Selection and constraint underlie irreversibility of tooth loss in cypriniform fishes

Abstract

The apparent irreversibility of the loss of complex traits in evolution (Dollo's Law) has been explained either by constraints on generating the lost traits or the complexity of selection required for their return. Distinguishing between these explanations is challenging, however, and little is known about the specific nature of potential constraints. We investigated the mechanisms underlying the irreversibility of trait loss using reduction of dentition in cypriniform fishes, a lineage that includes the zebrafish (Danio rerio) as a model. Teeth were lost from the mouth and upper pharynx in this group at least 50 million y ago and retained only in the lower pharynx. We identified regional loss of expression of the Ectodysplasin (Eda) signaling ligand as a likely cause of dentition reduction. In addition, we found that overexpression of this gene in the zebrafish is sufficient to restore teeth to the upper pharynx but not to the mouth. Because both regions are competent to respond to Eda signaling with transcriptional output, the likely constraint on the reappearance of oral teeth is the alteration of multiple genetic pathways required for tooth development. The upper pharyngeal teeth are fully formed, but do not exhibit the ancestral relationship to other pharyngeal structures, suggesting that they would not be favored by selection. Our results illustrate an underlying commonality between constraint and selection as explanations for the irreversibility of trait loss; multiple genetic changes would be required to restore teeth themselves to the oral region and optimally functioning ones to the upper pharynx.

Keywords: Astyanax mexicanus; transgenic.

Fig. 1.

Regional loss of eda expression is associated with reduction of dentition in the zebrafish lineage. (A) Teeth were lost from the mouth and upper pharynx in the zebrafish (D. rerio) lineage after its divergence from that of the Mexican tetra (A. mexicanus). (B) eda expression in upper (arrowhead) and lower (arrow) jaws of Astyanax. (C and D) eda expression in upper (arrowhead) and lower (arrow) pharynx of Astyanax. (E) Absence of eda expression in the mouth of Danio. (F and G) eda expression in the lower (arrow) but not upper pharynx of Danio. All eda expression shown is in mesenchyme, as confirmed by sectioning (Fig. S1). Abbreviations: e, eye; hpf, hours postfertilization; lpt, lower pharyngeal teeth; m, mouth; ot, oral teeth; upt, upper pharyngeal teeth; y, yolk. (Scale bars, 50 μm.)

Fig. 2.

The nkt mutation in eda results in early arrest of pharyngeal tooth development. (A and D) Ventral views of alizarin stained 7-dpf larvae showing complete absence of teeth in an nkt mutant homozygote. A single tooth is sometimes formed on one side of these mutants (n = 4 of 19). (B and E) pitx2 is expressed in presumptive pharyngeal tooth epithelium (arrows) in the nkt mutant homozygote (n = 5 of 5), heterozygote (n = 11 of 11), and wild-type homozygote (n = 5 of 5). (C and F) dlx2b is expressed in pharyngeal tooth germs (arrows) of wild-type homozygotes (n = 3 of 3) and nkt heterozygotes (n = 12 of 12), but its expression is lacking in the pharynx of the nkt mutant homozygote (n = 5 of 5). Abbreviations: cb5, fifth ceratobranchial bone; e, eye; hpf, hours postfertilization; n, notochord. (Scale bars, 50 μm.)

Fig. 3.

Ectopic Eda expression restores upper pharyngeal teeth to the zebrafish. Lower pharyngeal teeth (arrows) in lateral (A) and transverse (B) views of wild-type alizarin red-stained larvae. (C and D) bmp2b expression is limited to tooth germs (arrows) of the lower pharynx in wild-type. Upper pharyngeal teeth (arrowheads) in lateral (E) and transverse (F) views of ef1α:eda transgenic zebrafish. (G) bmp2b expression is induced in the upper pharyngeal epithelium (arrowhead) by ectopic eda expression and becomes limited to tooth germs (arrowhead) at later stages (H). Abbreviations: cb5, fifth ceratobranchial; cl, cleithrum; cp, chewing pad; d, dentary bone; n, notochord; y, yolk. (Scale bars, 50 μm.)

Fig. 4.

The upper of pharynx of adult zebrafish is only partially restored to the ancestral condition by ectopic eda expression. (A and D) Dorsal views of alizarin-stained pharyngeal arches with fifth ceratobranchials removed to show location of ectopic teeth (arrowheads in D) posterior to upper gill arch elements and dorsal to fifth ceratobranchials. Approximate position of the chewing pad is indicated by dotted lines. (E) Calcified tissue uniting ectopic teeth resembles upper pharyngeal toothplates of A. mexicanus (B). (C and F) Transverse sections (approximate plane of section indicated in D) reveal teeth in the vicinity of the palatal organ and projecting into the pharyngeal cavity (arrowheads in C) as well as lateral to the chewing pad (arrowhead in F). Lower pharyngeal teeth in (C and F) indicated by arrows. Abbreviations: cb, ceratobranchial; cp, chewing pad; eb, epibranchial; gr, gill raker; mpf, months postfertilization; pb, pharyngobranchial; pc, pharyngeal cavity; po, palatal organ; tp, toothplate. (Scale bars, 50 μm.)

Fig. 5.

Competence to respond to Eda signaling is distributed throughout the oropharyngeal cavity. (A and B) Confocal imaging of GFP fluorescence and C-F) in situ hybridization analysis of gfp RNA expression in NF-κB reporter zebrafish. Oral expression is limited to neuromasts and pharyngeal expression to lower tooth germs (arrow) in fish lacking the ef1α:eda transgene (A, C, and E). Presence of this transgene (B, D, and F) induces reporter expression throughout the oropharyngeal cavity, including in lower (arrow) and upper (arrowhead) pharyngeal tooth germs. Abbreviations: dpf, days postfertilization; e, eye; mc, Meckel’s cartilage; nm, neuromast. (Scale bars, 50 μm.)

Fig. 6.

The irreversibility of cypriniform dentition reduction results from the action of both selection and constraint. Left side indicates locations of competence to respond to Eda signaling and to do so with tooth production in an extant larval cypriniform. Right side indicates “forbidden” morphologies that represent reversal of cypriniform dentition reduction. The Upper morphology does not exist in nature because of the complexity of selection required to restore ancestral pharyngeal function. The Lower morphology does not exist because of constraint on the ability to produce oral teeth.