Craniofacial development of vertebrates depends largely on neural crest contribution and each subdomain of the crest-derived ectomesenchyme follows its specific genetic control. The rat small eye (rSey) involves a mutation in the Pax-6 gene and the external feature of rSey homozygous embryos exhibits craniofacial defects in ocular and frontonasal regions. In order to identify the mechanism of craniofacial development, we examined the cranial morphology and migration of cephalic crest cells in rSey embryos. The chondrocranial defects of homozygous rSey embryos primarily consisted of spheno-orbital and ethmoidal anomalies. The former defects appeared to be brought about by the lack of the eye. In the ethmoid region, the nasal septum and the derivative of the medial nasal prominence were present, while the rest of the nasal capsule, as well as the nasal and lachrymal bones, were totally absent except for a pair of cartilaginous rods in place of the nasal capsule. This suggests that the primary cranial defect is restricted to the lateral nasal prominence derivatives. Dil labeling revealed the abnormal migration of crest cells specifically from the anterior midbrain to the lateral nasal prominence in homozygous rSey embryos. Pax-6 was not expressed in the crest cells but was strongly expressed in the frontonasal ectoderm. To determine whether or not this migratory defect actually resides in environmental cues, normal midbrain crest cells from wild-type embryos were labeled with Dil and were orthotopically injected into host rSey embryos. Migration of the donor crest cells into the lateral nasal prominence was abnormal in homozygous host embryos, while they migrated normally in wild-type or heterozygous embryos. Therefore, the cranial defects in rSey homozygous embryos are due to inappropriate substrate for crest cell migration towards the lateral nasal prominence, which consistently explains the cranial morphology of homozygous rSey embryos.

In this study, we investigated the process of supernumerary upper incisor formation in the Pax6 mutant rat, rSey 2 /rSey 2 , which exhibits a facial cleft between the medial nasal and maxillary processes. Histological investigation and epithelial labeling studies of wild type rat embryos indicated that the upper incisor develops by fusion of two primary dental placodes (PDPs) in the medial nasal process with a contribution from the epithelium of the maxillary process. In the rSey 2 /rSey 2 embryo, both PDPs are formed but they stay apart, then subsequently these PDPs independently develop into upper incisor tooth buds. In order to examine if the failure of the two placodes to fuse is due to the cleft between the maxillary and medial nasal processes, maxillary and medial nasal process fusion was inhibited with a barrier in wild type embryos. This resulted in the maintenance of the two distinct PDPs. These results demonstrate that fusion of the facial processes reduces the number of odontogenic placodes and is required to assemble all components at one site for rat upper incisor formation. The results also provide further insight into the mechanism of supernumerary incisor formation in human cleft lip conditions. Developmental Dynamics 235:2134 -2143, 2006.

Developmental process of rat maxillary incisor has been studied through histological analysis and investigation of tooth-related gene expression patterns at initial tooth development. The tooth-related genes studied here are fibroblast growth factor-8 (Fgf-8), pituitary homeobox gene-2 (Pitx-2), sonic hedgehog (Shh), muscle segment homeobox-1 (Msx-1), paired box-9 (Pax-9) and bone morphogenetic protein-4 (Bmp-4). The genes are expressed in oral epithelium and/or ectomesenchyme at the stage of epithelial thickening to the early bud stage of tooth development. Both the histological observation and tooth-related gene expression patterns during early stage of maxillary incisor development demonstrate that dual odontogenic origins aligned medio-laterally in the medial nasal process develop, subsequently only single functional maxillary incisor dental placode forms. The cascade of tooth-related gene expression patterns in rat maxillary incisor studied here is quite similar to those of the previous studies in mouse mandibular molar, even though the origins of oral epithelium and ectomesenchyme involved in development of maxillary incisor and mandibular molar are different. Thus, we conclude that maxillary incisor and mandibular molar share a similar signaling control of Fgf-8, Pitx-2, Shh, Msx-1, Pax-9 and Bmp-4 genes at the stage of oral epithelial thickening to the early bud stage of tooth development.

Neural crest (NC) is a transient structure that gives rise to various types of tissues. Many NC cells are pluripotent in the sense that their progeny can generate more than one derivative. However, the potentiality to differentiate into certain derivatives, such as cartilage and bone, seems to be specified with respect to the neuraxial levels at which the NC generates. In order to compare the differentiation potentiality of different regions of head NC, the derivatives of forebrain and midbrain mouse NC have been investigated in vitro using explant cultures of neuroepithelial fragments. From morphology and expression of specific markers, the midbrain crest cultures obviously generated earlier and were greater in number of neuronal cells than were the forebrain ones. Moreover, collagen type II positive cells were detected in the midbrain but not in the forebrain crest cultures. Finally, pigment cells were only observed in the forebrain cultures. The results suggest that the forebrain and midbrain crest cells have a different potentiality to differentiate.

Objective:Excess treatment of 13-cis-RA (Accutane®) on pregnant women induces craniofacial malformation found in infants. However, the effect of folic acid on 13-cis-RA-induced cellular damages of developing midfacial processes is still unknown. The purpose of this study was to investigate the pretreatment effect of folic acid (FA) on 13-cis-RA-induced cellular damage in developing midfacial processes in rat embryos.Materials and Methods:The rat embryos at developing midfacial processes were performed by whole embryo culture in vitro, in the presence of 13-cis-RA (20 µM) with or without pre-treatment of FA (100 µM). The midfacial morphogenesis score, PCNA and TUNEL assay staining were evaluated for morphogenesis, cell proliferation and apoptosis of the midfacial processes, respectively.Results:The 13-cis-RA-treated embryos at 24h showed atrophy of midfacial processes with significantly decreased morphogenesis score and cell proliferation, and increased apoptotic cell death. In contrast, the embryos pre-treated with FA for 18h, followed by 13-cis-RA treatment for 24h (FA-RA) showed significantly greater morphogenesis score, increased cell proliferation and lower apoptotic cell death compared to those of the 13-cis-RA-treated embryos.Conclusion:The results suggest that FA reduced the teratogenic effects of 13-cis-RA on midfacial process tissue. Future investigations regarding the anti-teratogenic mechanism of FA on the prevention of damages in midface processes induced by 13-cis-RA on pregnant woman are warranted.