OBJECTIVE: This study aims to study the expression patterns of ectodysplasin (EDA) and ectodysplasin receptor (EDAR) during the early development of zebrafish and provide a foundation for further research of the Eda signaling pathway in tooth development. METHODS: Total RNA was extracted from zebrafish embryos at 48 hours postfertilization (hpf) and then reverse transcribed for cDNA library generation. The corresponding RNA polymerase was selected for the synthesis of the digoxin-labeled antisense mRNA probe of zebrafish pharyngeal tooth specific marker dlx2b and Eda signaling-associated genes eda and edar in vitro. The three sequences were ligated into a pGEMT vector with a TA cloning kit, and polymerase chain reaction (PCR) was applied to linearize the plasmid. The resultant PCR sequences were used as templates for synthesizing Dig-labeled mRNA probe dlx2b, eda, and edar. Zebrafish embryos were collected at 36, 48, 56, 60, 72, and 84 hpf, then whole mount in situ hybridization was performed for the detection of eda and edar expression patterns. Then, their expression patterns at 72 hpf were compared with the expression pattern of dlx2b. RESULTS: The mRNA antisense probes of dlx2b, eda, and edar were successfully obtained. The positive signals of eda and edar were observed in zebrafish pharyngeal tooth region at 48-72 hpf and thus conform to the signals of dlx2b in the positive regions. CONCLUSIONS The ligand eda and edar, which are associated with the Eda signaling pathway, are strongly expressed only at the pharyngeal tooth region in zebrafish from tooth initiation to the morphogenesis stage. Thus, the Eda signaling pathway may be involved in the regulation of the early development of zebrafish pharyngeal teeth.
Animals ; Ectodysplasins ; Edar Receptor ; Odontogenesis ; Receptors, Ectodysplasin ; Zebrafish

Objective: To detect gene mutation in patients with hypohidrotic ectodermal dysplasia (HED) by using whole exome sequencing, to analyze the pathogenicity of the mutations, and to provide reference for the genetic diagnosis of HED patients. Methods: Peripheral blood genomic DNA was extracted from each of the HED patients and their family members collected in Peking University School and Hospital of Stomatology from August 2016 to August 2021. Whole exome sequencing and sanger sequencing were performed to detect gene mutations. Functions of the rare variants after the database filtering were analyzed by bioinformatics tools. Results: Three reported mutations of ectodysplasin A (EDA) gene (c.2T>C, c.161A>G, c.467G>A) and a mutation of ectodysplasin A receptor (EDAR) gene (c.871G>A) were detected by whole genome sequencing in four HED patients, and were verified by Sanger sequencing in four HED families. The EDAR gene mutation founded in this research was reported in HED patients for the first time. Bioinformatics tools predicted that the mutations of EDA gene detected in this study were highly species conserved and disease-causing. The combined annotation dependent depletion (CADD) scores of EDA gene mutations c.2T>C, c.161A>G and c.467G>A were 22.5, 26.3 and 25.5 respectively, and the genomic evolutionary rate profiling (GERP) scores were 2.16, 2.26 and 2.18 respectively. The EDAR gene mutation c.871G>A detected in this study was species conserved and possibly disease-causing. The CADD and GERP scores of EDAR gene mutation c.871G>A were 22.0 and 1.93 respectively. Conclusions: Three reported mutations of EDA gene and a previously unreported mutation of EDAR gene were detected in four HED families. Different mutations of EDA gene and EDAR gene could make different influence on the protein function and lead to the occurrence of HED.
Ectodermal Dysplasia/genetics* ; Ectodermal Dysplasia 1, Anhidrotic/genetics* ; Edar Receptor/genetics* ; Humans ; Mutation ; Pedigree ; Whole Exome Sequencing