Retinoic acid expands the evolutionarily reduced dentition of zebrafish

Abstract

Zebrafish lost anterior teeth during evolution but retain a posterior pharyngeal dentition that requires retinoic acid (RA) cell-cell signaling for its development. The purposes of this study were to test the sufficiency of RA to induce tooth development and to assess its role in evolution. We found that exposure of embryos to exogenous RA induces a dramatic anterior expansion of the number of pharyngeal teeth that later form and shifts anteriorly the expression patterns of genes normally expressed in the posterior tooth-forming region, such as pitx2 and dlx2b. After RA exposure, we also observed a correlation between cartilage malformations and ectopic tooth induction, as well as abnormal cranial neural crest marker gene expression. Additionally, we observed that the RA-induced zebrafish anterior teeth resemble in pattern and number the dentition of fish species that retain anterior pharyngeal teeth such as medaka but that medaka do not express the aldh1a2 RA-synthesizing enzyme in tooth-forming regions. We conclude that RA is sufficient to induce anterior ectopic tooth development in zebrafish where teeth were lost in evolution, potentially by altering neural crest cell development, and that changes in the location of RA synthesis correlate with evolutionary changes in vertebrate dentitions.

Figure 1.

Exogenous RA treatment expands tooth development. A, B) Ventral view of the pharyngeal region of 4-dpf alizarin red-stained zebrafish larvae, anterior to the left. A) DMSO control exhibiting a pair of well-formed teeth on each side of the ventral, posterior pharynx. B) Specimen treated exogenously with 6 × 10⁻⁷ M RA between 24 and 36 hpf, with ectopic teeth positioned more anteriorly than in wild type. Additional teeth located more dorsally are not visible in this focal plane. C, D) Oblique ventrolateral view of the head of 4-dpf dlx2b:GFP reporter larvae. C) Control embryo with GFP expression in developing tooth germs. D) RA-treated larva with anteriorly expanded supernumerary foci of GFP expression. E, F) Confocal micrographs oriented as in A and B, with a single plane of labeled cell nuclei (gray) in the ventral pharynx superimposed on an extended-focus composite of dlx2b:GFP reporter expression (green). G, H) Transverse sections in planes indicated in E and F. G) Control larva with dlx2b:GFP expression in tooth germs developing normally, ventral to the lumen of the pharynx (dotted line). H) RA-treated specimen with tooth germs located both dorsal and ventral to the pharyngeal lumen. Arrows indicate selected teeth or tooth germs. Scale bars = 50 μm.

Figure 2.

RA expands pitx2, dlx2b, and hoxb5a expression. Lateral (A, B, E–H, K, L) or ventral (C, D, I, J, M, N) view of mRNA in situ hybridizations, anterior to the left, with normal site of tooth formation indicated (arrow). A) Control embryo expressing pitx2 in the posterior pharyngeal region at 48 hpf. B) Embryo treated with exogenous RA exhibits an anterior expansion of pitx2 (arrowhead). C) Horizontal confocal fluorescence section of pharyngeal epithelial and tooth germ pitx2 expression in control. D) Identical section after RA treatment with pitx2 expression expanded anteriorly in the pharyngeal epithelium (arrowhead). E) Embryo implanted with a control bead (asterisk) and a wild-type pattern of pitx2 expression. F) RA bead-implanted embryo with pitx2 expression extended more anteriorly (arrowhead). G) Expression of hoxb5a mRNA in the posterior brain and pharyngeal region (arrow) at 56 hpf. H) RA-treated embryo with anterior expansion of pharyngeal hoxb5a expression (arrowhead). I) Horizontal section of hoxb5a expression surrounding control tooth germs (arrow). J) RA-exposed embryo with hoxb5a expression expanded anteriorly (arrowhead). K) dlx2b expression in a control embryo at 72 hpf. L) RA-treated embryo showing dlx2b expression expanded anteriorly (arrowhead). M) 96 hpf larva with dlx2b (green) and hoxb5a (red) expression in the posterior pharynx in and around developing teeth. N) Larva after RA treatment with both dlx2b and hoxb5a expression expanded anteriorly (arrowheads). Scale bars = 100 μm.

Figure 3.

RA tooth expansion correlates with cartilage disruption. Ventral views of the 4-dpf pharyngeal region in alcian blue, alizarin red double-stained larvae, anterior to the left (arrows indicate selected teeth). A) Control zebrafish larva with a pair of well-formed teeth attached to the ossifying fifth ceratobranchial cartilages. B) Larva treated with RA from 24 hpf exhibiting supernumerary teeth and severe cartilage loss. C) Larva showing normal tooth development with a control bead positioned nearby. D–F) Larvae with RA-coated bead implantation at 24 hpf exhibit a range of cartilage deformation and supernumerary tooth phenotypes ranging from relatively severe (D) to relatively mild (E, arrow indicates ectopic midline tooth). F) Closeup view showing supernumerary teeth in proximity to an RA bead. Scale bars = 50 μm.

Figure 4.

RA alters late migratory/early postmigratory cranial neural crest organization and gene expression. Lateral views of 30-hpf embryos, anterior to the left (arrows indicate normal tooth-forming region). A) Control fli1:GFP reporter expression. B) RA-treated embryos at with disorganized cell arrangements at 24–30 hpf. C, D) Magnified views from embryos in A and B. E) Control dlx2a mRNA expression. F) RA-treated embryo with down-regulated dlx2a. G) Control crestin mRNA expression. H) RA-treated embryo with crestin expression upregulated in the anterior pharyngeal region (arrowhead). Scale bar = 100 μm.

Figure 5.

Gene expression analysis of CNC cell subtype markers and potential RA target genes. Lateral views of 52- to 56-hpf embryos, anterior to the left (arrows indicate normal tooth-forming region). A) Control sox9a mRNA expression at 56 hpf. B) RA-treated embryo with up-regulation of sox9a expression in the pharyngeal region (arrowhead) at 24–56 hpf. C) Control foxd3 mRNA expression. D) RA-treated embryo with more widespread pharyngeal foxd3 expression (arrowhead). E) Control mitfa mRNA expression marking developing melanocytes (double arrow). Differentiated melanocytes are also visible (arrowhead). F) RA-treated embryo with similar levels of mitfa expression but a disorganized-appearing melanocyte pattern (arrowhead). G) Control lef1 mRNA expression. H) RA-treated embryo with up-regulation of pharyngeal lef1 expression (arrowhead). I) Control prdm1a mRNA expression, restricted primarily to the developing tooth germ. J) RA-treated embryo showing strong prdm1a up-regulation (arrowhead). Scale bar = 100 μm.

Figure 6.

Multispecies comparison of the early larval pharyngeal dentition. Ventral (A, C, E, G) and 45°-rotated, ventrolateral (B, D, F, H) views of alizarin red-stained larva, visualized as 3D projections of confocal z stacks, anterior to the left. A, B) Zebrafish at 4 dpf with a bilateral pair of well-formed teeth located in the ventral, posterior pharynx. C) Zebrafish larva at 4 dpf, after exposure to RA beginning at 24 hpf, exhibiting supernumerary teeth that are anterior to normal positions, yet retaining a pattern of growth that is close to bilaterally symmetrical. For example, one bilateral pair of teeth (a/a′) is located closer to the midline than a nearby pair (b/b′). D) A ventrolateral view of the individual from C reveals dorsoventral bilateral symmetry as well: e.g., the more anterior labeled pair (a/a′) is located more ventrally than the more posterior pair (b/b′). Teeth near the b/b′ position were often found dorsal to the pharyngeal lumen (Fig. 1H). E, F) Mexican tetra larva at 3 dpf with 2 pairs of dorsal pharyngeal teeth located anteriorly to a single pair of ventral teeth. G, H) Medaka larva at 5 dpf with 5 pairs of anterodorsal teeth and 2 pairs of posteroventral teeth. Scale bars = 50 μm.

Figure 7.

Comparison of retinaldehyde dehydrogenase expression between zebrafish and medaka. Dorsal views of approximately stage-matched zebrafish and medaka embryos, anterior to the left, labeled for aldh1a2 mRNA expression by in situ hybridization. A, C) Zebrafish aldh1a2 mRNA expression visualized at 24 hpf (A) and 36 hpf (C) in the posterior pharyngeal region near the location where pharyngeal tooth germs will later form (arrows). B, D) Stage 25 (B) and 27 (D) medaka embryos with no detectable aldh1a2 mRNA expression in any part of the pharyngeal region (arrows).