Gene expression profiling and 3′ untranslated region (3′UTR) length in differentiating adherent neural stem (ANS) cells. RNAseq data were obtained from ANS cells at T0 (proliferating), T1, T2 and T3 (three timepoints of GABAergic differentiation). (A) Cluster analyses and heat Map of differentially expressed coding mRNAs, relevant to the control of cell cycle exit (in green) and early neuronal differentiation (in yellow). The color-code of the raw Z-scores is shown on the right. (B,C) Venn diagrams summarizing the number of transcripts showing shortening (B) or lengthening (C) during inhibitory neuron differentiation, comparing proliferating (T0) with differentiating (T1, T2, T3) ANS cells. (D,E) Technical validation of 3′UTR lengthening. A scheme illustrating the strategy used to quantify the relative abundance of the long vs. common forms of 3′UTR is shown in (D). Ten selected 3′UTRs were examined by Real-Time qPCR analysis (in E). Eight of them showed changes in the relative abundance consistent with the RNAseq data. See also for a full list. *p < 0.05.

Pes1 and Gng2 transcript variants in the mouse embryonic brain. Detection and localization of variants of Pes1 and Gng2 transcripts in the mouse embryonic cortex and ganglionic eminence (GE) of the forebrain, to reveal differential abundance in proliferating vs. differentiating regions. (A) Scheme showing the Gng2 and Pes1 gene organization, the location of the alternative polyA+ sites and of the probes used to detect all variants (common) or the long-variants. Scale is shown on the right. (B–D) Coronal sections of normal mouse embryonic forebrain at the age E14.5 subjected to two-colors fluorescent in situ hybridization (FISH) to determine the localization and relative abundance of variants of the Gng2 mRNAs. At least 10 sections were stained, obtained from two brain specimens. Representative images of two-colors FISH to detect all variants (red, in B) or the long variant (green, in C) Gng2 transcripts in the basal brain primordium. A merged image is shown in (D). (E) Histogram and profile of the relative fluorescent intensity of the images in (B–D), from the area indicated in (D; white rectangle, subdivided in ventricular zone(VZ) and subventricular/mantle zone (SVZ/MZ). At least two areas/slide were considered. The results relative to the VZ or the SVZ/MZ are shown as histogram. Gng2-all variants is indicated with red bars and lines, the Gng2-long variant is indicated with green bars and lines. Note the relative increase in the relative fluorescent detected in the SVZ/MZ area. (F,G) Images of the expression and localization of Dlx2 (F) and Dlx5 (G) mRNAs by ISH (from www.genepaint.org) marking, respectively, proliferating progenitors and committed/early differentiating GABAergic neurons. The position of the VZ and SVZ/MZ is indicated in (F). (H–J) Representative images of two-colors FISH to detect all variants (green, in F) or the long variant (red, in G) Pes1 transcripts in the cortical primordium. A merge image is shown in (H). (K) Histogram and profile of the relative fluorescent intensity of the cortical primordium, from the areas indicated in (H; (white rectangle, subdivided inVZ/SVZ and cortical plate (CP)). At least two areas/slide were considered. The results relative to the VZ/SVZ or the CP are shown as histogram. Pes1-all variants are indicated with green bars and lines, Pes1-long variant is indicated with red bars and lines. Note the relative increase in the relative fluorescence detected in the CP. Asterisks indicate statistical significance, **p < 0.01, ***p < 0.001.

Differential expression of RNA binding protein (RBP) of the Elavl family in differentiating ASN cells. (A) Expression of Elavl1, Elavl2, Elavl3 and Elavl4 RBPs in proliferating (T0) vs. differentiating (T1, T2, T3) ASN cells, by RNAseq analysis. (B) Plotting of the same data as in (A) showing differential expression of Elavl3 mRNA in proliferating vs. differentiating ASN cells, by RNAseq analysis. (C) RealTime qPCR analyses on independent RNA samples to quantify the relative abundance of Elavl3 mRNA in ASN cells at T0, T1 and T2. Results are expressed as relative abundance, T2 is made = 1. *p < 0.05.

Effect of depletion of Elavl3 in differentiating ANS cells. (A) RealTime qPCR analysis to detect the abundance of Elavl3 mRNA in ANS cells treated with the control (black) or anti-Elavl3 (white) siRNA. (B) Western blot analysis of ANS cells treated with the control siRNA or with the anti-Elavl3 siRNA, to confirm the downmodulation of Elavl3 protein (top). Staining with anti-actin on the same blot is used as loading control (bottom). (C) Real Time qPCR analysis to detect the relative abundance of the longer vs. the common variants of Pes1, Hnrnpa0, Gng2 and Tex2 mRNAs, comparing siRNA-treated vs. control-treated ANS cells, maintained in differentiation conditions. (D) Real-Time qPCR analysis to detect the mRNA abundance of the differentiation genes GAD1, Tubβ3, NeuN and Map2 in ANS cells treated with the control (black bars) or anti-Elavl3 (open bars) siRNA. *p < 0.05.