Overexpression Of CoupTFI In Cortical Progenitor Cells Partially Rescues The Cortical Phenotype Of Foxc1-Mutants.

<p>Cresyl Violet staining of E14.5 coronal forebrain sections (A–D). Quantification of dorsal forebrain length at E14.5 (E). Tbr1 (green) and Ctip2 (red) immunohistochemistry of the dorsal cortex at E14.5 (F–I). Quantification of Ctip2 and Tbr1 cell number at E14.5 (J). Pax6 (red) immunohistochemistry of the dorsal cortex at E14.5 (K–N) Quantification of Pax6 cell number at E14.5 (O). Tbr2 (red) immunohistochemistry of the dorsal cortex at E14.5 (P–S). Quantification of Tbr2 cell number at E14.5 (T). Sections are counterstained with DAPI (blue) (F–I, K–N, P–S). Scale bar: 500 µm in A–D; 50 µm in F–I, K–N, P–S. E, J, O, P were analyzed by one way ANOVA: E: F_(3,12) = 40.87, p<0.001; J: F_(3,12) = 62.28, p<0.001; F_(3,12) = 48.8, p<0.001; O: F_(3,12) = 74.38, p<0.001; P: F_(3,11) = 76.71, p<0.001. *p<0.05, **p<0.01; ***p<0.001 and indicate significance for Bonferroni’s Multiple Comparison Test posthoc analysis. Asterisks directly above the bar indicate significance from untreated control; within group differences are indicated by connected lines.</p

CoupTFI And CoupTFII Expression Is Not Downregulated Or Misexpressed In The Cortex Of Foxc1-Mutants At E14.5.

<p><i>In situ</i> hybridization of coronal sections of control (A, C, E, G, I, K, M, O) and Foxc1^H/L (B, D, F, H, J, L, N, P) at E14.5 with CoupTFI probe (A–H) and CoupTFII probe (I–P). Higher magnification panels in E, F, K, L correspond to boxed regions in G, H, O, P. Scale bar: 500 µm in AF, I–N, 100 µm in G, H, O, P. Arrow in L, N points to the cortical hem. * in D, F, L, N indicates the ventral progenitor population. MZ: marginal zone.</p

Overexpression Of CoupTFI In Cortical Progenitor Cells Increases Early Neuorgenesis.

<p>Tbr1 (green) and Ctip2 (red) immunohistochemistry of the dorsal cortex at E12.5 (A–D). Sections are counterstained with DAPI (blue). Quantificaiton of Ctip2 and Tbr1 cell number at E12.5 (E). Quantification of the Q-fraction at E12.5 (F) and E14.5 (G). E–G were analyzed by one way ANOVA: E: Ctip2 F_(3,11) = 35.5, p<0.001; Tbr1: F_(3,12) = 66.1, p<0.001; F: F_(3,12) = 5.2, p<0.001; G: F_(3,12) = 54.6, p<0.001. *p<0.05, **p<0.01; ***p<0.001 and indicate significance for Bonferroni’s Multiple Comparison Test posthoc analysis. Asterisks directly above the bar indicate significance from untreated control; within group differences are indicated by connected lines.</p

CoupTFI Is Required For The RA Mediated Rescue Of Foxc1-Mutants Animals.

<p>Cresyl Violet staining of E14.5 coronal forebrain sections of untreated and RA treated (E10–E14.5) embryos (A–H). Asterisk in D, H signifies the medial wall. Pax6 (red) immunohistochemistry of the dorsal cortex at E14.5 of untreated and RA treated (E10–E14.5 embryos) (I–P). Tbr2 (red) immunohistochemistry of the dorsal cortex at E14.5 of untreated and RA treated (E10–E14.5) embryos (Q–X). Tbr1 (green) and Ctip2 (red) immunohistochemistry of the dorsal cortex at E14.5 of untreated and RA treated (E10–E14.5) embryos (Y–FF). Sections are counterstained with DAPI (blue) (I–FF). Scale bar: 500 µm in A–H, 50 µm in I–FF. VZ: ventricular zone; SVZ: subventricular zone; PC: progenitor cell; CP: cortical plate.</p

CoupTFI Is Required For The RA Mediated Rescue Of Foxc1-Mutants Animals.

<p>Quantification of dorsal forebrain length at E14.5 (A), Pax6 cell number at E14.5 (B), Tbr2 cell number at E14.5 (C), Ctip2 cell number at E14.5 (D), Tbr1 cell number at E14.5 (E), area of the counting window (F), density of DAPI⁺ cells in the counting window (G). Error bars represent SEM. A–G were analyzed by two way ANOVA: A: genotype (F_(3,25) = 55.86, p<0.001), treatment (F_(1,25) = 27.84, p<0.001), interaction (F_(3,25) = 49.98, p<0.001); B: genotype (F_(3,26) = 9.5, p<0.001), treatment (F_(1,26) = 48.66, p<0.01), interaction (F_(3,26) = 11.67, p<0.001); C: genotype (F_(3,27) = 19.23, p<0.001), treatment (F_(1,27) = 8.71, p<0.01), interaction (F_(3,27) = 14.55, p<0.001); D: genotype (F_(3,26) = 40.83, p<0.001), treatment (F_(1,26) = 18.45, p<0.001), interaction (F_(3,26) = 14.75, p<0.001); E: genotype (F_(3,26) = 47.99, p<0.001), treatment (F_(1,26) = 48.66, p<0.001), interaction (F_(3,26) = 14.91, p<0.001); F: genotype (F_(3,24) = 24.77, p<0.001), treatment (F_(1,24) = 8.158, p<0.01), interaction (F_(3,24) = 13.38, p<0.001); G: genotype (F_(3,25) =10.33, p<0.001), treatment (F_(1,25) =0.53, p = 0.47), interaction (F_(3,25) = 1.62, p = 0.2). *p<0.05, ***p<0.001 and indicate significance for Bonferroni’s Multiple Comparison Test posthoc analysis. Asterisks directly above the bar indicate significance from untreated control; within group differences are indicated by connected lines.</p

Sampling Window Used For Cell Counts.

<p>The location of the 150 µm sampling window in the medial-lateral dimension of the cortex spanning from the ventricular to pial surface of the dorsal cortex is identified by the black box in this coronal forebrain section of an E14.5. Scale bar = 500 µm.</p

Transcribed enhancers during ESC neural commitment.

<p>The table lists the non-annotated CAGE promoters or promoters annotated to ncRNAs active in ESCs, NESCs or both cell types (maintaining active transcription during ESC neural commitment), located inside ESC-specific, NESC-specific or common enhancer regions.</p><p>Transcribed enhancers during ESC neural commitment.</p

Change in gene expression profile during ESC neural induction.

<p>A) Global unsupervised clustering performed on the entire pool of 19,204 genes indicates that, at the transcriptional level, ESCs and NESCs are two distinct cell populations. Red boxes highlight reproducible clusters that are strongly supported by data (Bootstrap Probability value ≥95%). B) Heat map of the subset of 2,413 genes that change their expression levels in ESCs as compared to NESCs (at False Discovery Rate <0.01 and absolute FC level ≥2). Expression levels are presented as row-wise standardized values (log₂ fold change). C) Functional enrichment of the 2,413 differentially expressed genes obtained using DAVID GO annotation. Upper plot reports the GO categories associated to genes up-regulated in ESCs, while in the lower plots are the functional enrichment of genes over-expressed during neural induction, i.e. in NESCs.</p

Genome-wide mapping of epigenetically-defined promoters and enhancers in ESCs and NESCs.

<p>A) Heat maps showing the distribution of promoter (left) and enhancer (right) regions in a window of ±5-kb from CAGE-mapped TSSs in ESCs and NESCs. Promoter islands are defined as H3K4me3^+/high/me1^-/low, enhancers as H3K4me1^+/high/me3^-/low. Promoter islands are clustered around CAGE-mapped TSSs, whereas enhancers are spread out. B) Average profile of single H3K4me3 (orange) and H3K4me1 (green) peaks around the TSS, in a ±3-kb. Normalized read count means histone modification read count per million mapped reads. C) Epigenetic state of CAGE promoters in ESCs. The histogram shows the fraction of ESC-specific, down-regulated, up-regulated and NESC-specific promoters with the epigenetic signature of active (H3K4me3⁺, red) or poised (H3K4me3⁺/H3K27me3⁺, grey) promoter. The most part of up-regulated and NESC-specific promoters are poised for transcription in ESCs. D) Pie-diagrams showing the fraction of common and cell-specific total enhancers mapped in ESCs and NESCs. E) Analysis of putative TFBS enrichment within cell-specific enhancers as determined by HOMER tool. ESC-specific enhancers showed enrichment of binding motifs for the pluripotency TFs, as observed for cell-specific promoters, and for ETS family factors. NESC-specific enhancers were enriched for ETS-family, RFX-family and Jun/AP1 factors.</p

Networks of genes associated to NESC-specific and up-regulated CAGE promoters.

<p>The networks visually represent the connections between the genes associated to NESC-specific (up) and up-regulated CAGE promoters (down). Most of the genes are included in the regulatory pathways of axonal guidance signaling, ESC pluripotency and signal transduction. Purple arrows indicate the connections between genes based on the Ingenuity Knowledge Base dataset (dotted or solid lines for indirect and direct relationships respectively). Then, genes included in IPA canonical pathways (CP) are indicated by grey arrows. The shape of the gene symbol indicates the corresponding protein function, while the color (from white to red) represents the CAGE expression level of the promoter associated to the gene (for NESC-specific promoters) or its ratio between ESCs and NESCs (for upregulated CAGE promoters). For a complete IPA legend see <a href="http://ingenuity.force.com/ipa/articles/Feature_Description/Legend" target="_blank">http://ingenuity.force.com/ipa/articles/Feature_Description/Legend</a></p