Rab8, Rab11, and Rab35 coordinate lumen and cilia formation during zebrafish left-right organizer development

An essential process during Danio rerio's left-right organizer (Kupffer's Vesicle, KV) formation is the formation of a motile cilium by developing KV cells which extends into the KV lumen. Beating of motile cilia within the KV lumen directs fluid flow to establish the embryo's left-right axis. However, the timepoint at which KV cells start to form cilia and how cilia formation is coordinated with KV lumen formation have not been examined. We identified that nascent KV cells form cilia at their centrosomes at random intracellular positions that then move towards a forming apical membrane containing cystic fibrosis transmembrane conductance regulator (CFTR). Using optogenetic clustering approaches, we found that Rab35 positive membranes recruit Rab11 to modulate CFTR delivery to the apical membrane, which is required for lumen opening, and subsequent cilia extension into the lumen. Once the intracellular cilia reach the CFTR positive apical membrane, Arl13b-positive cilia extend and elongate in a Rab8 dependent manner into the forming lumen once the lumen reaches an area of 300 mu m(2). These studies demonstrate the need to acutely coordinate Rab8, Rab11, and Rab35-mediated membrane trafficking events to ensure appropriate timing in lumen and cilia formation during KV development. Author summaryCilia are microtubule-based structures that extend from the cell surface to sense and transmit extracellular cues to the cell body. Defective cilia result in a series of conditions known as ciliopathies. Although the mechanisms of cilia formation have been elucidated in vitro, how a cilium is made in vivo during embryogenesis is less studied. We take advantage of the Zebrafish's organ of asymmetry, also known as the Kupffer's Vesicle (KV), as a model to study cilia formation during vertebrate embryogenesis. In the KV, cilia and lumen formation occur concurrently, making it a great model to study how these processes are coordinated. Here we show that KV precursor cells (dorsal forerunner cells) form cilia within the cell volume before extending into the lumen of the KV. To determine the molecular mechanisms of cilia formation during KV development, we focus on three Rab GTPases, Rab11, Rab8 and Rab35, that have been implicated in cilia or lumen formation independently in vitro. We use optogenetic clustering in parallel with morpholino depletion approaches to show that these three Rab GTPases coordinate specific events that govern cilia and lumen formation during KV development.

Automated summary

Cilia and lumen formation during Danio rerio's left-right organizer (Kupffer's Vesicle, KV) development are acutely coordinated by Rab8, Rab11, and Rab35-mediated membrane trafficking events. Nascent KV cells form cilia at random intracellular positions before extending into the forming lumen, which requires the recruitment of Rab11 by Rab35 positive membranes to modulate CFTR delivery to the apical membrane. Once the lumen reaches a certain size, Arl13b-positive cilia elongate in a Rab8 dependent manner into the lumen.