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. 2013 Nov 21;503(7476):360-4.
doi: 10.1038/nature12632. Epub 2013 Sep 22.

Nanog, Pou5f1 and SoxB1 Activate Zygotic Gene Expression During the Maternal-To-Zygotic Transition

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Free PMC article

Nanog, Pou5f1 and SoxB1 Activate Zygotic Gene Expression During the Maternal-To-Zygotic Transition

Miler T Lee et al. Nature. .
Free PMC article

Abstract

After fertilization, maternal factors direct development and trigger zygotic genome activation (ZGA) at the maternal-to-zygotic transition (MZT). In zebrafish, ZGA is required for gastrulation and clearance of maternal messenger RNAs, which is in part regulated by the conserved microRNA miR-430. However, the factors that activate the zygotic program in vertebrates are unknown. Here we show that Nanog, Pou5f1 (also called Oct4) and SoxB1 regulate zygotic gene activation in zebrafish. We identified several hundred genes directly activated by maternal factors, constituting the first wave of zygotic transcription. Ribosome profiling revealed that nanog, sox19b and pou5f1 are the most highly translated transcription factors pre-MZT. Combined loss of these factors resulted in developmental arrest before gastrulation and a failure to activate >75% of zygotic genes, including miR-430. Our results demonstrate that maternal Nanog, Pou5f1 and SoxB1 are required to initiate the zygotic developmental program and induce clearance of the maternal program by activating miR-430 expression.

Figures

Figure 1
Figure 1. Characterization of the zygotic transcriptome
a. Embryos showing the effects of α-amanitin, U1U2 morpholino (U1U2 MO) and cycloheximide (CHX). b. Sequencing read density across oep. Intronic signal increases with zygotic expression in total RNA. c. Expression histogram of zygotic genes. d. Maternal (M) but not zygotic factors (Z1) can activate transcription upon splice or translation inhibition. e. Metagene of read density across exon-intron boundaries in first-wave genes. U1U2 MO shows enriched intron signal (purple). f. Biplot comparing expression in wild type and U1U2 MO. Points above 5 RPKM in U1U2 MO are considered first-wave genes.
Figure 2
Figure 2. Identification of Nanog, SoxB1 and Pou5f1 as zygotic gene regulators
a. Schematic illustrating ribosome profiling. b. Rank plot showing translation levels pre-MZT. Sequence-specific transcription factors are highlighted. c. Embryos with combined loss of Nanog+SoxB1, Nanog+Pou5f1 or triple LOF arrest similar to α-amanitin and are rescued with mRNA injection. d. Ribosome footprints for h1m, sox19b and nanog in wild type and Nanog MO + SoxB1 MO. sox19b and nanog are highly depleted in the MO conditions. e. Biplots comparing wild type and MO ribosome footprints and input mRNA.
Figure 3
Figure 3. Transcriptome-wide effects of loss of Nanog, SoxB1 and Pou5f1
a. Biplots showing widespread gene expression loss in the triple LOF at 6hpf. b. Donut plots showing global effects of LOF. Percentages show the combined effect for strictly zygotic and maternal+zygotic (M+Z) gene groups. c. In situ hybridization shows expression defects in LOF embryos, which is rescued by mRNA injection.d. Heatmap showing first-wave zygotic genes in single and combined LOF conditions (N, Nanog MO; S, SoxB1 MO; P, MZpou5f1). Patterns shown are regulation by Nanog predominantly; SoxB1/Pou5f; or Nanog in combination with SoxB1/Pou5f1.
Figure 4
Figure 4. miR-430 expression is regulated by Nanog
a. Northern blot shows miR-430 is severely reduced in Nanog LOF and nearly undetectable in the triple LOF b. RNA-Seq read levels of the pri-mir-430 polycistron in wild type and LOF. c. Bar plot of total miR-430 aligning reads. d. First-wave genes are highly bound by Nanog. e. Nanog binding across the miR-430 region (top panel) and a zoomed region where reads are preferentially aligned to the 5’ end. Binding profiles show a strong peak between two precursors. pre-miR-430a, b and c are marked in red.
Figure 5
Figure 5. miR-430 activity is abrogated by Nanog LOF
a. In situ showing degradation of miR-430 target cd82b at 6hpf in wild type, compared to stabilization in MZdicer (lacking miR-430 activity). b. cd82b is stabilized in the Nanog-SoxB1 LOF embryo, indicating loss of miR-430 activity. The effect is rescued with injection of nanog and soxB1 mRNA. c. Cumulative plots showing stabilized expression of miR-430 targets in MZdicer and LOF embryos, compared to wild type. P values are for two-sided Wilcoxon rank sum tests comparing each miR-430 target group to non-targets.

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