Molecular mechanisms for thyroid hormone-induced remodeling in the amphibian digestive tract: a model for studying organ regeneration

During amphibian metamorphosis the digestive tract is extensively remodeled under the control of epithelial-connective tissue interactions. At the cellular level, larval epithelial cells undergo apoptosis, while a small number of stem cells appear, actively proliferate, and then differentiate to form adult epithelium that is analogous to its mammalian counterpart. Therefore the amphibian digestive tract is a unique model system for the study of postembryonic organ regeneration. As amphibian intestinal remodeling can be triggered by thyroid hormone (TH), the molecular mechanisms involved can be studied from the perspective of examining the expression cascade of TH response genes. A number of these genes have been isolated from the intestine of Xenopus laevis. Recent progress in the functional analysis of this cascade has shed light on key molecules in intestinal remodeling such as matrix metalloproteinase-11, sonic hedgehog, and bone morphogenetic protein-4. These genes are also thought to play key roles in organogenesis and/or homeostasis in both chick and mammalian digestive tract, suggesting the existence of conserved mechanisms underlying such events in terrestrial vertebrates. In this article, we review our recent findings in this field, focusing on the development of adult epithelium in the X. laevis intestine.     

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.     

Isolation of connective-tissue-specific genes involved in <Emphasis Type="Italic">Xenopus</Emphasis> intestinal remodeling: thyroid hormone up-regulates Tolloid/BMP-1 expression

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.     

Thyroid-hormone-dependent and fibroblast-specific expression of BMP-4 correlates with 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.     

Evolutionary insights into postembryonic development of adult intestinal stem cells

In the adult vertebrate intestine, multi-potent stem cells continuously generate all of the epithelial cells throughout the adulthood. While it has long been known that the frog intestine is formed via the development of adult intestinal stem cells during thyroid hormone (TH)-dependent metamorphosis, the basic structure of the adult intestine is formed by birth in mammals and it is unclear if the subsequent maturation of the intestine involves any changes in the intestinal stem cells. Two recent papers showing that B lymphocyte-induced maturation protein 1 (Blimp1) regulates postnatal epithelial stem cell reprogramming during mouse intestinal maturation support the model that adult intestinal stem cells are developed during postembryonic development in mammals, in a TH-dependent process similar to intestinal remodeling during amphibian metamorphosis. Since the formation of the adult intestine in both mammals and amphibians is closely associated with the adaptation from aquatic to terrestrial life during the peak of endogenous TH levels, the molecular mechanisms by which the adult stem cells are developed are likely evolutionally conserved.     

Recent progress in the study of brown adipose tissue

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.     

Thyroid hormone-induced expression of Sonic hedgehog correlates with adult epithelial development during remodeling of the Xenopus stomach and intestine

Sonic hedgehog (Shh) was isolated from the Xenopus laevis intestine as an early thyroid hormone (TH) response gene. To investigate possible roles of TH-upregulated expression of Shh during metamorphosis, we raised a polyclonal antibody against Xenopus Shh and immunohistochemically examined the relationship between Shh expression and the larval-to-adult intestinal remodeling at the cellular level. Our results indicate that the epithelial-specific expression of Shh in the intestine spatiotemporally correlates well with active proliferation and/or initial differentiation of the secondary (adult) epithelial primordia that originate from stem cells, but not with apoptosis of the primary (larval) epithelium. Given the similar transformations of the stomach during metamorphosis, we also analyzed Shh expression in this organ and found similar correlations in the stomach, although the position of the adult epithelial primordia and their final differentiation in the stomach are different from those in the intestine. Furthermore, we show here that Shh expression is organ-autonomously induced by TH and its correlation with the adult epithelial development is reproduced in vitro in both the intestine and the stomach. More importantly, addition of recombinant Shh protein to the culture medium results in developmental anomalies of both organs. However, differentiation of the adult epithelium is more severely inhibited by exogenous Shh in the intestine than in the stomach. These results suggest that TH-upregulated expression of Shh plays important roles in the postembryonic gastrointestinal remodeling, but its roles are at least partially different between the intestine and the stomach.     

Spatial and temporal expression pattern of a novel gene in the frog Xenopus laevis: correlations with adult intestinal epithelial differentiation during metamorphosis

We report the cloning of a novel gene (ID14) and its expression pattern in tadpoles and adults of Xenopus laevis. ID14 encodes a 315-amino acid protein that has a signal peptide and a nidogen domain. Even though several genes have a nidogen domain, ID14 is not the homolog of any known gene. ID14 is a late thyroid hormone (TH)-regulated gene in the tadpole intestine, and its expression in the intestine does not begin until the climax of metamorphosis, correlating with adult intestinal epithelial differentiation. In contrast, ID14 is expressed in tadpole skin and tail and is not regulated by TH. In situ hybridization revealed that this putative extracellular matrix protein is expressed in the epithelia of the tadpole skin and tail and in the intestinal epithelium after metamorphosis. In the adult, ID14 is found predominantly in the intestine with weak expression in the stomach, lung, and testis. Its exclusive expression in the adult intestinal epithelial cells makes it a useful marker for developmental studies and may give insights into cell/cell interactions in intestinal metamorphosis and adult intestinal stem cell maintenance.     

Models of epithelial histogenesis

Epithelial tissues emerge from coordinated sequences of cell renewal, specialization and assembly. Like corresponding immature tissues, adult epithelial tissues are provided by stem cells which are responsible for tissue homeostasis. Advances in epithelial histogenesis has permitted to clarify several aspects related to stem cell identification and dynamics and to understand how stem cells interact with their environment, the so-called stem cell niche. The development and maintenance of epithelial tissues involves epithelial-mesenchymal signalling pathways and cell-matrix interactions which control target nuclear factors and genes. The tooth germ is a prototype for such inductive tissue interactions and provides a powerful experimental system for the study of genetic pathways during development. Clonogenic epithelial cells isolated from developing as well mature epithelial tissues has been used to engineer epithelial tissue-equivalents, e.g. epidermal constructs, that are used in clinical practise and biomedical research. Information on molecular mechanisms which regulate epithelial histogenesis, including the role of specific growth/differentiation factors and cognate receptors, is essential to improve epithelial tissue engineering.     

A niche for adult neural stem cells

The adult mammalian brain harbors multipotent stem cells, which reside and participate in specialized niches that support self-renewal and differentiation. The first cellular and molecular elements of the stem cell niche in the adult brain have been identified and include cell-cell interactions and somatic cell signaling, the vasculature, the extracellular matrix and basal lamina. Furthermore, regulation at the epigenetic level via chromatin modification and remodeling is an integral aspect of stem cell biology. Understanding the in vivo stem cell niche will provide a framework for the elucidation of stem cell function in the adult brain.     

Stem cell niches in mammals

Stem cells safeguard tissue homeostasis and guarantee tissue repair throughout life. The decision between self-renewal and differentiation is influenced by a specialized microenvironment called stem cell niche. Physical and molecular interactions with niche cells and orientation of the cleavage plane during stem cell mitosis control the balance between symmetric and asymmetric division of stem cells. Here we highlight recent progress made on the anatomical and molecular characterization of mammalian stem cell niches, focusing particularly on bone marrow, tooth and hair follicle. The knowledge of the regulation of stem cells within their niches in health and disease will be instrumental to develop novel therapies that target stem cell niches to achieve tissue repair and re-establish tissue homeostasis.     

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.     

Adipocyte lineage cells contribute to the skin stem cell niche to drive hair cycling

In mammalian skin, multiple types of resident cells are required to create a functional tissue and support tissue homeostasis and regeneration. The cells that compose the epithelial stem cell niche for skin homeostasis and regeneration are not well defined. Here, we identify adipose precursor cells within the skin and demonstrate that their dynamic regeneration parallels the activation of skin stem cells. Functional analysis of adipocyte lineage cells in mice with defects in adipogenesis and in transplantation experiments revealed that intradermal adipocyte lineage cells are necessary and sufficient to drive follicular stem cell activation. Furthermore, we implicate PDGF expression by immature adipocyte cells in the regulation of follicular stem cell activity. These data highlight adipogenic cells as skin niche cells that positively regulate skin stem cell activity, and suggest that adipocyte lineage cells may alter epithelial stem cell function clinically.     

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.     

One of the duplicated matrix metalloproteinase-9 genes is expressed in regressing tail during anuran metamorphosis

The drastic morphological changes of the tadpole are induced during the climax of anuran metamorphosis, when the concentration of endogenous thyroid hormone is maximal. The tadpole tail, which is twice as long as the body, shortens rapidly and disappears completely in several days. We isolated a cDNA clone, designated as Xl MMP-9TH, similar to the previously reported Xenopus laevis MMP-9 gene, and showed that their Xenopus tropicalis counterparts are located tandemly about 9 kb apart from each other in the genome. The Xenopus MMP-9TH gene was expressed in the regressing tail and gills and the remodeling intestine and central nervous system, and induced in thyroid hormone-treated tail-derived myoblastic cultured cells, while MMP-9 mRNA was detected in embryos. Three thyroid hormone response elements in the distal promoter and the first intron were involved in the upregulation of the Xl MMP-9TH gene by thyroid hormone in transient expression assays, and their relative positions are conserved between X. laevis and X. tropicalis promoters. These data strongly suggest that the MMP-9 gene was duplicated, and differentiated into two genes, one of which was specialized in a common ancestor of X. laevis and X. tropicalis to be expressed in degenerating and remodeling organs as a response to thyroid hormone during metamorphosis.