Imaging Cell Division, Cell Motility and Small GTPase Activation during Zebrafish Eye Morphogenesis
Chi-Bin Chien, Ph.D.
University of Utah, Salt Lake City, UT
David Mahoney Neuroimaging Program
December 2006, for 3 years
Imaging Zebrafish Provides a Window to See Eye Changes During Development
Utah investigators will use molecular imaging techniques to view the morphogenesis (structural growth) of the developing eye in zebrafish, to learn how the eye develops normally and how development can go awry in the presence of specific genetic mutations.
During development of the central nervous system, including the eye, areas turn inside out, push back inside themselves, and fold. Studying development of the eye in zebrafish, which are translucent, affords the opportunity to observe normal development of the eye and to determine how manipulation of specific genes involved in eye diseases derails normal development.
The investigators will develop cellular imaging methods to simultaneously visualize different eye structures over time, using fluorescent proteins. Then they will apply these imaging and data analysis techniques to understand normal eye morphogenesis. They will characterize underlying cell movements and changes in shape, determine where and when cellular proliferation occurs, and detect when and where specific molecular switches that help control changes in shape, called GTPases, are activated. Thereafter, they will apply these same techniques to image zebrafish with specific genetic mutations that are known to affect eye malformation, but whose functions are not yet understood. Comparing morphogenesis in the normal and genetically mutated zebrafish should reveal how these genes disrupt normal eye development.
Significance: Development of these cellular imaging techniques is anticipated to improve understanding of normal eye development and how this is altered by specific genes. Additionally, this approach is anticipated to be applicable to studies of the developing cerebral cortex in zebrafish, and eventually to understanding the functions of genes that may alter its normal development.
Imaging Cell Division, Cell Motility, and Small GTPase Activation During Zebrafish Eye Morphogenesis
For the embryonic neural tube to develop into the proper form of the adult central nervous system (CNS), it must undergo extensive growth, evagination, invagination, and folding—in short, morphogenesis. Despite being crucial for brain anatomy and function, CNS morphogenesis has been little studied. The developing eye is a particularly good model of morphogenesis, for several reasons. The optic vesicle exhibits many complex movements as it forms the mature eye cup. The neural retina is a well-understood, laminar structure. Finally, the eye is the most accessible region of the CNS, easy both to observe and to manipulate experimentally. To study eye morphogenesis, we use the zebrafish embryo. Not only does the embryo's transparency afford extraordinary optical access, but it is also easy to deliver optical probes, and to perform many molecular and genetic perturbations. The project has three main goals, all directed at understanding the cellular basis of eye morphogenesis.
First, we are developing methods to image tissue and cellular behavior in the developing zebrafish eye. We simultaneously image different structures using fusion constructs of green and red fluorescent proteins. We have already imaged chromatin, cell membranes, and apical polarity, and will add markers for activation of PI-3-kinase and small GTPase activity. In preliminary experiments, we have acquired four-dimensional movies encompassing the entire volume of the optic vesicle, tracked from 12 to 24 hours postfertilization—the entire period of morphogenesis. We are also using photoactivatable fluorescent proteins to track the behavior of specific clusters of retinal progenitor cells. Visualization and analysis of these large datasets are challenging, and we are working with collaborators in the University of Utah's Scientific Computing Institute to develop new algorithms and software.
Second, we will apply these tools to understand the morphogenesis of the normal (wildtype) zebrafish eye. We will study the period beginning immediately after evagination, and proceeding through formation of the definitive eye cup—a period that has been heretofore little studied. We will characterize the underlying cell movements and cell shape changes; determine where and when proliferation occurs; determine when apicobasal polarity is established; and search for specific patterns of small GTPase activation. These experiments will reveal in detail the cellular behaviors that lead to formation of the normal eye cup.
Finally, we will begin to apply our imaging tools to understand how eye formation can go awry, using some of the many zebrafish mutants with known eye formation defects. As a starting point, we will look at three sets of mutants. bashful (laminin 1) and other mutants in laminin genes show abnormal interactions between the lens and neural retina. nagie oko (pals1/MAGUK p55) is a mutant defective in a component of the apical polarity complex that is known to have defects in retinal lamination after 24 hpf, but we suspect that it also has earlier defects in cell movement or behavior. Third, we will analyze no isthmus (pax2a) as an example of a mutant in which the optic stalk fails to close properly (coloboma). In the long run, we plan to use similar analyses to understand the function of human disease genes whose malfunction is known to lead to eye formation defects.
In summary, we will develop and begin to use cellular and molecular imaging tools that will allow us to understand zebrafish eye morphogenesis in exquisite cellular detail. We will start to understand the mechanisms of CNS morphogenesis, a basic process of vertebrate neural development. Furthermore, we will have a clear path forward to developing zebrafish models of human eye development diseases.
CNS morphogenesis is crucial for proper brain development, but is poorly understood—we know relatively little about the patterns of cell behavior, migration, and shape change that lead to the normal shape of the brain.
Our lab studies the embryonic zebrafish eye, which affords extraordinary optical access and allows molecular and genetic perturbations. It is an excellent model as many defects of brain morphogenesis also affect the eye. Our central hypothesis is that eye morphogenesis is instructed by extrinsic and intrinsic influences, which drive localized changes in cell shape and motility, mediated by activation of Rho-family small GTPases.
We have three main goals. First, to develop methods to image patterns of mitosis, cell polarity, cell shape changes, and activation of the PI-3-kinase pathway and small GTPases in live developing zebrafish embryos. Second, to apply these methods to characterize the normal morphogenesis of the zebrafish eye, to understand the normal patterns of cell division, migration, and shape changes that underlie its normal development, and to start to understand the molecular signals that drive these cellular behaviors. Third, to begin to study eye morphogenesis in known mutants. We have already found that zebrafish mutant for the laminin alpha-1 gene show striking defects in morphogenesis of the lens and neural retina, and show underlying defects in apicobasal polarity.
We will use two strategies: whole-eye imaging (imaging every cell in the eye) as well as cluster imaging (imaging a few labeled cells within the context of the eye). Whole-eye labeling uses early mRNA injection or stable transgenes to drive expression of fluorescent protein fusions that mark cell nuclei, cell membranes, polarity markers, or GTPase activity. Cluster labeling uses mRNA or transgenes for photoactivatable markers such as Kaede or PA-GFP. Live embryos are then embedded in low-melt agarose and imaged continuously for 12 hours using a fast laser-scanning confocal microscope (Olympus FV1000). Sample data can be viewed at the Chien lab website.
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