RESUMEN
Belting singers receiving care at the speech therapy service mentioned they find it easier to sing in American English than in Brazilian Portuguese. OBJECTIVE: to assess and compare the production of vowels and consonants of excerpts from musicals in Portuguese and English. METHODS: a phonological and phonetic analysis of the same excerpt from musicals was performed in both languages. RESULTS: We have observed that the predominance of fricatives in English and plosives in Portuguese explains the fluidity of singing in English and a greater disruption of speech fluidity in Portuguese. CONCLUSION: therefore, belting in English is easier than it is in Portuguese.
Asunto(s)
Canto , Acústica del Lenguaje , Humanos , Portugal , Habla , FonéticaRESUMEN
A lack of knowledge about rabbit herbage intake during grazing limits the development of organic rabbit production. This study describes rabbit herbage intake under a wide range of grazing conditions and characterises the factors that decrease rabbit herbage intake and daily weight gain. It was conducted with growing rabbits reared in moving cages with 0.4 m2 of grazing area per rabbit. Rabbits grazed on pastures dominated by legumes (LEG) or grass and forbs (GRF) and received 60 g/day per rabbit of a complete pelleted feed. Three trials were performed in winter, summer and spring. Mean herbage allowance was 27% higher in LEG (62.3 g dry matter (DM)/kg metabolic weight (MW), equal to kg0.75) than in GRF (49.2 g DM/kg MW). Herbage intake varied greatly (36.3±18.0 g DM/kg MW) among trials and was higher in LEG than in GRF (39.5v.34.1 g DM/kg MW). For both pasture types, herbage intake was logarithmically related to herbage allowance and plateaued around 75 g DM/kg MW. Crude protein and digestible energy (DE) intake differed by pasture type and season. Mean CP intake was 40% higher in LEG (15.0 g/kg MW) than in GRF (10.7 g/kg MW). In summer, mean DE intake was 27% higher in LEG than in GRF but no significant differences in DE intake were found between LEG and GRF in winter and spring. Maximum DE intake plateaued near 1000 kJ/kg MW. Daily weight gain was always higher for rabbits grazing LEG (mean=22.6 g) than GRF (mean=16.0 g). Weight gain was significantly related to CP intake, whereas DE intake had no significant effect. Meeting the objective of mean daily weight gain of 20 g requires herbage intake of 32 and 50 g DM/kg MW in LEG and GRF, respectively. Therefore, according to the herbage use efficiency observed in our experiments, herbage allowance must reach 42 and 78 g DM/kg MW in LEG and GRF, respectively. When herbage allowance is lower, rabbits cannot meet the CP intake (13 g/kg MW) required for this weight gain objective.
Asunto(s)
Alimentación Animal , Dieta/veterinaria , Ingestión de Energía , Fabaceae , Poaceae , Conejos/fisiología , Aumento de Peso , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Peso Corporal , Femenino , LactanciaRESUMEN
Tendon grafts are a component of the therapeutic arsenal for managing chronic flexor tendons injuries in the hand, especially during two-stage Hunter reconstruction. The purpose of this anatomical study was to compare the strength of the Pulvertaft weave versus the step-cut suture used for flexor tendon reconstruction to determine their role in early active mobilization. We performed a biomechanical study with cadaver specimens. Thirty-four hands were randomized and the tendons from both hands were equally assigned to each group. A comparison of the Pulvertaft weave (group 1) versus the step-cut suture (group 2) using the flexor digitorum profundus from the fourth finger and the longus palmaris was carried out. The main variable was the failure load in both repair groups. We also evaluated the cross-sectional area (CSA) and the tensile strength of the repairs. Thirty hands were included in our study. There was no significant difference in the failure load between the two groups (116N for group 1 versus 103N for group 2, P=0.2). The CSA was significantly smaller in the step-cut group compared to Pulvertaft group (19.8mm2 versus 35mm2, P<0.01). The tensile strength was significantly higher in the step-cut group than in the Pulvertaft group (5.3N/mm2 versus 3.4N/mm2, P<0.01). Early active mobilization requires a minimum repair strength of 75N. In our study, the step-cut suture appears strong enough and thin enough to decrease the fibrosis, which would lead to better functional results. No other study of this type has been published. The specimens in which the repair strength was less than 75N all involved a thin, weak longus palmaris. Other biomechanical studies should be done to define the anatomical criteria required for use of the palmaris longus tendon. The step-cut suture seems to be strong enough and thin enough to provide sufficient proximal attachment during flexor tendon reconstruction to allow early active mobilization.
Asunto(s)
Técnicas de Sutura , Tendones/cirugía , Resistencia a la Tracción , Cadáver , Humanos , Distribución AleatoriaRESUMEN
Given the very recent investment in research on organic rabbit production, many knowledge gaps remain. Simulation models based on data from experiments and farms may help generate general principles for organic rabbit production. Our goals were to (i) develop a model to simulate intake regulation and growth of rabbits raised on pastures, (ii) validate this model under a diversity of conditions and (iii) conduct a simulation experiment to predict the potential to decrease the supply of complete feed by increasing the grazing area per rabbit. The model developed (PASTRAB) simulates organic rabbit fattening on pastures in four main submodels that represent dynamics of (i) herbage standing biomass, fill and feed values; (ii) intake of herbage, complementary feed (i.e. complete pellets, cereal-legume grain mixtures) and hay as regulated by herbage allowance, fill and feed values of feedstuffs and rabbit physiological parameters; (iii) conversion of rabbit intake into live weight gain; and (iv) rabbit mortality. The model also calculates gross margin per rabbit sold. Model accuracy was assessed by considering the fit between observed and predicted herbage intake, which was low, with a relative root mean square error (rRMSE) of 51% and 66% on grass-based and legume-based pastures, respectively. However, the standard deviations of observed herbage intake were similar to the root mean square error of predicted herbage intake, indicating that it would have been difficult to improve model calibration. The fit between observed and predicted rabbit live weight was acceptable, with an rRMSE of 11% and 10% for grass-based and legume-based pastures, respectively. Simulated scenarios showed that a decrease in complementary feed combined with an increase in the grazing area per rabbit had little impact on average daily growth and gross margin per rabbit but increased herbage use efficiency. With 90 g of complementary feed per day and grazing of 0.4 m²/rabbit per day, herbage use efficiency was 22%, with average daily growth of 21.6 g/day and gross margin of 18.80 /rabbit. With no complementary feed and grazing of 1.2 m²/rabbit per day, average daily growth decreased (19.2 g/day), but herbage use efficiency reached 100% and gross margin reached 19.20 /rabbit. We used PASTRAB in participatory workshops with farmers so that the latter could explore adaptations to their current practices. Overall, farmers considered the model predictions realistic, and some of them decided to adapt some of their management practices immediately after the workshops.
Asunto(s)
Alimentación Animal , Poaceae , Conejos/fisiología , Crianza de Animales Domésticos , Animales , Fabaceae , Conejos/crecimiento & desarrollo , Conejos/metabolismo , Aumento de PesoRESUMEN
We describe graph machines, an alternative approach to traditional machine-learning-based QSAR, which circumvents the problem of designing, computing and selecting molecular descriptors. In that approach, which is similar in spirit to recursive networks, molecules are considered as structured data, represented as graphs. For each example of the data set, a mathematical function (graph machine) is built, whose structure reflects the structure of the molecule under consideration; it is the combination of identical parameterised functions, called "node functions" (e.g. a feedforward neural network). The parameters of the node functions, shared both within and across the graph machines, are adjusted during training with the "shared weights" technique. Model selection is then performed by traditional cross-validation. Therefore, the designer's main task consists in finding the optimal complexity for the node function. The efficiency of this new approach has been demonstrated in many QSAR or QSPR tasks, as well as in modelling the activities of complex chemicals (e.g. the toxicity of a family of phenols or the anti-HIV activities of HEPT derivatives). It generally outperforms traditional techniques without requiring the selection and computation of descriptors.
Asunto(s)
Gráficos por Computador , Simulación por Computador , Relación Estructura-Actividad Cuantitativa , Fármacos Anti-VIH/farmacología , Pruebas de Carcinogenicidad/métodos , Redes Neurales de la Computación , Fenoles/toxicidadRESUMEN
Organic agriculture is developing worldwide, and organic rabbit production has developed within this context. It entails raising rabbits in moving cages or paddocks, which enables them to graze grasslands. As organic farmers currently lack basic technical information, the objective of this article is to characterize herbage intake, feed intake and the growth rate of rabbits raised on grasslands in different environmental and management contexts (weather conditions, grassland type and complete feed supplementation). Three experiments were performed with moving cages at an experimental station. From weaning, rabbits grazed a natural grassland, a tall fescue grassland and a sainfoin grassland in experiments 1, 2 and 3, respectively. Rabbit diets were supplemented with a complete pelleted feed limited to 69 g dry matter (DM)/rabbit per day in experiment 1 and 52 g DM/rabbit per day in experiments 2 and 3. Herbage allowance and fiber, DM and protein contents, as well as rabbit intake and live weight, were measured weekly. Mean herbage DM intake per rabbit per day differed significantly (P<0.001) between experiments. It was highest in experiment 1 (78.5 g DM/day) and was 43.9 and 51.2 g DM/day in experiments 2 and 3, respectively. Herbage allowance was the most significant determinant of herbage DM intake during grazing, followed by rabbit metabolic weight (live weight0.75) and herbage protein and fiber contents. Across experiments, a 10 g DM increase in herbage allowance and a 100 g increase in rabbit metabolic weight corresponded to a mean increase of 6.8 and 9.6 g of herbage DM intake, respectively. When including complete feed, daily mean DM intakes differed significantly among experiments (P<0.001), ranging from 96.1 g DM/rabbit per day in experiment 2 to 163.6 g DM/rabbit per day in experiment 1. Metabolic weight of rabbits raised on grasslands increased linearly over time in all three experiments, yielding daily mean growth rates of 26.2, 19.2 and 28.5 g/day in experiments 1, 2 and 3, respectively. The highest growth rate was obtained on the sainfoin grassland despite lower concentrate supplementation. Thus, it seems possible to reduce complete feed supplementation without reducing animal performance. This possibility requires improving our knowledge about organic rabbit production systems and especially grazing and animal health management.
Asunto(s)
Dieta/veterinaria , Ingestión de Alimentos/fisiología , Pradera , Herbivoria/fisiología , Poaceae , Conejos/crecimiento & desarrollo , Conejos/fisiología , Alimentación Animal , Animales , Fibras de la Dieta/análisis , Suplementos DietéticosRESUMEN
Studies were carried out on the embryo of the amphibian Pleurodeles waltl to investigate the potential role of the N-linked oligosaccharides of the ectodermal cell membrane in the neural induction process. Glycopeptidase F (GPase F) was used to cleave N-linked oligosaccharides on presumptive ectoderm. Removal of oligosaccharide moieties from ectoderm membrane glycoconjugates completely inhibited natural neural induction in vitro. On the other hand, Swainsonine (Sw) and 1-deoxynojirimycin (dNM), specific inhibitors of enzymes involved in glycosylation, provoked strong and persistent changes in the structure of the N-linked oligosaccharides of presumptive ectoderm but did not prevent neuralisation of treated ectoderm. We conclude that N-linked carbohydrates are implicated in the phenomenon of neural induction. However, the structural integrity of N-linked carbohydrates of target tissue is not itself critical in this process. The existence of specific carbohydrates on presumptive ectoderm was still questioned as receptors of neural signal.
Asunto(s)
Ectodermo/fisiología , Sistema Nervioso/embriología , Oligosacáridos/metabolismo , Pleurodeles/embriología , 1-Desoxinojirimicina , Alcaloides , Amidohidrolasas , Animales , Ectodermo/química , Glucosamina/análogos & derivados , Manosidasas/antagonistas & inhibidores , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa , SwainsoninaRESUMEN
Recent evidence indicates that oligodendrocytes originate initially from the ventral neural tube. We have documented in chick embryos the effect of early ventralization of the dorsal neural tube on oligodendrocyte differentiation. Notochord or floor plate grafted at stage 10 in dorsal position induced the development of oligodendrocyte precursors in the dorsal spinal cord. In vitro, oligodendrocytes differentiated from medial but not intermediate neural plate explants, suggesting that the ventral restriction of oligodendrogenesis is established early. Furthermore, quail fibroblasts overexpressing the ventralizing signal Sonic Hedgehog induced oligodendrocyte differentiation in both the intermediate neural plate and the E4 dorsal spinal cord. These results strongly suggest that the emergence of the oligodendrocyte lineage is related to the establishment of the dorso-ventral polarity of the neural tube.
Asunto(s)
Inducción Embrionaria , Notocorda/fisiología , Oligodendroglía/citología , Proteínas/fisiología , Transactivadores , Animales , Biomarcadores , Células Cultivadas , Embrión de Pollo , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Proteínas Hedgehog , Mesodermo/fisiología , Notocorda/trasplante , Oligodendroglía/metabolismo , Proteínas/genética , Codorniz , Somitos/fisiología , Médula Espinal/metabolismo , Factores de TiempoRESUMEN
We have analyzed the transduction pathways involved in the triggering of neural induction, in amphibian embryos, in vivo. Using a plasmid construction, we have targetted the bioluminescent calcium probe aequorin to the plasma membrane of ectoderm cells of the amphibian Pleurodeles waltl before gastrulation. We have demonstrated that the in vivo triggering of neural induction depends on the activation of calcium-dependent pathways and involves L-type calcium channels. Furthermore, on excised ectoderm taken at the gastrula stage, we show that noggin, a protein currently considered as one of the natural inducers, also activates L-type calcium channels. This activation represents the first necessary event to determine cells of the dorsal ectoderm toward the neural pathway.
Asunto(s)
Canales de Calcio/metabolismo , Sistema Nervioso/embriología , Sistema Nervioso/metabolismo , Pleurodeles/embriología , Pleurodeles/metabolismo , Aequorina/metabolismo , Animales , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/efectos de los fármacos , Canales de Calcio Tipo L , Proteínas Portadoras , Dihidropiridinas/farmacología , Ectodermo/metabolismo , Gástrula/metabolismo , Transporte Iónico/efectos de los fármacos , Nimodipina/farmacología , Proteínas/farmacología , Transducción de SeñalRESUMEN
In our laboratory we use Urodeles (Pleurodeles waltl) and Anurans (Xenopus laevis) to perform comparative studies on neural determination. Urodeles are a good embryological system to study early events in ontogenesis since they present several advantages: slow development, external localization of chordamesoderm at the beginning of gastrulation, large size of cells, diploid genome, etc. I have focused this overview-report on the main findings on Pleurodeles neurogenesis. The determination of the two neural lineages (neuronal and astroglial) appears during gastrulation as a consequence of (a) permissive event(s) activated through a Ca(++)-dependent transducing pathway. This signaling-pathway involves L-type Ca++ channels. The activation of this Ca++ transduction route is sufficient to activate both neuronal and glial structural specific genes, via direct activation of "immediate early genes". The specification of neuronal functional differentiation depends on additional factors of chordamesoderm origin acting during gastrulation and later on. At the early neurula stage, in the neural plate, 20% of progenitor cells present a neuronal fate, 80% are at least bipotential and generate mixed clones (neurons and astrogliocytes). The issue of the state of "commitment" of the precursor cells (competent ectoderm) and the identification of specifying molecules (from Spemann organizer) are underway in Pleurodeles and Xenopus.
Asunto(s)
Sistema Nervioso Central/embriología , Urodelos/embriología , Animales , Canales de Calcio/fisiología , Ectodermo/citología , Pleurodeles/embriología , Proteína Quinasa C/metabolismo , Xenopus laevis/embriologíaRESUMEN
Unlike biologists from several European countries, most French embryologists did not work from the onset on problems associated with the Spemann-Mangold organizer, though they were fully aware of the importance of the discovery. They preferred to stay on other original topics, but their later work was of course influenced by the induction concepts. The exploration of secondary inductions in various organ formations was flourishing after 1950. As far as primary induction is concerned, two exceptions must be stressed: Vintemberger, who, before World War II, worked on the frog organizer for a few years, and especially Capuron (1968), who repeated Spemann and Mangold's fundamental experiment on a large scale. Then, from 1980 on, a series of studies dealing with the neural induction concept focused on studies of the gastrula ectoderm itself, was undertaken, mainly in Toulouse University by Duprat and her colleagues, and in Paris-6 University by Boucaut and his colleagues.
Asunto(s)
Embriología/historia , Inducción Embrionaria , Organizadores Embrionarios , Anfibios/embriología , Animales , Señalización del Calcio , Francia , Historia del Siglo XX , Modelos BiológicosRESUMEN
Initial development of astroglial phenotype has been studied in vitro in an amphibian embryo (Pleurodeles waltI), to document the differentiation potentialities acquired by neural precursor cells isolated at the early neurula stage. In particular, we sought to determine whether interactions between neuroepithelial cells and the inducing tissue, the chordamesoderm, are required beyond this stage to specify precursor cells along glial lineages. Glial cell differentiation was documented by examining the appearance of glial fibrillary acidic protein (GFAp), a specific marker of astroglial lineages. Cells expressing GFAp-immunoreactivity differentiated rapidly, after 48 hours of culture, from cultivated neural plate cells, irrespective of the presence or absence of the inducing tissue. The widespread expression of Pleurodeles GFAp protein in neural plate cultures, in which CNS precursor cells develop alone in a simple saline medium, showed that prolonged contact with chordamesodermal cells was not necessary for the emergence of the astroglial phenotype. In addition, the initial development of astroglial phenotype has been defined in vivo. The first detectable GFAp-immunoreactivity was visualized in the neural tube of stage-24 embryos, a stage corresponding to 2-3 days in culture, defining radial glial cell end-feet. Thus, dissociation and culture of neural precursor cells did not appear to modify the onset of astroglial differentiation. At stage 32, GFAp-immunoreactivity was observed over the entire length of radial glial fibers and was also evidenced in mitotic cells located in the ventricular zone, suggesting that radial glial cells were not all post-mitotic.
Asunto(s)
Astrocitos/fisiología , Neuronas/fisiología , Pleurodeles/embriología , Animales , Especificidad de Anticuerpos , Astrocitos/inmunología , Astrocitos/ultraestructura , Biomarcadores , Diferenciación Celular , Epitelio/fisiología , Epitelio/ultraestructura , Proteína Ácida Fibrilar de la Glía/inmunología , Técnicas In Vitro , Filamentos Intermedios/inmunología , Filamentos Intermedios/fisiología , Filamentos Intermedios/ultraestructura , Mitosis , Cresta Neural/fisiología , Cresta Neural/ultraestructura , Neuronas/inmunología , Neuronas/ultraestructuraRESUMEN
At the late gastrula-early neurula stage some embryonic neuroblasts from neural plate and neural fold present apparently as a consequence of neural induction, the capability to develop in vitro into different neuronal subpopulations (cholinergic, dopaminergic, noradrenergic, somatostatinergic and some other peptidergic subpopulations without ongoing influences from the chordamesoderm (Duprat et al., 1987). Using the same in vitro model system, the aim of the present work was to delineate the abilities of these neuroblasts to develop GABAergic traits. The initial appearance and development of GABAergic phenotype has been quantitated by assaying the activity of glutamic acid decarboxylase (GAD). GAD activity was undetectable at the early gastrula stage (stage 8a) and was slightly measurable at the early neurula stage (stage 14- onset of the culture). It increased subsequently over the next 14 days in vitro. The temporal pattern of appearance and development of GAD activity in culture was in agreement with that observed in vivo. Immunocytochemical studies showed that GABA-like immunoreactivity was expressed in vitro in a subpopulation of neurons. Thus the developmental program for GAD expression and GABA phenotype maturation is acquired at least in some neuronal precursors. These data together with previously reported results on the expression of cholinergic, catecholaminergic and peptidergic phenotypes demonstrate that different neuronal subpopulations emerge near the end of gastrulation i.e. immediately after neural induction. The embryonic origin of this neuroblast heterogeneity remains to be determined.
Asunto(s)
Sistema Nervioso/embriología , Neuronas/fisiología , Pleurodeles/embriología , Salamandridae/embriología , Ácido gamma-Aminobutírico/fisiología , Animales , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Gástrula/fisiología , Glutamato Descarboxilasa/metabolismo , Inmunohistoquímica , Cinética , Sistema Nervioso/citología , Factores de Tiempo , Ácido gamma-Aminobutírico/análisisRESUMEN
The mechanisms involved in the first step of neurogenesis, i.e. neural induction, are poorly understood, particularly in terms of the signalling pathway. In a recent work it has been shown that in urodeles the activation of L-type calcium channels is sufficient to trigger neural induction. In order to substantiate a possible role of this channel in early development in anurans, we have detailed the kinetics of the expression and the localization of the alpha 1 subunit of L-type calcium channel in the early stages of Xenopus laevis embryogenesis using immunological techniques. We observed that the expression of the alpha 1 subunit started during blastulation, where a cytoplasmic labeling was observed. At the onset of gastrulation alpha 1 was targeted to the plasma membrane of the dorsal and the ventral ectoderm. Some labeling was found in the mesoderm but never in the endoderm. This expression seems to be general, since similar results have been obtained in anurans (Xenopus) and in urodeles (Pleurodeles). In addition, we found that the alpha 0 subunit of the G(o) protein is expressed simultaneously and strictly colocalized with the alpha 1 subunit of the L-type calcium channel. The role of this channel and its regulation by G(o) protein during early neurogenesis is discussed.
Asunto(s)
Canales de Calcio/metabolismo , Sistema Nervioso/embriología , Xenopus laevis/embriología , Animales , Canales de Calcio/fisiología , Ectodermo/metabolismo , Inducción Embrionaria , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Proteínas de Unión al GTP/inmunología , Proteínas de Unión al GTP/metabolismo , Inmunohistoquímica , Mesodermo/metabolismo , Sistema Nervioso/crecimiento & desarrolloRESUMEN
Using antibodies against homeoprotein XIHbox 1 from Xenopus laevis, we have detected a new embryonic protein with a much larger molecular weight, 115 kDa. Antibodies fractionated according to their affinity for 3 different domains of the XIHbox 1 protein were used to show that this new protein is related to the C-terminal region of XIHbox 1 protein, downstream from the homeodomain. By immunohistochemistry, the protein was shown to be localized in nuclei of embryonic cells. On SDS-polyacrylamide gels, the 115 kDa protein appears as a set of closely spaced bands whose pattern varies with the stage of development and with the parental origin of the embryos. The protein could be extracted from embryos in a multiprotein complex of approximately 600 kDa. In contrast, the 18 and 27 kDa proteins predicted from the sequence of cloned cDNA to be transcribed and translated from the XIHbox 1 gene could not be detected, suggesting that they are rare or unstable in embryos. These data suggest that the new protein is involved in the development of Xenopus embryos, with a function possibly related to that of the homeoprotein XIHbox 1.
Asunto(s)
Anticuerpos , Embrión no Mamífero/química , Proteínas de Homeodominio/inmunología , Proteínas/análisis , Proteínas de Xenopus , Xenopus laevis/embriología , Animales , Especificidad de Anticuerpos , Western Blotting , Embrión no Mamífero/ultraestructura , Proteínas de Homeodominio/química , Proteínas de Homeodominio/genética , Inmunohistoquímica , Peso Molecular , Proteínas/químicaRESUMEN
Changes in activity of the Na+, K+ ATPase of maturing Pleurodeles waltl were followed by measuring the resting potential in presence or absence of the specific inhibitor dihydroouabain. Corresponding currents were measured in voltage clamp conditions to eliminate the differences in resting potential at the origin and at the end of the meiotic maturation process. Our data confirm previous results obtained on Xenopus, indicating that the Na+,K+ pump activity disappears from the plasma membrane during progesterone-induced maturation and can be reactivated by an increase in internal Ca2+ triggered by ionomycin. Moreover we show by ultrastructural histochemistry that these modulations are likely to depend on the internalization and reinsertion of the transporter into the plasma membrane.
Asunto(s)
Calcio/farmacología , Meiosis , Oocitos/enzimología , Pleurodeles/fisiología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Membrana Celular/fisiología , Electrofisiología , Histocitoquímica , Potenciales de la Membrana/efectos de los fármacos , Oocitos/fisiología , Progesterona/farmacologíaRESUMEN
As an immediate consequence of neural induction during gastrulation, some neuroectodermal cells acquire the ability to develop a number of specific neuronal and astroglial features, without requiring subsequent chordamesodermal cues. Thus, cholinergic, dopaminergic, noradrenergic, gabaergic, somatostatinergic, enkephalinergic, etc. traits are expressed in cultures of neural plate and neural fold isolated from amphibian late gastrulae immediately after induction and cultured in a defined medium. These results strongly suggest that at the late gastrula stage, the neural precursor population does not yet constitute a homogeneous set of cells. It was of interest to know the origin of this heterogeneity. Is it a direct result of the process of neural induction itself, stochastic phenomena being involved or not at the cellular level, or does it reflect a pre-existing heterogeneity in the presumptive ectoderm? At the early gastrula state, presumptive ectoderm can be neuralized consecutively to its dissociation into single cells. Using this experimental model, we have demonstrated by means of immunological probes that neuralized presumptive ectodermal cells, without any intervention of the chordamesoderm (natural inducing tissue), can develop autonomously into glial and neuronal lineages. These data suggest the existence of diverse predispositions of presumptive ectodermal cells. Competent ectoderm seems to be a heterogeneous structure with cells presenting distinct neural predispositions that can emerge as a consequence of a permissive inductive signal without real specificity (such as a target tissue dissociation). Moreover, such a differentiated neuronal population includes neurons of the GABAergic and enkephalinergic phenotypes but not of the cholinergic, catecholaminergic, somatostatinergic, etc. phenotypes. These data show that the developmental program of ectodermal cells induced without interaction with the chordamesoderm appears restricted compared to the naturally induced ectoderm. Experiments are now under way to analyze such sequential neural events.
Asunto(s)
Anfibios/embriología , Ectodermo/fisiología , Sistema Nervioso/embriología , Animales , Agregación Celular , Diferenciación Celular , Ectodermo/citología , Embrión no Mamífero/fisiología , Modelos BiológicosRESUMEN
The ability of the ectodermal cells to be induced and to differentiate toward neural tissue, called neural competence, is acquired shortly before gastrulation and lost during late gastrula stages in Pleurodeles waltl embryos. We have examined ectodermal cells' neural competence in relation to the evolution of the density of L-type calcium channels using the fluorescent labelled dihydropyridine probe (STBodipy-DHP). We find that the appearance of dihydropyridine sensitive calcium channels (L-type Ca2+ channels) is correlated with the acquisition of neural competence by the ectoderm cells. The highest density of these channels is reached when competence of the ectoderm is optimal. Conversely, the decrease of L-type Ca2+ channel density occurs simultaneously with the normal loss of competence. In addition, we show that these channels are functional since stimulation by S(-)-Bay K 8644 triggered an increase in [Ca2+]i revealed by fluorescence measurements using Fluo-3. This increase in [Ca2+]i is a function of the L-type Ca2+ channels' density. We propose that the molecular basis of the gain and loss of neural competence is linked to the presence of L-type Ca2+ channels in ectodermal cell membranes of Pleurodeles waltl embryos.
Asunto(s)
Compuestos de Boro , Canales de Calcio/análisis , Calcio/metabolismo , Dihidropiridinas , Ectodermo/metabolismo , Colorantes Fluorescentes , Pleurodeles/embriología , Ácido 3-piridinacarboxílico, 1,4-dihidro-2,6-dimetil-5-nitro-4-(2-(trifluorometil)fenil)-, Éster Metílico/farmacología , Animales , Blastocisto/metabolismo , Canales de Calcio Tipo L , Diferenciación Celular , Gástrula/metabolismo , Microscopía FluorescenteRESUMEN
A cDNA library of erythrocyte mRNAs was established from immature red blood cells of the adult amphibian, Pleurodeles waltlii (urodele; salamander). The cDNA clones corresponding to the four adult globin chains were first identified and characterized by positive selection and the cDNAs derived from the two (major and minor) alpha-globin chains sequenced. The sequences presented contain both the complete 3'-noncoding region and the coding region of both chains, with the exception of the first nine codons of the minor alpha-chain, and a portion of the 5'-noncoding region of the major chain. The amino acid sequences of the encoded alpha-globin polypeptides have been deduced and compared with those of Xenopus laevis and of man. These comparative studies suggest that the alpha-globins of Pleurodeles waltlii and Xenopus laevis may have diverged from a common ancestral gene at the time when mammalian and amphibian lines diverged, and that they then evolved separately. Duplication of the alpha-gene, which is responsible for the polypeptide heterogeneity, appears to have occurred earlier in Pleurodeles waltlii than in Xenopus laevis.
Asunto(s)
Clonación Molecular , Genes , Globinas/genética , Pleurodeles/genética , ARN Mensajero/genética , Salamandridae/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN/análisis , Enzimas de Restricción del ADN , Sustancias Macromoleculares , ARN Mensajero/aislamiento & purificaciónRESUMEN
Neuron-substratum interactions regulating axon growth in the developing central nervous system of the rat have been studied by means of an in vitro bioassay: the tissue section culture. We have previously shown that purified chicken sensory or sympathetic neurons grown on natural substrata consisting of cryostat sections of neonatal rat spinal cord elaborate numerous long neurites [Sagot et al. (1991) Brain Res. 543, 25-35]. Perturbation experiments, in which neuron-substratum interactions are modified by antibodies and peptides, have allowed us to analyse some of the molecular determinants which control neurite outgrowth in this system. Antibodies directed against the beta 1-integrin subunit, one of the neuronal receptors for extracellular matrix molecules, reduced the percentage of growing neurons by about 30% and the length of neurites by about 50%. In contrast, antibodies directed against laminin-1 or fibronectin, two extracellular matrix proteins transiently expressed in various areas of the developing central nervous system, were unable to block neurite outgrowth. Paradoxically, a peptide containing the IKVAV sequence, which mimics an active sequence of the laminin alpha 1 chain responsible for neurite extension, also blocked neurite outgrowth on neonatal spinal cord substrata. These results indicate that integrin receptors containing the beta 1 subunit may play a role in regulating axon growth in the developing nervous system. Among the putative extracellular matrix ligands for these receptors, laminin and fibronectin do not appear as prominent candidates in the neonatal spinal cord. However, our data also suggest that the developing central nervous system may contain neurite outgrowth-promoting proteins carrying the IKVAV sequence, different from laminin-1.