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National Institutes of Health

Eunice Kennedy Shriver National Institute of Child Health and Human Development

2019 Annual Report of the Division of Intramural Research

Building the Zebrafish Lateral Line System

Ajay Chitnis
  • Ajay Chitnis, MBBS, PhD, Head, Section on Neural Developmental Dynamics
  • Damian E. Dalle Nogare, PhD, Staff Scientist
  • Gregory Palardy, BS, Research Technician
  • Chongmin Wang, MS, Research Technician
  • Pritesh Krishnakumar, PhD, Postdoctoral Fellow
  • Uma Neelathi, PhD, Postdoctoral Fellow
  • Micheal Hilzendeger, BS, Postbaccalaureate Intramural Research Training Award Fellow
  • Jon Schrope, BS, Postbaccalaureate Intramural Research Training Award Fellow
  • Haille Soderholm, BS, Postbaccalaureate Intramural Research Training Award Fellow

Cells divide, move, adhere, and interact with their neighbors and their environment to determine the formation of multicellular organ systems with unique fates, morphologies, function, and behavior. Our goal is to understand how such interactions determine the self-organization of cell communities in the nervous system of the zebrafish embryo. The lateral line is a mechano-sensory system that helps sense the pattern of water flow over the fish and amphibian body; it consists of sensory organs called neuromasts that are distributed in a stereotypic pattern over the body surface. Each neuromast has sensory hair cells at its center, surrounded by support cells, which serve as progenitors for the production of more hair cells during growth and for the regeneration of neuromasts. The development of this superficial sensory system in zebrafish is spearheaded by the posterior lateral line primordia (pLLp), groups of about 150 cells formed on either side of a day-old embryo near the ear. Cells in the primordia migrate collectively under the skin to the tip of the tail, as they divide and reorganize to form nascent neuromasts, which are deposited sequentially from its trailing end. Their journey is easily observed in live transgenic embryos with fluorescent primordium cells. Furthermore, a range of genetic and cellular manipulations can be used to investigate gene function and morphogenesis in the system. Understanding the self-organization of this relatively simple and accessible system in zebrafish will help elucidate the broader principles that determine cell fate specification, morphogenesis, and collective cell migration in the developing vertebrate nervous system.

Self-organization of the zebrafish lateral line primordium

Formation of the posterior lateral line system in zebrafish is pioneered by the pLLp. While leading cells in the pLLp have a relatively mesenchymal morphology, trailing cells are more epithelial; they have distinct apical/basal polarity and reorganize to sequentially form nascent neuromasts or protoneuromasts. The pLLp 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. Thus, 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.

Wnts (Wingless/Integrated) and Fgfs (fibroblast growth factors) are evolutionarily conserved secreted proteins that allow cells to communicate with their neighbors via distinct signaling pathways to influence various aspects of their neighbor's behavior, fate, shape, and capacity to proliferate. Wnt and Fgf signaling systems coordinate morphogenesis and migration of the primordium. Thus, 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 the cells 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 helps determine collective migration of the pLLp 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.

The interactions between the leading Wnt system and the trailing Fgf system provide a useful framework for understanding the self-organization of neuromast formation and deposition by the migrating pLLp; however, many questions remain unanswered. The Wnt and Fgf signaling systems act simply as a means of communication between cells, and it remains unclear what molecular mechanisms the systems regulate to specifically determine morphogenesis of epithelial rosettes and the collective migration of primordium cells. Furthermore, the mechanics of collective migration in the primordium remains poorly understood, specifically, how the pull of leading cells, which migrate in response to chemokine cues in their path, determines the Fgf–dependent collective migration of trailing cells in the primordium. The summary above suggests that morphogenesis of epithelial rosettes during the assembly of nascent neuromasts is entirely dependent on Fgf signaling. However, it has been observed that, in the absence of collective migration mediated by chemokines in the leading cells, the trailing cells in the primordium come together to form one or two large rosettes. These and other observations related to the changes in the number and size of epithelial rosettes in the presence and absence of collective migration suggest that primordium cells have an inherent potential to form epithelial rosettes and that the formation of rosettes can be influenced by a variety of signaling systems and/or by migratory behavior of leading cells. We built agent-based models using the NetLogo programming environment to visualize how both signaling and mechanical interactions could contribute to periodic formation of neuromasts in the migrating primordium.

A sheath of motile cells supports collective migration of the zebrafish posterior lateral line primordium under the skin.

The zebrafish pLLp migrates in a channel formed by the underlying horizontal myoseptum and somites, and the overlying skin. While cells in the leading part of the pLLp are flat and have a more mesenchymal morphology, cells in the trailing part progressively reorganize to form epithelial rosettes, called protoneuromasts. The epithelial cells extend basal cryptic lamellipodia in the direction of migration in response to both chemokine and Fgf signals. We showed that, in addition to these cryptic lamellipodia, the core tall epithelial cells are in fact surrounded by a population of flat motile cells, which extend actin-rich migratory processes apposed to the overlying skin. These thin cells wrap around the protoneuromasts, forming a continuous sheath of cells around the apical and lateral surface of the pLLp. The processes extended by these cells are highly polarized in the direction of migration, and such directionality, like that of the basal lamellipodia, is dependent on Fgf signaling. Consistent with interactions of sheath cells with the overlying skin contributing to migration, removal of the skin stalls migration. However, this is accompanied by some surprising changes; there is a profound change in the morphology of the sheath cells, with directional superficial lamellipodia being replaced by undirected blebs or ruffles. Furthermore, removal of the skin not only affects underlying lamellipodia, it simultaneously alters the morphology and behavior of the deeper basal cryptic lamellipodia, even though these cells do not directly contact the skin. Directional actin-rich protrusions on both the apical and basal surface and migration are completely and simultaneously restored upon regrowth of the skin over the pLLp. We suggest that this system utilizes a circumferential sheath of motile cells to allow the internal epithelial cells to migrate collectively in the confined space of the horizontal myoseptum and that elastic confinement provided by the overlying skin is essential for effective collective migratory behavior of primordium cells.


  1. Chitnis A, Dalle Nogare D. Time-lapse imaging beyond the diffraction limit. Methods 2018;150:32-41.
  2. Dalle Nogare D, Natesh, N, Chitnis AB. A sheath of motile cells supports collective migration of the Zebrafish posterior lateral line primordium under the skin. BioRxiv 2019;783043.


  • Hari Shroff, PhD, Laboratory of Molecular Imaging and Nanomedicine, NIBIB, Bethesda, MD


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