Detangling the evolutionary developmental integration of dentate jaws: evidence that a p63 gene network regulates odontogenesis exclusive of mandible morphogenesis.

Vertebrate jaws and dentitions fit and function together, yet the genetic processes that coordinate cranial and dental morphogenesis and evolution remain poorly understood. Teeth but not jaws fail to form in the edentate p63-/- mouse mutant, which we used here to identify genes important to odontogenesis, but not jaw morphogenesis, and that may allow dentitions to change during development and evolution without necessarily affecting the jaw skeleton. With the working hypothesis that tooth and jaw development are autonomously controlled by discreet gene regulatory networks, using gene expression microarray assays validated by quantitative reverse-transcription PCR we contrasted expression in mandibular prominences at embryonic days (E) 10-13 of mice with normal lower jaw development but either normal (p63+/- , p63+/+ ) or arrested (p63-/- ) tooth development. The p63-/- mice showed significantly different expression (fold change ≥2, ≤-2; P ≤ 0.05) of several genes. Some of these are known to help regulate odontogenesis (e.g., p63, Osr2, Cldn3/4) and/or to be targets of p63 (e.g., Jag1/2, Fgfr2); other genes have no previously reported roles in odontogenesis or the p63 pathway (e.g., Fermt1, Cbln1, Pltp, Krt8). As expected, from E10 to E13, few genes known to regulate mandible morphogenesis differed in expression between mouse strains. This study newly links several genes to odontogenesis and/or to the p63 signaling network. We propose that these genes act in a novel odontogenic network that is exclusive of lower jaw morphogenesis, and posit that this network evolved in oral, not pharyngeal, teeth.

Technique: imaging earliest tooth development in 3D using a silver-based tissue contrast agent.

Looking in microscopic detail at the 3D organization of initiating teeth within the embryonic jaw has long-proved technologically challenging because of the radio-translucency of these tiny un-mineralized oral tissues. Yet 3D image data showing changes in the physical relationships among developing tooth and jaw tissues are vital to understand the coordinated morphogenesis of vertebrate teeth and jaws as an animal grows and as species evolve. Here, we present a new synchrotron-based scanning solution to image odontogenesis in 3D and in histological detail using a silver-based contrast agent. We stained fixed, intact wild-type mice aged embryonic (E) day 10 to birth with 1% Protargol-S at 37°C for 12-32 hr. Specimens were scanned at 4-10 µm pixel size at 28 keV, just above the silver K-edge, using micro-computed tomography (µCT) at the Canadian Light Source synchrotron. Synchrotron µCT scans of silver-stained embryos showed even the earliest visible stages of tooth initiation, as well as many other tissue types and structures, in histological detail. Silver stain penetration was optimal for imaging structures in intact embryos E15 and younger. This silver stain method offers a powerful yet straightforward approach to visualize at high-resolution and in 3D the earliest stages of odontogenesis in situ, and demonstrates the important of studying the tooth organ in all three planes of view.

Jaw growth in the absence of teeth: the developmental morphology of edentulous mandibles using the p63 mouse mutant.

Mammalian tooth and jaw development must be coordinated well enough that these systems continue to function together properly throughout growth, thus optimizing an animal's survival and fitness, as well as a species' success. The persistent question is how teeth and jaws remain developmentally and functionally viable despite sometimes monumental evolutionary changes to tooth and jaw shape and size. Here we used the p63 mouse mutant to test the effect of tooth development - or the lack thereof - on normal mandible developmental morphology. Using 3D geometric morphometrics, we compared for the first time mandible shape among mice with normal tooth and jaw development against p63 double knock-out mice, with failed tooth development but apparently normal jaw development. Mandible shape differed statistically between toothless (p63(-/-) ) and toothed (p63(+/-) , p63(+/+) ) mice as early as embryonic day (E) 18. As expected, most of the shape difference in the p63(-/-) mandibles was due to underdeveloped alveolar bone related to arrested odontogenesis in these E18-aged mice. Mandible shape did not differ statistically between p63(+/-) and p63(+/+) adult mice, which showed normal tooth development. Our results support the idea of a gene regulatory network that is exclusive to the mandible and independent of the dentition. This study also underscores the biomechanical impact of the teeth on the developing alveolar bone. Importantly, this work shows quantitatively that the p63 mutant is an apt model with which to study mandible morphogenesis in isolation of odontogenesis to clarify developmental relationships between the teeth and jaws.