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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
  • Raul Rojas, PhD, Postdoctoral Fellow
  • Chongmin Wang, MS, Research Technician
  • Kyeong-Won Yoo, PhD, Postdoctoral Fellow
  • Shana Spindler, PhD, Postdoctoral Fellow
  • Katherine Somers, BS, Postbaccalaureate Intramural Research Training Award Fellow
  • Swetha Rao, Summer Student

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 fates 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 to visualize how local interactions between cells leads to the emergence of patterned neural development in the growing embryo.

Atoh1a expression must be restricted by Notch signaling for effective morphogenesis of the posterior lateral line primordium in zebrafish.

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 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 its trailing end to establish the posterior lateral line system. The migrating primordium contains three to four "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 they mature, the proneuromasts also form center-oriented epithelial rosettes. Eventually, mature proneuromasts are deposited from the trailing end of the migrating pLLp and sensory nerves of the lateral line ganglion innervate the sensory hair cells.

Understanding the self-organization of the pLLp system and how its developmental fails under specific experimental conditions provides an informative context in which to clarify the broader mechanisms regulating organogenesis in the developing embryo. It is proving to be an especially attractive system in which to probe how interaction between distinct signaling pathways contributes to self-organization and how specification of cell fate and morphogenesis is integrated within a developing sensory organ. The neuromasts are archetypical sense organs, whose development and morphogenesis has remarkable similarities to diverse sensory organs such as the ommatidia in the Drosophila eye and hair cells of the mammalian ear.

A Wnt-dependent FGF signaling center at the leading end of the pLLp initiates formation of "proneuromasts" by facilitating the reorganization of cells into epithelial rosettes and by initiating atoh1a expression. Expression of atoh1a endows proneuromast cells with the potential to become sensory hair cells. However, lateral inhibition mediated by Delta-Notch signaling restricts atoh1a expression and hair cell progenitor fate to a central cell while the surrounding cells are allowed to become support cells. We have shown that, as atoh1a expression becomes established in the central cell, it drives expression of fgf10 and the Notch ligand deltaD while inhibiting expression of fgfr1. As a source of FGF10, the central cell activates the FGF pathway in neighboring cells, ensuring that they form stable epithelial rosettes. At the same time, DeltaD activates Notch in neighboring cells, inhibiting atoh1a expression and ensuring that these cells are specified as supporting cells. In mind bomb mutants, loss of Notch-mediated lateral inhibition prevents atoh1a expression from being restricted to a central cell in a maturing proneuromast. Instead, atoh1a expression progressively expands to surrounding cells, which are specified as hair cell progenitors instead of support cells. Unregulated atoh1a expression reduces FGFR1 expression, eventually resulting in attenuated FGF signaling, which prevents effective maturation of epithelial rosettes in the pLLp.

Surprisingly, the progressive expansion of atoh1a expression not only expands the number hair progenitors and reduces FGF signaling in the migrating primordium, it also eventually leads to catastrophic fragmentation of the pLLp. In the past year, our studies showed that atoh1a-expressing cells express e-cadherin at a relatively low level. As a consequence, expanding atoh1a expression reduces e-cadherin expression in the migrating primordium while leaving n-cadherin expression slightly expanded or unchanged. The reduction of e-cadherin expression is likely to contribute to reduced cohesion and fragmentation of the pLLp.

In the neuromasts, central hair-cell precursors expressing atoh1a are surrounded by support cells that do not express atoh1a. Furthermore, a population of non-sensory cells surrounds the support cells. Our studies suggest that while the central atoh1a-expressing hair cell progenitors in maturing neuromasts express only n-cadherin, non-sensory cells express only e-cadherin. As support cells express both n-cadherin and e-cadherin, they can form effective adhesive interactions with both the outer non-sensory cells that express only e-cadherin and inner hair cell precursors that express only n-cadherin. In this manner, support cells serve as a "glue" holding cells expressing only e-cadherin or n-cadherin together in both maturing and deposited neuromasts.

Failure of Notch signaling allows prospective support cells that surround the central atoh1a-expressing cell to start expressing atoh1a, which prevents them from expressing e-cadherin. This prevents the cells from effectively adhering to surrounding non-sensory cells that express only e-cadherin. We believe that this also contributes to fragmentation of the pLLp when Notch signaling is lost. We developed a simple computational model of the migrating pLLp to illustrate how replacement of prospective support cells that normally express both e-cadherin and n-cadherin by atoh1a-expressing cells expressing only n-cadherin eventually contributes to fragmentation of the pLLp.

Modeling migration of the lateral line primordium

The pLLp migrates from the otic vesicle to the tip of the tail in zebrafish. Its migration is determined by the chemokine signaling system. While cells in the leading two thirds express the chemokine receptor cxcr4b, cells in the trailing third express another receptor, cxcr7b. The polarized expression of these chemokine receptors is essential for effective caudal migration along a relatively uniform stripe of expression of the chemokine sdf1a, a diffusible ligand that can bind to these receptors. While cells expressing cxcr4b have the potential to respond to sdf1a and move in a direction with a high concentration of the chemokine, cells expressing cxcr7b cannot. They bind, internalize, and degrade sdf1a but do not respond by driving movement of the cells. We have developed a computational model of pLLp based on predicted emergent mechanical properties of the pLLp. The model serves as an effective tool for visualizing how polarized expression of cxcr7b and cxcr4b is likely to contribute to directed migration of the pLLp. We showed how the model can be used to effectively predict how the pLLp will behave under a variety of experimental conditions under which chemokine receptor expression is altered in the pLLp system.

Additional Funding

  • K99 award to Miho Matsuda


  • Matsuda M, Chitnis AB. Atoh1a expression must be restricted by Notch signaling for effective morphogenesis of the posterior lateral line primordium in zebrafish. Development. 2010 137:3477-3487.


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