Diaphanous gene mutation affects spiral cleavage and chirality in snails.

Shuichi Asakawa,Shin Umeda,Miho Shimizu,Reiko Kuroda,Yuji Hosoiri,Futaba Ichikawa,Hiromi Takahashi,Kohei Fujikura,Masanori Abe

doi:10.1038/srep34809

Abstract

L-R (left and right) symmetry breaking during embryogenesis and the establishment of asymmetric body plan are key issues in developmental biology, but the onset including the handedness-determining gene locus still remains unknown. Using pure dextral (DD) and sinistral (dd) strains of the pond snail Lymnaea stagnalis as well as its F2 through to F10 backcrossed lines, the single handedness-determining-gene locus was mapped by genetic linkage analysis, BAC cloning and chromosome walking. We have identified the actin-related diaphanous gene Lsdia1 as the strongest candidate. Although the cDNA and derived amino acid sequences of the tandemly duplicated Lsdia1 and Lsdia2 genes are very similar, we could discriminate the two genes/proteins in our molecular biology experiments. The Lsdia1 gene of the sinistral strain carries a frameshift mutation that abrogates full-length LsDia1 protein expression. In the dextral strain, it is already translated prior to oviposition. Expression of Lsdia1 (only in the dextral strain) and Lsdia2 (in both chirality) decreases after the 1-cell stage, with no asymmetric localization throughout. The evolutionary relationships among body handedness, SD/SI (spiral deformation/spindle inclination) at the third cleavage, and expression of diaphanous proteins are discussed in comparison with three other pond snails (L. peregra, Physa acuta and Indoplanorbis exustus).

Highlights

The symmetry breaking event during early embryogenesis is a fundamental step for the correct positioning and morphogenesis of internal organs
We have shown that the manipulation transfers the zygotic expression of nodal and pitx to the mirror image position
In the equal cleaving gastropods such as Lymnaea stagnalis (Ls). stagnalis, all four cells are of equal size at the 4-cell stage

Summary

Introduction

The symmetry breaking event during early embryogenesis is a fundamental step for the correct positioning and morphogenesis of internal organs. Spiral deformation (SD) and spindle inclination (SI) were uniquely observed only in the dominant dextral embryos during the third cleavage, and they were shown to be strongly linked to the gene that determines the snail chirality[21,22] Based on these results and using pure dextral (DD) and sinistral (dd) strains as well as novel dextral and sinistral lines of F10 congenic snails (with 99.9% sinistral- and 0.1% dextral-derived genomes22), we here identify the diaphanous-related formin, Lsdia[1], as the handedness-determining gene and elucidate the mechanisms that establish the L-R body plan in Lymnaea. We have cloned and sequenced dia homologous genes of the pond snails, L. peregra, Physa acuta and Indoplanorbis exustus, and compared the type of diaphanous proteins, SD/SI at the third cleavage and body handedness

Methods

Results

Conclusion