DVL1variants and C-terminal deletions have differential effects on craniofacial development and WNT signaling

Abstract

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.1519ΔT, p.Trp507Glyfs*142) on chicken craniofacial development. Misexpression of wt (wt) or mutant hDVL1variants in vivo caused upper beak shortening (wtDVL1n=8/14;DVL11519ΔT12/13). At early stages of development, theDVL11519ΔTinhibited frontonasal mass narrowing, chondrogenesis, and proliferation. To test whether the phenotypes were caused due to the abnormal C-terminal peptide inDVL11519ΔT, we designed two additional constructs. TheDVL11519*(DVL1507*) retains first 30 amino acids of the C-terminus whileDVL11431*(DVL1477*) removes the entire C-terminus.DVL11519*injected embryos had normal beaks whileDVL11431*caused high mortality and the phenotypes were like theDVL11519ΔT. In frontonasal micromass cultures, bothDVL11519ΔTandDVL11431*inhibited skeletogenesis while theDVL11519*resembled wtDVL1andGFPcultures. In luciferase assaysDVL11519ΔT,DVL11519*andDVL11431*weakly activated the WNT canonical and non-canonical JNK-PCP pathways compared to wtDVL1. Furthermore, we observed that variant DVL1507*fsis 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-DVL11519ΔTavian 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)DVL1variant 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 theDVL1C-terminus are sufficient for normal development.