Understanding the fate of adult-generated neurons and the mechanisms that influence them requires consistent labeling and tracking of large numbers of stem cells. We generated a nestin-CreER^T2/R26R-yellow fluorescent protein (YFP) mouse to inducibly label nestin-expressing stem cells and their progeny in the adult subventricular zone (SVZ) and subgranular zone (SGZ). Several findings show that the estrogen ligand tamoxifen (TAM) specifically induced recombination in stem cells and their progeny in nestin-CreER^T2/R26R-YFP mice: 97% of SGZ stem-like cells (GFAP/Sox2 with radial glial morphology) expressed YFP; YFP+ neurospheres could be generatedin vitroafter recombinationin vivo, and maturing YFP+ progeny were increasingly evident in the olfactory bulb (OB) and dentate gyrus (DG) granule cell layer. Revealing an unexpected regional dissimilarity in adult neurogenesis, YFP+ cells accumulated up to 100 d after TAM in the OB, but in the SGZ, YFP+ cells reached a plateau 30 d after TAM. In addition, most SVZ and SGZ YFP+ cells became neurons, underscoring a link between nestin and neuronal fate. Finally, quantification of YFP+ cells in nestin-CreER^T2/R26R-YFP mice allowed us to estimate, for example, that stem cells and their progeny contribute to no more than 1% of the adult DG granule cell layer. In addition to revealing the dynamic contribution of nestin-expressing stem cells to adult neurogenesis, this work highlights the utility of the nestin-CreER^T2/R26R-YFP mouse for inducible gene ablation in stem cells and their progenyin vivoin the two major regions of adult neurogenesis.

HLA-G is a nonclassical class I MHC molecule with an unknown function and with unusual characteristics that distinguish it from other class I MHC molecules. Here, we demonstrate that HLA-G forms disulfide-linked dimers that are present on the cell surface. Immunoprecipitation of HLA-G from surface biotinylated transfectants using the anti-β2-microglobulin mAb BBM.1 revealed the presence of an ≈78-kDa form of HLA-G heavy chain that was reduced by using DTT to a 39-kDa form. Mutation of Cys-42 to a serine completely abrogated dimerization of HLA-G, suggesting that the disulfide linkage formed exclusively through this residue. A possible interaction between the HLA-G monomer or dimer and the KIR2DL4 receptor was also investigated, but no interaction between these molecules could be detected through several approaches. The cell-surface expression of dimerized HLA-G molecules may have implications for HLA-G/receptor interactions and for the search for specific receptors that bind HLA-G.

The adult mammalian olfactory bulb (OB) receives a continuing influx of new interneurons. Neuroblasts from the subventricular zone (SVZ) migrate into the OB and differentiate into granule cells and periglomerular cells that are presumed to integrate into the synaptic circuits of the OB. We have used retroviral infection into the SVZ of mice to label adult-generated granule cells and follow their differentiation and integration into OB circuitry. Using synaptic markers and electron microscopy, we show new granule cells integrating into the reciprocal circuitry of the external plexiform layer (EPL), beginning at 21 d postinfection (dpi). We further show that synapses are formed earlier, beginning at 10 dpi, on the somata and basal dendrites of new cells in the granule cell layer (GCL), before dendritic elaboration in the EPL. In the EPL, elaborate dendritic arbors with spines are first evident at 14 dpi. The density of spines increases from 14 to 28 dpi, and then decreases by 56 dpi. Despite the initial appearance of dendritic spines at 14 dpi in the EPL, no expression of presynaptic or postsynaptic markers is seen until 21 dpi. These data suggest that adult-generated granule cells are first innervated by centrifugal or mitral/tufted cell axon collaterals in the GCL and that these inputs may contribute to their differentiation, maturation, and synaptic integration into the dendrodendritic local circuits found in the EPL.

We generated a knockout mouse for the neuronal-specific β-tubulin isoform Tubb3 to investigate its role in nervous system formation and maintenance. Tubb3-/- mice have no detectable neurobehavioral or neuropathological deficits, and upregulation of mRNA and protein of the remaining β-tubulin isotypes results in equivalent total β-tubulin levels in Tubb3-/- and wild-type mice. Despite similar levels of total β-tubulin, adult dorsal root ganglia lacking TUBB3 have decreased growth cone microtubule dynamics and a decreased neurite outgrowth rate of 22% in vitro and in vivo. The effect of the 22% slower growth rate is exacerbated for sensory recovery, where fibers must reinnervate the full volume of the skin to recover touch function. Overall, these data reveal that, while TUBB3 is not required for formation of the nervous system, it has a specific role in the rate of peripheral axon regeneration that cannot be replaced by other β-tubulins.