Abstract
Purpose: :
The purpose of this study is to (1) evaluate changes in calcium (Ca2+) during cone death in a pde6c-/- zebrafish model, and to (2) assess the "bystander effect" in a cone population comprised of wild type and mutant photoreceptors. In the pde6c-/- zebrafish the cone phosphodiesterase gene is mutated, and it is suggested that an influx of Ca2+ causes the cones to die aoptotically. However, the spatial and temporal characteristics of this Ca2+ increase have never been examined in a live animal. This study will follow changes in Ca2+ within the photoreceptors before and during apoptosis.Retinal degeneration is often associated with a phenomenon known as the "bystander effect" in which the death of mutant cells triggers neighboring healthy cells to die. Little is known about the requirements for this spread of apoptosis, and this study proposes to test the ability of a chimeric cone population to undergo the bystander effect.
Methods: :
A zebrafish model lacking cone phosphodiesterase (pde6c) is used for both studies. Changes in Ca2+ are monitored using genetically encoded, fluorescent Ca2+ probes and multiphoton microscopy. Analysis is done while cones mature and die in the pde6c-/- fish. Indicators include the GCaMP family of probes and the TN-XXL FRET-based indicator.To test the potential for a mixed cone population to display a bystander effect, labeled pde6c-/- cells are injected into wild type embryos. Within these chimeric fish the health of wild type cones is evaluated as pde6c-/- cones die apoptotically. Variations in mosaicism allow for a detailed analysis of the percentage of mutant cells required to initiate a bystander effect, and dissemination of this effect is quantified.
Results: :
The pde6c-/- zebrafish are blind, and their cones die apoptotically between 3 and 5 days post fertilization. Preliminary experiments have defined a timeline of 8 hours over which a pde6c-/- cone will go from healthy in appearance to a rounded apoptotic body. This time course will be used to inform further studies on Ca2+ and the bystander effect. Preliminary studies of each of these have been initiated. GCAMP2 has been expressed in WT cones and decreases in fluorescence in response to light have been detected in outer segments.
Conclusions: :
Currently there is no clear understanding of the role of Ca2+ in photoreceptor degeneration. This work will provide an in vivo measure of Ca2+ changes and their importance during apoptosis. Further, this study will help to establish the potential for bystander cell death in a population of cone photoreceptors and increase our understating of the timing and propagation of apoptosis in a cell population.
Keywords: retinal degenerations: cell biology • apoptosis/cell death • cell survival