MECHANISMS GOVERNING THE GENERATION AND TIMING OF LEFT-RIGHT ASYMMETRIES IN THE EMBRYONIC ZEBRAFISH

Abstract

Left-right patterning is the process of generating directed asymmetries within the viscera of vertebrates during early embryogenesis, occurring before specific organ development has begun. The key regulator of asymmetric organogenesis is Nodal, a TGF-β signaling ligand necessary for organ laterality and development. Nodal is typically expressed in the left lateral plate mesoderm as the result of cilia-driven asymmetric fluid-flow in the left right organizer. D. rerio (commonly zebrafish) maintains this paradigm and is an ideal model organism for studying the process of breaking left-right asymmetry. While the use of a ciliated tissue to generate and sense asymmetric fluid-flow is conserved in vertebrate development, the mechanisms by which this flow creates and restricts left-sided Nodal signaling remain unclear. This study aims to define the timings and gene functions necessary for proper left-right patterning in the zebrafish embryo. We describe the expression profiles of southpaw (nodal-related 3, spaw), charon (dand5), and lefty1(lft1) as the key signaling genes involved in breaking left-right symmetry between the 6-somite stage (6ss) and 12ss. Critically, Kupffer's vesicle, the left-right organizer of zebrafish, establishes right-biased charon between 6ss and 8ss, in a manner that is dependent on the proper cilia motility and sensation of fluid-flow. Embryos that cannot sense fluid-flow obtain elevated levels of Charon, which significantly delays Spaw signaling and the initiation of laterality in the lateral plate mesoderm. Conversely, we find loss of the ciliogenesis master regulator transcription factor Foxj1 decreases charon expression in Kupffer's vesicle, effectively decreasing the Spaw signaling threshold necessary for spaw propagation in both the left and right lateral plate mesoderm. We also characterize the potential for embryos to reset their spaw laterality following improper or weak spaw initiation. Finally, we identify the zebrafish homolog of polycystic kidney disease 1-like-1 and examine its role in left-right patterning in Kupffer's vesicle.