Nr-CAM and TAG-1 are expressed in distinct populations of developing precerebellar and cerebellar neurons.

Nr-CAM and TAG-1 interact at the floor-plate during the formation of spinal cord commissural projections [Stoeckli, E.T., Landmesser, L.T., Sci. 274 (1995) 1123-1133; Fitzli, D., Stoeckli, E.T., Kunz, S., Siribour, K., Rader, C., Kunz, B., Kozlov, S.V., Buchstaller, A., Lane, R.P., Suter, D.M., Dreyer, W.J., Sonderegger, P., J. Cell. Biol. 149 (2000) 951-968]. We report here the spatio-temporal patterns of expression of these two adhesion molecules during the development of the lower brainstem (medulla and pons) and cerebellum. Nr-CAM and Tag-1 label distinct populations of precerebellar neurons at key steps of their development. Nr-CAM expression starts at E11.5-E12 in the floor-plate, that constitutes an intermediate target during axon outgrowth and nuclear migration of precerebellar neurons. At E13-E14, it is expressed in both floor-plate and inferior olivary nuclei (ION) neurons before being strictly restricted to ION neurons from E15 onwards. Furthermore Nr-CAM, which is widely expressed in the cerebellum during embryonic development, becomes strictly confined to Purkinje and Golgi cells in postnatal cerebellum, suggesting a possible role of Nr-CAM for the maturation or stabilization of the synaptic contacts, in particular between climbing fibers and Purkinje cells. On the other hand, Tag-1 is expressed by migrating neurons that will form the lateral reticular and basilar pontine nuclei. These results emphasize the possibility that TAG-1/Nr-CAM interactions are also involved in the development of the cerebellar system (precerebellar and cerebellar neurons). However, the pattern of cerebellar expression of TAG-1--early migrating Purkinje cells up to E14 and external granular cells--prevents the implication of this adhesion molecule in the organization of extracerebellar projections.

Netrin-1 acts as a survival factor via its receptors UNC5H and DCC.

The membrane receptors DCC and UNC5H have been shown to be crucial for axon guidance and neuronal migration by acting as receptors for netrin-1. DCC has also been proposed as a dependence receptor inducing apoptosis in cells that are beyond netrin-1 availability. Here we show that the netrin-1 receptors UNC5H (UNC5H1, UNC5H2, UNC5H3) also act as dependence receptors. UNC5H receptors induce apoptosis, but this effect is blocked in the presence of netrin-1. Moreover, we demonstrate that UNC5H receptors are cleaved in vitro by caspase in their intracellular domains. This cleavage may lead to the exposure of a fragment encompassing a death domain required for cell death induction in vivo. Finally, we present evidence that during development of the nervous system, the presence of netrin-1 is crucial to maintain survival of UNC5H- and DCC-expressing neurons, especially in the ventricular zone of the brainstem. Altogether, these results argue for a role of netrin-1 during the development of the nervous system, not only as a guidance cue but as a survival factor via its receptors DCC and UNC5H.

[Formation of the boundary between the midbrain and the hindbrain: involvement of Otx2 and Gbx2 genes]. / Formation de la frontière entre cerveaux moyen et postérieur: implication des gènes Otx2 et Gbx2.

We have studied the neuromeric organisation of the mesencephalic-metencephalic (mes-met) territory of the avian neural tube using chick/quail transplantation experiments and analysing the expression of various regulatory genes in chimeric and normal embryos. Homotopic grafts demonstrate the presence of an interneuromeric boundary separating the mesencephalic and cerebellar territories (the mes-met or midbrain/hindbrain boundary). This boundary is characterised from HH10 onwards by the confrontation of the Otx2-Wnt1 and Gbx2-Fgf8 expressing domains, while En2 and Pax2 genes are expressed at both sides of the mes-met boundary. The evolution of the position of the Otx2/Gbx2 boundary with respect to the vesicles and constriction observed within the mes-met domain between stages HH10 and HH20, allows us to redefine the fate map of this region and to propose a new nomenclature for HH10. Transplantation between the prosencephalic neuroepithelium and the mes-met domain shows the possibility of inducing a mes-met phenotype within the two caudal-most prosomeres, preceded by its characteristic genetic cascade. The induction selectively takes place along the boundary between the graft (Otx2 positive) and the host cerebellar territory (expressing high levels of Gbx2); this includes the induction inside the graft of a new Otx2/Gbx2 boundary. Conversely, no induction is ever observed when the graft is confronted to the host Otx2 expressing domain. Although Fgf8 may be involved in the inductive events, our data strongly suggest that confrontation between Otx2 and Gbx2 is essential as an organiser of the mes-met domain.

Floor plate and netrin-1 are involved in the migration and survival of inferior olivary neurons.

During their circumferential migration, the nuclei of inferior olivary neurons translocate within their axons until they reach the floor plate where they stop, although their axons have already crossed the midline to project to the contralateral cerebellum. Signals released by the floor plate, including netrin-1, have been implicated in promoting axonal growth and chemoattraction during axonal pathfinding in different midline crossing systems. In the present study, we report experiments that strongly suggest that the floor plate could also be involved in the migration of inferior olivary neurons. First, we show that the pattern of expression of netrin receptors DCC (for deleted in colorectal cancer), neogenin (a DCC-related protein), and members of the Unc5 family in wild-type mice is consistent with a possible role of netrins in directing the migration of precerebellar neurons from the rhombic lips. Second, we have studied mice deficient in netrin-1 production. In these mice, the number of inferior olivary neurons is remarkably decreased. Some of them are located ectopically along the migration stream, whereas the others are located medioventrally and form an atrophic inferior olivary complex: most subnuclei are missing. However, axons of the remaining olivary cell bodies located in the vicinity of the floor plate still succeed in entering their target, the cerebellum, but they establish an ipsilateral projection instead of the normal contralateral projection. In vitro experiments involving ablations of the midline show a fusion of the two olivary masses normally located on either side of the ventral midline, suggesting that the floor plate may function as a specific stop signal for inferior olivary neurons. These results establish a requirement for netrin-1 in the migration of inferior olivary neurons and suggest that it may function as a specific guidance cue for the initial steps of the migration from the rhombic lips and then later in the development of the normal crossed projection of the inferior olivary neurons. They also establish a requirement for netrin-1, either directly or indirectly, for the survival of inferior olivary neurons.

The embryonic cerebellum contains topographic cues that guide developing inferior olivary axons.

The formation of the olivocerebellar projection is supposed to be regulated by positional information shared between pre- and postsynaptic neurons. However, experimental evidence to support this hypothesis is missing. In the chick, caudal neurons in the inferior olive project to the anterior cerebellum and rostral ones to the posterior cerebellum. We here report in vitro experiments that strongly support the existence of anteroposterior polarity cues in the embryonic cerebellum. We developed an in vitro system that was easily accessible to experimental manipulations. Large hindbrain explants of E7.5-E8 chick embryos, containing the cerebellum and its attached brainstem, were plated and studied using axonal tracing methods. In these cultures, we have shown that the normal anteroposterior topography of the olivocerebellar projection was acquired, even when the cerebellar lamella was detached from the brainstem and placed again in its original position. We also found that, following various experimental rotations of the anteroposterior axis of the cerebellum, the rostromedian olivary neurons still project to the posterior vermis and the caudolateral neurons to the anterior vermis, that now have inverted locations. Thus, the rotation of the target region results in the rotation of the projection. In addition, we have shown that the formation of the projection map could be due to the inability of rostromedian inferior olivary axons to grow in the anterior cerebellum. All these experiments strongly indicate that olivocerebellar fibers recognize within their target region polarity cues that organize their anteroposterior topography, and we suggest that Purkinje cells might carry these cues.

The caudal limit of Otx2 gene expression as a marker of the midbrain/hindbrain boundary: a study using in situ hybridisation and chick/quail homotopic grafts.

Segmentation of the neural tube has been clearly shown in the forebrain and caudal hindbrain but has never been demonstrated within the midbrain/hindbrain domain. Since the homeobox-containing gene Otx2 has a caudal limit of expression in this region, we examined, mainly in chick embryos, the possibility that this limit could represent an interneuromeric boundary separating either two cerebellar domains or the mesencephalic and cerebellar primordia. In situ hybridisation with chick or mouse Otx2 probes showed the existence of a transient Otx2-negative area in the caudal mesencephalic vesicle, between stages HH10 and HH17/18 in chick, and at embryonic day 9.5 in mice. The first post-mitotic neurons of the mesencephalon sensu stricto, as labelled with an anti-beta-tubulin antibody, overlay the Otx2-positive neuroepithelium with a perfect match of the caudal limits of these two markers at all embryonic stages analysed (until stage HH20). Chick/quail homotopic grafts of various portions of the midbrain/hindbrain domain have shown that the progeny of the cells located in the caudal mesencephalic vesicle at stage HH10 are found within the rhombomere 1 as early as stage HH14. Furthermore, our results indicate that the cells forming the HH20 constriction (coinciding with the caudal Otx2 limit) are the progeny of those located at the caudal Otx2 limit at stage HH10 (within the mesencephalic vesicle). As a result, the Otx2-positive portion of the HH10 mesencephalic vesicle gives rise to the HH20 mesencephalon, while the Otx2-negative portion gives rise to the HH20 rostral rhombomere 1. Long-survival analysis allowing the recognition of the various grisea of the chimeric brains strongly supports the view that, as early as stage HH10, the caudal limit of Otx2 expression separates mesencephalic from isthmo/cerebellar territories. Finally, this study revealed unexpected rostrocaudal morphogenetic movements taking place between stages HH10 and HH16 in the mediodorsal part of the caudal Otx2-positive domain.

Further observations on the susceptibility of diencephalic prosomeres to En-2 induction and on the resulting histogenetic capabilities.

It has been previously shown by chick/quail heterotopic grafts that En-2 expression and a mesencephalic phenotype can be induced within the avian primordial prosencephalic vesicle, although the induction appeared restricted to the caudal forebrain. The present experiments were aimed at further analyzing the competence of the prosencephalic neuroepithelium. Different types of grafts were performed between chick and quail embryos: (i) caudal forebrain grafts positioned in the midbrain/hindbrain junction (the En-2-positive domain); (ii) En-2-positive grafts integrated at different levels of the forebrain. In both cases, the grafts were transplanted either with a normal orientation or after inversion of their rostro-caudal axis. The chimeric embryos were analyzed at stages HH19-24 for expression of En-2 and Pax-6 homeobox-containing genes, normally expressed in the meso-isthmo-cerebellar and prosencephalic domains, respectively. A cytoarchitectonic analysis of grafted and surrounding host tissue was also performed at later developmental stages in chimeric embryos with caudal forebrain grafts. Our results show that the caudal diencephalon, including the prospective territories for prosomeres 1 and 2, is competent to express En-2 when in close contact to the En-2 polarizing region, whereas the more rostral neuroepithelium, including the prospective territories for the third prosomere and telencephalon, does not change its fate under similar conditions. The ectopic-induced neuroepithelium can develop mesencephalon, but also isthmus and cerebellum according to its site of integration rostrally or caudally to the mesencephalic/isthmo-cerebellar boundary. Our data also show that within the competent diencephalon, the induced En-2 expression can be arrested at the P1/P2 interneuromeric boundary. This arrest appears to be directionally oriented as it only takes place when the induction is produced within prosomere 1 but not when it comes from prosomere 2. These data can be considered as resulting from either a possible oriented permissiveness of cells which form the boundary separating prosomeres 1 and 2, or of a different permissiveness of the cells composing these two caudal prosomeres.

Transforming growth factor-beta 3, glial cell line-derived neurotrophic factor, and fibroblast growth factor-2, act in different manners to promote motoneuron survival in vitro.

Developing chick motoneurons depend on as yet unidentified factors from the periphery and the central nervous system for their survival. Using cultures of purified embryonic motoneurons, we show that basic fibroblast growth factor (FGF-2) or transforming growth factor-beta 3 (TGF beta 3) each have only low survival-promoting activity when tested alone, but act synergistically to keep motoneurons alive for at least 3 days. Glial cell line-derived neurotrophic factor (GDNF), another member of the TGF beta family, was itself sufficient to maintain a population of motoneurons. However, its effect was not significantly increased by the addition of FGF-2. These results suggest that FGF-2, TGF beta 3, and GDNF, which are all present in the environment of developing motoneurons, may act different mechanisms as physiological survival factors for this population of central neurons.

Survival of newly postmitotic motoneurons is transiently independent of exogenous trophic support.

We compared the survival requirements of early- and late-born motoneurons from E5 chicken spinal cord. Density gradient centrifugation followed by immunopanning using SC1 antibody allowed us to purify two size classes of motoneuron. Large motoneurons retained by 6.8% metrizamide were shown by BrdU labeling in ovo to be born on average 1.5 d earlier than the small motoneurons recovered from the metrizamide pellet. Large motoneurons were both biochemically and functionally more mature: they expressed higher levels of choline acetyltransferase and low-affinity neurotrophin receptor, and had an acute requirement for trophic support from muscle-derived factors. After 24 hr in culture in basal medium, all early-born motoneurons died, whereas 60% of late-born motoneurons survived. Small motoneurons can develop into large motoneurons in ovo, suggesting that they represent a general transitional stage in motoneuron development. Our results suggest that a defined period elapses between birth of a motoneuron and its acquisition of trophic dependence, possibly corresponding to the time required for target innervation. This property may have important consequences for the timing and regulation of developmental motoneuron death.

Neurotrophic activity of a homeobox peptide.

Homeoproteins are well known for their role in defining the shape of organs during early development. The late expression of some homeogenes in the nervous system suggests that they might have other, additional functions, possibly in neurite growth and target recognition. The 60 amino acid-long peptide corresponding to the homeobox of Antennapedia (pAntp) translocates through the membrane of neurons in culture and reaches their nuclei. This process is followed by an enhanced morphological differentiation of the neurons. Internalization by neurons is four-fold that observed with fibroplasts. This difference is abolished upon treatment with Endo-N which specifically cleaves alpha,2-8 bonds in polysialic acid. To understand the mode of action of the peptide, we constructed three mutants modified in their capacity to specifically bind promoters and/or to translocate through the cell membrane. The biological properties of the mutants demonstrate that the neurotrophic action of pAntp requires its internalization and integrity of its specific DNA-binding capacity.

Neurotrophic activity of the Antennapedia homeodomain depends on its specific DNA-binding properties.

In previous reports we have demonstrated that the 60-aa peptide corresponding to the homeodomain of the Drosophila protein Antennapedia (pAntp) translocates through the membrane of neurons in culture, accumulates in neuronal nuclei, and promotes neurite growth. To analyze the importance of specific pAntp DNA-binding properties in this phenomenon we have constructed three mutant versions of pAntp that differ in their ability to translocate through the membrane and to bind specifically the cognate sequence for homeodomains present in the promoter of HoxA5. We demonstrate that removing two hydrophobic residues of the third helix inhibits pAntp internalization and suppresses its neurotrophic activity. We also show that pAntp neurotrophic activity is lost when mutations are introduced in positions preserving its penetration and nuclear accumulation but abolishing its capacity to bind specifically the cognate DNA-binding motif for homeoproteins. Our results strongly suggest that pAntp neurotrophicity requires both its internalization and its specific binding to homeobox cognate sequences. We propose that homeoproteins might regulate important events in the morphological differentiation of the postmitotic neuron.

Motoneuron survival factors: biological roles and therapeutic potential.

The existence of factors that promote motoneuron survival in the spinal cord at critical stages of development was first deduced 50 yr ago. The large amount of work that has been put into characterizing such factors reflects both their biological importance and the hope that such molecules may be used therapeutically to slow motoneuron death in pathologies such as the spinal muscular atrophies and amyotrophic lateral sclerosis. Since 1990, several factors have been shown to have in vitro and/or in vivo activities that suggest they play a role in regulating motoneuron survival. Their physiological functions during motoneuron development are probably different and complementary. Several of them seem reasonable candidates for preclinical development, but many crucial experiments remain to be done.

Neurotrophic activity of an homeobox peptide.

The sixty aminoacid-long peptide corresponding to the homeobox of Antennapedia (pAntp) translocates through the membrane of neurons in culture and reaches their nuclei. This process is followed by an enhanced morphological differentiation of the neurons. Internalization by neurons is 4-fold that observed with fibroblasts. This difference is abolished upon treatment with Endo-N which specifically cleaves alpha, 2-8 bounds in polysialic acid (PSA). To understand the mode of action of the peptide, the authors constructed three mutants modified in their capacity to specifically bind promoters and/or to translocate through the cell membrane. The biological properties of the mutants demonstrate that the neurotrophic action of pAntp requires its internalization and the integrity of its specific DNA-binding capacity.

[Growth and survival factors of spinal motoneurons]. / Facteurs de croissance et de survie des motoneurones spinaux.

The development of motoneurons in the spinal cord is strongly dependent on their interactions with their target tissue, skeletal muscle, and with other cells of the central nervous system. The molecular nature of these interactions has remained obscure for many years. However, over the last few years, known growth factors have been shown to have biological activity on the survival of motoneurons, at least in culture. The factors that have been studied are members of the FGF family (fibroblast growth factors), the TGF-beta family (transforming growth factor-beta), CNTF (ciliary neurotrophic factor) and CDF-LIF (cholinergic development factor-leukaemia inhibitory factor). There are also strong reasons to suppose that at least one member of the neurotrophin family (the family that contains Nerve Growth Factor) is involved in motoneuron development. A more detailed analysis of the biological role of each of these factors should not only enlighten us as to the importance of cell-cell interactions in development of the motoneuron, but also open the way to attempts to slow motoneuron death in pathological situations, either in animals or in man.

[Neurotrophic activity of homeopeptide]. / Activité neurotrophique d'un homéopeptide.

The sixty aminoacid-long peptide corresponding to the homeobox of Antennapedia (pAntp) translocates through the membrane of neurons in culture and reaches their nuclei. This process is followed by an enhanced morphological differentiation of the neurons. Internalization by neurons is 4-fold that observed with fibroblasts. This difference is abolished upon treatment Endo-N which specifically cleaves alpha, 2-8 bounds in polysialic acid (PSA). To understand the mode of action of the peptide, we constructed three mutants modified in their capacity to specifically bind promoters and/or to translocate through the cell membrane. The biological properties of the mutants demonstrate that the neurotrophic action of pAntp requires its internalization and the integrity of its specific DNA-binding capacity.

Antennapedia homeobox peptide enhances growth and branching of embryonic chicken motoneurons in vitro.

Spinal motoneuron development is regulated by a variety of intrinsic and extrinsic factors. Among these, a possible role for homeoproteins is suggested by their expression in the motoneuron at relatively late stages. To investigate their possible involvement in motoneuron growth, we adapted a novel technique recently developed in this laboratory, based on the ability of the 60 amino acid-long homeobox of Antennapedia (pAntp) to translocate through the neuronal membrane and to accumulate in the nucleus (Joliot, A. H., C. Pernelle, H. Deagostini-Bazin, and A. Prochiantz. 1991. Proc. Natl. Acad. Sci. USA. 88:1864-1868; Joliot, A. H., A. Triller, M. Volovitch, C. Pernelle, and A. Prochiantz. 1991. New Biol. 3:1121-1134). Motoneurons from E5 chicken spinal cord were incubated with pAntp, purified by panning on SC1 antibody and plated on polyornithine/laminin substrata without further addition of pAntp. After 24 h, neurite outgrowth was already extensive in controls. In cultures of motoneurons that had been preincubated with 10(-7) M pAntp, neurite length was doubled; a similar effect was obtained using postnatal muscle extracts. Morphological analysis using a neurofilament marker specific for axons indicated that the homeobox peptide enhances primarily axonal elongation and branching. To test the hypothesis that the biological activity of pAntp involves its specific attachment to cognate homeobox binding sites present in the genome, we generated a mutant of pAntp called pAntp40P2, that was still able to translocate through the motoneuron membrane and to reach the nucleus, but had lost the specific DNA-binding properties of the wild-type peptide. Preincubation of pAntp40P2 with purified motoneurons failed to increase neurite outgrowth. This finding raises the possibility that motoneuron growth is controlled by homeobox proteins.

Control of neuronal morphogenesis by homeoproteins: consequences for the making of neuronal networks.

To test whether homeoproteins can act as genetic regulators in the processes of neurite growth, branching, guidance, and connectivity, the 60 amino acid homeodomain of Antennapedia was introduced in embryonic neurons in primary culture. It was hoped that this homeopeptide would bind to specific promoters and thus behave as a competitive inhibitor of endogenous homeoproteins. The introduction of the homeodomain in the nerve cells was made easy by its unexpected capability to translocate through the membranes and to accumulate within the nuclei. The presence of the homeodomain within the cells correlated with an increase in neurite growth and branching. The absence of activity of mutant peptides, still internalized but unable to bind with high affinity to homeoprotein cognate binding sites, strongly suggested that endogenous homeoproteins modulate neurite outgrowth and branching. Moreover, the efficient internalization of the homeobox peptide by live cells in culture raises the possibility that, in addition to their well-established role as cell-autonomous transcription factors, some homeoproteins may also exert paracrine functions. We examine how these hypotheses could modify our current views on the establishment and plasticity of neuronal networks.

Neurotrophic factors in development and plasticity of spinal neurons.

Factors affecting neuronal growth may be considered to fall into two major categories: those required for neuronal survival during development or following a lesion, and those which enhance growth or regeneration of axonal or dendritic processes. We briefly review here some recent studies on the former in spinal cord development and plasticity as an introduction to other papers in the session on Factors controlling Neural Growth, and then present in more detail work on factors affecting motoneuron development in vitro. The neurotrophins are a closely-related family of basic neurotrophic factors including nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) and neurotrophins -3, -4 and -5 that enhance neuronal survival by binding to surface receptors whose major components are the trk tyrosine kinases and p75NGF-R. Only the latter has been studied in the context of spinal cord neuroplasticity: its levels on motoneurons are up-regulated following central or peripheral trauma, although its function there remains unknown. Much evidence exists for the existence of 'motoneuron growth factors' involved in regulation of survival and development of spinal motoneurons. Following a critical comparison of techniques for their purification, we review results obtained in vitro and in vivo using known growth factors such as ciliary neurotrophic factor (CNTF), basic fibroblast growth factor (bFGF) and transforming growth factor (TGF/ß1). Although none of them satisfies all the criteria for the embryonic 'motoneuron growth factor', CNTF is of potential interest for reducing motoneuron loss in pathological situations.

Antennapedia homeobox as a signal for the cellular internalization and nuclear addressing of a small exogenous peptide.

In a previous study we demonstrated that a homeobox peptide corresponding to the 60 amino acid long DNA-binding region of the Drosophila antennapedia homeo-protein was capable of crossing the plasma membrane of cells in culture. This finding has led us to investigate whether chimeric molecules encompassing the homeobox would behave in a similar manner. We demonstrate here that a peptide of 93 amino acids composed of the homeobox and of the C terminus of Rab3, a small GTP-binding protein, crosses the membrane of myoblasts, myotubes and neurons and is conveyed to their nuclei. This transport is highly efficient, is observed in all the cells present in the culture and occurs at 37 degrees C and 12 degrees C without quantitative peptide degradation. Beyond its theoretical implications for our current views on cellular interactions, this finding could have technical repercussions on the development of drugs with intracellular targets.

Levels of mRNA coding for motoneuron growth-promoting factors are increased in denervated muscle.

Partial denervation of skeletal muscle induces sprouting of axons remaining within the muscle, possibly as a result of increased synthesis by denervated muscle fibers of motoneuron growth-promoting factors. Direct verification of this hypothesis has not been possible because the molecules responsible are not unambiguously characterized. We used Xenopus oocytes as a functional assay for mRNAs coding for secreted growth factors: preparations of mRNA from innervated and denervated neonatal muscle were injected into oocytes. Three days later, oocytes injected with denervated muscle mRNA expressed increased levels of nicotinic acetylcholine receptor and voltage-dependent sodium channels at their membrane. Proteins secreted by the same oocytes were tested for their effects on (i) neurite outgrowth from embryonic chicken ventral spinal cord neurons; (ii) survival in mixed culture of embryonic chicken motoneurons identified using the SC1 antibody; and (iii) survival of embryonic motoneurons purified by panning on SC1 antibody. In all three assays, media conditioned by oocytes injected with mRNA from denervated muscle contained significantly higher levels of biological activity than did those from oocytes injected with innervated muscle mRNA or water. mRNA was prepared from muscle at different times after denervation: a maximal increase was obtained already after 1 day, consistent with an involvement in sprouting. Synthesis of motoneuron growth-promoting factors is thus regulated by denervation in a parallel fashion to that of other key components of the neuromuscular junction.