Conserved roles for cytoskeletal components in determining laterality

Abstract

SUMMARY Consistently-biased left-right (LR) patterning is required for the proper placement of organs including the heart and viscera. The LR axis is especially fascinating as an example of multi-scale pattern formation, since here chiral events at the subcellular level are integrated and amplified into asymmetric transcriptional cascades and ultimately into the anatomical patterning of the entire body. In contrast to the other two body axes, there is considerable controversy about the earliest mechanisms of embryonic laterality. Many molecular components of asymmetry have not been widely tested among phyla with diverse bodyplans, and it is unknown whether parallel (redundant) pathways may exist that could reverse abnormal asymmetry states at specific checkpoints in development. To address conservation of the early steps of LR patterning, we used the Xenopus laevis (frog) embryo to functionally test a number of protein targets known to direct asymmetry in plants, fruit fly, and rodent. Using the same reagents that randomize asymmetry in Arabidopsis, Drosophila, and mouse embryos, we show that manipulation of the microtubule and actin cytoskeleton immediately post-fertilization, but not later, results in laterality defects in Xenopus embryos. Moreover, we observed organ-specific randomization effects and a striking dissociation of organ situs from effects on the expression of left side control genes, which parallel data from Drosophila and mouse. Remarkably, some early manipulations that disrupt laterality of transcriptional asymmetry determinants can be subsequently “rescued” by the embryo, resulting in normal organ situs. These data reveal the existence of novel corrective mechanisms, demonstrate that asymmetric expression of Nodal is not a definitive marker of laterality, and suggest the existence of amplification pathways that connect early cytoskeletal processes to control of organ situs bypassing Nodal. Counter to alternative models of symmetry breaking during neurulation (via ciliary structures absent in many phyla), our data suggest a widely-conserved role for the cytoskeleton in regulating left-right axis formation immediately after fertilization of the egg. The novel mechanisms that rescue organ situs, even after incorrect expression of genes previously considered to be left-side master regulators, suggest LR patterning as a new context in which to explore multi-scale redundancy and integration of patterning from the subcellular structure to the entire bodyplan.

From cytoskeletal dynamics to organ asymmetry: a nonlinear, regulative pathway underlies left–right patterning

“…For example, in studying proteins with roles in early embryonic asymmetries [188], and in particular cytoskeletal proteins with a conserved role in determining embryonic laterality [86,87], the number of embryos with reversed organs is actually smaller than the number with incorrect Nodal expression, particularly for intracellular motor protein family myosins (table 1). This suggests that the pathway from Nodal to organ situs is not as linear as had been assumed: having incorrect Nodal expression does not actually mean that an embryo's organs will be reversed, despite Nodal's established role as a determinant or 'master regulator' of LR situs.…”

“…This model has been linked to data on birth defects and epithelial morphogenesis in humans and mice [104]. As in Xenopus, the mouse model has also revealed that a-tubulin organization is critical for asymmetry, via studies of the protein Mahogunin [84,86,87,105,106].…”

“…Recent functional data revealed conserved mechanisms in a range of organisms from plants to mammals, which establish asymmetry without a ciliated structure, or long before it forms; indeed, many phyla including some vertebrates determine their LR axis very early after fertilization [80][81][82][83][84][85][86][87][88][89][90]. Even mouse embryos are known to exhibit molecular and functional asymmetries (e.g.…”

“…Secondly, the conservation of phenotypes obtained from mutations in cytoskeletal and cytoskeleton-associated proteins exhibited in frog, compared to those of plants [84,86], snails [87] and Drosophila [86], demonstrate the need for physical signals upstream of gene-regulatory networks (GRNs): for example, the organ-specific effects of myosins on Drosophila laterality were replicated in frog [86], transcending the differences in molecular LR pathways between vertebrates and invertebrates and raising the question of how the cytoskeleton generates chirality. Finally, the apparent disconnect between the asymmetric expression of Nodal and subsequent organ situs observed in mouse [105] and replicated in frog [86] highlight a conserved role for the cytoskeleton not only in laterality, but in the ability to correct earlier defects in laterality between the point of expression of markers of laterality and the positioning of visceral organs.…”

“…Our own work in Xenopus has demonstrated a number of key features that point to the importance of the cytoskeleton at multiple points in laterality. Firstly, fundamental cytoskeletal proteins such as tubulins and myosins are functionally important for normal embryonic laterality at the very earliest points of embryonic development, immediately post-fertilization [84,86]. The cytoskeleton appears to be required for the normal early localization of asymmetric components such as ion channels [84,95,97], a mechanism widely conserved to many somatic cell types [139,140].…”