DLX3-Dependent Regulation of Ion Transporters and Carbonic Anhydrases is Crucial for Enamel Mineralization.

Patients with tricho-dento-osseous (TDO) syndrome, an ectodermal dysplasia caused by mutations in the homeodomain transcription factor DLX3, exhibit enamel hypoplasia and hypomineralization. Here we used a conditional knockout mouse model to investigate the developmental and molecular consequences of Dlx3 deletion in the dental epithelium in vivo. Dlx3 deletion in the dental epithelium resulted in the formation of chalky hypomineralized enamel in all teeth. Interestingly, transcriptomic analysis revealed that major enamel matrix proteins and proteases known to be involved in enamel secretion and maturation were not affected significantly by Dlx3 deletion in the enamel organ. In contrast, expression of several ion transporters and carbonic anhydrases known to play an important role in enamel pH regulation during maturation was significantly affected in enamel organs lacking DLX3. Most of these affected genes showed binding of DLX3 to their proximal promoter as evidenced by chromatin immunoprecipitation sequencing (ChIP-seq) analysis on rat enamel organ. These molecular findings were consistent with altered pH staining evidenced by disruption of characteristic pH oscillations in the enamel. Taken together, these results show that DLX3 is indispensable for the regulation of ion transporters and carbonic anhydrases during the maturation stage of amelogenesis, exerting a crucial regulatory function on pH oscillations during enamel mineralization. © 2016 American Society for Bone and Mineral Research.

In vivo impact of Dlx3 conditional inactivation in neural crest-derived craniofacial bones.

Mutations in DLX3 in humans lead to defects in craniofacial and appendicular bones, yet the in vivo activities related to Dlx3 function during normal skeletal development have not been fully elucidated. Here we used a conditional knockout approach to analyze the effects of neural crest deletion of Dlx3 on craniofacial bones development. At birth, mutant mice exhibit a normal overall positioning of the skull bones, but a change in the shape of the calvaria was observed. Molecular analysis of the genes affected in the frontal bones and mandibles from these mice identified several bone markers known to affect bone development, with a strong prediction for increased bone formation and mineralization in vivo. Interestingly, while a subset of these genes were similarly affected in frontal bones and mandibles (Sost, Mepe, Bglap, Alp, Ibsp, Agt), several genes, including Lect1 and Calca, were specifically affected in frontal bones. Consistent with these molecular alterations, cells isolated from the frontal bone of mutant mice exhibited increased differentiation and mineralization capacities ex vivo, supporting cell autonomous defects in neural crest cells. However, adult mutant animals exhibited decreased bone mineral density in both mandibles and calvaria, as well as a significant increase in bone porosity. Together, these observations suggest that mature osteoblasts in the adult respond to signals that regulate adult bone mass and remodeling. This study provides new downstream targets for Dlx3 in craniofacial bone, and gives additional evidence of the complex regulation of bone formation and homeostasis in the adult skeleton.

Neural crest deletion of Dlx3 leads to major dentin defects through down-regulation of Dspp.

During development, Dlx3 is expressed in ectodermal appendages such as hair and teeth. Thus far, the evidence that Dlx3 plays a crucial role in tooth development comes from reports showing that autosomal dominant mutations in DLX3 result in severe enamel and dentin defects leading to abscesses and infections. However, the normal function of DLX3 in odontogenesis remains unknown. Here, we use a mouse model to demonstrate that the absence of Dlx3 in the neural crest results in major impairment of odontoblast differentiation and dentin production. Mutant mice develop brittle teeth with hypoplastic dentin and molars with an enlarged pulp chamber and underdeveloped roots. Using this mouse model, we found that dentin sialophosphoprotein (Dspp), a major component of the dentin matrix, is strongly down-regulated in odontoblasts lacking Dlx3. Using ChIP-seq, we further demonstrate the direct binding of Dlx3 to the Dspp promoter in vivo. Luciferase reporter assays determined that Dlx3 positively regulates Dspp expression. This establishes a regulatory pathway where the transcription factor Dlx3 is essential in dentin formation by directly regulating a crucial matrix protein.

Early regulative ability of the neuroepithelium to form cardiac neural crest.

The cardiac neural crest (arising from the level of hindbrain rhombomeres 6-8) contributes to the septation of the cardiac outflow tract and the formation of aortic arches. Removal of this population after neural tube closure results in severe septation defects in the chick, reminiscent of human birth defects. Because neural crest cells from other axial levels have regenerative capacity, we asked whether the cardiac neural crest might also regenerate at early stages in a manner that declines with time. Accordingly, we find that ablation of presumptive cardiac crest at stage 7, as the neural folds elevate, results in reformation of migrating cardiac neural crest by stage 13. Fate mapping reveals that the new population derives largely from the neuroepithelium ventral and rostral to the ablation. The stage of ablation dictates the competence of residual tissue to regulate and regenerate, as this capacity is lost by stage 9, consistent with previous reports. These findings suggest that there is a temporal window during which the presumptive cardiac neural crest has the capacity to regulate and regenerate, but this regenerative ability is lost earlier than in other neural crest populations.