During development of the CNS, neurons and glia are generated in a sequential manner. The mechanism underlying the later onset of gliogenesis is poorly understood, although the cytokineinduced Jak-STAT pathway has been postulated to regulate astrogliogenesis. Here, we report that the overall activity of Jak-STAT signaling is dynamically regulated in mouse cortical germinal zone during development. As such, activated STAT1/3 and STAT-mediated transcription are negligible at early, neurogenic stages, when neurogenic factors are highly expressed. At later, gliogenic periods, decreased expression of neurogenic factors causes robust elevation of STAT activity. Our data demonstrate a positive autoregulatory loop whereby STAT1/3 directly induces the expression of various components of the Jak-STAT pathway to strengthen STAT signaling and trigger astrogliogenesis. Forced activation of Jak-STAT signaling leads to precocious astrogliogenesis, and inhibition of this pathway blocks astrocyte differentiation. These observations suggest that autoregulation of the Jak-STAT pathway controls the onset of astrogliogenesis.During embryonic development, the generation of three major neural cell types (neurons, astrocytes, and oligodendrocytes) in the CNS occurs sequentially, whereby almost all neurons are generated before the appearance of glial cells 1,2 , with the exception of a few COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. NIH Public Access Author ManuscriptNat Neurosci. Author manuscript; available in PMC 2014 November 06. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript sites of postnatal and adult neurogenesis such as the subgranular zone (SGZ) of the hippocampus and the subventricular zone (SVZ) of the forebrain 3 . This strategy of building the CNS through sequential production of neurons and glia has become more comprehensible, as recent findings have demonstrated that glial cells are important in critical neuronal maturation processes such as axonal path finding and synapse formation [4][5][6] . It is conceivable that delayed or precocious production of glial cells may lead to inappropriate wiring, disorganization, and eventually, dysfunction of the CNS.The 'neurons-first, glia-second' differentiation theme for neural progenitors can be recapitulated in culture. Cortical neural progenitor stem cells isolated from relatively early embryonic stages (for example, mouse embryonic day (E) 10-11) give rise to neurons, not glial cells, after short-term culturing (fewer than 4 d), whereas cortical progenitors isolated from perinatal stages tend to differentiate into astrocytes under the same culture conditions 7 .In addition, both E10-11 cortical progenitors and embryonic stem cell-derived neural stem or progenitor cells (NSCs or NPCs) switch from being neurogenic to gliogenic over time in vitro 8,9 , suggesting that the molecular switch for the transition from neurogenesis to gliogenesis may be internally programmed in neural progenitors.S...

Mutations resulting in defective splicing constitute a significant proportion (30/62 [48%]) of a new series of mutations in the ATM gene in patients with ataxia-telangiectasia (AT) that were detected by the protein-truncation assay followed by sequence analysis of genomic DNA. Fewer than half of the splicing mutations involved the canonical AG splice-acceptor site or GT splice-donor site. A higher percentage of mutations occurred at less stringently conserved sites, including silent mutations of the last nucleotide of exons, mutations in nucleotides other than the conserved AG and GT in the consensus splice sites, and creation of splice-acceptor or splice-donor sites in either introns or exons. These splicing mutations led to a variety of consequences, including exon skipping and, to a lesser degree, intron retention, activation of cryptic splice sites, or creation of new splice sites. In addition, 5 of 12 nonsense mutations and 1 missense mutation were associated with deletion in the cDNA of the exons in which the mutations occurred. No ATM protein was detected by western blotting in any AT cell line in which splicing mutations were identified. Several cases of exon skipping in both normal controls and patients for whom no underlying defect could be found in genomic DNA were also observed, suggesting caution in the interpretation of exon deletions observed in ATM cDNA when there is no accompanying identification of genomic mutations.

No abstract

No abstract

Effective biological application of nanocrystalline semiconductor quantum dots continues to be hampered by the lack of easily implemented and widely applicable labeling chemistries. Here, we introduce two new orthogonal nanocrystal bioconjugation chemistries that overcome many of the labeling issues associated with currently utilized approaches. These chemistries specifically target either (1) the ubiquitous amines found on proteins or (2) thiols present in either antibody hinge regions or recombinantly introduced into other proteins to facilitate site-specific labeling. The amine chemistry incorporates aniline-catalyzed hydrazone bond formation, while the sulfhydryl chemistry utilizes nanocrystals displaying surface activated maleimide groups. Both reactive chemistries are rapidly implemented, yielding purified nanocrystal-protein bioconjugates in as little as 3 h. Following initial characterization of the nanocrystal materials, the wide applicability and strong multiplexing potential of these chemistries are demonstrated in an array of applications including immunoassays, immunolabeling in both cellular and tissue samples, in vivo cellular uptake, and flow cytometry. Side-by-side comparison of the immunolabeled cells suggested a functional equivalence between results generated with the amine and thiol-labeled antibody-nanocrystal bioconjugates in that format. Three-color labeling was achieved in the cellular uptake format, with no significant toxicity observed while simultaneous five-color labeling of different epitopes was demonstrated for the immunolabeled tissue sample. Novel labeling applications are also facilitated by these chemistries, as highlighted by the ability to directly label cellular membranes in adherent cell cultures with the thiol-reactive chemistry.

No abstract

No abstract

Neural stem cells (NSCs) were isolated from embryonic day 16 Sprague-Dawley rats and cultured in a novel serum-free stem cell medium that selected for the growth of NSCs and against the growth of GFAP(+) cells (astrocytes). NSCs maintained in culture for extended periods of time retained immunoreactivity for both nestin and PSA-NCAM, two markers characteristic of the stem cell phenotype. Moreover, using an oligodendrocyte (OL) specification medium, NSCs differentiated into OL as evidenced by their morphology and expression of multiple oligodendrocyte/myelin-specific markers. In addition, NSCs are capable of acquiring a neuronal phenotype as evidenced by expressing neuronal markers, such as neurofilament (NF) and NeuN when cultured in a defined medium for neurons indicating that these cells are also a good source of neuroblasts, which could be used to replace neuronal populations in the brain. We also showed successful propagation and differentiation of NSCs into OL after cryostorage, allowing for the later use of stored NSCs. The long-term goal of culturing NSCs and committed oligodendrocyte progenitors (OLP) is to obtain homogeneous populations for transplantation with the goal of remyelinating the myelin-deficient CNS. Our preliminary experiments carried out on normal and myelin deficient rats demonstrate that these cells survive and migrate extensively in both types of hosts. NSCs grafted as such, as well as cells derived from NSCs exposed to selective specification before grafting, are able to differentiate within the host brain. As expected, NSCs are capable of giving rise to astrocytes in a medium favoring this phenotype.