Spatial and temporal expression patterns of Xenopus Nkx-2.3 gene in skin epidermis during metamorphosis

Using PCR cloning, the mRNA of XNkx-2.3 gene, a Xenopus tinman homologue, was identified in a cDNA library prepared from thyroid hormone (T(3))-treated tadpole skin. Quantitative RT-PCR and RNase Protection Assay confirmed the expression of XNkx-2.3 in adult frog skin and its amount was similar to the amount found in heart. In situ hybridization indicated that XNkx-2.3 was expressed in the frog epidermis. Further analysis of XNkx-2.3 expression patterns demonstrates that it shares great similarities with a 63 kDa keratin, a well-characterized marker for skin maturation, in the following aspects. First, XNkx-2.3 was expressed in tadpole skin during metamorphosis (stages 55-59), but not in pre-metamorphic (stage 54) skin. Secondly, XNkx-2.3 expression in skin responded to T(3) stimulation because it could be precociously induced by T(3) at pre-metamorphic stage, both in tadpoles and in cultures of skin explants. Finally, the T(3)-induced appearance of XNkx-2.3 in head skin occurred earlier and at higher level than that in tail skin. These data suggest that XNkx-2.3 may be an important factor for skin maturation and may also serve as a good marker to indicate the maturation of Xenopus epidermis.     

Ultrastructural changes in the intestinal connective tissue of Xenopus laevis during metamorphosis

Ultrastructural changes in the intestinal connective tissue of Xenopus laevis during metamorphosis have been studied. Throughout the larval period to stage 60, the connective tissue consists of a few immature fibroblasts surrounded by a sparse extracellular matrix: few collagen fibrils are visible except close to the thin basal lamina. At the beginning of the transition from larval to adult epithelial form around stage 60, extensive changes are observed in connective tissue. The cells become more numerous and different types appear as the collagen fibrils increase in number and density. Through gaps in the thickened and extensively folded basal lamina, frequent contacts between epithelial and connective tissue cells are established. Thereafter, with the progression of fold formation, the connective tissue cells become oriented according to their position relative to the fold structure. The basal lamina beneath the adult epithelium becomes thin after stage 62, while that beneath the larval epithelium remains thick. Upon the completion of metamorphosis, the connective tissue consists mainly of typical fibroblasts with definite orientation and numerous collagen fibrils. These observations indicate that developmental changes in the connective tissue, especially in the region close to the epithelium, are closely related spatiotemporarily to the transition from the larval to the adult epithelial form. This suggests that tissue interactions between the connective tissue and the epithelium play important roles in controlling the epithelial degeneration, proliferation, and differentiation during metamorphic climax.     

Spatial, temporal, and hormonal regulation of epidermal keratin expression during development of the frog, Xenopus laevis.

To study the mechanism of hormone-induced keratin expression in the epidermis during Xenopus metamorphosis, a monospecific antibody was raised against a unique carboxy-terminal peptide of the 63-kDa keratin. Immunohistological analysis demonstrated that the onset of 63-kDa keratin expression showed distinct regional and temporal differences. The expression started at stage 54 in the hindlimb epidermis, at stage 57 in the head, and over 1 month later at stage 63 in the tail. The amount of 63-kDa keratin was further regulated during epidermal stratification and differentiation. The 63-kDa keratin was expressed first in basal epidermal cells before stratification began. The outer layer of the larval epidermis (periderm) did not express the 63-kDa keratin. As the cells moved out of basal layer, they stained more intensely with the anti-keratin antibody indicating that 63-kDa keratin synthesis is up-regulated during differentiation. Similar results were obtained with cultures of purified epidermal cells grown in high calcium conditions. Since we have shown that thyroid hormone (T3) induces 63-kDa keratin gene expression and hydrocortisone (HC) modulates T3 action we examined the effects of T3 and HC at the single cell level with the anti-keratin antibody. Immunostaining demonstrated that T3 alone and T3 plus HC increased the number of 63-kDa keratin-positive cells as well as the amount of 63-kDa keratin per cell. Unexpectedly these hormones had the same effects on head and tail epidermal cells even though the latter cells degenerate during metamorphosis. The major difference between tail and head cells was that the percentage 63-kDa keratin-producing cells was much greater in the head than in the tail.     

Differential antigen expression during metamorphosis in the tripartite olfactory system of the African clawed frog, Xenopus laevis

In the adult African clawed frog, Xenopus laevis, olfactory epithelium is housed in three separate nasal cavities: the principal cavity, the middle cavity, and the vomeronasal organ. The sensory epithelium in each of these cavities has distinct cellular features, and presumed physiological and behavioral functions, which arise during metamorphosis. Most notably, the middle cavity is formed de novo, and the principal cavity is transformed from a larval sensory epithelium with water exposure to an adult olfactory epithelium with air exposure. To understand the cellular nature of this plasticity more clearly, we characterized the staining patterns generated in the olfactory system of X. laevis with a new monoclonal antibody, anti-E7. The olfactory epithelium is first stained with anti-E7 during late embryonic development. Transection of the olfactory nerves during metamorphosis eliminates all staining and indicates that the staining is associated with mature or nearly mature olfactory receptor neurons. The antibody diffusely stains the vomeronasal organ throughout development and in adults. In the larval principal cavity, the olfactory receptor neurons are brightly stained, but this cellular staining is lost after metamorphosis. The mucus from Bowman's glands in the principal cavity, however, is intensely stained in adults. The middle cavity, throughout development and in adulthood, has the same staining characteristics as the larval principal cavity. Thus, the E7 antibody can distinguish the three areas of the olfactory epithelium, allowing measurement of sensory epithelium volume, and serves as an excellent marker for the changes in the sensory epithelium that occur during metamorphosis.     

Developmental changes of purinergic control of intestinal motor activity during metamorphosis in the African clawed frog, Xenopus laevis

Little is known about the purinergic regulation of intestinal motor activity in amphibians. Purinergic control of intestinal motility is subject to changes during development in mammals. The aim of this study was to investigate purinergic control of intestinal smooth muscle in the amphibian Xenopus laevis and explore possible changes in this system during the developmental phase of metamorphosis. Effects of purinergic compounds on mean force and contraction frequency in intestinal circular muscle strips from prometamorphic, metamorphic, and juvenile animals were investigated. Before metamorphosis, low concentrations of ATP reduced motor activity, whereas the effects were reversed at higher concentrations. ATP-induced relaxation was not inhibited by the P2-receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) but was blocked by the ecto-nucleotidase inhibitor 6-N,N-diethyl-d-beta,gamma-dibromomethylene ATP (ARL67256), indicating that an ATP-derived metabolite mediated the relaxation response at this stage. Adenosine induced relaxation before, during, and after metamorphosis, which was blocked by the A(1)-receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). The stable ATP-analog adenosine 5'-[gamma-thio]-triphosphate (ATPgammaS) and 2-methylthioATP (2-MeSATP) elicited contractions in the circular muscle strips in prometamorphic tadpoles. However, in juvenile froglets, 2-MeSATP caused relaxation, as did ATPgammaS at low concentrations. The P2Y(11)/P2X(1)-receptor antagonist NF157 antagonized the ATPgammaS-induced relaxation. The P2X-preferring agonist alpha-beta-methyleneadenosine 5'-triphosphate (alpha-beta-MeATP) evoked PPADS-sensitive increases in mean force at all stages investigated. This study demonstrates the existence of an adenosine A(1)-like receptor mediating relaxation and a P2X-like receptor mediating contraction in the X. laevis gut before, during, and after metamorphosis. Furthermore, the development of a P2Y(11)-like receptor-mediated relaxation during metamorphosis is shown.     

Inductive action of epithelium on differentiation of intestinal connective tissue of Xenopus laevis tadpoles during metamorphosis in vitro

The action of the epithelium on differentiation of connective tissue cells of Xenopus small intestine during metamorphosis was investigated by using culture and morphological techniques. Connective tissue fragments isolated from the small intestine at stage 57 were cultivated in the presence or absence of homologous epithelium. In the presence of the epithelium, metamorphic changes in the connective tissue were fully induced by hormones including thyroid hormone (T₃), as during spontaneous metamorphosis, whereas they were partially induced in the absence of the epithelium. Macrophage-like cells showing non-specific esterase activity in the connective tissue were much fewer in the absence of the epithelium than in the presence of it, and aggregates of fibroblasts possessing well-developed rough endoplasmic reticulum developed only in the presence of the epithelium. Just before the aggregation of the fibroblasts, the connective tissue close to the epithelium became intensely stained with concanavalin A (ConA) and wheat germ agglutinin (WGA). The present results indicate that the epithelium plays important roles in the differentiation of intestinal connective tissue cells, which in turn affect the epithelial transformation from larval to adult form during anuran metamorphosis. Thus, the tissue interaction between the epithelium and the connective tissue in the anuran small intestine is truly bidirectional.     

A causative role of stromelysin-3 in extracellular matrix remodeling and epithelial apoptosis during intestinal metamorphosis in Xenopus laevis

The matrix metalloproteinases are a family of proteases capable of degrading various components of the extracellular matrix. Expression studies have implicated the involvement of the matrix metalloproteinase stromelysin-3 (ST3) in tissue remodeling and pathogenesis. However, the in vivo role of ST3 has been difficult to study because of a lack of good animal models. Here we used intestinal remodeling during thyroid hormone-dependent metamorphosis of Xenopus laevis as a model to investigate in vivo the role of ST3 during postembryonic organ development in vertebrates. We generated transgenic tadpoles expressing ST3 under control of a heat shock-inducible promoter. We showed for the first time in vivo that wild type ST3 but not a catalytically inactive mutant was sufficient to induce larval epithelial cell death and fibroblast activation, events that normally occur only in the presence of thyroid hormone. We further demonstrated that these changes in cell fate are associated with altered gene expression in the intestine and remodeling of the intestinal basal lamina. These results thus suggest that ST3 regulates cell fate and tissue morphogenesis through direct or indirect ECM remodeling.     

A novel Xenopus laevis larval keratin gene, xlk2: its gene structure and expression during regeneration and metamorphosis of limb and tail

A novel cytokeratin (CK) gene, xlk2, was cloned from a cDNA library prepared from regenerating limbs of Xenopus larvae. The deduced amino acid sequence indicated that its product, XLK2, is a 48 kDa type I (acidic) CK and has a high similarity to CK13, 15, and 19 with the highest homology (58%) to mouse CK15. The gene of xlk2 exclusively expressed in basal cells of the bi-layered larval epidermis, but not in other cells in larvae and not in other periods of life. Its expression was down-regulated during spontaneous and thyroid hormone-induced metamorphosis. The basal cells of the apical epidermal cap (AEC) formed on the regenerate of larval limbs terminated the expression of xlk2, whereas those of the adjacent normal epidermis continued to express it. The AEC-basal cells did not re-express the gene in the regenerate. In contrast, the basal cells of the tail regenerate also once terminated the expression of xlk2, but was able to re-express xlk2 later, supporting a notion that the "de-differentiated" basal cells of the tail epidermal regenerate re-differentiate into larval normal epidermal cells.     

Hormonal regulation of adult type keratin gene expression in larval epidermal cells of the frog Xenopus laevis

Triiodothyronin (T3) is known to induce amphibian metamorphosis but other hormones such as glucocorticoids accelerate T3 action. The increase in plasma concentration of both T3 and glucocorticoids during metamorphic climax is correlated with the transformation of the epidermis from larval type (uncornified) to adult type (cornified). Previously we have shown that T3 induced adult-type 63 Kd keratin gene expression and cornification of the larval epidermis. In this study, we have examined the effects of T3 and hydrocortisone (HC) on the conversion of larval to adult epidermal cells in vitro. When larval epidermal cells were treated with both T3 and HC, they had a synergistic effect on adult-type keratin synthesis (both 63 Kd and 49 Kd keratins) and epidermal cornification. The synergistic effect between T3 and HC required a pretreatment with T3 for 3 days. During this time, addition of HC to cultures containing T3 did not change the amount of 63 Kd keratin mRNA. Thus, HC did not reduce the lag time for epidermal cells to respond to T3. After 4 days of hormone treatment, T3 increased the amount of 63 Kd keratin mRNA 9-fold while T3 and HC induced it 18-fold. When cultures were pretreated with T3 for 3 days, a 1 day treatment with HC was sufficient to obtain the synergistic effect. Thus the induction of 63 Kd keratin gene expression by T3 required a much longer lag (3 days) than the lag required for the synergistic action of T3 and HC (less than 1 day).(ABSTRACT TRUNCATED AT 250 WORDS)     

Ledgerline, a novel Xenopus laevis gene, regulates differentiation of presomitic mesoderm during somitogenesis

Segmentation of the vertebrate body via the sequential formation of somites is an important process in embryogenesis. This sequential process is governed by the activation and regulation of Notch-related molecular oscillators by fibroblast growth factor and retinoic acid (RA) signaling. In this study, we identified ledgerline, a novel gene of Xenopus laevis expressed specifically in the presomitic mesoderm. Knockdown of ledgerline using antisense morpholino oligonucleotides shifted the developing somite front and altered the expression of genes that regulate molecular oscillation, including Delta2, ESR5, Hairy2a, and Thylacine1. Knockdown of ledgerline also downregulated RALDH-2 expression. Injection of RARalpha-CA, a constitutively active mutant of the RA receptor RARalpha, subsequently reduced the altered Thylacine1 expression. These results strongly suggest that ledgerline is essential for mesodermal RA activity and differentiation of the presomitic mesoderm during Xenopus somitogenesis.     

Differential keratin gene expression during the differentiation of the cement gland of Xenopus laevis.

The expression of both epidermal and nonepidermal keratins has been detected in the cement gland of Xenopus laevis by antibody staining. Northern blot and in situ hybridizations with gene-specific probes indicated the expression of the nonepidermal keratin, XK endo B, and the embryonic epidermal keratin, XK70, in the cement gland. Furthermore, since explanted animal pole cells can be induced to differentiate into cement gland cells in vitro by incubation in NH4Cl, we have demonstrated the in vitro induction of XK endo B, maintenance of XK70, and repression of another embryonic epidermal keratin, XK81. This is the first report of keratin gene expression in the cement gland.     

Identification and characterization of a novel collagenase in Xenopus laevis: possible roles during frog development.

Matrix metalloproteinases (MMPs) participate in extracellular matrix remodeling and degradation and have been implicated in playing important roles during organ development and pathological processes. Although it has been hypothesized for > 30 years that collagenase activities are responsible for collagen degradation during tadpole tail resorption, none of the previously cloned amphibian MMPs have been biochemically demonstrated to be collagenases. Here, we report a novel matrix metalloproteinase gene from metamorphosing Xenopus laevis tadpoles. In vitro biochemical studies demonstrate that this Xenopus enzyme is an interstitial collagenase and has an essentially identical enzymatic activity toward a collagen substrate as the human interstitial collagenase. Sequence comparison of this enzyme to other known MMPs suggests that the Xenopus collagenase is not a homologue of any known collagenases but instead represents a novel collagenase, Xenopus collagenase-4 (xCol4, MMP-18). Interestingly, during development, xCol4 is highly expressed only transiently in whole animals, at approximately the time when tadpole feeding begins, suggesting a role during the maturation of the digestive tract. More importantly, during metamorphosis, xCol4 is regulated in a tissue-dependent manner. High levels of its mRNA are present as the tadpole tail resorbs. Similarly, its expression is elevated during hindlimb morphogenesis and intestinal remodeling. In addition, when premetamorphic tadpoles are treated with thyroid hormone, the causative agent of metamorphosis, xCol4 expression is induced in the tail. These results suggest that xCol4 may facilitate larval tissue degeneration and adult organogenesis during amphibian metamorphosis.     

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.     

Spatial and temporal localization of FGF receptors in Xenopus laevis

Summary Mesoderm formation is a result of cell-cell interactions between the vegetal and animal hemisphere and is thought to be mediated by inducing peptide growth factors including members of the FGF and TGF superfamilies. Our immunochemical study analyses the distribution of FGF receptors coded by the human flg gene during embryogenesis of Xenopus laevis. Immunostaining was detected in the dorsal and ventral ectoderm and also in the marginal zone of early cleavage, blastula and gastrula stages. Signals were very strong in the mid and late blastula (stage 8 and 9) and declined slightly in the early gastrula (stage 10). A dramatic decrease was observed up to the late gastrula (stage 11⁺). In stage 13 embryos, immunostaining was only found in cells around the blastopore. Isolated ectoderm cultured in vitro showed a similar temporal expression and decrease of the signal as the normal embryos. These results indicate that receptor expression is independent of the interaction of the animal cells with the vegetal part of the embryo. Of interest is the fact that the signal cannot only be found at or near the cell surface but also within the cell. This suggests the presence of an intracellular isoform of the receptor resulting from the endogenous expression of splice variants and the internalization of transmembrane receptor. Taken together our results suggest that the loss of competence (for bFGF around stage 10) is not directly correlated with the presence of receptors. The possible roles of heparan sulphate glucosaminoglycans (low affinity receptors) and control mechanisms in the intracellular signalling pathway downstream of the receptor level should be taken into consideration.