Differential expression of paralog RNA binding proteins establishes a dynamic splicing program required for normal cerebral cortex development

Abstract

Sam68 and SLM2 are paralog RNA binding proteins (RBPs) expressed in the cerebral cortex and display similar splicing activities. However, their relative functions during cortical development are unknown. We found that these RBPs exhibit an opposite expression pattern during development. Sam68 expression declines postnatally while SLM2 increases after birth, and this developmental pattern is reinforced by hierarchical control of Sam68 expression by SLM2. Analysis of Sam68:Slm2 double knockout (Sam68:Slm2dko) mice revealed hundreds of exons that respond to joint depletion of these proteins. Moreover, parallel analysis of single and double knockout cortices indicated that exons regulated mainly by SLM2 are characterized by a dynamic splicing pattern during development, whereas Sam68-dependent exons are spliced at relatively constant rates. Dynamic splicing of SLM2-sensitive exons is completely suppressed in the Sam68:Slm2dko developing cortex. Sam68:Slm2dko mice die perinatally with defects in neurogenesis and in neuronal differentiation, and develop a hydrocephalus, consistent with splicing alterations in genes related to these biological processes. Thus, our study reveals that developmental control of separate Sam68 and Slm2 paralog genes encoding homologous RBPs enables the orchestration of a dynamic splicing program needed for brain development and viability, while ensuring a robust redundant mechanism that supports proper cortical development.

Graphical Abstract

Figure 1.

SLM2 cortical expression steadily increases during development. (A, B) Confocal images of SLM2 (A) or Sam68 (B) staining (green) counterstained with Neurotrace to show all brain cells (blue) in the mouse cortex at P30. The cortical regions corresponding to layers I-VI are marked by dotted lines in the Neurotrace image. A scheme of the cortical region examined is shown on the left of the panels. Scale bar 50μm. (C) Confocal images of Sam68 and Slm2 mRNA (RNAscope probe). The bar graph represents the percentage of cells displaying Sam68 and Slm2 colocalization or stained by only one of the two. Scale bar 20 μm. Data represent the mean + standard error (SE) of at least three independent samples. Statistical analysis was performed independently for Sam68 and Slm2 probe expression by one way ANOVA Tukey's multiple comparisons test; ****P= 0.0001. (D, E) Confocal images of SLM2 (D) or Sam68 (E) and NeuN, CNPase, Iba1 or GFAP in the layer V of the sensory motor cortex. Scale bars 20 μm. (F, G) Quantitative q-PCR (F) and Western blot (G) analyses of Sam68 and SLM2 transcript and protein expression in the developing cortex. L34 was used for normalization of the q-PCR data (F). Data represent the mean + standard error (SE) of at least three independent samples. Statistical analysis was performed independently for Sam68 and SLM2 expression by one way ANOVA Tukey's multiple comparisons test; *P= 0.05, ***P= 0.001, ****P= 0.0001. Coomassie blue staining was performed as loading control of the western blot (G).

Figure 2.

SLM2 represses the expression of Sam68 in the adult cortex. (A) Schematic representation of the canonical full-length and the NMD-targeted splice variants of Sam68 (upper panel) and Slm2 (lower panel) genes. The position of the forward (red) and reverse (green) primers used for detection of the alternative end variants 1 and 2 by capillary RT-PCR analyses are indicated by arrows. (B, C) Quantitative capillary RT-PCR analyses of the percentage of NMD-targeted alternative end 2 mRNA variant of Sam68 (B) and Slm2 (C) in the wild-type cortex at the indicated developmental stages. (D) Quantitative capillary RT-PCR analyses of the percentage of NMD-targeted alternative end 2 mRNA variant of Sam68 in the wild-type and Slm2^ko cortex at the indicated developmental stages. (B–D) Data are expressed as alternative end 2/alternative end 1 + alternative end 2 and represent the mean + SE of at least three independent samples. Statistical analysis was performed by one way ANOVA with respect to the E15.5 ratio for each gene; *P= 0.05, **P= 0.01, ***P= 0.001, ****P= 0.0001. (E) Representative Western blot analysis of Sam68 protein expression in the wild-type and Slm2^ko cortex at P10 and P30. Bar graphs show the densitometric analysis of three independent samples. Statistical analysis was performed by Student's t-test; **P= 0.01, n.s.= not significant. (F) Schematic representation of the minigene encompassing the Sam68 genomic region between the canonical exon 9 and the NMD-associated exon 10. The different alternative splicing options are show by dotted lines. (G) Quantitative capillary RT-PCR analyses of the percentage of unstable mRNA in HEK293T cells transfected with the Sam68 minigene and Sam68-GFP or SLM2-GFP plasmid. Statistical analysis was performed by Student's t-test respect to control (GFP); **P= 0.01, ****P= 0.0001.

Figure 3.

Dynamic splicing regulation of common exon targets of Sam68 and SLM2 during cortical development. (A, B) RT-PCR analyses of the Nrxn1 exon 20 (A) and Stxbp5l exon 24 (B) splicing pattern in the wild-type cortex isolated at the indicated developmental ages. Graphs report the densitometric analysis of the ‘percent spliced in’ (PSI) of the alternative exon and represent the mean ± SE of five independent samples. (C, D) RT-PCR analyses of the Nrxn1 exon 20 (C) and Stxbp5l exon 24 (D) splicing pattern in the wild-type, Sam68^ko and Slm2^ko cortices isolated at the indicated developmental ages. Graphs report the densitometric analysis of the PSI of the alternative exon and represent the mean ± SE of five independent samples. (A–D) Statistical analyses were performed with one-way ANOVA, Tukey's multiple comparisons test; *P < 0.05; **P < 0.01***P < 0.001; ****P < 0.0001.

Figure 4.

Sam68 and SLM2 regulate a widespread splicing program in the developing cortex. (A) Representative RT-PCR analysis of the splicing pattern of Nrxn1 exon 20 and Stxbp5l exon 24 during development of the wild-type and Sam68:Slm2^dko cortex. (B) Pie chart of the splicing events and splicing patterns that are differentially regulated in the Sam68:Slm2^dko cortex. (C) Bar graph representation of the up- and down-regulated exon cassette, alternative last exon (ALE) and intron retention (IR) events in the Sam68:Slm2^dko cortex. (D–F) Representative RT-PCR analysis of the splicing pattern of Kif21a exon 30 (D), Arhgap12 exon 7a (E) and Kans1l exon 11 (F) during development of the wild-type cortex. (G–I) Representative RT-PCR analysis of the splicing pattern of Kif21a exon 30 (G), Arhgap12 exon 7a (H) and Kans1l exon 11 (I) in the wild-type, Sam68^ko, Slm2^ko and Sam68:Slm2^dko cortices at the indicated developmental stages. All graphs report the densitometric analysis of the PSI of the alternative exon and represent the mean ± SE of three independent samples. (D–I) Statistical analyses were performed with one-way ANOVA, Tukey's multiple comparisons test; *P< 0.05; **P< 0.01; ***P< 0.001; ****P< 0.0001.

Figure 5.

Combined ablation of Sam68 and SLM2 expression impairs neurogenesis. (A, B) Gene Ontology (GO) analysis of the genes regulated at splicing level in the Sam68:Slm2^dko cortex. Dots’ size in the graph is proportional to the number of genes in each category; dots’ color represents the enrichment grade, with red indicating high enrichment score and blue indicating low enrichment score. Gene Ratio indicates the number of the differentially regulated genes on the total of genes in the GO term. P-value was calculated with Classic Fisher's Exact test. (C–E) Representative images (C) and quantitative analysis of the cortical thickness from ventricle to pial surface (D) or of neuronal cells stained for SOX2 (E) in the cortex of wild-type, Sam68^ko, Slm2^ko and Sam68:Slm2^dko embryos at E18.5. (F, G) Representative images (F) and quantitative analysis (G) of immunofluorescence for the neural progenitor marker TBR2 in the cortex of wild-type, Sam68^ko, Slm2^ko and Sam68:Slm2^dko embryos at E18.5. Histograms show the data (mean ± SE) of three independent male embryos for each genotype used for the analyses. Statistical analyses were performed using the one-way ANOVA test; *P< 0.05; ***P< 0.001; ****P< 0.0001. Scale bar = 50 μm in (C) and 33 μm in (F).

Figure 6.

Combined ablation of Sam68 and SLM2 expression impairs neuronal maturation, dendritic arborization and synaptic organization of cortical neurons. (A) Representative images of cortical neurons of the indicated genotypes showing immunostaining with β-Tubulin and DAPI and the relative 3D-reconstructed structures (scale bar = 20 μm). (B–D) Box-and-whisker plots of soma area (B) and overall surface area (D) and graph of the distribution of the intersections at different distances from the soma (C) of individual cortical neurons of the indicated genotypes (7–10 neurons per culture were randomly selected, n = 4 mice for each experimental group). (E) Representative images of immunofluorescence for VGluT1 (red) and phalloidin (green) in cortical neurons of the indicated genotypes. (F) Box-and-whisker plots of the number of VGluT1 puncta (in an area of 400 μm²) on phalloidin positive neurons of the indicated genotypes (n = 5 mice for experimental group). In the box-and-whisker plots, the center line shows the median value, edges are upper and lower quartiles, whiskers show minimum and maximum values, and each point is an individual set of experiments. Data were analyzed by one-way ANOVA followed by a Bonferroni post hoc test, *P< 0.05, **P< 0.01, ***P< 0.001.

Figure 7.

3D-MicroCT virtual histology of embryo brains. (A–D) CT imaging of the head of E18.5 embryos of the indicated genotypes acquired along a virtual median sagittal plane (A), two coronal planes (B, C) sectioned according to the a and b axes defined in (A), and a transverse plane (D). Arrows indicate the lateral ventricles; the hatched area in (D) indicates the cortical region in a central cross section (scale bar = 1 mm). (E–G) The bar graphs indicate the volume of lateral ventricle (E), the volume of brain (F) and the volume of the total embryo (G); n = 4 mice for each experimental group. Data were analyzed by one-way ANOVA followed by a Bonferroni post hoc test,, *P< 0.05, ***P< 0.001, ****P< 0.0001.