Evolution of Developmental Control Mechanisms The formation and positioning of cilia in Ciona intestinalis embryos in relation to the generation and evolution of chordate left-right asymmetry

article i nfo Article history: In the early mouse embryo monocilia on the ventral node rotate to generate a leftward flow of fluid. This nodal flowisessentialfortheleft-sidedexpressionofnodalandpitx2,andforsubsequentasymmetricorganpatterning. Equivalentleft fluid flowhasbeenidentifiedinothervertebrates,includingXenopusandzebrafish,indicatingitis an ancient vertebrate mechanism. Asymmetric nodal and Pitx expression have also been identified in several invertebrates, including the vertebrates' nearest relatives, the urochordates. However whether cilia regulate this asymmetric gene expression remains unknown, and previous studies in urochordates have not identified any cilia prior to the larval stage, when asymmetry is already long established. Here we use Scanning and Trans- mission Electron Microscopy and immunofluorescence to investigate cilia in the urochordate Ciona intestinalis. We show that single cilia are transiently present on each ectoderm cell of the late neurula/early tailbud stage embryo, a time point just before onset of asymmetric nodal expression. Mapping the position of each cilium on these cells shows they are posteriorly positioned, something also described for mouse node cilia. The C. intestinalis cilia have a 9+0 ring ultrastructure, however we find no evidence of structures associated with motility such as dynein arms, radial spokes or nexin. Furthermore the 9+0 ring structure becomes disorganised immediately after the cilia have exited the cell, indicative of cilia which are not capable of motility. Our results indicate that although cilia are present prior to molecular asymmetries, they are not motile and hence cannot be operating in the same way as the flow-generating cilia of the vertebrate node. We conclude that the cilia may have a role in the development of C. intestinalis left-right asymmetry but that this would have to be in a sensory capacity, perhaps as mechanosensors as hypothesised in two-cilia physical models of vertebrate cilia- driven asymmetry.