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.

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.

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.

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.

Functional analysis of thyroid hormone receptor beta in Xenopus tropicalis founders using CRISPR-Cas.

Amphibians provide an ideal model to study the actions of thyroid hormone (TH) in animal development because TH signaling via two TH receptors, TRα and TRß, is indispensable for amphibian metamorphosis. However, specific roles for the TRß isoform in metamorphosis are poorly understood. To address this issue, we generated trß-disrupted Xenopus tropicalis tadpoles using the CRISPR-Cas system. We first established a highly efficient and rapid workflow for gene disruption in the founder generation (F0) by injecting sgRNA and Cas9 ribonucleoprotein. Most embryos showed severe mutant phenotypes carrying high somatic mutation rates. Utilizing this founder analysis system, we examined the role of trß in metamorphosis. trß-disrupted pre-metamorphic tadpoles exhibited mixed responsiveness to exogenous TH. Specifically, gill resorption and activation of several TH-response genes, including trß itself and two protease genes, were impaired. However, hind limb outgrowth and induction of the TH-response genes, klf9 and fra-2, were not affected by loss of trß Surprisingly, trß-disrupted tadpoles were able to undergo spontaneous metamorphosis normally, except for a slight delay in tail resorption. These results indicate TRß is not required but contributes to the timing of resorptive events of metamorphosis.

Organ Culture of the Xenopus Tadpole Intestine.

During Xenopus metamorphosis, most tadpole organs remodel from the larval to adult form to prepare for adaptation to terrestrial life. Organ culture serves as an important tool for studying larval-to-adult organ remodeling independent of the effects of other parts of the body. Here, I introduce a protocol for organ culture in vitro using the Xenopus laevis tadpole intestine before metamorphic climax. During culture in the absence of exogenous 3,3',5-triiodo-l-thyronine (T3), the most potent natural thyroid hormone, the intestine remains in its larval state without any metamorphic changes. In contrast, when T3 is added to the culture medium, the larval epithelium undergoes apoptosis, whereas adult stem cells appear, actively proliferate, and finally generate the differentiated adult epithelium within a week. At the same time, the surrounding nonepithelial tissues also develop. Thus, this culture model is useful for clarifying the control mechanisms of apoptosis in larval tissues, formation of adult stem cells, and cell proliferation and differentiation of adult tissues, all of which occur in harmony during natural metamorphosis. Moreover, a procedure for tissue recombination combined with organ culture provides a platform for investigating complex tissue interactions during organ remodeling. Such tissue recombination experiments will help to reveal the important role of nonepithelial tissues in larval epithelial apoptosis and/or adult stem cell development in the X. laevis intestine.

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.

Growth, Development, and Intestinal Remodeling Occurs in the Absence of Thyroid Hormone Receptor α in Tadpoles of Xenopus tropicalis.

During development in all vertebrates, thyroid hormone receptors (TRs) are expressed before as well as during and after the peak in plasma thyroid hormone (TH) levels. Previously, we established a role for unliganded TRα in gene repression and developmental timing using tadpoles of TRα knockout (TRαKO) frogs. Here, we examined the role of liganded TRα on growth, development, and intestinal remodeling during natural and TH-induced metamorphosis. Disrupted TRα had little effect on growth during the larval period, but after metamorphosis, TRαKO juveniles grew more slowly than wild-type (WT) juveniles. TRαKO tadpoles developed faster throughout premetamorphosis when TH was low or absent, and despite their decreased responsivity to exogenous TH, TRαKO tadpoles not only were able to complete TH-dependent metamorphosis but also did so earlier than WT tadpoles. In contrast to external morphology, larval epithelial cell apoptosis and adult cell proliferation of intestinal remodeling were delayed in TRαKO tadpoles. Also, TRαKO intestines did not shrink in length to the full extent, and fewer intestinal folds into the lumen were present in TRαKO compared with WT juveniles. Such delayed remodeling occurred despite higher premetamorphic expression levels of TH target genes important for metamorphic progression-namely, TRß, Klf9, and ST3. Furthermore, the decreased TH-dependent intestinal shrinkage was consistent with reduced TH response gene expression during natural and TH-induced metamorphosis. As in the TRα null mouse model, TRαKO frogs had statistically significant but surprisingly mild growth and development phenotypes with normal survival and fertility.

How thyroid hormone regulates transformation of larval epithelial cells into adult stem cells in the amphibian intestine.

In the amphibian intestine during metamorphosis, a small number of larval epithelial cells dedifferentiate into adult stem cells that newly form the adult epithelium analogous to the mammalian counterpart, while most of them undergo apoptosis. Because this larval-to-adult intestinal remodeling can be experimentally induced by thyroid hormone (TH) both in vivo and in vitro, TH response genes identified in the Xenopus intestine provide us valuable clues to investigating how adult stem cells and their niche are formed during postembryonic development. Their expression and functional analyses by using the culture and recent transgenic (Tg) techniques have shed light on key signaling pathways essential for intestinal stem cell development. The present review focuses on such recent findings and discusses the evolutionally conserved roles of TH in development or maintenance of the stem cells which are common to the terrestrial vertebrate intestines.

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.

A requirement for hedgehog signaling in thyroid hormone-induced postembryonic intestinal remodeling.

BACKGROUND: Intestinal remodeling during amphibian metamorphosis has long been studied as a model for the formation of the adult organs in vertebrates, especially the formation of adult organ-specific stem cells. Like all other processes during metamorphosis, this process is controlled by thyroid hormone (T3), which affects cell fate and behavior through transcriptional regulation of target genes by binding to T3 receptors (TRs). Earlier studies have shown that Sonic hedgehog (Shh) is induced by T3 in the developing adult stem cells and that the Shh receptor and other downstream components are present in the connective tissue and at lower levels in the muscles at the climax of intestinal remodeling. However, no in vivo studies have carried out to investigate whether Shh produced in the adult cells can regulate the connective tissue to promote intestinal maturation. RESULTS: We have addressed this issue by treating tadpoles with Shh inhibitor cyclopamine. We showed that cyclopamine but not the structurally related chemical tomatidine inhibited the expression of Shh response genes BMP4, Snai2, and Twist1. More importantly, we showed that cyclopamine reduced the cell proliferation of both the developing adult stem cells as well as cells in the other intestinal tissues at the climax of metamorphosis, leading to delayed/incomplete remodeling of the intestine at the end of metamorphosis. We further revealed that both Snai2 and Twist1 were strongly upregulated during metamorphosis in the intestine and their expression was restricted to the connective tissue. CONCLUSIONS: Our results suggest that Shh indeed signals the connective tissue whereby it can increase adult stem cell proliferation and promote formation of the adult intestine.

Thyroid hormone-regulated Wnt5a/Ror2 signaling is essential for dedifferentiation of larval epithelial cells into adult stem cells in the Xenopus laevis intestine.

BACKGROUND AND AIMS: Amphibian intestinal remodeling, where thyroid hormone (T3) induces some larval epithelial cells to become adult stem cells analogous to the mammalian intestinal ones, serves as a unique model for studying how the adult stem cells are formed. To clarify its molecular mechanisms, we here investigated roles of non-canonical Wnt signaling in the larval-to-adult intestinal remodeling during Xenopus laevis metamorphosis. METHODS/FINDINGS: Our quantitative RT-PCR (qRT-PCR) and immunohistochemical analyses indicated that the expressions of Wnt5a and its receptors, frizzled 2 (Fzd2) and receptor tyrosine kinase-like orphan receptor 2 (Ror2) are up-regulated by T3 and are spatiotemporally correlated with adult epithelial development in the X. laevis intestine. Notably, changes in morphology of larval absorptive epithelial cells expressing Ror2 coincide well with formation of the adult stem cells during metamorphosis. In addition, by using organ cultures of the tadpole intestine, we have experimentally shown that addition of exogenous Wnt5a protein to the culture medium causes morphological changes in the larval epithelium expressing Ror2 even in the absence of T3. In contrast, in the presence of T3 where the adult stem cells are formed in vitro, inhibition of endogenous Wnt5a by an anti-Wnt5a antibody suppressed the epithelial morphological changes, leading to the failure of stem cell formation. SIGNIFICANCE: Our findings strongly suggest that the adult stem cells originate from the larval absorptive cells expressing Ror2, which require Wnt5a/Ror2 signaling for their dedifferentiation accompanied by changes in cell morphology.

Involvement of androgen receptor in sex determination in an amphibian species.

In mice and humans, the androgen receptor (AR) gene, located on the X chromosome, is not known to be involved in sex determination. In the Japanese frog Rana rugosa the AR is located on the sex chromosomes (X, Y, Z and W). Phylogenetic analysis shows that the AR on the X chromosome (X-AR) of the Korean R. rugosa is basal and segregates into two clusters: one containing W-AR of Japanese R. rugosa, the other containing Y-AR. AR expression is twice as high in ZZ (male) compared to ZW (female) embryos in which the W-AR is barely expressed. Higher AR-expression may be associated with male sex determination in this species. To examine whether the Z-AR is involved in sex determination in R. rugosa, we produced transgenic (Tg) frogs carrying an exogenous Z-AR. Analysis of ZW Tg frogs revealed development of masculinized gonads or 'ovotestes'. Expression of CYP17 and Dmrt1, genes known to be activated during normal male gonadal development, were up-regulated in the ZW ovotestis. Testosterone, supplied to the rearing water, completed the female-to-male sex-reversal in the AR-Tg ZW frogs. Here we report that Z-AR is involved in male sex-determination in an amphibian species.

Expression profiling of intestinal tissues implicates tissue-specific genes and pathways essential for thyroid hormone-induced adult stem cell development.

The study of the epithelium during development in the vertebrate intestine touches upon many contemporary aspects of biology: to name a few, the formation of the adult stem cells (ASCs) essential for the life-long self-renewal and the balance of stem cell activity for renewal vs cancer development. Although extensive analyses have been carried out on the property and functions of the adult intestinal stem cells in mammals, little is known about their formation during development due to the difficulty of manipulating late-stage, uterus-enclosed embryos. The gastrointestinal tract of the amphibian Xenopus laevis is an excellent model system for the study of mammalian ASC formation, cell proliferation, and differentiation. During T3-dependent amphibian metamorphosis, the digestive tract is extensively remodeled from the larval to the adult form for the adaptation of the amphibian from its aquatic herbivorous lifestyle to that of a terrestrial carnivorous frog. This involves de novo formation of ASCs that requires T3 signaling in both the larval epithelium and nonepithelial tissues. To understand the underlying molecular mechanisms, we have characterized the gene expression profiles in the epithelium and nonepithelial tissues by using cDNA microarrays. Our results revealed that T3 induces distinct tissue-specific gene regulation programs associated with the remodeling of the intestine, particularly the formation of the ASCs, and further suggested the existence of potentially many novel stem cell-associated genes, at least in the intestine during development.

Thyroid hormone-induced cell-cell interactions are required for the development of adult intestinal stem cells.

The mammalian intestine has long been used as a model to study organ-specific adult stem cells, which are essential for organ repair and tissue regeneration throughout adult life. The establishment of the intestinal epithelial cell self-renewing system takes place during perinatal development when the villus-crypt axis is established with the adult stem cells localized in the crypt. This developmental period is characterized by high levels of plasma thyroid hormone (T3) and T3 deficiency is known to impair intestinal development. Determining how T3 regulates adult stem cell development in the mammalian intestine can be difficult due to maternal influences. Intestinal remodeling during amphibian metamorphosis resembles perinatal intestinal maturation in mammals and its dependence on T3 is well established. A major advantage of the amphibian model is that it can easily be controlled by altering the availability of T3. The ability to manipulate and examine this relatively rapid and localized formation of adult stem cells has greatly assisted in the elucidation of molecular mechanisms regulating their formation and further revealed evidence that supports conservation in the underlying mechanisms of adult stem cell development in vertebrates. Furthermore, genetic studies in Xenopus laevis indicate that T3 actions in both the epithelium and the rest of the intestine, most likely the underlying connective tissue, are required for the formation of adult stem cells. Molecular analyses suggest that cell-cell interactions involving hedgehog and BMP pathways are critical for the establishment of the stem cell niche that is essential for the formation of the adult intestinal stem cells.

Tissue-specific upregulation of MDS/EVI gene transcripts in the intestine by thyroid hormone during Xenopus metamorphosis.

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.

Establishment of intestinal stem cell niche during amphibian metamorphosis.

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.

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.