andp<0.001 for tissue specific and house keeping genes, respectively. (E) Schematic representation of the analysis performed in figure 1C shows two zebrafish TADs with five genes (top part) that have an ortholog in humans (lower part). Genes 1 and 3 form an example of a gene pair, which are present in zebrafish within the same TAD and are within 1MB distance in humans. This situation is classified as intra-TAD conservation. Gene pair 2 and 5 is within 1mb in the human genome, but present in neighboring TADs in zebrafish. This is classified as inter-TAD conservation. (F) Cumulative distributions of intra-and inter-TAD gene-pairs (black and red, resp.). The three sizeranges (S, M and L) are used to stratify the conservation-analyses to overcome differences in numbers of intra-and inter-TAD gene-pairs. (G) Barplots showing the enrichment of correlated genes, as indicated, based on three different Tomo-Seq datasets (red bars) within TADs as compared to all genes (grey bars). Analysis similar to Figure 1E. 

F

  borders o b s e r v e d e x p e c t e d o b s e r v e d e x p e c t e d o b s e r v e d e x p e c t e 


Figure S1 .

 S1 Figure S1. Zebrafish TADs recapitulate known features of TADs. Related to Figure1(A) Boxplot showing the distribution of H3K27ac ChIP-seq peaks and gene density in the A/B compartment, as indicated. The Wilcoxon rank sum test reveals significant difference between compartments for H3K27ac and gene density (p=1.7x10 -9  and p=7.1x10 -28 , respectively). (B) Boxplot showing the transcripts per million of genes in the A/B compartment, as indicated. Wilcoxon rank sum test p-value reveals significant difference (p=3.3x10 -37  ). (C) Barplot showing the overlap of TSSs from all EnsEMBL genes with TAD borders. Confidence intervals were obtained by 1000 circular permutations of the TAD borders (p<0.001). (D) Barplot showing the overlap of house-keeping and tissue specific TSSs, as indicated, with TAD borders. Empirical p-values were obtained by 1000 circular permutations of the TAD borders. p=0.018 and p<0.001 for tissue specific and house keeping genes, respectively. (E) Schematic representation of the analysis performed in figure1Cshows two zebrafish TADs with five genes (top part) that have an ortholog in humans (lower part). Genes 1 and 3 form an example of a gene pair, which are present in zebrafish within the same TAD and are within 1MB distance in humans. This situation is classified as intra-TAD conservation. Gene pair 2 and 5 is within 1mb in the human genome, but present in neighboring TADs in zebrafish. This is classified as inter-TAD conservation. (F) Cumulative distributions of intra-and inter-TAD gene-pairs (black and red, resp.). The three sizeranges (S, M and L) are used to stratify the conservation-analyses to overcome differences in numbers of intra-and inter-TAD gene-pairs. (G) Barplots showing the enrichment of correlated genes, as indicated, based on three different Tomo-Seq datasets (red bars) within TADs as compared to all genes (grey bars). Analysis similar to Figure1E. 


FigureFigure S4 .

 S4 Figure S2 Hi-C analysis shows loss and reestablishment of chromatin architecture throughout development. Related to Figure 2. (A) Schematic explanation of the insulation score. By sliding a square along the diagonal of a Hi-C matrix (shown here horizontally) the insulation score is calculated. At TAD borders there will be a segregation of interactions leading to a reduction in the score. TAD borders therefore show a local minimum in the insulation score. (B) Average insulation scores around 24hpf TAD borders throughout zebrafish development, as indicated (top panel). Bottom panel shows the insulation scores for the individual TAD borders for all four Hi-C datasets in a heatmap. (C) Upper panel shows the average insulation scores aligned to TAD borders for all the replicate Hi-C templates (2.25hpf, n = 2; 4hpf, n = 8; 8hpf, n = 9; 24hpf, n = 4). Bottom panel shows a heatmap quantifying the (dis)similarity of Hi-C templates of different timepoints. Pairwise difference between all templates is visualized as the Wilcoxon rank-sum test p-value of the insulation scores at the TAD borders. Hierarchical clustering shows the clustering of templates from the same timepoint. (D-G) Representative images of DAPI stained zebrafish embryos at indicated developmental stages. At 2.25hpf the embryos divide synchronously and the number of embryos showing a certain nuclear staining is indicated in the top left of the image. White arrows indicate nuclei in metaphase and blue arrows indicate nuclei in other cell cycle stages. (H) Motif count and orientation of CTCF binding sites inferred from 4hpf ATAC-seq relative to TAD borders. (I) MNase-seq read density surrounding ATAC-seq inferred CTCF binding sites at 4hpf (top panel) and 24hpf (bottom panel). CTCF binding sites are separated on their orientation, as indicated. 


Table S1 Primer sequences used for 4C-seq.

 S1 Related to STAR Methods 


	Table S1 view 1 view8 >chr24:3031279-3031965_fwd >chr5:35734021-35734441_fwd ACGTCGCTTATATCGGGATC TATCAGCAGCAGAAAAGATC >chr24:3031279-3031965_rev >chr5:35734021-35734441_rev GATGCCCTACACTCTCATTC AGGATTCCAAGGGTTTACAT	view 1 view8 24:3031279-3031965_fwd_BC1 5:35734021-35734441_fwd_BC1 24:3031279-3031965_fwd_BC2 5:35734021-35734441_fwd_BC2 24:3031279-3031965_rev 5:35734021-35734441_rev	primer sequences AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTACTATCAGCAGCAGAAAAGATC AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTACACGTCGCTTATATCGGGATC AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTtgTATCAGCAGCAGAAAAGATC AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTtgACGTCGCTTATATCGGGATC CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTAGGATTCCAAGGGTTTACAT CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTGATGCCCTACACTCTCATTC
	view 2 view9 >chr3:18723607-18724650_fwd >chr12:34629626-34629939_fwd CTTCCATATACATGCAGATC CATTCTTTAATCTTCAGATC >chr3:18723607-18724650_rev >chr12:34629626-34629939_rev AAAATTTTCATATTGGGGTG GCTCCATCATGTATTCGATT	view9 view 2 3:18723607-18724650_fwd_BC1 12:34629626-34629939_fwd_BC1 3:18723607-18724650_fwd_BC2 12:34629626-34629939_fwd_BC2 3:18723607-18724650_rev 12:34629626-34629939_rev	AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTACCATTCTTTAATCTTCAGATC AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTACCTTCCATATACATGCAGATC AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTtgCATTCTTTAATCTTCAGATC AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTtgCTTCCATATACATGCAGATC CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTGCTCCATCATGTATTCGATT CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTAAAATTTTCATATTGGGGTG
	view3 view10 >chr4:11210227-11210713_fwd >chr22:18522392-18522640_fwd ATGAATGAATCTGACTGATC ATAACCGTAACAAAGGGATC >chr4:11210227-11210713_rev >chr22:18522392-18522640_rev TGAAACAAGGATTATTCCTCA CCAATGAAATAGTGGAGAGC	view10 view3 4:11210227-11210713_fwd_BC1 22:18522392-18522640_fwd_BC1 4:11210227-11210713_fwd_BC2 22:18522392-18522640_fwd_BC2 4:11210227-11210713_rev 22:18522392-18522640_rev	AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTACATAACCGTAACAAAGGGATC AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTACATGAATGAATCTGACTGATC AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTtgATAACCGTAACAAAGGGATC AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTtgATGAATGAATCTGACTGATC CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTCCAATGAAATAGTGGAGAGC CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTTGAAACAAGGATTATTCCTCA
	view4 view11 (check distance to original viewpoint) view11 (check distance to original viewpoint) view4 >chr10:21909251-21909539_fwd 10:21909251-21909539_fwd_BC1 >chr6:32114060-32114600_fwd 6:32114060-32114600_fwd_BC1 AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTACTGTTAAAAATGAGCTGGATC AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTACAGAAACAAAAAGTTTAGATC AGAAACAAAAAGTTTAGATC 10:21909251-21909539_fwd_BC2 TGTTAAAAATGAGCTGGATC 6:32114060-32114600_fwd_BC2 AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTtgTGTTAAAAATGAGCTGGATC AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTtgAGAAACAAAAAGTTTAGATC >chr10:21909251-21909539_rev 10:21909251-21909539_rev >chr6:32114060-32114600_rev 6:32114060-32114600_rev CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTTGAAGATTGCCATTTTCTCT CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTACAGAGCCATTTATGCAGAC ACAGAGCCATTTATGCAGAC TGAAGATTGCCATTTTCTCT
	view12 view5 (check distance from viewpoint) view5 (check distance from viewpoint) view12 >chr3:28876830-28877096_fwd 3:28876830-28877096_fwd_BC1 AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTACATATTCAGTCTCCCAAGATC
	>chr22:37421871-37422581_fwd ATATTCAGTCTCCCAAGATC GCGACTGGACGCTATGGATC >chr3:28876830-28877096_rev >chr22:37421871-37422581_rev GGAGAGTAAATTATGGCACG	22:37421871-37422581_fwd_BC1 3:28876830-28877096_fwd_BC2 22:37421871-37422581_fwd_BC2 3:28876830-28877096_rev_BC2 22:37421871-37422581_rev	AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTtgATATTCAGTCTCCCAAGATC AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTACGCGACTGGACGCTATGGATC CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTGGAGAGTAAATTATGGCACG AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTtgGCGACTGGACGCTATGGATC CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTTTCTGCAATCCAAATTAGCT
	TTCTGCAATCCAAATTAGCT		
	view 6	view 6	
	>chr24:12689902-12691922_fwd	24:12689902-12691922_fwd_BC1	AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTACAGGCCTAATGAATGTAGATC
	AGGCCTAATGAATGTAGATC	24:12689902-12691922_fwd_BC2	AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTtgAGGCCTAATGAATGTAGATC
	>chr24:12689902-12691922_rev	24:12689902-12691922_rev	CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTCTTCAATCAGCATCCGTTTT
	CTTCAATCAGCATCCGTTTT		
	view7	view7	
	>chr21:13593987-13594356_fwd	21:13593987-13594356_fwd_BC1	AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTACAACCTGTGTGGGCCAGGATC
	AACCTGTGTGGGCCAGGATC	21:13593987-13594356_fwd_BC2	AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTtgAACCTGTGTGGGCCAGGATC
	>chr21:13593987-13594356_rev	21:13593987-13594356_rev	CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTCTGCCCTCTTATGCATATTT
	CTGCCCTCTTATGCATATTT