<i>Laf4</i> is expressed in the developing mouse cortex.

<p><i>In situ</i> hybridisation of <i>Laf4</i> in the mouse embryo at E11.5, E13.5 and E15.5 (A). Expression is observed in the brain and somites (arrowheads) at E11.5 and 13.5 (A). By E15.5 expression is observed in the developing cortex below the medial zone (MZ) in the cortical plate (CP) and subventricular zone (SVZ) (B). The boxed region in (A) is shown in (B) with a control sense riboprobe from an adjacent section. Scale bars = 2 mm in (A), 100 µM in (B).</p

Knockdown of <i>Laf4</i> causes a defect in the migration of cortical cells.

<p>E14 mouse brains were electroporated with shRNA-Laf4 or shRNA-scramble vectors with a control (IRES-GFP) or the Laf4-rescue-IRES-GFP vector and cultured for six days before re-sectioning for image analysis (A). The average percentage of GFP⁺ cells in 5 zones (example zones Z1–Z5 shown) spanning from the ventricle (V) to the pial border was quantified (B). Brains electroporated with shRNA-Laf4 construct show a significant reduction in GFP⁺ cells corresponding to migrating cells to the superficial layers of the cortex; this phenotype is almost completely reversed by co-electroporation with a Laf4-rescue-IRES-GFP construct. (<i>n</i> = 6 brains quantified for each vector combination; *<i>p</i><0.05, **<i>p</i><0.01 (2-way ANOVA Bonferroni’s multiple comparison test). Scale bar = 50 µM.</p

Knockdown of <i>Laf4</i> by shRNA.

<p>Quantitative RT-PCR of <i>Laf4</i> in N2a (A and B) and primary cortical cells (C and D). Comparative over-expression of HA-tagged Laf4 (Laf4-HA-IRES-GFP) and the shRNA control (Laf4-HA-rescue-IRES-GFP) occurs at similar levels in both cell types; data are shown versus endogenous <i>Laf4</i> (negative control HA-IRES-GFP vector) (A and C). Knockdown of endogenous <i>Laf4</i> in both cell types after transfection (B) or electroporation (D) with shRNA-Laf4 shows the recovery of <i>Laf4</i> expression using Laf4-HA-rescue-IRES-GFP. (E) Protein levels from over-expression of Laf4 (Laf4-HA-IRES-GFP), but not the control (Laf4-HA-rescue-IRES-GFP) construct is significantly knocked-down by the same shRNA<i>-</i>Laf4 in HeLa cells. The negative control shRNA (shRNA-scramble) has no effect on the levels of either endogenous <i>Laf4</i> (B and D) or exogenous over-expressed Laf4 (E). In all panels, quantification of three independent repeats in shown (*<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001, 1-way ANOVA Bonferroni’s multiple comparison test).</p

Identification of Laf4 as a potential transcriptional regulator of <i>Mdga2</i>.

<p>(A) qRT-PCR analysis of four genes originally shown to be up-regulated from microarray transcriptome profiling of FACS-sorted primary cortical cells over-expressing Laf4 (Laf4-HA-IRES-GFP) compared to an empty control (HA-IRES-GFP) vector. Significant up-regulation of <i>Gatad1, Mdga2, Ndufs</i> and <i>Scna3</i> was confirmed in <i>Laf4</i> over-expressing cells (<i>n</i> = 3 biological replicates *<i>p</i><0.05, **<i>p</i><0.01, 2-way ANOVA Bonferroni’s multiple comparison test). (B) ChIP from cortical cells transfected and FACS-sorted as in (A) was performed using an anti-HA antibody to HA-tagged Laf4-IRES-GFP or empty vector (HA-IRES-GFP). qPCR showed specific binding of Laf4 to <i>Mdga2</i> at the beginning of the coding region (primer pair 3 and 4 shown in (C)) and at the end of the first exon/beginning of the first intron (primer pair 4). Laf4 did not appear to bind to <i>Mdga1</i> for the primers tested. (<i>n</i> = 3 biological replicates; *<i>p</i><0.05, ***<i>p</i><0.001). 1-way ANOVA Bonferroni’s multiple comparison test used to compare each primer sets to primer 5 (Mdga2) or 3 (Mdga1) used as control primer set. (D) qRT-PCR analysis of <i>Mdga2</i> levels in cortical cells transfected with shRNA-Laf4 vector (*<i>p</i><0.01, 1-way ANOVA Bonferroni’s multiple comparison test).</p

Over-expression of <i>Mdga2</i> rescues the deficit of cortical cells migration generated by <i>Laf4</i> knockdown.

<p>(A) E14 mouse brains were electroporated with shRNA-Laf4 or shRNA-scramble vectors with the control IRES-GFP or an Mdga2-IRES-GFP vector and cultured for six days before re-sectioning for image analysis. The average percentage of GFP⁺ cells in 5 zones spanning from the ventricle (V) to the pial border was quantified (B). Co-electroporation of shRNA-Laf4 and Mdga2-IRES-GFP constructs results in rescue of the migration defect observed using shRNA-Laf4 alone. (<i>n</i> = 4 brains quantified for each vector combination; *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001, 2-way ANOVA Bonferroni’s multiple comparison test).</p

Differences in gene expression between WT and FTO-4 mice based on microarray data.

<p>Expression of the indicated genes in the cerebellum (A), hypothalamus (B), WAT (C) and gastrocnemius (D) of wildtype (WT, black bars) and FTO-4 (white bars) mice. Data are expressed as the fold change compared to wildtype. Significance was tested using Student's t-test to compare FTO-4 to WT. ***p<0.001, **p<0.01, *p<0.05.</p

Effect of FTO overexpression on m6A levels in RNA.

<p>FTO mRNA expression in wild-type (black bar) and FTO-4 (white bar) MEFs as the fold change compared to wild-type (A). m6A levels measured as a percentage of adenosine levels for mRNA and total RNA by LC/MS in wild-type (black bar) and FTO-4 (white bar) MEFs (B). Significance was tested using Student's t-tests to compare FTO-4 to WT data. **p<0.01.</p

Overview of potential m6A sites in obesity-related genes with changed expression in FTO-4 mice.

<p>Pie chart of obesity-related genes whose expression is changed in the indicated tissues of FTO-4 mice. Dark grey, m6A sites in their mRNA transcripts <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097162#pone.0097162-Meyer1" target="_blank">[23]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097162#pone.0097162-Dominissini1" target="_blank">[24]</a>. Light grey, no m6A sites <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097162#pone.0097162-Meyer1" target="_blank">[23]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097162#pone.0097162-Dominissini1" target="_blank">[24]</a>. The number of genes involved is indicated: see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097162#pone-0097162-t002" target="_blank">Table 2</a> for gene names.</p

Top 10 genes that showed the highest amount of overexpression in FTO-4 mice per tissue.

<p>Top 10 genes that showed the highest amount of overexpression in FTO-4 mice per tissue.</p

Top 10 genes that showed the highest amount of downregulation in FTO-4 mice per tissue.

<p>Top 10 genes that showed the highest amount of downregulation in FTO-4 mice per tissue.</p