Ectodermal dysplasias (ED) are a heterogeneous group of heritable disorders of the skin and its appendages characterized by the defective development of two or more ectodermal derivatives, including hair, teeth, nails, sweat glands, etc... . The spectrum of clinical manifestations is wide and may include additional manifestations from other ectodermal, mesodermal and endodermal structures. Overall prevalence of ectodermal dysplasias are unknown, but are considered rare diseases. It has been shown, that four genes, EDA, EDAR, EDARADD and WNT10A, account for 90% of hypohidrotic/anhidrotic ED cases. These genes are involved in the NFkB signaling pathway, a very important pathway involved in the correct development of ectodermal structures, as well as in the crosstalk between ectoderm and mesoderm during embryo development. Effective treatment for EDs is lacking, therefore creating animal models for in vivo drug screening is necessary. In close collaboration with the IMIB Telomerase Cancer and Aging group we are generating and characterizing wnt10a and eda zebrafish Knock Out (KO) models using CRIPR-Cas9 technology.

CRISPR-Cas9 technology, zebrafish egg microinjection and gene expression by qPCR.

Using CRISPR-Cas9 technology and microinjection in zebrafish eggs we have obtained wnt10a zebrafish mutant individuals. This mutant strain presents a 1 base pair deletion in the +60 position of the wnt10a coding sequence, generating a codon frameshift and a premature STOP codon in the amino acid 45, thus a nonfunctional WNT10a protein as a result. The expression of WNT10a signaling pathway target genes was analyzed in whole larvae samples at different time point across zebrafish larval development. The majority of the WNT10a target genes analyzed, such as sonic hedgehog a and b (shha and shhb) or bone morphogenetic protein 2a and 4 (bmp2a and bmp4) showed a decreased expression in WNT10a KO larvae compared to wild type across larval development. The expression of other target genes, such as msx1a or eda itself are not largely affected in WNT10a whole larvae samples. In parallel, and using the same CRISPR-Cas9 based strategy, we are generating eda mutant animals. So far, we have obtained several F1 mutant heterozygous fish lines with different editions of the EDA genomic locus and were are now obtaining the homozygous strains.

We have generated wnt10a zebrafish mutant line and are characterizing these animals. We are also obtaining eda KO strain, These new animal models of loss of function of genes that are affected in ED patients will be used for designing and performing medium-to-high-throughput compound screenings in order to find new drugs that revert the observed phenotype.