Abstract
Purpose: :
Progranulin-A (PgrnA) is a multi-functional protein, which in mammals plays roles in development, tumorigenesis and wound repair. In zebrafish, pgrnA expression is strongly induced following selective photoreceptor death and during photoreceptor regeneration. The purpose of this study was to determine the cellular expression and function of PgrnA both during retinal development and photoreceptor regeneration.
Methods: :
PgrnA was identified by expression profiling using Affymetrix GeneChip® Zebrafish Genome Arrays following the light-induced death of photoreceptors. To establish the cell type expression pattern of pgrnA , RNA in situ hybridization and immunocytochemistry (individually and in combination) were performed. To determine PgrnA function during retinal development, morpholino oligonucleotides were injected into early embryos to specifically inhibit PgrnA protein translation, which was documented by Western blot analysis. Retinal development was evaluated at 72 hours post fertilization (hpf) and 7 days post fertilization (dpf) in cryosections using markers of cell proliferation and cell type-specific antibodies.
Results: :
In adults following photoreceptor injury, pgrnA is upregulated both by microglia that are intrinsic to the retina and microglia that accumulate in the subretinal space overlying the depleted outer nuclear layer. In embryos, pgrnA is also expressed by microglia. Following knockdown of PgrnA protein, the overall development of morphant embryos is largely unaltered, however, differentiation of retinal neurons is nearly completely inhibited. At 72 hpf, a time when retinal differentiation in control animals is nearing completion, pgrnA morphant retinas consist of an undifferentiated neuroepithelium. At 7dpf, 3 or 4 days after the time when morpholino-dependent translation inhibition is lost, pgrnA morphant retinas are fully differentiated, revealing a PgrnA-specific recovery.
Conclusions: :
PgrnA is a microglia-specific signaling molecule, which in adults is induced by photoreceptor death and regeneration and in embryos regulates neuronal differentiation during development. This study highlights the fundamental roles that non-neuronal cells play following retinal injury and during retinal development.
Keywords: retinal development • retinal glia • regeneration