Oncostatin M regulates neural precursor activity in the adult brain.

The regulation of neural precursor cell (NPC) activity is the major determinant of the rate of neuronal production in neurogenic regions of the adult brain. Here, we show that Oncostatin M (Osm) and its receptor, OsmRß, are both expressed in the subventricular zone (SVZ) and that in contradistinction to leukemia inhibitory factor and ciliary neutrophic factor, Osm directly inhibits the proliferation of adult NPCs as measured by a decreased level of neurosphere formation in vitro. Similarly, intraventricular infusion of Osm dramatically decreases the pool of NPCs in both the SVZ and the hippocampus. In keeping with the inhibitory action of Osm, we reveal that mice lacking OsmRß have substantially more NPCs in the SVZ, the hippocampus and the olfactory bulb, demonstrating that endogenous Osm signaling is important for NPC homeostasis. Finally, we show that Osm can also inhibit clonal growth of glioblastoma-derived neurospheres, further supporting the close link between NPCs and tumor stem cells.

Mice carrying a R142C Notch 3 knock-in mutation do not develop a CADASIL-like phenotype.

CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy, MIM 125310) is a genetic vascular dementia disease that is linked to missense mutations, small in-frame deletions, and splice site mutations in the human Notch 3 gene. Here we describe the generation of a mouse knockin model for one of the most prevalent CADASIL mutations, an arginine to cysteine transition at position 141, R141C, which corresponds to mutation R142C in mouse NOTCH 3. CADASIL(R142C) mice show no apparent CADASIL-like phenotype after histological and MRI analysis. The NOTCH 3 (R142C) receptor is processed normally and does not appear to accumulate the ectodomain, which has been observed in CADASIL patients. We discuss possible reasons for the different outcomes of the same germline CADASIL mutation in mice and humans.

Characterization of Notch3-deficient mice: normal embryonic development and absence of genetic interactions with a Notch1 mutation.

The Notch signaling pathway is an evolutionarily conserved signaling mechanism and mutations in its components disrupt cell fate specification and embryonic development in many organisms. To analyze the in vivo role of the Notch3 gene in mice, we created a deletion allele by gene targeting. Embryos homozygous for this mutation developed normally and homozygous mutant adults were viable and fertile. We also examined whether we could detect genetic interactions during early embryogenesis between the Notch3 mutation and a targeted mutation of the Notch1 gene. Double homozygous mutant embryos exhibited defects normally observed in Notch1-deficient embryos, but we detected no obvious synergistic effects in the double mutants. These data demonstrate that the Notch3 gene is not essential for embryonic development or fertility in mice, and does not have a redundant function with the Notch1 gene during early embryogenesis.

A CADASIL-mutated Notch 3 receptor exhibits impaired intracellular trafficking and maturation but normal ligand-induced signaling.

Notch receptors are single transmembrane receptors that contain a large number of epidermal growth factor-like repeats (EGF repeats) in their extracellular domains. Mutations in the EGF repeats of the human Notch 3 receptor lead to the vascular dementia disease Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL). The vast majority of CADASIL mutations are missense mutations removing or inserting cysteine residues in the EGF repeats, but it is not yet clear whether these mutations primarily affect receptor trafficking, maturation, andor signaling. To address this issue, we have generated and analyzed stable cell lines expressing either wild-type murine Notch 3 (mNotch 3) or the mutant mNotch 3(R142C), which corresponds to the prevalent CADASIL form of Notch 3, Notch 3(R141C) in humans. We find that a lower proportion of mNotch 3(R142C) is expressed in the site 1-cleaved configuration, and that reduced amounts of mNotch 3(R142C) appear at the cell surface, as compared with wild-type mNotch 3. This observation is accompanied by a higher propensity for mNotch 3(R142C) to form intracellular aggregates, which may be a result of increased accumulation or slowed transport in the secretory pathway. In contrast to the impaired cell surface expression, mNotch 3(R142C) signals equally well in response to Delta 1 and Jagged 1 as wild-type mNotch 3. Taken together, these data suggest that trafficking and localization rather than signaling of mNotch 3 are affected in mNotch 3(R142C).

Notch signaling is involved in the regulation of Id3 gene transcription during Xenopus embryogenesis.

During Xenopus embryogenesis, XId3, a member of the Id helix-loop-helix protein family, is expressed in a large variety of differentiating tissues including epidermis, cement gland, brain, neural tube, neural crest cell derivatives, somites, and tailbud. Transcription of XId3 is mediated by several cis-regulatory elements including an enhancer of 440 bp located 870 bp upstream from the transcription initiation site. The enhancer activity in embryos was studied using transgenic methodology. A galactosidase reporter gene, driven by a regulatory element composed of the enhancer and a minimal promoter derived from the XId3 gene, was expressed in transgenic embryos with a profile that faithfully reproduced that of the endogenous XId3 gene. The pattern resulted from a synergistic effect between the enhancer and the promoter, and in vitro transactivation assays showed that transcription can be stimulated by Notch signaling. The presence of potential Su(H) binding sites, in both the enhancer and the promoter, suggests that these represent candidates for in vivo cis-regulatory elements. The data presented here suggest that Notch control of differentiation may involve activation of transcription of Id, a negative regulator of bHLH transcription factors.