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Cellular, Molecular and Genetic Analysis of Neural Fate in Zebrafish Embryos

Ajay Chitnis, MBBS, PhD
  • Ajay Chitnis, MBBS, PhD, Head, Section on Neural Developmental Dynamics
  • Damian E. Dalle Nogare, PhD, Postdoctoral Fellow
  • Hiromi Ikeda, PhD, Postdoctoral Fellow
  • Miho Matsuda, PhD, Postdoctoral Fellow
  • Gregory Palardy, BS, Research Technician
  • Kinneret Rand, PhD, Postdoctoral Fellow
  • Raul Rojas, PhD, Postdoctoral Fellow
  • Chongmin Wang, MS, Research Technician
  • Kyeong-Won Yoo, PhD, Postdoctoral Fellow

Our goal is to understand how the architecture of the mature nervous system emerges as a consequence of local interactions between cells during early development. We use a combination of cellular, molecular, genetic, and computational tools to understand how cells differentiate in distinct patterns in the various compartments of the zebrafish nervous system. We analyze zebrafish mutants and embryos microinjected with morpholinos or mRNA to alter gene function. We examine mechanisms involved in the division of the prospective neural tissue into compartments with distinct fate and examine how cell differentiation is regulated within each compartment. We use transgenic zebrafish lines with fluorescent protein expression to take advantage of the transparency of zebrafish embryos and watch morphogenesis and cell signaling in a living embryo. Genetic analysis allows us to identify regulatory networks essential for specific aspects of neural patterning, while cell-biological experiments identify trafficking events that are essential for regulating signaling. Finally, our group develops computer models of the genetic regulatory networks as a platform to integrate what has been learned through a combination of cellular, molecular, and genetic analysis, allowing us visualize how local interactions between cells leads to the emergence of patterned neural development in the growing embryo.

Notch-restricted atoh1 expression regulates cell fate and morphogenesis in the zebrafish lateral line system

Sensory nerves of the lateral line ganglion innervate the sensory hair cells in the neuromasts. Together, they form a part of a sensory system on the surface of fish that detects water flow. The neuromasts are archetypical sense organs whose development and morphogenesis has remarkable similarities to diverse sensory organs like ommatidia in the Drosophila eye and hair cells of the mammalian ear.

The posterior lateral line primordium (pLLp) is a cohesive collection of about a hundred cells. It migrates caudally under the skin in the zebrafish trunk and tail, periodically depositing neuromasts from it trailing end. The migrating pLLp contains 3 to 4 “proneuromasts” at various stages of maturation. As they mature, a sensory hair cell is specified at the center of the proneuromast. Support cells, which also serve as pool of progenitors, surround the hair cell. As the proneuromasts mature, they also form center-oriented epithelial rosettes (Hava et al.). Eventually, mature proneuromasts are deposited from the trailing end of the migrating pLLp and sensory nerves of the lateral line ganglion innervate their sensory hair cells. The surrounding progenitor cells in the neuromast eventually contribute to formation of additional sensory hair cells as the neuromasts mature.

We are attempting to understanding how a) new proneuromasts are initiated at the leading end of the pLLp, b) how sensory hair cells are specified at the center of these cells clusters, c) how they form center-oriented epithelial rosettes, d) how they are deposited from the trailing end of the migrating pLLp and, e) what regulates caudal migration of the pLLp. The very accessible, easily manipulated and lateral line system of the zebrafish embryo provides an attractive context to understand the broader mechanisms regulating organogenesis in the developing embryo. The system is proving to be especially amenable for understanding how the function of distinct signaling pathways is integrated and how specification of cell fate and morphogenesis is coordinated within a developing organ.

Previous studies showed that as stable proneuromasts are deposited from its trailing end, formation of new proneuromasts is initiated by Wnt-dependent FGF signaling center at the leading end of the migrating pLLp. This FGF signaling center initiates atoh1a expression, and Notch-mediated lateral inhibition restricts atoh1 expression to a central cell within maturing proneuromasts. We have now shown that the central atoh1a plays a critical role in regulating FGF signaling within maturing neuromasts at the trailing end of the migrating pLLp. atoh1a expression drives expression of FGF ligands like FGF10 while inhibiting expression of its receptor FGFR1. Normally, the Notch-signaling–dependent restriction of atoh1a expression allows the self-organization of a restricted FGF signaling center within maturing neuromasts as the central FGF–expressing cell activates FGF signaling in its neighbors. However, when Notch signaling fails, too many cells express atoh1. They begin to express FGF and shut off expression of its receptor, FGFR1. This eventually leads to failure of FGF signaling, unregulated Wnt signaling and collapse and disorganization of the migrating pLLp.

We have developed computer models to visualize how interactions between the FGF, Wnt, and Notch signaling systems normally regulate the self-organization of the lateral line system. Computational modeling illustrates how interaction between the FGF, Wnt, and Notch signaling systems and differential regulation of chemokine receptors could regulate cell fate, morphogenesis, and migration of the pLLp. The modeling also predicts a key role played by negative feedback in the self-organization of the lateral line system. We are carrying out experiments to test predictions of the computer models.

Additional Funding

  • JSPS award to Miho Matsuda

Publications

  • Hava D, Forster U, Matsuda M, Cui S, Link BA, Eichhorst J, Wiesner B, Chitnis A, Abdelilah-Seyfried S. Apical membrane maturation and cellular rosette formation during morphogenesis of the zebrafish lateral line. J Cell Sci 2009 136:197-206.

Contact

For more information, email chitnisa@mail.nih.gov.

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