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

Abstract

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 μm2. 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.

The centrosome – diverse functions in fertilization and development across species

“…However, the formation of cilia during human node formation is not fully annotated. It is possible that in humans, cilia are formed before the development of the node, as is the case for zebrafish embryos ( Aljiboury et al, 2023 ). Like in humans, the zebrafish LRO (Kupffer's vesicle, KV) is the first annotated ciliated organ in the developing embryo ( Essner et al, 2005 ).…”

“…Like in humans, the zebrafish LRO (Kupffer's vesicle, KV) is the first annotated ciliated organ in the developing embryo ( Essner et al, 2005 ). From our recent studies, we know that cilia are formed prior to KV lumen development ( Aljiboury et al, 2023 ). We found that ∼33% of KV precursor cells had cilia at the 90% epiboly stage, suggesting that cilia formation is a precursor to LRO formation.…”

“…Most studies that identified molecular mechanisms for intracellular primary cilia formation relied on in vitro cell culture ( Knödler et al, 2010 ; Kuhns et al, 2019 ; Westlake et al, 2011 ; a recent review is provided by Zhao et al, 2023 ). However, our recent study identified that the zebrafish LRO also uses an intracellular mechanism for motile cilia formation that involves the small GTPases Rab11 and Rab35 ( Aljiboury et al, 2023 ). What is yet to be resolved, however, is what determines when a cell uses intracellular versus extracellular mechanisms for cilia formation during development.…”