Isolation and characterization of Xenopus laevis homologs of the mouse inv gene and functional analysis of the conserved calmodulin binding sites

The homozygous inv （inversion of embryonic turning） mouse mutant shows situs inversus and polycystic kidney disease, both of which result from the lack of the inv gene. Previously, we suggested that inv may be important for the left-right axis formation, not only in mice but also in Xenopus, and that calmodulin regulates this inv protein function. Here, we isolated and characterized two Xenopus laevis homologs （Xinv-1 and Xinv-2） of the mouse inv gene, and performed functional analysis of the conserved IQ motifs that interact with calmodulin. Xinv-1 expresses early in development in the same manner as mouse inv does. Unexpectedly, a full-length Xenopus inv mRNA did not randomize cardiac orientation when injected into Xenopus embryos, which is different from mouse inv mRNA. Contrary to mouse inv mRNA, Xenopus inv mRNA with mutated IQ randomized cardiac orientation. The present study indicates that calmodulin binding sites （IQ motifs） are crucial in controlling the biological activity of both mouse and Xenopus inv proteins. Although mouse and Xenopus inv genes have a quite similar structure, the interaction with calmodulin and IQ motifs ofXenopus inv and mouse inv proteins may regulate their function in different ways.

Molecular analyses of Xenopus laevis Mesp-related genes

During vertebrate somitogenesis,somites bud off from the anterior end of the presomitic mesoderm(PSM).Meso-dermal posterior(Mesp)-related genes play essential roles in somitogenesis,particularly in the definition of the somite boundary position.Among vertebrates,two types of Mesp-related genes have been identified:Mesp1 and Mesp2 in the mouse;Meso-1 and Meso-2 in the chicken;Xl-mespa and Xl-mespb(also known as Thylacine1)in the African clawed frog(Xenopus laevis);and mesp-a and mesp-b in the zebrafish.However,the functional differences between two Mesp-related genes remain unknown.In the present study,we carried out comparative analyses of the Xl-mespa and Xl-mespb genes.The amino acid sequences of the Xl-mespa and Xl-mespb proteins showed a high level of similarity.The expression of Xl-mespa started broadly in the ventrolateral mesoderm and gradually shifted to a striped pattern of expression.In contrast,Xl-mespb showed a striped pattern of expression from the start.These expression profiles completely overlapped at the PSM during somitogenesis.To investigate the functional differ-ences between Xl-mespa and Xl-mespb in terms of target gene regulation,we carried out a luciferase assay using the murine Lunatic fringe(L-fng)promoter.Transcription of the L-fng promoter was activated more strongly by Xl-mespb than by Xl-mespa.This same pattern was observed for the murine Mesp-related proteins.These results suggest that the functional differences between the two types of Mesp-related genes are evolutionally conserved in vertebrates.