Building the Posterior Lateral Line system In Zebrafish Embryos

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

BACKGROUND Formation of the Posterior Lateral Line system in zebrafish is pioneered by the posterior Lateral Line (pLL) primordium, a group of about 150 cells that forms near the ear. While leading cells in the pLL primordium have a relatively mesenchymal morphology, trailing cells are more epithelial; they have distinct apical basal polarity and they reorganize to sequentially form nascent neuromasts or protoneuromasts. The pLL primordium begins migration toward the tip of the tail at about 22 hours post fertilization (hpf). Proliferation adds to the growth of the primordium, nevertheless, as the primordium migrates, the length of the column of cells undergoing collective migration progressively shrinks as cells stop migrating and are deposited from the trailing end: cells that were incorporated into protoneuromasts are deposited as neuromasts, while cells that were not, are deposited between neuromasts as interneuromast cells. Eventually, the primordium ends its migration a day later after depositing 5-6 neuromasts and by resolving into 2-3 terminal neuromasts. Establishment of polarized Wnt and FGF signaling systems coordinates morphogenesis and migration of the primordium: Wnt signaling dominates at the leading end and is thought to determine the relatively mesenchymal morphology of leading cells, while FGF signaling dominates in the trailing end. There, FGF determines reorganization of groups of trailing cells to form rosettes as they constrict at their apical ends. Furthermore, FGF signaling determines the specification of a central cell in each rosette as a sensory hair cell progenitor and it helps determine collective migration of the pLL primordium cells. Wnt signaling promotes its own activity and at the same time drives expression of fgf3 and fgf10. However, leading cells do not respond to these FGF ligands because Wnt signaling simultaneously promotes expression of intracellular inhibitors of the FGF receptor. Instead, the FGFs activate FGF receptors and initiate FGF signaling at the trailing end of the primordium, where Wnt signaling is weakest. There, FGF signaling determines expression of the diffusible Wnt antagonist Dkk1b, which counteracts Wnt signaling to help establish stable FGF responsive centers. Once established, the trailing FGF signaling system coordinates morphogenesis of nascent neuromasts by simultaneously promoting the reorganization of cells into epithelial rosettes and by initiating expression of factors that help specify a sensory hair cell progenitor at the center of each forming neuromast. Over time, the leading domain with active Wnt signaling shrinks closer to the leading edge and additional FGF signaling centers form sequentially in its wake, each associated with formation of additional protoneuromasts. SOX2 STABILIZES MATURING EPITHELIAL ROSETTES IN THE ZEBRAFISH POSTERIOR LATERAL LINE PRIMORDIUM Greg Palardy, Sana Fatma, Abhishek Mukherjee, Chongmnin Wang and Ajay Chitnis Protoneuromasts are formed within the migrating Posterior Lateral Line primordium, starting from its trailing end, as clusters of cells sequentially reorganize to form epithelial rosettes, each around a central Atoh1a expressing cell specified as a sensory hair cell progenitor. Their formation serves as a model for understanding more broadly the steps that determine the self-organization of sensory organs. Protoneuromast formation is initiated in Fgf signaling domains that are periodically established in a trailing zone of the migrating primordium in response to Fgfs produced by Wnt active cells in a leading zone. The local promotion of Wnt activity coupled coupled with longer range inhibition of Wnt by Fgf-dependent Dkk1b expression facilitates periodic establishment of the Fgf signaling centers that initiate formation of nascent protoneuromasts. Progressive restriction of an initially broad Wnt signaling domain to a smaller leading zone allows new Fgf signaling-dependent protoneuromasts to form in the wake of the shrinking Wnt system. However, the Wnt inhibitor Dkk1b is not expressed in these maturing neuromasts raising a question about what inhibits Wnt signaling in the trailing neuromasts. We now show that Sox2 is expressed in nascent and maturing protoneuromasts in a pattern that is complementary to domains with Wnt signaling activity. Furthermore, Sox2 functions in a partially redundant manner with Sox1a and Sox3, to inhibit Wnt signaling. This helps keep Wnt activity restricted to a leading zone, which we suggest is essential for effective stabilization of maturing protoneuromasts in the trailing zone. Together with past observations, this study helps define a key third step in the periodic self-organization of neuromasts in the primordium: first, a step that polarizes Wnt activity in the primordium, a second pattern forming step that generates periodic Fgf signaling centers in the context of polarized Wnt activity, and a third involving Sox2 which helps stabilize nascent neuromasts formed in the earlier pattern forming stage. SIGNALING AND MECHANICS INFLUENCE THE NUMBER AND SIZE OF EPITHELIAL ROSETTES IN THE MIGRATING ZEBRAFISH POSTERIOR LATERAL LINE PRIMORDIUM Abhishek Mukherjee, Michael Hilzendeger, Damian Dalle Nogare, Megan Schupp, Maryam Bolouri and Ajay Chitnis Protoneuromasts are formed within the migrating primordium, starting from its trailing end as clusters of cells apically constrict and form epithelial rosettes. Their formation is promoted by Fgf signaling centers that form periodically in the wake of a shrinking Wnt active domain that inhibits epithelial rosette formation and that progressively shrinks toward the leading end of the primordium. However, the precise number and size of epithelial rosettes is not strictly dependent on a prepattern of Fgf signaling activity as it is broadly influenced by the balance of mechanical interactions that promote or oppose formation of epithelial rosettes. When chemokine-dependent migration of leading cells is compromised, the resulting slowing of the primordium is accompanied by the fusion of epithelial rosettes to form fewer larger rosettes. However, such fusion is not observed when Fgf signaling, responsible for migration of trailing cells, is inhibited to slow primordium migration. These observations can be accounted for by a mechanics-based model, where local interactions associated with apical constriction and cell adhesion promote aggregation, while tension along the length of the primordium, influenced by the relative efficacy of leading and trailing cell migration, opposes such aggregation. We describe the development of a Cellular Potts model which allows us to explore how the relative speed of leading versus trailing cells, as well as changes in cell adhesion and mechanical coupling, differentially regulated by Wnt and Fgf signaling, can influence the pattern of neuromast formation and deposition by the migrating primordium. Our studies illustrate how signaling and mechanics cooperate to coordinate self-organization of morphogenesis in the migrating primordium.