Characterization of a new mouse line triggering transient oligodendrocyte progenitor depletion.

Oligodendrocyte progenitor cells (OPC) are the main proliferative cells in the healthy adult brain. They produce new myelinating oligodendrocytes to ensure physiological myelin remodeling and regeneration after various pathological insults. Growing evidence suggests that OPC have other functions. Here, we aimed to develop an experimental model that allows the specific ablation of OPC at the adult stage to unravel possible new functions. We generated a transgenic mouse expressing a floxed human diphtheria toxin receptor under the control of the PDGFRa promoter, crossed with an Olig2Cre mouse to limit the recombination to the oligodendrocyte lineage in the central nervous system. We determined a diphtheria toxin dose to substantially decrease OPC density in the cortex and the corpus callosum without triggering side toxicity after a few daily injections. OPC density was normalized 7 days post-treatment, showing high repopulation capacity from few surviving OPC. We took advantage of this strong but transient depletion to show that OPC loss was associated with behavioral impairment, which was restored by OPC recovery, as well as disruption of the excitation/inhibition balance in the sensorimotor cortex, reinforcing the hypothesis of a neuromodulatory role of OPC in the adult brain.

Periodic notch inhibition by lunatic fringe underlies the chick segmentation clock.

The segmented aspect of the vertebrate body plan first arises through the sequential formation of somites. The periodicity of somitogenesis is thought to be regulated by a molecular oscillator, the segmentation clock, which functions in presomitic mesoderm cells. This oscillator controls the periodic expression of 'cyclic genes', which are all related to the Notch pathway. The mechanism underlying this oscillator is not understood. Here we show that the protein product of the cyclic gene lunatic fringe (Lfng), which encodes a glycosyltransferase that can modify Notch activity, oscillates in the chick presomitic mesoderm. Overexpressing Lfng in the paraxial mesoderm abolishes the expression of cyclic genes including endogenous Lfng and leads to defects in segmentation. This effect on cyclic genes phenocopies inhibition of Notch signalling in the presomitic mesoderm. We therefore propose that Lfng establishes a negative feedback loop that implements periodic inhibition of Notch, which in turn controls the rhythmic expression of cyclic genes in the chick presomitic mesoderm. This feedback loop provides a molecular basis for the oscillator underlying the avian segmentation clock.

Notch signalling acts in postmitotic avian myogenic cells to control MyoD activation.

During Drosophila myogenesis, Notch signalling acts at multiple steps of the muscle differentiation process. In vertebrates, Notch activation has been shown to block MyoD activation and muscle differentiation in vitro, suggesting that this pathway may act to maintain the cells in an undifferentiated proliferative state. In this paper, we address the role of Notch signalling in vivo during chick myogenesis. We first demonstrate that the Notch1 receptor is expressed in postmitotic cells of the myotome and that the Notch ligands Delta1 and Serrate2 are detected in subsets of differentiating myogenic cells and are thus in position to signal to Notch1 during myogenic differentiation. We also reinvestigate the expression of MyoD and Myf5 during avian myogenesis, and observe that Myf5 is expressed earlier than MyoD, consistent with previous results in the mouse. We then show that forced expression of the Notch ligand, Delta1, during early myogenesis, using a retroviral system, has no effect on the expression of the early myogenic markers Pax3 and Myf5, but causes strong down-regulation of MyoD in infected somites. Although Delta1 overexpression results in the complete lack of differentiated muscles, detailed examination of the infected embryos shows that initial formation of a myotome is not prevented, indicating that exit from the cell cycle has not been blocked. These results suggest that Notch signalling acts in postmitotic myogenic cells to control a critical step of muscle differentiation.

Role of growth factors in shaping the developing somite.

In the vertebrate embryo, the lateral somite gives rise to limb bud and body wall muscles whereas the medial somite generates the axial musculature. We show that in chick embryos, this polarity along the medio-lateral axis is achieved through the antagonistic influences of the lateral plate and the medial neural tube. Bone morphogenetic protein 4 (BMP4) mediates the lateralising signal delivered by the lateral plate and is counteracted locally by Noggin expressed in the medial dermomyotome; Noggin expression in the somite is regulated by the Wntl protein which is expressed in the dorsal neural tube and mediates the medialising effect of the neural tube. Therefore, somite medio-lateral patterning results from a signalling cascade in which Wnt1 produced by the neural tube promotes noggin expression in the medial somite which in turn antagonises lateral plate-derived BMP4. This mechanism could lead to the establishment of a BMP4 activity gradient that would produce appropriate BMP4 signalling to generate medial and lateral somite patterning.

Noggin acts downstream of Wnt and Sonic Hedgehog to antagonize BMP4 in avian somite patterning.

In the vertebrate embryo, the lateral compartment of the somite gives rise to muscles of the limb and body wall and is patterned in response to lateral-plate-derived BMP4. Activation of the myogenic program distinctive to the medial somite, i.e. relatively immediate development of the epaxial muscle lineage, requires neutralization of this lateral signal. We have analyzed the properties of molecules likely to play a role in opposing lateral somite specification by BMP4. We propose that the BMP4 antagonist Noggin plays an important role in promoting medial somite patterning in vivo. We demonstrate that Noggin expression in the somite is under the control of a neural-tube-derived factor, whose effect can be mimicked experimentally by Wnt1. Wnt1 is appropriately expressed in the neural tube. Furthermore, we show that Sonic Hedgehog is able to activate ectopic expression of Noggin resulting in the blocking of BMP4 specification of the lateral somite. Our results are consistent with a model in which Noggin activation lies downstream of the SHH and Wnt signaling pathways.

Antibody against Bacillus thuringiensis phosphatidylinositol-phospholipase C: some examples of its potential uses.

We have purified the phosphatidylinositol-phospholipase C enzyme from the bacterium Bacillus thuringiensis. This enzyme is able to release in soluble form molecules which are anchored to membranes via a glycan-phosphatidylinositol group. It exhibits a molecular weight of 33-35 kDa. We raised polyclonal antisera against the molecule and used them in immunoblot as well as radioimmunoassays for enzyme detection. This last technique should facilitate monitoring of chromatographic steps during enzyme purification. We coupled antibodies to Sepharose beads in order to remove the enzyme from incubation media. This reagent also proved to be particularly useful in control experiments designed to ascertain that the observed release of molecules is due to the action of the phosphatidylinositol-phospholipase C enzyme and not to spontaneous release or to cleavage by nonspecific hydrolases. A search for cross-reactive molecules in other bacterial strains or mammalian tissues gave negative results. This leads to the conclusion that a great diversity exists between phosphatidylinositol-phospholipases C, even among different bacterial strains.

Post-translation modifications of neural cell surface molecules.

Neural cells seem to express a relatively low number of adhesion molecules at their surface. The hypothesis of cell surface modulation postulates that a repertoire of specificities and binding affinities might result from alteration over time of the amount, distribution or chemical properties of a particular kind of molecules. We are describing two post-translational modifications; glycosylation and glypiation; which affect the neural cell adhesive molecule (N-CAM). We produced a monoclonal antibody recognizing only the highly sialyled of N-CAM and reported some of its applications. We also showed that N-CAM-120 is anchored to the membranes via a complex glycane-phosphatidylinositol.

Cell membrane, but not circulating, carcinoembryonic antigen is linked to a phosphatidylinositol-containing hydrophobic domain.

Carcinoembryonic antigen is present in the cell membrane of most tumors of colorectal origin and in the plasma of patients with colorectal cancer and other malignancies. In this paper we demonstrate that carcinoembryonic antigen can be released from HT-29 cells by phosphatidylinositol specific phospholipase C. Triton X-114 phase separation shows that phospholipase C converts the antigen into a water soluble protein. In addition, plasma carcinoembryonic antigen behaves as the cleaved antigen in phase separation experiments. This strongly suggests that carcinoembryonic antigen is attached to cell membranes by a glycosyl-phosphatidylinositol anchor and that it can be released in vivo by enzymatic cleavage of the hydrophobic tail.