Quantitative and functional interrogation of parent-of-origin allelic expression biases in the brain.

The maternal and paternal genomes play different roles in mammalian brains as a result of genomic imprinting, an epigenetic regulation leading to differential expression of the parental alleles of some genes. Here we investigate genomic imprinting in the cerebellum using a newly developed Bayesian statistical model that provides unprecedented transcript-level resolution. We uncover 160 imprinted transcripts, including 41 novel and independently validated imprinted genes. Strikingly, many genes exhibit parentally biased--rather than monoallelic--expression, with different magnitudes according to age, organ, and brain region. Developmental changes in parental bias and overall gene expression are strongly correlated, suggesting combined roles in regulating gene dosage. Finally, brain-specific deletion of the paternal, but not maternal, allele of the paternally-biased Bcl-x, (Bcl2l1) results in loss of specific neuron types, supporting the functional significance of parental biases. These findings reveal the remarkable complexity of genomic imprinting, with important implications for understanding the normal and diseased brain.

Proliferative and transcriptional identity of distinct classes of neural precursors in the mammalian olfactory epithelium.

Neural precursors in the developing olfactory epithelium (OE) give rise to three major neuronal classes - olfactory receptor (ORNs), vomeronasal (VRNs) and gonadotropin releasing hormone (GnRH) neurons. Nevertheless, the molecular and proliferative identities of these precursors are largely unknown. We characterized two precursor classes in the olfactory epithelium (OE) shortly after it becomes a distinct tissue at midgestation in the mouse: slowly dividing self-renewing precursors that express Meis1/2 at high levels, and rapidly dividing neurogenic precursors that express high levels of Sox2 and Ascl1. Precursors expressing high levels of Meis genes primarily reside in the lateral OE, whereas precursors expressing high levels of Sox2 and Ascl1 primarily reside in the medial OE. Fgf8 maintains these expression signatures and proliferative identities. Using electroporation in the wild-type embryonic OE in vitro as well as Fgf8, Sox2 and Ascl1 mutant mice in vivo, we found that Sox2 dose and Meis1 - independent of Pbx co-factors - regulate Ascl1 expression and the transition from lateral to medial precursor state. Thus, we have identified proliferative characteristics and a dose-dependent transcriptional network that define distinct OE precursors: medial precursors that are most probably transit amplifying neurogenic progenitors for ORNs, VRNs and GnRH neurons, and lateral precursors that include multi-potent self-renewing OE neural stem cells.

Selection and validation of microarray candidate genes from subregions and subnuclei of the brain.

DNA array technology now allows an enormous amount of expression data to be obtained. For large-scale gene profiling enterprises, this is of course welcome. However, the scientist interested in follow-up studies of a handful of differentially expressed genes may find it hard to sift through the vast datasets to pinpoint genes with the most desirable and reliable behaviors. Here, we present the methodology we have employed to discover genes differentially expressed in the adult mouse brain. We first used Affymetrix microarrays to compare gene expression from five different brain regions: the amygdala, cerebellum, hippocampus, olfactory bulb, and periaqueductal gray. Second, we identified genes differentially expressed within three distinct amygdala subnuclei. In this case, the tissue was microdissected by laser-capture to minimize contamination from adjacent subnuclei, and extracted RNA was subjected to three rounds of linear amplification prior to hybridization to the microarrays. To select candidate genes, we developed a custom algorithm to identify those genes with the most robust changes in expression across different replicate samples. Confirmation of expression patterns with in situ hybridization uncovered further criteria to consider in the selection process.

Transient expression of the bHLH factor neurogenin-2 marks a subpopulation of neural crest cells biased for a sensory but not a neuronal fate.

Lineage-tracing experiments have indicated that some premigratory neural crest cells (NCCs) are pleuripotent, generating sensory and sympathetic neurons and their associated glia. Using an inducible Cre recombinase-based fate mapping system, we have permanently marked a subpopulation of NCCs that expresses Ngn2, a bHLH transcription factor required for sensory neurogenesis, and compared its fate to the bulk NCC population marked by expression of Wnt1. Ngn2(+) progenitors were four times more likely than Wnt1(+) NCCs to contribute to sensory rather than sympathetic ganglia. Within dorsal root ganglia, however, both Ngn2- and Wnt1-expressing cells were equally likely to generate neurons or glia. These data suggest that Ngn2 marks an NCC subpopulation with a predictable fate bias, early in migration. Taken together with previous work, these data suggest that NCCs become restricted to sensory or autonomic sublineages before becoming committed to neuronal or glial fates.