Figure 2. Increasing the levels of all core histones delays onset of transcription and gastrulation.

(A) Schematic representation of experimental procedure. Histone cocktail (HC) containing ~5800 genomes worth of histones, or BSA was injected into the yolk of 1-cell embryos and qPCR and NanoString analysis was carried out at stages around genome activation. Orange crosses represent the timing of stages used for the analysis. (B) Expression of mxtx2 and fam212aa was analyzed by qPCR at early 1K, high, and oblong stage in uninjected, BSA-injected and HC-injected embryos. Bar graphs show the same data, focusing on high stage. Error bars represent SEM (n ≥ 13). ***p<0.001 (two-tailed Student’s t test, compared to BSA control). (C) Expression of 53 zygotically expressed genes was analyzed by NanoString analysis at high stage in uninjected, BSA-injected and HC-injected embryos. Mean counts of three independent biological replicates are shown. Location of mxtx2 and fam212aa counts is indicated (See Figure 2—figure supplement 2 for more details). (D) Relative expression level of mxtx2 and fam212aa at high stage, for embryos injected with BSA, HC, and HC minus H3, H4, H2A, or H2B. Error bars represent SEM (n = 7). ***p<0.001 (ordinary one-way ANOVA). (E) Brightfield images of embryos that were not injected, injected with BSA, or injected with HC. Boxed images represent the onset of gastrulation. Scale bar shown for the uninjected 2-cell embryo applies to all treatments except for 24 hpf embryos which have a different scale bar. All scale bars represent 250 μm. hpf, hours post-fertilization. (F) Bar graph shows the quantification of the extra time it takes embryos to start gastrulation upon injecting BSA, HC, or HC minus one histone, compared to uninjected embryos. Error bars represent SEM (n = 27 for BSA, n = 25 for HC, n = 7 for HC minus one histone experiments). ***p<0.001 (ordinary one-way ANOVA with Tukey’s multiple comparison test). In B and D, mRNA levels are normalized to the expression of eif4g2α.

DOI: http://dx.doi.org/10.7554/eLife.23326.006

Figure 2—figure supplement 1. Increasing the levels of all core histones delays onset of transcription.

(A) Expression of nnr, vox, sox19a, and grhl3 was analyzed by qPCR at early 1K, high, and oblong stage in uninjected, BSA-injected and HC-injected embryos. Bar graphs show the same data, focusing on high stage. Error bars represent SEM (n ≥ 13). ***p<0.001 (two-tailed Student’s t-test, compared to BSA control). (B) Staging by morphology was verified by cell counting. Each data point represents a single embryo. Error bars represent SEM. (C) Confocal microscope images of transgenic fish line Tg(h2afz:h2afz-GFP) injected with Cy5 conjugated to H4. Arrow points at chromatin in dividing cell. Scale bar, 20 μm. (D) Relative expression level of nnr, vox, sox19a, and grhl3 at high stage, for embryos injected with BSA, HC, and HC minus H3, H4, H2A, or H2B. Error bars represent SEM (n = 7). *p<0.05; **p<0.01; ***p<0.001 (ordinary one-way ANOVA). In A and D, expression is normalized to the expression of eif4g2α.

DOI: http://dx.doi.org/10.7554/eLife.23326.008

Figure 2—figure supplement 2. Increasing the levels of all core histones delays onset of transcription for a large number of genes.

NanoString’s nCounter technology was used to analyze changes in gene expression upon HC injection for a large number of genes (see Materials and methods for more details). From a custom probe set with 84 zygotically expressed genes and 12 controls genes (see Figure 2—source data 1), 53 genes that are induced at high stage were used for analysis. mRNA was collected from high stage embryos that were uninjected, BSA-injected and HC-injected (n = 3). (A) Fold difference in expression of twelve control genes, for embryos injected with BSA or HC compared to uninjected. Control genes are maternally provided and were previously shown to be stable from 512 cell to dome stage in NanoString analysis (data not shown). Error bars represent SEM (n = 3). No significant difference was detected between BSA or HC-injected in control genes (Ordinary one-way ANOVA with Tukey’s multiple comparison test), showing that there are no differences in total RNA amount between samples. (B) Proportion of genes affected by BSA or HC injection at high stage in NanoString analysis. Error bars represent SEM (n = 3). A large proportion of genes are down-regulated in HC-injected embryos (86%) compared to BSA-injected embryos (4%). (C) Mean counts of three independent biological replicates in NanoString analysis for uninjected, BSA-injected and HC-injected embryos compared to a high (left) and 1K (right) standard from uninjected embryos. While uninjected and BSA-injected are statistically similar to the high standard, HC-injected is statistically similar to the 1K standard. Thus, HC-injected embryos that are developmentally at high stage, are transcriptionally delayed by one developmental stage. Error bars represent SEM (n = 3). ***p<0.001 (Ordinary one-way ANOVA with Tukey’s multiple comparison test). (D) Fold difference in mRNA counts for HC-injected and BSA-injected compared to uninjected embryos at high stage for all genes that were analyzed by qPCR in this study. Error bars represent SEM (n = 3). *p<0.05; **p<0.01; ***p<0.001 (two-tailed Student’s t-test ratio paired, compared to BSA-injected).

DOI: http://dx.doi.org/10.7554/eLife.23326.009