Nakamura Y , de Paiva Alves E , Veenstra GJ , Hoppler S .

BB/I003746/1 Biotechnology and Biological Sciences Research Council , BB/M001695/1 Biotechnology and Biological Sciences Research Council

Xla Wt + wnt8a MO (Fig.2 C)
Xla Wt + wnt8a MO (Fig.2 C)
Xla Wt + wnt8a MO (Fig.2 D)

	Fig. 1. β-catenin ChIP-seq analysis of early gastrulae. (A) Experimental design of β-catenin ChIP-seq analysis. Early gastrulae were collected and fixed. Following chromatin shearing, β-catenin antibodies were used to selectively precipitate DNA fragments bound by β-catenin- containing protein complexes. Subsequently, the precipitated DNA fragments were sequenced. (B) Genome view of example β-catenin-target gene hoxd1. Note clear β-catenin ChIP-seq peaks (β-peaks) downstream (to the left) of the hoxd1 locus. (C) Scatter plot combining peak calling analysis by SPP (considering signal strength, applying False Discovery Rate (FDR) ≤ 0.1) and MACS2 (considering fold change, applying p ≤ 0.01) software, with black dots indicating 10,638 β-peaks reproducibly called (applying an Irreproducible Discovery Rate (IDR) ≤ 0.01). (D,E) β-peaks are associated with sequences throughout the genome (D) but enriched close to and just upstream (putative promoter) of the transcription start site (TSS) of nearby genes (E, analysed in 500 bp bins). Pie chart (in D) showing percentage of β-peak according to their location relative to TSS (within 1 kb, 1-5 kb, 5-10 kb, 10-50 kb, over 50 kb upstream or downstream of TSS as indicated by the colour code). (F) Heatmap illustrating genome-wide association of β-peaks with histone modifications and transcription co-factor-binding sites indicative of cis-regulatory modules (CRMs, such as promoters and enhancers) in patterns that can be clustered into ten groups. Each horizontal line represents the 5 kb downstream and upstream region of ChIP-seq data around a β-peak. (G) Enriched motifs from de novo motif search of sequences under β-peaks. Note identification of consensus TCF/LEF-binding but also other known transcription factor- binding motifs. Statistical significance (e-values) and the number of β-peaks are indicated below each motif logo. The analysis of motif distribution shows central enrichment of motifs within β-peak regions (500 bp window).
	Fig. 2. RNA-seq analysis to identify wnt8a-regulated genes. (A) Experimental design to identify wnt8a-regulated genes. wnt8a MO and standard control MO (CoMO) were microinjected into the ventral marginal zone (VMZ) of four-cell stage embryos (prospective endogenous wnt8a-expressing and ventral mesoderm tissue). For the re-instatement experiment, wnt8a MO was co-injected together with a DNA construct driving exogenous Wnt8a (CSKA-wnt8a) in the same tissue. Eventually three biological replicates per experimental sample were sequenced. The experimental conditions were optimized by comparing the morphology of uninjected (i), CoMO-injected (ii), wnt8a MO-injected (iii) and wnt8a MO- and CSKA-wnt8a DNA co-injected embryos (iv), as well as expected changes to expression of candidate genes (Fig. S2). (B) Venn diagrams of genes that are positively (top) or negatively (bottom) regulated by wnt8a signalling identified by Generalized Linear Model (GLM) statistical analysis (FDR < 0.1, see the supplementary materials and methods) of RNA-seq results. 41 genes were identified with reduced expression in the wnt8a knockdown (blue, compared to uninjected and CoMO-injected controls) and 274 genes with increased expression when Wnt8a expression was re-instated (green, compared to wnt8a knockdown). A shortlist of 14 positively wnt8a-regulated genes (listed on the left) was selected for further analysis by the overlap between these two groups of genes (see Table S2 for full gene lists). 18 genes with increased expression were identified in the wnt8a knockdown (amber) and 193 genes with reduced expression when Wnt8a expression was reinstated (purple) with one gene (atp12a) in the overlap, therefore apparently negatively regulated by wnt8a. (C) Validation of RNA-seq-discovered candidate genes by RT-qPCR. Transcripts collected from embryos microinjected into all four blastomeres with wnt8a MO (yellow bars) and wnt8a MO co- injected with CSKA-wnt8a DNA (pink bars) were compared to control (CoMO-injected, blue). All 14 positively wnt8a-regulated candidate genes of the shortlist were confirmed; but not atp12a, which had been suggested to be negatively regulated. Note varying extent of dependence on wnt8a function in different genes. *p < 0.1; **p < 0.05; ns, not significant (p ≥ 0.1)(two-tailed Student’s t-test). Error bars represent s.d. of two biological replicates. (D) Vegetal view of early gastrulae (with dorsal up) of control- (uninjected) and wnt8a MO- injected embryos. Note expression of wnt8a-regulated genes in similar but not always identical pattern as wnt8a. Also note reduced expression to varying extent in wnt8a MO- injected embryos.
	Fig. 3. Integrating β-catenin ChIP-seq and RNA-seq analysis to identify direct wnt8a/β- catenin target genes. (A) Venn diagram illustrating overlap between genes near β-peaks (red disc) and the positively wnt8a-regulated genes (as in Fig. 2B). Note that from among the longlist of 274 potential wnt8a-regulated genes, 179 are associated with identified β-peaks (amber border around lens-shaped area), representing the longlist of probable direct wnt8a/β-catenin target genes. Also note that all but one (xmcl2) of the validated shortlist of positively wnt8a-regulated genes are among those and therefore represent the short list of 13 direct wnt8a/β-catenin target genes (yellow). Also note that the majority of gene loci near β-peaks are not correlated with wnt8a-regulated genes and conversely, that more than a third of wnt8a-regulated genes in our longlist are not associated with identified β-peaks (most likely representing indirect wnt8a target genes). (B) β-catenin ChIP-qPCR of identified β-peaks of our shortlist in chromatin extracted from control (uninjected) and wnt8a MO- injected embryos. Note β-catenin association is reduced in wnt8a loss-of-function experiment in most of the analysed 15 identified β-peaks (IgG antibodies were used as control; error bars represent s.e.m. of three to five biological replicates). (C) Luciferase assays of reporter constructs containing sequences near identified β-peaks of wnt8a-regulated genes. Error bars represent s.d. of three biological replicates. (D) Gene Ontology analysis suggests that β-peak-associated genes tend to encode predominantly transcription factors and also cell-to- cell signalling components, and to function in developmental processes; with different emphasis between wnt8a-regulated (amber) and non-regulated genes (red). (E) DNA occupancy level of β-catenin around the peak summit shows higher enrichment in direct wnt8a/β-catenin target gene loci (purple: shortlist, amber: longlist) compared to non-wnt8a- regulated genes (red). Read density was analysed using HOMER (bin size 100 bp). (F) TCF/LEF consensus motif is enriched under all 58 β-peaks associated with all 13 shortlisted wnt8a/β-catenin target genes.
	Fig. 4. β-catenin recruitment is not sufficient for transcriptional regulation. (A,B) Maternally activated Wnt/β-catenin signalling regulates transcription of only context-specific maternal Wnt/β-catenin target genes. Experimental enhancement of maternal Wnt signalling, by injection of Wnt8a mRNA at the two- to four-cell stage, increases expression of maternal Wnt targets sia1 and nodal3.1 when analysed at the MBT compared to uninjected control (A). In contrast, expression levels of wnt8a target genes remain unchanged. However, β-catenin binding increases at both maternal Wnt target and zygotic wnt8a-regulated target loci at the 1000-cell stage (B). Note reduction of β-catenin binding following injection of axin mRNA indicates that maternally regulated endogenous β-catenin associates with not only maternal Wnt target genes but also zygotic wnt8a target genes. (C,D) Zygotically activated β-catenin controls expression of only zygotic wnt8a targets. wnt8a MO or CSKA-wnt8a DNA were injected at the two- to four-cell stage and gene expression and β-catenin binding were analysed at the early gastrula stage. Knockdown of wnt8a reduces and zygotic activation of Wnt8a signalling increases expression of the wnt8a target hoxd1, as a control. While the wnt8a knockdown or overexpression does not affect expression of maternal Wnt-regulated genes (C), over-activation of Wnt8a signalling increases β-catenin binding to some maternal Wnt-regulated loci (D) but not to the well-characterized direct maternal Wnt target genes sia1 and nodal3.1. Error bars represent s.d. and s.e.m. of three biological replicates for RT-qPCR and ChIP-qPCR, respectively.
	Fig. 5. BMP or FGF signalling is required for wnt8a target gene expression but not for β-catenin recruitment. (A) BMP signalling is required for context-specific transcriptional regulation by wnt8a signalling, but only of some wnt8a target genes. Two- to four-cell stage embryos were injected with BMP antagonist noggin (nog) mRNA. CSKA-wnt8a DNA was injected additionally to reinstate Wnt8a expression (as endogenous wnt8a expression is itself regulated by BMP signalling). Expression was analysed by RT-qPCR at the early gastrula stage. When BMP signalling is blocked, expression of BMP-dependent genes remains reduced even when Wnt8a expression is reinstated. (B) FGF signalling is required for context-specific transcriptional regulation by wnt8a signalling, but only of some wnt8a target genes. Embryos were treated with FGFR inhibitor SU5402 from the 1,000/2,000-cell stage through the early gastrula and injected where indicated with CSKA-wnt8a DNA at the two- to four-cell stages (to reinstate Wnt8a expression as endogenous wnt8a expression is itself regulated by FGF signalling). When FGF signalling is inhibited, expression of FGF- dependent genes remains reduced, even when Wnt8a expression is reinstated. (C) wnt8a target genes can therefore be classified into BMP- or FGF-dependent genes. Note some genes belong to both groups and others are neither BMP- nor FGF-dependent. (D,E) in situ hybridisation shows expression of msx1 (D) and hoxd1 (E) in cross-sections and lateral views (small images) of control uninjected and experimentally manipulated embryos as indicated, with dorsal right. (F) BMP signalling is not required for wnt8a-regulated β-catenin recruitment to BMP-dependent wnt8a target gene loci. Embryos were treated as in A and analysed with β-catenin ChIP-qPCR at the early gastrula stage. (G) FGF signalling is not essential for wnt8a-regulated β-catenin recruitment to FGF-dependent wnt8a target gene loci. Embryos were treated as in B and analysed with β-catenin ChIP-qPCR at the early gastrula stage. Uninjected, untreated embryos were used as controls in A,B, and D-G. *p < 0.1; **p < 0.05 with two-tailed Student’s t-test. Error bars represent s.d. of four biological replicates (in A,B) or s.e.m. of three biological replicates (in F,G). Note that wnt8a gene expression was decreased by BMP or FGF pathway inhibition (wnt8a in blue bars in A,B) but restored by coinjection of CSKA-wnt8a DNA (wnt8a in orange bars in A,B) compared to controls (wnt8a in yellow bars in A,B) and that higher wnt8a expression levels in green bars reflect both expression of endogenous wnt8a and expression from CSKA-wnt8a DNA, resulting in upregulation of several wnt8a target genes (in A,B).
	Fig. 6. Model for context-specific Wnt/β-catenin target gene regulation. (A) In previous concept established from studies of individual genes, Wnt signalling specifically controls β-catenin recruitment to the Wnt-response element (WRE) of context-specific target genes and leads to their transcription (e.g. gene B in the context-X). (B) In the revised concept from our studies, Wnt-regulated β-catenin recruitment takes place at numerous loci. Transcriptional activation at those loci is conditional on context-specific mechanisms (e.g. context-X-specific mechanism for gene B in the context-X).

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