Patterning by heritage in mouse molar row development

Abstract

It is known from paleontology studies that two premolars have been lost during mouse evolution. During mouse mandible development, two bud-like structures transiently form that may represent rudimentary precursors of the lost premolars. However, the interpretation of these structures and their significance for mouse molar development are highly controversial because of a lack of molecular data. Here, we searched for typical tooth signaling centers in these two bud-like structures, and followed their fate using molecular markers, 3D reconstructions, and lineage tracing in vitro. Transient signaling centers were indeed found to be located at the tips of both the anterior and posterior rudimentary buds. These centers expressed a similar set of molecular markers as the "primary enamel knot" (pEK), the signaling center of the first molar (M1). These two transient signaling centers were sequentially patterned before and anterior to the M1 pEK. We also determined the dynamics of the M1 pEK, which, slightly later during development, spread up to the field formerly occupied by the posterior transient signaling center. It can be concluded that two rudimentary tooth buds initiate the sequential development of the mouse molars and these have previously been mistaken for early stages of M1 development. Although neither rudiment progresses to form an adult tooth, the posterior one merges with the adjacent M1, which may explain the anterior enlargement of the M1 during mouse family evolution. This study highlights how rudiments of lost structures can stay integrated and participate in morphogenesis of functional organs and help in understanding their evolution, as Darwin suspected long ago.

Fig. 1.

Reduction of the lower cheek teeth during mouse evolution and their pattern during mouse ontogeny. (A) Dentition in the adult mouse is considerably reduced. Each jaw quadrant comprises only one incisor (In) and three molars (M1, M2, M3); a large toothless diastema occurs at the place of missing canine and premolar teeth. (B–D) Evolution of the mouse lower molars from a common ancestor of lagomorphs and rodents. (D) Two premolars (called P3 and P4) were lost in the mouse lineage (B). (E and F) The two current interpretations of mouse lower molar development. The “rudimentary buds hypothesis” of mouse lower molar row development (E) has a basis in descriptive morphological studies and evolutionary data: Two rudimentary premolar buds (MS and R2) are the first tooth primordia, which sequentially develop in the cheek region of mandible, before and in front of molars. These buds’ progression is stopped by apoptosis. (F) In the classic view, the first molar (M1) is the first tooth primordium that appears in the cheek region of the mandible. Afterward, the other molars (M2, M3) are sequentially added.

Fig. 2.

Three Shh signaling centers are sequentially patterned in the cheek region of the mandible. (A) The time-table shows in colors (blue, red, yellow) the presence of a distinct signal patch in the cheek region of mandible of the Shh-EGFP mice according to their age (ED) and body weight (mg). Gray represents the mandibles with weak or indistinct signal. Blue, red, and yellow boxes represent the mandibles with the signal patch in the cheek region at ED 12.7, 13.3 + 13.7, and 14.3, respectively. The mandibles hybridized with Shh antisense probe were sectioned frontally to show dental epithelium (in a rectangle), which was reconstructed in 3D (Shh signal in color according to the time-table). The Shh signaling centers correspond to the respective morphological structures MS, R2, and M1 bud. (B) Time-lapse microscopy beginning at ED 12.7 (90 mg), culture time in picture corner. Blue, red, or yellow arrowhead designates three successive EGFP patches; the white arrowhead designates a necrotic zone. An overlay of the pictures at 0, 31, and 60 h shows three distinct EGFP signals.

Fig. 3.

MS and R2 display typical pEK markers. Localization of several pEK markers in whole mandibles (ED 12.7, 80–90 mg) or dissociated dental epithelium from the lower cheek tooth region (ED 13.3, 130–140 mg; and ED 14.3, 220–230mg), as revealed by in situ hybridization with Shh, Edar, p21, Fgf4, and Bmp4 antisense probes of CD1 embryos or by X-gal staining of TOPGAL embryos carrying a Wnt pathway reporter transgene (41). The residual signal is indicated by white letters. (Scale bars, 250 μm.)

Fig. 4.

Sequential patterning of the cheek teeth in mouse mandible. (A) Time-lapse microscopy pictures of Shh-EGFP mandibles cultured after DiI microinjection at ED 12.7, 90 mg (in MS) or at ED 13.3, 145 mg (in R2), (culture time in picture corner). Note the pEK of the M1 appears posteriorly to the DiI label. The R2 label is later overlapped by the anteriorly extending M1pEK. (B) A series of ED 14.0 dissociated dental epithelia hybridized with Shh antisense probe document the secondary anterior extension of the M1 Shh-signaling at the bud-cap transition. (C) A model of integration of the premolar rudiments in patterning of mouse molars. (Arrows) The influence of a tooth primordium on the newly rising one. (D) A two-step working model of mouse tooth row evolution.