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 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. EPIDERMAL CONFINEMENT IS ESSENTIAL FOR COLLECTIVE MIGRATION DETERMINED BY SUPERFICIAL MIGRATORY CELLS THAT FORM A SHEATH AROUND DEEPER CELLS OF THE ZEBRAFISH POSTERIOR LATERAL LINE PRIMORDIUM The zebrafish posterior lateral line primordium migrates in a channel between the skin and somites, periodically depositing neuromasts. Its migration depends on the coordinated movement of its relatively mesenchymal leading cells and epithelialized trailing cells, which form neuromasts. We now describe a third category of flat primordium cells that wrap around deeper epithelialized cells to form a superficial migratory sheath, and that extend polarized lamellipodia to migrate along the overlying skin. Polarization of lamellipodia extended by both superficial cells and deeper neuromast-forming cells depends on Fgf signaling. Furthermore, removal of the overlying skin has similar effects on superficial and deep cells: their lamellipodia are lost, blebs appear instead, and collective migration fails. Covering the skinned embryos with agarose does not lead to a recovery of polarized lamellipodia extended by either superficial or deeper cells. On the other hand, covering the primordium with Matrigel, a proprietary substrate that contains components of the extracellular matrix, did result in recovery lamellipodia, however, these protrusions were not coordinately polarized. These observations suggest that interaction with extracellular matrix associated with the skin play an important role in the emergence of lamellipodia, but that confinement by the skin provides additional constraints or components required for determination of polarized protrusions. Together, these observations highlight the broad role confinement by the skin plays in the determining directed migration of the posterior lateral line primordium, which has emerged as a powerful model for studying collective migration in vivo.