Spatiotemporal expression and transcriptional perturbations by long noncoding RNAs in the mouse brain - PubMed (original) (raw)

. 2015 Jun 2;112(22):6855-62.

doi: 10.1073/pnas.1411263112.

Abigail F Groff, Martin Sauvageau, Zachary Trayes-Gibson, Diana B Sanchez-Gomez, Michael Morse, Ryan D Martin, Lara E Elcavage, Stephen C Liapis, Meryem Gonzalez-Celeiro, Olivia Plana, Eric Li, Chiara Gerhardinger, Giulio S Tomassy, Paola Arlotta, John L Rinn

Spatiotemporal expression and transcriptional perturbations by long noncoding RNAs in the mouse brain

Loyal A Goff et al. Proc Natl Acad Sci U S A. 2015.

Abstract

Long noncoding RNAs (lncRNAs) have been implicated in numerous cellular processes including brain development. However, the in vivo expression dynamics and molecular pathways regulated by these loci are not well understood. Here, we leveraged a cohort of 13 lncRNAnull mutant mouse models to investigate the spatiotemporal expression of lncRNAs in the developing and adult brain and the transcriptome alterations resulting from the loss of these lncRNA loci. We show that several lncRNAs are differentially expressed both in time and space, with some presenting highly restricted expression in only selected brain regions. We further demonstrate altered regulation of genes for a large variety of cellular pathways and processes upon deletion of the lncRNA loci. Finally, we found that 4 of the 13 lncRNAs significantly affect the expression of several neighboring proteincoding genes in a cis-like manner. By providing insight into the endogenous expression patterns and the transcriptional perturbations caused by deletion of the lncRNA locus in the developing and postnatal mammalian brain, these data provide a resource to facilitate future examination of the specific functional relevance of these genes in neural development, brain function, and disease.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

LncRNAs are temporally regulated in the developing and adult brain. β-Gal staining of lncRNA heterozygous mutant brain coronal sections at E14.5 (A, C, E, and G) and adult (B, D, F, and H) stages. (A) Lincenc1 is expressed in the VTel and in the IZ and lateral CP (Inset) of the DTel at E14.5. (B, Upper Right) In the adult brain, Lincenc1 is expressed in the primary somatosensory cortex, more specifically in layers II/III of the S1 S1Tr and layer IV in the S1BF. (Lower Right) β-Gal staining also shows expression in the hippocampus (Left) and the dentate gyrus. (C and D) At E14.5, Eldr is expressed in the VTel (C), but in the adult brain it is expressed in scattered cells in the upper layers of the neocortex as well as in layer VI cells lining the subcortical white matter (arrowheads) (D, Left, Right, and Inset). (E) No β-gal staining could be detected in Tug1 +/− brains at E14.5, except in the choroid plexus. (F, Left, Right, and Inset) In adult brains, Tug1 expression was detected in scattered cells in the neocortex. (G and H) β-Gal staining indicates expression of Peril in the VTel at E14.5 (G) and in the ependymal zone of the ventricles in the adult brain (H, Right and Inset). Images are representative of staining performed on two animals. CP, cortical plate; DG, dentate gyrus; DTel, dorsal telencephalon; Hip, hippocampus; IZ, intermediate zone; LV, lateral ventricle; Ncx, neocortex; S1BF, primary somatosensory cortex barrel field; S1Tr, primary somatosensory of the trunk; Str, striatum; VTel, ventral telencephalon; VZ/SVZ, ventricular zone/subventricular zone. [Scale bars: 500 μm (A_–_H, lower magnification panels), 100 μm (B, D, F, H, higher magnification right panels and inset).]

Fig. 2.

Fig. 2.

LncRNAs are expressed in distinct and specific regions in the adult brain. (A) β-gal staining of a coronal section from Ptgs2os2 +/− adult brain shows expression in layers II/III and IV of the neocortex (Ncx) and in CA1, CA2 and CA3 regions of the hippocampus. (B) β-gal staining shows Crnde is expressed in the hypothalamus and the thalamus. (C) Kantr is expressed mostly in scattered cells in the neocortex (Upper Right Inset) and in the CA1 of the hippocampus (Lower Right Inset). (D) Celrr is expressed at high levels in the substantia nigra, and β-gal (green) colocalizes with TH+ (magenta) dopaminergic neurons. Images are representative of staining performed on two animals. β-gal, β-galactosidase; CA, cornu ammonis; Hip, hippocampus; Ncx, neocortex; SN, substantia nigra; TH, tyrosine hydroxylase. [Scale bars: 500 μm (A_–_D, lower magnification left panels), 200 μm (A_–_D, higher magnification right panels), 20 μm (D, b-gal/TH-labeled confocal images).]

Fig. 3.

Fig. 3.

Cis effects of lncRNA deletion in the developing and adult brain. (A) Scatter plots of average lacZ expression (fragments per kilobase of exon per million fragments mapped, FPKM) in lncRNA KO versus endogenous lncRNA expression averaged across WT samples for all embryonic and adult samples. The line y = x is a reference for perfect correlation. (B) Summary of lncRNAs that significantly regulate their closest protein-coding neighbor. LncRNA mutant strains are shown in parenthesis. The x axis shows gene start distance (in kilobases) from the lncRNA transcriptional start site. The y axis shows log2-fold change of expression levels between WT and lncRNA KO. Genes in quadrants III and IV are down-regulated in the KO. The lncRNA is shown in blue, and the closest protein-coding neighbor is shown in red. The orientation of each gene is indicated with arrows. (C) Example of a _cis_-region plot. The x axis shows gene start distance (in megabases) from the lncRNA transcriptional start site. Here we show a truncated region spanning only a 2-Mb window rather than 4 Mb around the lncRNA of interest, Tug1. The y axis shows the test statistic (Cuffdiff2). Red color indicates significant differential expression between WT and KO. (D) Table of significance: P values for every _cis_-region plot. The five highlighted conditions have regional effects with significantly differentially regulated neighboring genes.

Fig. 4.

Fig. 4.

LncRNAs at the Pou3f3 genomic locus are specifically and dynamically regulated in the developing and adult brain. (A) Pou3f3 genomic locus and targeting strategy of the adjacent lncRNAs Pantr1 and Pantr2. The RNA-seq representative read density profiles for E14.5 and adult brains collected from Pantr1 −/− and Pantr2 −/− mice confirm deletion of each respective lncRNA compared with WT samples. (B) RNA-seq expression estimates (average from triplicates) in E14.5 and adult brains collected from WT, Pantr1 −/−, and Pantr2 −/− mutant mice for Pantr1, Pou3f3, and Pantr2. (C) β-Gal staining of lncRNA Pantr1 +/− (Left) and Pantr2 +/− (Right) adult brain coronal sections shows strong expression of Pantr1 in the CA1 region of the hippocampus (Pantr1, Lower Left), in both upper (II/III, IV) and deep (V, VI) layers of the neocortex, and in the caudate putamen, whereas Pantr2 is expressed most strongly in the medial habenula and at lower levels in the lateral habenula, layer V of the neocortex, and the CA1 of the hippocampus (Pantr2, Lower Right). Images are representative of staining performed on two animals. CA, cornu ammonis; CPu, caudate putamen; Hip, hippocampus; Hyp, hypothalamus; LHb, lateral habenular nucleus; MHb, medial habenular nucleus; Ncx, neocortex; O, stratum oriens of the cornu ammonis; P, stratum pyramidale of the cornu ammonis; R, stratum radiatum of the cornu ammonis; Th, thalamus. [Scale bars: 500 μm (lower magnification whole brain panels), 200 μm (higher magnification middle and bottom panels).]

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