Mireille Fauquet

PEA-15 mediates cytoplasmic sequestration of ERK MAP kinase.

The ERK 1/2 MAP kinase pathway controls cell growth and survival and modulates integrin function. Here, we report that PEA-15, a protein variably expressed in multiple cell types, blocks ERK-dependent transcription and proliferation by binding ERKs and preventing their localization in the nucleus. PEA-15 contains a nuclear export sequence required for its capacity to anchor ERK in the cytoplasm. Genetic deletion of PEA-15 results in increased ERK nuclear localization with consequent increased cFos transcription and cell proliferation. Thus, PEA-15 can redirect the biological outcome of MAP kinase signaling by regulating the subcellular localization of ERK MAP kinase.

Cell lineages in peripheral nervous system ontogeny: medium-induced modulation of neuronal phenotypic expression in neural crest cell cultures.

Neural crest, taken from cephalic and trunk levels of quail embryos, was grown in vitro in conventional tissue culture medium (Dulbeccos modified Eagles medium containing 15% fetal calf serum and either 2 or 15% chick embryo extract (CEE] or in a chemically defined serum- and CEE-free medium. Depending on the conditions employed, different types of neuronal or neuronlike cells developed in the cultures. Thus, in medium containing 15% CEE, adrenergic cells (identified by tyrosine hydroxylase immunoreactivity and catecholamine histofluorescence) emerged after 5-6 days. These cells lacked tetanus toxin binding sites and did not react with an antibody directed against 70-kDa neurofilament protein. In the fully defined medium, a neuronal cell type exhibiting neurofilament and substance P (SP) immunoreactivity differentiated from noncycling precursors within 1 or 2 days of culture. If serum was added to the medium, the neurites disintegrated and the neuronal cells ultimately died. By sequentially culturing neural crest, first in the wholly synthetic medium for 1-3 days and then in the conventional medium supplemented with serum and 15% CEE, the disappearance of the SP-positive neurons was followed, several days later, by the emergence of adrenergic cells. The majority of these cells and/or their precursors were found to undergo cell division in culture. We conclude that the cells expressing the adrenergic phenotype (characteristic of the sympathetic nervous system) and those displaying SP immunoreactivity, comparable to a category of neurons in dorsal root and cranial sensory ganglia, derive from distinct sets of precursors. Our results reinforce the contention, deduced from in ovo transplantation experiments (see N. M. Le Douarin, (1984) In Cellular and Molecular Biology of Neuronal Development (I. Black, Ed.), pp. 3-28. Plenum, New York), that at least two lineages, from which sensory and autonomic cell types are derived respectively, are segregated early during neural crest ontogeny and have extremely different survival and trophic requirements.

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.

Cloning of Quail Tyrosine Hydroxylase: Amino Acid Homology with Other Hydroxylases Discloses Functional Domains

Abstract: A cDNA clone containing the entire coding region of quail tyrosine hydroxylase (TH) has been isolated and analyzed. Comparison with rat and human THs and phenylalanine hydroxylases reveals several highly conserved domains. Two of them, shared by all these hydroxylases, are localized in the central and C‐terminal parts of the molecules, and most probably include the active site. Two others are found only in the TH molecules. One contains putative sites of phosphorylation and is implicated in the posttranslational regulation of the enzyme. The second highly preserved domain, consisting of a stretch of 21 amino acids, is presumably associated with an important feature of the enzyme that remains to be identified.

Coexpression of somatostatin-like immunoreactivity and catecholaminergic properties in neural crest derivatives: comodulation of peptidergic and adrenergic differentiation in cultured neural crest

In the avian embryo, somatostatin-like immunoreactivity (SLI) and adrenergic characteristics appear virtually simultaneously in the developing sympathetic nervous system and adrenal medulla. We have used double-labeling techniques to show that both properties coexist in the same cells. In the quail, not only do all somatostatin-containing cells in the adrenosympathetic system exhibit tyrosine hydroxylase immunoreactivity and possess catecholamines (CA), but this coexistence of the peptidergic and adrenergic phenotypes is already present very early in ontogeny. However, not all adrenergic cells express SLI. The development of sympathoadrenal precursors can be followed in vitro. Adrenergic precursor cells, obtained from the migrating neural crest, differentiate in culture into neuron-like cells that contain SLI and CA. This coexpression can be regulated by the same factors. For instance, corticosterone and progesterone increase SLI content and CA production in the neural crest cell cultures. The ontogeny of the autonomic lineage is discussed in the light of these results.

Environmentally Directed Nerve Cell Differentiation: In Vivo and In Vitro Studies

Publisher Summary By using the quail–chick marker system to follow the migration and differentiation of neural crest cells, the neural axis is regionalized at an early stage into “adrenergic” and “cholinergic” sections, from which arise respectively the sympathetic and parasympathetic/enteric ganglioblasts of the autonomic nervous system. This regionalization does not correspond to an irreversible determination of the crest cells because, under certain experimental conditions, cholinergic neurons can develop from the “adrenergic” region of the crest and vice versa. The phenotypic expression of the presumptive ganglion cells appears to be responsive to the environmental conditions they encounter during and/or after their migration. A study of the nature of the factors required to obtain cholinergic and/or adrenergic differentiation has been undertaken by culturing explants of “pure” neural crest from both “cholinergic” and “adrenergic” regions of the neural axis. Results clearly show that the tissue environment in which the neurons develop determines their final phenotype and is responsible for the observed distribution of catecholaminergic cells in the dorsal trunk structures and cholinergic cells in the splanchnopleural derivatives.

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.

Evolution of Acetylcholinesterase Transcripts and Molecular Forms During Development in the Central Nervous System of the Quail

Abstract: We studied the expression of acetylcholinesterase (AChE) in the nervous system (cerebellum, optic lobes, and neuroretina) of the quail at different stages of development, from embryonic day 10 (E10) to the adult. Analyzing AChE mRNAs and AChE molecular forms, we observed variations in the following: (a) production of multiplemRNA species (4.5 kb, 5.3 kb, and 6 kb); (b) translation and/or stability of the AChE protein; (c) production of active and inactive AChE molecules; (d) production of amphiphilic and nonamphiphilic AChE forms; and (e) proportions of tetrameric G4, dimeric G2, and monomeric G1 forms. The large transcripts present distinct temporal patterns and disappear in the adult, which possesses only the 4.5‐kb mRNA; these changes are unlikely to be related to those observed for the AChE protein, because all transcripts seem to encode the same catalytic subunit (type T). In addition, the levels of mRNA and AChE are not correlated in the three regions, especially at the adult stage. The proportion of inactive AChE was found to be markedly higher at the hatching period (E16) than at earlier stages (E10 and E13) or in the adult. The G4 form is pre‐dominant already at E10, and in the adult its proportion reaches 80% of the activity in the cerebellum and optic lobes, and 65–70% in the neuroretina. This form is largely nonamhiphilic in embryonic tissues, but it becomes progressively more amphiphilic with development. Thus, the different processing and maturation steps appear to be regulated in an independent manner and potentially correspond to physiologically adaptative mechanisms.

Difference in the expression of asymmetric acetylcholinesterase molecular forms during myogenesis in early avian dermomyotomes and limb buds in ovo and in vitro.

The A12 (asymmetric) form of acetylcholinesterase (AChE) is generally considered to be synthesized in leg muscle tissues by myotubes under neural influence, but not by myoblasts. We have examined the expression of the different molecular forms of AChE in explants of developing limb buds and dermomyotomes (the myogenic part of the somites) obtained from 3-day-old chick and quail embryos, either directly after removal or during in vitro culture. We describe a muscular differentiation of both territories in vitro, leading to the formation of myotubes which are morphologically similar to the class of early muscle cells described by Bonner and Hauschka (1974). In vivo the A12 form is present in quail dermomyotomes which are almost entirely composed of mononucleated poorly differentiated cells; in contrast, it is absent from similar cells in chick dermomyotomes and from limb buds in both species. This shows that in the case of quail embryos the appearance of the A12 form precedes the fusion of myoblasts into myotubes. In both species, dermomyotome explants express asymmetric and globular forms of the enzyme during muscular differentiation in vitro, whereas limb buds synthesize only globular forms. After surgical removal of neural tube and/or neural crest at 2 days in ovo, the biosynthesis of the A forms in quail dermomyotomes is not suppressed and is consequently not dependent upon prior connection of the dermomyotomes to central neurons or upon the presence of autonomic precursors. Since limb bud muscle cells derive from somites our results raise questions concerning the differentiation of migrating somitic cells in this territory where a neural influence appears necessary to induce the biosynthesis of asymmetric AChE forms.

Selective photocleavage of single-stranded nucleic acids by cyclobisintercaland molecules

Irradiation of mixtures of a single-stranded circular plasmid and of a double-stranded supercoiled DNA in presence of the cyclobisintercaland compounds 2 or 3 shows that these reagents effect the selective photocleavage of the single-stranded entity. Furthermore, 2 also cleaves tRNAasp preferentially at single-stranded domains.