Cell cycle dependent mechanisms triggering lumen formation in vivo

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SUMMARY STATEMENT: In humans and other vertebrates, motile cilia located in an organ of asymmetry play an important role in cardiac left-right development. Evidence from model organisms, such as in zebrafish organ of asymmetry, (Kupffer’s Vesicle, KV) indicates that conserved cilia-driven leftward flow establishes left- right signals to regulate target genes to control asymmetric heart morphogenesis. While events downstream of leftward flow have received much attention, little is known about how the organ of asymmetry is formed and the biology of the ciliated cells that generate fluid flow. This project addresses the broad question: How do ciliated cells develop into a functional polarized organ? To address this question we are bringing together cell biology and developmental biology to investigate how the cytokinetic bridge establishes apical polarity and a lumen in vivo. We propose that this occurs through a sequential process that starts with cell division and placement of the cytokinetic midbody, which marks a site for where the apical membrane should be placed. Cytokinetic bridge resolution (a.k.a. abscission) results in the separation of two daughter cells following mitosis allowing for the cell to initiate ciliogenesis. This process has important implications in embryogenesis, and broad implications in the role of cytokinesis in developing cellular diversity. While abscission has been examined in vitro, little has been done to examine the role of cytokinesis/abscission in epithelial establishment and de novo lumen formation in vivo. Thus, our work will test the overall hypothesis that cell division is an essential process that initiates lumen formation, ciliogenesis, and subsequently tissue morphogenesis. Here we propose to examine in the zebrafish KV a requirement for abscission in the transition of progenitor-mesenchymal-like migratory cells to epithelial-ciliated cells (tested in Aim 1). For instance, does cell division trigger KV-specific apical polarity protein expression and does division contribute to how cells are patterned to form a KV? We propose that following cytokinesis, daughter cells stay interconnected by a cytokinetic bridge while apical polarity is established. This process requires targeted membrane traffic into the cytokinetic bridge. During this time, the two daughter cells position themselves so that the cytokinetic bridge is placed where an apical membrane and lumen will form. Once the bridge is cleaved, a lumen is initiated (Aim 2) and KV cells can form primary cilia (Aim 3). We will use photoconversion to track cell fate following division, and laser ablation or optogenetics to determine whether abscission timing is important for apical polarity, cilia formation, and lumenogenesis. These studies will identify important mechanisms for de novo tissue morphogenesis.