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.