Plasticity in neural crest cell differentiation.

The neural crest is a pluripotent population of cells that are endowed with migratory capacities. It has long been known that the differentiation pathway taken by cells derived from the neural crest is largely controlled by the microenvironment to which they home after their migration phase, indicating a high degree of plasticity in their developmental fate. Recent progress has been made concerning the factors which influence survival, growth and differentiation of selected sets of precursors in each embryonic site colonised by derivatives of the neural crest.

A surface protein expressed by avian myelinating and nonmyelinating Schwann cells but not by satellite or enteric glial cells.

Searching for specific markers of neural crest-derived cell lineages, we immunized mice with glycoproteins purified from adult quail peripheral myelin. We obtained a monoclonal antibody that reacts with myelin and peripheral glial cells. This antibody, to Schwann cell myelin protein (SMP), is specific for the membranes of all Schwann cells, irrespective of whether they are associated with myelinated nerves. SMP persists on Schwann cells in long-term cultures in vitro, but is absent from satellite cells of peripheral ganglia, both in vivo and in vitro. The antigen (a protein doublet of Mr 75,000-80,000) is present in, but not restricted to, the myelin lamellae, since it is distributed along the whole myelinating Schwann cell membrane. In the CNS, SMP appears as a single band of Mr 80,000. SMP is first detectable by immunofluorescence at E6 in the quail, which is at least 6 days earlier than the first appearance of already described markers related to myelination.

Genetic and epigenetic control in neural crest development.

The neural crest is a fascinating structure of the vertebrate embryo; its ontogeny includes a transient period during which its component cells undergo an epithelio-mesenchymal transition and become migratory. This phase was shown recently to be controlled by the 'Slug' gene which belongs to the 'Snail' family of Drosophila transcription factors. After homing to specific sites in the embryo, the crest-derived cells produce a large variety of phenotypes. Recent advances have shown that during migration most crest cells exhibit various degrees of pluripotentiality, some being already committed to a single and definite fate. Moreover, several lines of evidence point to the existence of totipotent stem cells in the neural crest, the progeny of which become progressively diversified through a combination of intrinsic and extrinsic influences. The latter have been documented by the disruption of several neurotrophin genes, which results in severe deficiencies of selected subsets of neural crest derivatives. The neural crest has also been shown to play an important role in the development of the vertebrate head and hypobranchial region. The genetic control of this process depends on the activity of developmental genes, among which the vertebrate Hox genes are essential, particularly at the rhombencephalic level.

The developmental potentials of the caudalmost part of the neural crest are restricted to melanocytes and glia.

The avian spinal cord is characterized by an absence of motor nerves and sensory nerves and ganglia at its caudalmost part. Since peripheral sensory neurons derive from neural crest cells, three basic mechanisms could account for this feature: (i) the caudalmost neural tube does not generate any neural crest cells; (ii) neural crest cells originating from the caudal part of the neural tube cannot give rise to dorsal root ganglia or (iii) the caudal environment is not permissive for the formation of dorsal root ganglia. To solve this problem, we have first studied the pattern of expression of ventral (HNF3beta) and dorsal (slug) marker genes in the caudal region of the neural tube; in a second approach, we have recorded the emergence of neural crest cells using the HNK1 monoclonal antibody; and finally, we have analyzed the developmental potentials of neural crest cells arising from the caudalmost part of the neural tube in avian embryo in in vitro culture and by means of heterotopic transplantations in vivo. We show here that neural crest cells arising from the neural tube located at the level of somites 47-53 can differentiate both in vitro and in vivo into melanocytes and Schwann cells but not into neurons. Furthermore, the neural tube located caudally to the last pair of somites (i.e. the 53rd pair) does not give rise to neural crest cells in any of the situations tested. The specific anatomical aspect of the avian spinal cord can thus be accounted for by limited developmental potentials of neural crest cells arising from the most caudal part of the neural tube.

Ovarian carcinoma cells are effectively transfected by polyethylenimine (PEI) derivatives.

As a prerequisite to nonviral gene therapy approaches of ovarian carcinoma, we evaluated the possibility of transfecting established tumor cell lines (SKOV3, IGROV1) as well as primary mesothelial and tumor cells by various polyethylenimine (PEI) derivatives. Several PEI-based vectors were able to effectively transfect these cells, as shown by high luciferase expression levels (10(8) to 10(9) relative light units per milligram of cell protein) that corresponded with 25-50% of green fluorescent protein-positive cells after 24 hours. However, unpredictable differences were observed among the vectors and cell types that a posteriori justified the screening procedure. We also showed that cells that were not transfected after the first experiment remained transfectable in a subsequent transfection experiment to a level similar to that of the initial population. This experiment does not support the emergence of a transfection-resistant cell population and opens the door to multiple therapeutic gene deliveries. Although efficacy and cell targeting still remain to be improved, PEI derivatives appear to be promising molecules for the development of nonviral gene therapy of ovarian carcinoma.

A monoclonal antibody directed against quail tyrosine hydroxylase: description and use in immunocytochemical studies on differentiating neural crest cells.

Catecholamine (CA) synthesis is one of the phenotypic traits expressed by some neural crest-derived cells in vivo and in vitro. In the present study, we have evidenced, in quail embryos, the expression of the first enzyme of CA metabolism, tyrosine hydroxylase (TOH), using a monoclonal antibody raised against the quail enzyme. This antibody also recognizes TOH from chick and pleurodele, but not from several mammalian species (rat, human). We have also investigated the extent to which TOH-positive cells, differentiated in neural crest cultures, express structural neuronal markers and display vasoactive intestinal polypeptide (VIP) and substance P (SP) immunoreactivity. Double-immunolabeling experiments show that, in vitro, half of the population of TOH-positive cells exhibits tetanus toxin binding sites but none of them are recognized by a neurofilament antibody. On the other hand, some TOH-positive cells contain VIP or SP. These observations suggest that under our culture conditions autonomic neural crest precursors differentiate only into immature sympathoblasts, but are able to synthesize peptides in addition to CA.

In vivo and in vitro expression of vasoactive intestinal polypeptide-like immunoreactivity by neural crest derivatives.

Qualitative and quantitative in vivo studies were performed on the development of the neuropeptide vasoactive intestinal polypeptide (VIP) in the peripheral nervous system of quail embryos. VIP-like immunoreactivity (VIPLI) was found by radioimmunoassay (RIA) from the sixth day of embryonic life onward in the sympathetic chain, the esophagus and duodenum, and from day 15 of incubation onward in the adrenal glands and the nodose ganglia. By using immunocytochemistry, we identified cells expressing VIPLI in sensory spinal ganglia of 13- to 15-day-old embryos. In neural crest cultures, cells expressing the VIP phenotype differentiated constantly under various culture conditions, in contrast to other phenotypes which had specific medium requirements, i.e. adrenergic cells or substance P-containing neurons.

Quail neural crest cells transformed by Rous sarcoma virus can be established into differentiating permanent cell cultures.

Quail neural crest cells derived from the truncal neural primordium, infected in vitro by Rous sarcoma virus (RSV) in January 1978, were induced to multiply and have been established into permanent cultures. These cultures contain cells that differentiate into melanocytes, neuron-like cells and flat cells. About 50% of these different cell types are tetanus-toxin positive. Electrophysiological studies have shown that some cells can generate action potentials similar to those reported in quail neural crest primary cultures. Taken together these data show that the RSV-transformed quail neural crest permanent cultures are composed of stem cells which can differentiate into cell types specific for neural crest.

Quox 1 homeobox protein is expressed in postmitotic sensory neurons of dorsal root ganglia.

The expression of vertebrate homeoproteins has been extensively studied in a variety of normal and cancerous tissues, but little is known on the role of vertebrate homeoproteins in the proliferation and differentiation of cells from these tissues. In the present study, we investigate the relationship between Quox 1 protein (a quail homeodomain containing protein) expression and the proliferation and differentiation of quail dorsal root ganglia (DRG) and neural crest cells. In vivo [3H]TdR labeling experiments demonstrate that the postmitotic sensory neuroblasts appear before the formation of the ganglion, and that more than half of sensory neuroblasts from DRG have already terminated their proliferation in embryos of 2 days of incubation (E2). All DRG neurons have completely ceased to proliferate from E6.5 onwards. By means of immunocytochemistry, we observe that Quox 1 protein is accumulated exclusively in all bipolar neurons in culture of DRG from E9-E11, and in all postmitotic sensory-like neuroblasts during in vitro cell differentiation of the neural crest. The Quox 1 immunoreactive neurons express simultaneously neurofilaments or substance P, and they are never labeled by anti-bromodeoxyuridine. These observations together with the morphology of Quox 1 positive cells, demonstrate that Quox 1 protein is expressed in the postmitotic sensory neurons of DRG. Our previous experiments have shown that between E4 and E6, the accumulation of Quox 1 protein increases in DRG in vivo, but decreases in the central nervous system in which cell proliferation decreases (Xue et al., (1993) Mech. Dev. 43, 149-158). Taken together, our results show that the accumulation of Quox 1 protein in DRG is tightly linked to the increase in the number of postmitotic neurons, whereas in the central nervous system the level of expression of Quox 1 seems concomitant with the extent of cell proliferation.

Developmental potentialities of cells derived from the truncal neural crest in clonal cultures.

The developmental potentialities of single truncal neural crest derived cells were analysed in clonal cultures. The clone-forming ability and differentiation potential of crest cells migrating through the somitic mesoderm of 3-day-old embryos (E3) and of non-neuronal cells of dorsal root ganglia taken at E6-14 were compared. Since most of the cells present in the sclerotomal and rostral parts of the somite at E3 become later on incorporated into the spinal ganglia, one can consider that these two cell populations represent the same derivatives of the trunk neural crest at different developmental stages. After 10 days in vitro, the size of clones and their phenotypic composition varied noticeably, revealing a certain heterogeneity in the founder cell populations in terms of developmental potencies. Clones obtained from migrating neural crest cells at E3 were often large (greater than 1000 cells) and many of them contained neuronal and non-neuronal cells. Dorsal root ganglion cells produced mostly small clones (less than 100 cells) in which only non-neuronal (i.e. glial) phenotypes were expressed. Therefore, both the capacity for proliferation and the differentiation ability of cloned neural crest derived cells decrease considerably with increasing embryonic age. This is even more striking if these results are compared with those obtained previously in our laboratory with single cells cultures of E2 cephalic neural crest. In the latter case, both clone sizes and cellular diversity within the colonies were much higher than with E3 truncal crest and dorsal root ganglia (DRG) non-neuronal cells. The second result of the present work concerns the differentiation of the dormant autonomic neuronal precursors of the DRG. It has been established previously that the non-neuronal cells of the DRG include adrenergic precursors than can differentiate in mass culture of dissociated DRG cells. We show that these cells never differentiate in clonal cultures but depend upon the cell density of the culture. This suggests that cell to cell interaction between crest derived cells are critical in eliciting the differentiation of the adrenergic phenotype.

Quox 1 homeobox protein is expressed in postmitotic sensory neurons of dorsal root ganglia

The expression of vertebrate homeoproteins has been extensively studied in a variety of normal and cancerous tissues, but little is known on the role of vertebrate homeoproteins in the proliferation and differentiation of cells from these tissues. In the present study, we investigate the relationship between Quox 1 protein (a quail homeodomain containing protein) expression and the proliferation and differentiation of quail dorsal root ganglia (DRG) and neural crest cells. In vivo [3H]TdR labeling experiments demonstrate that the postmitotic sensory neuroblasts appear before the formation of the ganglion, and that more than half of sensory neuroblasts from DRG have already terminated their proliferation in embryos of 2 days of incubation (E2). All DRG neurons have completely ceased to proliferate from E6.5 onwards. By means of immunocytochemistry, we observe that Quox 1 protein is accumulated exclusively in all bipolar neurons in culture of DRG from E9-E11, and in all postmitotic sensory-like neuroblasts during in vitro cell differentiation of the neural crest. The Quox 1 immunoreactive neurons express simultaneously neurofilaments or substance P, and they are never labeled by anti-bromodeoxyuridine. These observations together with the morphology of Quox 1 positive cells, demonstrate that Quox1 protein is expressed in the postmitotic sensory neurons of DRG. Our previous experiments have shown that between E4 and E6, the accumulation of Quox 1 protein increases in DRG in vivo, but decreases in the central nervous system in which cell proliferation decreases (Xue et al., (1993) Mech. Dev. 43, 149-158). Taken together, our results show that the accumulation of Quox 1 protein in DRG is tightly linked to the increase in the number of postmitotic neurons, whereas in the central nervous system the level of expression of Quox 1 seems concomitant with the extent of cell proliferation.

The tyrosine hydroxylase gene is expressed in endoderm and pancreas of early quail embryos.

The initial expression of the gene encoding tyrosine hydroxylase (TH) was studied in the trunk of quail embryos by in situ hybridization. We detected the presence of quail TH mRNA on embryonic day 3.5 (E3.5) in the sympathetic ganglia and aortic plexus, both neural crest derived structures. In contrast, the TH gene was expressed much earlier in the endodermal layer of E2 embryos, i.e. from the 8-somite stage onwards. TH mRNA was found also in the pancreatic bud, an endoderm-derived structure. The TH protein and catecholamines were subsequently looked for in these structures. TH immunoreactivity was found in cells of E2 explanted endoderm, but no catecholamine histofluorescence was observed before or after a few days in culture. TH-positive cells were also detected in cultures of pancreatic rudiments, explanted from E3 to E6 quail embryos. We suggest that the TH-positive cells of the endoderm are the progenitors of the catecholaminergic cells of the pancreas and of the enterochromaffin cells of the gut. The hypothesis that the TH-positive cells of the endoderm are involved in the expression of the catecholaminergic phenotype by neural crest cells is discussed.

[Effect of actinomycin D and cycloheximide on planarian regeneration after repeated amputation]. / Effets de l'actinomycine D et de la cycloheximide sur la régénération des planaires après des amputations répétées

A Planaria deprived of its head twice at an interval of a few days can regenerate in the presence of actinomycin D, even if the second amputation takes place 13 days after the first one, but it cannot regenerate in the presence of cyclohemixide. This indicates that at the moment of the second amputation, stable RNAs are available for regeneration in the tissues behind the level of section, whereas proteins have to be synthesized in situ in the blastema.

[The effect of repeated amputations on planarian regeneration in the presence of the heat-stable toxin from Bacillus thuringiensis]. / Effets d'amputations répétées sur la régénération des Planaires, en présence de la toxine thermostable de Bacillus thuringiensis

The exotoxin of Bacillus thuringiensis, which is an inhibitor of RNA synthesis, inhibits Planarian regeneration. Planarians which have been cut twice at the same level are able to regenerate after the second section in the presence of the toxin. This indicates that the first amputation stimulates a synthesis of stable RNAs which thus are available for regeneration at the moment of the second section.

Migration and differentiation of neural crest cells and their derivatives: in vivo and in vitro studies on the early development of the avian peripheral nervous system.

After a period of extensive migration through the vertebrate embryo, neural crest cells differentiate into a great variety of cell types, including all the elements of the peripheral nervous system. We have studied crest cell migration in quail-chick chimeras in which quail cells can be identified by means of a stable natural nuclear marker. The results of interspecific grafts of neural primordium, performed systematically at different levels of the neuraxis, have established the sites of origin of the principal peripheral ganglia. In addition, they suggest that the cholinergic and adrenergic phenotypes are not predetermined in the neural crest before migration, but are the result of multiple cellular interactions. Furthermore, the phenotype expression of young differentiating autonomic ganglia is labile and can be modified if the latter are subjected to an appropriate cellular environment by grafting into a younger host embryo. The results of experiments in which fragments of neural crest, sensory and autonomic ganglia were transplanted are presented in terms of a model of crest cell-line segregation. As a step towards the analysis of the cellular interactions occurring during autonomic neuron differentiation, we have also studied neuronal development in tissue cultures of neural crest, taken from the cranial and trunk levels of quail embryos and grown in the presence or absence of other embryonic tissues. The results confirm that both levels of the crest are potentially able to give rise to cells that can make acetylcholine and catecholamines. However, whereas acetylcholine-synthesizing ability is apparently a very early feature of autonomic neuron precursors, the ability to produce catecholamines is acquired later as a result of interactions with other cell types, in particular with mesenchymal derivatives. Although production of both neurotransmitters can be considerably stimulated by associating crest with any of several young embryonic rudiments, only when trunk crest is cultured with the sclerotomal moiety of the somite is biochemical differentiation accompanied by extensive morphological and cytochemical neuronal maturation.

A monoclonal antibody recognizing a common antigen on neurons and fibroblasts in chicken and quail.

A monoclonal antibody, FiN1, obtained by immunization of a mouse with homogenates of embryonic quail nodose ganglia, was found to react with a surface antigenic determinant, both in quail and chick, present on practically all neurons of the spinal cord and of the peripheral nervous system and on a subpopulation of fibroblasts. An ontogenetic study performed on tissue sections, cell suspensions and cultures showed that FiN1 defines a differentiation marker which appears relatively late in development, during the second half of embryonic life, and persists after hatching. The onset and evolution of its expression during development varies in a tissue-specific manner.

Transient blocking of both B7.1 (CD80) and B7.2 (CD86) in addition to CD40-CD40L interaction fully abrogates the immune response following systemic injection of adenovirus vector.

Blockade of the CD40-CD40L and CD80/CD86-CD28 costimulatory pathways represents a strategy to inhibit the immune response against Ad vectors designed for gene therapy applications. Since most previous studies have used a CTLA4-Ig fusion molecule binding to both CD80 and CD86, the respective roles of these B7 molecules remained undefined. We have studied the effect of blocking monoclonal Abs (mAbs) directed against the costimulatory molecules CD40L, CD80 and CD86, alone or in different combinations, on the humoral and cellular immune responses against Ad. Groups of mice were transiently treated with each combination of blocking mAbs upon systemic injection of a first Ad vector. Combinations of anti-CD80 + anti-CD86 or anti-CD40L + anti-CD86 mAbs resulted in strong inhibition of the immune response against Ad. Using either of these mAb pairs, a second vector could be administered 1 month after the first injection but with lower efficiency than in naive animals. Thus, CD86 stands as the pivotal B7 molecule involved in the development of the immune response against Ad. However, only the blockade of both CD80 and CD86 in addition to CD40L fully inhibited the humoral and cellular responses against the Ad vector, such that readministration after 1 month was as efficient as in naive animals. At the time of readministration, treated animals had regained their ability to mount a normal immune response to the second Ad vector, showing that tolerance was not induced.