Takashi Hasebe

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.— Ishizuya‐Oka, A.,Hasebe, T., Buchholz, D. R., Kajita, M., Fu, L., Shi, Y.‐B. The origin of the adult intestinal stem cells induced by thyroid hormone in Xenopus laevis. FASEB J. 23, 2568–2575 (2009)

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.

The development of the adult intestinal stem cells: Insights from studies on thyroid hormone-dependent amphibian metamorphosis

Adult organ-specific stem cells are essential for organ homeostasis and repair in adult vertebrates. The intestine is one of the best-studied organs in this regard. The intestinal epithelium undergoes constant self-renewal throughout adult life across vertebrates through the proliferation and subsequent differentiation of the adult stem cells. This self-renewal system is established late during development, around birth, in mammals when endogenous thyroid hormone (T3) levels are high. Amphibian metamorphosis resembles mammalian postembryonic development around birth and is totally dependent upon the presence of high levels of T3. During this process, the tadpole intestine, predominantly a monolayer of larval epithelial cells, undergoes drastic transformation. 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. We and others have studied the T3-dependent remodeling of the intestine in Xenopus laevis. Here we will highlight some of the recent findings on the origin of the adult intestinal stem cells. We will discuss observations suggesting that liganded T3 receptor (TR) regulates cell autonomous formation of adult intestinal progenitor cells and that T3 action in the connective tissue is important for the establishment of the stem cell niche. We will further review evidence suggesting similar T3-dependent formation of adult intestinal stem cells in other vertebrates.

Shh/BMP-4 Signaling Pathway Is Essential for Intestinal Epithelial Development During Xenopus Larval-to-Adult Remodeling

During amphibian larval‐to‐adult intestinal remodeling, progenitor cells of the adult epithelium actively proliferate and differentiate under the control of thyroid hormone (TH) to form the intestinal absorptive epithelium, which is analogous to the mammalian counterpart. We previously found that TH–up‐regulated expression of bone morphogenetic protein‐4 (BMP‐4) spatiotemporally correlates with adult epithelial development in the Xenopus laevis intestine. Here, we aimed to clarify the role of BMP‐4 in intestinal remodeling. Our reverse transcriptase‐polymerase chain reaction and in situ hybridization analyses indicated that mRNA of BMPR‐IA, a type I receptor of BMP‐4, is expressed in both the developing connective tissue and progenitor cells of the adult epithelium. More importantly, using organ culture and immunohistochemical procedures, we have shown that BMP‐4 not only represses cell proliferation of the connective tissue but promotes differentiation of the intestinal absorptive epithelium. In addition, we found that the connective tissue‐specific expression of BMP‐4 mRNA is up‐regulated by sonic hedgehog (Shh), whose epithelium‐specific expression is directly induced by TH. These results strongly suggest that the Shh/BMP‐4 signaling pathway plays key roles in the amphibian intestinal remodeling through epithelial–connective tissue interactions. Developmental Dynamics 235:3240–3249, 2006.

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. STEM CELLS 2011;29:154–161

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.

Spatio-Temporal Expression Profile of Stem Cell- Associated Gene LGR5 in the Intestine during Thyroid Hormone-Dependent Metamorphosis in Xenopus laevis

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.

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.

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.

Improved Cre reporter transgenic Xenopus

We have produced and characterized improved transgenic reporter lines for detection of Cre recombinase activity during Xenopus development. Improvements include choice of fluorophores, which make these Cre reporter lines generally suitable for lineage tracing studies. We also include data for several new parameters affecting survival and transgenesis efficiency using the recently developed meganuclease method of frog transgenesis. These transgenic frogs express cyan fluorescent protein (CFP) under control of the ubiquitous promoter CMV, where CFP is replaced by DsRed2 (a red fluorescent protein) in the presence of Cre. Three independent, high expression, Cre‐sensitive lines have been identified that maintain robust fluorophore expression across generations and lack DsRed2 expression in the absence of Cre. A novel use of these lines is to indelibly mark embryonic blastomeres by Cre mRNA injection for permanent fate mapping. Similarly, transgenically expressed Cre under control of tissue‐specific promoters will allow detailed analysis of cell lineage relationships throughout embryogenesis, metamorphosis, and adulthood. Developmental Dynamics 238:2401–2408, 2009.