Fig 1.
Hoxd gene expression in mouse and chick limb buds.
(A, B) WISH analysis of E12.5 mouse and HH28 (equivalent to E12.25 to E12.5) chick FL and HL buds with expression of Hoxd gene and Col2a1 or Aggrecan, which are markers for chondrocyte differentiation. (A, left) Schemes showing the morphologies of FL (red) and HL (yellow) in adult mice. (A, right) Expression of Hoxd gene in FL buds are comparable to those in HL buds. The expression domain of Col2a1 (white arrowheads) corresponds to a low-Hoxd-expression region leading to the future mesopodium. (B, left) Schemes representing morphologies of FL (red) and HL (yellow) buds in chicken. (B, right) Expression of Hoxd gene in proximal HL is significantly reduced and restricted to the presumptive fibula. (C, D) RNA-seq profiles of Hoxd gene in microdissected proximal and distal domains from either E12.5 mouse (C) or HH30 (equivalent to E13 to E13.5) chick (D) FL and HL buds. Expression level of Hoxd12 was slightly stronger in mouse proximal FL than in proximal HL (red arrow in C), a difference more pronounced in chick (red arrow in D). Right limbs in (A, B) are oriented proximally to the bottom and distally to the top. The y axis represents the strand-specific RNA-seq read counts, normalized by the total number of million mapped reads. Col2a1, collagen type II alpha 1 chain gene; E, embryonic day; FL, forelimb; HH, Hamburger–Hamilton stage; HL, hindlimb; RNA-seq, RNA sequencing; WISH, whole-mount in situ hybridization.
Fig 2.
Conserved bimodal regulation at the chick HoxD locus.
(A, B) 4C-seq tracks showing contacts established by mouse Hoxd11 (A) and chick Hoxd10-11 (B) viewpoints in mouse and chick proximal and distal cells from FL and HL at E12.5 and HH30, respectively. (A) The interactions between Hoxd11 to and around the CS39 region were mainly observed in proximal cells, whereas those between Hoxd11 and either island III or Prox, which are hallmarks of the C-DOM activity, were increased in the distal region. (B) The contacts extend up to the predicted borders of the two TADs located on either side of the HoxD cluster (C-DOM, opened arrowheads; T-DOM, closed arrowheads). In addition to the interactions between Hoxd10-11 and CS39, contacts were also observed with CS93 in proximal FL bud cells. These contacts are decreased in proximal HL bud cells in which Hoxd expression is strongly reduced (red arrows). 4C-seq, circular chromosome conformation capture sequencing; C-DOM, centromeric regulatory domain; E, embryonic day; FL, forelimb; HH, Hamburger–Hamilton stage; HL, hindlimb; TAD, topologically associating domain; T-DOM, telomeric regulatory domain.
Fig 3.
Differential enhancer activities of mouse and chick CS93 in FL and HL buds.
(A) Genomic coordinates and sequence alignment using either the bat or the chick sequence onto the mouse genome and schematics summarizing the enhancer activities for each of the identified sequences [6,17] (this work). Neither murine BAR116 nor CS9 showed any enhancer activity in limbs [6,17], whereas the bat BAR116 displayed different patterns between mouse FL and HL [6]. The sequences of both the bat BAR116 (Myoluc2, GL429772: 6,606,808–6,608,652) and the chick CS93 (galGal5, chr7:16,104,952–16,105,803) were aligned with BLAT onto the mouse genome. (B) (Left) Genomic coordinates of either the chick CS93 (green rectangle) or the chick 2-kb region used in the enhancer assay (blue domain). The 2-kb sequence contains the chick CS93 region and the region of high interactions with the Hoxd10 to Hoxd11 region in proximal FL bud cells at HH30. (Middle) Conservation plot of mouse CS93 and bat BAR116 using the 2-kb region of chick CS93 as a reference. The peaks represent a conservation higher than 50%. Pink regions are conserved noncoding sequences. (Right) The sequence similarity obtained from mVista tools shows the highest conservation of the chick CS93 with the bat BAR116 sequences. (C, D) Enhancer activities of mouse CS93 (C) and the 2-kb region of chick CS93 (D) in mouse FL and HL buds E12.5. The lacZ expression pattern (C) showed that mouse CS93 has an enhancer activity in the proximal region of developing limb buds at E12.5. In contrast to the mouse, the 2-kb region of chick CS93 (D) showed differential enhancer activity between FL and HL buds at E12.5, as was also reported for the bat BAR116 sequence. The numbers of lacZ-positive embryos over total transgene integrated are indicated. BAR116, Bat Accelerated Region 116; E, embryonic day; FL, forelimb; HH, Hamburger–Hamilton stage; HL, hindlimb.
Fig 4.
Premature termination of T-DOM activity in chick HL buds.
(A, B) Comparison of H3K27ac and H3K27me3 ChIP-seq profiles in either whole, proximal, or distal FL and HL buds at HH19 (equivalent to mouse E9.5), HH20 (equivalent to mouse E10), and HH28. (A) In chick HL bud, enrichment of H3K27ac at region a in T-DOM was initially detected at HH19, whereas it was significantly decreased at HH20. Few H3K27ac marks were scored in region b in HL bud at both HH19 and HH20, as compared with those in FL buds. At HH28, the accumulation of H3K27ac marks was quite low in both the HoxD cluster and the T-DOM region in proximal HL when compared to distal FL cells, whereas the profiles of H3K27ac in the distal region where Hoxd genes are strongly expressed were similar between FL and HL buds at HH28. (B) In FL and HL buds at HH20, several C-DOM regions were decorated by H3K27me3. In contrast, T-DOM was not labeled in FL buds at this stage, nor had H3K27me3 marks started to accumulate around CS39 in HL buds. In proximal HL buds where Hoxd expression was reduced, H3K27me3 enrichment was observed at the HoxD cluster and over T-DOM when compared to proximal FL buds. Both regions a and b in T-DOM were enriched in H3K27me3. Enrichment (y axis) of ChIP is shown as the log2 ratio of the normalized number of reads between ChIP and input samples. C-DOM, centromeric regulatory domain; ChIP, chromatin immunoprecipitation; ChIP-seq, ChIP sequencing; FL, forelimb; H3K27ac, acetylation of histone H3 lysine 27; H3K27me3, trimethylation of H2K27; HH, Hamburger–Hamilton stage; HL, hindlimb; T-DOM, telomeric regulatory domain.
Fig 5.
Chromatin conformation and bound CTCF sites at the mouse and chick HoxD locus.
(A) Hi-C heat map data adapted from [19] at a 40-kb resolution. The black lines demarcate the TADs in mouse distal FLs at E12.5. (B) CHi-C heat maps at 5-kb resolution by using either chick FL or HL buds at HH20. (C) Comparison of bound CTCF and site orientations at the HoxD cluster between mouse distal FL at E12.5 (top) and chick FL bud at HH20 (bottom). Open and closed arrowheads indicate the orientations of the CTCF motifs. (D) Subtraction of the CHi-C matrices shown in (B), with FL bud cells in red and HL bud cells in blue. The black lines demarcate the TADs. The green rectangle is enlarged in (E). (E, F) Subtraction of the CHi-C matrices shown in (B) between the HoxD cluster and the area from region a to region b within T-DOM at a 5-kb resolution. A decrease in contacts is detected between the HoxD cluster and the CS93 region in HLs (black rectangle in E), which corresponds to the reduction in H3K27ac levels seen in HL cells at HH20 (F). ChIP-seq profiles of CTCF and H3K27ac from FL and HL buds at HH20 are shown in red and blue, respectively. Open and closed arrowheads indicate the orientation of the CTCF motifs. Enrichments (y axis) of CTCF and H3K27ac ChIP are shown at the normalized 1x sequencing depth or the log2 ratio of the normalized number of reads between ChIP and input samples, respectively. CHi-C, capture Hi-C; ChIP, chromatin immunoprecipitation; ChIP-seq, ChIP sequencing; CTCF, CCCTC-binding factor; E, embryonic day; FL, forelimb; H3K27ac, acetylation histone H3 lysine 27; Hi-C, high-throughput chromosome conformation capture; HH, Hamburger–Hamilton stage; HL, hindlimb; TAD, topologically associating domain.
Fig 6.
Hoxa13 expression in chicken limb buds.
(A) Expression patterns of Hoxa13 and Hoxd11 and mRNA steady-state levels in chick FL and HL buds from HH20 to HH22. A stronger expression of Hoxa13 is observed in chick HL bud when compared to FL bud (top). mRNA level of Hoxd11 increases in FL bud as development proceeds yet seems to decrease in HL bud (bottom). Expression levels are normalized to Gapdh and shown as fold change relative to FL bud at HH20–21. Error bars indicate standard deviation of three biological replicates. NS, p > 0.05; *p < 0.05; **p < 0.01, Welch two-sample t test. (B) Expression of Hoxa13 and Hoxd11 in mouse FL and HL buds from E10.5 to E10.75. mRNA levels of both genes in FL and HL buds increase as development proceeds. Expression levels are normalized to Gapdh and shown as fold change relative to FL buds at E10.5. Error bars indicate standard deviation of two or four biological replicates. *p < 0.05; NS, p > 0.05, Welch two-sample t test. For both A and B, individual numerical values of RT-qPCR are given in S1 Table. E, embryonic day; FL, forelimb; HH, Hamburger–Hamilton stage; HL, hindlimb; RT-qPCR, quantitative reverse transcription PCR.
Fig 7.
Different effects of a T-DOM deletion on FL and HL buds.
(A) The HoxDDel(attp-SB3) and HoxDDel(Mtx-Ttn) alleles are deletions of about 1 Mb or 2.1 Mb, respectively, including T-DOM (left, dashed line). Hoxd11 expression in E12.5 FLs and HLs from either control (HoxDDel[8–13]/+) animals (indicated as “+/Δ”) or mutant (HoxDDel[attp-SB3]/Del[8–13], HoxDDel[Mtx-Ttn]/Del[8–13], HoxDDel[8–13]/+;HoxC−/−, HoxDDel[attp-SB3]/Del[8–13];HoxC−/−) littermates (indicated as “Del(attp-SB3)/Δ,” “Del(Mtx-Ttn)/Δ,” “+/Δ/HoxC−/−,” and “Del(attp-SB3)/Δ/HoxC−/−,” respectively). In Del(attp-SB3)/Δ mutants, Hoxd11 expression is dramatically reduced in proximal FLs (arrowhead) but remains robust in proximal HLs (arrowhead). In HoxDDel(Mtx-TiE2) mutants, Hoxd11 expression is abrogated in both proximal FL and HL buds (arrowhead). The absence of both T-DOM and the HoxC cluster does not affect Hoxd11 expression. (B) Hi-C data adapted from [19] showing the two TADs on either side of the HoxD cluster and the TAD next to T-DOM. The 4C profiles represent contacts established by Hoxd11 in proximal FL and HL buds from control or Del(attp-SB3)/Δ mutant animals. In mutant cells lacking T-DOM (tracks 2 and 4), additional contacts between Hoxd11 and the neighboring TAD are scored. The shaded region (red arrows) shows the domains in which increased contacts are detected in mutant proximal HL versus proximal FL buds. (C) Enlargement of 4C profiles shown in (B), DNaseI HS profiles using E11.5 embryos and potential limb enhancer regions (pink rectangles) identified by using the Limb-Enhancer Genie tool. Potential HEs are shown by red arrows. (D) Mouse HE1 is active in the proximal FL and HL buds and in the trunk at E12.5. The number indicates stained embryos over total number of integrations. 4C, circular chromosome conformation capture; 4C-seq, 4C sequencing; E, embryonic day; FL, forelimb; HE, hidden enhancer; Hi-C, high-throughput chromosome conformation capture; HL, hindlimb; HS, hypersensitive sites; PFL, proximal FL; PHL, proximal HL; TAD, topologically associating domain; T-DOM, telomeric regulatory domain.
Fig 8.
Model of TAD boundaries at the mouse and chicken HoxD cluster.
(A, B) TAD boundaries at the HoxD locus in mouse (A) and chick (B) limb buds. (A) In the mouse, the boundary is dynamic and moves along a few genes within a window determined by a series of CTCF sites. Accordingly, T-DOM enhancers interact with promoters up to Hoxd11 (green arrow in A). (B) In chick, the boundary appears slightly displaced toward the Hoxd13 locus. This latter situation may enable T-DOM enhancers to interact with Hoxd13 more efficiently in chick than in murine limb bud cells (green arrow in B). Black and white arrowheads indicate the orientation of CTCF motifs. C-DOM, centromeric regulatory domain; CTCF, CCCTC-binding factor; TAD, topologically associating domain; T-DOM, telomeric regulatory domain.