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 Δ T , p.Trp507Glyfs*142) on chicken craniofacial development. Misexpression of wt (wt) or mutant h DVL1 variants in vivo caused upper beak shortening (wt DVL1 n=8/14; DVL1 ^{1519 Δ T} 12/13). At early stages of development, the DVL1 ^{1519 Δ T} inhibited frontonasal mass narrowing, chondrogenesis, and proliferation. To test whether the phenotypes were caused due to the abnormal C-terminal peptide in DVL1 ^{1519 Δ T} , we designed two additional constructs. The DVL1 ^1519* (DVL1 ^507* ) retains first 30 amino acids of the C-terminus while DVL1 ^1431* (DVL1 ^477* ) removes the entire C-terminus. DVL1 ^1519* injected embryos had normal beaks while DVL1 ^1431* caused high mortality and the phenotypes were like the DVL1 ^{1519 Δ T} . In frontonasal micromass cultures, both DVL1 ^{1519 Δ T} and DVL1 ^1431* inhibited skeletogenesis while the DVL1 ^1519* resembled wt DVL1 and GFP cultures. In luciferase assays DVL1 ^{1519 Δ T} , DVL1 ^1519* and DVL1 ^1431* weakly activated the WNT canonical and non-canonical JNK-PCP pathways compared to wt DVL1 . Furthermore, we observed that variant DVL1 ^507*fs is stalled in the nucleus similar to hDVL1 ^477* , possibly due to the abnormal C-terminus interfering with the nuclear export sequence. wtDVL1 and DVL1 ^507* were distributed in nucleus and the cytoplasm. Our RS- DVL1 ^1519ΔT 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.