Biomedical subjects

J F Brunet

Publications and source records attributed to J F Brunet.

At least 37 records · Page 2Linked to original sources

PubMed1997

Defects in sensory and autonomic ganglia and absence of locus coeruleus in mice deficient for the homeobox gene Phox2a.

Phox2a is a vertebrate homeodomain protein expressed in subsets of differentiating neurons. Here, we show that it is essential for proper development of the locus coeruleus, a subset of sympathetic and parasympathetic ganglia and the VIIth, IXth, and Xth cranial sensory ganglia. In the sensory ganglia, we have identified two differentiation blocks in Phox2a-/- mice. First, the transient expression of dopamine-beta-hydroxylase in neuroblasts is abolished, providing evidence that Phox2a controls noradrenergic traits in vivo. Second, the expression of the GDNF receptor subunit Ret is dramatically reduced, and there is a massive increase in apoptosis of ganglion cells, which are known to depend on GDNF in vivo. Therefore, Phox2a appears to regulate conventional differentiation traits and the ability of neurons to respond to essential survival factors.

Animals↗

PubMed1997

Expression and interactions of the two closely related homeobox genes Phox2a and Phox2b during neurogenesis.

Recent evidence suggests that specific families of homeodomain transcription factors control the generation and survival of distinct neuronal types. We had previously characterized the homeobox gene Phox2a, which is expressed in differentiating neurons of the central and peripheral autonomic nervous system as well as in motor nuclei of the hindbrain. Targeted deletion of the Phox2a gene affects part of the structures in which it is expressed: the locus coeruleus, visceral sensory and parasympathetic ganglia and, as we show here, the nuclei of the IIIrd and IVth cranial nerves. We now report on the characterization of Phox2b, a close relative of Phox2a, with an identical homeodomain. Phox2a and Phox2b are co-expressed at most sites, therefore suggesting a broader role for Phox2 genes in the specification of the autonomic nervous system and cranial motor nuclei than revealed by the Phox2a knock-out mice. A detailed analysis of the relative timing of Phox2a and Phox2b expression at various sites suggests positive cross-regulations, which are substantiated by the loss of Phox2b expression in cranial ganglia of Phox2a-deficient mice. In the major part of the rhombencephalon, Phox2b expression precedes that of Phox2a and starts in the proliferative neuroepithelium, in a pattern strikingly restricted on the dorsoventral axis and at rhombomeric borders. This suggests that Phox2b links early patterning events to the differentiation of defined neuronal populations in the hindbrain.

Amino Acid Sequence↗

PubMed1996

The expression pattern of the transcription factor Phox2 delineates synaptic pathways of the autonomic nervous system.

Many transcription factors, and most prominently among them, homeodomain proteins, are expressed in specific groups of cells in the developing nervous system in patterns that suggest their involvement in neural fate determination. How various aspects of neural identity are controlled by such transcription factors, or sets of them, is still mostly unknown. It has been shown previously that Phox2 is such a homeodomain protein, expressed exclusively in differentiated groups of neurons or their precursors, and that its expression correlated with that of the noradrenaline synthesis enzyme dopamine-beta-hydroxylase. Here we confirm this striking correlation at the single-cell level with the use of an anti-Phox2 antibody. Moreover, we uncover a second, nonmutually exclusive correlative clue to the Phox2 expression pattern: a high proportion of Phox2-expressing cells are involved in, or located in areas involved in, synaptic circuits, i.e., that of the medullary control reflexes of autonomic functions. This suggests that Phox2 could be involved in the establishment of these circuits.

Animals↗

PubMed1996

Otlx2, an Otx-related homeobox gene expressed in the pituitary gland and in a restricted pattern in the forebrain.

The vertebrate nervous system forms by the specification of, successively, neuroepithelial regions and cell groups. One of the proposed major histogenic steps is the subdivision of the neural tube in compartments along its caudorostral and dorsoventral axis. This event is reflected, and may be directed, by the restricted expression of many transcription factors. Here, we report on the isolation of a new homeobox gene of the paired superclass, Otlx2, whose early expression pattern in the mesencephalon and prosencephalon is congruent with proposed neuromeric models of brain morphogenesis. In addition, its late embryonic and postnatal expression, in clear continuity with the earlier pattern, suggests a role in the neuronal differentiation and the histogenesis of several prosencephalic and mesencephalic areas. Finally, Otlx2 is expressed from the earliest morphogenetic events in the Rathke's pouch, the anlage of the adenohypophysis, an expression site shared by a very close homologue, Otlx1/Ptx1/P-Otx.

Amino Acid Sequence↗

PubMed1995

Barx1, a new mouse homeodomain transcription factor expressed in cranio-facial ectomesenchyme and the stomach.

In the process of cloning murine proteins capable of binding to a regulatory module of the Ncam promoter, we isolated a novel homeobox gene, Barx1, the first vertebrate member of the structural subclass defined by Drosophila BarH1. Here we report its sequence, chromosomal localisation and embryonic expression pattern. Barx1 was strongly expressed in restricted areas of head and neck mesenchyme and in the wall of the developing stomach and at weaker levels in the proximal fore- and hindlimbs. At embryonic day 10.5, expression in the head region is detected in spatially restricted areas of the first and second branchial arches, before any apparent cellular or morphological differentiation. Later in development, all expressing tissues in this region, which include the mesenchyme underlying the olfactory epithelium, the primary and secondary palate, the molar tooth papillae and the stroma of the submandibular gland, appear derived from ectomesenchyme of neural crest origin. At day 16.5, all locations other than the developing molars had become Barx1-negative. An intriguing feature is the restriction of Barx1 expression to the molars suggesting a role in the differentiation of molars from incisors. Barx1 already marks the future stomach region of the primitive gut at embryonic day 9.5 and is present in the mesenchymal wall of the stomach up to day 16.5. These results thus direct a search for its function to a number of inductive epithelial-mesenchymal interactions during craniofacial development and to stomach organogenesis.

Amino Acid Sequence↗

PubMed1995

Differential regulation of transcription factor gene expression and phenotypic markers in developing sympathetic neurons.

We have examined the regulation of transcription factor gene expression and phenotypic markers in developing chick sympathetic neurons. Sympathetic progenitor cells first express the bHLH transcriptional regulator Cash-1 (a chicken achaete-scute homologue), followed by coordinate expression of Phox2, a paired homeodomain protein, and GATA-2, a zinc finger protein. SCG10, a pan-neuronal membrane protein, is first detected one stage later, followed by the catecholaminergic neurotransmitter enzyme tyrosine hydroxylase (TH). We have used these markers to ask two questions: (1) is their expression dependent upon inductive signals derived from the notochord or floor plate?; (2) does their sequential expression reflect a single linear pathway or multiple parallel pathways? Notochord ablation experiments indicate that the floor plate is essential for induction of GATA-2, Phox2 and TH, but not for that of Cash-1 and SCG10. Taken together these data suggest that the development of sympathetic neurons involves multiple transcriptional regulatory cascades: one, dependent upon notochord or floor plate-derived signals and involving Phox2 and GATA-2, is assigned to the expression of the neurotransmitter phenotype; the other, independent of such signals and involving Cash-1, is assigned to the expression of pan-neuronal properties. The parallel specification of different components of the terminal neuronal phenotype is likely to be a general feature of neuronal development.

Animals↗

PubMed1994

Chemolabeling of frozen cerebral tissue proteins and immunopurified products with biotin and digoxigenin: physicochemical characteristics of biotinylated and digoxigeninated products.

Biotinylation and digoxigenination have been compared for labeling proteins from a total frozen tissue extract and from products of immunopurification with anti-RAR beta (retinoic acid receptor beta). The detection of biotinylated and digoxigeninated proteins was found to be easier and more sensitive than detection of silver-stained proteins after two-dimensional electrophoresis. Although biotinylated or digoxigeninated proteins can be detected with avidin conjugates or anti-digoxigenin antibodies, they can also be detected with specific antibodies such as anti-RAR beta antibodies. Previously, coimmunoprecipitates could be visualized only by radioactive amino acid incorporation in cell culture, whereas biotinylation and digoxigenination enable the study of specific protein expression in frozen tissues by immunoprecipitation and the visualization of coimmunoprecipitates. Chemolabeling presents the two major advantages of limiting the use of radioisotopes and allowing the use of frozen tissues in all types of protein expression studies.

Albumins↗

PubMed1993

Immunodetection of endogenous opioid peptides in human brain tumors and associated cyst fluids.

The antitumorigenic effects of endogenous opioid peptides and their presence in extracerebral tumors are well documented. In this study, methionine-enkephaline (met-enkephalin) was measured by radioimmunoassay in 108 glial and nonglial brain tumors and in 44 associated cyst fluids. By immunohistochemistry, the distribution of the peptide and its precursor, preproenkephalin A, was also analyzed. Met-enkephalin and preproenkephalin were detected in the cytoplasm and cell processes of all tumors. Moreover, for neuroectodermal tumors (i.e., gliomas, gangliogliomas, and dysembryoplastic neuroepithelial tumors), a strong inverse correlation (P < 0.0001) was observed between the met-enkephalin levels and the degree of malignancy (242.9, 148.3, 55.3, and 30.3 pg/mg protein for grade 1, 2, 3, and 4, respectively). When compared to normal tissue, this differential expression mainly results from a decrease in the opioid peptide content in high-grade neuroectodermal tumors. Meningiomas and cerebral metastases displayed low met-enkephalin levels, similar to those of grade 4 neuroectodermal tumors. Large amounts of met-enkephalin were found in all cyst fluids. These data suggest that the endogenous opioid system is an integral component of brain tumors and that met-enkephalin may represent a useful malignancy marker in neuroectodermal tumors.

Brain Neoplasms↗

PubMed1993

Identification and characterization of an anti-tyrosine kinase factor in cystic gliomas.

In view of the frequent activation of the epidermal growth factor receptor (EGF-R) in gliomas and autocrine hypothesis, we searched for 'EGF-like' factor(s) in cystic fluids (CFs) associated with gliomas. Membranes of A431 cells, which overexpress EGF-R, were used to explore such activity in 20 CFs. In all cases CFs induced inhibition of EGF-R phosphorylation. Biochemical analysis revealed an anti-tyrosine kinase activity which was identified as a 18 kDa proteic factor. Effectiveness at high dilution and anti-proliferative effect on living cells in culture suggest that this factor may be involved in the negative regulation of glial oncogenesis.

Astrocytoma↗

PubMed1993

The mouse homeodomain protein Phox2 regulates Ncam promoter activity in concert with Cux/CDP and is a putative determinant of neurotransmitter phenotype.

Transcriptional regulation of the gene encoding the cell adhesion receptor NCAM (neural cell adhesion molecule), a putative effector molecule of a variety of morphogenetic events, is likely to involve important regulators of morphogenesis. Here we identify two mouse homeodomain proteins that bind to an upstream regulatory element in the Ncam promoter: Cux, related to Drosophila cut and human CDP, and Phox2, a novel protein with a homeodomain related to that of the Drosophila paired gene. In transient transfection experiments, Cux was found to be a strong inhibitor of Ncam promoter activity, and this inhibition could be relieved by simultaneously overexpressing Phox2. These results suggest that the Ncam gene might be a direct target of homeodomain proteins and provide a striking example of regulatory cross-talk between homeodomain proteins of different classes. Whereas the expression pattern of Cux/CDP includes many NCAM-negative sites, Phox2 expression was restricted to cells also expressing Ncam or their progenitors. The localisation data thus strongly reinforce the notion that Phox2 plays a role in transcriptional activation of Ncam in Phox2-positive cell types. In the peripheral nervous system, Phox2 was strongly expressed in all ganglia of the autonomic nervous system and more weakly in some cranial sensory ganglia, but not in the sensory ganglia of the trunk. Phox2 transcripts were detected in the primordia of sympathetic ganglia as soon as they form. Phox2 expression in the brain was confined to spatially restricted domains in the hindbrain, which correspond to the noradrenergic and adrenergic nuclei once they are identifiable. All Phox2-expressing components of the peripheral nervous system are at least transiently adrenergic or noradrenergic. In the developing brain, Phox2 was expressed at all known locations of (nor)adrenergic neurones and of their precursors. These results suggest that Phox2, in addition to regulating the NCAM gene, may be part of the regulatory cascade that controls the differentiation of neurons towards this neurotransmitter phenotype.

Amino Acid Sequence↗

PubMed1993

A possible link between cell adhesion receptors, homeodomain proteins and neuronal identity.

The orderly arrangement of neuronal cell bodies and axonal projections generated during nervous system development requires precise spatio-temporal control of the expression and activity of cell adhesion receptors. Recent evidence suggests that homeobox genes, an important class of developmental control genes, many of which are preferentially expressed in developing nervous tissue, play prominent roles in the regulation of expression of these molecules. We have characterised two mouse homeobox genes, named Cux and Phox2, the products of which bind to and regulate the promoter of the Ncam gene in vitro. Its highly specific expression pattern suggests that Phox2, besides regulating Ncam, may be determinant of the noradrenergic phenotype.

Animals↗

PubMed1992

NCAM: structural diversity, function and regulation of expression.

NCAM is a large family of structurally closely related proteins with cell-cell adhesive properties and a temporo-spatially regulated expression throughout development. This review covers recent work on NCAM with an emphasis on the still open questions of the full extent of structural diversity and the mechanism whereby it arises, the chemistry and functional consequences of the binding event and the intricacies of the developmental regulation of NCAM, all of which have ramifications in its likely role as an effector of morphogenesis.

Animals↗

PubMed1991

Identification of a peptide specific for Aplysia sensory neurons by PCR-based differential screening.

In order to identify genes specific for the sensory neurons of Aplysia, a miniaturized differential screening method based on the polymerase chain reaction and applicable to small amounts of tissue was used. One messenger RNA was isolated that is expressed in every mechanoreceptor sensory cluster of the Aplysia central nervous system. This messenger RNA encodes a peptide that seems to function as an inhibitory cotransmitter. The peptide selectively inhibits certain postsynaptic cells but not others and thereby allows the sensory neurons to achieve target-specific synaptic actions.

Animals↗

PubMed1989

Novel structures CTLA-2 alpha and CTLA-2 beta expressed in mouse activated T cells and mast cells and homologous to cysteine proteinase proregions.

Differential screening of a subtracted cDNA library led to the detection of two distinct but homologous mouse cDNA, called CTLA-2 alpha and CTLA-2 beta. The corresponding transcripts have a tissue distribution restricted to T lymphocytes, where they are inducible upon activation, and to mast cells. The open-frame regions of both cDNA encode proteins homologous to cysteine proteinase precursors, remarkably, however, only to the proregion of these. The ctla-2 alpha and ctla-2 beta genes both map to the C1 band of mouse chromosome 13. Sequence comparisons suggest that the proregion of an ancestor proteinase gene evolved to the ctla-2 genes by successive duplications, first to autonomy, then to amplification. These results raise the question of the possible role of cysteine proteinase proregions, of cysteine proteinases themselves and of inhibitors thereof in activated T lymphocytes; from a different point of view, they also show that some protease proregions may have evolved as autonomous modules.

Amino Acid Sequence↗

PubMed1989

Developmentally regulated expression of the neural cell adhesion molecule (NCAM) by mouse thymocytes.

The expression of the neural cell adhesion molecule (NCAM) has been investigated during thymus ontogeny. NCAM mRNA was readily detectable at day 19 of gestation, the youngest age studied. Its level declined after birth to become undetectable at 3 weeks of age. Cell surface expression of NCAM protein was detected on 14% of day 15 fetal thymocytes and peaked during the perinatal period, when around 40% of the thymocytes expressed low to medium levels of NCAM. At postnatal day 2, the vast majority of the NCAM+ cells were also CD4+ and CD8+. At embryonic day 15, NCAM appeared also to be expressed by CD4- thymocytes since 14% of the cells were already NCAM+ whereas CD4 was virtually undetectable. In frozen section of the newborn thymus, surface staining for NCAM was present on a subpopulation of cells in the cortex, rare in the medulla and absent from the sub-capsular area. In conjunction with other cell adhesion molecules, NCAM could play a role in cell interactions during thymic development.

Age Factors↗

PubMed1988

A differential molecular biology search for genes preferentially expressed in functional T lymphocytes: the CTLA genes.

One approach to the isolation of molecules involved in T cell-mediated cytolysis stems from the postulate of a possible correlation between molecular phenotype and molecular functional involvement. Accordingly, CTL-specific molecules have been looked for, using a strategy based on the differential screening of a subtracted cDNA library. This led to the isolation and characterization of the following structures, expressed mostly (but no exclusively) in CTLs and inducible upon lymphocyte activation: CTLA-1 and CTLA-3 (serine-proteases), CTLA-4 (a member of the Ig superfamily) and CTLA-2 alpha and beta (homologues to the proregion of cysteine-proteases). The theoretical and practical limitations and the prospects of this type of approach are discussed.

Animals↗

PubMed1987

CTLA-1 and CTLA-3 serine esterase transcripts are detected mostly in cytotoxic T cells, but not only and not always.

We and other investigators previously reported the cloning of CTLA-1 (or CCP-1) and CTLA-3 (or H Factor) serine esterase-related transcripts preferentially expressed in cytolytic T lymphocytes. We extended the survey of the tissue specificity of these molecules. Two main sets of results were obtained. First, both CTLA-1 and CTLA-3 transcripts could be found in the various cytolytic T cells tested, although in widely different amounts, and in some cases just at the threshold of detection. Secondly, these transcripts were not found in most of the other cells tested, including in some natural cytotoxic cells and in activated cytotoxic macrophages; however, they could be detected in mast cells for CTLA-1 and in some noncytotoxic lymphocytes for CTLA-3. Thus, the CTLA-1 and CTLA-3 serine esterase products are most probably not required for macrophage or natural cytotoxicity; their presence cannot be taken as characteristic of cytotoxic T cells; and a discussion about their relevance to T cell-mediated cytotoxicity should take into account their widely different amounts from one cytotoxic T cell to another.

Animals↗

PubMed1986

Self-sparing of long-term in vitro-cloned or uncloned cytotoxic T lymphocytes.

At least some long-term in vitro-cultured cytotoxic T cell clones and uncloned cell populations are able, in the presence of Con A, to lyse other cells, to be lysed by other cells, but not to lyse themselves. This as-yet-unexplained result may have implications as to the mechanism of T cell-mediated cytotoxicity.

Clone Cells↗