Functional enhancers in the HDAC9-TWIST1 locus characterized using zebrafish and mice.

(A) A scheme of the HDAC9-TWIST1 locus. UCSC genome browser track represents genes and black bars represents enhancer candidate sequences. (B) Zebrafish whole-mount in situ hybridization of twist1b shows head, pectoral fin and branchial arch expression at 72 hpf. (C) Mouse whole-mount in situ hybridization of Twist1 shows limb and branchial arch expression along the anterior-posterior axes at E11.5. (D) Mouse whole-mount in situ hybridization of Hdac9 shows no expression in the limb and branchial arch at E11.5. (E–H) Tissue-specific enhancers in zebrafish embryos at 72 hpf. Tissue-specific GFP expression is indicated by red arrows: (E) branchial arch; (F) pectoral fin and branchial arch; (G) pectoral fin and branchial arch; (H) pectoral fin, otic vesicle and branchial arch. (I-N) Tissue-specific enhancers in mouse E11.5 embryos. Tissue-specific LacZ expression is indicated by red arrows: (I, I') pharyngeal arch 2; (J, J') whole limb bud; (K, K') posterior limb bud and branchial arch (known as HDAC eExon19); (L, L') dorsal and anterior limb bud (known as HDAC eExon18); (M, M') the base of the limb bud; (N, N') distal and posterior limb bud. The dashed boxes highlight tissue-specific expression. The numbers in the bottom left of the embryo pictures indicate the number of embryos showing this expression pattern from the total number of LacZ-stained embryos. Abbreviations: Pectoral fin (PF), branchial arch (BA), otic vesicle (OV), forelimb (FL), hindlimb (HL), mandibula (Md), pharyngeal arch 2 (PA2). Images K and L were taken with permission from Birnbaum et al. [4].

Segmental analysis of eTw-5 and eTw-11 enhancers in zebrafish embryos at 72 hpf.

(A) eTw-5 was divided into three overlapping segments: Segment 1, segment 2 and segment 3. The UCSC genome browser (http://genome.ucsc.edu) conservation track shows that segment 2 is most conserved between humans and fish. (B) A graph displaying the number of embryos presenting GFP expression in the pectoral fin and branchial arch tissues from all live embryos at 72 hpf. (C) Zebrafish enhancer assay results for eTw-5 segments. The full sequence of the p300 ChIP-seq peak drove GFP expression in the pectoral and caudal fins, branchial arch and somitic muscles. Segment 1 did not drive GFP expression, segment 2 drove GFP expression in the fins and branchial arch and segment 3 drove GFP expression mostly in somitic muscles and to some extent in the branchial arch. (D) eTw-11 was divided into four overlapping segments (segments 1–4). The UCSC genome browser (http://genome.ucsc.edu) conservation track shows that segment 3, 4 are conserved from zebrafish to humans. (E) A graph displaying the number of embryos with GFP expression in the pectoral fin and branchial arch tissues from all live embryos at 72 hpf. (F) Zebrafish enhancer assay results for eTw-11 segments: the full sequence of the p300 ChIP-seq peak drove GFP expression in the base of the pectoral fin, in the otic vesicle and branchial arch. Segments 1, 3 and 4 did not drive GFP expression, while segment 2 drove dominant GFP expression in the base of the pectoral fin and the branchial arch.

Functional enhancers in the HDAC9-TWIST1 locus characterized using zebrafish.

(A) eTw-1 drives GFP expression in the caudal fin. (B) eTw-2 drives GFP expression in the branchial arch. (C) eTw-5 drives GFP expression in the pectoral fin, branchial arch and somitic muscles. (D) eTw-6 drives GFP in the pectoral fin and branchial arch. Top: lateral view. Bottom: ventral view. (E) eTw-8 drives GFP in expression in the branchial arch. (F) eTw-11 drives GFP expression in the base of the pectoral fin, in the branchial arch and in the otic vesicle. Top: lateral view. Bottom: dorsal view.

Additional segmental analysis of eTw-5 enhancer function in zebrafish embryos at 72 hpf.

(A) The eTw-5 segment 2 was divided into segments 2a and 2b. (B) A graph displaying the number of embryos presenting GFP expression in the pectoral and caudal fins and branchial arch out of all live embryos at 72 hpf. (C) Zebrafish enhancer assay results for eTw-5 segments: segment 2a drives GFP expression in the epidermis surrounding the head and segment 2b drives GFP expression in the pectoral and caudal fins.

Segmental analysis of eTw-6 enhancer function in zebrafish embryos at 72 hpf.

(A) The eTw-6 enhancer that drives GFP expression in the pectoral and branchial arch was divided into four overlapping segments (1–4). The UCSC genome browser (http://genome.ucsc.edu) conservation track shows that segment 4 which contains Hdac9 exon 18 is conserved between humans and fish. (B) A graph displaying the number of embryos with GFP expression in the pectoral fin, branchial arch, brain and specific neurons out of all live embryos at 72 hpf. (C) Zebrafish enhancer assay results for eTw-6 segments: segment 1 and segment 4 did not drive GFP expression, while segments 2 and segment 3 drove GFP expression in neurons near the eyes that project to the trunk.

Analysis of eTw-8 enhancer function in zebrafish embryos at 72 hpf.

(A) eTw-8 was divided into three segments (1–3). The UCSC genome browser (http://genome.ucsc.edu) conservation track shows that segment 3 is the most evolutionarily conserved. (B) A graph displaying the number of embryos with GFP expression in the branchial arch and pectoral fin tissues out of all live embryos at 72 hpf. (C) Zebrafish enhancer assay results for eTw-8 segments: The full sequence of the p300 ChIP-seq peak drove GFP expression in the branchial arch, while segment 3 drove similar GFP expression in the branchial arch.