Design of specific hardware to obtain embryos and maintain adult urodele amphibians aboard a space station.

The study of the influence of weightlessness on fertilization and embryonic development of a vertebrate is of importance in the understanding of basic embryogenesis and in the preparation of the future exploration of space. Accordingly, specific hardware was designed to perform experiments on board the MIR space station with an amphibian vertebrate model, taking into account the biological requirements and the multiple constraints of a long-term mission. This paper describes the biological uses and presents the technological specifications of the device developed under CNES management. The hardware was adapted to and is compatible with biological requirements as confirmed by three experiments performed in space on board the orbital MIR station.

Contrasting influences of the organizer and induction concepts on the scientific activity of French embryologists.

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.

Pleurodeles waltl, amphibian, Urodele, is a suitable biological model for embryological and physiological space experiments on a vertebrate.

Pleurodeles waltl (amphibian, Urodele) is an appropriate biological model for space experiments on a vertebrate. One reason for interest in this animal concerns the study of the effects of absence of gravity on embryonic development. First, after mating (on Earth) the females retain live, functional sperm in their cloacum for up to 5 months, allowing normal in vivo fertilisation after hormonal stimulation. Second, their development is slow, which allows analyses of all the key stages of ontogenesis from the oocyte to swimming tailbud embryos or larvae. We have performed detailed studies and analyses of the effects of weightlessness on amphibian Pleurodeles embryos, fertilised and allowed to develop until the swimming larvae stage. These experiments were performed in space during three missions on the MIR-station: FERTILE I, FERTILE II and NEUROGENESIS respectively in 1996, 1998 and 1999. We show that in microgravity abnormalities appeared at specific stages of development compared to 1g-centrifuge control embryos and 1g-ground control embryos. In this report we describe abnormalities occurring in the central nervous system. These modifications occur during the neurulation process (delay in the closure of the neural tube and failure of closure of this tube in the cephalic area) and at the early tailbud stage (microcephaly observed in 40% of the microgravity-embryos). However, if acephalic and microcephalic embryos are not taken into account, these abnormalities did not disturb further morphological, biochemical and functional development and the embryos were able to regulate and a majority of normal hatching and swimming larvae were obtained in weightlessness with a developmental time-course equivalent to that of 1g-centrifuge control embryos (on the MIR station) and 1g-ground control embryos.

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.

Activation of Pax6 depends on somitogenesis in the chick embryo cervical spinal cord.

Pax6 is a paired-type homeobox gene expressed in discrete regions of the central nervous system. In the spinal cord of 7- to 10-somite-stage chicken embryos, Pax6 is not detected within the caudal neural plate, but is progressively upregulated in the neuroepithelium neighbouring each newly formed somite. In the present study, we accumulate data suggesting that this initial activation of Pax6 is controlled via the paraxial mesoderm in correlation with somitogenesis. First, we observed that high levels of Pax6 expression occur independently of the presence of SHH-expressing cells when neural plates are maintained in culture in the presence of paraxial mesoderm. Second, grafting a somite caudally under a neural plate that has not yet expressed the gene induces a premature activation of Pax6. Furthermore, after the graft of a somite, a period of incubation corresponding to the individualization of a new somite in the host embryo produces an appreciable activation of Pax6. Conversely, Pax6 expression is delayed under conditions where somitogenesis is retarded, i.e., when the rostral part of the presomitic mesoderm is replaced by the same tissue isolated more caudally. Finally, Pax6 transcripts disappear from the neural tube when a somite is replaced by presomitic mesoderm, suggesting that the somite is also involved in the maintenance of Pax6 expression in the developing spinal cord. All together these observations lead to the proposal that Pax6 activation is triggered by the paraxial mesoderm in phase with somitogenesis in the cervical spinal cord.

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.

A novel Xenopus mix-like gene milk involved in the control of the endomesodermal fates.

Here we describe a novel Xenopus homeobox gene, milk, related by sequence homology and expression pattern to the vegetally expressed Mix.1. As is the case with Mix.1, milk is an immediate early response gene to the mesoderm inducer activin. milk is expressed at the early gastrula stage in the vegetal cells, fated to form endoderm, and in the marginal zone fated to form mesoderm. During gastrulation, expression of milk becomes progressively reduced in the involuting mesodermal cells but is retained in the endoderm, suggesting that it may play a key role in the definition of the endo-mesodermal boundary in the embryo. Overexpression of milk in the marginal zone blocks mesodermal cell involution, represses the expression of several mesodermal genes such as Xbra, goosecoid, Xvent-1 or Xpo and increases the expression of the endodermal gene, endodermin. In the dorsal marginal zone, overexpression of milk leads to a severe late phenotype including the absence of axial structures. Ectopic expression of milk in the animal hemisphere or in ectodermal explants induces a strong expression of endodermin. Taken together, we propose that milk plays a role in the correct patterning of the embryo by repressing mesoderm formation and promoting endoderm identity.

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.

L-type calcium channel activation controls the in vivo transduction of the neuralizing signal in the amphibian embryos.

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.

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.

What mechanisms drive neural induction and neural determination in urodeles?

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.

Antibodies directed against the beta 1-integrin subunit and peptides containing the IKVAV sequence of laminin perturb neurite outgrowth of peripheral neurons on immature spinal cord substrata.

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.

Amphibian tail regeneration in space: effect on the pigmentation of the blastema.

In Urodele amphibians, the tail regenerates after section. This regeneration, including tissues as different as bone (vertebrae), muscle, epidermis and central nervous system (spinal cord), was studied in adult Pleurodeles sent aboard the russian satellite Bion 10 and compared with tail regeneration in synchronous controls. Spinal cord, muscle and cartilage regeneration occurred in space animals as in synchronous controls. One of the most important differences between the two groups was the pigmentation of the blastemas: it was shown in laboratory, to be not due to a difference in light intensity.

The pleurodele, an animal model for space biology studies.

Pleurodeles waltl, an Urodele amphibian is proposed as a model for space biology studies. Our laboratory is developing three types of experiments in space using this animal: 1) in vivo fertilization and development ("FERTILE" project); 2) influence of microgravity and space radiation on the organization and preservation of specialized structures in the neurons and muscle cells (in vitro; "CELIMENE" PROJECT); 3) influence of microgravity on tissue regeneration (muscle, bone, epidermis and spinal cord).

A novel TGF-beta-like gene, fugacin, specifically expressed in the Spemann organizer of Xenopus.

Using a differential screening strategy, we have cloned a novel Xenopus gene, fugacin, related to the transforming growth factor beta superfamily. Transcripts were detected primarily in the dorsal marginal zone of late blastula. Thereafter, they became highly localized to the blastopore lip of early gastrula and were not observed at later stages. This gene, which is most homologous to the mouse gene nodal, displays a new pattern of cysteine residues. These findings highlight the potential role of these growth factors during early vertebrate development.

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.

The homeobox-containing gene XANF-1 may control development of the Spemann organizer.

At the beginning of gastrulation the homeobox-containing gene, XANF-1, is expressed at a low level throughout the animal hemisphere of Xenopus laevis embryos, with a local maximum of expression in the region of the dorsal blastopore lip. By the end of gastrulation expression ceases everywhere except in the most anterior part of the neurectoderm. We have investigated the functions of this gene by microinjecting XANF-1 mRNA in the blastomeres of the 32-cell stage embryo and have observed the following effects. First, microinjections of the mRNA in the animal blastomeres and the blastomeres of the marginal zone elicited massive migration of cells to the interior of the embryo at the early gastrula stage. Second, overexpression of XANF-1 in the ventral marginal zone (VMZ) resulted in the appearance of an additional centre of gastrulation movements and the formation of a secondary axis. In addition we showed that synthetic XANF-1 mRNA was able to cause dorsal-type differentiation in VMZ explants extirpated from the microinjected embryos at the beginning of gastrulation. These results suggest that XANF-1 may control the main functions of cells of the Spemann organizer.

Notochord and floor plate stimulate oligodendrocyte differentiation in cultures of the chick dorsal neural tube.

The regionalization of oligodendrocyte potentialities and the cellular interactions leading to the expression of the oligodendrocyte phenotype have been analyzed in the embryonic chick spinal cord. Dorsal and ventral regions of the spinal cord of 4-day-old embryos (E4) were cultivated separately. Oligodendrocyte differentiation was monitored at various times after explantation, using specific oligodendrocyte markers. After 2 weeks, several hundreds of differentiated oligodendrocytes were invariably observed in ventral cultures whereas significant numbers of oligodendrocytes failed to develop in dorsal spinal cord cultures. However, the E7 dorsal spinal cord was found to produce large numbers of oligodendrocytes, indicating that the ventral restriction of oligodendrocyte potentialities is transient. To test whether ventrally derived signals might influence oligodendrocyte differentiation, E4 dorsal spinal cord microexplants were cocultivated with notochord segments or with floor plate tissue. Numerous oligodendrocytes were found in dorsal explants associated with either tissue, notochord or floor plate, but not in dorsal explants cultivated alone, indicating that cells competent to be induced along the oligodendrocyte phenotype exist in the dorsal spinal cord. These results show that oligodendrocyte differentiation potentialities are initially restricted to the ventral spinal cord and suggest that ventrally derived signals from notochord and floor plate influence oligodendrocyte differentiation in the embryonic spinal cord.

[Fibroblast growth factors (FGF-2) and delayed involution of the posterior limbs of the slow-worm embryo (Anguis fragilis, L.)]. / Facteur de croissance des fibroblastes (FGF-2) et retard d'involution des membres postérieurs de l'embryon d'orvet (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.

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.