Anne-Marie Duprat

Induction of oligodendrocyte progenitors in the trunk neural tube by ventralizing signals: effects of notochord and floor plate grafts, and of sonic hedgehog.

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.

Noggin upregulates Fos expression by a calcium‐mediated pathway in amphibian embryos

In amphibia, noggin, one of the neural inducers expressed in the Spemann organizer, acts by neutralizing the effects of bone morphogenetic protein‐4 (BMP‐4). It is shown that noggin is able to activate L‐type calcium channels. The fos proto‐oncogene is known to be induced within minutes by calcium signaling. Here it is reported that in animal cap explants of the amphibian Pleurodeles waltl, noggin can induce upregulation of a FOS‐related protein in a calcium‐dependent manner. Specific inhibition of the dihydropyridine sensitive L‐type calcium channels blocked both calcium influx and the induction of FOS‐related protein. When animal cap explants were treated with caffeine in order to release calcium from an internal store or with a specific agonist of the L‐type calcium channels, FOS‐related protein could be detected in cell nuclei by 5 or 15 min, respectively. Additionally, the calcium calmodulin kinase inhibitor, KN62, could block the upregulation of FOS‐related protein induced by agents that increased intracellular calcium ([Ca2+]i). The present results suggest that transcription factors from the FOS family are downstream targets of neural inducer noggin.

Differentiation in microgravity of neural and muscle cells of a vertebrate (amphibian)

The CELIMENE space experiment (CELulles en Impesanteur: Muscle Et Neurone Embryonnaires) was devoted to the study of the influence of gravity on the differentiation, the organisation and the maintenance of the highly specialised nervous system and muscular system. CELIMENE was carried out during the first flight of the IBIS hardware (Instrument for BIology in Space) with the fully automatic space mission PHOTON 10 in February 1995. Using the amphibian Pleurodeles waltl as a vertebrate model, in vitro experiments involved immunocytochemical detection of glial-, neuronal- and muscle-specific markers, and neurotransmitters in cells developed under conditions of microgravity compared with 1g controls, on-board and on the ground. We observed that the altered gravity did not disturb cell morphogenesis or differentiation.

Extracellular matrix and neural determination during amphibian

Abstract Previous studies have shown the presence of a network of extracellular matrix on the inner surface of the ectoderm of the early gastrulae Pleurodeles waltlii . Similar dense networks have been described by other authors in another Urodele species and in Anurans. It now seems well established that this extracellular material (ECM) has important functions in the mechanisms of morphogenetic cell movements during gastrulation. The results reported here provide evidence that this ECM is not implicated in the transmission of the neuralizing signal at the gastrula stage, and therefore is not involved in the process of neural determination.

Does gravity influence the early stages of the development of the nervous system in an amphibian

As a result of previous studies using hypergravity (centrifuge) or virtual microgravity (clinostat), it was proposed that gravity was involved in embryonic development, i.e., in the establishment of the embryonic polarities and the body plan pattern which subsequently direct morphogenesis and organogenesis of the central nervous system and of sensory organs. Recent experiments were performed in space using sounding rockets and orbiting space-modules to ascertain whether gravity is indeed required for embryogenesis in Invertebrates and Vertebrates. Eggs fertilised in vivo or in vitro in microgravity showed some abnormalities during embryonic development but were able to regulate and produce nearly normal larvae.

An Elevation of Internal Calcium Occurring Via L-Type Channels Mediates Neural Induction in the Amphibian Embryo

In amphibians, neural induction takes place during gastrulation, as a consequence of an interaction between the chordamesoderm (inductive tissue) and the ectoderm (target tissue). The mechanism of neural induction has been the subject of many investigations more than 60 years (Saxen, 1989; Duprat et al. 1990; Westenbroeck et al. 1990). Although the natural inducer still remains unidentified, numerous, apparently unrelated, substances have been found to act as inducers (Tiedemann and Born, 1978; Saxen, 1989). Recently an endogeneous soluble protein, noggin, has been shown to have neural inducing activity in Xenopus (Lamb et al. 1993). It has been also suggested that the inhibition of the signal transduced by the activin type II -receptor leads to neuralization (Hemmati-Brivanlou and Melton, 1994). Follistatin, that blocks activin activity by direct binding of activin protein, can induce neural tissue in vivo (Hemmati-Brivanlou et al. 1994). Furthermore, it has also been demonstrated that the inducing signal from the chordamesoderm is recognized at the level of the plasma membrane of the target tissue (Tiedemann and Born, 1978; Takata et al. 1981; Gualandris et al. 1985). Therefore it has become important to define the mechanism of transduction of the neuralizing signal. Using Xenopus embryos, it has been suggested that activation of protein kinase C (PKC) by phorbol esters, leads to neural induction in a limited part of ectodermal expiants (Davids et al. 1987; Otte et al. 1988, 1989). An increase in cAMP dependent protein kinase (PKA) activity during neural induction, has also been observed (Otte et al. 1989), suggesting a cross-talk between PKA and PKC., during this process. These data suggest that PKC pathway is activated by neural induction to initiate neural-specific gene expression. Additional support for a role of a PKC pathway in neural induction is provided by the phorbol ester (TPA) induction of the neural-specific src + mRNA in dorsal competent ectoderm of Xenopus embryo (Collett and Steele, 1992, 1993).

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.

Stimulating effect of the divalent cation ionophore A 23187 on in vitro neuroblast differentiation; comparative studies with myoblasts.

The effects of the divalent cation ionophore A 23187 and papaverine on the in vitro differentiation of isolated embryonic cells from young neurulae (Pleurodeles waltlii) were observed. These experiments suggest that an intracellular increase or decrease of the divalent cation concentration (Ca++) stimulates or disturbs the morphological differentiation of already determined embryonic cells from neurulae but does not change their developmental pathway.

Structural alteration of the target plasma membrane affects reception but not expression of the neural inductive signal

We have previously reported that incubation of presumptive neuroectoderm in a solution of lectin (soybean agglutinin or garden pea agglutinin at 50 micrograms ml-1, 30 min) prior to its association with blastoporal lip inhibits neural induction (Duprat et al., 1982). We have also shown that incubation of presumptive ectoderm immediately after its association with blastoporal lip does not prevent neural induction. The same pattern of fluorescence on the ectodermal surface was observed when incubation with lectin was carried out before or after association of ectoderm with blastoporal lip. Although a particular molecular organization of the plasma membrane of the target ectoderm appears to be essential for reception or initiation of neural induction, the subsequent transmission and expression of this neural information does not appear to be affected by structural modifications of the target membrane. Similar studies performed using the inducer Con A indicated that the experimentally induced signal differed from the natural one.

Alterations in lateral lipid mobility in the plasma membrane of urodelean ectodermal cells during gastrulation

The mobility characteristics of lipids were studied in the plasmalemma of dissociated presumptive ectodermal cells from embryos of Pleurodeles Waltl at different stages of development, from early blastula to early neurula, using a Fluorescence Recovery After Photobleaching technique (FRAP), after incorporation of the lipophilic fluorescent probe 5N-(hexadecanoyl)-aminofluoresceine (HEDAF) into the cell plasma membrane. At all stages of development, fluorescence recovery was found to extrapolate to 100%, which suggested that the lipid phase in these plasma membranes can be regarded as dynamically homogeneous (no immobilized fraction). It appears as a continuum over a wide cell surface area, in which lipids are free to move laterally. The lateral diffusion coefficient of the probe, obtained from statistical analysis of the fluorescence recovery data, was found to decrease significantly from blastula to gastrula, slightly increasing at the neurula stage. These changes in the dynamic properties of the lipid probe HEDAF during gastrulation suggest that the lipid phase of the plasma membrane of these ectodermal cells undergo structural changes. The results lend support to the idea that the plasma membrane of these cells is actively involved in the morphogenetic movements which characterize the development of the embryo.