Left-Right Organizer Cellular and Molecular Dynamics Drive Early Cardiac Asymmetries - PROJECT SUMMARY/ABSTRACT Abnormal development of the left-right (LR) axis during embryogenesis can manifest as a broad spectrum of severe congenital heart diseases (CHDs), a group of conditions that affects 1% of all infants and in which the cardiac structure is disrupted. The LR axis is established at the Left-Right Organizer (LRO), a transient embryonic structure conserved across several vertebrate species. The Brueckner lab has discovered that transcriptional and morphological left-right asymmetry requires motile cilia in LRO “pit cells” that generate force sensed by mechanosensing/calcium-signaling immotile cilia in LRO “crown cells.” However, how the LRO develops and the mechanisms by which transcript symmetries are first broken are not fully understood. Advancing knowledge of LRO formation and function will inform our understanding of the etiology of laterality defect-associated CHDs. The goal of this proposal is to define the cellular subpopulations that constitute the LRO and establish how they initiate LR asymmetry of the developing embryo. Based on preliminary findings, the central hypothesis of this proposal is that the mouse LRO forms from two transcriptionally dis- tinct cell populations (crown and pit) that develop further heterogeneities over time, including a newly-identified MMP21 (matrix metalloprotease 21)-positive pit cell subpopulation required to es- tablish the earliest LRO transcriptional asymmetries. To test this hypothesis, this project will utilize the mouse LRO as a model for human LR development, as evolutionary and genetic studies have revealed motile cilia as key drivers of LR asymmetry in both species. Aim 1 will investigate LRO structure and em- bryonic origins, utilizing single cell and spatial transcriptomic data, followed by in vivo validation, to identify and characterize the origins and transcriptional markers of LRO cell subpopulations. Aim 2 will investigate LRO function and expand upon the currently incomplete model of LR symmetry breaking to include the MMP21 mRNA and protein. This aim will use hybridization chain reaction in situ hybridization (HCR-FISH) and immunofluorescence (IF) experiments in mouse embryos with mutations in ciliary components and LRO genes. Long-term, this work will have both basic and translational research implications, and results from this project will provide a comprehensive view of the origins and roles of rare LRO cell populations that are essential to the proper inception of cardiac morphogenesis. Upon completion of this fellowship, the applicant will have received training in mouse genetics, early vertebrate development, scRNA-seq and spatial tran- scriptomic data generation and analysis, and advanced microscopy image analysis, as well as strengthened her skills in mentoring, scientific communication, research design, and critical thinking, thus providing her with the expertise necessary for a successful academic career in independent early developmental biology research.
