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.2012 Apr;19(4):692-702.
doi: 10.1038/cdd.2011.144. Epub 2011 Nov 18.

RP58/ZNF238 directly modulates proneurogenic gene levels and is required for neuronal differentiation and brain expansion

Affiliations

RP58/ZNF238 directly modulates proneurogenic gene levels and is required for neuronal differentiation and brain expansion

C Xiang et al. Cell Death Differ.2012 Apr.

Abstract

Although neurogenic pathways have been described in the developing neocortex, less is known about mechanisms ensuring correct neuronal differentiation thus also preventing tumor growth. We have shown that RP58 (aka zfp238 or znf238) is highly expressed in differentiating neurons, that its expression is lost or diminished in brain tumors, and that its reintroduction blocks their proliferation. Mice with loss of RP58 die at birth with neocortical defects. Using a novel conditional RP58 allele here we show that its CNS-specific loss yields a novel postnatal phenotype: microencephaly, agenesis of the corpus callosum and cerebellar hypoplasia that resembles the chr1qter deletion microcephaly syndrome in human. RP58 mutant brains maintain precursor pools but have reduced neuronal and increased glial differentiation. Well-timed downregulation of pax6, ngn2 and neuroD1 depends on RP58 mediated transcriptional repression, ngn2 and neuroD1 being direct targets. Thus, RP58 may act to favor neuronal differentiation and brain growth by coherently repressing multiple proneurogenic genes in a timely manner.

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Figures

Figure 1
Figure 1
Loss ofRP58 produces a novel postnatal microencephalic phenotype with a thin neocortex and absence of corpus callosum. (a–d) Dorsal views at the same magnification of control (a andc) andRP58 cKO (b andd) brains at postnatal (P) day 8 and 15. Note the reduced size of the cerebral (ctx) and cerebellar (cb) cortices in mutant brains. The black arrows indicate the cerebellar vermis, which is lost inRP58 mutants. (e–j) Histological analyses of control (e,g andi) andRP58 mutant (f,h andj) cortices at E15.5 and P2 after Nissl staining, seen in a coronal section (e andf) and sagittal sections (g–j) near the midline. Mutant cortices lack a midline-crossing corpus callosum (Cc, arrows in panele) and have a thin cerebral cortex as compared with the controls (g–j). Scale bars=0.5 cm: panelsa–d; 100 μm: panelse andf; 200 μm: panelsg andh; 500 μm: panelsi andj
Figure 2
Figure 2
Loss ofRP58 impairs normal neurogenesis. (a) Top: Sagittal sections of E18.5 cortices were stained with Nissl (top row) or subjected toin situ hybridization for the probes indicated on the left. Note the decrease of markers expressed in layer V, IV and II–IV in theRP58 cKO (compare with Supplementary Figure 4 for theRP58 KO). The asterisks show ectopicER81 expression in the VZ and residual faintcux2 expression in the cortical upper layers. Scale bar=100 μm. Bottom: Sagittal sections of control andRP58 cKO E18.5 cortices showing altered localization of CTIP2+ cells and strong reduction of SATB2+ cells in the mutant cortices. Nuclei are labeled with the Hoechst dye. Scale bar=200 μm. (b) Top: Analyses of neuronal-specific MAP2 labeling in control andRP58 mutant cortices at E14.5. Note loss of neurons in the mutant cortices. Scale bar=100 μm. Bottom: Sagittal sections of control andRP58 cKO P8 cortices showing reduction in thickness and loss of NeuN-expressing neurons in the mutant brain. Scale bar=200 μm. (c) Analyses of the density and morphology of Nestin+ and GFAP+ labeled cells by confocal microscopy in P2 control andRP58 mutant cortices focusing on the subventricular zone (SVZ) and cortical plate (CP). Scale bar=30 μm. (d) Analysis of the number of MAP2+ cells in E14.5 acutely dissociated cortices. After dissociation, cells were plated 2 h before fixation and immunostaining. The number of MAP2+ cells at E14.5 is reduced in theRP58 mutant cerebral cortex when compared with the control cortex. The experiment was performed on three independent litters. The asterisk denotes significant change (P<0.01). (e) Western blot analysis of the levels of cell type-specific markers in P0 control andRP58 mutant cortices. RP58 expression is lost in the mutants, which have increased Nestin and GFAP levels, and decreased MAP2 levels. Tubulin is shown as a loading control. (f) Quantification of the expression levels of multiple markers of radial glial cells (glast, blbp) and of intermediate neurogenic progenitors (INPs) (svet1, math2, satb2, cux2 andtbr2) in cortical cells at E14.5. Analyses were performed by quantitative RT-PCR and values were normalized to the expression of the housekeeping genegapdh. (g) Localization of the changes in the expression ofRP58 (top row) andtbr2 (bottom row) mRNAs in E14.5 control and mutant cortices byin situ hybridization. Note thattbr2 is expressed in cells in the VZ (asterisk) as well as in the SVZ (arrow). Scale bar=100 μm
Figure 3
Figure 3
Loss of RP58 leads to increased glial differentiation. (a) The origin of the astrocytes in the cortical VZ/SVZ was determined in BrdU birth-dating experiments: dividing cells were labeled with a BrdU pulse at E16.5 and examined for BrdU and GFAP expression at P2. BrdU+/GFAP+ cell counting was performed on sections from three different brains for each genotype and indicated that more astrocytes were produced at E16.5 in the RP58 mutant mice as compared with the control cortex. The asterisk denotes significant change (P<0.05). Scale bar=20 μm. (b) E14.5 cerebral cortices were dissociated and grown as primary spheres at clonal density. Primary spheres were then dissociated and differentiated for 3–4 days. The number of neurons (Tuj1+ cells) and astrocytes (GFAP+ cells) was counted for each differentiation assay. Three independent experiments (three different litters) were analyzed. Neuronal differentiation is highly reduced in the RP58 mutant in favor of glial differentiation. The asterisk denotes significant change (P<0.05). Scale bar=100 μm
Figure 4
Figure 4
Deregulation of progenitor pools was revealed by cell sorting inRP58 mutant cortices. (a) FACS graph showing separation of E14.5 cortical cells depending on their expression of CD133 (Prominin1) and EGFP, the latter driven from thetbr2 regulatory region intbr2: EGFP mice. The subpopulations of cells include the following: Group-A: CD133+/EGFP−-containing RGCs in the VZ; Group-B: double positives, containing INPs in the SVZ; and Group-C: CD133−/EGFP+, containing differentiating/differentiated cortical neurons. (b) Characterization of the three cell populations described in panela by expression of key molecular markers of RGCs (blbp,glast,pax6), INPs (tbr2) and neurons (tbr1,MAP2). (c) Quantification of the pool size of A and B cells in control andRP58 KO cortices. A small increase in A cells and a slight decrease in B cells were observed in the mutant cortices at E14.5. The asterisks denote significant changes (P<0.05 for A cells,P<0.01 for B cells). (d) Loss of RP58 leads to an increased number of neocortical neurospheres. E14.5 cerebral cortices were dissociated and grown as primary spheres at clonal density (less than 10 cells/μl). After 7 days, primary spheres were dissociated and challenged to form secondary spheres also at clonal density. The number of primary and secondary neurospheres was assessed visually in triplicate assays for each experiment. Three independent experiments with three different litters were analyzed. A two-tailedt-test was applied for statistical analysis. The asterisks denote significant changes (P<0.001)
Figure 5
Figure 5
Loss of RP58 leads to deregulation of proneurogenic gene expression. Analyses of the expression levels ofRP58, and key RGC and INP regulators in sorted cell populations in control andRP58 mutant cortices at E14.5. Note (1) loss ofRP58 expression in the mutant and (2) the consistent increase ofpax6,ngn2 andneuroD1 in C cells. The diagrams are from a representative experiment. Experiments were performed in triplicates from three independent litters, with similar results
Figure 6
Figure 6
RP58 directly regulatesngn2 andneuroD1 expression. (a) A schematic diagram showing the localization of two putative RP58-binding sites and the primers (arrrows) used in the ChIP–PCR in panelsb andc. Thengn2 gene is indicated in green. The putative RP58-binding sites (BS1 and BS2) in the 5′ngn2 genomic region are indicated by red boxes. (b) ChIP analyses of RP58 recruitment and presence of trimethylated histone H3K27 (H3K27me3) as a marker of repressed loci in control andRP58 mutant cortices using primers specific to thengn2 genomic region encompassing the RP58-binding site BS2 at chr3:127336163+127336391 (see scheme in panela). Note that the histograms show the fold enrichment of WT/KO. (c) Specific and preferential binding of RP58 to thengn2 genomic region in the differentiated C cell populations. This experiment was performed on cells sorted from two independent litters, with primers encompassing the same binding site BS2, with similar results. Note that the background thresholds obtained with a ChIP experiment using an isotyped IgG are set at 1 (black bars). (d) Top: A schematic diagram showing thengn2-luciferase construct (ngn2-luc plasmid:ngn2 regulatory region containing two RP58-binding sites) used for the data presented in panelse andf. Bottom: Sequences of the two RP58-binding sites, BS1 and BS2, in thengn2 promoter region. Mutant versions of BS1 and BS2, and their sequence orientations are noted. (e) Transcriptional repression of anngn2 regulatory region driving the expression of firefly luciferase by RP58. HEK293T cells were transfected with thengn2-luc plasmid (see panela).Ngn2-luc was transfected alone or with increasing doses of an RP58-expressing plasmid. Luciferase activity was assayed 48 h after transfection. Shown is a representative experiment of three independent biological replicates with similar results. The asterisks denote significant changes (P<0.001). (f) Mutation of RP58-binding sites impairs its repression of thengn2 promoter. Thengn2-luc constructs containing no (WT), 1 or 2 mutant (MUT) RP58-binding sites were co-transfected into HEK293T cells together with an RP58-expressing plasmid. Luciferase activity was normalized as percentage of the same reporter activity in the absence of exogenous RP58. Shown is a representative experiment of three independent biological replicates with similar results. A statistically significant difference of activity was observed between BS1-MUT and BS2-MUT (P<0.01), as well as between the single mutant BS1-MUT and the double mutant BS1-MUT+BS2-MUT (P<0.01). (g) A schematic diagram showing the localization of the primers (arrows) used in the ChIP–PCR in panelsh andi. TheneuroD1 gene is indicated in yellow. The unique consensus RP58-binding site (BS) in the proximal 5′neuroD1 genomic region is indicated by a red box. (h) ChIP analyses of RP58 recruitment and presence of trimethylated histone H3K27 (H3K27me3) as a marker of repressed loci in control andRP58 mutant cortices using primers specific to theneuroD1 genomic region encompassing the RP58-binding site. Note that the histograms show the fold enrichment of WT/KO. (i) Specific and preferential binding of RP58 to theneuroD1 genomic region in cells of the progenitor B cell population. Note that the background thresholds obtained with a ChIP experiment using an isotyped IgG are set at 1 (black bars). (j) Top: A schematic diagram showing theneuroD1-luciferase construct used for the data presented in panelsk andl. Bottom: Sequence of the RP58-binding site BS in theneuroD1 promoter region. The mutant version of BS and sequence orientation is noted. (k) Transcriptional repression by RP58 of aneuroD1 regulatory region driving the expression of firefly luciferase. HEK293T cells were transfected with theneuroD1-luc plasmid (neuroD1 regulatory region containing one RP58-binding site driving aluciferase reporter gene).NeuroD1-luc was transfected alone or with increasing doses of an RP58-expressing plasmid. Shown is a representative experiment of three independent biological replicates with similar results. The asterisks denote significant changes (P<0.001). (l) Mutation of RP58-binding site impairs its repression of theneuroD1 promoter. TheneuroD1-luc constructs containing the WT or mutant BS were co-transfected into HEK293T cells together with an RP58-expressing plasmid. Luciferase activity was normalized as percentage of the same reporter activity in the absence of exogenous RP58. Shown is a representative experiment of three independent biological replicates with similar results
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