Disruption of mir430 transcription bodies does not substantially impact development.

a, Schematic representation of the maternal-to-zygotic transition in zebrafish embryos. b,c, Visualization of elongating RNAPII (Ser2P) with Fabs in WT embryos at the 256-cell (256c), 1,024-cell (1,024c), Oblong and Sphere stages (b) and in WT and mir430^−/− embryos at the 128-cell stage (c). Shown are representative micrographs of individual nuclei, extracted from a spinning disk confocal microscopy timelapse. d, Schematic representation of a nucleus in WT, mir430^−/− and mir430^−/− with injected MiR430 embryos. e, Approach to assess rescue of miR430 activity, as previously described²⁴. The expression of eGFP encoded by an mRNA with three perfect target sites for miR430 is compared with the expression of RFP encoded by an mRNA without such sites (Methods). f, Schematic representation of expected eGFP and RFP expression in embryos with active and inactive MiR430 microRNA activity. g, Representative micrographs showing eGFP and RFP expression in WT, mir430^+/−, mir430^−/− and mir430^−/ with injected MiR430 embryos at 24 hpf. N = 3 biologically independent experiments. h, Rescue of miR430 activity was assessed in different genotypes at 24 hpf. Normalized eGFP signal in WT, mir430^+/− and mir430^−/− embryos without (left) and with (right) injected MiR430. N = 3 biologically independent experiments, n = 30 (WT without injected MiR430), n = 53 (mir430^+/− without injected MiR430), n = 31 (mir430^−/− without injected MiR430), n = 45 (WT with injected MiR430), n = 74 (mir430^+/− with injected MiR430), n = 27 (mir430^−/− with injected MiR430). Kruskal–Wallis tests were performed (without injected MiR430: χ² = 67.892, d.f. = 2, P value = 1.809 × 10⁻¹⁵; with injected MiR430: χ² = 2.5872, d.f. = 2, P value = 0.2743). When this test was statistically significant (P value < 0.05), pairwise comparisons with Bonferroni correction were performed using a pairwise Wilcoxon rank-sum test. A comparison was considered significant when adjusted P value was <0.05, and adjusted P values were reported using WT as reference. i, Rescue of miR430 activity as assessed by epiboly progression. Shown are representative micrographs of embryos at late epiboly stage in different genotypes. The misregulation of yolk internalization in mir430^−/− embryos is indicated. j, Time at which epiboly is completed in different genotypes without (left) and with (right) injected MiR430 RNA. N = 3 (without injected MiR430) and N = 4 (with injected MiR430) biologically independent experiments, n = 19 (WT without injected MiR430), n = 34 (mir430^+/− without injected MiR430), n = 19 (mir430^−/− without injected MiR430), n = 27 (WT with injected MiR430), n = 53 (mir430^+/− with injected MiR430), n = 28 (mir430^−/− with injected MiR430). Kruskal–Wallis tests were performed (without injected MiR430: χ² = 38.379, d.f. = 2, P value = 4.635 × 10⁻⁹; with injected MiR430: χ² = 0.00080597, d.f. = 2, P value = 0.9996). When this test was statistically significant (P value < 0.05), pairwise comparisons with Bonferroni correction were performed using a pairwise Wilcoxon rank-sum test. A comparison was considered significant when adjusted P value was <0.05, and adjusted P values were reported using WT as reference. k, Time at which Kupffer’s vesicle appears in different genotypes without (left) and with (right) injected MiR430. N = 3 (without injected MiR430) and N = 4 (with injected MiR430) biologically independent experiments, n = 18 (WT without injected MiR430), n = 27 (mir430^+/− without injected MiR430), n = 14 (mir430^−/− without injected MiR430), n = 25 (WT with injected MiR430), n = 44 (mir430^+/− with injected MiR430), n = 26 (mir430^−/− with injected MiR430). Kruskal–Wallis tests were performed (without injected MiR430: χ² = 26.038, d.f. = 2, P value = 2.218 × 10⁻⁶; with injected MiR430: χ² = 2.543, d.f. = 2, P value = 0.2804). When this test was statistically significant (P value < 0.05), pairwise comparisons with Bonferroni correction were performed using a pairwise Wilcoxon rank-sum test. A comparison was considered significant when adjusted P value was <0.05, and adjusted P values were reported using WT as reference. l, Larvae at 48 hpf for different genotypes. Representative micrographs are shown. The malformation of trunk morphology and eye, the development of heart oedema, and the appearance of blisters at the tail tip in mir430^−/− embryos are indicated (red arrowheads). Source numerical data are available in Source data.

Source data

Disruption of mir430 transcription bodies causes widespread misregulation of gene expression that recovers over time.

a, Volcano plots showing upregulated and downregulated genes in mir430^−/−+ inj MiR430 (n = 3 biologically independent samples) versus WT (n = 3 biologically independent samples) embryos at 256-cell, 1,024-cell, Oblong and Sphere stages. Wald test with Benjamini–Hochberg correction was performed, and genes with adjusted P values <0.01 were considered significantly differentially expressed. Genes whose coverage is shown in b are shown in black in the left-most plot. b, Coverage plot of irx7 (upregulated) and tmpob (downregulated) in WT and mir430^−/− + inj MiR430. The single strata visualize the labelling degree of the reads. c, Distribution of upregulated and downregulated genes across the genome. The mir430 locus on chromosome (Chr) 4 is shown in green and an expansion of its surrounding sequence is shown at the bottom of the panel. d, Alluvial plots showing the overlap between downregulated (left) and upregulated (right) genes at different stages of ZGA. e, Difference in average gene expression between mir430^−/− + inj MiR430 and WT embryos across stages for those genes that were identified to be differentially expressed at 256-cell stage (n = 242 downregulated genes and n = 716 upregulated genes). Boxplots show median, quartiles, minimum and maximum, and 1.5× interquartile range. Individual points represent outliers. Source numerical data are available on GEO (GSE248237).

Characterization of downregulated genes.

a, Average expression level in transcripts per million (TPM) in WT embryos for all genes (n = 32,428), non-expressed genes (n = 9,545), non-differentially expressed (DE) genes (n = 19,426) and upregulated (n = 716) and downregulated (n = 242) genes (mir430^−/− + inj MiR430 versus WT at 256-cell stage). Boxplots show median, quartiles, minimum and maximum, and 1.5× interquartile range. Outliers are not shown. b, Representative images of a DNA-FISH experiment for upregulated genes (green), downregulated genes (magenta) and mir430 (yellow) in a nucleus of a WT embryo at the 256-cell stage. N = 3. Nuclei were also stained with DAPI (blue). Images shown are maximum intensity projections. c,d, Schematic representation of oligopaint probe design for upregulated and downregulated genes (c) and the mir430 locus (d). Primers used for qPCR amplification and their complementarity within a probe are shown. Probe sequences are reported in Supplementary Table 2. Fwd, forward; Rev, reverse. e, Distributions of 3D distances of upregulated (up) genes from the mir430 locus (green) and downregulated (down) genes from the mir430 locus (red) in WT embryos. Quantification of one biological replicate was performed. N = 19 embryos, n = 643 nuclei. Source numerical data are available on GEO (GSE248237).

Loss of mir430 transcription bodies causes premature gene activation.

a, Time of induction in WT embryos for the 716 genes that are upregulated in mir430^−/− + inj MiR430 versus WT embryos (at 256-cell stage). Expression at 256-cell stage is used as a reference. The genes are split into four groups based on when they are induced (n = 446 induced genes at 1,024-cell stage; n = 51 induced genes at Oblong; n = 83 genes induced at Sphere; n = 136 not induced). The percentage of genes in each group is indicated. Boxplots show median, quartiles, minimum and maximum, and 1.5× interquartile range. Individual points represent outliers. b, Scatterplot representing the fold change in expression between mir430^−/− + inj MiR430 and WT embryos at 256-cell stage on the y axis, and between 1,024-cell stage and 256-cell stage in WT embryos on the x axis. All upregulated genes are shown in grey, and genes induced at 1,024-cell stage (62%) are shown in blue. c, Heatmap of chromatin accessibility at 256-cell stage of the 716 genes that are upregulated in mir430^−/− + inj MiR430 embryos compared with WT embryos at the 256-cell stage, and—for comparison—the 716 most expressed genes in WT embryos at the 256-cell stage. Genes are ranked by accessibility. d, Graph showing the fraction of promoters of the 716 upregulated (in mir430^−/− + inj MiR430 versus WT at the 256-cell stage) and the 716 most expressed genes (in WT at 256-cell stage) amongst the x% most accessible promoters at 256-cell stage in WT embryos. The distribution of non-expressed genes as well as the non-differentially expressed genes (mir430^−/− + inj MiR430 versus WT at 256-cell stage) are shown for comparison. e, Scatterplot representing the enrichment ratio (log₂) of transcription factor motifs in the promoters (TSS ± 2 kb) of upregulated genes compared with the promoters of not expressed genes (y axis) and the percentage of promoters of the upregulated genes that have the motif (x axis). Only motifs whose corresponding protein is translated during early development²⁷ were considered, and only motifs with a P value < 0.05 and E-value ≤ 10 are shown. Motifs corresponding to the three pluripotency factors Nanog, Sox19b and Pou5f3 (POU5F1, Pou5f1, Pou5f1::Sox2, POU5F1B, POU2F1, POU2F1::SOX2) are labelled in red. Source numerical data are available in Source data and on GEO (GSE248237 and GSE130944).

Source data

Loss of mir430 transcription bodies causes ectopic transcription bodies to go into elongation.

a, Visualization of RNAPII-Ser2P and RNAPII-Ser5P with Fabs in WT and mir430^−/− + inj MiR430 embryos at 512- and 1,024-cell stages. Shown are representative micrographs of individual nuclei. See Extended Data Fig. 6 for complete cell cycles. N = 3 biologically independent experiments. b, Quantification of Ser5P-positive/Ser2P-negative (red), Ser5P-positive/Ser2P-positive (yellow) and Ser5P-negative/Ser2P-positive (green) transcription bodies during the cell cycle at 512-cell and 1,024-cell stages as shown in a. Number (upper panel) and percentage (lower panel) are shown. N = 3 biologically independent experiments, number of nuclei (n) analysed at each timepoint, in each cell cycle and in each condition (WT and mir430^−/− + inj MiR430) are reported. Source numerical data are available in Source data.

Source data

CDK9 sequestration in mir430 transcription bodies inhibits transcription elongation elsewhere.

a, Visualization of CDK9 and RNAPII-Ser2P by immunofluorescence in WT nuclei at 256-cell stage. Nuclei are labelled with DAPI. b–d, Quantification of the enrichment of CDK9 and RNAPII-Ser2P in the two large transcription bodies. The enrichment (observed versus expected signal intensity) (b), the percentage of total nuclear signal present in the two large transcription bodies (c) and the percentage of the total nuclear area occupied by the two large transcription bodies (d) are shown. N = 3 biologically independent experiments, n = 214 nuclei. Wilcoxon rank-sum test was performed to assess if the log₂ of the ‘Enrichment of signal in transcription bodies (two-sided)’, the ‘percentage of nuclear signal in two transcription bodies’ (one-sided) and the ‘percentage of nuclear area occupied by two transcription bodies (one-sided)’ are significantly different than zero (V = 23,005 for all tests). A test was considered significant when it had a P value <0.05. Boxplots show median, quartiles, minimum and maximum, and 1.5× interquartile range. Individual points represent outliers. See Methods for a detailed description of quantification. e, Volcano plots showing upregulated and downregulated genes in WT + inj cdk9 mRNA (n = 3 biologically independent samples) versus WT (n = 3 biologically independent samples) embryos at the 256-cell stage (top) and the 1,024-cell stage (bottom). Wald test with Benjamini–Hochberg correction was performed, and genes with adjusted P values <0.01 were considered significantly differentially expressed. f, Visualization of RNAPII-Ser2P with Fabs, and miR430 RNA with MOVIE in WT, WT + inj cdk9 mRNA and mir430^−/− + inj MiR430 embryos across stages. Shown are representative micrographs of individual nuclei at 256-cell and 1,024-cell stages, extracted from a spinning disk confocal microscopy timelapse. See Extended Data Figs. 7–9 for complete cell cycles from 128-cell to 1,024-cell stages. g, Quantification of ectopic transcription bodies as shown in b. N = 3 biologically independent experiments; n = 101, 184, 290 and 378 at 128-cell, 256-cell, 512-cell and 1,024-cell stages in WT; n = 43, 82, 127 and 179 at 128-cell, 256-cell, 512-cell and 1,024-cell stages in WT + inj cdk9; n = 45, 65, 97 and 152 at 128-cell, 256-cell, 512-cell and 1,024-cell stages in mir430^−/− + inj MiR430, respectively. Boxplots show median, quartiles, minimum and maximum, and 1.5× interquartile range. Individual points represent outliers. See Extended Data Fig. 10 and Methods for a detailed description of quantification. h, Model for the role of transcription bodies in transcription regulation. In WT nuclei, two large transcription bodies are nucleated by the mir430 locus. They sequester CDK9 and potentially other factors that are required for pause release, thereby stalling transcription elsewhere in the nucleus in the initiation state. The specific disruption of the two mir430 transcription bodies leads to a redistribution of CDK9, which results in pause release and the upregulation of genes elsewhere in the nucleus. Source numerical data are available in Source data and on GEO (GSE248237).

Source data