Induction and development of neural crest in Xenopus laevis.

Neural crest cells are a migratory embryonic cell population that form at the border between the neural plate and the future epidermis. This border, the neural plate border, corresponds to the neural fold. The neural fold surrounds the entire neural plate, but only the lateral and posterior portions of the fold give rise to neural crest cells, while the anterior neural fold differentiates as forebrain. This review focuses on neural crest development in Xenopus laevis embryos, and analyzes aspects of the induction of the neural crest in Xenopus, summarizing available information relating to the expression of several genes in the neural crest. Two models for neural crest induction are discussed. In the first model, the neural crest is induced by the interaction between the neural plate and the epidermis. In the second, the specification of the neural plate border arises as a consequence of a gradient of BMP activity. The role of posteriorizing signals on neural crest specification is also discussed. Finally, we propose that the specification and differentiation of the neural crest is controlled by a cascade of transcription factors, encoded and expressed from a hierarchy of genes. A set of extracellular signals establishes the positional information in the ectoderm, which activates Prepattern genes (Gli, Xiro, Zic, Dlx, etc.) across extended and overlapping domains. A local combination of these genes at the neural plate border activates the cascade of neural crest specification, while different sets of genes are activated at both sides of the neural folds (in the epidermis and the neural plate). The genes activated in regions adjacent to the neural plate border have an inhibitory effect on the neural crest transcription program.

Hypoglycemic effect of the water extract of Smallantus sonchifolius (yacon) leaves in normal and diabetic rats.

The hypoglycemic effect of the water extract of the leaves of Smallantus sonchifolius (yacon) was examined in normal, transiently hyperglycemic and streptozotocin (STZ)-induced diabetic rats. Ten-percent yacon decoction produced a significant decrease in plasma glucose levels in normal rats when administered by intraperitoneal injection or gastric tube. In a glucose tolerance test, a single administration of 10% yacon decoction lowered the plasma glucose levels in normal rats. In contrast, a single oral or intraperitoneal administration of yacon decoction produced no effect on the plasma glucose levels of STZ-induced diabetic rats. However, the administration of 2% yacon tea ad libitum instead of water for 30 days produced a significant hypoglycemic effect on STZ-induced diabetic rats. After 30 days of tea administration, diabetic rats showed improved body (plasma glucose, plasma insulin levels, body weight) and renal parameters (kidney weight, kidney to body weight ratio, creatinine clearance, urinary albumin excretion) in comparison with the diabetic controls. Our results suggest that yacon water extract produces an increase in plasma insulin concentration.

Changes in the expression of small intestine extracellular matrix proteins in streptozotocin-induced diabetic rats.

Diabetes mellitus is characterized by anatomical and functional alterations of the intestinal tract. However, the aetiology of these disturbances remains unclear. The aim of the present work was to investigate the effects of diabetes on the expression of laminin-1 and fibronectin in the small intestine of Streptozotocin (STZ)-induced diabetic rats. The Western immunoblotting of the extracts from the small intestine revealed that experimental diabetes resulted in a marked increase in the intensity of the bands corresponding to laminin-1 and fibronectin. Immunohistochemical studies demonstrated a strong labelling to these two extracellular matrix (ECM) proteins in the small intestine of diabetic rats, mainly localized in the smooth muscle layer. These results occur together with a thickening of the basement membrane (BM) of the smooth muscle cells, demonstrated by transmission electron microscopy (TEM). We propose that the accumulation of ECM proteins in the smooth muscle layer may be an effect mediated by hyperglycaemia, since insulin treatment of diabetic rats reversed this accumulation. These results could provide information on the potential role of the ECM in the intestine, an organ which is known to exhibit important alterations in diabetes.

Changes in liver gangliosides in streptozotocin-induced diabetic rats.

Diabetes mellitus is associated with various structural and functional liver abnormalities that affect the glycogen and lipid metabolisms. The effects of streptozotocin-induced diabetes and of insulin supplementation to Sprague-Dawley diabetic rats on ganglioside patterns in liver were determined. Diabetic livers showed a tendency to hepatomegaly 3 weeks after STZ-induction of diabetes. The concentration of total gangliosides in diabetic and non-diabetic livers was similar, but the concentration of total gangliosides in the liver of insulin-stabilized rats was slightly increased. Bidimensional TLC chromatographic analysis of gangliosides isolated from normal diabetic and insulin-stabilized diabetic livers showed quantitative and qualitative changes. In comparison with normal controls, the densitometric analyses of diabetic liver ganglioside patterns had increased amounts of GM3, GM1, GD1b, and GT1b gangliosides, while GM2 could not be detected. The hepatic ganglioside pattern of insulin-stabilized diabetic rats was partially restored, resembling the profile of normal rats. The activity of GalNAcT, GalT-2 and SialT-4 transferases was measured in liver microsomal fractions of the different groups of animals. Diabetic rats showed an increased activity of GalNAcT and a decrease in the activity of GalT-2 and SialT-4 compared with the controls. The enzymatic activities found in insulin-treated rats showed a tendency to return to the values observed in normal control animals. The results evidenced that streptozotocin-induced diabetes affects the liver ganglioside pattern and the ganglioside synthesis enzyme activity. The alterations found in ganglioside metabolism could represent one of the earliest changes associated with the diabetic pathology.

Inhibition of the synthesis of glycosphingolipid by a ceramide analogue (PPMP) in the gastrulation of Bufo arenarum.

In the present study the role of glycosphingolipids (GSL) in amphibian development was investigated. We analysed the de novo synthesis of neutral GSL and gangliosides through the initial stages of Bufo arenarum embryo development and their participation during gastrulation using 1-phenyl-2-palmitoyl-3-morpholino-1-propanol (PPMP), a potent inhibitor of glucosylceramide synthase. Ganglioside synthesis began at the blastula stage and reached a maximum during gastrulation (stages 10-12) while neutral GSL synthesis showed a slight gradual increase, the former being quantitatively more significant than the latter. Ganglioside synthesis was reduced by 90% while neutral GSL synthesis was inhibited by 65% when embryos at blastula stage were cultured for 24 h in 20 microM PPMP. The depletion of GSL from amphibian embryos induced an abnormal gastrulation in a dose-dependent manner. We found that PPMP had a pronounced effect on development since no embryos exhibited normal gastrulation; their developmental rate either slowed down or, more often, became totally arrested. Morphological analysis of arrested embryos revealed inhibition of the gastrulation morphogenetic movements. Analysis of mesodermal cell morphology in those embryos showed a severe decrease in the number and complexity of cellular extensions such as filopodia and lamellipodia. Mesodermal cells isolated from PPMP-treated embryos had very low adhesion percentages. Our results suggest that glycosphingolipids participate in Bufo arenarum gastrulation, probably through their involvement in cell adhesion events.

Effect of gap junction uncoupling in full-grown Bufo arenarum ovarian follicles: participation of cAMP in meiotic arrest.

The aim of the present study was to determine the presence of the connexins Cx43, Cx32 and Cx26 in Bufo arenarum ovarian follicles during the breeding season as well as to analyse the possible alterations in the meiotic process when connexins are blocked by specific antibodies. Western blot analysis revealed that the Cx43 and Cx32 proteins were present but not Cx26. We demonstrated that the anti-Cx43 and anti-Cx32 antibodies produced the uncoupling of the gap junctions. When these junctions are blocked the maturation process is triggered in the oocytes. We determined that dbcAMP exerts an inhibitory effect on the maturation induced by the uncoupling of the gap junctions when the oocytes are injected or pretreated with this metabolite. We propose the idea that cAMP is the regulatory molecule in meiotic arrest in this amphibian species.

Participation of the GM1 ganglioside in the gastrulation of anuran amphibian Bufo arenarum.

In the present paper we established the ganglioside composition of the blastula and gastrula stages of the anuran amphibian Bufo arenarum, two relevant stages characterized by dynamic changes in morphology and cellular rearrangements. Densitometric studies evidenced that GD1a and GT1b were the more abundant gangliosides of the blastula embryos whereas GM1 and GM2 were the predominant species in gastrula embryos. Analysis of ganglioside abundance indicates that the "a" and "b" synthesis pathways perform similar biosynthetic activities in the blastula stage, in contrast to the gastrula stage in which a marked predominance of the "a" pathway occurred. The spatio-temporal expression of GM1 and of polygangliotetraosyl ceramides (pGTC) was investigated by wholemount immunocytochemistry using cholera toxin B subunit (CTB) and an affinity purified human anti-GM1 antibody. The pGTC were detected as GM1 after treatment with neuraminidase. Blastomeres from the inner surface of the blastocoelic roof (BCR) of blastula embryos were GM1 and pGTC positive. At midgastrula stage, embryos showed an increased labeling on the inner surface of BCR. To establish whether the GM1 ganglioside was involved in the gastrulation processes, CTB, anti-GM1 antibodies and anti-GM1 Fab' fragments were microinjected into the blastocoel cavity of blastula embryos. Treatment with the probes blocked gastrulation. Scanning electron microscopy analysis of blocked embryos revealed that mesodermal cell migration, radial interdigitation, and convergent extension movements were affected. The blocking of gastrulation was correlated with the absence of fibronectin and EP3/EP4 on the inner surface of blastocoelic roof of CTB- or anti-GM1 treated embryos. Results show that the GM1 ganglioside is differentially expressed by embryonic cells and participates in the morphogenetic processes of amphibian gastrulation. J. Exp. Zool. 286:457-472, 2000.

Comparative study of vitellogenesis in the anuran amphibians Ceratophrys cranwelli (Leptodactilidae) and Bufo arenarum (Bufonidae).

The present study analyses, by transmission electron microscopy, vitellogenesis in two anuran amphibian families: Leptodactilidae (Ceratophrys cranwelli) and Bufonidae (Bufo arenarum). These differ in the type of stimulus that sets off their reproductive period, pluvial changes being the trigger in C. cranwelli and temperature increase in B. arenarum. We found that vitellogenesis follows an endocytic pathway that involves membranous structures (coated pits, coated vesicles, endosomes and multivesicular bodies). This process results in a fully grown yolk platelet of similar structure in both species. Despite the above similarity, a distinctive feature in B. arenarum was that the multivesicular bodies exhibited condensed proteins together with lipid droplets, the latter remaining as such even in the primordial yolk platelet. In C. cranwelli, however, lipids droplets were only found attached to the primordial yolk platelet. The coexistence of lipid droplets together with proteins in the nascent precursor yolk platelets observed in B. arenarum is similar to that found in B. marinus. This fact might constitute a characteristic feature of the Bufonidae family.

Evidence for the presence and participation of 85-75 KDa extracellular matrix components in cell interactions of Bufo arenarum gastrulation.

We studied the presence and distribution of the extracellular materials (ECM), obtained by mild embryonic dissociation through nondenaturing and denaturing PAGE, immunoblotting and immunocytochemical wholemount in the gastrulation of anuran amphibian Bufo arenarum. The SDS-PAGE, under reducing conditions, revealed the protein profile of the ECM which comprised six bands. The Western immunoblotting effected with antibodies against fibronectins (FN) of Xenopus laevis, Ambystoma mexicanum and Bufo arenarum revealed that the 210 and 190 KDa bands (EP1-EP2) present in the ECM were identified as FN. Polyclonal antibodies against the 85-75 KDa polypeptides (EP3-EP4) were obtained and used throughout this study. The distribution of FN and EP3-EP4 was comparatively studied in the blastocoelic roof (BCR) of stage 10.5 Bufo arenarum, Xenopus laevis and Ambystoma mexicanum embryos. In the anurans, FN appeared as a network of fine fibrils apparently oriented at random, while in Ambystoma, FN appeared as a complex anastomosing network of oriented fibrils. EP3-EP4 were found in Bufo and in Xenopus both in the intercellular contact zones and in the cellular periphery. No linear arrangements of these proteins were observed. Few, if any, EP3-EP4 were found on the BCR of Ambystoma mexicanum. At stage 11, EP3-EP4, which showed a dramatic increase at the chordomesoderm-neuroectoderm junction in Bufo arenarum embryos, appeared as an amorphous material. For the purpose of analyzing the role of EP3-EP4 during Bufo arenarum gastrulation, anti-EP3-EP4 antibodies and anti-EP3-EP4 Fab fragments were microinjected into the blastocoel cavity of stage 9 embryos, an event that cause severe alterations in the gastrulation process. Convergent extension of the dorsal marginal zone and the epiboly of the BCR were the most strongly affected events. Results show that EP3-EP4 are required for normal Bufo arenarum gastrulation.

Amphibian Bufo arenarum vitronectin-like protein: its localization during oogenesis.

In the present study, we analyzed the localization of vitronectin-like protein in oocytes during oogenesis as well as in the serum and liver tissue of the amphibian Bufo arenarum. Vitronectin-like protein was purified from serum by heparin-affinity chromatography and showed to have the two biological properties in common with most animal vitronectins (VN): heparin binding activity and an RGD-dependent cell-spreading activity. SDS-PAGE of vitronectin-like protein revealed that it consists of two bands of 64 kDa and 72 kDa, while immunoblotting analyses showed that this protein strongly cross-reacts with two monoclonal antibodies against human VN. No immunofluorescent staining of vitronectin-like protein was observed in previtellogenic oocytes (stages I and II). In vitellogenic oocytes (stages III, IV and V) fluorescence was observed in the cortical cytoplasm localized in yolk platelets, extending concomitantly with the vitellogenic process. When we examined the yolk platelet formation pathway by immunoelectron microscopy, gold particles indicated that vitronectin-like protein was located on the yolk platelet precursors: multivesicular bodies and primordial yolk platelets. Gold particles also were seen sparsely distributed in all oocyte investing layers. The mean serum vitronectin-like protein concentration in amphibian animals was 127.8 +/- 11.6 micrograms/ml in adult males and 181.5 +/- 14.3 micrograms/ml in adult females. Serum vitronectin-like protein of males and females was susceptible to hormonal stimulation (17-beta estradiol). These results suggest that vitronectin-like protein is stored in the yolk platelets and may be involved in the later events of amphibian development.

Alteration of morphogenetic cell interactions by 1-beta-D-arabinofuranosylcytosine in Bufo arenarum embryo.

Amphibian gastrulation was used as a model system to study the action of the nucleoside 1-beta-D-arabinofuranosylcytosine (Ara-C) on the early events of amphibian morphogenesis. Ara-C inhibits both glycoprotein and glycolipid synthesis and interferes with DNA synthesis. Thus, it is useful to investigate the importance of the cell surface and the nucleous during Bufo arenarum morphogenesis. Living embryos were incubated with Ara-C at blastula and gastrula stages. Treated-embryos undergo abnormal gastrulation, most of the embryos exogastrulate, although some do not gastrulate at all. This antimetabolite did not interfere with neural induction, as partial exogastrulae developed a small neural tube. We have proven that Area-C disturbs the typical intercellular organization and inhibits the radial intercalation of the blastocoelic roof. The mesodermal migration is the most affected morphogenetic process. The results described in this paper demonstrate that the timing of gastrulation movements strongly involves the participation of surface and extracellular molecules in cell recognition and cell interaction but does not involve a significant increase in cell division rate and can also occur in the absence of the cell division.

Alteration of morphogenetic cell interactions by 1-beta-D-arabinofuranosylcytosine in Bufo arenarum embryo.

Amphibian gastrulation was used as a model system to study the action of the nucleoside 1-beta-D-arabinofuranosylcytosine (Ara-C) on the early events of amphibian morphogenesis. Ara-C inhibits both glycoprotein and glycolipid synthesis and interferes with DNA synthesis. Thus, it is useful to investigate the importance of the cell surface and the nucleous during Bufo arenarum morphogenesis. Living embryos were incubated with Ara-C at blastula and gastrula stages. Treated-embryos undergo abnormal gastrulation, most of the embryos exogastrulate, although some do not gastrulate at all. This antimetabolite did not interfere with neural induction, as partial exogastrulae developed a small neural tube. We have proven that Area-C disturbs the typical intercellular organization and inhibits the radial intercalation of the blastocoelic roof. The mesodermal migration is the most affected morphogenetic process. The results described in this paper demonstrate that the timing of gastrulation movements strongly involves the participation of surface and extracellular molecules in cell recognition and cell interaction but does not involve a significant increase in cell division rate and can also occur in the absence of the cell division.