Article Figures & Data
Figures
- Figure 1. Chd5 deficiency in NSCs leads to premature activation.
(A) Representative phase contrast images of wild-type (+/+) and Chd5-deficient (Chd5−/−) postnatal day 1 (P1) NSCs grown as adherent cultures (upper images) and neurosphere cultures (lower images). Scale bars = 100 μm (upper images) and 200 μm (lower images). (B) Quantification of the size of wild-type and Chd5-deficient NSC neurospheres expressing empty vector (EV) or Chd5 cDNA (Chd5). Data are represented as mean ± SD (n = 3). ns, no significance; **<0.01; ***<0.001; Tukey's multiple comparison test. (C) Flow cytometry of EdU-incorporated populations of wild-type and Chd5-deficient NSCs. Data are represented as mean ± SD (n = 4). ***<0.001; unpaired t test. (D, E) Flow cytometry of Egfr-positive and Cd133-positive populations in wild-type and Chd5-deficient NSCs. Data are represented as mean ± SD (n = 3). ns, no significance; *<0.05; unpaired t test. (F, G) Immunofluorescent images and Western blots of wild-type and Chd5-deficient NSCs, assessed for Chd5 (red), nestin (green), and DAPI nuclear signal (blue) (left panels), or assessed for Pax6, nestin, and β-actin (actin) expression (right panels). Scale bar = 50 μm. (H, I) Representative immunofluorescent images of wild-type and Chd5-deficient NSCs, assessed for vimentin (red), nestin (green), and DAPI nuclear signal (blue), and corresponding distribution of mean signal intensities over pixels of nestin and vimentin expression in randomly selected fields (4–5 fields per sample, n = 2–3). Scale bar = 50 μm.
- Figure S1. Disrupted dynamics of early stages of differentiation perturbs neural cell fate specification.
(A) Representative immunofluorescent images of wild-type (+/+) and Chd5-deficient (Chd5−/−) NSCs that were undifferentiated (0 h) or differentiated (for 3, 6, 12, and 24 h) and assessed for vimentin (red), nestin (green), and DAPI nuclear signal (blue). Scale bar = 50 μm. (B, C) Distribution and comparison of mean signal intensities over pixels of nestin expression in randomly selected fields (4–5 fields per sample) of wild-type (upper panels) and Chd5-deficient (lower panels) NSCs at the time points, indicated in representative images in Fig S1A. Data are represented as mean ± SD (n = 2). ns, no significance; **<0.01; ****<0.0001; Tukey’s multiple comparisons test. (D) Temporal representation of mean signal intensities over pixels of nestin expression of wild-type (blue) and Chd5-deficient (gray) NSCs, as plotted in Fig S1B and C. Data are represented as mean ± SD (n = 2). (E) Immunofluorescent images of wild-type and Chd5-deficient NSCs that were differentiated for 48 h and assessed for Gfap (red), Map2 (green), and DAPI nuclear signal (blue). Scale bar = 50 μm. (F) Western blots of P1 neonatal wild-type and Chd5-deficient cortices and assessed for Chd5, Map2, and β-actin (actin) expression. Arrow indicates the band with a predicted molecular weight. (G) Western blots of undifferentiated wild-type and Chd5-deficient NSCs (0 h post-differentiation) and differentiated cells (3-h post-differentiation), assessed for Tbr2, Tbr1, and β-actin (actin) expression. Arrow indicates the band with a predicted molecular weight.
- Figure 2. Chd5-deficient NSCs generate excessive astrocytes at the expense of neurons.
(A, B) Representative immunofluorescent images of wild-type (+/+) and Chd5-deficient (Chd5−/−) NSCs (upper panels) that were differentiated for 4 d (left panels) and 7 d (right panels) and analyzed for Gfap (red), Map2 (green), and DAPI nuclear signal (blue), with corresponding quantification (lower panels) of Map2+ neurons and Gfap+ astrocytes in randomly selected fields (10–20 fields per sample). Data are represented as mean ± SD (n = 2–3). ****<0.0001; unpaired t test. Scale bar = 50 μm. (C, D) Representative immunofluorescent images of wild-type NSCs transduced with empty vector (+/+;EV) or Chd5 cDNA (+/+;Chd5) (left panels) and Chd5-deficient NSCs transduced with empty vector (Chd5−/−;EV) or Chd5 cDNA (Chd5−/−;Chd5) (right panels), which were differentiated for 7 d and analyzed for Gfap (red), Map2 (green), and DAPI nuclear signal (blue) (upper panels), with corresponding quantification of Map2+ neurons and Gfap+ astrocytes (lower panels) in randomly selected fields (5–8 fields per sample). Data are represented as mean ± SD (n = 2). *<0.05; ***<0.001; unpaired t test. Scale bar = 50 μm. (E) Distribution of Map2+ neurons and Gfap+ astrocytes of cultures of wild-type and Chd5-deficient NSCs 4 and 7 d post-differentiation; representation of data shown in Fig 2A and B. Numbers indicate mean percentage of each population. n indicates the total number of cells analyzed. (F) Distribution of Map2+ neurons and Gfap+ astrocytes of cultures of wild-type and Chd5-deficient NSCs expressing EV or Chd5 cDNA (Chd5) and subject to differentiation for 7 d; representation of data shown in Fig 2C and D. Numbers indicate mean percentage of each population. n indicates the total number of cells analyzed.
- Figure S2. Nuclear DNA signal is altered in Chd5-deficient NSCs.
(A) Representative immunofluorescent images of wild-type (+/+) and Chd5-deficient (Chd5−/−) NSCs that were undifferentiated (0 h) or differentiated (for 6, 12, 24 and 48 h) and assessed for DAPI nuclear signal (left panels) and corresponding grayscale images (right panels). Dashed rectangles indicate the zoomed-in regions, shown in insets. Scale bar = 50 μm (main image) and 10 μm (insets). (B) Temporal representation of mean signal intensities over pixels of DAPI nuclear signal of wild-type (blue) and Chd5-deficient (gray) NSCs, as plotted in Fig S2C and D. Data are represented as mean ± SD (n = 2). (C, D) Distribution and comparison of mean signal intensities over pixels of DAPI signal in randomly selected fields (9–16 fields per sample) of wild-type (upper panels) and Chd5-deficient (lower panels) NSCs at all time points, indicated in representative images in Fig S2A. Data are represented as mean ± SD (n = 2). ns, no significance; ****<0.0001; Tukey’s multiple comparisons test.
- Figure S3. Chd5-deficient NSCs have alterations in histone distribution.
(A) Schematic illustration of H2B clustering pattern analysis of wild-type (+/+) and Chd5-deficient (Chd5−/−) NSCs that were undifferentiated (0 h) or differentiated (for 3 h). Series of z-stacks of high-resolution structured illumination microscopy immunofluorescent images (left panel) were segmented and analyzed for detection of histone H2B spots in each nucleus (upper right panel). Spot properties (i.e., sizes and mean signal intensities over pixels) of H2B spots were assessed. Images with detected spot information were subsequently transformed into binary grayscale images (bottom right panel), and clustering patterns (i.e., distance to first closest neighbor, distance second closest neighbor, and number of neighbors) among H2B spots were further examined. (B) Quantification of spot properties of wild-type and Chd5-deficient NSCs that were undifferentiated (0 h) or differentiated (for 3 h). Data are represented as mean ± SD. n indicates the total number of cells analyzed. ns, no significance; ****<0.0001; Tukey’s multiple comparisons test. (C) Quantification of clustering patterns of wild-type and Chd5-deficient NSCs that were undifferentiated (0 h) or differentiated (for 3 h). Data are represented as mean ± SD. n indicates the total number of cells analyzed. ns, no significance; ***<0.005; ****<0.0001; Tukey’s multiple comparisons test. (D) Immunofluorescent images of undifferentiated wild-type and Chd5-deficient NSCs (left panels) assessed for H3K27Ac (red) and DAPI nuclear signal (blue). Scale bar = 50 μm. (E) Quantification of pre-rRNA and 18S rRNA of wild-type and Chd5-deficient NSCs. Data are represented as mean ± SD (n = 2–3). **<0.005; unpaired t test. (F) Western blots of undifferentiated wild-type and Chd5-deficient NSCs (0 h post-differentiation) and differentiated cells (24 h post-differentiation), assessed for Rps6, Rpl7a, and β-actin (actin) expression.
- Figure 3. Chd5 deficiency leads to compromised expression of the repressive histone mark H3K27me3 and up-regulation of ribosomal protein genes.
(A, B) Immunofluorescent images of undifferentiated wild-type (+/+) and Chd5-deficient (Chd5−/−) NSCs (left panels) assessed for H3K27me3 (red), nestin (green), and DAPI nuclear signal (blue), and Western blots (right panels) probed for H3K27me3, H3K9me3, Histone H3, Ezh2, and β-actin (actin). Scale bar = 50 μm. (C, D) Representative immunofluorescent images of Chd5-deficient NSCs transduced with control shRluc (Chd5−/−; shRluc), shUtx (Chd5−/−; shUtx), and shJmjd3 (Chd5−/−; Jmjd3) that were differentiated for 7 d and analyzed for Gfap (red), Map2 (green), and DAPI nuclear signal (blue) (upper panels), with corresponding quantification (lower panels). Data are represented as mean ± SD (n = 3). *<0.05; **<0.01; ****<0.0001; Tukey's multiple comparison test. Scale bar = 50 μm. (E) Distribution of Map2+ neurons and Gfap+ astrocytes in cultures of wild-type and Chd5-deficient NSCs expressing control shRluc, shUtx, and shJmjd3 that were differentiated for 7 d; representation of data shown in Fig 3C. Numbers indicate mean percentage of each population. n indicates the total number of cells analyzed. (F) Comparison of steady-state mRNA levels of wild-type and Chd5-deficient NSCs. Mean relative log10-TPM expression values of two replicates for each condition are displayed. Differentially expressed genes are shown in red and ribosomal protein genes are marked with blue circles. Embedded pie chart indicates the fraction of up-regulated ribosomal protein genes (n = 46) among the total number of ribosomal protein genes (n = 84). See also Tables S1 and S2. (G) GO terms displaying significant enrichment (>fivefold) of differentially expressed genes in Chd5-deficient NSCs. See also Table S2. TPM, transcripts per million mapped.
- Figure 4. Precocious surge of protein synthesis disrupts dynamic translational regulation of the proneural factor Mash1 during early stages of neural differentiation.
(A, B) Representative immunofluorescent images of wild-type (+/+) and Chd5-deficient (Chd5−/−) NSCs (left panels), showing OP-puro–labeled nascent peptides (red) and DAPI nuclear signal (blue), with corresponding distribution and quantification of mean signal intensities over pixels of OP-puro signal (right panels) in randomly selected fields (5 fields per sample). Data are represented as mean ± SD (n = 2). ****<0.0001; unpaired t test. Scale bar = 50 μm. (C, D) Representative immunofluorescent images of wild-type and Chd5-deficient undifferentiated (0 h post-differentiation) and differentiated (3, 6, 12, and 24-h post-differentiation) NSCs (left panels), and each image was assessed for OP-puro–labeled nascent peptides (green) and DAPI nuclear signal (blue), with temporal representation of corresponding mean signal intensities over pixels of OP-puro signal at all time points in randomly selected fields (2–6 fields per sample), as plotted in Fig S3A and B. Data are represented as mean ± SD (n = 2). Scale bar = 50 μm. (E) Western blots of undifferentiated wild-type and Chd5-deficient NSCs (0 h post-differentiation) and differentiated cells (3-h post-differentiation), assessed for phosphorylated eIF4E at serine residue 209 (p-eIF4E), unmodified total eIF4E (eIF4E), phosphorylated eIF2α at serine residue 51 (p-eIF2α), unmodified total eIF2α, and β-actin (actin) expression. (F) Representative immunofluorescent images of wild-type and Chd5-deficient NSCs (left panels) that were undifferentiated (0 min) or differentiated (for 60, 120, and 180 min) and assessed for Mash1 (green) and DAPI nuclear signal (blue). Dashed rectangles indicate the zoomed-in regions, shown in insets. (G) Quantification of corresponding frequency of Mash1+ population, displaying distinct nuclear Mash1 expression, in randomly selected fields (5–7 fields per sample) (right panels). Data are represented as mean ± SD (n = 2). Scale bar = 50 μm (main image) and 10 μm (insets). (H) Western blots of wild-type and Chd5-deficient NSCs, assessed for Mash1 and β-actin (actin) expression at indicated time points. (I) Quantification of Ascl1 (i.e., transcript of Mash1) in total input and polysome fractions of wild-type and Chd5-deficient NSCs. Data are represented as mean ± SD (n = 2). *<0.05; **<0.01; ***<0.005; ****<0.0001; Tukey's multiple comparison test.
- Figure S4. Disrupted dynamic translational regulation of proneural factor Mash1.
(A, B) Distribution and comparison of mean signal intensities over pixels of OP-puro signal in randomly selected fields (2–6 fields per sample) of wild-type (upper panels) and Chd5-deficient (lower panels) NSCs at all time points, as shown in representative images in Fig 4C. Data are represented as mean ± SD (n = 2). ns, no significance; ****<0.0001; Tukey’s multiple comparisons test. (C) Comparison of the Mash1+ populations between wild-type and Chd5-deficient NSCs at each time point. Data are represented as mean ± SD (n = 2). ns, no significance; *<0.05; ***<0.001; unpaired t test. (D) Temporal representation of the Mash1+ populations of wild-type (left panel) and Chd5-deficient (right panel) NSCs at the time points, as indicated in Fig 4F. Data are represented as mean ± SD (n = 2). ns, no significance; *<0.05; **<0.01; ****<0.0001; Tukey’s multiple comparisons test.
Supplementary Materials
