Identification of a distant cis-regulatory element controlling pharyngeal arch-specific expression of zebrafish gdf6a/radar

Abstract

Skeletal formation is an essential and intricately regulated part of vertebrate development. Humans and mice deficient in growth and differentiation factor 6 (Gdf6) have numerous skeletal abnormalities, including joint fusions and cartilage reductions. The expression of Gdf6 is dynamic and in part regulated by distant evolutionarily conserved cis-regulatory elements. radar/gdf6a is a zebrafish ortholog of Gdf6 and has an essential role in embryonic patterning. Here, we show that radar is transcribed in the cells surrounding and between the developing cartilages of the ventral pharyngeal arches, similar to mouse Gdf6. A 312 bp evolutionarily conserved region (ECR5), 122 kilobases downstream, drives expression in a pharyngeal arch-specific manner similar to endogenous radar/gdf6a. Deletion analysis identified a 78 bp region within ECR5 that is essential for transgene activity. This work illustrates that radar is regulated in the pharyngeal arches by a distant conserved element and suggests radar has similar functions in skeletal development in fish and mammals.

Figure 1

Expression of radar in the pharyngeal arch cartilages. (A–D & O lateral; E–L ventral). The expression of radar, sox9a, sox9b, and gdf5 in ventral arches is evident by in situ hybridization at 77 hpf. sox9a (B,F,J) is detected in cartilage while sox9b (C, G,K) is localized to the epithelial sheath surrounding cartilages (K). gdf5 is expressed in the jaw joint (white arrow, H) and medially in posterior arches, and at the basihyal (black arrow; D,H,L) (Chiang et al., 2001; Yan et al., 2005)(D,H,L). radar expression is detected in the jaw and along the ventral midline (E and arrows in I). M–N. Transverse sections detecting radar and gdf5 transcript. Both are expressed in the jaw joint (paired ventral staining in M and N) while only gdf5 is expressed dorsally in the pharynx (N). O. High resolution whole-mount imaging shows radar is detectable between posterior arch pharyngeal cartilages (arrow) (lateral view; left = anterior). P. Sagittal section shows that radar is expressed surrounding medial hypobranchial cartilages. Q–S. Sagittal sections showing sox9a, sox9b, and gdf5 transcripts. gdf5 and radar are coexpressed near the midline around ceratohyals and hypobranchials though radar extends more ventrally below hypobranchials. Abbreviations: bh, basihyal; ch, ceratohyal; hb1, hypobranchial 1; hb2, hypobranchial 2; hb3, hypobranchial 3; ep, ethmoid plate; e, eye; m, mouth opening.

Figure 2

Analysis of pharyngeal arch organization in wild-type and radar mutant larvae. A and B. Lateral and ventral view of alcian blue stained 5 days post fertilization (dpf) wild-type larvae. C. High magnification of dotted box area in panel A noting normal articulation of ceratohyals (at joint indicated by black arrow) and normal positions of ceratobranchials (red arrows) D. Camera lucida image outlining the alcian blue stained cartilages in panel B. E. Collagen-2α1 staining of the third arch in 5 dpf wild-type larvae to visualize ceratobranchial, basibranchial, and hypobranchial. F and G. Lateral and ventral view of alcian blue radar^s327 mutant. H. High magnification of region in panel F demarcated by dotted box showing abnormal articulation of ceratohyals (black arrow) and more sharply angled ceratobranchials (red arrows). I. Camera lucida image of the alcian blue stained cartilages in E. J. Collagen-2α1 staining of third arch in 5 dpf mutant larvae reveals morphological abnormalities of the hypobranchial (asterix) and hypobranchial/ceratobranchial joint (arrow). ch, ceratohyals; cb, ceratobranchials; bb; basibranchial; hb, hypobranchials.

Figure 3

Gdf6 noncoding evolutionarily conserved regions (ECRs) with mammal/fish conservation, and flanking genes in fish and mammals. A. Inter-fish comparison of zebrafish, medaka and Fugu radar loci, showing arrangement of fish/mammal ECRs and flanking genes (not to scale). There are 5 noncoding ECRs with mammal/fish conservation dispersed throughout the vertebrate Gdf6 locus as identified by Pipmaker alignments to zebrafish BAC CH211-216g21. Two of these conserved elements are located within the radar intron while the other 3 are 3′ of the radar transcriptional start site. The zebrafish/human ECR alignments ranged in size from 109 bp to 227 bp (see Table 1). B: ECRs 1–5 and flanking genes near mouse and human Gdf6. C: Comparison of gene order in zebrafish and medaka gdf6b regions, and a segment of human 8q23. The zebrafish map is truncated at left due to a scaffold break (not shown). The fish eny2 and trhr paralogs are given suffixes –a and –b in accordance with their linkage to gdf6a or gdf6b.

Figure 4

ECR5 is highly conserved amongst vertebrates. A University of California Santa Cruz Genome Browser (Kent et al., 2002) screenshot illustrating the cross species conservation pertaining to the 312 bp in size radar ECR5 (red) in the zebrafish. The 312 bp sequnce contains most of a conservation block evident by the PhastCons Track (blue), which denotes regions with statistically significant conservation based (Siepel et al., 2005). There are high levels of sequence conservation among fishes (zebrafish, fugu, tetraodon), the frog (Xenopus tropicalis), and mammals (opossum, mouse, human). (green).

Figure 5

ECR5 drives transgene expression in a subset of the pharyngeal arches in context of the minimal cFOS promoter. A. Lateral brightfield image of ECR5 transgenic zebrafish larva at 4 days post fertilization (dpf). B. Fluorescent image of ECR5 transgenic at 4dpf showing transgene expression in a subset of the pharyngeal arches. The faint line of signal dorsal to the arches was due to autofluorescence and not transgene expression, as revealed by staining with anti-GFP antibody (not shown). C. Overlay of brightfield and fluorescent ECR5 transgene expression. D. Ventral view of ECR5 transgene expression (green). Anterior is at top right. Wheat germ agglutinin labeling of cartilage (red) shows that transgene expression does not overlap with mature cartilage of the flanking ceratohyals. E. Immunohistochemistry for GFP on a ECR5 transgenic 4dpf embryo sagittal section shows that transgene (brown) is not expressed in internal chondrocytes within cartilage elements but is restricted to perichondrium and cells between elements. Abbreviations: e, eye; m, mouth; h, heart; ch, ceratohyal cb1, ceratobranchial 1.

Figure 6

Deletion analysis identified a subregion necessary for ECR5 transgene expression in the zebrafish. Four 78 bp deletions were engineered into the 312 bp ECR5 construct and analyzed for transgene expression in stable transgenics. This result suggests that Deletion B contains an element(s) required for transgene expression. Numbers of lines with GFP expression in pharyngeal arches, relative to total numbers of transgenic lines analyzed for each construct are shown at right.

Figure 7

ClustalW alignment of deletion B region illustrating conservation. The region within deletion B is highly conserved amongst vertebrates. Consensus bases are shaded gray. In silico analysis using TRANSFAC Match analysis identified putative binding sites (bars) for selected transcription factors previously reported to have roles in pharyngeal arch cartilage formation.