Dkk1 inhibition normalizes limb phenotypes in a mouse model of Fzd2 associated omodysplasia Robinow syndromes

ABSTRACT

FRIZZLED-2 (FZD2) is a transmembrane Wnt ligand receptor. We previously identified a pathogenic human FZD2 variant, encoding for a protein with a premature stop and loss of 17 amino acids. This includes a portion of the consensus DISHEVELLED binding sequence required for Wnt signal transduction. Patients with this variant exhibited FZD2-associated autosomal dominant Robinow Syndrome. To model this variant, we utilized zygote microinjection and i-GONAD-based CRISPR/Cas9-mediated genome editing to generate an allelic series in the mouse. Embryos mosaic for humanized Fzd2^W553* knock-in exhibited cleft palate and shortened limbs, consistent with FZD2^W548* patient phenotypes. We also generated two germline mouse alleles with small deletions, Fzd2^D3 and Fzd2^D4. Homozygotes for each allele survive embryonic development at normal ratios but exhibit a highly penetrant cleft palate phenotype, shortened limbs compared to wild-type and perinatal lethality. Fzd2^D4 craniofacial tissues indicated decreased canonical WNT signaling. In utero treatment with IIIC3a (DKK inhibitor) normalized the limb lengths in Fzd2^D4 homozygotes. The in vivo replication represents an approach to further investigate the mechanism of FZD2 phenotypes and validates the utility of CRISPR knock-in mice as a tool for demonstrating pathogenicity of human genetic variants. We also present evidence for a potential therapeutic intervention.