Gentsch GE , Owens NDL , Smith JC .

Wellcome Trust

             Wnt signaling pathway
             BMP signaling pathway
             nodal signaling pathway

	Graphical abstract
	Figure S1. Dynamics and Architecture of ZGA in X. tropicalis, Related to Figure 1. (A) Progression of ZGA from the 32-cell to the late gastrula stage based on (i) whole gene body (fulllength) occupancy of RNAPII (i.e., RNAPII was enriched across entire gene bodies; see Transparent Methods). Co-aligned: Active histone marks H3K4me1 (ii) and H3K36me3 (iii) (Hontelez et al., 2015) and intronic signal from the high time-resolution profile of poly(A) RNA (iv) (Owens et al., 2016). The horizontal dotted line separates RNAPII-engaged (RNAPII+) from non-engaged (RNAPII-) genes as detected until the late gastrula stage. The vertical dotted lines in the poly(A) RNA plot indicate the developmental time points of the MBT (white) and the late gastrula stage (black), respectively. (B) Transcript feature levels (mean +/- SD) during the maternal-to-zygotic transition. Asterisk, polyadenylation immediately after fertilization (Collart et al., 2014) transiently increased the intronic signal obtained from the poly(A) RNA samples. (C) Venn diagram of zygotic genes detected by full-length RNAPII occupancy or reduced exonic or intronic transcript counts upon blocking RNAPII-mediated transcription with α-amanitin. (D) Pairwise Spearman’s correlations (Rs) of enrichment values resulting from RNAPII profiling and 4sU tagging to detect zygotic genes at indicated developmental stages. (E) Plots of the number of active and newly activated genes (i) or newly activated genes versus the developmental time (i), the number of completed cell divisions (ii) or formed blastomeres (iii). (F) Alluvial diagram of spatio-temporal ZGA including maternally inherited RNA transcripts of genes not activated by the late gastrula stage. Tissue-specificity inferred from regional transcript enrichments along the animal-vegetal or the dorso-ventral or both axes (Blitz et al., 2017). (G) Numbers of genes with full-length RNAPII occupancy at indicated developmental stages. Examples of ubiquitously (black) and tissue-specifically (orange) expressed genes are listed to the right. (H) RNAPII dynamics at the Cys2-His2 [C2H2] zinc finger (ZF) cluster from the 32-cell to the early neurula stage. (I) Expression dynamics of C2H2 ZF genes normalized to maximal transcript levels recorded between fertilization and 23.5 hpf (Owens et al., 2016). (J) Phylogenetic tree of the C2H2 ZF genes shown in (H,I). Maternal VegT (mVegT), outgroup TF of this phylogenetic tree. (K) Beeswarm boxplots showing various metrics of the zygotic/maternal genes (i) and mRNA (ii) during ZGA. Asterisks, significant Wilcoxon rank-sum tests against maternal and post-MBT activated genes and corresponding effect sizes (reffect): # introns, p <2.1e-13, reffect 0.07-0.43; Ø intron (kb), p <7.9e-7, reffect 0.07-0.42; CDS (kb), p <3e-7, reffect 0.05-0.27; mRNA (kb), p <7e-11, reffect 0.06-0.32; 5’ UTR (kb), p <0.015, reffect 0.03-0.15; and 3’ UTR (kb), p <1.4e-6, reffect 0.04-0.21. (L) Meta-profiles (mean +/- SD) of RNAPII (separated by developmental stage) and input (negative control) densities at zygotic genes. Abbreviations: 32, 32-cell; 128, 128-cell; 1K, 1,024-cell; MBT, mid-blastula transition; mG, mid-gastrula; lG, late gastrula; eN, early neurula; 4sU, 4-thiouridine; Mdn, median; TPM, transcripts per million.
	Figure S2. Effect of Canonical Wnt, Nodal and BMP Signals on ZGA, Related to Figure 2. (A) Poly(A) RNA profiles of the C2H2 ZF cluster (Figure S1H) for indicated control and LOFs at the MBT. (B) Graphical explanations of figure panels (C-F). (C-F) Summary (i) and temporal resolution (ii) of gene mis-regulations upon the LOF of Nodal (C) or BMP (D) signaling or maternal Pou5f3/Sox3 (mPou5f3/Sox3) (E) or VegT (mVegT) (F). Percentages only refer to the down-regulated genes (by ≥1/3 compared to control expression level) among all zygotic genes with the same range of expression ratios along the animal-vegetal or the dorso-ventral axes.
	Figure S3. Relationship between Canonical Wnt, Nodal and BMP to Control Regional ZGA, Related to Figure 3. (A-C,E,H-J,L) Scatter plots of relative (% to control) transcript levels between indicated LOFs with each dot (gene) color-coded according to a third attribute: (A,H,I) relative (% to control) transcript levels, (B) synergy factors between single inductive signals, and (C,E,J,L) regional expression ratios between opposite ends of the indicated axis. (G,N) Venn diagram of down-regulated genes by indicated LOFs. (D,F,K,M) Beeswarm boxplots of regional expression (as measured along the indicated axes) depending on increased Wnt-Nodal (D,F) or Nodal-BMP (K,M) synergy. (O) Coverage and enrichment of Smad and β-catenin-associated DNA motifs (SMAD and bHSH motifs) at endogenous binding sites of β-catenin, Smad1, Smad2 (Gentsch et al., 2018b) at indicated developmental stages (color-coded).
	Figure 1. Dynamics and Architecture of ZGA in X. tropicalis(A) (i) Genome-wide profiling of RNAPII and total RNA (Owens et al., 2016) to determine temporal ZGA dynamics. (ii) Complementary approach to detect transcriptionally active genes by α-amanitin-induced loss and 4sU enrichment of nascent (zygotic) transcripts.(B) Progression of ZGA from the 32-cell to the late gastrula stage based on (i) whole gene body (full-length) occupancy of RNAPII (i.e., RNAPII was enriched across entire gene bodies; see Transparent Methods). Co-aligned: (ii) High time-resolution of total RNA, separated by intron- and exon-derived signals, from fertilization to the late gastrula stage, and (iii) enrichments of 4sU-tagged RNA at the MBT and the mid-gastrula stage. Numbers below RNAPII heatmap represent counts of active (blue) and activated (red) genes at the indicated developmental stages. The horizontal dotted line separates RNAPII-engaged (RNAPII+) from non-engaged (RNAPII−) genes as detected until the late gastrula stage. The vertical dotted lines in the total RNA plots indicate the developmental time points of the MBT (white) and the late gastrula stage (black), respectively.(C) 2D space-filling (Hilbert) curves showing RNAPII recruitment to chromosomes (Chr) at the 128-cell stage and the MBT. A few zygotic genes are highlighted as being active (ON) or not (OFF) based on their engagement with RNAPII.(D) Alluvial diagram of spatiotemporal ZGA. Tissue specificity inferred from regional transcript enrichments along the animal-vegetal or the dorsoventral axes or both (Blitz et al., 2017).(E) ZGA-associated enrichment of biological processes. The statistical significance of enrichment (hypergeometric p-value) an the number of activated genes associated with each biological process are visualized by bubble size and color, respectively.(F) Beeswarm boxplots of maternal and/or zygotic gene sizes. *, p < 1.9 × 10−7 (Wilcoxon rank-sum test against maternal and post-MBT activated genes); reffect 0.06 (“MBT” vs “Egg”) - 0.48 (“128” vs “mG”).(G) Maternal/zygotic contribution to the transcriptome deduced from full-length RNAPII occupancy and maternally inherited RNA.Abbreviations: 32, 32-cell; 128, 128-cell; 1K, 1,024-cell; MBT, midblastula transition; mG, mid-gastrula; lG, late gastrula; 4sU, 4-thiouridine; Mdn, median; TPM, transcripts per million. See also Figure S1 and Tables S1 and S2.
	Figure 2. Spatiotemporal ZGA Regulated by Canonical Wnt, Nodal, and BMP Signals(A) Spatiotemporal ZGA and nuclear localization of signal mediators β-catenin (canonical Wnt), Smad2 (Nodal), and Smad1 (BMP) (Faure et al., 2000, Larabell et al., 1997, Schohl and Fagotto, 2002) from the 32-cell to the late gastrula stage.(B) Morphological phenotypes of single and combined signal LOFs at the late gastrula and the mid-tail bud stage. Left (“control”) pictures are taken from Gentsch et al. (2018b). Bullet points, failed blastopore formation. Arrowheads, excessive neural fold formation. Scale bar, 0.5 mm.(C) Heatmap (i) and bar graph summary (ii) of ZGA mis-regulated in various LOF embryos: α-amanitin, positive control; control MO, negative control. Abbreviations: B, BMP; N, Nodal; W, canonical Wnt; mPS, maternal Pou5f3/Sox3; mV, maternal VegT; 4x zT, four zygotic T-box TFs (zygotic VegT, Eomes, Tbxt and Tbxt.2).(D) Biological processes enriched with mis-regulated and control (not down-regulated or unaffected by the loss of maternal TFs or signaling) sets of zygotic genes under indicated LOFs. The statistical significance of enrichment (hypergeometric p-value) and the number of zygotic genes are visualized by bubble size and color, respectively.(E) Summary (i) and temporal resolution (ii) of Wnt LOF effects on regional ZGA. Percentages only refer to the down-regulated genes among all zygotic genes with similar expression ratios along the animal-vegetal or the dorsoventral axes.See also Figure S2, Tables S1 and S3, and Video S1.
	Figure 3. Uniform Gene Expression across Dorsoventral Axis Achieved by Wnt/BMP Synergy(A, C, E, F, G, and J) Scatterplots of relative (% to control) transcript levels between indicated LOFs with each dot (gene) color-coded according to a third attribute: (A and B) relative (% to control) transcript levels, (E and F) synergy factors between single inductive signals, and (G and J) regional expression ratios between opposite ends of the indicated axis.(B and D) Venn diagram of down-regulated genes by indicated LOFs.(H and K) Beeswarm boxplots of regional expression (as measured along the indicated axes) depending on increased Wnt-BMP synergy.(I) Whole-mount in situ hybridization (WMISH) of tbxt and eomes transcripts under various LOFs. Control and Nodal LOF pictures are from Gentsch et al. (2018b).See also Figure S3 and Table S3.
	Figure 4. Canonical Wnt, Nodal, and BMP Signals Induce the Majority of Regional ZGA(A) Total percentage of active (zygotic only and maternal-zygotic) genes mis-regulated by Wnt, Nodal, and/or BMP LOF.(B) Graphical explanations of figure panels (C) and (D).(C and D) Summary (i) and temporal resolution (ii) of minimal (C) and maximal (D) transcript levels of active genes (separated by regional expression along the primary body axes) detected among Wnt, Nodal, and/or BMP LOFs. Percentages only refer to the down-regulated (C) or up-regulated (D) genes among all zygotic genes with the same range of expression ratios along the animal-vegetal or dorsoventral axes.(E) Exponential activation of gradually longer genes before the MBT (bulk ZGA) when cell divisions occur at rapid and nearly constant intervals (∼20 min at 28°C). Sequential induction of the canonical Wnt, Nodal, and BMP pathway is critical to high percentages of regional ZGA (as measured along the two primary body axes within the indicated halves and quadrants of an early gastrula embryo): e.g., ∼89% or ∼98% of gene expression enriched in the dorsal half or vegetal-dorsal quadrant, respectively.

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