Biomedical subjects

A M Duprat

Publications and source records attributed to A M Duprat.

At least 37 records · Page 2Linked to original sources

PubMed1995

115 kDa protein from Xenopus laevis embryos recognized by antibodies directed against the Xenopus homeoprotein XIHbox 1.

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.

Animals↗

PubMed1995

Regulation of Na+, K+ ATPase activity during meiotic maturation of Pleurodeles waltl oocytes. Role of calcium.

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.

Animals↗

PubMed1995

[Fibroblast growth factors (FGF-2) and delayed involution of the posterior limbs of the slow-worm embryo (Anguis fragilis, L.)].

Administration at doses of 1,800 ng to 6,200 ng of basic fibroblast growth factor (bFGF), around the posterior limb buds of young slow-worm (Anguis fragilis, L.) embryos developing in cultured eggs has, after 4 to 6 days, in several embryos (10 among 15, treated at stage of allantoic bud, from 3 to 9 mm long), delayed the involution of these posterior limbs: their anlagen are larger than the control ones and histological examination shows an increase, between 20% and 30%, of cell density in the mesodermal component of the limb bud. These results bring into light the possibility to control, to some degree, the regressive evolution of the limbs.

Animals↗

PubMed1995

[Fibroblast growth factor (FGF-2) and delayed involution of the posterior limbs of the slow-worm embryo (Anguis fragilis, L.)].

Animals↗

PubMed1995

Expression of L-type Ca2+ channel during early embryogenesis in Xenopus laevis.

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.

Animals↗

PubMed1994

Increased internal Ca2+ mediates neural induction in the amphibian embryo.

The molecular mechanism of neural induction is still unknown and the identity of the natural inducer remains elusive. It has been suggested that both the protein kinase C and cAMP signal transduction pathways may be involved in mediating its action. Here we provide evidence that Ca2+ is implicated in the process of transduction of the neuralizing signal. We find that an increase in intracellular Ca2+ concentration [Ca2+]i occurs during neural induction provoked in vitro by the lectin Con A in Pleurodeles waltl embryo. We demonstrate that specific L-type Ca2+ channel agonists also trigger neural induction. Conversely, noninducing lectins do not raise [Ca2+]i. Ryanodine and caffeine trigger neural induction. An increase in [Ca2+]i was also observed after treatment with the phorbol 12-myristate 13-acetate, which has been reported to be inductive. The [Ca2+]i increase triggered by phorbol ester and Con A was abolished by staurosporine and by L-type Ca2+ channel antagonists. Our findings demonstrate that the [Ca2+]i increase occurs via L-type Ca2+ channels. We suggest an amplification of this increase by a Ca(2+)-induced Ca2+ release mechanism which involves intracellular ryanodine-sensitive stores. We propose that Ca(2+)-dependent processes controlled by protein kinase C are implicated in the regulation of gene expression in response to neural induction.

Alkaloids↗

PubMed1994

In vivo fertilization and development in microgravity using pleurodele ("ZEUS" project).

The objectives of this experiment are to perform natural fertilization and to achieve embryonic development in microgravity. Pleurodeles waltl, an urodele amphibian, is considered by CNES and NASA to be suitable experimental material for achieving in vivo fertilization in space. Previously inseminated females can be embarked in the Frog Environmental Unit (FEU) developed by NASA. Laying of eggs will be provoked by hormonal stimulation in flight and development will be followed. Various technical problems have been resolved in laboratory experiments and during parabolic flights : the time of hormone stimulation after insemination, choice of hormone guaranteeing [correction of guarenteing] 95% success, other factors conditioning [correction of conditionning] the laying, experimental procedures to study developmental kinetics at phenotypic levels, and selection of cellular and molecular markers of development.

Animals↗

PubMed1993

Lineage analysis of early neural plate cells: cells with purely neuronal fate coexist with bipotential neuroglial progenitors.

To investigate the lineage relationships of neurons and astroglial cells early in central nervous system development, we have analyzed the progeny of neural plate cells in an amphibian embryo (Pleurodeles waltl). A fluorescent tracer, lysinated rhodamine-dextran, was iontophoretically injected into individual precursor cells in various areas of the early neural plate. The phenotypes of clonally related cells were identified in the hindbrain and spinal cord by morphological and immunohistochemical criteria 12 days later, at larval stages. We found that the large majority of clones (83%) contained both neurons and astroglial cells, whereas the remainder (17%) were homogeneous and were only composed of neurons. We never observed purely astroglial clones. These results clearly demonstrate the predominance of bipotential progenitors in the neural plate. Interestingly, the progenitors with a restricted neuronal fate were always located along the intermediate axes of the neural plate, while mixed progenitors were found in all areas examined. The analysis of migratory paths has shown that sister cells first migrated together along radial pathways without dispersion along the rostrocaudal axis. From larval stages, some neurons migrated away from the original clonal cohort along dorsoventral and ventrodorsal tangential routes, but only after they had reached the border between the intermediate and marginal zones.

Animals↗

PubMed1993

Experimentally provoked neural induction results in an incomplete expression of neuronal traits.

In the amphibian embryo, the ectoderm becomes a neural structure during gastrulation as a result of an interaction with the dorsal mesoderm. From that time onward, neurectodermal cells have the ability to express in vitro a large variety of mature phenotypes without further interaction with the inducing tissue. The neuralization of the ectoderm can be reproduced in a variety of experimental situations that do not involve the dorsal mesoderm. In this study we have analyzed biochemically the extent to which artificial inductions mimic the natural inducing process. Making use of antibodies specific to different neurotransmitter pathways, we have shown that the repertoire of the phenotypes expressed by experimentally induced neurons is always restricted compared to those obtained after induction in vivo. Only a limited number of generic neuronal characteristics are expressed. These results suggest that the expression of a complete neuronal phenotype normally involves a sequence of inductive events that can be experimentally uncoupled.

Animals↗

PubMed1992

Cells from the early chick optic nerve generate neurons but not oligodendrocytes in vitro.

We have recently described neuronal potentialities in neuroepithelial cells of the embryonic chicken optic nerve (Giess et al., Proc. Natl. Acad. Sci. USA, 87 (1990), 1643-1647). To further investigate the developmental repertoire of optic nerve cells, oligodendroglial development was studied in cultures of optic nerve explanted at various developmental stages. Oligodendrocyte differentiation was analyzed using antibodies directed against galactocerebrosides (Gal-C) and against sulfatides. Optic nerves removed at embryonic days 5 and 6 (E5-E6) never gave rise in culture to differentiated oligodendrocytes, even after 3 weeks in vitro. In contrast, in cultures of optic nerves removed from E7 or older embryos, cells expressing both oligodendrocyte markers were rapidly and invariably observed. Absence of oligodendrocytes before E7 was not due to culture conditions being inadequate to support the differentiation of early precursors along this pathway, since neuroepithelial cells from E2 and E4 trunk neural tube cultivated in the same conditions expressed Gal-C after respectively 16 and 10 days. These results demonstrate that the optic nerve territory is initially devoid of oligodendrocyte potentialities. Whether oligodendrocyte precursors that, around E7, populate the optic nerve are induced by a specific developmental signal occurring at this stage or migrate from outside the optic nerve remains to be determined.

Animals↗

PubMed1992

Hemopexin is synthesized in peripheral nerves but not in central nervous system and accumulates after axotomy.

In adult mammals, injured axons regrow over long distances in peripheral nerves but fail to do so in the central nervous system. Analysis of molecular components of tissue environments that allow axonal regrowth revealed a dramatic increase in the level of hemopexin, a heme-transporting protein, in long-term axotomized peripheral nerve. In contrast, hemopexin did not accumulate in lesioned optic nerve. Sciatic nerve and skeletal muscle, but not brain, were shown to be sites of synthesis of hemopexin. Thus, hemopexin expression, which can no longer be considered to be liver-specific, correlates with tissular permissivity for axonal regeneration.

Amino Acid Sequence↗

PubMed1991

Expression of the guanine nucleotide-binding protein Go correlates with the state of neural competence in the amphibian embryo.

The nucleotide-binding protein Go is a transducing molecule closely associated with neural structures in vertebrates. Because of the potential importance of molecules of this type during the first step of neurogenesis, we have investigated the kinetics of expression of Go in the amphibian (Pleurodeles waltl) embryo, focusing our attention on the stages corresponding to the acquisition of neural competence by presumptive ectoderm and to the process of neural induction. Using affinity-purified IgGs directed against the alpha subunit of Go, Go-like immunoreaction (GoLI) is first detected at the midblastula stage in some animal cap (future ectodermal) cells just before they have attained competence to be neuralized. At the early gastrula stage, GoLI is almost exclusively expressed by neural-competent tissue as a whole, with no obvious difference between the dorsal (prospective neural) and the ventral (prospective epidermal) ectoderm. The expression of GoLI is therefore related to the state of competence of the tissue rather than to its fate. At the early neurula stage, immediately following neural induction, the expression of GoLI persists essentially in that part of ectoderm that has been diverted from epidermal differentiation towards the neural pathway; in the ventral ectoderm, as neural competence is lost GoLI disappears. Furthermore, in the neurectoderm, only approximately 70% of the cells conserve GoLI, demonstrating that immediately following neural induction the population of neurectodermal cells is not homogeneous.

Animals↗

PubMed1991

Removal of N-linked oligosaccharides of presumptive ectoderm impairs neural induction in Pleurodeles waltl.

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.

1-Deoxynojirimycin↗

PubMed1990

Are neuronal precursor cells committed to coexpress different neuroactive substances in early amphibian neurulae?

Considering the initial expression of neurotransmitters and neuropeptides immediately after neural induction in amphibian embryos, we previously pointed out that a neuronal cell population emerges from neural plate (NP) and neural fold (NF) expressing very early specific cholinergic, catecholaminergic, GABAergic and peptidergic traits. The purpose of the present work was to investigate the extent to which the neuroblasts that are present in the neurectoderm immediately after gastrulation are committed to give rise to multiple subsets of neurons containing various combinations of neuroactive transmitters rather than to different subpopulations of neurochemically homogeneous neurons. By means of double immunocytochemical localization with a monoclonal TOH-antibody and polyclonal antibodies against GABA or somatostatin, no coexistence of neurotransmitters and neuropeptide was ever found in neuronal subpopulations arising in vitro from NP or NF. The early emergence, under the same conditions, of distinct neuronal subpopulations as a consequence of neural induction strongly suggests that, at the gastrula stage, the neural precursor population most probably does not constitute a homogeneous set of cells.

Animals↗

PubMed1990

Modulation of neural commitment by changes in target cell contacts in Pleurodeles waltl.

In amphibian development, neural structures arise from the presumptive ectoderm at the gastrula stage by an inductive interaction with the chordamesoderm. It has been previously reported that early gastrula presumptive ectoderm can be neuralized when it is dissociated into single cells. A similar result is reported here with regard to Pleurodeles waltl presumptive ectoderm. Using this experimental model system we demonstrate: first, that neuronal and glial lineages can be specified from the presumptive ectoderm without any intervention of the natural inducing tissue; and second, that whereas rupture of cell-cell contacts evoked neural induction, dissociation immediately followed by reaggregation reduces the neuralizing response, pointing toward an active role played by cell-cell contacts of presumptive ectodermal cells in the modulation of neural commitment.

Animals↗

PubMed1990

Extracellular matrix: an immunological and biochemical (CAT and TOH activity) survey of in vitro differentiation of isolated amphibian neuroblasts.

After neural induction certain cells in the neuroepithelium immediately acquire the property to express certain neural phenotypes (Duprat et al., 1984, 1987). However, the activity of almost all the specific enzymes involved in the biosynthesis of neurotransmitters is considerably higher when neurectodermal cells are cultured with chordamesodermal cells than when they are cultured alone. The stimulating effects of chordamesoderm do not appear to be due to diffusible factors (Duprat et al., 1985b). The present study was designed to investigate the role of extracellular matrix components in neuronal cell differentiation. We showed that the extracellular matrix cannot replace chordamesoderm in stimulating the biochemical differentiation of neuroblasts, although fibronectin and especially laminin stimulate morphological differentiation. We suggest that interaction between neuronal and non-neuronal cells plays an important part in functional biochemical differentiation, whereas the molecules of extracellular matrix are important for morphological differentiation.

Animals↗

PubMed1990

Neuronal potentialities of cells in the optic nerve of the chicken embryo are revealed in culture.

Neuronal potentialities in neuroepithelial cells of the chicken embryonic optic nerve were studied in culture by using neurofilament antibodies as neuronal markers. Embryonic day-4 and -5 (E4 and E5) optic stalks were explanted in vitro. Within the first few days of culture, numerous morphologically identifiable neurons extending long neurites developed. These neurons and their processes were specifically labeled with neurofilament antibodies. Similar results were obtained by explanting only the medial portion of E7 optic stalks away from possibly contaminating cerebral or retinal tissue. To determine whether neuronal potentialities persisted at later embryonic stages, cultures of dissociated optic stalks were established at E11, E15, and E18. Neurons labeled with the various neurofilament antibodies appeared in all cultures of E11 and E15 optic stalks. However, typical neurons could not be recognized in cultures of E18 optic nerves. These results indicate that cells with neuronal potentialities are present in the embryonic optic nerve from early stages of development and persist until at least E15. Since the adult optic nerve is devoid of nerve cell bodies, our observations are consistent with the hypothesis that axons of retinal ganglion cells, which course through the optic stalk, repress neuronal potentialities within a subpopulation of precursor cells during normal development.

Animals↗

PubMed1990

From presumptive ectoderm to neural cells in an amphibian.

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.

Amphibians↗