Fig. 4

Domain requirement of Dvl2 for its involvement in convergent extension and membrane distribution during neurulation. (A) Schematic diagram of the genomic structure of the endogenous wild-type Dvl2 allele, the targeted Dvl2 null allele (Dvl2 KO), the two wild-type Dvl2 BAC transgenes, Dvl2-EGFP1 and Dvl2-EGFP2, and three mutant Dvl2 BAC transgenes: δDIX-EGFP, δDEP-EGFP and dsh1-EGFP. Although Dvl2-EGFP1 had an EGFP cassette fused in-frame to the last codon of Dvl2 and a LoxP site inserted into intron 2 for genotyping purpose (see Materials and methods), Dvl2-EGFP2 contained an additional LoxP site inserted in the 3' flanking gene Acadvl (acylcoenzyme A dehydrogenase, very long chain). δDIX-EGFP and δDEP-EGFP deletion mutant transgenes were generated by removing sequence encoding amino acids 67-159 and 442-736, respectively. dsh1-EGFP point mutation was generated by replacing AAG with ATG to introduce a K→M substitution at amino acid 446. Lower panel showed sequence alignment of the wild-type Dvl2-EGFP and the modified dsh1-EGFP BAC transgenes, as well as endogenous mouse Dvl1, Dvl2, Dvl3, Xenopus XDsh, Drosophila Dsh and the dsh1 mutation identified in fly. Although Dvl2-EGFP1, Dvl2-EGFP2 (B) and δDIX-EGFP (F) can fully rescue the neural tube closure defects in Dvl1^-/-; Dvl2^-/- mutants, δDEP-EGFP transgenes completely failed to rescue the neural tube defects in Dvl1^-/-; Dvl2^-/- mutants (C). Confocal analysis indicated that although δDIX-EGFP was primarily localized to the membrane (G,H), δDEP-EGFP was evenly distributed in the cytoplasm (D,E).