Endogenous and reporter expression of dlx2b in developing zebrafish tooth germs.

(A) Ventral view with anterior to the left of a 120 hpf larval head labeled with alizarin red S (magenta) to show mineralizing tissues and DAPI (white) to stain nuclei and thus provide a cellular context. The tooth-forming region in the posterior pharynx is indicated (box), highlighting the approximate zoom and cropping of the subsequent panels, in which the right-side (bottom) tooth germs are labeled. (B) Optical section of dlx2b mRNA in situ hybridization (green) at 72 hpf with the 4V1 tooth germ indicated. (C) Expression from the dlx2bKI reporter line showing the 3V1 tooth germ at a morphogenesis stage and 4V1 at cytodifferentiation. (D) GFP expression in the dlx2b4kb reporter, additionally stained with alizarin red S to show developing tooth mineralization. CB5 = fifth ceratobranchial cartilage (onto which all of the teeth eventually attach).

Location and sequence of the dlx2b minimal tooth enhancer MTE.

(A) Diagram of the 5’ end of the zebrafish dlx2b gene, including the 5’ UTR and 817 bp of upstream non-coding sequence. The location of the identified 215 bp minimal tooth enhancer MTE is indicated. The black bars below the gene schematic represent regions of homology with the stickleback, X. tropicalis, and human genomes (UCSC Genome Browser Zv9/danRer7). (B) Sequence of MTE aligned with those of other species. Predicted possible transcription factor binding sites with high conservation are shaded.

Tooth germ reporter expression.

Ventral view close-up of one side of the tooth forming region with the tooth germs 4V1 and 3V1 indicated. The dlx2bKI reporter line (A), the dlx2b4kb line (B), and dlx2bMTE (C), all exhibit GFP expression primarily in the inner dental epithelium (e) but also somewhat in the dental mesenchyme (m), whereas no GFP expression was observed in the dlx2b744-674 truncated reporter (D).

Conserved transcription factor binding sites are required for MTE function.

Zebrafish larvae injected transiently with either the dlx2bMTE GFP reporter construct (A) or the dlx2bMTEmDFCA construct with the predicted Dlx, FoxA, Cebp, and Ap1 binding sites all mutated (B). Arrows indicate the location of the tooth-forming region.

MTE is required for proper tissue-specific expression in developing tooth germs.

(A) In dlx2bKI at 78 hpf, GFP expression appears much stronger in the inner dental epithelium (e) than in the dental mesenchyme (m) in both 4V1 and 3V1 tooth germs. (B) In dlx2bKIΔMTE at the same stage, the expression pattern appears reversed, with strong GFP signal in the dental mesenchyme and little to none in the epithelium.

Summary of results and model of possible MTE enhancer function.

The left side (A, B) represents the Tol2-based transgene experiments at a remote locus and the right side (C, D) the knock-in experiments at the dlx2b locus. The top of the diagram (A, C) depicts the normal MTE sequence and the bottom (B, D) the mutated versions. MTE is sufficient to drive a mostly-epithelial tooth germ expression pattern even at a remote locus (A) but when mutated at a remote locus (B), there are perhaps no other nearby enhancers to compensate and thus all tooth germ expression is lost. In contrast, at the dlx2b locus, MTE may be a primary driver of epithelial tooth germ expression (C), but when mutated, one or more other cis-regulatory elements (diamond) maintain expression, but in a more mesenchymal pattern (D).

Transgenic lines used in this study.

The dlx2bKI allele.

(A) Schematic diagram of the Mbait-hs-eGFP plasmid insertion into the dlx2b locus. Sequences at the 5’ (B) and 3’ (C) end of the insertion.

Sequences mutated to test the necessity of various predicted/possible transcription factor binding sites.

Expression of TFs in and near 4V1.

(A-D) expression of dlx2b in 4V1 (arrow) at 56 (A) and 72 hpf (C). (B,D) Transverse sections of A and C respectively. The arrowhead in B points to the 4V1 tooth germ. (E-L) expression of dlx2a (E,F); foxa2 (G,H); cebpa (I,J) and AP-1/jun (K,L) at 56 hpf. The arrow in E and the arrowhead in F denote the expression of dlx2a in 4V1 and more lateral mesenchyme (M-T) expression of dlx2a (E,F); foxa2 (G,H); cebpa (I,J) and AP-1/jun (K,L) at 72 hpf. The arrow in M and the arrowhead in N denote the expression of dlx2a in 4V1 and lateral mesenchyme. The arrowhead in R points to the location of expression of cebpa in the 4V1 tooth germ.

Expression of cebpa.

(A) mRNA in situ hybridization. (B) GFP expression from an individual heterozygous for the cebpaKI allele. For both methods, expression is strongest in the basal part of the inner dental epithelium.

Sequence of the deletion in the dlx2bKIΔMTE allele.

Summary of reporter constructs used in this study.

The dlx2bMTE line is from construct #9. Mutation sequences are shown in Fig. S2.