Zebrafish biosensors to analyze vertebrate lens development

In recent years, the zebrafish (Danio rerio) has proved to be an excellent model organism for the analysis of many evolutionary conserved steps of vertebrate organogenesis, including the formation of specific eye segments. Moreover, zebrafish are a cost-effective vertebrate system for high-throughput approaches, such as large-scale genetic and small molecule screening. Several tools are available to perform reverse genetics analyses, such as gene over-expression and knock-down strategies, as well as a series of transgenic/conditionally tractable lines and mutants displaying specific ocular disorders affecting, for example, the lens. One of the key advantages of the zebrafish over other vertebrate models is its suitability for in vivo time- lapse (4D) imaging. This allows qualitative and quantitative characterization of morphogenetic events and cell dynamics, as well as the dissection of signaling pathways activated at specific developmental stages. In this context, our laboratory is currently generating a series of transgenic zebrafish lines, each one responsive to specific signaling pathways which are active during vertebrate development. These lines allow a 4D analysis of signaling events by in vivo color-based visualization of tissues and organs responding to a specific pathway at a given time point. At present, we are validating a group of fish lines expressing green or red fluorescent proteins when the following pathways are activated: Wnt, Bmp, Tgf- beta, Shh, Notch, cAMP, Stat3 and Hypoxia. Our goal is to analyze these lines in relation to eye formation, and in particular to lens development. This task will be performed by time-lapse confocal microscopy focused on the eye region, as well as by fluorescent whole-mount in situ hybridization with lens markers combined with probes for the signaling reporters. The same transgenic lines will be also pharmacologically targeted as well as crossed with available lens-specific mutants. This detailed dissection of specific time points and anatomical districts, where single or multiple signals are activated during eye formation and lens development, will help to simultaneously evaluate multiple roles and cross-talk of the pathways involved. This approach will also shed light on current knowledge about micro-environmental factors, whose role in eye formation and lens development is still poorlycharacterized or controversial.