Sp5 and Sp8 are coexpressed in axial progenitors and are required for posterior development.

A. Alcian blue (cartilage) and Alizarin red (bone) staining of E18.5 skeletons.

B. Fluorescent in situ hybridization analysis of Nkx1-2, Tbxt, Sp5 and Sp8 expression in E7.75 wildtype embryos. Midline sagittal sections reveal that Sp5 and Tbxt share an anterior boundary in the caudal epiblast (arrowhead). Abbreviations: N, node; PS, primitive streak.

C. Whole mount fluorescent in situ hybridization analysis and optical cross sections of E8.5 wild type embryos for Tbxt (green), Sp5 (Red), Sp8 (purple) and Dapi (grey). The schematic on the left illustrates the axial levels at which optical sections were taken through the node (a), anterior PS/node streak border (b), mid primitive streak (C) and posterior PS (d).

D. UMAP plots of single-cell RNAseq analysis of E8.25 mouse embryos showing Tbxt, Sox2, Sp5, and Sp8 transcripts (Pijuan-Sala et al., 2019).

E. Heatmaps depicting mean scaled gene expression of Tbxt, Sox2, Sp5, and Sp8 in differentiating ESCs exposed to combinatorial Bmp, Fgf, RA, Wnt and Tgfβ signals (Yeo et al., 2020).

Sp5/8 regulates the Wnt3a/β-catenin pathway.

A. Over Representation Analysis (ORA) of 564 downregulated genes in E8.5 Sp5/8 dko mutants. Color scale reflects adjusted P-value, circle size represents gene number.

B. Heat map of log2 z-scores of normalized counts showing differentially expressed genes (P≤0.05; FC ≥0.5).

C. Venn diagram illustrating the intersection between Sp5 ChIPseq and Sp5/8 dko RNA-seq analyses. 613 genes including Wnt3a and Tbxt are direct targets of Sp5.

D. Venn diagram showing 197 differentially expressed genes in common between the Sp5/8 dko and Wnt3a ko transcriptomes.

Sp5/8 are integral components of the core regulatory circuitry of axial progenitors.

A. Four-way Venn diagram illustrating differentially expressed genes common to Sp5/8 dko, Cdx tko (Amin et al., 2016), Tbxt ko (Koch et al., 2017), and Wnt3a ko transcriptomes. Key regulators are highlighted in bold.

B. Four-way Venn diagram illustrating direct target genes bound combinatorially by Tbxt, Flag-Sp5, Flag-Sp8, and/or Cdx2. ChIP-seq was performed in NMC populations differentiated in vitro from CHIR-treated ESCs or, in the case of Cdx2, EpiSCs (Amin et al., 2016).

C. Sp5/8, Tbxt and Cdx TFs form self-sustaining autoregulatory loops that collectively maintain a Wnt3a/Fgf positive feedback loop to drive NMC self-renewal and axial elongation.

Sp5/8 regulate axial progenitors and the expression of niche factors.

A-C. Whole mount immunofluorescence analysis showing Tbxt (green) and Sox2 (red) protein expression in the posterior region of E8.5 control (a), Sp5/8 dko (b) and T-cre; Sp8gof (c) mutants. The bracket indicates the length of the primitive streak, the asterisk depicts the NSB.

D-I. Midline sagittal sections taken through the primitive streak of flat mounted E8.5 control (d-f) and Sp5/8 dko embryos (g-i) processed for Tbxt (green), and Sox2 (red) IF (Dapi, grey).

J-X. HCR analysis of Nkx1-2 (magenta), Cdx1 (red), Cdx2 (green), Tbx6 (green), and Msgn1 (red) expression in E8.5 control (j-n), Sp5/8 dko (o-s) and T-cre; Sp8gof (t-x) embryos.

Y-NN. Chromogenic WISH of E8.5 control (y-bb) and Sp5/8 dko (cc-ff), E8.5 control (gg-jj) and T-cre; Sp8gof (kk-nn) with DIG-labeled Wnt3a (y, cc, gg, kk), Fgf4 (z, dd, hh, ll), Fgf8 (aa, ee, ii, m) and Fgf17 (bb, ff, jj, nn) riboprobes. (y-ff) dorsal views, (gg-nn) lateral views.

Identification of a novel Wnt3a downstream enhancer.

A. Integrative Genomics Viewer analysis of ChIP-seq peaks for Cdx2 (Amin et al., 2016), Flag-Sp5, Flag-Sp8, Tbx6, β-catenin, and Tbxt (Koch et al., 2017) at Wnt3a. Two putative downstream enhancers are denoted as E1 (red bar, referred to as Wnt3aDE in text) and E2.

B. ChIP-seq tracks derived from (Xiang et al., 2020) and (Yang et al., 2019) showing H3K27ac deposition at both the promoter and putative enhancer region of Wnt3a in the mesendodermal and primitive streak lineages.

C. scATAC-seq data of E8.5 wild type mouse embryos showing accessibility across Wnt3a

D. (Argelaguet et al., 2022).

E. scATAC-seq data from wild type (WT) and Tbxt knockout (KO) mouse embryos at E8.5 showing reduced accessibility at Wnt3aDE in the absence of Tbxt (Argelaguet et al., 2022).

The Wnt3aDE is a WRE necessary for expression of Wnt3a and the Wnt3a-Tbxt autoregulatory loop.

A. Luciferase reporter assays of Wnt3a enhancer constructs: 0.47kb E1 (Wnt3aDE), 0.8kb E2 and the 1.8kb E1+E2, in differentiating ESCs treated with CHIR and Fgf to generate NMCs. Data are depicted as Mean+/- SD (n=3). Y-axis, relative luciferase units.

B. Sp5 overexpression enhances Wnt3aDE-luc activity in differentiating i3xFlag-Sp5 ES cells treated with bFGF (12ng/ml), CHIR(3μM), or bFGF+CHIR +/- Dox to induce 3xFlag-Sp5.

C. Luciferase assays of Wnt3aDE-luc deletion constructs. Schematic depicts the relative locations of Sp5, Cdx2, Tcf/Lef, and T-box binding motifs in the deletion constructs.

D. (Top) Schematic showing polarized Wnt3a expression (green) during gastruloid elongation. (Bottom) HCR analysis of Wnt3a (green), Tbxt (red), and Cdx2 (cyan) expression in WT(R1) and Wnt3aλ1DE gastruloids one (D4) and two (D5) days after CHIR treatment, respectively. Dapi, grey.

Sp5/8 regulate Tcf7 and Tle occupancy at WREs.

A. Metaplots and heatmaps of Tcf7 and Tle binding profiles at sites of Sp5 and Tcf7 co-occupancy in WT and Sp5/8 dko NMCs.

B. Identification of potential sites of Sp5/8-dependent Tcf exchange. 286 WREs defined by Sp5 and Tcf7 co-occupancy in wt cells display reduced Tcf7, and increased Tle, peak intensities in Sp5/8 dko cells. Only peaks with a log2 fold change > 0.58 and a P-value ≤ 0.05 were included in this analysis.

C. IGV genome tracks of RNA-seq and ChIPseq at select Wnt target genes in +/+ and Sp5/8-/-(dko) mutants. Top rows in each set depict RNAseq expression data obtained from E8.5 +/+ and Sp5/8-/- embryos. Bottom rows illustrate ChIP-seq data for Flag-Sp5 (in iF-Sp5 cells), Tcf7 and Tle performed in +/+ and Sp5/8-/- EBs. β-catenin, Tcf7l1, Lef1 ChIP data from (Blassberg et al., 2022). Tbxt (a), Wnt3a (b), Msgn1 (c), Cdx2 (d), Fgf8 (e) and Tbx6 (f) loci are shown. ChIP-qPCR is shown for Tbxt, Wnt3a, and Msgn1.

Model: Sp5/8 regulate Tcf exchange to promote Wnt target gene transcription.

Sp5 is induced by Wnt ligand. Elevated levels of Sp5 promote the dynamic exchange of repressive Tcf7l1-Tle complexes for activating Tcf7-β-catenin complexes at Wnt Response Elements (WREs). We speculate that the binding of Sp5/8 to DNA and to Tcf7 enhances the activation of Wnt target genes by modulating Tcf7-β-catenin binding kinetics at WREs. In the absence of Sp5/8, repressive Tcf7l1-Tle replace activating Tcf7-β-catenin complexes, resulting in the down regulation of Wnt target gene expression.

Gene Set Enrichment Analysis (GSEA) of differentially expressed genes in Sp5/8 dko bulk-RNA seq. Related to Figs 1 and 2.

A. Schematic representation of E8.5 control and Sp5/8 dko or Wnt3a-/-mutants, with the dashed box illustrating the posterior termini dissected for RNA extraction and bulk-RNA sequencing (n=3 for controls and Sp5/8 dko mutants).

B. Principal component analysis of control (n=3) and Sp5/8dko (n=3) samples used for bulk-RNA seq analysis.

C-D. Treemap visualization of significantly enriched GO terms identified through GSEA analysis of Sp5/8 dko bulk RNA-seq differentially expressed downregulated genes (E) and upregulated genes (F). Each rectangle denotes a GO term, with similar terms grouped by color to reflect semantic similarity. Rectangle areas are proportional to adjusted p-values (-log10 transformed).

D. Principal component analysis of control (n=3) and Wnt3a-/- (n=3) samples used for bulk-RNA seq analysis.

Comparisons of Sp5/8dko differentially expressed genes to Cdx-tko and Tbxt ko. Related to Fig.2 and 3.

A. Venn diagram showing the overlap of DEGs between the Sp5/8 dko and Cdx tko (Amin et al., 2016). Select downregulated genes are featured, NMC-relevant genes are highlighted in bold.

B. GO biological processes terms of downregulated genes shared between Sp5/8 dko and Cdx tko datasets.

C. Venn diagram showing the overlap of DEGs between Sp5/8 dko and Tbxt ko (Koch et al., 2017). Key down regulated genes are highlighted.

D. GO biological processes terms of downregulated genes shared between Sp5/8 dko and Tbxt ko datasets.

RNA in situ expression of NMC genes in T-Cre-Sp8GOF and Sp5/8 dko embryos. Related to Fig.4.

A-B. Two color whole mount in situ hybridization of E8.5 embryos for Sox2 (orange) and Tbxt (purple) (A) and Nkx1-2 (orange) and Uncx4.1(purple) (B) in control and T-Cre; Sp8GOF embryos. Lateral and dorsal views are shown A and lateral view in B.

C-D. Lateral view of whole mount fluorescent in situ hybridization analysis for transcripts of Wnt8a (yellow) in Sp5/8dko embryos (C) and T-Cre; Sp8GOF embryos at E8.5 (D).

Whole mount fluorescent in situ hybridization analysis of RA pathway genes. Related to Fig.4.

Lateral view of embryos processed for Cyp26a1 (green; a, b, e, f) and Aldh1a2 (red, c, d, e, f) in E8.5 control and Sp5/8dko embryos.

Characterization of Wnt3aDE using comparative genomics. Related to Fig.5.

A. Comparative genomics analysis of the mouse Wnt3a locus using Vista tools (Frazer et al., 2004). The conserved Wnt3aDE is boxed.

B. Motif annotations from CIIIDER annotated against the 466 bp putative Wnt3aDE sequence (Gearing et al., 2019). Wnt3aDE is identified as -15696 intergenic peak (mm9; Chr11: 59057304-59057626 upstream of Arf1(see Supp. Data file-1).

Generation of Wnt3aDDEand Sp5/8 dko ESCs using CRISPR/cas9 technology. Related to Fig. 6 and 7.

A. Schematic view of Wnt3aDDE ko ES cell generation using CRISPR-Cas9 mediated deletion. Two guides (see Supp. data file-7) were designed to delete 1.1kb encompassing the 0.47kb Wnt3aDE. Genotyping oligos were designed to amplify a 1.4kb fragment in wildtype and 0.3kb fragment in Wnt3aDDE ko ESCs. PCR genotyping gel shows deletion of Wnt3aDEin two different clones.

B-C. Generation of Sp5/8dko ES cells. B. Schematic of Sp5 ko using CRISPR-Cas9 mediated deletion in Exon-2 using two guides designed to delete 616 bases encoding the Zn-finger domains of Sp5. ES cells were differentiated as EBs and treated with bFGF+CHIR for 24h. Sp5 protein was not detected in D3 cell extracts. C. Schematic view of Sp8 ko using CRISPR-Cas9 mediated deletion in Exon-3 using two guides designed to delete the coding region. Random sequence was inserted by NHEJ repair and these ES cells failed to express Sp8 protein in D3 EBs.

Comparative analyses of Flag-Sp5, Tcf7, and Tle ChIP-seq data sets. Related to Fig.7.

A. Metaplots and heat maps depicting 28,154 Tcf7 ChIP-seq peaks and 6,506 Tle peaks in WT and Sp5/8dko EBs.

B. (Left)Venn diagram illustrating the overlap between Flag-Sp5 and Tcf7 ChIP-seq peak sets. A total of 3,471 shared putative WREs, corresponding to 1168 unique genes, were identified. (Right) Venn diagram showing the intersection of the 1,168 genes associated with shared Flag-Sp5 and Tcf7 peaks and genes differentially expressed (up- or downregulated) in Sp5/8dko bulk-RNA-seq.

C. (Left) Western blot analysis depicting Tcf7 and Lef1 protein expression in NMCs differentiated in vitro from WT, Sp5/8 dko, Sp5GOF and Sp8GOF EBs. (Right) Tle protein expression in WT and Sp5/8 dko NMCs differentiated as EBs. YY1, loading control.

D. Quantitative PCR time course showing log fold change of RNA expression of Tle1-4, Tcf7l1, Tcf7, Cdx2, Tbxt, and Wnt3a in Sp5/8 dko versus WT EBs. RNA expression was normalized to Gapdh for all genes.

Motif and Over-representation analyses of F-Sp5, Tcf7, and Tle ChIPseq datasets. Related to Fig. 7.

A-B. Meme-ChIP motif analysis of peak sequences from Tcf7 (A) and Tle (B) ChIP-seq data in WT and Sp5/8dko cells. In Tcf7 ChIP-seq, the TCF/LEF motif was the most significantly enriched motif in both WT and Sp5/8dko. In Tle ChIP-seq, Znf motif and Tbx motif were most enriched in WT and Sp5/8dko, respectively, with additional enrichment of TCF/LEF and SP/KLF motifs in Sp5/8dko only.

C-D. Over-representation analysis (ORA) using WEB-based GEne SeT Analysis Toolkit on 266 overlapping genes corresponding to 286 F-Sp5 and Tcf7 overlapping WREs (as shown in Fig. 7B). Enrichment results are shown for (A) Gene Ontology (GO) Biological Process terms and (B) KEGG pathways.