OR08-3 Chimera Analysis of Pomc Neuron Function Utilizing Combinations of Fluorescently Tagged Wild-Type and Arcuate Nucleus-Specific Pomc Deficient Mouse Embryos

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: OR08-Obesity: Novel Mechanisms of Body Weight Regulation
Basic
Saturday, June 15, 2013: 11:30 AM-1:00 PM
Presentation Start Time: 12:00 PM
Room 301 (Moscone Center)
Miho Yamashita*1, Veronica Otero-Corchon1, Thomas L Saunders1, Lev Fedorov2 and Malcolm James Low1
1University of Michigan, Ann Arbor, MI, 2Oregon Health & Science University, Portland, MI
Hypothalamic proopiomelanocortin (POMC) neurons in the arcuate nucleus (ARC) are heterogeneous for their afferent and efferent signaling properties. Discrete or partially overlapping subpopulations respond to leptin, insulin and 5-HT and utilize GABA or glutamate as a neurotransmitter. Little is known about the functional consequences of this diversity at the systems level. To address the physiological function of subpopulations of POMC neurons in an unbiased fashion, we are using somatic chimeras in which each mouse has stochastically determined juxtapositions of genetically distinct POMC neurons. As proof of principle, we first generated a series of chimeras by aggregation of morulae pairs derived from transgenic mice expressing either a green (EGFP) or red (tDimer-dsRed) fluorescent protein in ARC POMC neurons and pituitary corticotrophs and melanotrophs. Nine chimeras exhibited unique distribution patterns of fluorescently labeled cells in the pituitary gland and throughout the ARC, with no cellular co-expression of the two fluorescent tags.  The percentage of neuronal chimerism ranged from 10-90%. We next crossed a mutant ArcPomcfneo allele containing a neomycin cassette in the vicinity of the neuronal-specific upstream enhancers, which silences Pomc expression in the ARC but not pituitary, to the transgenic strain with cell autonomous expression of EGFP in all POMC cells. These mice were used to generate a novel embryonic stem (ES) cell line that is homozygous for both the ArcPomcfneo and PomcEGFPtg alleles. Initial production of chimeras by the aggregation of these ES cells with morulae from PomcdsRedtg/tg mice was inefficient, but demonstrated the expected result of mixed neuronal populations in the ARC; green neurons that lack POMC peptide immunoreactivity and red neurons that normally express POMC. Microinjection of the ArcPomcfneo/fneo:PomcEGFPtg/tg ES cells into blastocysts derived from the PomcdsRedtg/tg mice mice has yielded 13 chimeras to date. Each mouse is being phenotyped longitudinally by evaluations of body weight, fat mass, length, food intake, refeeding response, locomotor activity, O2 consumption, and corticosterone, terminating with a detailed histological analysis of the ARC and POMC projections throughout the brain. A multivariate analysis will determine whether the number and spatial localization of subpopulations of Pomc expressing and non-expressing POMC neurons can predict the final metabolic and obesity outcome of the mice.

Nothing to Disclose: MY, VO, TLS, LF, MJL

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm

Sources of Research Support: NIH grant DK066604 awarded to MJL.