Molecular characterization of retinal stem cells and their niches in adult zebrafish

Abstract

Background: The persistence in adult teleost fish of retinal stem cells that exhibit all of the features of true 'adult stem cells'--self-renewal, multipotency, and the capacity to respond to injury by mitotic activation with the ability to regenerate differentiated tissues--has been known for several decades. However, the specialized cellular and molecular characteristics of these adult retinal stem cells and the microenvironmental niches that support their maintenance in the differentiated retina and regulate their activity during growth and regeneration have not yet been elucidated.

Results: Our data show that the zebrafish retina has two kinds of specialized niches that sustain retinal stem cells: 1) a neuroepithelial germinal zone at the interface between neural retina and ciliary epithelium, called the ciliary marginal zone (CMZ), a continuous annulus around the retinal circumference, and 2) the microenvironment around some Müller glia in the differentiated retina. In the uninjured retina, scattered Müller glia (more frequently those in peripheral retina) are associated with clusters of proliferating retinal progenitors that are restricted to the rod photoreceptor lineage, but following injury, the Müller-associated retinal progenitors can function as multipotent retinal stem cells to regenerate other types of retinal neurons. The CMZ has several features in common with the neurogenic niches in the adult mammalian brain, including access to the apical epithelial surface and a close association with blood vessels. Müller glia in the teleost retina have a complex response to local injury that includes some features of reactive gliosis (up-regulation of glial fibrillary acidic protein, GFAP, and re-entry into the cell cycle) together with dedifferentiation and re-acquisition of phenotypic and molecular characteristics of multipotent retinal progenitors in the CMZ (diffuse distribution of N-cadherin, activation of Notch-Delta signaling, and expression of rx1, vsx2/Chx10, and pax6a) along with characteristics associated with radial glia (expression of brain lipid binding protein, BLBP). We also describe a novel specific marker for Müller glia, apoE.

Conclusion: The stem cell niches that support multi-lineage retinal progenitors in the intact, growing and regenerating teleost retina have properties characteristic of neuroepithelia and neurogenic radial glia. The regenerative capacity of the adult zebrafish retina with its ability to replace lost retinal neurons provides an opportunity to discover the molecular regulators that lead to functional repair of damaged neural tissue.

Figure 1

Molecular characterization of postembryonic neurogenesis in the CMZ of the zebrafish retina. A) and B) Larval zebrafish (4 dpf). A) Mitotic retinal progenitor cells in the CMZ (arrow) immunoreactive for PCNA (green). B) rx1 (magenta) in the CMZ (arrow) and in differentiated cones in the outer nuclear layer (ONL). L, lens; GCL, ganglion cell layer, INL, inner nuclear layer. Scale bar = 50 μm. C) – J) Two-month-old juvenile zebrafish retinas. C) Cells in peripheral CMZ are double-labeled (white arrow) with rx1 (magenta) and PCNA (green). D) Cells in peripheral CMZ are double-labeled (white arrow) with vsx2 (magenta) and PCNA (green). E) Amacrine cells in the INL and ganglion cells in the GCL express pax6a, and many PCNA⁺ cells in the CMZ are double-labeled with pax6a (white arrow). F) Cells in the peripheral CMZ co-express rx1 (magenta) and pax6a (green). Scale bar = 50 μm. G) Cells in the CMZ (arrow) express notch1a (magenta). H) PCNA⁺ cells (green) in the CMZ, nonpigmented cells in the adjacent ciliary margin and Müller glia (arrows) in the differentiated retina adjacent to the CMZ are BLBP⁺. BLBP-immunoreactivity gradually disappears from Müller glia more centrally. I) Immunoreactivity for SSEA-1/LeX (green) around the circumferential blood vessel (BV) and in a narrow band of the inner plexiform layer, IPL. J) Immunoreactivity for N-cadherin protein (Cdh, magenta) completely surrounds the cell bodies of PNCA⁺ (green) mitotic cells in the CMZ and cells in the adjacent non-pigmented ciliary epithelium. In the laminated retina, N-cadherin is localized to the zonula adherens junctions of the outer limiting membrane (arrowhead) and synaptic layers. An isolated PCNA⁺ cell in the inner nuclear layer (arrow) is an INL progenitor in the rod lineage. Nuclei are counterstained with DAPI (blue).

Figure 2

Definition of CMZ regions for the analysis of regionalized gene expression. Schematic drawing of histological landmarks that define four areas in the CMZ: peripheral, middle, central-inner and central-outer (shaded regions). The outer plexiform layer (OPL) divides central-inner from central-outer. The definitive ganglion cell layer (GCL) derives from the middle CMZ, reflecting their early birth order. Progenitors in central-inner CMZ are destined for the inner nuclear layer (INL) and the central-outer region produces cone photoreceptors in the outer nuclear layer (ONL). Arrows, blood vessels; RPE, retinal pigmented epithelium; OLM, outer limiting membrane; IPL, inner plexiform layer; M, Müller glial cell.

Figure 3

Injury-induced proliferating retinal progenitors express rx1, vsx2, and pax6a. A) – D) Histology of the heat-lesioned retina at 1 and 3 dpl; the boxed area in A) is magnified in B). Cell loss is mainly confined to the retinal pigmented epithelium (RPE) and photoreceptors in the outer nuclear layer, ONL. The CMZ (arrow in A) is at the junction between the neural retina and the ciliary epithelium (CE), which is continuous anteriorly with the iris epithelium (IE). Scale bar (A) = 150 μm. C) At 3 dpl double cones immunoreactive with zpr1 (red) are missing from the lesioned area and elongated nuclei appear in the inner nuclear layer (white arrows). Counterstained with DAPI (blue). D) By 3 dpl, radial fibers of Müller glia in the inner nuclear layer, INL, are visible in the region of the lesion (black arrows), indicative of reactive gliosis. Scale bar = 50 μm. D) Proliferating cells in the inner and outer nuclear layers of the retina in the lesioned area (asterisks) at 5 dpl incorporated BrdU (green) injected at 4 dpl. E) Injury-induced proliferating cells are also immunoreactive for proliferating cell nuclear antigen, PCNA (green) and are associated with radial fibers of Müller glia (magenta, anti-human GFAP). Note: the commercial polyclonal GFAP antibody used here is not selective for GFAP in zebrafish but labels other intermediate filaments (data not shown). In contrast, the monoclonal zrf1, which was generated against zebrafish proteins, selectively labels zebrafish GFAP [97]. Scale bar = 50 μm. F) – L) A 4 h or 24 h pulse of BrdU at 4 or 5 dpl labels clusters of nuclei (magenta) that extend between the INL and ONL and express rx1 (F) and vsx2 (J) as visualized by in situ hybridization (red). DAPI (blue); zrf1 (green, anti-zebrafish GFAP). Scale bar = 25 μm. Higher magnification views of rx1 (G,H, I) and vsx2 (K, L) in BrdU⁺ progenitors enclosed in a basket of zrf1⁺ Müller glial fibers.

Figure 4

Molecular profile of the injury-induced proliferating retinal progenitors is similar to retinal stem cells in the CMZ. A) At 4 dpl deltaC expression (dlc; magenta) is upregulated in the CMZ (arrow) and in cells in the INL beneath the lesion (asterisks). Scale bar = 150 μm. B) At 4 dpl, cells in the CMZ (arrow) and lesioned area labeled with a 2-hour pulse of BrdU (green) also express deltaC (magenta). C) notch1b (n1b; green) and D) notch3 (n3; green) are also up-regulated but are generally not co-expressed with deltaC (magenta). E) At 4 dpl, N-cadherin immunoreactivity (Cdh2; magenta) is strongly up-regulated in the lesioned area (asterisks) and diffusely localized. Inset: zrf1, anti-zebrafish-GFAP (green) in radial fibers of Müller glia in the lesioned retina (7 dpl) co-localizes with N-cadherin immunoreactivity (magenta). F) At 7 dpl, BrdU⁺ nuclei (green) associate with Müller glial radial fibers that are strongly immunoreactive for N-cadherin (magenta). DAPI (blue). G) At 3 dpl, activated Müller glia confined to the lesioned region (asterisks) express BLBP (magenta), a marker of immature Müller glia, and they are mitotically active (PCNA⁺, green). H) Radial fibers of injury-activated Müller glia are zrf1⁺ (blue). Proliferating Müller nuclei are PCNA⁺ (green) and many have migrated to the apical surface (the former ONL where photoreceptors are missing) and are BLBP⁺ (magenta).

Figure 5

Injury-induced retinal progenitors regenerate cone photoreceptors within a week. A) At 4 dpl, proliferating cells (BrdU; magenta) fill the lesioned area (asterisks) in which double cones immunoreactive for zpr1 (green) are missing. Scale bar = 250 μm. B) By 7 dpl, some retinal progenitors that were labeled with BrdU (magenta) at 4 dpl have begun to differentiate into cones and are double-labeled with zpr1 (green). Boxed areas are shown at higher magnification in the insets; double-labeled cells are white. C) By 31 dpl fully differentiated, regenerated cone photoreceptors (zpr1; green) are labeled with BrdU (magenta) injected at 3 dpl (double-labeled white nuclei are indicated by black arrows). Unidentified BrdU⁺ nuclei are seen in the inner retina. D) Cocktail of riboprobes to cone opsins (green) identifies BrdU⁺ (magenta), regenerated cones at 31 dpl (double-labeled white nuclei are indicated by black arrows). BrdU⁺ rod nuclei (magenta) are in the inner part of the outer nuclear layer, ONL. E) Müller glia in a transgenic zebrafish Tg(gfap:GFP)^mi2001 are labeled with anti-GFP (green) and co-express apoE (magenta, in situ hybridization) in their cell bodies. F) At 4 dpl, most BrdU⁺ proliferating nuclei are in the outer nuclear layer, ONL, but a few apoE⁺ Müller glial cells are also BrdU⁺ (inset). Radial fibers of Müller glia are labeled with zpr1/anti-GFAP (blue).

Figure 6

A) Time course of regeneration of cone and rod photoreceptors. The number of BrdU⁺ rod and cone photoreceptors per section at 31 dpl is plotted as a function of time of BrdU injection (in days post-lesion). Each bar is the mean number per section calculated by combining data from counts on two retinas and 4 to 17 sections per retina. The data were pooled after an ANOVA showed that there was no significant difference between the average counts between retinas at each time point. The error bars represent one SEM. Comparison of means with a single factor ANOVA showed that slightly more cones are born at 3 dpl than at 4 dpl (p < 0.05), fewer at 7 dpl (p < 0.001) and almost none at 10 dpl (data not shown). In contrast, significantly fewer rods than cones are born at 3 and 4 dpl (p < 0.001), but many more rods than cones are born at 7 (p < 0.001) and 10 dpl (data not shown). B) Most of the proliferating cells at 2 dpl are Müller glia. Fish with heat lesioned retinas were exposed to a 4 h pulse of BrdU at 2, 3 or 4 dpl. Retinas were processed for in situ hybridization with an apoE probe to label cell bodies of Müller glia and for BrdU immunocytochemistry. Each bar represents one lesion, and 2 to 9 sections were counted for each lesion. All BrdU⁺ nuclei were counted and scored for apoE. The mean number of BrdU⁺ nuclei per section is given above each bar. Statistical analysis of the data by single factor ANOVA indicates that the percent apoE⁺ cells in the BrdU⁺ population at 2 dpl greater than at 3 dpl (p < 0.01) or at 4 dpl (p < 0.01) but 3 dpl does not differ from 4 dpl.

Figure 7

Stem cell niches in zebrafish retina. The germinal zone at the boundary between neural retina and ciliary epithelium (CE) is a circumferential wedge of neuroepithelial cells (in red) called the ciliary marginal zone (CMZ). Multipotent retinal stem cells span the width of the retinal epithelium adjacent to the CE and more restricted retina progenitors give rise sequentially to 1) retinal ganglion cells (GC), 2) amacrine cells (AC), 3) bipolar (BP) and horizontal cells (HC), and 4) cone photoreceptors. The CMZ is separated from the retinal pigmented epithelium (RPE) by a narrow subretinal space. The ora collection blood vessel (BV) encircles the retina at the CMZ; other blood vessels of the hyaloid circulation lie along the vitreal surface. Müller glia (blue) span the width of the retina and create a niche that supports retinal progenitors of the last-born retinal neuron in the 5) rod photoreceptor lineage (green). Expression of specific markers is shown for retinal progenitors/stem cells and associated cell types. Retinal injury induces a reorganization of the Müller cell/rod lineage niche to produce a regeneration niche (purple) that mirrors the CMZ niche in patterns of cellular organization and gene expression.