Gentsch GE , Spruce T , Owens NDL , Smith JC .

U117597140 RCUK | Medical Research Council (MRC), FC001-157 RCUK | Medical Research Council (MRC), FC001-157 Cancer Research UK (CRUK), FC001-157 Wellcome Trust (Wellcome), Wellcome Trust

	Fig 1. Characterisation of pCRMs instructing ZGA. a Timeline (hpf, hours post-fertilisation, at 25 °C) of the maternal-to-zygotic transition and earliest signalling events (nuclear accumulation of Wnt, Nodal and Bmp signal mediators β-catenin, Smad2 and Smad1, respectively) during early X. tropicalis development up to the late gastrula embryo (12 hpf) with ∼40,000 (40 K) cells. b Signal transduction pathway causing signal mediators to enter the nucleus and engage with pCRMs (e.g., marked by RNAPII). c Snapshot of RNAPII recruitment to pCRMs of the zygotic gene locus tbxt from the 32-cell to the late gastrula stage. The underlying DNA sequence of RNAPII+ pCRMs are used to discover enriched DNA motifs de novo (illustrated as coloured arrows for one RNAPII+ pCRM). d Spearman correlations (Rs) of RNAPII binding levels across ∼27,000 pCRMs (Supplementary Data 2) between the indicated developmental stages. e Temporal enrichment (Z-score) of consensus DNA motifs known to be recognised by indicated TF families among RNAPII+ pCRMs. f MBT-staged heat map of DNase-probed chromatin accessibility, RNAPII binding and H3K4me1 marking (n = 2 biologically independent samples) across ∼17,500 pCRMs (Supplementary Data 3) grouped by sequence conservation levels (phastCons) and sorted by the statistical significance of pCRM accessibility. Abbreviations used for the developmental timeline: 32, 128 and 1 K, 32-, 128- and 1024-cell stage; MBT, mid-blastula transition; eG, mG and lG, early, mid- and late gastrula stage
	Fig 2. Search for ZGA-critical proteins based on their significantly enriched DNA recognition motifs at accessible and engaged (RNAPII+/H3K4me1+) pCRMs and their high translation frequency before the MBT. a Maternal protein concentrations in the egg13 versus ribosome footprint (translation) levels at the 8-cell stage14. Most frequently translated representatives of various TF families are labelled. b Matching canonical pCRM-enriched DNA motifs (sorted by statistical significance) with frequently translated TFs and signal mediators. c WMIHC of Sox3 protein in control and Sox3 loss-of-function (LOF) embryos at the 64-cell and blastula stage. Nuclear accumulation of Sox3 protein was detected in both the animal (An) and vegetal (Vg) hemisphere of control embryos. Scale bar, 0.5 mm. d Graphical illustration of protein levels (derived from mass spectrometry data42) and nuclear localisations (mainly derived from WMIHC, see references below) of selected TFs and signal mediators based on our and previously published results: Sox3 (this study and ref. 50), mPouV (Pou5f3.2 and Pou5f3.3) and (zygotic) Pou5f3.1 (deduced from transcript data15), mVegT and zVegT16, β-catenin10,17, Smad1 (this study and refs. 10,18) and Smad2 (this study and refs. 10,18). Shaded boxes indicate periods of non-nuclear protein localisation. Arrows indicate tissue movements of gastrulation. Abbreviations used for the developmental timeline: 8 and 32, 8-cell and 32-cell stage; MBT, mid-blastula transition; and eG, early gastrula stage
	Fig 3. Chromatin engagement of TFs and signal mediators during the maternal-to-zygotic transition. a Chromatin profiling (ChIP-Seq) of selected TFs and signal mediators from the 32- or 1024-cell stage (32, 1 K) to the early (eG) and late (lG) gastrula and the early tailbud (eTailbud) stage. In all subsequent figure panels, the chromatin factors and developmental stages profiled are consistently colour-coded as illustrated here. The excerpt of multiple chromatin tracks shows the binding of maternal TFs (Sox3, Foxh1 and VegT) and signal mediators (β-catenin, Smad2 and Smad1) to the siamois2 (sia2 and sia2l) super-enhancer at the 1024-cell stage (see Supplementary Fig. 3a for the temporal progression of chromatin engagement to the siamois2 and ventx super-enhancers). b Bubble plot shows significantly enriched biological processes associated with zygotic super-enhancer+ genes (i.e., genes possessing engaged super-enhancers ≤5 kb from their active TSS at indicated developmental stages). c, f Meta-plots (mean [solid line] ± s.d. [dotted line or polygon]) summarise the level of RNAPII (c) or signal mediator (f) engagement across 2000 pCRMs most frequently occupied by the indicated TFs or signal mediators at the 1024-cell and early gastrula stage, respectively. The pie chart next to each meta-plot shows the percentage of these TF+ or signal mediator+ pCRMs bound (ChIP ≥2x input tag density) by RNAPII or signal mediators, respectively. See Supplementary Note 1 for percentage numbers. d Biplot of principal component (PC) 1 (accounting for 40% variance) and 2 (28% variance) shows the relationship of TF and signal mediator binding levels across ∼12,500 highly engaged pCRMs (compiled from the 2000 pCRMs with the highest DNA occupancy levels detected per protein and developmental stage) over several developmental stages. Note that developmental time (arrow) separates these profiles best. e Biplot of PC1 (60% variance) and PC2 (18% variance) for the temporal progression of RNAPII and TF binding levels across the same set of pCRMs as in d. g Heat map shows the statistical significance (hypergeometric p-value) of finding TF- and signal-specific DNA consensus motifs (y-axis) across 2000 pCRMs most frequently occupied by the indicated TFs or signal mediators (x-axis)
	Fig 4. Ectopic expression of the muscle determinant MyoD reveals the effect of co-expressed TFs on chromatin engagement and gene expression. a Experimental design: Ectopic expression of the MyoD-HA mRNA construct injected into the animal hemisphere followed by the genome-wide chromatin profiling (ChIP-Seq; n = 2 biologically independent samples) of MyoD-HA, Sox3 and RNAPII in early gastrula embryos. b Biplot of principal component (PC) 1 (accounting for 50% variance) and 2 (23% variance) shows the relationship of Sox3 (triangle), RNAPII (circle) and MyoD-HA (square) binding levels across MyoD+ and/or Sox3+ pCRMs. Arrows show the normal temporal dynamics of Sox3 and RNAPII binding (black dash-dotted line) and the MyoD-enforced (red dotted line) changes to them (red solid line) at early gastrula stage. Fill colour of symbols represents the developmental stage as indicated, while line colours refer to whether MyoD-HA was expressed (red) or not (black). Abbreviations used for the developmental timeline: 1 K, 1024-cell stage; eG and lG, early and late gastrula stage. c Heat map of the DNA occupancies of 3845 pCRMs and enriched DNA motifs sorted by the significance of MyoD-HA-enforced changes (p∆) to Sox3 binding levels. d, f Snapshot of chromatin co-recruitment to the super-enhancers of canonical MyoD and Sox3 target genes actc1 (d) and sox2 (f), respectively. e Heat map shows the significance of DNA motif enrichments (y-axis) at 10,000 pCRMs most frequently occupied by the indicated proteins in uninjected and MyoD-HA injected embryos (x-axis). g Experimental design: Animal cap assay to quantify MyoD- or Sox3-enforced transcription. h RT-qPCR results from the animal cap assay. Error bars, mean + s.d. (n = 2 biologically independent samples)
	Fig 5. Profiling chromatin for Sox3 in different anterior–posterior compartments of the central nervous system (CNS). a Experimental design: Genome-wide Sox3 profiling of head, trunk and bud dissected from early tailbud embryos after tissue fixation. b Snapshot of Sox3 binding to HoxD cluster in the head, trunk and bud. Note the differential binding of Sox3 with ‘anterior’, ‘middle’ and ‘posterior’ Hox genes being preferentially occupied in head, trunk and bud, respectively. c Heat map shows the significance of DNA motif enrichments (y-axis) across 10,000 pCRMs most frequently occupied in each chromatin profile (x-axis). Profiles are grouped depending on whether the cells expressing the TF of interest preferentially contribute to the anterior or posterior compartment. Abbreviations used for the developmental timeline: lG, late gastrula; and eTailbud, early tailbud. d Anatomical map of TF expression at the posterior end of an early tailbud embryo to explain the co-enrichment of ‘posterior’ TF-specific DNA recognition motifs in c. Sox3-expressing cells of the posterior neural tube originate from the chordoneural hinge (CNH) and posterior wall of the neurenteric canal (PNC) expressing Brachyury and Brachyury/Tbx6, respectively21. Both neural tube and PNC are also exposed to the expression of Cdx such as Cdx4101
	Fig 6. Synergistic relationship between maternal Pou5f3 (mPouV) and Sox3. a Morphological phenotypes caused by single and combined LOFs of Sox3 and mPouV when control embryos reached mid-gastrula and mid-tailbud stage. See also Supplementary Movie 1. b Phenotypical rescue of mPouV/Sox3 LOF embryos by the co-injection of both X. laevis Pou5f3.3 and Sox2 mRNA alongside mCherry mRNA as a tracer. c Experimental design: Profiling the poly(A) RNA transcriptome (n = 3 biologically independent samples) over three consecutive developmental stages under indicated conditions. Abbreviations used for the developmental timeline: MBT, midblastula transition; lB, late blastula; and eG, early gastrula. d Biplot of PC1 (accounting for 86% variance) and PC2 (8% variance) shows the relationship of developmental stage-specific poly(A) RNA transcriptomes of control and α-amanitin-injected embryos in biological triplicates (#1–3). e Detection of 3687 zygotic genes with reduced transcript levels (≥ 50%, FDR ≤10%) in α-amanitin-injected embryos. These genes were used as reference for all other LOFs. Dots in scatterplot are coloured according to the maternal contribution56 to the transcript level of each of these zygotic genes. f Scatterplot of transcript fold changes (FCs) caused by mPouV and mPouV/Sox3 LOFs with dots coloured according to the ratio of FCs. Numbered dots refer to some developmentally relevant genes listed in e. g Early gastrula-staged WMISH: mir427, animal view; ventx, lateral view, ventral side facing right; and tbxt, dorsal view. Numbers in the right bottom corner of each image refer to the count of embryos detected with the displayed WMISH staining among all embryos analysed per condition and in situ probe. Scale bars (a, b, g), 0.5 mm
	Fig 7. Signal-induced regionalisation of ZGA depends on maternal TFs. a Transcriptional comparison of zygotic genes between the LOFs of maternal PouV/Sox3 and Nodal signalling. Dots are coloured according to the normal ratio of transcript levels (regional expression79) across the animal-vegetal (An:Vg) or dorso-ventral (D:V) axis. Numbered dots refer to genes listed in Fig. 6e. b Venn diagram of genes downregulated by the mPouV/Sox3 LOF (orange) or the LOF of single signal transduction pathways (black). c Early gastrula-staged WMISH for tbxt and eomes under indicated LOFs. Scale bar, 0.5 mm. d Quantification of tbxt and eomes transcript levels in control and mPouV/Sox3 LOF animal caps with or without stimulated Nodal signalling. Error bars, mean + s.d. (n = 2 biologically independent samples). Two-tailed Student’s t-test: *p = 0.014; **p = 0.005; and NS, not significant (p ≥ 0.02). e Bar graphs show the percentage (and number) of downregulated zygotic genes (% ZGA under indicated LOFs) grouped by the normal ratio of transcript levels (regional expression79) across the animal-vegetal (An:Vg) and dorso-ventral (D:V) axis
	Fig 8. Maternal pluripotency TFs mPouV and Sox3 remodel ∼40% of the accessible chromatin landscape to contribute to ∼25% of ZGA. a Used approach to reveal the effect of mPouV/Sox3 on chromatin accessibility (DNase-Seq) and chromatin composition (ChIP-Seq) around the MBT (n = 2 biologically independent samples). b Double-logarithmic volcano plot shows massive chromatin accessibility (DNase cleavage) reductions caused by mPouV/Sox3 LOF. pCRMs (dots) with significant accessibility changes (FDR ≤10%) are marked in red. c Violin plots show the comparison of chromatin accessibility (DNase cleavages) between uninjected and mPouV/Sox3 LOF embryos at all and affected (FDR ≤10%) pCRMs. Wilcoxon test: *p < 2.2 × 10−16. d Normalised meta-plots (top row, mean ± s.d.) and heat maps (bottom row) show the level of chromatin accessibility, chromatin engagements and RNA across accessible pCRMs in uninjected and mPouV/Sox3 LOF embryos. RNA was profiled at and beyond the MBT as shown in Fig. 6c. In the heat map, the pCRMs are sorted and grouped by significantly reduced DNase cleavages under mPouV/Sox3 LOF: red group, affected (FDR ≤10%) and blue group, unaffected (FDR >10%). These groups are represented in the meta-plots. Each heat map under mPouV/Sox3 LOF are followed by a heat map showing the statistical significance (Wald test) of changes (p∆) caused by mPouV/Sox3 LOF. e Heat map shows the occurrence of DNA motifs at accessible pCRMs sorted and grouped as in d. f Pie charts summarise the distribution of distances (kb) to nearest zygotic TSSs of affected (top pie chart) and unaffected (bottom pie chart) pCRMs. g Panel compares the effect of mPouV/Sox3 LOF on chromatin accessibility and RNAPII-mediated gene expression. Plot to the left shows the localisation of accessible pCRMs (affected, dot coloured in orange to red with FDR decreasing from 10%; and unaffected, grey dot) relative to the zygotic TSSs that are active by the MBT92 and produce enough RNA transcripts to show significant ≥ two-fold reductions upon α-amanitin injection (Fig. 6e). Gene loci are sorted by mPouV/Sox3 LOF-induced transcript fold changes as shown in the log-scaled bar graph to the right
	Fig 9. Pioneering activity of mPouV/Sox3 initiates extensive chromatin remodelling, such as the chromatin looping of distal pCRMs with promoters. a Used approach to reveal the effect of mPouV/Sox3 on chromatin conformation between 30 promoters and distal pCRMs (next-generation capture-C; n = 3 biologically independent samples) at the MBT. b Bar graph shows the number of normalised promoter contacts (mean + s.d.; n = 3 biologically independent samples) derived from non-redundant capture-reporter FLASH88 reads (Supplementary Fig. 12b) for each promoter captured with one or two probes under control condition (Supplementary Data 10). c Violin plots compare the number of promoter contacts with accessible pCRMs between uninjected and mPouV/Sox3 LOF embryos. The comparison is stratified into pCRMs with stable and lost accessibility upon mPouV/Sox3 LOF and shown for both all and affected (FDR ≤10%) promoter contacts. Wilcoxon tests and effect size estimates: *p = 5 × 10–5 and reffect = 0.12 (small effect); **p = 5 × 10−30 and reffect = 0.34 (medium effect); and ***p = 5 × 10−10 and reffect = 0.78 (large effect). d Superimposed line tracks show promoter contact frequencies, chromatin accessibilities and DNA occupancies of various chromatin components (Smad2, β-catenin, H3K4me1 and RNAPII) at the cdc25b gene locus between control (uninjected) and mPouV/Sox3 LOF embryos. The RNA track is split into a high (0–12) and low (0–0.01) expression window. Note that the low-expression window shows that locally transcribed non-coding super-enhancer RNA depend on mPouV/Sox3 as well as the gene cdc25b. Heat maps (p∆) below each superimposed line plot show the statistical significance (Wald test) of changes caused by mPouV/Sox3 LOF. The footer highlights the occurrences of canonical POU/SOX motifs (black filled rectangles) at accessible pCRMs (±50 bp from the accessibility centre) and one strongly affected pCRM with an arrowhead. Asterisks on the p∆ heat map mark significant (FDR ≤10%) reductions to pCRM accessibility. pCRMs are boxed in and their frequency of contacts with the cdc25b promoter are illustrated with an arc of varying strength. Boxes of affected pCRM and arcs of promoter contacts are coloured orange
	Fig 10. Maternal Pou5f3/Sox3-initiated chromatin remodelling to prime the first transcriptional response to inductive signals during ZGA. a, b Superimposed line tracks show promoter contact frequencies, chromatin accessibilities and DNA occupancies of various chromatin components (Smad2, H3K4me1 and RNAPII) at the Nodal-responsive mesoderm determinants tbxt (a) and eomes (b) between control (uninjected) and mPouV/Sox3 LOF embryos. Heat maps (p∆) below each superimposed line track show the statistical significance (Wald test) of changes caused by mPouV/Sox3 LOF. The footer highlights the occurrences of canonical POU/SOX motifs (black filled rectangles) at accessible pCRMs (±50 bp from the accessibility centre) and one strongly affected pCRM with an arrowhead. Asterisks on the p∆ heat map mark significant (FDR ≤10%) reductions to pCRM accessibility. pCRMs are boxed in and their frequency of contacts with the promoter are illustrated with an arc of varying strength. Boxes of affected pCRM and arcs of promoter contacts are coloured orange. c Stacked bar graphs summarise the correlation of unaffected and significantly reduced (FDR ≤10%) pCRM accessibility with RNAPII-mediated expression of all, signal responsive and non-responsive zygotic genes in mPouV/Sox3 LOF embryos at the MBT. These correlations and corresponding numbers (placed on the stacked bars) are shown for pCRM-gene associations and zygotic genes. TSS-centric maps of reduced chromatin accessibility are shown for signal responsive and non-responsive genes in Supplementary Figs. 15–17. d Model of chromatin pioneering and opportunistic engagement to predefine first zygotic responses to inductive signals
	Fig. 1. Characterisation of pCRMs instructing ZGA. a Timeline (hpf, hours post-fertilisation, at 25 °C) of the maternal-to-zygotic transition and earliest signalling events (nuclear accumulation of Wnt, Nodal and Bmp signal mediators β-catenin, Smad2 and Smad1, respectively) during early X. tropicalis development up to the late gastrula embryo (12 hpf) with ∼40,000 (40 K) cells. b Signal transduction pathway causing signal mediators to enter the nucleus and engage with pCRMs (e.g., marked by RNAPII). c Snapshot of RNAPII recruitment to pCRMs of the zygotic gene locus tbxt from the 32-cell to the late gastrula stage. The underlying DNA sequence of RNAPII+ pCRMs are used to discover enriched DNA motifs de novo (illustrated as coloured arrows for one RNAPII+ pCRM). d Spearman correlations (Rs) of RNAPII binding levels across ∼27,000 pCRMs (Supplementary Data 2) between the indicated developmental stages. e Temporal enrichment (Z-score) of consensus DNA motifs known to be recognised by indicated TF families among RNAPII+ pCRMs. f MBT-staged heat map of DNase-probed chromatin accessibility, RNAPII binding and H3K4me1 marking (n = 2 biologically independent samples) across ∼17,500 pCRMs (Supplementary Data 3) grouped by sequence conservation levels (phastCons) and sorted by the statistical significance of pCRM accessibility. Abbreviations used for the developmental timeline: 32, 128 and 1 K, 32-, 128- and 1024-cell stage; MBT, mid-blastula transition; eG, mG and lG, early, mid- and late gastrula stage
	Fig. 2. Search for ZGA-critical proteins based on their significantly enriched DNA recognition motifs at accessible and engaged (RNAPII+/H3K4me1+) pCRMs and their high translation frequency before the MBT. a Maternal protein concentrations in the egg13 versus ribosome footprint (translation) levels at the 8-cell stage14. Most frequently translated representatives of various TF families are labelled. b Matching canonical pCRM-enriched DNA motifs (sorted by statistical significance) with frequently translated TFs and signal mediators. c WMIHC of Sox3 protein in control and Sox3 loss-of-function (LOF) embryos at the 64-cell and blastula stage. Nuclear accumulation of Sox3 protein was detected in both the animal (An) and vegetal (Vg) hemisphere of control embryos. Scale bar, 0.5 mm. d Graphical illustration of protein levels (derived from mass spectrometry data42) and nuclear localisations (mainly derived from WMIHC, see references below) of selected TFs and signal mediators based on our and previously published results: Sox3 (this study and ref. 50), mPouV (Pou5f3.2 and Pou5f3.3) and (zygotic) Pou5f3.1 (deduced from transcript data15), mVegT and zVegT16, β-catenin10,17, Smad1 (this study and refs. 10,18) and Smad2 (this study and refs. 10,18). Shaded boxes indicate periods of non-nuclear protein localisation. Arrows indicate tissue movements of gastrulation. Abbreviations used for the developmental timeline: 8 and 32, 8-cell and 32-cell stage; MBT, mid-blastula transition; and eG, early gastrula stage
	Fig. 3. Chromatin engagement of TFs and signal mediators during the maternal-to-zygotic transition. a Chromatin profiling (ChIP-Seq) of selected TFs and signal mediators from the 32- or 1024-cell stage (32, 1 K) to the early (eG) and late (lG) gastrula and the early tailbud (eTailbud) stage. In all subsequent figure panels, the chromatin factors and developmental stages profiled are consistently colour-coded as illustrated here. The excerpt of multiple chromatin tracks shows the binding of maternal TFs (Sox3, Foxh1 and VegT) and signal mediators (β-catenin, Smad2 and Smad1) to the siamois2 (sia2 and sia2l) super-enhancer at the 1024-cell stage (see Supplementary Fig. 3a for the temporal progression of chromatin engagement to the siamois2 and ventx super-enhancers). b Bubble plot shows significantly enriched biological processes associated with zygotic super-enhancer+ genes (i.e., genes possessing engaged super-enhancers ≤5 kb from their active TSS at indicated developmental stages). c, f Meta-plots (mean [solid line] ± s.d. [dotted line or polygon]) summarise the level of RNAPII (c) or signal mediator (f) engagement across 2000 pCRMs most frequently occupied by the indicated TFs or signal mediators at the 1024-cell and early gastrula stage, respectively. The pie chart next to each meta-plot shows the percentage of these TF+ or signal mediator+ pCRMs bound (ChIP ≥2x input tag density) by RNAPII or signal mediators, respectively. See Supplementary Note 1 for percentage numbers. d Biplot of principal component (PC) 1 (accounting for 40% variance) and 2 (28% variance) shows the relationship of TF and signal mediator binding levels across ∼12,500 highly engaged pCRMs (compiled from the 2000 pCRMs with the highest DNA occupancy levels detected per protein and developmental stage) over several developmental stages. Note that developmental time (arrow) separates these profiles best. e Biplot of PC1 (60% variance) and PC2 (18% variance) for the temporal progression of RNAPII and TF binding levels across the same set of pCRMs as in d. g Heat map shows the statistical significance (hypergeometric p-value) of finding TF- and signal-specific DNA consensus motifs (y-axis) across 2000 pCRMs most frequently occupied by the indicated TFs or signal mediators (x-axis)
	Fig. 4. Ectopic expression of the muscle determinant MyoD reveals the effect of co-expressed TFs on chromatin engagement and gene expression. a Experimental design: Ectopic expression of the MyoD-HA mRNA construct injected into the animal hemisphere followed by the genome-wide chromatin profiling (ChIP-Seq; n = 2 biologically independent samples) of MyoD-HA, Sox3 and RNAPII in early gastrula embryos. b Biplot of principal component (PC) 1 (accounting for 50% variance) and 2 (23% variance) shows the relationship of Sox3 (triangle), RNAPII (circle) and MyoD-HA (square) binding levels across MyoD+ and/or Sox3+ pCRMs. Arrows show the normal temporal dynamics of Sox3 and RNAPII binding (black dash-dotted line) and the MyoD-enforced (red dotted line) changes to them (red solid line) at early gastrula stage. Fill colour of symbols represents the developmental stage as indicated, while line colours refer to whether MyoD-HA was expressed (red) or not (black). Abbreviations used for the developmental timeline: 1 K, 1024-cell stage; eG and lG, early and late gastrula stage. c Heat map of the DNA occupancies of 3845 pCRMs and enriched DNA motifs sorted by the significance of MyoD-HA-enforced changes (p∆) to Sox3 binding levels. d, f Snapshot of chromatin co-recruitment to the super-enhancers of canonical MyoD and Sox3 target genes actc1 (d) and sox2 (f), respectively. e Heat map shows the significance of DNA motif enrichments (y-axis) at 10,000 pCRMs most frequently occupied by the indicated proteins in uninjected and MyoD-HA injected embryos (x-axis). g Experimental design: Animal cap assay to quantify MyoD- or Sox3-enforced transcription. h RT-qPCR results from the animal cap assay. Error bars, mean + s.d. (n = 2 biologically independent samples)
	Fig. 5. Profiling chromatin for Sox3 in different anterior–posterior compartments of the central nervous system (CNS). a Experimental design: Genome-wide Sox3 profiling of head, trunk and bud dissected from early tailbud embryos after tissue fixation. b Snapshot of Sox3 binding to HoxD cluster in the head, trunk and bud. Note the differential binding of Sox3 with ‘anterior’, ‘middle’ and ‘posterior’ Hox genes being preferentially occupied in head, trunk and bud, respectively. c Heat map shows the significance of DNA motif enrichments (y-axis) across 10,000 pCRMs most frequently occupied in each chromatin profile (x-axis). Profiles are grouped depending on whether the cells expressing the TF of interest preferentially contribute to the anterior or posterior compartment. Abbreviations used for the developmental timeline: lG, late gastrula; and eTailbud, early tailbud. d Anatomical map of TF expression at the posterior end of an early tailbud embryo to explain the co-enrichment of ‘posterior’ TF-specific DNA recognition motifs in c. Sox3-expressing cells of the posterior neural tube originate from the chordoneural hinge (CNH) and posterior wall of the neurenteric canal (PNC) expressing Brachyury and Brachyury/Tbx6, respectively21. Both neural tube and PNC are also exposed to the expression of Cdx such as Cdx4101
	Fig. 6. Synergistic relationship between maternal Pou5f3 (mPouV) and Sox3. a Morphological phenotypes caused by single and combined LOFs of Sox3 and mPouV when control embryos reached mid-gastrula and mid-tailbud stage. See also Supplementary Movie 1. b Phenotypical rescue of mPouV/Sox3 LOF embryos by the co-injection of both X. laevis Pou5f3.3 and Sox2 mRNA alongside mCherry mRNA as a tracer. c Experimental design: Profiling the poly(A) RNA transcriptome (n = 3 biologically independent samples) over three consecutive developmental stages under indicated conditions. Abbreviations used for the developmental timeline: MBT, midblastula transition; lB, late blastula; and eG, early gastrula. d Biplot of PC1 (accounting for 86% variance) and PC2 (8% variance) shows the relationship of developmental stage-specific poly(A) RNA transcriptomes of control and α-amanitin-injected embryos in biological triplicates (#1–3). e Detection of 3687 zygotic genes with reduced transcript levels (≥ 50%, FDR ≤10%) in α-amanitin-injected embryos. These genes were used as reference for all other LOFs. Dots in scatterplot are coloured according to the maternal contribution56 to the transcript level of each of these zygotic genes. f Scatterplot of transcript fold changes (FCs) caused by mPouV and mPouV/Sox3 LOFs with dots coloured according to the ratio of FCs. Numbered dots refer to some developmentally relevant genes listed in e. g Early gastrula-staged WMISH: mir427, animal view; ventx, lateral view, ventral side facing right; and tbxt, dorsal view. Numbers in the right bottom corner of each image refer to the count of embryos detected with the displayed WMISH staining among all embryos analysed per condition and in situ probe. Scale bars (a, b, g), 0.5 mm
	Fig. 7. Signal-induced regionalisation of ZGA depends on maternal TFs. a Transcriptional comparison of zygotic genes between the LOFs of maternal PouV/Sox3 and Nodal signalling. Dots are coloured according to the normal ratio of transcript levels (regional expression79) across the animal-vegetal (An:Vg) or dorso-ventral (D:V) axis. Numbered dots refer to genes listed in Fig. 6e. b Venn diagram of genes downregulated by the mPouV/Sox3 LOF (orange) or the LOF of single signal transduction pathways (black). c Early gastrula-staged WMISH for tbxt and eomes under indicated LOFs. Scale bar, 0.5 mm. d Quantification of tbxt and eomes transcript levels in control and mPouV/Sox3 LOF animal caps with or without stimulated Nodal signalling. Error bars, mean + s.d. (n = 2 biologically independent samples). Two-tailed Student’s t-test: *p = 0.014; **p = 0.005; and NS, not significant (p ≥ 0.02). e Bar graphs show the percentage (and number) of downregulated zygotic genes (% ZGA under indicated LOFs) grouped by the normal ratio of transcript levels (regional expression79) across the animal-vegetal (An:Vg) and dorso-ventral (D:V) axis
	Fig. 8. Maternal pluripotency TFs mPouV and Sox3 remodel ∼40% of the accessible chromatin landscape to contribute to ∼25% of ZGA. a Used approach to reveal the effect of mPouV/Sox3 on chromatin accessibility (DNase-Seq) and chromatin composition (ChIP-Seq) around the MBT (n = 2 biologically independent samples). b Double-logarithmic volcano plot shows massive chromatin accessibility (DNase cleavage) reductions caused by mPouV/Sox3 LOF. pCRMs (dots) with significant accessibility changes (FDR ≤10%) are marked in red. c Violin plots show the comparison of chromatin accessibility (DNase cleavages) between uninjected and mPouV/Sox3 LOF embryos at all and affected (FDR ≤10%) pCRMs. Wilcoxon test: *p < 2.2 × 10−16. d Normalised meta-plots (top row, mean ± s.d.) and heat maps (bottom row) show the level of chromatin accessibility, chromatin engagements and RNA across accessible pCRMs in uninjected and mPouV/Sox3 LOF embryos. RNA was profiled at and beyond the MBT as shown in Fig. 6c. In the heat map, the pCRMs are sorted and grouped by significantly reduced DNase cleavages under mPouV/Sox3 LOF: red group, affected (FDR ≤10%) and blue group, unaffected (FDR >10%). These groups are represented in the meta-plots. Each heat map under mPouV/Sox3 LOF are followed by a heat map showing the statistical significance (Wald test) of changes (p∆) caused by mPouV/Sox3 LOF. e Heat map shows the occurrence of DNA motifs at accessible pCRMs sorted and grouped as in d. f Pie charts summarise the distribution of distances (kb) to nearest zygotic TSSs of affected (top pie chart) and unaffected (bottom pie chart) pCRMs. g Panel compares the effect of mPouV/Sox3 LOF on chromatin accessibility and RNAPII-mediated gene expression. Plot to the left shows the localisation of accessible pCRMs (affected, dot coloured in orange to red with FDR decreasing from 10%; and unaffected, grey dot) relative to the zygotic TSSs that are active by the MBT92 and produce enough RNA transcripts to show significant ≥ two-fold reductions upon α-amanitin injection (Fig. 6e). Gene loci are sorted by mPouV/Sox3 LOF-induced transcript fold changes as shown in the log-scaled bar graph to the right
	Fig. 9. Pioneering activity of mPouV/Sox3 initiates extensive chromatin remodelling, such as the chromatin looping of distal pCRMs with promoters. a Used approach to reveal the effect of mPouV/Sox3 on chromatin conformation between 30 promoters and distal pCRMs (next-generation capture-C; n = 3 biologically independent samples) at the MBT. b Bar graph shows the number of normalised promoter contacts (mean + s.d.; n = 3 biologically independent samples) derived from non-redundant capture-reporter FLASH88 reads (Supplementary Fig. 12b) for each promoter captured with one or two probes under control condition (Supplementary Data 10). c Violin plots compare the number of promoter contacts with accessible pCRMs between uninjected and mPouV/Sox3 LOF embryos. The comparison is stratified into pCRMs with stable and lost accessibility upon mPouV/Sox3 LOF and shown for both all and affected (FDR ≤10%) promoter contacts. Wilcoxon tests and effect size estimates: *p = 5 × 10–5 and reffect = 0.12 (small effect); **p = 5 × 10−30 and reffect = 0.34 (medium effect); and ***p = 5 × 10−10 and reffect = 0.78 (large effect). d Superimposed line tracks show promoter contact frequencies, chromatin accessibilities and DNA occupancies of various chromatin components (Smad2, β-catenin, H3K4me1 and RNAPII) at the cdc25b gene locus between control (uninjected) and mPouV/Sox3 LOF embryos. The RNA track is split into a high (0–12) and low (0–0.01) expression window. Note that the low-expression window shows that locally transcribed non-coding super-enhancer RNA depend on mPouV/Sox3 as well as the gene cdc25b. Heat maps (p∆) below each superimposed line plot show the statistical significance (Wald test) of changes caused by mPouV/Sox3 LOF. The footer highlights the occurrences of canonical POU/SOX motifs (black filled rectangles) at accessible pCRMs (±50 bp from the accessibility centre) and one strongly affected pCRM with an arrowhead. Asterisks on the p∆ heat map mark significant (FDR ≤10%) reductions to pCRM accessibility. pCRMs are boxed in and their frequency of contacts with the cdc25b promoter are illustrated with an arc of varying strength. Boxes of affected pCRM and arcs of promoter contacts are coloured orange
	Fig. 10. Maternal Pou5f3/Sox3-initiated chromatin remodelling to prime the first transcriptional response to inductive signals during ZGA. a, b Superimposed line tracks show promoter contact frequencies, chromatin accessibilities and DNA occupancies of various chromatin components (Smad2, H3K4me1 and RNAPII) at the Nodal-responsive mesoderm determinants tbxt (a) and eomes (b) between control (uninjected) and mPouV/Sox3 LOF embryos. Heat maps (p∆) below each superimposed line track show the statistical significance (Wald test) of changes caused by mPouV/Sox3 LOF. The footer highlights the occurrences of canonical POU/SOX motifs (black filled rectangles) at accessible pCRMs (±50 bp from the accessibility centre) and one strongly affected pCRM with an arrowhead. Asterisks on the p∆ heat map mark significant (FDR ≤10%) reductions to pCRM accessibility. pCRMs are boxed in and their frequency of contacts with the promoter are illustrated with an arc of varying strength. Boxes of affected pCRM and arcs of promoter contacts are coloured orange. c Stacked bar graphs summarise the correlation of unaffected and significantly reduced (FDR ≤10%) pCRM accessibility with RNAPII-mediated expression of all, signal responsive and non-responsive zygotic genes in mPouV/Sox3 LOF embryos at the MBT. These correlations and corresponding numbers (placed on the stacked bars) are shown for pCRM-gene associations and zygotic genes. TSS-centric maps of reduced chromatin accessibility are shown for signal responsive and non-responsive genes in Supplementary Figs. 15–17. d Model of chromatin pioneering and opportunistic engagement to predefine first zygotic responses to inductive signals

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