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.     

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-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.     

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.     

Transformation of the intestinal epithelium of the larval anadromous sea lamprey Petromyzon marinus L. during metamorphosis

The events in the transformation of the intestine of the larval lamprey into the adult intestine were followed through the seven (1–7) stages of metamorphosis in anadromous Petromyzon marinus L. Light and electron-microscope observations demonstrated that the processes of degeneration, differentiation, and proliferation are involved in the transformation. In the anterior intestine, degeneration of cells and the extrusion of others into the lumen results in the disappearance of secretory (zymogen) cells and the decline in numbers of endocrine and ciliated cells. Larval absorptive cells, with a prominent brush border, are believed to dedifferentiate into unspecialized columnar cells with few microvilli. Degeneration and removal of cells occurs by both autophagy and heterography and cells extruded into the lumen in the anterior intestine are phagocytosed by epithelial cells of the posterior intestine. The loss of epithelial cells during transformation results in the folding and degradation of parts of the basal lamina and in an extensive widening of the lateral intercellular spaces in all parts of the intestine. As metamorphosis is a nontrophic period of the lamprey life cycle, the possible morphological effects of starvation on the intestinal epithelium are discussed. The development of longitudinal folds is a consequence of the events of metamorphic transformation of the intestinal mucosa. Although an interaction between the epithelium and the underlying tissues is believed to be importent, the actual mechanism of fold development is unknown. The intestinal epithelium of adult lampreys develops from surviving cells of the larval (primary) epithelium. Unlike the situation in amphibians, there does not appear to be a group (nest) of undifferentiated larval cells which differentiate into the adult (secondary) epithelium. Instead, in lampreys, columnar cells that persist through the degradative processes seem to be the source of absorptive and ciliated cells and probably are responsible for mucous and secretory cells. Preliminary observations indicate that the intestinal epithelium of feeding adults is specialized into an anterior region which liberates a secretion, absorbs lipid, and possesses the machinery for ion transport. A posterior region absorbs lipid, secretes mucus, and likely is involved in some protein absorption.     

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.     

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.     

Thyroid Hormone Regulation of Adult Intestinal Stem Cell Development: Mechanisms and Evolutionary Conservations

The adult mammalian intestine has long been used as a model to study adult stem cell function and tissue renewal as the intestinal epithelium is constantly undergoing self-renewal throughout adult life. This is accomplished through the proliferation and subsequent differentiation of the adult stem cells located in the crypt. The development of this self-renewal system is, however, poorly understood. A number of studies suggest that the formation/maturation of the adult intestine is conserved in vertebrates and depends on endogenous thyroid hormone (T3). In amphibians such as Xenopus laevis, the process takes place during metamorphosis, which is totally dependent upon T3 and resembles postembryonic development in mammals when T3 levels are also high. During metamorphosis, the larval epithelial cells in the tadpole intestine undergo apoptosis and concurrently, adult epithelial stem/progenitor cells are formed de novo, which subsequently lead to the formation of a trough-crest axis of the epithelial fold in the frog, resembling the crypt-villus axis in the adult mammalian intestine. Here we will review some recent molecular and genetic studies that support the conservation of the development of the adult intestinal stem cells in vertebrates. We will discuss the mechanisms by which T3 regulates this process via its nuclear receptors.     

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).

     

Neurotrophin receptors and enteric neuronal development during metamorphosis in the amphibian<Emphasis Type="Italic"> Xenopus laevis</Emphasis>

During metamorphosis, the frog intestine goes through a dramatic shortening with extensive apoptosis and regeneration in the epithelial layer and connective tissue. Our aim was to study changes in the enteric nervous system represented by one inhibitory (vasoactive intestinal polypeptide; VIP) and one excitatory (substance P, neurokinin A; SP/NKA) nerve population and concomitant changes in neurotrophin receptor occurrence during this development in the gut of Xenopus laevis adults and tadpoles at different stages of metamorphosis (NF stages 57–66). Sections were incubated with antibodies against the neurotrophin Trk receptors and p75^NTR, and the neurotransmitters VIP and SP/NKA. Trk-immunoreactive nerves increased dramatically but transiently in number during early metamorphic climax. Nerves immunoreactive for p75^NTR were present throughout the gut, decreased in number in the middle intestine during climax, and increased in the large intestine during late metamorphosis. The percentage of VIP-immunoreactive nerves did not change during metamorphosis. SP/NKA-immunoreactive nerves were first apparent at NF stages 61–62 in the middle intestine and increased in the stomach and large intestine during metamorphosis. Endocrine cells expressing SP/NKA increased in number in stomach, proximal, and middle intestine during metamorphic climax. Thus, neurotrophin receptors are expressed transiently in neurons of the enteric nervous system during metamorphosis in Xenopus laevis and SP/NKA innervation is more abundant in the intestine of the postmetamorphic frog than in the tadpole.This study was supported by grants from the Swedish Research Council to S. Holmgren     

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.     

Ultrastructural changes in the connective tissue of the gastric region during the metamorphosis of Xenopus laevis

Development of the gastric connective tissue of Xenopus laevis during metamorphosis was investigated by electron microscopy. Throughout the larval period to stage 60, the layer of connective tissue underlying the gastric epithelium consists of immature fibroblasts surrounded by a sparse extracellular matrix. At the beginning of the transition from the larval to the adult epithelial form, at about stage 60, extensive changes occur in the connective tissue. The number of cells suddenly increses and different cell types appear. Numerous contacts between epithelial and connective tissue cells are established through random gaps in the thickened basal lamina. During stages 62–63, just after the beginning of the morphogenesis of adult-type glands, the basal lamina lining the glandular epithelium becomes thinner, and the number of contacts decreases rapidly except near the tips of the glands. After the glandular cells begin to produce zymogen granules at stage 64, contacts become rare. From stage 63, when the muscularis mucosae develops, until the completion of metamorphosis, the connective tissue consists mainly of typical fibroblasts. Outside the muscularis mucosae, the fibroblasts of the lamina propria are aligned in parallel with the curvature of the glands. These observations indicate that developmental changes in the connective tissue are closely related spatiotemporally to those of the epithelial transition from larval to adult form during metamorphic climax. Although some changes are similar to those in the intestine (Ishizuya-Oka and Shimozawa, '87b), others are specific to the gastric region, which suggests that connective tissue may have a role in organ-specific differentiation of the gastric epithelium.     

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.     

Developmental changes in keratin patterns during epidermal maturation

The biochemical maturation of the epidermis of Xenopus laevis was examined through an identification of the keratins expressed at selected stages of development. The keratin patterns obtained were compared to those observed in the adult epidermis and two Xenopus non-epidermal, epithelial cell lines. The keratins expressed during development can be grouped into three classes: (1) keratins which are restricted to the embryonic epidermis (58 and 59 kDa); (2) keratins which are prominent during development, but become minor components of the adult epidermis (47, 48, and 60 kDa); and (3) keratins which accumulate during development to become the major keratins of the adult epidermis (49, 53, 56, and 63 kDa). The embryo-specific keratins are present at all developmental stages prior to metamorphosis which we have investigated, but disappear when the epidermis keratinizes during metamorphosis. Both class 1 and 2 keratins, while undetectable or minor components of the adult skin, are present in the two non-epidermal cell lines. In contrast, the class 3 keratins show little overlap with the keratins of these cell lines. All of the class 3 keratins appear after hatching with the exception of the 53-kDa keratin which is present at the earliest developmental stage which we have examined. All of the major keratins of the adult epidermis accumulate as metamorphosis proceeds, while the embryo-restricted keratins are gradually lost.