Abstract
BackgroundThe zebrafish retina maintains two populations of stem cells: first, the germinal zone or ciliary marginal zone (CMZ) contains multipotent retinal progenitors that add cells to the retinal periphery as the fish continue to grow; second, radial glia (Müller cells) occasionally divide asymmetrically to generate committed progenitors that differentiate into rod photoreceptors, which are added interstitially throughout the retina with growth. Retinal injury stimulates Müller glia to dedifferentiate, re-enter the cell cycle, and generate multipotent retinal progenitors similar to those in the CMZ to replace missing neurons. The specific signals that maintain these two distinct populations of endogenous retinal stem cells are not understood.ResultsWe used genetic and pharmacological manipulation of the β-catenin/Wnt signaling pathway to show that it is required to maintain proliferation in the CMZ and that hyperstimulation of β-catenin/Wnt signaling inhibits normal retinal differentiation and expands the population of proliferative retinal progenitors. To test whether similar effects occur during regeneration, we developed a method for making rapid, selective photoreceptor ablations in larval zebrafish with intense light. We found that dephosphorylated β-catenin accumulates in Müller glia as they re-enter the cell cycle following injury, but not in Müller glia that remain quiescent. Activation of Wnt signaling is required for regenerative proliferation, and hyperstimulation results in loss of Müller glia from the INL as all proliferative cells move into the ONL.Conclusionsβ-catenin/Wnt signaling is thus required for the maintenance of retinal progenitors during both initial development and lesion-induced regeneration, and is sufficient to prevent differentiation of those progenitors and maintain them in a proliferative state. This suggests that the β-catenin/Wnt cascade is part of the shared molecular circuitry that maintains retinal stem cells for both homeostatic growth and epimorphic regeneration.
Highlights
The zebrafish retina maintains two populations of stem cells: first, the germinal zone or ciliary marginal zone (CMZ) contains multipotent retinal progenitors that add cells to the retinal periphery as the fish continue to grow; second, radial glia (Müller cells) occasionally divide asymmetrically to generate committed progenitors that differentiate into rod photoreceptors, which are added interstitially throughout the retina with growth
We found that activation of Wnt signaling by inhibition of Glycogen synthase kinase 3β (GSK3β) with 1-azakenpaullone, which has been previously used predominantly in cell culture, works well in zebrafish in vivo and phenocopies the effects on eye development seen in axin1-deficient fish [20,21]. 1azakenpaullone is highly selective for GSK3β, though it can inhibit cyclin-dependent kinases (CDKs) at higher concentrations [25]
As the phenotype that we see is an increase in proliferation rather than an decrease that would be expected if 1-azakenpaullone was inhibiting CDKs, and since the effects of 1-azakenpaullone can be blocked with dominant-negative TCF which is downstream of GSK3β in the Wnt signaling cascade, we conclude that the primary effect of 1-azakenpaullone in our system is inhibition of GSK3β to stimulate the β-catenin/Wnt cascade
Summary
The zebrafish retina maintains two populations of stem cells: first, the germinal zone or ciliary marginal zone (CMZ) contains multipotent retinal progenitors that add cells to the retinal periphery as the fish continue to grow; second, radial glia (Müller cells) occasionally divide asymmetrically to generate committed progenitors that differentiate into rod photoreceptors, which are added interstitially throughout the retina with growth. The fish retina increases in size from a proliferating neuroepithelium residing at the perimeter of the neural retina adjacent to the ciliary epithelium, termed the ciliary marginal zone (CMZ) [1,2] These stem cells produce multipotent progenitors capable of differentiating into all retinal cell fates. The normally quiescent Müller glial cells within the differentiated central regions of the teleost retina dedifferentiate and reenter the cell cycle to produce multipotent progenitors that replace the lost cells [2,3,4,5,6] It is not clear whether the cell-cell signals that transiently maintain the regenerative stem cell pool following injury are the same as those involved in maintaining the permanent stem cell compartment in the CMZ. Wnt signaling has been implicated in the limited, non-regenerative, Müller-glial proliferation that occurs following injury in the murine retina [17,18], and was recently suggested to be necessary and sufficient for Müller glial proliferation in the regenerating adult zebrafish retina [19]
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