Date of Award
obesity, leptin receptor, ventromedical hypothalamic nucleus
The ventromedial hypothalamic nucleus (VMH) plays an important role in the regulation of food intake, glucose metabolism, and body weight. However, in contrast to other hypothalamic nuclei that are also known to regulate energy homeostasis, there is a paucity of nucleus-specific marker genes for the VMH that can be used to label its constituent neurons. This represents a significant impediment to the application of molecular approaches for analyzing VMH circuitry and function. Thus, we conducted a microarray screen in order to identify VMH-specific genes that could be used to label populations of VMH neurons. Laser-capture microdissection was used to isolate RNA from the VMH and from two adjacent hypothalamic nuclei known to play a role in energy balance, the arcuate (ARC) and dorsomedial hypothalamic nucleus (DMH). Amplified RNA from these three nuclei were intercompared to identify genes with VMH-enriched expression. The top 12 VMH marker gene candidates were screened by real-time PCR, and three genes (Cerebellin 1, PACAP, and a novel gene we characterized, LBH2) were examined by in situ hybridization for further validation and examination of their subnuclear expression profile. One of the VMH markers, steroidogenic factor 1 (SF-1), is an orphan nuclear receptor with few known target genes. As this transcription factor is responsible for proper developmental formation of the VMH and also for normal energy homeostasis, we endeavored to determine whether any of the VMH marker genes may be regulated by SF- 1. The expression of 4 markers was significantly altered in VMH neurons of SF-1 2 knockout mice, and this result was confirmed by an in situ hybridization study of cerebellin 1 expression in brain-specific SF-1 knockout mice. One of the VMH markers was a previous undescribed gene that we further characterized and named LBH2 owing to its similarity to a presumed transcription factor called limb bud and heart (LBH). To further examine the expression of this gene in the VMH, and to begin to describe VMH neuronal populations, we created LBH2-GFP BAC transgenic mouse line expressing GFP under the control of the LBH2 promoter. These mice were validated for correct expression of the transgene and examined for overlap with other populations of interest in the VMH, including estrogen receptor-alpha neurons and leptin-responsive neurons. Finally, we used a TK+ strain of PRV Bartha2001 to trace neuronal inputs from AgRP neurons in the ARC using AgRP-cre mice. Using this technique, it could be determined that the dorsomedial and intermediate VMH sends significant outputs to this population of ARC neurons. This technique, together with the marker genes discovered, may now be used to identify and catalog individual VMH neuronal subsets that project to these neurons. In conclusion, we have discovered a set of marker genes for the VMH using lasercapture microdissection coupled with cDNA microarray analysis. This combination of techniques represents a powerful approach for the identification of genes enriched in specific, anatomically-defined brain regions. The discovery of multiple genes regulated by SF-1 also suggests this technique may be useful for identifying nucleus-specific transcriptional networks. The VMH-enriched genes identified here, in conjunction with LBH2-GFP mice and other transgenic animals, will provide a basis for a full 3 characterization of VMH neurons, and will prove greatly useful for future neuroanatomic and transgenic-based studies of this important nucleus.
Segal, Jeremy P., "A Molecular Dissection of the Ventromedial Hypothalamic Nucleus" (2006). Student Theses and Dissertations. 5.