Establishment of dlx3b Mutant Zebrafish Lines Resembling TDO Syndrome

Objectives: Mutations of distal-less 3 (DLX3) gene are responsible for human tricho-dento-osseous syndrome (TDO), a human autosomal-dominant disease with defects in tooth, hair, and bone. Tooth defects in TDO patients are characterized by hypoplastic and hypomaturation amelogenesis imperfecta and taurodontism. However, the exact mechanisms of mutant DLX3 on tooth development remains largely unknown. Our previous in vitro studies found that mutant DLX3 disrupts the activation function of wild-type DLX3 on enamel proteins Amelx, Enam, Klk4, and Odam. In this study, we aimed to establish dlx3b mutant zebrafish models for in vivo investigation of the molecular mechanisms underlying TDO disease.
Methods: 1) Whole mount in situ hybridization was performed to detect expression of dlx3b during different stages of wild-type zebrafish tooth development. Antisense probes were amplified with T7 primers using constructed dlx3b plasmid. 2) To generate a mutant zebrafish line resembling Dlx3 mutation in human TDO syndrome, the target site of transcription activator-like effector nucleases (TALENs) was designed just at the 3’-terminal of dlx3b homeodomain. Embryos were injected with mRNA encoding the specific designed TALENs, raised, screened, and mated.
Results: 1) Expression of dlx3b was identified at early morphogenesis, late morphogenesis, early cytodifferentiation, and late cytodifferentiation stages of the first generation teeth in zebrafish embryos. 2) Sequencing results confirmed that multiple deletions were induced at the target site, and 3 stable mutant dlx3b zebrafish lines were obtained by germline transmission. In these three fish lines, the protein sequence of Dlx3b was mutated into F180QfsX33, K182FfsX7, and F180LfsX7, respectively.
Conclusions: Stable mutant dlx3b zebrafish lines resembling the DLX3 mutations responsible for human TDO syndrome were first established using TALENs, facilitating for further in vivo investigations of the mechanic mechanisms underlying tooth defects of TDO patients.