Many internal organs, including the heart, digestive organs and parts of the brain, develop left-right (LR) asymmetries that are essential for their function. Defects in LR development cause complex congenital heart disease and other developmental abnormalities that significantly impact the first year of human life. Rapid progress has been made in identifying genes that regulate LR development. However, the fundamental developmental mechanisms that are controlled by these genes are poorly understood. Using several experimental approaches, we have shown that Kupffer's vesicle (KV) is a transient embryonic organ of asymmetry in zebrafish, essential for the earliest known steps in LR development. KV cells have motile cilia that move fluid in a LR asymmetric direction, analogous to nodal cilia in mice. 1 of the advantages in zebrafish is that we know the embryonic lineages, Dorsal Forerunner Cells (DFC) that give rise to ciliated cells. We developed a technique (DFCMO) to knockdown gene function specifically in DFC/KV cells, allowing us to study the specific developmental and cellular functions of genes in the ciliated cells while avoiding the pleiotrophic phenotypes common in mouse and zebrafish laterality mutants. This technique has revealed a unique role for a transcription factor, no tail, and a dynein molecule in DFC/KV cells. The proposed research uses a combination of laterality mutants (both previously described and novel), newly cloned genes, morpholinos, transgenics, lineage-mapping embryological techniques, a mutant screen, and a novel DFCMO technique. Unique experimental advantages in zebrafish will allow us to discover the mechanisms that control 3 fundamental, but poorly understood steps in LR development: regulation of KV organogenesis and generation of LR asymmetric fluid flow (Aim 1), transmission of LR information from the cilia-dependent flow in the KV to lateral cells (Aim 2), and posterior to anterior transmission of LR information through lateral plate mesoderm (Aim 3). This research will uncover developmental mechanisms that are essential in LR development and that will be applicable to fundamental questions in organogenesis.
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