DVL1 variants and C-terminal deletions have differential effects on craniofacial development and WNT signaling

Robinow Syndrome (RS) is a rare disease characterized by craniofacial malformations and limb shortening linked with mutations in seven WNT pathway genes. Our objective was to investigate the functional effects of frameshift mutations the intracellular adaptor protein, Dishevelled (DVL1; c.1519{Delta}T, p.Trp507Glyfs*142) on chicken craniofacial development. Misexpression of wt (wt) or mutant hDVL1 variants in vivo caused upper beak shortening (wtDVL1 n=8/14; DVL11519{Delta}T 12/13). At early stages of development, the DVL11519{Delta}T inhibited frontonasal mass narrowing, chondrogenesis, and proliferation. To test whether the phenotypes were caused due to the abnormal C-terminal peptide in DVL11519{Delta}T, we designed two additional constructs. The DVL11519* (DVL1507*) retains first 30 amino acids of the C-terminus while DVL11431* (DVL1477*) removes the entire C-terminus. DVL11519* injected embryos had normal beaks while DVL11431* caused high mortality and the phenotypes were like the DVL11519{Delta}T. In frontonasal micromass cultures, both DVL11519{Delta}T and DVL11431* inhibited skeletogenesis while the DVL11519* resembled wtDVL1 and GFP cultures. In luciferase assays DVL11519{Delta}T, DVL11519*and DVL11431* weakly activated the WNT canonical and non-canonical JNK-PCP pathways compared to wtDVL1. Furthermore, we observed that variant DVL1507*fs is stalled in the nucleus similar to hDVL1477*, possibly due to the abnormal C-terminus interfering with the nuclear export sequence. wtDVL1 and DVL1507* were distributed in nucleus and the cytoplasm. Our RS-DVL11519{Delta}T avian model recapitulates the broad face and jaw hypoplasia and demonstrates defects in both branches of WNT signaling. This is the first study to clarify the role of abnormal C-terminus in ADRS and to recognize the importance of an uncharacterized C-terminal sequence. Summary StatementFunctional and biochemical studies on chicken embryos with the Robinow syndrome (RS) DVL1 variant demonstrate defects in skeletogenesis and both branches of WNT signaling. This is the first study to establish a link between the RS facial defects and the mutated C-terminal sequence. We identified first 30 amino acids of the DVL1 C-terminus are sufficient for normal development.