The objective of this study was to utilize three-dimensional (3D) semi-landmarks & geometric morphometrics (GM) to quantify shape variation in the maxillo-mandibular complex of mice on prolonged diets of equivalent nutrition but different consistency. Forty three wild type mice (22 males; 21 females) were divided into two groups. For 15 generations, the control group (6 males; 6 females) was fed a standard hard pellet diet, whereas the experimental group (6 males; 6 females) was fed a soft powder diet of the same nutritional value. After the 15th generation, 19 more mice were added to the sample group but were placed in opposing sample categories (soft vs. hard). Mouse heads were collected at 6-weeks of age and fixed in 4% paraformaldehyde for 48h. Specimens were scanned using Skyscan 1076 MicroCT scanner at 18-micron resolution (55 kV, 150 mA, 0.5 mm Al filter) & reconstructed as series of 8-bit grayscale images. Segmentation & surface generation from microCT data was performed. Seventy landmarks were placed to measure 3D changes and subsequently analyzed by GM to determine shape variation.

Subtle morphological changes were noted in the skull, mandible, and temporomandibular joint (TMJ) using principle components analysis (PCA) and canonical variates analysis (CVA). For example, soft diet mice possessed shorter mandibular body lengths and wider condylar necks. In addition, sexual dimorphic differences were observed in the TMJ.

Thus, we demonstrated the impact of changing diet consistency over multiple generations on the morphology of the mouse maxillo-mandibular complex using 3D GM analysis.

Alagille Syndrome is a multisystem genetic disorder characterized by chronic cholestasis,cardiovascular anomalies, ocular abnormalities, skeletal defects, and characteristic facial features. Mutations in Jagged1 (Jag1), a key ligand in the Notch signaling pathway, are associated with the majority of cases. The Notch signaling pathway plays important roles in the development and homeostasis of most, if not all tissues. However, the roles of Jag1 and the Notch signaling pathway in craniofacial and dental development remain unclear. This study aimed to elucidate the roles of Notch signaling pathway in craniofacial and dental development using a mouse model of Alagille Syndrome (Jag1Ndr/Ndr mice, which possess a missense mutation (H268Q) in Jag1). Embryonic and postnatal mouse specimens were collected at various stages from Jag1Ndr/Ndr mice. Micro-computed tomography (microCT) and three-dimensional (3D) Geometric Morphometric Analysis (GMA) were performed on adult skulls to analyze changes in craniofacial morphology. Hematoxylin and eosin (H&E) staining on histological sections was performed on control and Jag1Ndr/Ndr mice at embryonic day (E)14.5, E16.5, postnatal (P)0, and P7 to analyze tooth development. 3D GMA showed variations in the coronoid process, zygomatic process, and the area where parietal, occipital, and squamosal bones intersect in Jag1Ndr/Ndr mutant mice. In addition, mutants had more convex crania from both frontal and lateral views and a longer snout. Our histological analysis of tooth development showed defects in cell-matrix adhesion (ameloblast-enamel matrix) and cell-cell adhesion (ameloblast-stratum intermedium) at P7. MicroCT analysis of P7 and adult molars supported our histological findings and revealed changes in tooth morphology. Disruption in Notch signaling due to a missense mutation (H268Q) in Jag1 led to: 1) specific changes in craniofacial morphology; 2) defective cell-matrix and cell-cell attachments during tooth development; 3) possible changes in tooth structures due to abnormal tooth development and mineralization. Our study demonstrates for the first time that Notch signaling is essential for specific changes in craniofacial morphology and proper dental development.

Neural crest mesenchyme (NCM) controls species-specific pattern in the craniofacial skeleton but how this cell population accomplishes such a complex task remains unclear. To elucidate mechanisms through which NCM directs skeletal development and evolution, we made chimeras from quail and duck embryos, which differ markedly in their craniofacial morphology and maturation rates. We show that quail NCM, when transplanted into duck, maintains its faster timetable for development and autonomously executes molecular and cellular programs for the induction, differentiation, and mineralization of bone, including premature expression of osteogenic genes such as Runx2 and Col1a1. In contrast, the duck host systemic environment appears to be relatively permissive and supports osteogenesis independently by providing circulating minerals and a vascular network. Further experiments reveal that NCM establishes the timing of osteogenesis by regulating cell cycle progression in a stage- and species-specific manner. Altering the time-course of D-type cyclin expression mimics chimeras by accelerating expression of Runx2 and Col1a1. We also discover higher endogenous expression of Runx2 in quail coincident with their smaller craniofacial skeletons, and by prematurely over-expressing Runx2 in chick embryos we reduce the overall size of the craniofacial skeleton. Thus, our work indicates that NCM establishes species-specific size in the craniofacial skeleton by controlling cell cycle, Runx2 expression, and the timing of key events during osteogenesis.