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. 2018 May 10;7:e36998. doi: 10.7554/eLife.36998

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© 2018, Chau et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Figure 5. — (A) MA plot displaying genes encoding transcription factors in E8.5 and E10.5 neuroepithelium. Each dot represents a single gene. Red dots denote differentially expressed genes identified by Cuffdiff (q<0.05). Genes below blue line (y = 0) enriched in E8.5. Myc (arrow) expression is ~10 fold higher in E8.5 epithelium (FPKM: E8.5=28.73, E10.5 = 2.76). (B) MYC expression was enriched in E8.5 neuroepithelium. Once downregulated at E10.5, MYC expression remained low throughout cortical development. Scale bar = 20 μm. (C, D) GSEA of E8.5 versus E10.5 neuroepithelium for gene sets containing genes up-regulated by MYC and whose promoters are bound by MYC (C), and E-box containing MYC target genes (D). Broad Institute Molecular Signatures Database Identifiers: DANG_MYC_TARGETS_UP (C), BENPORATH_MYC_TARGETS_WITH _EBOX (D). Each line represents a single gene in the gene set; genes on the right side enriched in E8.5. (E) Quantification of nucleolar volume of E8.5 embryos treated with vehicle control or KJ-Pyr-9 for 24 hr. Each data point represents one nucleolus. ***p≤0.001, Welch’s t-test. Sample size, vehicle: n = 140 from three embryos; KJ-Pyr-9: n = 140 from three embryos. (F) Quantification of nucleolar volume of Myc^-/- compared to controls (wild type and heterozygous littermates) in E8.5 neuroepithelium. *p≤0.05 Unpaired t-test. Sample size, controls: n = 238 from five embryos; Myc^-/-: n = 97 from two embryos. (G) Immunostaining shows overexpression of MYC (red) in the developing cortex of E12.5 MYC-OE (right) embryos from the Nestin-cre x StopFLMYC cross. TUJ1 (green) staining labels neurons. (H) Functional annotation clustering of the 105 MYC-OE enriched genes shows overrepresentation of genes encoding ribosome constituents. The top five enriched functional clusters are shown. (I, J) GSEA of WT versus MYC-OE apical progenitors for gene sets involved in ribosome biogenesis. Broad Institute Molecular Signatures Database Identifiers: KEGG_RIBOSOME (I), and GO_RIBOSOME_ BIOGENESIS (J). Each line represents a single gene in the gene set, genes on the right side are enriched in MYC-OE. (K) Heatmap of the 43 ribosomal protein genes that are differentially expressed between MYC-OE and WT apical progenitors (* q < 0.3, **q < 0.1). All ribosomal proteins are more highly expressed in MYC-OE. Red and green indicate relatively higher and lower expression, with gene FPKM values log2 transformed. (L) MA plot displaying genes encoding ribosomal proteins in E13.5 apical progenitors. Each dot represents a single gene. Red dots denote differentially expressed genes as identified by Cuffdiff (q<0.1). Genes above blue line (y = 0) are enriched in MYC-OE. (M) Quantification of nucleolar volume of WT and MYC-OE (Foxg1-cre driven) forebrain progenitors at E11.5. Each data point represents one nucleolus. ****p≤0.0001, Welch’s t-test. Sample size, WT: n = 194 from four embryos; MYC-OE: n = 144 from three embryos. (N) Quantification of nucleolar volume of WT and MYC-OE (Nestin-cre driven) apical progenitors at E13.5. Each data point represents one nucleolus. ***p≤0.001, Welch’s t-test. Sample size, WT: n = 234 from five embryos; MYC-OE: n = 248 from five embryos.

Figure 5—figure supplement 1. — (A) MA plot displaying genes encoding transcription factors in E8.5 and E10.5 neuroepithelium. Each dot represents a single gene. Red dots denote differentially expressed genes identified by Cuffdiff (q<0.05). Genes below blue line (y = 0) enriched in E8.5. Myc (arrow) expression is ~10 fold higher in E8.5 epithelium (FPKM: E8.5=28.73, E10.5 = 2.76). (B) MYC expression was enriched in E8.5 neuroepithelium. Once downregulated at E10.5, MYC expression remained low throughout cortical development. Scale bar = 20 μm. (C, D) GSEA of E8.5 versus E10.5 neuroepithelium for gene sets containing genes up-regulated by MYC and whose promoters are bound by MYC (C), and E-box containing MYC target genes (D). Broad Institute Molecular Signatures Database Identifiers: DANG_MYC_TARGETS_UP (C), BENPORATH_MYC_TARGETS_WITH _EBOX (D). Each line represents a single gene in the gene set; genes on the right side enriched in E8.5. (E) Quantification of nucleolar volume of E8.5 embryos treated with vehicle control or KJ-Pyr-9 for 24 hr. Each data point represents one nucleolus. ***p≤0.001, Welch’s t-test. Sample size, vehicle: n = 140 from three embryos; KJ-Pyr-9: n = 140 from three embryos. (F) Quantification of nucleolar volume of Myc^-/- compared to controls (wild type and heterozygous littermates) in E8.5 neuroepithelium. *p≤0.05 Unpaired t-test. Sample size, controls: n = 238 from five embryos; Myc^-/-: n = 97 from two embryos. (G) Immunostaining shows overexpression of MYC (red) in the developing cortex of E12.5 MYC-OE (right) embryos from the Nestin-cre x StopFLMYC cross. TUJ1 (green) staining labels neurons. (H) Functional annotation clustering of the 105 MYC-OE enriched genes shows overrepresentation of genes encoding ribosome constituents. The top five enriched functional clusters are shown. (I, J) GSEA of WT versus MYC-OE apical progenitors for gene sets involved in ribosome biogenesis. Broad Institute Molecular Signatures Database Identifiers: KEGG_RIBOSOME (I), and GO_RIBOSOME_ BIOGENESIS (J). Each line represents a single gene in the gene set, genes on the right side are enriched in MYC-OE. (K) Heatmap of the 43 ribosomal protein genes that are differentially expressed between MYC-OE and WT apical progenitors (* q < 0.3, **q < 0.1). All ribosomal proteins are more highly expressed in MYC-OE. Red and green indicate relatively higher and lower expression, with gene FPKM values log2 transformed. (L) MA plot displaying genes encoding ribosomal proteins in E13.5 apical progenitors. Each dot represents a single gene. Red dots denote differentially expressed genes as identified by Cuffdiff (q<0.1). Genes above blue line (y = 0) are enriched in MYC-OE. (M) Quantification of nucleolar volume of WT and MYC-OE (Foxg1-cre driven) forebrain progenitors at E11.5. Each data point represents one nucleolus. ****p≤0.0001, Welch’s t-test. Sample size, WT: n = 194 from four embryos; MYC-OE: n = 144 from three embryos. (N) Quantification of nucleolar volume of WT and MYC-OE (Nestin-cre driven) apical progenitors at E13.5. Each data point represents one nucleolus. ***p≤0.001, Welch’s t-test. Sample size, WT: n = 234 from five embryos; MYC-OE: n = 248 from five embryos.