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.     

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.     

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.     

The balance of two opposing factors Mad and Myc regulates cell fate during tissue remodeling

Cell proliferation and differentiation are two distinct yet coupled processes in development in diverse organisms. Understanding the molecular mechanisms that regulate this process is a central theme in developmental biology. The intestinal epithelium is a highly complex tissue that relies on the coordination of cell proliferation within the crypts and apoptosis mainly at the tip of the villi, preservation of epithelial function through differentiation, and homeostatic cell migration along the crypt-villus axis. Small populations of adult stem cells are responsible for the self-renewal of the epithelium throughout life. Surprisingly, much less is known about the mechanisms governing the remodeling of the intestine from the embryonic to adult form. Furthermore, it remains unknown how thyroid hormone (T3) affects stem cell development during this postembryonic process, which is around birth in mammals when T3 level increase rapidly in the plasma. Tissue remodeling during amphibian metamorphosis is very similar to the maturation of the mammalian organs around birth in mammals and is regulated by T3. In particular, many unique features of Xenopus intestinal remodeling during metamorphosis has enabled us and others to elucidate how adult stem cells are formed during postembryonic development in vertebrates. In this review, we will focus on recent findings on the role of Mad1/c-Myc in cell death and proliferation during intestinal metamorphosis and discuss how a Mad1–c-Myc balance controls intestinal epithelial cell fate during this T3-dependent process.     

Molecular and cytological analyses reveal distinct transformations of intestinal epithelial cells during <Emphasis Type="Italic">Xenopus</Emphasis> metamorphosis

Background

The thyroid hormone (T3)-induced formation of adult intestine during amphibian metamorphosis resembles the maturation of the mammalian intestine during postembryonic development, the period around birth when plasma T3 level peaks. This process involves de novo formation of adult intestinal stem cells as well as the removal of the larval epithelial cells through apoptosis. Earlier studies have revealed a number of cytological and molecular markers for the epithelial cells undergoing different changes during metamorphosis. However, the lack of established double labeling has made it difficult to ascertain the identities of the metamorphosing epithelial cells.

Results

Here, we carried out different double-staining with a number of cytological and molecular markers during T3-induced and natural metamorphosis in Xenopus laevis. Our studies demonstrated conclusively that the clusters of proliferating cells in the epithelium at the climax of metamorphosis are undifferentiated epithelial cells and express the well-known adult intestinal stem cell marker gene Lgr5. We further show that the adult stem cells and apoptotic larval epithelial cells are distinct epithelial cells during metamorphosis.

Conclusions

Our findings suggest that morphologically identical larval epithelial cells choose two alternative paths: programmed cell death or dedifferentiation to form adult stem cells, in response to T3 during metamorphosis with apoptosis occurring prior to the formation of the proliferating adult stem cell clusters (islets).

     

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.     

Cytological and Morphological Analyses Reveal Distinct Features of Intestinal Development during Xenopus tropicalis Metamorphosis

Background

The formation and/or maturation of adult organs in vertebrates often takes place during postembryonic development, a period around birth in mammals when thyroid hormone (T3) levels are high. The T3-dependent anuran metamorphosis serves as a model to study postembryonic development. Studies on the remodeling of the intestine during Xenopus (X.) laevis metamorphosis have shown that the development of the adult intestine involves de novo formation of adult stem cells in a process controlled by T3. On the other hand, X. tropicalis, highly related to X. laevis, offers a number of advantages for studying developmental mechanisms, especially at genome-wide level, over X. laevis, largely due to its shorter life cycle and sequenced genome. To establish X. tropicalis intestinal metamorphosis as a model for adult organogenesis, we analyzed the morphological and cytological changes in X. tropicalis intestine during metamorphosis.

Methodology/Principal Findings

We observed that in X. tropicalis, the premetamorphic intestine was made of mainly a monolayer of larval epithelial cells surrounded by little connective tissue except in the single epithelial fold, the typhlosole. During metamorphosis, the larval epithelium degenerates and adult epithelium develops to form a multi-folded structure with elaborate connective tissue and muscles. Interestingly, typhlosole, which is likely critical for adult epithelial development, is present along the entire length of the small intestine in premetamorphic tadpoles, in contrast to X. laevis, where it is present only in the anterior 1/3. T3-treatment induces intestinal remodeling, including the shortening of the intestine and the typhlosole, just like in X. laevis.

Conclusions/Significance

Our observations indicate that the intestine undergoes similar metamorphic changes in X. laevis and X. tropicalis, making it possible to use the large amount of information available on X. laevis intestinal metamorphosis and the genome sequence information and genetic advantages of X. tropicalis to dissect the pathways governing adult intestinal development.     

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.     

Differential regulation of two histidine ammonia-lyase genes during <Emphasis Type="Italic">Xenopus</Emphasis> development implicates distinct functions during thyroid hormone-induced formation of adult stem cells

Background

Organ-specific, adult stem cells are essential for organ-homeostasis and tissue repair and regeneration. The formation of such stem cells during vertebrate development remains to be investigated. Frog metamorphosis offers an excellent opportunity to study the formation of adult stem cells as this process involves essentially the transformations of all larval tissues/organs into the adult form. Of particular interest is the remodeling of the intestine. Early studies in Xenopus laevis have shown that this process involves complete degeneration of the larval epithelium and de novo formation of adult stem cells through dedifferentiation of some larval epithelial cells. A major advantage of this metamorphosis model is its total dependence on thyroid hormone (T3). In an effort to identify genes that are important for stem cell development, we have previously carried out tissue-specific microarray analysis of intestinal gene expression during Xenopus laevis metamorphosis.

Results

We report the detailed characterization of one of the genes thus identified, the histidine ammonia-lyase (HAL) gene, which encodes an enzyme known as histidase or histidinase. We show that there are two duplicated HAL genes, HAL1 and HAL2, in both Xenopus laevis and Xenopus tropicalis, a highly related but diploid species. Interestingly, only HAL2 is highly upregulated by T3 and appears to be specifically expressed in the adult intestinal progenitor/stem cells while HAL1 is not expressed in the intestine during metamorphosis. Furthermore, when analyzed in whole animals, HAL1 appears to be expressed only during embryogenesis but not metamorphosis while the opposite appears to be true for HAL2.

Conclusions

Our results suggest that the duplicated HAL genes have distinct functions with HAL2 likely involved in the formation and/or proliferation of the adult stem cells during metamorphosis.

     

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.     

Temporal and spatial expression of an intestinal Na+/PO43− cotransporter correlates with epithelial transformation during thyroid hormone-dependent frog metamorphosis

The amphibian intestine has two morphologically distinct structures during development. Early embryogenesis generates a simple tube-like intestine in the tadpole whereas after thyroid hormone (T₃)-dependent metamorphosis a newly remodeled adult intestine is formed similar to that of higher vertebrates. This change requires a drastic transformation of the epithelial layer. We have isolated a Na⁺/PO₄³⁻ cotransporter gene that may contribute to this transformation. The deduced amino acid sequence of this gene shows a high degree of homology to the mammalian renal NA⁺/PO₄³⁻ cotransporters, which have little or no expression in organs other than the kidney. The frog gene is highly expressed and regulated by T₃ in the intestine with little expression and/or regulation by T₃ in most other organs. Its mRNA is restricted to the differentiated epithelial cells both in tadpoles and postmetamorphic frogs. Interestingly, its expression is low in premetamorphic tadpoles, but up-regulated when metamorphosis is initiated by endogenous T₃. As the larval epithelium undergoes programmed cell death (apoptosis), the mRNA level drops to a minimum. Subsequently, the gene is reactivated at the tip region of the newly formed adult intestinal folds and a crest-trough polarity of expression is established by the end of metamorphosis. This temporal regulation profile is also reproduced when premetamorphic tadpoles are treated with T₃ to induce precocious intestinal remodeling. These results suggest a possible role of the Na⁺/PO₄³⁻ cotransporter during metamorphosis and demonstrate that the adult epithelial cell differentiation pattern is established in the direction of crest-to-trough of the intestinal fold, concurrent with the epithelial morphogenic process. Dev Genet 20:53–66, 1997. © 1997 Wiley-Liss, Inc.     

Stem cells and lineages of the intestine: a developmental and evolutionary perspective

The intestine consists of epithelial cells that secrete digestive enzymes and mucus (gland cells), absorb food particles (enterocytes), and produce hormones (endocrine cells). Intestinal cells are rapidly turned over and need to be replaced. In cnidarians, mitosis of differentiated intestinal cells accounts for much of the replacement; in addition, migratory, multipotent stem cells (interstitial cells) contribute to the production of intestinal cells. In other phyla, intestinal cell replacement is solely the function of stem cells entering the gut from the outside (such as in case of the neoblasts of platyhelminths) or intestinal stem cells located within the midgut epithelium (as in both vertebrates or arthropods). We will attempt in the following to review important aspects of midgut stem cells in different animal groups: where are they located, what types of lineages do they produce, and how do they develop. We will start out with a comparative survey of midgut cell types found across the animal kingdom; then briefly look at the specification of these cells during embryonic development; and finally focus on the stem cells that regenerate midgut cells during adult life. In a number of model systems, including mouse, zebrafish and Drosophila, the molecular pathways controlling intestinal stem cells proliferation and the specification of intestinal cell types are under intensive investigation. We will highlight findings of the recent literature, focusing on aspects that are shared between the different models and that point at evolutionary ancient mechanisms of intestinal cell formation.     

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.     

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.