The question of how the epidermal appendages form during development and which pathways regulate the differentiation of the stratified epidermis has been central to epithelial biology. Transcription is one of the most important regulatory mechanisms controlling the stepwise program of epidermal differentiation. Our research efforts have focused in characterizing the regulation and function of Dlx3 homeobox transcription factor, a member of the murine Dlx family, with essential roles in epidermal, osteogenic and placental development. Dlx homeodomain proteins are a family of transcription factors that play crucial roles in developmental processes and differentiation of individual tissues. The Dlx3 gene is broadly expressed in the branchial arches and embryonic ectoderm, as well as in the tooth, hair follicle and interfollicular epidermis. To elucidate the functional role of the Dlx3 homeodomain protein during early development and during epidermal differentiation, we are currently in the process of determining the target genes and the Dlx3 interacting factors necessary to exert the transcription regulatory function. For the comprehensive analysis of Dlx3 gene expression throughout development, we generated a knock-in mouse carrying the reporter gene beta-galactosidase (LacZ) under the control of the endogenous Dlx3 promoter. Whole-mount or local X-gal staining shows the temporal and spatial expression of the Dlx3 gene in various tissues and organs including hair, teeth and bone. Targeted deletion of Dlx3 results in embryonic developmental arrest around day 9.5-10, associated with a gross failure of the placenta to undergo proper morphogenesis. It was not possible to assess the effects of Dlx3 loss of function on epidermal differentiation, since embryonic death occurs significantly earlier than the onset of epidermal stratification (E15.5). Recent work also showed that Dlx3 induction coincides with the onset of commitment to osteogenic lineage, as reflected by the induction of bone-related differentiation genes. Although Dlx3 had been implicated in skeletal development in the human autosomal dominant Tricho-Dento-Osseous syndrome, where the mutation is associated with defects in craniofacial and endochondral bone, a direct role for Dlx3 in bone formation had not been identified. In a collaborative effort with Dr. Lian's laboratory, a regulatory role for Dlx3 in bone-specific transcriptional control during osteoblast development was demonstrated. During embryonic development and organ formation, a series of signals between epithelial cells and underlying mesenchymal cells, leads to the formation of a variety of appendages/organs. Anomalies in epithelial/mesenchymal-derived organs are characteristics of a group of about 150 human heritable pathological disorders defined as ectodermal dysplasias. DLX3 is among the few genes for which mutations have been directly linked with ectodermal dysplasias. The importance of Dlx3 in the patterning and development of ectodermal structures derived from epithelial-mesenchymal interactions during embryogenesis (i.e. tooth, hair) is corroborated by the effects of DLX3 mutations in patients with the autosomal dominant Tricho-Dento-Osseous syndrome. Recent research on the mechanistic role of Dlx3 in epidermal development and appendage formation has led to the important finding that Dlx3 is a direct target of the p63. This work presents the crucial finding that besides autosomal mutations in either DLX3 or P63 genes being associated with human ectodermal dysplasias, misregulation of DLX3 function is directly involved in the pathogenesis of ectodermal human syndromes associated to p63 molecular lesions. Furthermore, utilizing mouse models, we have determined that the selective ablation of Dlx3 in the epidermis results in complete alopecia owing to failure of the hair shaft and inner root sheath to form, which is caused by the abnormal differentiation of the cortex. Significantly, we elucidated the regulatory cascade that positions Dlx3 downstream of Wnt signaling and as an upstream regulator of other transcription factors that regulate hair follicle differentiation.Taken together with hair follicle abnormalities in humans with Tricho-Dento-Osseous (TDO) syndrome, our results establish that Dlx3 is essential for hair morphogenesis, differentiation and cycling programs. During development, Dlx3 is also expressed in teeth and bone. 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. We have recently demonstrated 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 downregulated in odontoblasts lacking Dlx3. Using ChIP-seq, we further demonstrate the direct binding of Dlx3 to the Dspp promoter in vivo. This establishes a regulatory pathway where the transcription factor Dlx3 is essential in dentin formation by directly regulating a crucial matrix protein during tooth development.

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National Institute of Arthritis and Musculoskeletal and Skin Diseases
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Duverger, Olivier; Ohara, Takahiro; Bible, Paul W et al. (2017) DLX3-Dependent Regulation of Ion Transporters and Carbonic Anhydrases is Crucial for Enamel Mineralization. J Bone Miner Res 32:641-653
Duverger, Olivier; Beniash, Elia; Morasso, Maria I (2016) Keratins as components of the enamel organic matrix. Matrix Biol 52-54:260-265
Peled, Alon; Sarig, Ofer; Samuelov, Liat et al. (2016) Mutations in TSPEAR, Encoding a Regulator of Notch Signaling, Affect Tooth and Hair Follicle Morphogenesis. PLoS Genet 12:e1006369
Kim, Jin-Chul; Duverger, Olivier; Hwang, Joonsung et al. (2015) Epidermal stem cells in the isthmus/infundibulum influence hair shaft differentiation: evidence from targeted DLX3 deletion. J Invest Dermatol 135:299-301
Bible, Paul W; Kanno, Yuka; Wei, Lai et al. (2015) PAPST, a User Friendly and Powerful Java Platform for ChIP-Seq Peak Co-Localization Analysis and Beyond. PLoS One 10:e0127285
Duverger, Olivier; Ohara, Takahiro; Shaffer, John R et al. (2014) Hair keratin mutations in tooth enamel increase dental decay risk. J Clin Invest 124:5219-24
Duverger, Olivier; Morasso, Maria I (2014) To grow or not to grow: hair morphogenesis and human genetic hair disorders. Semin Cell Dev Biol 25-26:22-33
Isaac, J; Erthal, J; Gordon, J et al. (2014) DLX3 regulates bone mass by targeting genes supporting osteoblast differentiation and mineral homeostasis in vivo. Cell Death Differ 21:1365-76
Duverger, Olivier; Isaac, Juliane; Zah, Angela et al. (2013) In vivo impact of Dlx3 conditional inactivation in neural crest-derived craniofacial bones. J Cell Physiol 228:654-64
Okano, Junko; Levy, Clara; Lichti, Ulrike et al. (2012) Cutaneous retinoic acid levels determine hair follicle development and downgrowth. J Biol Chem 287:39304-15

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