Biomedical subjects

J F Brunet

Publications and source records attributed to J F Brunet.

At least 19 recordsLinked to original sources

PubMed2004

Early acquisition of typical metabolic features upon differentiation of mouse neural stem cells into astrocytes.

Specific metabolic features, such as glutamate reuptake, have been associated with normal functions of mature astrocytes. In this study, we examined whether these characteristics are acquired together with classical phenotypic markers of differentiated astrocytes. Differentiation of E14 mouse neurospheres into astrocytes was induced by the addition of fetal bovine serum (FBS). Degree of differentiation was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence for both GFAP and nestin. Neural stem cells expressed nestin but not GFAP, while differentiated astrocytes were immunopositive for GFAP but displayed low levels of nestin expression. A strong increase in the expression of the glutamate transporter GLAST and the monocarboxylate transporter MCT1 accompanied phenotypic changes. In addition, active glutamate transport appeared in differentiated astrocytes, as well as their capacity to increase aerobic glycolysis in response to glutamate. Leukemia inhibitory factor (LIF) and ciliary neurotrophic factor, but not interleukin-6, triggered the expression of phenotypic and morphological characteristics of astrocytes. In addition, exposure to LIF led to the appearance of metabolic features typically associated with astrocytes. Altogether, our results show that acquisition of some specific metabolic features by astrocytes occurs early in their differentiation process and that LIF represents a candidate signal to induce their expression.

Animals↗

PubMed2003

Aquaporin 1 and aquaporin 4 expression in human brain after subarachnoid hemorrhage and in peritumoral tissue.

Aquaporins (AQPs) are a protein family of water channels which facilitate the water flux through the plasmatic membranes. The expression of AQPs has been described in rat brain by several studies. Despite recent reports that have shown an over-expression of AQP1 and 4 in human tumoral cells, little is known about AQP expression in human brain. The purpose of this study was to investigate the expression of AQP1 and AQP4 in human brain after subarachnoid hemorrhage (SAH) and in peritumoral tissue by western blot and immunohistochemistry. The results showed a marked increase of the expression of AQP1 and AQP4. This over-expression occurred on the astrocytic processes and polarization on astrocytic end-feet was lost. No expression was observed on neuronal cells. This study is the first demonstration of the induction of AQP1 and AQP4 on reactive astrocytes in an acute brain injury, such as SAH. These results reinforce the hypothesis that AQPs may be involved in the dynamics of brain edema formation or resolution. Further studies are needed to understand their functional role.

Aquaporin 1↗

PubMed2001

Isolation of multipotent neural precursors residing in the cortex of the adult human brain.

Multipotent precursors able to generate neurons, astrocytes, and oligodendrocytes have previously been isolated from human brain embryos and recently from neurogenic regions of the adult human brains. The isolation of multipotent neural precursors from adult human should open new perspectives to study adult neurogenesis and for brain repair. The present study describes the in vitro isolation from adult human brains of a progenitor responsive to both epidermal and basic fibroblast growth factors that forms spheres as it proliferates. Single spheres derived from various regions of the brain generate in vitro neurons, astrocytes, and oligodendrocytes. The clonal origin of the spheres was revealed by genomic viral insertion using lentiviral vector. Interestingly, this vector appears to be a potent tool for gene transfer into human neural progeny. Ninety-six percent of the spheres investigated were multipotent. Multipotent precursors were isolated from all brain regions studied, including the temporal and the frontal cortex, the amygdala, the hippocampus, and the ventricular zone. This study is the first evidence that primitive precursors such as multipotent precursors exist in the adult human cortex and can reside far from the ventricles. Neurogenesis derived from adult human progenitors differ to murine neurogenesis by the requirement of laminin for oligodendrocyte generation and by the action of basic-fibroblast growth factor and platelet derived growth factor that prevented the formation of oligodendrocytes and neurons. Moreover, the differentiation of human adult precursors seems to differ from fetal ones: adult precursors do not necessitate the removal of mitogen for differentiation. These results indicate that the study of adult multipotent precursors is a new platform to study adult human neurogenesis, potentially generate neural cells for transplantation, and design protocols for in vivo stimulation.

Adolescent↗

PubMed2000

Specification of the central noradrenergic phenotype by the homeobox gene Phox2b.

The closely related homeobox genes Phox2a and Phox2b are expressed in all central and peripheral noradrenergic neurons. Our previous results have shown that Phox2a controls the differentiation of the main noradrenergic center of the brain, the locus coeruleus, but leaves unaffected the other noradrenergic centers. Here, we report that Phox2b has a wider and overlapping role, in that it is required for the differentiation of all noradrenergic centers in the brain, including the locus coeruleus. Together with the previously reported lack of dopamine-b-hydroxylase and tyrosine hydroxylase expression in the peripheral nervous system of Phox2b knock-out embryos, our present findings make Phox2b a master regulator of all central and peripheral noradrenergic differentiation. We discuss the nonredundancy of Phox2 genes and their complex partnership with the bHLH transcription factor Mash1, which is also required for the differentiation of most noradrenergic cell types.

Adrenergic Fibers↗

PubMed2000

Role of the target in the pathfinding of facial visceral motor axons.

Axon navigation depends, in part, on guidance cues emanating from the target. We have investigated the possible role of the target in the pathfinding of visceral motor axons to cranial parasympathetic ganglia. Mice homozygous for a tau-LacZ transgene targeted in the Phox2a locus lack the sphenopalatine ganglion, which is the normal target of visceral motor axons of the facial nerve. We found that in these mutants, facial visceral motor axon pathfinding was disrupted, and some axons were misrouted to an alternative parasympathetic ganglion. Moreover, the absence of correct facial visceral motor pathways was concomitant with defects in the pathfinding of rostrally-projecting sympathetic axons.

Animals↗

PubMed2000

The homeodomain protein CePHOX2/CEH-17 controls antero-posterior axonal growth in C. elegans.

An essential aspect of a neuron's identity is the pattern of its axonal projections. In C. elegans, axons extend either longitudinally or circumferentially in response to distinct molecular cues, some of which have been identified. It is currently unclear, however, how the differential capacity to respond to these cues is transcriptionally implemented in distinct neuronal subtypes. Here, we characterise a C. elegans paired-like homeobox gene, CePhox2/ceh-17, expressed in five head neurons, ALA and the 4 SIAs, all of which project axons towards the tail along the lateral and sublateral cords. Abrogation of ceh-17 function, while leaving intact many phenotypic traits of these neurons, disrupts their antero-posterior axonal elongation beyond the mid-body region. Conversely, ectopic expression of ceh-17 in the mechanoreceptors, several of which are known to pioneer their tract, leads to exaggerated longitudinal axonal outgrowth. Thus, ceh-17 is a novel gene involved in fasciculation-independent longitudinal axonal navigation.

Amino Acid Sequence↗

PubMed2000

The Phox2b transcription factor coordinately regulates neuronal cell cycle exit and identity.

In the vertebrate neural tube, cell cycle exit of neuronal progenitors is accompanied by the expression of transcription factors that define their generic and sub-type specific properties, but how the regulation of cell cycle withdrawal intersects with that of cell fate determination is poorly understood. Here we show by both loss- and gain-of-function experiments that the neuronal-subtype-specific homeodomain transcription factor Phox2b drives progenitor cells to become post-mitotic. In the absence of Phox2b, post-mitotic neuronal precursors are not generated in proper numbers. Conversely, forced expression of Phox2b in the embryonic chick spinal cord drives ventricular zone progenitors to become post-mitotic neurons and to relocate to the mantle layer. In the neurons thus generated, ectopic expression of Phox2b is sufficient to initiate a programme of motor neuronal differentiation characterised by expression of Islet1 and of the cholinergic transmitter phenotype, in line with our previous results showing that Phox2b is an essential determinant of cranial motor neurons. These results suggest that Phox2b coordinates quantitative and qualitative aspects of neurogenesis, thus ensuring that neurons of the correct phenotype are generated in proper numbers at the appropriate times and locations.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Control of hindbrain motor neuron differentiation by the homeobox gene Phox2b.

Motor neurons are a widely studied model of vertebrate neurogenesis. They can be subdivided in somatic, branchial and visceral motor neurons. Recent studies on the dorsoventral patterning of the rhombencephalon have implicated the homeobox genes Pax6 and Nkx2.2 in the early divergence of the transcriptional programme of hindbrain somatic and visceral motor neuronal differentiation. We provide genetic evidence that the paired-like homeodomain protein Phox2b is required for the formation of all branchial and visceral, but not somatic, motor neurons in the hindbrain. In mice lacking Phox2b, both the generic and subtype-specific programs of motoneuronal differentiation are disrupted at an early stage. Most motor neuron precursors die inside the neuroepithelium while those that emigrate to the mantle layer fail to switch on early postmitotic markers and to downregulate neuroepithelial markers. Thus, the loss of function of Phox2b in hindbrain motor neurons exemplifies a novel control point in the generation of CNS neurons.

Animals↗

PubMed1999

The homeobox gene Phox2b is essential for the development of autonomic neural crest derivatives.

The sympathetic, parasympathetic and enteric ganglia are the main components of the peripheral autonomic nervous system, and are all derived from the neural crest. The factors needed for these structures to develop include the transcription factor Mash1, the glial-derived neurotrophic factor GNDF and its receptor subunits, and the neuregulin signalling system, each of which is essential for the differentiation and survival of subsets of autonomic neurons. Here we show that all autonomic ganglia fail to form properly and degenerate in mice lacking the homeodomain transcription factor Phox2b, as do the three cranial sensory ganglia that are part of the autonomic reflex circuits. In the anlagen of the enteric nervous system and the sympathetic ganglia, Phox2b is needed for the expression of the GDNF-receptor subunit Ret and for maintaining Mash1 expression. Mutant ganglionic anlagen also fail to switch on the genes that encode two enzymes needed for the biosynthesis of the neurotransmitter noradrenaline, dopamine-beta-hydroxylase and tyrosine hydroxylase, demonstrating that Phox2b regulates the noradrenergic phenotype in vertebrates.

Animals↗

PubMed1999

Deconstructing cell determination: proneural genes and neuronal identity.

Vertebrates express scores of bHLH proteins during neural development. Earlier studies inspired by the established role of "proneural" genes in fly neurogenesis, as well as by the vertebrate bHLH myogenic program, focused on the reconstruction of bHLH gene cascades, which are thought to control successive steps leading to neuronal differentiation. Little attention has been paid thus far to the relationship between the diversity of neural bHLH genes and the diversity of neuronal phenotypes. This article reviews recent evidence that, akin to their fly counterparts, vertebrate neural bHLH genes probably confer not only "generic" neuronal properties, but also neuronal type-specific properties, inextricably linking neural determination and the specification of neuronal identity. We also speculate on the relations between positional information and gene activity, and on the evolutionary significance of the diversity of bHLH genes.

Animals↗

PubMed1999

Specification of neurotransmitter identity by Phox2 proteins in neural crest stem cells.

We have investigated the specification of noradrenergic neurotransmitter identity in neural crest stem cells (NCSCs). Retroviral expression of both wild-type and dominant-negative forms of the paired homeodomain transcription factor Phox2a indicates a crucial and direct role for this protein (and/or the closely related Phox2b) in the regulation of endogenous tyrosine hydroxylase (TH) and dopamine-beta hydroxylase (DBH) gene expression in these cells. In collaboration with cAMP, Phox2a can induce expression of TH but not of DBH or of panneuronal genes. Phox2 proteins are, moreover, necessary for the induction of both TH and DBH by bone morphogenetic protein 2 (BMP2) (which induces Phox2a/b) and forskolin. They are also necessary for neuronal differentiation. These data suggest that Phox2a/b coordinates the specification of neurotransmitter identity and neuronal fate by cooperating environmental signals in sympathetic neuroblasts.

Adrenal Glands↗

PubMed1999

Transcriptional control of neurotransmitter phenotype.

The specification of neurotransmitter phenotype is an important aspect of neuronal fate determination. Recent studies have begun to define essential transcriptional regulators involved in controlling the mode of neurotransmission in vertebrates and invertebrates, and to examine their regulation by cell-extrinsic factors. An emerging concept is that the control of transmitter choice is intimately linked to that of other aspects of the neuronal phenotype.

Animals↗

PubMed1999

Induction of the epibranchial placodes.

The cranial sensory ganglia, in contrast to those of the trunk, have a dual embryonic origin arising from both neurogenic placodes and neural crest. Neurogenic placodes are focal thickenings of ectoderm, found exclusively in the head of vertebrate embryos. These structures can be split into two groups based on the positions that they occupy within the embryo, dorsolateral and epibranchial. The dorsolateral placodes develop alongside the central nervous system, while the epibranchial placodes are located close to the top of the clefts between the branchial arches. Importantly, previous studies have shown that the neurogenic placodes form under the influence of the surrounding cranial tissues. In this paper, we have analysed the nature of the inductive signal underlying the formation of the epibranchial placodes. We find that epibranchial placodes do not require neural crest for their induction, but rather that it is the pharyngeal endoderm that is the source of the inductive signal. We also find that, while cranial ectoderm is competent to respond to this inductive signal, trunk ectoderm is not. We have further identified the signalling molecule Bmp7 as the mediator of this inductive interaction. This molecule is expressed in a manner consistent with it playing such a role and, when added to ectoderm explants, it will promote the formation of epibranchial neuronal cells. Moreover, the Bmp7 antagonist follstatin will block the ability of pharyngeal endoderm to induce placodal neuronal cells, demonstrating that Bmp7 is required for this inductive interaction. This work answers the long standing question regarding the induction of the epibranchial placodes, and represents the first elucidation of an inductive mechanism, and a molecular effector, underlying the formation of any primary sensory neurons in higher vertebrates.

Animals↗

PubMed1998

Noradrenergic-specific transcription of the dopamine beta-hydroxylase gene requires synergy of multiple cis-acting elements including at least two Phox2a-binding sites.

Dopamine beta-hydroxylase (DBH) catalyzes the conversion of dopamine to noradrenaline and is selectively expressed in noradrenergic and adrenergic neurons and neuroendocrine cells. Recent data from this laboratory showed that a paired-like homeodomain (HD) protein, Phox2a, interacts with the HD-binding site residing within a composite promoter of the human DBH gene, designated domain IV, in a cell-specific manner and directly controls noradrenergic-specific DBH promoter activity. In this report, we demonstrate that three additional protein-binding sites (i.e., domains I, II, and III) between domain IV and the TATA box are critical for intact DBH promoter activity in noradrenergic cells and that they activate DBH transcription in a highly concerted manner. Transient transfection assays of mutant DBH reporter constructs indicated that domain I was active in every cell line tested, whereas domain III was preferentially active in DBH-positive cells. Remarkably, mutation of domain II was associated with inactivation of DBH promoter activity exclusively in DBH-positive cell lines, defining it as another noradrenergic-specific promoter element. The cell-specific profile of the promoter function of these sequence motifs was further supported by in vitro DNA-binding studies and Southwestern analysis. Furthermore, competition and antibody supershift assays show that transcription factors Sp1 and AP2 are the cognate nuclear factors interacting with domains I and III, respectively. Parallel evidence indicates that domain II is another Phox2a-binding site, demonstrating at least two binding sites for this factor in the upstream DBH promoter. Strikingly, four tandem copies of domain II increased the promoter activity of a minimal DBH promoter by 100- to 200-fold in DBH-positive cell lines without compromising cell specificity. Cotransfection of Phox2a-expression vector dramatically increased the activity of the multiple domain II promoter only in DBH-negative cell lines, confirming that domain II is responsive to Phox2a. Collectively, this study emphasizes a critical role of Phox2a as well as its functional synergism with other transcription factors (e.g., CREB, AP2, and Sp1) in transcriptional activation of the DBH gene.

Animals↗

PubMed1998

A single rostrocaudal colonization of the rodent intestine by enteric neuron precursors is revealed by the expression of Phox2b, Ret, and p75 and by explants grown under the kidney capsule or in organ culture.

The colonization of the rodent gastrointestinal tract by enteric neuron precursors is controversial due to the lack of specific cellular markers at early stages. The transcription factor, Phox2b, is expressed by enteric neuron precursors (Pattyn et al. Development 124, 4065-4075, 1997). In this study, we have used an antiserum to Phox2b to characterize in detail the spatiotemporal expression of Phox2b in the gastrointestinal tract of adult mice and embryonic mice and rats. In adult mice, all enteric neurons (labeled with neuron-specific enolase antibodies), and a subpopulation of glial cells (labeled with GFAP antibodies), showed immunoreactivity to Phox2b. In embryonic mice, the appearance of Phox2b-immunoreactive cells was mapped during development of the gastrointestinal tract. At Embryonic Days 9.5-10 (E9.5-10), Phox2b-labeled cells were present only in the stomach, and during subsequent development, labeled cells appeared as a single rostrocaudal wave along the gastrointestinal tract; at E14 Phox2b-labeled cells were present along the entire length of the gastrointestinal tract. Ret and p75 have also been reported to label migratory-stage enteric neuron precursors. A unidirectional, rostral-to-caudal colonization of the gastrointestinal tract of embryonic mice by Ret- and p75-immunoreactive cells was also observed, and the locations of Ret- and p75-positive cells within the gut were very similar to that of Phox2b-positive cells. To verify the location of enteric neuron precursors within the gut, explants from spatiotemporally defined regions of embryonic intestine, 0.3-3 mm long, were grown in the kidney subcapsular space, or in catenary organ culture, and examined for the presence of neurons. The location and sequence of appearance of enteric neuron precursors deduced from the explants grown under the kidney capsule or in organ culture was very similar to that seen with the Phox2b, Ret, and p75 antisera. Previous studies have mapped the rostrocaudal colonization of the rat intestine by enteric neuron precursors using HNK-1 as a marker. In the current study, all HNK-1-labeled cells in the gastrointestinal tract of rat embryos showed immunoreactivity to Phox2b, but HNK-1 cells comprised only a small subpopulation of the Phox2b-labeled cells. In addition, in rats, Phox2b-labeled cells were present in advance of (more caudal to) the most caudal HNK-1-labeled cells by 600-700 microm in the hindgut at E15. We conclude that the neural crest cell population that arises from the vagal level of the neural axis and that populates the stomach, midgut, and hindgut expresses Phox2b, Ret, and p75. In contrast, the sacral-level neural crest cells that populate the hindgut either do not express, or show a delayed expression of, all of the known markers of vagal- and trunk-level neural crest cells.

Animals↗

PubMed1998

Paired-like homeodomain proteins, Phox2a and Phox2b, are responsible for noradrenergic cell-specific transcription of the dopamine beta-hydroxylase gene.

Recently, a murine paired-like homeobox gene, Phox2a, has been identified whose product is critical for the development of several major noradrenergic neuron populations, including the locus coeruleus. In noradrenergic neurons, dopamine beta-hydroxylase (DBH) is a hallmark protein and catalyzes the conversion of dopamine to noradrenaline. Our previous studies have shown that a composite promoter (domain IV), residing at -185 to -150 bp upstream of the transcription start site, is critical for DBH transcription and is comprised of multiple cis-acting elements, including a cyclic AMP response element, a YY1 binding site, and two core motifs of the homeodomain (HD)-binding site. Here, we show that the HD-binding site residing within domain IV is a noradrenergic-specific cis-acting element. In contrast, the cyclic AMP response element is active in all cell lines tested. We provide evidence that Phox2a is expressed only in DBH-positive cell lines and interacts with the HD-binding site. Forced expression of Phox2a robustly activates DBH promoter activity in DBH-negative cell lines (>10-fold), but increased it only marginally (<50%) in DBH-positive cell lines. Furthermore, another protein factor with an identical HD, Phox2b, also activates DBH transcription with an efficiency comparable to that of Phox2a. In contrast, neither Phox2a nor Phox2b was able to transactivate tyrosine hydroxylase transcription, indicating that these transcription factors differentially activate catecholamine-synthesizing gene transcription. Together with the Phox2a knockout experiment, the studies described here make Phox2a and Phox2b the first strong candidate transcription factors for determining a neurotransmitter phenotype in vertebrates.

Animals↗

PubMed1998

Control of noradrenergic differentiation and Phox2a expression by MASH1 in the central and peripheral nervous system.

Mash1, a mammalian homologue of the Drosophila proneural genes of the achaete-scute complex, is transiently expressed throughout the developing peripheral autonomic nervous system and in subsets of cells in the neural tube. In the mouse, targeted mutation of Mash1 has revealed a role in the development of parts of the autonomic nervous system and of olfactory neurons, but no discernible phenotype in the brain has been reported. Here, we show that the adrenergic and noradrenergic centres of the brain are missing in Mash1 mutant embryos, whereas most other brainstem nuclei are preserved. Indeed, the present data together with the previous results show that, except in cranial sensory ganglia, Mash1 function is essential for the development of all central and peripheral neurons that express noradrenergic traits transiently or permanently. In particular, we show that, in the absence of MASH1, these neurons fail to initiate expression of the noradrenaline biosynthetic enzyme dopamine beta-hydroxylase. We had previously shown that all these neurons normally express the homeodomain transcription factor Phox2a, a positive regulator of the dopamine beta-hydroxylase gene and that a subset of them depend on it for their survival. We now report that expression of Phox2a is abolished or massively altered in the Mash1-/- mutants, both in the noradrenergic centres of the brain and in peripheral autonomic ganglia. These results suggest that MASH1 controls noradrenergic differentiation at least in part by controlling expression of Phox2a and point to fundamental homologies in the genetic circuits that determine the noradrenergic phenotype in the central and peripheral nervous system.

Animals↗

PubMed1997

Mouse Otlx2/RIEG expression in the odontogenic epithelium precedes tooth initiation and requires mesenchyme-derived signals for its maintenance.

The mouse Otlx2 gene is a new member of the paired-like family of homeobox genes whose human homologue, RIEG, is involved in Rieger syndrome, an autosomal-dominant disorder. One of the cardinal features of Rieger syndrome is dental hypoplasia, indicating that Otlx2/RIEG activity is essential for normal tooth development. Here, we analyzed the expression of Otlx2 during mouse tooth development and studied its regulation in dental explants. Otlx2 expression distinguishes stomatodeal from other ectoderm as early as Embryonic Day 8.5, well before tooth initiation. Thereafter, its craniofacial expression becomes restricted to the tooth-forming areas and to the epithelial components of molar and incisor primordia. Although Otlx2 induction precedes the specification of odontogenic mesenchyme, tissue recombination experiments show that the maintenance of its expression requires signals from the mesenchyme and that dental mesenchyme has the capacity to induce ectopic expression of Otlx2 in nondental epithelium. Finally, we compare Otlx2 expression with that of the recently identified homeodomain transcription factor Barx1 expressed in molar mesenchyme. Their strictly complementary expression patterns in the epithelial and mesenchymal components suggest that both genes participate in the reciprocal tissue interactions which are a hallmark of odontogenesis.

Animals↗