Biology subjects

Ononiwu, F.

Publications and source records attributed to Ononiwu, F..

3 recordsLinked to original sources

bioRxiv2025

Functionally Essential and Structurally Diverse: Insights into the zebrafish Left-Right Organizers Cilia via Optogenetic IFT88 Perturbation and Volume Electron Microscopy

In the zebrafish left-right organizer (LRO), the Kupffers Vesicle (KV), cilia extend from all cells into the fluid-filled lumen, but their structural diversity and contribution to morphogenesis remain incompletely defined. We hypothesized that cilia are required for KV development and may exist in distinct structural subtypes. Using a newly engineered transgenic line (sox17:Cry2-GFP), we optogenetically disrupted the intraflagellar transport protein IFT88 in KV progenitors via blue light-induced clustering of CIB1-RFP-IFT88. This perturbation impaired ciliogenesis and disrupted lumen formation, supporting a critical role for cilia in KV morphogenesis. To assess ciliary architecture, we used volume electron microscopy (vEM) to generate a high-resolution 3D ultrastructural map of mature KVs. Only 70.1% of cilia retained both mother and daughter centrioles, suggesting centriole elimination may occur in this tissue. Among centrioles present, 33.9% had distal appendages, 91.8% had subdistal appendages, and only 5.08% exhibited rootlet fibers. Cilia were also associated with membrane-bound vesicles, including spatially biased ciliary-associated vesicles (CaVs) and dense vesicles (CaDVs). These findings demonstrate that KV cilia are structurally diverse and spatially patterned, revealing a previously unappreciated level of complexity in LRO organization and providing new insight into how ciliary specialization may contribute to left-right axis specification.

developmental biology↗

bioRxiv2024

Temporal and anteriorly positioned mitotic zones drive asymmetric microtubule patterns needed for Left-Right Organizer development.

Cellular proliferation is vital for tissue development, including the Left-Right Organizer (LRO), a transient organ critical for establishing the vertebrate LR body plan. This study investigates cell redistribution and the role of specific progenitor cells in LRO formation, focusing on cell lineage and behavior. Using zebrafish as a model, we mapped all mitotic events in Kupffers Vesicle (KV), revealing an FGF-dependent, anteriorly enriched mitotic pattern. With a KV-specific fluorescent microtubule (MT) line, we observed that mitotic spindles align along the KVs longest axis until the rosette stage, spindles that form after spin, and are excluded from KV. Early aligned spindles assemble cytokinetic bridges that point MT bundles toward a tight junction where a rosette will initially form. Post-abscission, repurposed MT bundles remain targeted at the rosette center, facilitating actin recruitment. Additional cells, both cytokinetic and non-cytokinetic, are incorporated into the rosette, repurposing or assembling MT bundles before actin recruitment. These findings show that initial divisions are crucial for rosette assembly, MT patterning, and actin remodeling during KV development.

developmental biology↗

bioRxiv2022

Rab8-, Rab11-, and Rab35-dependent mechanisms coordinating lumen and cilia formation during Left-Right Organizer development

An essential process during Danio rerios left-right organizer (Kupffers Vesicle, KV) development is for the majority of developing KV cells to form a motile cilium that extend into the KV lumen. Left-right beating of motile cilia within the KV lumen directs fluid flow to establishment the embryos left-right axis. However, when KV cells start to form cilia and how cilia formation is coordinated with KV lumen formation has 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 lumenal cavity. 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 m2. 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.

cell biology↗