Mok GF , Folkes L , Weldon SA , Maniou E , Martinez-Heredia V , Godden AM , Williams RM , Sauka-Spengler T , Wheeler GN , Moxon S , Münsterberg AE .

BB/N007034/1 Biotechnology and Biological Sciences Research Council , MR/R000549/1 Medical Research Council

	Fig. 1: Transcriptional profiling of developing somites. a Schematic representation of HH14 chick embryo with presomitic mesoderm (PSM), epithelial somite (ES), maturing somite (MS) and differentiated somite (DS) dissected for RNA-seq and ATAC-seq, in triplicate. b–d Volcano plots showing enriched genes (log fold change >1.5) comparing PSM with ES, ES with MS, and MS with DS. e Heat map showing GO terms associated with PSM or DS enriched genes. f Clusters of highly correlated genes identified for myoblast differentiation and cartilage condensation. g Wnt, FGF and retinoic acid (RA) signalling pathways are shown in heat map. h Heat map showing k-means linear enrichment clustering across PSM, ES, MS and DS. i Dimension reduction algorithm t-SNE used to map top genes in each cluster determined by k-means. j Cluster B and k Cluster I are shown with some genes labelled to identify key transcription factors and signalling components.
	Fig. 2: Genome-wide profile of chromatin accessibility dynamics during somite development. a Correlation heat maps of accessible chromatin regions (ATAC-seq peak sites) comparing PSM and ES, b ES and MS and c MS and DS. d MA plots of significantly differential peak sites (pink) comparing PSM with ES, e ES with MS and f MS with DS. g Bar plot showing proportions of total genome sequence of peaks in PSM, ES, MS and DS (error bars = SD). Nearly half of all peaks lie within 50 kb of the promoter and TSS and half are in intergenic and intron regions. h GO terms associated with enriched transcription factors in DS compared to PSM. For enriched GO terms, p values were obtained from a modified Fisher exact test. i Number of transcription factor binding sites identified within 2 kb upstream of differentially expressed genes in DS compared to PSM. j Motif enrichment analysis performed using Homer. Transcription factor motifs identified as enriched in PSM accessible regions and k in DS accessible regions. Percentage indicates increase of motifs identified in either PSM or DS for each motif.
	Fig. 3: Differential footprints identified for CDX2 and LEF1 during somite development a Gene expression from mRNA-seq (error bars = SEM, n = 3) for CDX2. b Tn5 insertion frequency across all accessible regions containing at least one CDX2 motif, at nucleotide resolution in PSM, ES, MS and DS reveals the presence of a footprint centred on the CDX2 motif. Differential footprinting for CDX2 motif comparing PSM and ES, c PSM and MS, and d PSM and DS. e Gene expression for LEF1 (error bars = SEM, n = 3). f Differential footprinting for LEF1 motif comparing PSM and ES, g PSM and MS, and h PSM and DS. Similar GO terms for genes associated with (i) CDX2 footprints and (j) LEF1 footprints. For enriched GO terms, p values were obtained from a modified Fisher exact test. k, l Protein–protein network analysis using STRING database. Interactions between genes identified with (k) CDX2 and (l) LEF1 footprints in an accessible region, within 10 kb upstream or downstream. Highlighted in red are genes correlated with embryonic morphogenesis in CDX2-associated genes and animal organ morphogenesis in LEF1-associated genes. m Venn diagram of CDX2-associated genes against LEF1-associated genes identified only four common genes—Msgn1, Sall4, Spry1 and DDC.
	Fig. 4: Chromatin accessibility and differential footprints for HoxA cluster. a Gene expression from mRNA-seq for HoxA cluster (error bars = SEM, n = 3) in paraxial mesoderm regions. b Genome browser views of ATAC-seq profile across the HoxA cluster. ATAC and RNA profiles are shown in green for PSM, in blue for ES, in yellow for MS and in red for DS. Grey boxes indicate intergenic accessible regions and transcription factor footprints identified within those regions. Genome-wide differential footprinting for (c) HoxA2, (d) HoxA5, (e) HoxA10 and (f) HoxA11 between PSM and DS.
	Fig. 5: Identification of a regulatory element for TCF15. a ATAC-seq profile at the TCF15 locus. Grey boxes indicate putative enhancers identified (TCF15 Enh-1 and TCF15 Enh-2). RARA footprint identified within TCF15 Enh-2. Mutant reporter sequence for TCF15 Enh-2 RARA mutant. b TCF15 Enh-1 (n = 15/15) and c TCF15 Enh-2 (n = 9/9) reporter expression in presomitic mesoderm (Psm), notochord (Nc), somites (So) and lateral plate mesoderm (Lpm). d Combined TCF15 Enh-1/Enh-2 reporter expression (n = 9/9). e Transverse sections of embryo in c immunostained for Citrine showing TCF15 Enh-2 expression in somites and lateral plate mesoderm, white dashed line in c indicates location of section, representative of (n = 4/4). Nuclei stained with DAPI (blue). f TCF15 Enh-2 Citrine reporter with RARA binding site mutation displays lack of expression in somites (n = 6/6). Epigenome engineering using dCas9-Krab with (g) control scrambled sgRNAs resulted in no change (n = 8/9) and (h) sgRNAs targeting endogenous RARA binding site led to loss of TCF15 expression (n = 6/8) as shown by wholemount in situ hybridisation. i Percentage of embryos with normal (blue) or reduced (orange) TCF15 in situ expression after electroporation of control scrambled sgRNA with dCas9-Krab or sgRNAs targeting TCF15 Enh-2 RARA binding site with dCas9-Krab. All scale bars = 500 μm except for e scale bar = 100 μm.
	Fig. 6: Identification of a regulatory element for MEOX1. a ATAC-seq profile at MEOX1 locus. Grey box indicates putative enhancer identified. FOXO1 and ZIC3 footprints identified within enhancer element. Mutant reporter sequence for MEOX1 Enh ZIC/FOXO mutant. b MEOX1 Enh reporter expression in presomitic mesoderm (Psm) and in somites (So) (n = 13/13). c Transverse sections of embryo in b immunostained for Citrine showing MEOX1 Enh expression in epithelial somites, white dashed line in b indicates location of section, representative of (n = 4/4). Nuclei stained with DAPI (blue). d Still photographs from a time-lapse movie of MEOX1 Enh with the primitive streak (Ps) indicated. Fluorescent activity first observed in prospective paraxial mesoderm (Pm) at HH6 and continuous expression in the presomitic mesoderm (Psm) at HH7 prior to expression in somites (So) at HH8 and HH10 (n = 3/3). e FOXO1 and ZIC3 binding site mutation in MEOX1 Enh Citrine reporter led to loss of expression (n = 6/7). Epigenome engineering using dCas9-Krab with (f) control scrambled sgRNAs resulted in no change (n = 6/7) and (g) sgRNAs targeting endogenous FOXO1 and ZIC3 binding sites led to loss of MEOX1 expression (n = 9/11) as shown by wholemount in situ hybridisation. h Percentage of embryos with normal (blue) or reduced (orange) MEOX1 expression after injection and electroporation of control scrambled sgRNA with dCas9-Krab or sgRNAs targeting MEOX1 Enh FOXO1 and ZIC3 binding sites with dCas9-Krab. i Protein–protein network analysis using STRING database. Interactions between genes identified with MEOX1 footprints in an accessible region, within 10 kb upstream or downstream. Highlighted in red are genes correlated with anatomical structural development based on GO analysis. j RT-qPCR on somites dissected from wild-type (WT) embryos, or embryos electroporated with control scrambled sgRNA and dCas9-Krab, or sgRNAs targeting MEOX1 Enh FOXO1 and ZIC3 binding sites with dCas9-Krab. Statistical significance was determined by a two-tailed Student’s t test. **p = 0.001–0.01; *p = 0.01–0.1; ns not significant. All scale bars = 500 μm except for c scale bar = 100 μm.
	Fig. 7: Evolutionary conservation of MEOX1 enhancer and regulatory mechanism across vertebrates. a Genomic alignment of chick MEOX1 locus, ~10 kb. Exons and introns are represented by blue boxes and lines. Clustered green vertical lines indicate sequence identity between different species. The height of bars indicates extent of conservation in different species: turkey, zebra finch, American alligator, Chinese softshell turtle, Lizard, Human, Mouse, Zebrafish, Nile tilapia, Stickleback, Medaka, Fugu, Lamprey and Xenopus tropicalis, as indicated on the left of each row. Grey shading indicates the location of the MEOX1 Enh. b Expression of the conserved human MEOX1 Enh reporter in presomitic mesoderm, somites and lateral plate mesoderm in a HH9 chick embryo (n = 6/6). c Mutation of FOXO1 and ZIC3 binding sites in the human MEOX1 Enh Citrine reporter led to loss of expression (n = 4/4). d Chick MEOX1 Enh reporter injected into 1 cell of a Xenopus laevis 2-cell embryo shows Citrine expression in paraxial mesoderm (St14, white arrowheads (n = 6/6)) and in early somites and elongated myofibres (St25 (n = 6/6), St33 (n = 6/6), St42 (n = 6/6), white arrowheads). A-P anterior–posterior. Scale bars for b, c = 500 μm, d scale bar = 250 μm.

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