E-GEOD-38668 - Translational profiling of hypocretin neurons identifies Lhx9 as necessary for normal development of the hypocretinergic system
Released on 22 March 2013, last updated on 2 June 2014
The immense molecular diversity of neurons challenges our ability to deconvolve the relationship between the genetic and the cellular underpinnings of neuropsychiatric disorders. Hypocretin (orexin) containing neurons of the lateral hypothalamus are clearly essential for the normal regulation of sleep and wake behaviors, and have been implicated in feeding, anxiety, depression and reward. However, little is known about the molecular phenotypes of these cells, or the mechanism of their specification. We have generated a Hcrt bacTRAP line for comprehensive translational profiling of these neurons in vivo. From this profile, we have identified 188 transcripts, as enriched in these neurons, in additions to thousands more moderately enriched or nominally expressed. We validated many of these at the RNA and protein level, including the transcription factor Lhx9. Lhx9 protein is found in a subset of these neurons, and ablation of these gene results in a 30% loss of Hcrt neuron number, and a profound hypersomnolence in mice. This data suggests that Lhx9 may be important for specification of some Hcrt neurons, and the subsets of these neurons may contribute to discrete sleep phenotypes. Four independent TRAP replicates were collected, and total RNA from both the immunoprecipitate(IP) and unbound(total) fractions were seperately amplified and hybridized. Unbound fractions are provided to serve as total RNA controls for the tissue. Biological replicates are GCRMA normalized within groups. Following averaging of replicates, we recommend further global normalization between groups, using affymetrix biotinylated controls, to correct for any broad biases in scanning and hybridization. Finally for many analyses, we also recommend filtering to remove those probesets with low IP/Total fold change values from each cell type(see PMID:20962086). Researchers can contact us for spreadsheets where these additional steps have been completed.
transcription profiling by array
Joseph D Dougherty <email@example.com>, Afua Akuffo, Brie Wamsley, David Holtzman, Han Yuan, Jasbir Dalal, Jee H Roh, Joseph Dougherty, Nathaniel Heintz, Samir Shah
Translational profiling of hypocretin neurons identifies candidate molecules for sleep regulation. Dalal J, Roh JH, Maloney SE, Akuffo A, Shah S, Yuan H, Wamsley B, Jones WB, Strong Cde G, Gray PA, Holtzman DM, Heintz N, Dougherty JD. , Europe PMC 23431030