Reference Genes across Nine Brain Areas of Wild Type and Prader-Willi Syndrome Mice: Assessing Differences in Igfbp7, Pcsk1, Nhlh2 and Nlgn3 Expression

Abstract

Prader−Willi syndrome (PWS) is a complex neurodevelopmental disorder caused by the deletion or inactivation of paternally expressed imprinted genes at the chromosomal region 15q11−q13. The PWS-critical region (PWScr) harbors tandemly repeated non-protein coding IPW-A exons hosting the intronic SNORD116 snoRNA gene array that is predominantly expressed in brain. Paternal deletion of PWScr is associated with key PWS symptoms in humans and growth retardation in mice (PWScr model). Dysregulation of the hypothalamic−pituitary axis (HPA) is thought to be causally involved in the PWS phenotype. Here we performed a comprehensive reverse transcription quantitative PCR (RT-qPCR) analysis across nine different brain regions of wild-type (WT) and PWScr mice to identify stably expressed reference genes. Four methods (Delta Ct, BestKeeper, Normfinder and Genorm) were applied to rank 11 selected reference gene candidates according to their expression stability. The resulting panel consists of the top three most stably expressed genes suitable for gene-expression profiling and comparative transcriptome analysis of WT and/or PWScr mouse brain regions. Using these reference genes, we revealed significant differences in the expression patterns of Igfbp7, Nlgn3 and three HPA associated genes: Pcsk1, Pcsk2 and Nhlh2 across investigated brain regions of wild-type and PWScr mice. Our results raise a reasonable doubt on the involvement of the Snord116 in posttranscriptional regulation of Nlgn3 and Nhlh2 genes. We provide a valuable tool for expression analysis of specific genes across different areas of the mouse brain and for comparative investigation of PWScr mouse models to discover and verify different regulatory pathways affecting this complex disorder.

Keywords: Igfbp7; Nhlh2; Nlgn3; PWS-critical region; Pcsk1; Pcsk2; Prader–Willi syndrome; RT-qPCR; SNORD116; brain; brain regions; gene expression; posttranscriptional regulation; reference genes; transcriptome.

Figure 1

Mouse brain regions and expression of selected reference gene candidates. (A) Schematic representation of a sagittal section of an adult mouse brain with nine analyzed regions (colored and labelled). Areas that were not analyzed are shown in gray. (B) The resulting Cq values of the selected eight reference gene candidates in all nine brain regions analyzed (mean ± SD). Wild-type bars are depicted in dark gray, PWScr^m+/p— in white.

Figure 2

Identification of the reference gene panel. (A,C) Expression stability ranking across nine brain regions of reference gene candidates in WT and PWScr^m+/p− mice, respectively, determined by four different stability ranking methods. Avg. ranking: shows the calculation of the resulting average ranking of the candidate genes (highlighted in gray; bottom row). (B,D) Average ranking position of each of the genes (lower average rank/green is more stable than higher average rank/red) across the nine brain regions of WT (B) and PWScr^m+/p− (D) mice, respectively. (E) Average expression stability ranking positions of gene candidates across the nine brain regions in WT (top) and PWScr^m+/p− (bottom) mice. (F) Average ranking of expression stability between the two genotypes (WT and PWScr^m+/p−) within each of the nine brain regions for the reference gene candidates. (G) Compilation of reference gene panel. Regions: average ranking of genes for expression stability within regions of genotypes; genotypes: average ranking of expression stability across investigated genotypes within each brain region; combination: recommended panel of reference genes, i.e., the three highest ranked genes, highlighted in gray.

Figure 3

Expression analysis of Pcsk1, Pcsk2, Igfbp7 and Nhlh2. (A) Comparative expression analysis of Igfbp7, (B) Pcsk1, (C) Pcsk2, and (D) Nhlh2 genes relative to reference gene panel in nine brain regions of male mice P28 (WT in gray, PWScr^m+/p− in white). Mean ± SD of three biological replicates (six for WT hypothalamus); * p ≤ 0.05, ** p ≤ 0.01, unpaired two-samples t-test. (E) Northern blot hybridization analysis of Snord116 and Snord115 expression in brain areas and tissues of WT mice (left and middle panels, respectively); and PWScr^m+/p− (right panel) animals. As a loading control, tRNAs (negative image of an ethidium bromide-stained gel) is shown at the bottom.

Figure 4

Effect of Snord116 knock-out on expression and alternative splicing of Nlgn3 in mouse brain. (A) Putative complementarity of Snord116 (red) with exon 3 of Nlgn3 pre-mRNA (gray). 3′-splicing site (acceptor) indicated in yellow. Schematic representation of alternative splicing of Nlgn3 mRNA into isoform 1 (including exon 3) and isoform(s) 2 (excluding exon 3), respectively. Forward and reverse primers for the designed RT-qPCR assays are depicted as black arrows under the exons. (B) Expression levels of total Nlgn3, (C) Nlgn3 isoform 1, and (D) Nlgn3 isoform 2 relative to the reference gene panel in nine brain regions of WT and PWScr^m+/p− mice are depicted with gray and white bars, respectively, as Mean ± SD of three biological replicates (six for WT hypothalamus); * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, unpaired two-samples t-test.