A role for suppressed incisor cuspal morphogenesis in the evolution of mammalian heterodont dentition

Abstract

Changes in tooth shape have played a major role in vertebrate evolution with modification of dentition allowing an organism to adapt to new feeding strategies. The current view is that molar teeth evolved from simple conical teeth, similar to canines, by progressive addition of extra "cones" to form progressively complex multicuspid crowns. Mammalian incisors, however, are neither conical nor multicuspid, and their evolution is unclear. We show that hypomorphic mutation of a cell surface receptor, Lrp4, which modulates multiple signaling pathways, produces incisors with grooved enamel surfaces that exhibit the same molecular characteristics as the tips of molar cusps. Mice with a null mutation of Lrp4 develop extra cusps on molars and have incisors that exhibit clear molar-like cusp and root morphologies. Molecular analysis identifies misregulation of Shh and Bmp signaling in the mutant incisors and suggests an uncoupling of the processes of tooth shape determination and morphogenesis. Incisors thus possess a developmentally suppressed, cuspid crown-like morphogenesis program similar to that in molars that is revealed by loss of Lrp4 activity. Several mammalian species naturally possess multicuspid incisors, suggesting that mammals have the capacity to form multicuspid teeth regardless of location in the oral jaw. Localized loss of enamel may thus have been an intermediary step in the evolution of cusps, both of which use Lrp4-mediated signaling.

Fig. 1.

Grooves in incisors of mutant mice. (A and B) Grooved incisors are found in Lrp4^hypo/hypo (B), whereas there are no grooves in wild-type laboratory mice (Mus musculus; A). (C and D) Cross sections of incisors showed that grooves were caused by lack of enamel (D). (E and F) The first sign of grooves was at postnatal day 5 (P5) in Lrp4^hypo/hypo mice (arrow in F). (G–J) Shh expression was downregulated at the presumptive groove region in Lrp4^hypo/hypo mice at birth (arrowhead in H) whereas strong Ptc1 and Gli1 expression was observed in a similar region in wild-type (arrowhead in I and J). (M–P) Downregulation of Bmp4 (arrowhead in N) and Bmp7 (arrowhead in P) expression was observed in ameloblasts in Lrp4^hypo/hypo mice at birth. (K, L, Q, and R) Ptc^mes/mes (K and L) and K14-Noggin (Q and R) mice showed labial grooves that were caused by the lack of enamel. (S) Molar enamel–free zone in wild-type laboratory mice at P2. (T) Reduction in Bmp4 expression was observed at enamel-free zones at P2 (arrowhead). (U and V) Enamel-free zone marker gene, Slit1 was expressed at the presumptive groove region in Lrp4^hypo/hypo mice at birth (arrow in V), whereas very faint Slit1 expression could be detected in similar regions in wild-type (arrow in U). (A–D, K, L, Q, and R) Images of incisors obtained from 3-month-old animals. (W and X) Incisors of 2-year-old wild-type laboratory mouse. Three-dimensional reconstructions (B, K, Q, and W) and cross-section (C, D, L, R, and X) based on micro-CT scans and SEM images (A) of maxillary incisors. Developing upper incisors (E–J, M–P, U, and V) and lower molars (S and T). Lrp4^hypo/hypo (B, D, F, H, N, P, and V) and wild-type mice (A, C, E, G, I, J, M, O, and S–U). Ameloblasts are outline in blue (M–P).

Fig. 2.

Grooved incisors in various wild-type rodents, rabbits, and fish. (A–C) Grooved incisors are found in jumping mice (Zapus hudsonius; A), cane rat (Thryonomys swinderianus; B), and rabbit (Oryctolagus cuniculus; C). (D–F) Cross-sections of incisors showed that grooves were caused by lack of enamel. Multiple grooves were found in cane rat and in Lrp4^hypo/hypo mice (arrowheads in B, E, and I). Three-dimensional reconstructions (A–C) and cross-section (D and F) based on micro-CT scans, SEM images (I), and stereomicroscopic images (E) of maxillary incisors. (G and H) Enamel-free zone in cichlid fishes (Cyathochromis obliquidens; arrowhead in H). (A–F and I) All images of incisors were obtained from 3-month-old animals.

Fig. 3.

Folding enamel/dentin in rodent incisors. Incisors with grooves caused by folded enamel/dentin in wild-type rodent species (Chilean climbing mouse; A and C) and Andean swamp rat; B and D). Three-dimensional reconstructions (A and B) and cross-section (C and D) based on micro-CT scans of maxillary incisors.

Fig. 4.

Cusp-like structure and molar-type roots in mammalian incisors. (A and B) Lrp4 null mutants showed folding of enamel/dentin on the labial sides of incisors at P1 (arrowheads in B). Molar root-like structures were also found at the lingual side of incisors in Lrp4 null mutants (arrow in B). (C) Slit1 a marker of tertiary enamel knot and the enamel-free zones, was found in the tooth epithelium corresponding to the folded enamel/dentin (arrowheads in C). Ameloblasts are outlined in red (C). (D–F) Expression of enamel knot marker genes (Wnt10b; D) and (p21; E), and Lrp4 expression (F) in lower incisors at E13.5 in wild type. (G and H) Extra cusps in upper molar of Lrp4 null mutant (arrowhead in H). (I–L) Extra Fgf4 expression domain was found in second lower molar (J) and first lower molars (L) of Lrp4 null mice. (M and N) Some incisors showed a gap at the lingual side in Lrp4 null mutants (N). (O) Ptc2 expression was observed at the gap (arrowheads). (P and Q) Barx1 expression in lower incisor (P) and lower molar (Q) of Lrp4 null mutant at P1. (R–U) Multicuspid incisors of shrews (Sorex arcticus; R and S), flying lemurs (Galeopterus variegates; T), and great fruit-eating bats (Artibeus lituratus; U). (S) Cross-section based on micro-CT scans of maxillary incisors, showing folded enamel/dentin with enamel-free zone (arrowhead). (V) Diagram of evolutionary mutant/modification series; incisor of Lrp4 null mutant (Left), Lrp4^hypo/hypo (Center), and wild-type (Right) mice. (A–H and M–Q) Frontal sections. Wild-type mice (A, D–G, I, K, and M) and Lrp4 null mutant (B, C, H, J, L, and N–Q).