Translating Ribosomal Affinity Purification Followed By Rnaseq (TRAP-Seq) Identifies Core Genes That Are Differentially Expressed in the GnRH Neuron Transcriptome
Presentation Number: SUN 489
Date of Presentation: April 2nd, 2017
Laura L Burger*, Elizabeth R Wagenmaker and Suzanne M Moenter
University of Michigan, Ann Arbor, MI
The GnRH neuron is at the nexus of fertility control, but it is difficult to examine their transcripts in situ, as these cells are few and widely dispersed. We used Translating Ribosomal Affinity Purification (TRAP1) with RNAseq to examine mRNAs of mature GnRH neurons in adult male and female mice in the presence (intact) or absence (gonadectomized [GDX]) of negative gonadal feedback. The ribosomes in GnRH neurons were tagged with eGFP by crossing GnRH-CRE mice with Rosa flox/stop eGFP-Rpl10a mice. Preoptic area (POA) punches were collected from a 2mm brain slice from: intact males, diestrus females, and males and females 7 days post GDX. Mice were 80-90d old at tissue harvest. POA punches from 5-7 mice were pooled to provide adequate RNA for cDNA library creation. For each treatment, there were 3-4 biological replicates (each pooled sample is a replicate). GFP-labeled polysomes from GnRH neurons were isolated from the total RNA by immunoprecipitation. Each biological replicate contains a pair of RNA samples: one refined for GnRH neuron transcripts and the other depleted. cDNA libraries were created with SMARTer v4 Ultra Low Kit (Clontech), and 50 base, paired-end sequencing using an Illumina Hi-Seq platform was done by the University of Michigan DNA Sequencing Core. Analysis was done by our Bioinformatics Core using Cufflinks/CuffDiff for expression quantitation and normalization, and for differential expression using UCSC mm10.fa as the reference genome sequence. The threshold for gene differential expression (enriched vs de-enriched in the GnRH neuron) was set at a fold change >1.5 or <0.66, with false discovery rate of p≤0.05. There were ~3000 genes differentially expressed in GnRH neuron-enriched vs depleted mRNA. A core of ~800 genes were differentially expressed in all 4 treatments. These included enrichment for Gnrh1 (~60 fold across treatments), and the transcripts: Six1, Six6, Otx1, Otx2, and Vax1 that have been reported to be critical for GnRH neuron and/or gonadotrope development. Also enriched in GnRH neuron RNAs were genes, such as Rab25, Epcam, Tmprss6 and others, that are epithelial markers, and suggest the olfactory placodal origin of the GnRH neuron. Notably, genes associated with astroglia (e.g., Slc25a18, Acsbg1, Ttpal, Gfap) were either not differentially expressed or were de-enriched in GnRH neurons. GnRH neurons were also de-enriched, as expected, for Ar and Esr1 in all treatments. Esr2 was not enriched in GnRH RNA; this may be due to low expression in both refined and depleted RNAs. Pgr was not differentially regulated. Of two major neuromodulators of GnRH neuron activity, excitatory kisspeptin and inhibitory GnIH, only receptors for the latter (Npffr1) were significantly enriched (all 4 groups). Knowledge of the GnRH neuron translatome and its regulation will help generate hypotheses for studying physiology in these cells and may be extended to disease states, such as PCOS.
Nothing to Disclose: LLB, ERW, SMM