Mechanisms of Left Right Organizer Development

Summary Statement In vertebrates, motile cilia within the Left-Right Organizer (LRO) are pivotal for a developing organism’s left right axis formation, such as cardiac left-right development. Evidence from model organisms, like zebrafish LRO, highlights conserved cilia-driven leftward flow crucial for regulating target genes controlling asymmetric heart morphogenesis. LRO cells are made up of both motile and non-motile cilia with one population required for fluid flow generation and the other potentially for fluid sensing. Open questions that exist are: What are the mechanisms by which cells determine whether to develop motile or non-motile cilia within an LRO? What are the specific roles played by each of these cilia populations in development? However, answering these questions in mammalian LRO development is hindered by technical challenges, limiting real-time analysis of spatial cell arrangements, cytoskeletal characterization, cilia assembly, and transcriptional landscape exploration. We aim to address these challenges using Danio rerio (zebrafish) LRO to test our hypothesis: spatial and temporal cell division regulation guides intracellular and cellular remodeling essential for LRO maturation and function. Our R35 application encompasses two projects. Project 1: Assessing the impact of each LRO cell division event on LRO development. Here we will investigate cell redistribution mechanisms and the dominance of specific progenitor cells in LRO formation, addressing cell lineage and cell behavior questions. Additionally, comprehensive gene expression analysis across LRO developmental stages will identify key genes and pathways guiding LRO development. We will employ cell tracking, microtubule labeling with laser ablation, and transcriptomic profiling to understand mitotic events crucial for LRO development. Project 2: Characterizing mechanisms involved in microtubule pattern formation and reorganization during LRO development in relation to actin reorganization, tight and adherens junctions, and cilia formation. Utilizing live cell imaging, molecular and genetic manipulations, array tomography and AI, we will characterize LRO cilia localization and structure at rosette and lumen stages to identify models for LRO development events and fluid flow sensing. Success in addressing our outlined objectives in unraveling the temporal and spatial mechanisms coordinating cell division, intracellular remodeling, gene expression, polarity formation, junctional protein formation, and cilia ultrastructure during LRO development will position us to define how an LRO is assembled and provide novel models that can be tested for how other ciliated tissues develop. Project Number: 1R35GM158119-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Heidi Hehnly | Institution: SYRACUSE UNIVERSITY, SYRACUSE, NY | Award Amount: $2,055,625 | Activity Code: R35 | Study Section: Maximizing Investigators' Research Award C Study Section[MRAC] View on NIH RePORTER: https://reporter.nih.gov/project-details/11089879