development during intestinal remodeling.

During amphibian metamorphosis the small intestine is remodeled from larval to adult form, analogous to the mammalian intestine. The larval epithelium mostly undergoes apoptosis, while a small number of stem cells appear, actively proliferate, and differentiate into the adult epithelium possessing a cell-renewal system. Because amphibian intestinal remodeling is completely controlled by thyroid hormone (T3) through T3 receptors (TRs), it serves as an excellent model for studying the molecular mechanism of the mammalian intestinal development. TRs bind T3 response elements in target genes and have dual functions by interacting with coactivators or corepressors in a T3-dependent manner. A number of T3 response genes have been isolated from the Xenopus laevis intestine. They include signaling molecules, matrix metalloproteinases, and transcription factors. Functional studies have been carried out on many such genes in vitro and in vivo by using transgenic and culture technologies. Here we will review recent findings from such studies with a special emphasis on the adult intestinal stem cells, and discuss the evolutionarily conserved roles of T3 in the epithelial cell-renewal in the vertebrate intestine.

Sonic hedgehog and bone morphogenetic protein-4 signaling pathway involved in epithelial cell renewal along the radial axis of the intestine.

The organogenesis of the digestive tract proceeds according to the positional information along the cephalo-caudal, dorsal-ventral and left-right axes of the embryonic body and the radial axis of the tract during development. Among them the radial axis, which corresponds to the crypt-villus axis in the adult small intestine, is essential for a rapid cell renewal of the epithelium throughout adulthood and is important from the clinical viewpoint. All of the adult intestinal epithelial cells originate from multipotent stem cells localized in the basal region of the crypt. Descendants of the stem cells, as they migrate up or down along the crypt-villus axis, actively proliferate, differentiate and finally undergo apoptosis. Recently, there has been a growing body of evidence that the Wnt and Notch signaling pathways are involved in cell proliferation and cell fate determination, respectively, during the epithelial cell renewal. However, the molecular mechanisms by which the radial axis is established and/or is maintained to enable the epithelial cell renewal have not yet been fully understood, and their clarification is urgently needed for stem cell therapies. In the amphibian intestine during metamorphosis, stem cells analogous to the mammalian ones appear and newly form the epithelium that undergoes the cell renewal along the radial axis by the inductive action of thyroid hormone. Thus, this animal model provides us with a good opportunity to clarify the molecular mechanisms of radial axis formation. By using the Xenopus laevis intestine, we found that sonic hedgehog (Shh), which is secreted by the stem cells, induces bone morphogenetic protein-4 (BMP-4) in subepithelial fibroblasts and that both Shh and BMP-4 are involved in the development of the cell-renewable epithelium. In this review, we highlight the molecular aspects of the cell renewal of the adult intestinal epithelium and propose important roles of the Shh/BMP-4 signaling pathway in the establishment and/or maintenance of the radial axis common to the human intestine.