Chiral blastomere arrangement dictates zygotic left–right asymmetry pathway in snails

Abstract

Most animals display internal and/or external left-right asymmetry. Several mechanisms for left-right asymmetry determination have been proposed for vertebrates and invertebrates but they are still not well characterized, particularly at the early developmental stage. The gastropods Lymnaea stagnalis and the closely related Lymnaea peregra have both the sinistral (recessive) and the dextral (dominant) snails within a species and the chirality is hereditary, determined by a single locus that functions maternally. Intriguingly, the handedness-determining gene(s) and the mechanisms are not yet identified. Here we show that in L. stagnalis, the chiral blastomere arrangement at the eight-cell stage (but not the two- or four-cell stage) determines the left-right asymmetry throughout the developmental programme, and acts upstream of the Nodal signalling pathway. Thus, we could demonstrate that mechanical micromanipulation of the third cleavage chirality (from the four- to the eight-cell stage) leads to reversal of embryonic handedness. These manipulated embryos grew to 'dextralized' sinistral and 'sinistralized' dextral snails-that is, normal healthy fertile organisms with all the usual left-right asymmetries reversed to that encoded by the mothers' genetic information. Moreover, manipulation reversed the embryonic nodal expression patterns. Using backcrossed F(7) congenic animals, we could demonstrate a strong genetic linkage between the handedness-determining gene(s) and the chiral cytoskeletal dynamics at the third cleavage that promotes the dominant-type blastomere arrangement. These results establish the crucial importance of the maternally determined blastomere arrangement at the eight-cell stage in dictating zygotic signalling pathways in the organismal chiromorphogenesis. Similar chiral blastomere configuration mechanisms may also operate upstream of the Nodal pathway in left-right patterning of deuterostomes/vertebrates.

Far from solved: A perspective on what we know about early mechanisms of left–right asymmetry

“…This proposal is also not consistent with published data on the timing of involvement of physiological events in the LR pathway (Yost, 1991;Fukumoto et al, 2005a;Fukumoto et al, 2005b;Adams et al, 2006;Danilchik et al, 2006;Vandenberg and Levin, 2010). primitive streak (Gardner, 1997(Gardner, , 2001, this work shows that the mouse embryo is not exempt from the importance of very early events for LR asymmetry. Importantly, this is the same outcome as seen in the experiments involving micromanipulation of blastomeres in snail (Kuroda et al, 2009) and C. elegans (Wood and Kershaw, 1991) embryos, providing more evidence that there are conserved mechanisms for establishing LR asymmetry across phyla. While much work remains to be done to characterize other aspects of this laterality phenotype, it is clear that events in the mouse taking place 7 days before the appearance of the ciliated node can influence aspects of asymmetry.…”

“…The links between intracellular (cytoskeleton-driven) polarity and LR asymmetry have grown stronger: recent data from snails, frogs and mice all suggest that a chiral cytoskeleton is established extremely early during development (Danilchik et al, 2006;Kuroda et al, 2009;Gardner, 2010), a remarkable example of a widely-conserved system that has relevance to invertebrate and even plant systems (Doss, 1978;Hashimoto, 2002;Abe et al, 2004;Levin, 2006;Oviedo and Levin, 2007). Interestingly, the role of a MTOC as the initiator of LR asymmetry is common to all three main models in the field, a largely unacknowledged common ground among the many interesting controversies about downstream amplification and coordination mechanisms.…”

“…The rapid redistribution of serotonin among blastomeres (Fukumoto et al, 2005b) leads to stable, specific gene expression cascades on the left side; a similar situation occurs in C. elegans, where the NSY-5 gap junction protein establishes transient connections for physiological signals that specify the permanent asymmetry in the structure and function of the AWC olfactory neurons (Chuang et al, 2007). Similarly, as very early cytoskeletal biases in Xenopus ultimately result in asymmetries of visceral organs , the transduction of chiral cytoskeletal activity into asymmetric Nodal induction has recently been demonstrated in snail embryos; alterations in spindle orientations by means of mechanical manipulations in early cleavage stage embryos alter the Nodal signaling pathway as well as handedness of shell coiling (Kuroda et al, 2009). Thus, while details of the early steps by which physiological asymmetries result in consistently different genetic programs on the left and right sides differ among phyla, a remarkably wide conservation is seen in the logic and molecular nature of the control systems by which biophysical, epigenetic signals are transformed into stable transcriptional states.…”

A twisting story: how a single gene twists a snail? Mechanogenetics

“…The maternal inheritance is a crucial advantage as the chirality is determined at the early developmental stage. Recently, we proved that chirality is determined by a single locus (Hosoiri et al 2003;Kuroda, 2014;Kuroda et al 2009;Shibazaki et al 2004), although it had been postulated based on the early 20th century work (Boycott et al 1930;Sturtevant, 1923). Secondly, snails are hermaphrodite, reproducing offspring by self-and cross-fertilization, which is advantageous in tracing genetic traits.…”

“…As the species had not been well studied compared with Lymnaea peregra (Freeman & Lundelius, 1982) except for the detailed observation of development (Meshcheryakov, 1990), we studied the breeding behaviour (Hosoiri et al 2003), correlation of cleavage pattern and organismal morphology (Shibazaki et al 2004), cytoplasm injections to the 1-cell embryos of opposite chirality (Kuroda, 2014), cytoskeletal dynamics during spiral cleavages (Shibazaki et al 2004), expression of nodal-Pitx genes (Kuroda et al 2009), creation of mirrorimage healthy animals by twisting blastomeres at the third cleavage (Kuroda et al 2009), etc. We also constructed backcrossed congenic animals (Hosoiri et al 2003;Kuroda, 2014;Kuroda et al 2009;Shibazaki et al 2004) and carried out positional cloning in order to identify the handedness determining gene(s).…”