Isolation and nucleotide sequence analysis of the of Rhinella arenarum ß-catenin: an mRNA and protein expression study during the larval stages of the digestive tract development.

ß-catenin interacts with several proteins mediating key biological processes, such as cadherin-mediated cell-cell adhesion as well as signal transduction. This work was done to establish the molecular basis and regulation of the formation pattern of cadherin/ß-catenin-mediated adherens junctions, using an animal model of unknown gene sequence, the toad Rhinella arenarum. A Rhinella arenarum ß-catenin homolog was isolated from larval tissue, their sequence compared and analyzed with those of eight other vertebrates using bioinformatics tools. The mRNA and protein expression levels of ß-catenin were determined during the development of Rhinella arenarum digestive tract both by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) and immunohistochemistry-morphometry respectively. Using Xenopus laevis frog specific primers, a fragment 539 bp of Rhinella arenarum toad ß-catenin cDNA was obtained and sequenced. The resulting putative sequence of 177 amino acids showed high similarity at the amino acid level (97%) when compared to other six vertebrates (Xenopus laevis, Xenopus tropicalis, Mus musculus, Rattus norvegicus, Bos taurus and Homo sapiens), with sequences and structural domains characteristic of catenins. Subsequently, using primers specifically designed for Rhinella arenarum nucleotide sequence, ß-catenin-mRNA increasing levels were found during the Rhinella arenarum metamorphosis. Finally, increasing ß-catenin protein expression during development has confirmed the specificity the detection of Rhinella arenarum ß-catenin. Summarizing, we have isolated and sequenced a ß-catenin-homologue sequence from the Rhinella arenarum toad, which is highly conserved between species, and following we have detected ß-catenin mRNA and protein levels during their digestive tract development.

T3 regulates E-cadherin, and ß- and α-catenin expression in the stomach during the metamorphosis of the toad Rhinella arenarum.

The metamorphosis of Rhinella arenarum was induced precociously for 5 days, then blocked for 3 months to evaluate the role of thyroid hormones as modulators of morphoregulatory molecules such as E-cadherin, and ß- and α-catenin during epithelium remodeling. We then performed an immunohistochemical and morphometric study of these molecules in the larval stomach. We show that 3,5,3'-triiodothyronine exerts a positive regulatory effect on E-cadherin and ß- and α-catenin expression in stomach epithelium. This suggests continuous synthesis of E-cadherin, and ß- and α-catenin; synthesis essentially is thyroid hormone-independent during premetamorphosis and early prometamorphosis, but it becomes thyroid hormone-dependent during metamorphic climax.

Role of E-cadherin in epithelial architecture maintenance.

Morphogenesis and architecture of a developing epithelium is controlled by both cell shape and contacts, mediated by spatially and temporally regulated cell adhesion molecules. The authors study if E-cadherin functions as a key factor of epithelial adhesion and epidermal architecture in vivo. They apply whole-mount digital deconvolution microscopy to evaluate three-dimensional (3D) E-cadherin expression during skin morphogenesis of Rhinella arenarum and in a cell adhesion alteration model. Results show morphogenetic changes in the 3D E-cadherin spatiotemporal expression pattern correlated with the increase of E-cadherin and in the number of cells with hexagonal geometry. Alterations in junction-protein phosphorylation showed drastic loss of E-cadherin and beta-catenin in cell-cell contacts and the increase of cytoplasm and nuclear beta-catenin in epidermis, suggesting the activation of the beta-catenin signal pathway. Surprisingly, no changes in cell shape and skin architecture were registered, suggesting that epidermal E-cadherin appears to be involved in signaling rather than cell contact maintenance in vivo.

Expression of cell adhesion molecules in the normal and T3 blocked development of the tadpole's kidney of Bufo arenarum (Amphibian, Anuran, Bufonidae).

Cell adhesion molecules act as signal transducers from the extracellular environment to the cytoskeleton and the nucleus and consequently induce changes in the expression pattern of structural proteins. In this study, we showed the effect of thyroid hormone (TH) inhibition and arrest of metamorphosis on the expression of E-cadherin, beta-and alpha-catenin in the developing kidney of Bufo arenarum. Cell adhesion molecules have selective temporal and spatial expression during development suggesting a specific role in nephrogenesis. In order to study mechanisms controlling the expression of adhesion molecules during renal development, we blocked the B. arenarum metamorphosis with a goitrogenic substance that blocks TH synthesis. E-cadherin expression in the proximal tubules is independent of thyroid control. However, the blockage of TH synthesis causes up-regulation of E-cadherin in the collecting ducts, the distal tubules and the glomeruli. The expression of beta-and alpha-catenin in the collecting ducts, the distal tubules, the glomeruli and the mesonephric mesenchyme is independent of TH. TH blockage causes up-regulation of beta-and alpha-catenin in the proximal tubules. In contrast to E-cadherin, the expression of the desmosomal cadherin desmoglein 1 (Dsg-1) is absent in the control of the larvae kidney during metamorphosis and is expressed in some interstitial cells in the KClO4 treated larvae. According to this work, the Dsg-1 expression is down-regulated by TH. We demonstrated that the expression of E-cadherin, Dsg-1, beta-catenin and alpha-catenin are differentially affected by TH levels, suggesting a hormone-dependent role of these proteins in the B. arenarum renal metamorphosis.

Expression of cell adhesion molecules in the normal and T3 blocked development of the tadpole's kidney of Bufo arenarum (Amphibian, Anuran, Bufonidae) / Expressão das moléculas de adesão celular no desenvolvimento normal e com a inibição do hormônio tireoidea do rim nas larvas do Bufo arenarum (Amphibia, Anura, Bufonidae)

Cell adhesion molecules act as signal transducers from the extracellular environment to the cytoskeleton and the nucleus and consequently induce changes in the expression pattern of structural proteins. In this study, we showed the effect of thyroid hormone (TH) inhibition and arrest of metamorphosis on the expression of E-cadherin, β-and α-catenin in the developing kidney of Bufo arenarum. Cell adhesion molecules have selective temporal and spatial expression during development suggesting a specific role in nephrogenesis. In order to study mechanisms controlling the expression of adhesion molecules during renal development, we blocked the B. arenarum metamorphosis with a goitrogenic substance that blocks TH synthesis. E-cadherin expression in the proximal tubules is independent of thyroid control. However, the blockage of TH synthesis causes up-regulation of E-cadherin in the collecting ducts, the distal tubules and the glomeruli. The expression of β-and α-catenin in the collecting ducts, the distal tubules, the glomeruli and the mesonephric mesenchyme is independent of TH. TH blockage causes up-regulation of β-and α-catenin in the proximal tubules. In contrast to E-cadherin, the expression of the desmosomal cadherin desmoglein 1 (Dsg-1) is absent in the control of the larvae kidney during metamorphosis and is expressed in some interstitial cells in the KClO4 treated larvae. According to this work, the Dsg-1 expression is down-regulated by TH. We demonstrated that the expression of E-cadherin, Dsg-1, β-catenin and α-catenin are differentially affected by TH levels, suggesting a hormone-dependent role of these proteins in the B. arenarum renal metamorphosis.

Moléculas de adesão celular atuam como tradutores do ambiente extracelular para o citoesqueleto e o núcleo e, conseqüentemente, induzindo mudanças no padrão da expressão das proteínas estruturais. Neste estudo, observamos os efeitos da inibição do hormônio tireóidea (TH) e detenção da metamorfose na expressão da E-caderina, β- e α- catenina no desenvolvimento do rim do Bufo arenarum. As moléculas de adesão celular durante o desenvolvimento têm uma expressão temporal e espacial seletiva, sugerindo um papel específico na nefrogênese. Com o propósito de estudar os mecanismos de controle da expressão das moléculas de adesão durante o desenvolvimento renal, bloqueou-se a metamorfose do B. arenarum com uma substancia goitrogênica que bloqueia a síntese de TH. A expressão da E-caderina nos tubos proximais é independente do controle da tireóide. Entretanto, o bloqueio da síntese de TH provoca uma sobre elevação da E-caderina nos dutos coletores, nos tubos distais e nos glomérulos. A expressão da β- e α-catenina nos dutos coletores, nos tubos distais, nos glomérulos e no mesênquima mesonéfrico é independente da TH. O bloqueio da TH causa uma sobre-regulação da β- e α-catenina nos tubos proximais. Em contraste com a E-caderina, a expressão da caderina desmossomal demogloína 1 (Dsg-1) é ausente no controle durante a metamorfose da fase larval dos rins e se expressa em algumas células intersticiais nas larvas tratadas com KClO4. De acordo com este trabalho, a expressão Dsg-1 é subregulada pela TH. Demonstramos que a expressão da E-caderina, Dsg-1, β-catenina e α-catenina são afetadas de forma diferencial pelos níveis de TH, sugerindo um dependência hormonal destas proteínas na metamorfose renal do B. arenarum.

3D automatic quantification applied to optically sectioned images to improve microscopy analysis.

New fluorescence microscopy techniques, such as confocal or digital deconvolution microscopy, allow to easily obtain three-dimensional (3D) information from specimens. However, there are few 3D quantification tools that allow extracting information of these volumes. Therefore, the amount of information acquired by these techniques is difficult to manipulate and analyze manually. The present study describes a model-based method, which for the first time shows 3D visualization and quantification of fluorescent apoptotic body signals, from optical serial sections of porcine hepatocyte spheroids correlating them to their morphological structures. The method consists on an algorithm that counts apoptotic bodies in a spheroid structure and extracts information from them, such as their centroids in cartesian and radial coordinates, relative to the spheroid centre, and their integrated intensity. 3D visualization of the extracted information, allowed us to quantify the distribution of apoptotic bodies in three different zones of the spheroid.

CAS role in the brain apoptosis of Bufo arenarum induced by cypermethrin.

CAS might have a key role in the apoptosis induced by toxins, acting as anti-apoptotic factor, stimulating the cellular proliferation and the cell contact stabilization. To start to elucidate their role in the brain apoptosis of Bufo arenarum induced by cypermethrin (CY), the expression patterns of CAS and several cell adhesion molecules (CAMs) were established. Bufo arenarum tadpoles of the control and acute bioassay survival at different doses (39, 156, 625 and 2,500 microg CY/L) and times (24, 48, 72 and 96 h) of CY treatment were fixed in Carnoy, embedded in paraffin and sectioned. CAS and CAMs expression was determined by immunofluorescence and immunohistochemistry, respectively. When the bioassay starts, CAS increases suggesting a proliferative or regenerative effect, but decreases when the doses and/or the biocide exposure time increases, suggesting compromise of the cellular cycle control and trigger of an apoptotic wave. However, these neurotoxic mechanisms should not involve degradation of N-cadherin and alpha-catenin, in contrast of beta-catenin and axonal N-CAM180, at least in the initial apoptotic phase. Additionally, an adhesion compensatory mechanism by N-CAM180 is observed in the neuron cell body. These results suggest a dual role of CAS in the cellular cycle control during the CY-induced apoptosis: induction of cell proliferation and stabilization of the cell-cell junctions by modulating CAMs expression.

CAS role in the brain apoptosis of Bufo arenarum induced by cypermethrin

CAS might have a key role in the apoptosis induced by toxins, acting as anti-apoptotic factor, stimulating the cellular proliferation and the cell contact stabilization. To start to elucidate their role in the brain apoptosis of Bufo arenarum induced by cypermethrin (CY), the expression patterns of CAS and several cell adhesion molecules (CAMs) were established. Bufo arenarum tadpoles of the control and acute bioassay survival at different doses (39, 156, 625 and 2,500 microg CY/L) and times (24, 48, 72 and 96 h) of CY treatment were fixed in Carnoy, embedded in paraffin and sectioned. CAS and CAMs expression was determined by immunofluorescence and immunohistochemistry, respectively. When the bioassay starts, CAS increases suggesting a proliferative or regenerative effect, but decreases when the doses and/or the bbiocide exposure time increases, suggesting compromise of the cellular cycle control and trigger of an apoptotic wave. However, these neurotoxic mechanisms should not involve degradation of N-cadherin and alpha-catenin, in contrast of beta-catenin and axonal N-CAM180, at least in the initial apoptotic phase. Additionally, an adhesion compensatory mechanism by N-CAM180 is observed in the neuron cell body. These results suggest a dual role of CAS in the cellular cycle control during the CY-induced apoptosis: induction of cell proliferation and stabilization of the cell-cell junctions by modulating CAMs expression.

CAS role in the brain apoptosis of Bufo arenarum induced by cypermethrin

CAS might have a key role in the apoptosis induced by toxins, acting as anti-apoptotic factor, stimulating the cellular proliferation and the cell contact stabilization. To start to elucidate their role in the brain apoptosis of Bufo arenarum induced by cypermethrin (CY), the expression patterns of CAS and several cell adhesion molecules (CAMs) were established. Bufo arenarum tadpoles of the control and acute bioassay survival at different doses (39, 156, 625 and 2,500 microg CY/L) and times (24, 48, 72 and 96 h) of CY treatment were fixed in Carnoy, embedded in paraffin and sectioned. CAS and CAMs expression was determined by immunofluorescence and immunohistochemistry, respectively. When the bioassay starts, CAS increases suggesting a proliferative or regenerative effect, but decreases when the doses and/or the bbiocide exposure time increases, suggesting compromise of the cellular cycle control and trigger of an apoptotic wave. However, these neurotoxic mechanisms should not involve degradation of N-cadherin and alpha-catenin, in contrast of beta-catenin and axonal N-CAM180, at least in the initial apoptotic phase. Additionally, an adhesion compensatory mechanism by N-CAM180 is observed in the neuron cell body. These results suggest a dual role of CAS in the cellular cycle control during the CY-induced apoptosis: induction of cell proliferation and stabilization of the cell-cell junctions by modulating CAMs expression.(AU)

Apoptotic cell death in the central nervous system of Bufo arenarum tadpoles induced by cypermethrin.

Tadpoles of the toad Bufo arenarum treated with cypermethrin (CY) at concentrations above 39 mug CY/L showed dose-dependent apoptotic cell death in immature cells of the central nervous system as demonstrated by morphometric analysis, the TUNEL method, and DNA fragmentation assay. Light-and electron-microscopic studies showed structural alterations in the intermediate and marginal layers of the brain. Immature cerebral tissue showed cellular shrinkage, nuclear fragmentation and increase of intercellular spaces. In this study we demonstrated high toxicity of CY to larval stages of Bufo arenarum. Our results show that doses lower than those used in routine insecticide applications can cause massive apoptosis in the immature cells of the central nervous system. These results coincide with our previous studies in Physalaemus biligonigerus, confirming the severe toxic effects of CY to the central nervous system of anuran species from Argentina. This may increase the mortality index in wild animals and contribute to the loss of biodiversity in our agroecosystems. We postulate that CY induces apoptosis in central nervous system cells of Bufo arenarum tadpoles by specific neurotoxic mechanisms.

Alterations induced by E-cadherin and beta-catenin antibodies during the development of Bufo arenarum (Anura-Bufonidae).

E(epithelial)-cadherin is a member of a calcium-dependent family of cell surface glycoproteins involved in cell-cell adhesion and morphogenesis. Catenins are a large family of proteins that connect the cadherins to the cytoskeleton. They are important for cadherin function and for transducing signals involved in specification of cell fate during embryogenesis. The best characterized catenins include alpha-, beta-, gamma-, and p120-catenin. Using specific antibodies, we studied the expression and distribution of E-cadherin, and alpha- and beta-catenin in developmental stages of Bufo arenarum toad. The three proteins were found co-localized in stages 19 to 41 of development. Surprisingly, E-cadherin was the only of these three proteins found earlier than stage 19. To test whether E-cadherin and beta-catenin have a functional role in Bufo arenarum embryogenesis, stage 17 whole embryos were incubated with anti-E-cadherin and beta-catenin antibodies. Both anti-E-cadherin and anti-beta-catenin antibodies induced severe morphological alterations. However, while alterations produced by the anti-beta-catenin antibody, showed some variability from the most severe (neural tube and notochord duplication) to a simple delay in development, the alterations with anti-E-cadherin were homogeneous. These observations suggest a critical role for E-cadherin and beta-catenin in the early embryonic development of the Bufo arenarum toad. Our results are consistent with the developmental role of these proteins in other species. One of the most surprising findings was the blockage with the anti-beta-catenin antibodies on later embryo stages, and we hypothesize that the partial axes duplication could be mediated by the notochord induction.

Expression of polysialic acid, alpha- and beta-cantenins in adult toad testis in hibernation stage and after gonadotrophin--releasing hormone (GnRH) treatment.

The Polysialic Acid (PSA), glycosydic moiety of the Neural Cell Adhesion Molecule (N-CAM), and alpha- and beta-Catenins, which mediate interaction between Cadherins and cytoskeletal proteins, participate in cell adhesion phenomena in numerous organs and tissues. We have performed an immunohistochemical analysis, in hibernating toad testis and in GnRH-reactivated hibernating animals. In hibernating toads we could demonstrate PSA-immunoreactivity (PSA-IR) within the seminiferous tubules, in clusters of primary spermatocytes, spermatids and spermatozoa, in follicular and Sertoli cells. PSA-IR was seen in peritubular, Leydig and efferent duct cells. In GnRH-treated toads PSA-IR persists in primary spermatocyte groups. alpha-Catenin is localized in the basal laminae of seminiferous tubules and in Leydig cells of hibernating toads. This did not change after hormonal treatment. In hibernating toads, beta-Catenin was detected only in Leydig cells and within seminiferous tubules on basal spermatocystes and limiting spermatozoa clusters. In GnRH-treated toads, the beta-Catenin-IR was less intense in Leydig cells and vanished within seminiferous tubules.

Expression of N-CAM-180 and N-cadherin during development in two South-American anuran species (Bufo arenarum and Hyla nana).

Cadherins and N-CAM are Ca++-dependent and Ca++-independent cell adhesion molecules respectively. These molecules play a key role in morphogenesis and histogenesis. We determined the spatiotemporal pattern of N-cadherin and N-CAM-180 kDa expression by immunohistochemistry during development in two South-American anuran species (Bufo arenarum, toad and Hyla nana, frog). Both N-cadherin and N-CAM were not detectable during early developmental stages. Expression of N-cadherin appeared between the inner and the outer ectoderm layers at stages 19-20. At stages 24-25, N-cadherin was expressed in the neural tube and the heart. In early tadpoles, N-cadherin expression increased along with the central nervous system (CNS) morphogenesis, and reached its maximum level at metamorphic climax stage. N-Cadherin expression was not uniformly distributed. At stage 42, olfactory placodes and retina expressed N-cadherin. Contrary to N-CAM, the strongly myelinated cranial nerves were not labeled. N-Cadherin was present in several mesoderm derivatives such as the notochord, heart and skeletal muscle. The non-neural ectoderm and the endoderm were always negative. Expression of N-CAM appeared first in the neural tube at stages 24-25 and the level of expression became uniform from pre-metamorphic to metamorphic climax tadpoles. At this latter stage, a clear N-CAM immunolabeling appeared in the nerve terminals of pharynx and heart. N-Cadherin and N-CAM were found mainly co-expressed in the CNS from early tadpole to metamorphic climax tadpole. Our results show that the expression of N-CAM and N-cadherin is evolutionary conserved. Their increased expression during late developmental stages suggests that N-CAM and N-cadherin are involved in cell contact stabilization during tissue formation.

Paraquat tolerance and alteration of internal gill structure of Scinax nasica tadpoles (Anura: Hylidae).

Paraquat acute tolerance, and histological and ultrastructural response analysis of gills were evaluated in Scinax nasica tadpoles at stages 25-26. The treated tadpoles were able to survive with a mortality rate similar to the controls in media containing 6.48 to 10.80 mg PQ/L. At 18.00 mg PQ/L survival rate was about 85%; at 30.00 and 50.00 mg PQ/L an incremented mortality rate was detected. Their internal gills appear to have a critical role in adaptation to exposure to PQ. The LC-50 values found helped us conclude that increasing time of exposure to PQ produces a decrease of tadpole survival rate. With light microscopy, the apical ramification gill tufts exhibited a dose response increase of the total volume. At the ultrastructural level, an increase in the intercellular spaces and alteration in gill epithelium organization were the most notable changes. Furthermore, the capillaries showed enlarged endothelial cells, irregular nuclei, and numerous cytoplasmatic projections.