Alcama is a novel marker of multipotent retinal stem cells in adult zebrafish. (A) PCNA⁺ progenitors (red) in the CMZ and nGFP⁺ immature Müller glia (arrowheads) in the INL express Rx1 (magenta). (B) GFP⁺ Müller glia express rlbp1a transcripts (magenta). (C) Retinal progenitors and immature Müller glia express BLBP (white/magenta). (D) Alcama (white/magenta) in PCNA⁺ (green) cells in the CMZ (boxed region shown in the overlay in the middle panel). Differentiating RGCs are indicated by asterisks. (E) Alcama⁺ cells (white/magenta) in the INL are nGFP⁺ immature Müller glia (boxed regions are shown at higher magnification in the insets at bottom right). (F) Alcama (white/magenta) colocalizes with a GFP⁺ Müller glial process (boxed regions shown at higher magnification in the insets at the upper left). Scale bars: 10 μm.

Müller glia partially dedifferentiate and express retinal progenitor markers in response to photoreceptor lesions. (A-D) PCNA (green), BLBP (cyan) and Rx1 (white/magenta) in control (ctrl) retina. Higher magnifications of the boxed regions are shown on the right. (A) Cones are Rx1⁺ (asterisks, UV cone nuclei). (B) By 1 hpl, Rx1 is reduced in UV cones and BLBP is upregulated in Müller glia. (C) At 8 hpl, cones collapse (asterisk) and Müller glia express BLBP and Rx1 (boxed region). A PCNA⁺ microglia or rod precursor (arrowhead) can be seen in the ONL. (D) At 1 dpl, Müller glia are weakly PCNA⁺/BLBP⁺/Rx1⁺. (E,F) Injury-induced nGFP in lesioned area (asterisks) at 4 and 8 hpl and in immature Müller glia (arrowheads). Zpr1 (magenta), which recognizes Arrestin 3a and specifically labels red/green cones. Higher magnifications of the boxed regions are shown in insets. (C) Reduced rlbp1a transcript (white) by 4 hpl and no detectable signal at 1 dpl. Scale bars: 10 μm (A-D,G); 100 μm (E,F).

Injury-induced Müller glia and retinal progenitors are distinct cell populations. (A) By 2 dpl, Müller glial nuclei (nGFP⁺, green) move to the OLM (dotted line). (B) nGFP⁺ (anti-GFP, green)/pH3⁺ (magenta) mitotic Müller glia nuclei at the OLM. nGFP is diffuse (arrows). Higher magnifications of the boxed regions are shown in insets. (C) nGFP⁺(green)/PCNA⁺ (red)/Rx1⁺ (white/magenta) progenitors at 3 dpl. (D) Alcama (white/magenta); nGFP. (E) Alcama (white/magenta) colocalizes with GFP. Scale bars: 20 μm (A,B,D,E); 5 μm (C).

Injury-induced Müller glia divide asymmetrically once to generate neurogenic clusters. (A) Sequential labeling with EdU and BrdU. Two predicted alternative results are depicted: if Müller glia (MG) divide only once, they incorporate only EdU (green); if they undergo multiple divisions, they incorporate both EdU and BrdU (green+magenta=white). (B) Some nGFP⁺ (green) nuclei in flat-mounted retinas at 42 hpl are EdU⁺ (red). (C) Pair of EdU⁺ (red) nuclei at 42 hpl, of which only one is PCNA⁺ (magenta). (D) Optical z-stack projection at 72 hpl: clusters of BrdU⁺ nuclei (magenta) with few EdU⁺ nuclei (green). Right panels: single optical section of EdU⁺/nGFP⁺/BrdU^- nucleus (arrows). (E) Lateral view of BrdU⁺ nuclei (magenta) clusters with a single EdU⁺ (green)/BrdU-nucleus, reconstructed from optical Z-stack. Right panel: BrdU⁺ clusters (magenta outline). (F) Cluster with single EdU⁺ cell (white/red) at 72 hpl. (G) Cluster of weakly EdU⁺ cells (white/red) with one strongly EdU⁺ cell. (H) Cluster with one PCNA^-/strongly GFP⁺ (green) cell (arrows) and PCNA⁺ (magenta)/weakly GFP⁺ progenitors. Scale bars: 10 μm.

Induction of neuroepithelial markers in Müller glia is delayed after ouabain. (A) Control (ctrl) HuC/D⁺ neurons. (B) HuC/D immunoreactivity is absent in the GCL and reduced in the INL at 1 dpi. (C) PCNA (green), BLBP (cyan) and Rx1 (magenta) staining at 2 dpi. Right panel: BLBP⁺/Rx1⁺ Müller glia. Scattered PCNA⁺/BLBP^-/Rx1^- cells in the INL and GCL are microglia. (D) At 3 dpi, nGFP⁺/PCNA⁺ (red)/Rx1⁺ (white/magenta) Müller glial nuclei are apically displaced. (E) At 3 dpi, pH3⁺ nuclei (magenta) are apical in the INL but not at the OLM (dotted line). (F) Alcama⁺ (white/magenta) Müller glia radial processes at 5 dpi. (G) At 5 dpi, nGFP⁺/HuC/D⁺ (red) regenerated neurons (higher magnification of boxed area shown in inset). Scale bars: 20 μm (A-G); 2 μm (inset in C).

Reduction of N-cadherin function interferes with formation of neurogenic clusters after light lesions. (A) N-cadherin (white/magenta) at OLM (between arrowheads) in unlesioned (ctrl) retina. (B) At 3 dpl, N-cadherin (white/magenta) in Müller glia and progenitors (green). (C) RT-PCR products of cdh2 digested with BsmBI. (D,E) N-cadherin (white/magenta) in the ONL at 3 dpl in cdh2^+/m117; mi2002 (D) and cdh2^+/m117; mi2004 (E). (F) Neurogenic clusters (PCNA⁺; green) in sib retinas at 3 dpl. (G) PCNA⁺ (green)/BLBP⁺ (red)/Rx1⁺ (white/magenta) progenitors in cdh2^+/m117 hets at 3 dpl. (H) Counts of PCNA⁺ progenitors in the INL and ONL at 3 dpl. ^*P<0.005, ^**P<0.0005. (I) Nuclei of nGFP⁺/Alcama⁺ (white/magenta) Müller glia in the INL and nGFP⁺/Alcama^- progenitors in the ONL at 3 dpl in cdh2^+/m117; mi2004. (J) pH3⁺ (magenta) nuclei at 3 and 5 dpl in sibs. (K) pH3⁺ (magenta) nuclei at 3 and 5 dpl in cdh2^+/m117 hets. (L) Fraction of pH3⁺ cells in INL and ONL after lesion. ^*P<0.0001. Box plots: median, 25th and 75th percentiles; whiskers show maximum and minimum data points. Scale bars: 20 μm.

Reduction of N-cadherin function interferes with formation of neurogenic clusters after light lesions. (A) N-cadherin (white/magenta) at OLM (between arrowheads) in unlesioned (ctrl) retina. (B) At 3 dpl, N-cadherin (white/magenta) in Müller glia and progenitors (green). (C) RT-PCR products of cdh2 digested with BsmBI. (D,E) N-cadherin (white/magenta) in the ONL at 3 dpl in cdh2^+/m117; mi2002 (D) and cdh2^+/m117; mi2004 (E). (F) Neurogenic clusters (PCNA⁺; green) in sib retinas at 3 dpl. (G) PCNA⁺ (green)/BLBP⁺ (red)/Rx1⁺ (white/magenta) progenitors in cdh2^+/m117 hets at 3 dpl. (H) Counts of PCNA⁺ progenitors in the INL and ONL at 3 dpl. ^*P<0.005, ^**P<0.0005. (I) Nuclei of nGFP⁺/Alcama⁺ (white/magenta) Müller glia in the INL and nGFP⁺/Alcama^- progenitors in the ONL at 3 dpl in cdh2^+/m117; mi2004. (J) pH3⁺ (magenta) nuclei at 3 and 5 dpl in sibs. (K) pH3⁺ (magenta) nuclei at 3 and 5 dpl in cdh2^+/m117 hets. (L) Fraction of pH3⁺ cells in INL and ONL after lesion. ^*P<0.0001. Box plots: median, 25th and 75th percentiles; whiskers show maximum and minimum data points. Scale bars: 20 μm.

Activation of microglia at early stages after light lesion.
In the unlesioned retina, resident microglia labeled with the specific marker 4C4 (magenta) are at the boundaries of the INL and the GCL. At 8 hpl activated microglia migrate into the ONL and infiltrate through the subretinal space (SR) from the choroid circulation; they actively phagocytose photoreceptor debris at 1 to 2 dpl. Scale: 20 μm.

Müller glia partially dedifferentiate but fail to reenter the cell cycle when photoreceptor degeneration is confined to loss of UV cones.
(A) Immunochemistry with double cone marker, zpr1 (magenta) in retinal cryosection of light-lesioned mi2004 fish with inducible nGFP (green) at 3 dpl; ON, optic nerve. Muller glia in dorsal (D) peripheral retina, where zpr1+ double cones are not destroyed by the intense light (bracket), re-express the nGFP reporter. Immature Muller glia at the ventral (V) margin adjacent to the CMZ express nGFP (arrow). (B) Immunocytochemistry for PCNA (magenta) in a retinal cryosection. PCNA+ neurogenic clusters at 3 dpl in the central retina where all cone subtypes are destroyed (asterisks). Inset, higher magnification of the boxed region. Progenitors in the CMZ are PCNA+ (arrows). (C) Whole, flat-mounted retina of light-lesioned (3 dpl) Tg(sws1:GFP;sws2:mCherry) fish. Blue (red) and UV cones (green) and red-green double cones (not shown) are missing in the central lesioned region bounded by dotted lines. UV cones (green) are ablated from most of the retina, including dorsal. (D) By 42 dpl both UV cones (green) and blue cones (red) have regenerated within the central region (dotted line). UV cones fail to regenerate in the regions where blue cones (along with red/ green double cones, not shown) were spared: Boxed area dorsal to the lesion shown at higher magnification in the upper right, shows organized rows of surviving blue cones (red), but very few UV cones (green). Regenerated cones in central retina are identifiable because they fail to recreate the precisely organized cone mosaic pattern (boxed area in lower right; also see Supplementary Figure S5). Scales: 100 μm, A, B; 200 μm C, D; 20 μm, D (high magnification).

Injury-induced Muller glia reenter the cell cycle and complete cell division between 36 and 42 hpl.
(A) PCNA (magenta) immunocytochemistry on flat-mounted retina from a light lesioned mi2004 fish with inducible nGPF (green) at 36 hpl; some nGFP+ Muller glia express PCNA (arrow). (B) Flat-mounted, unlesioned retina from mi2002 fish with gfap:GFP reporter, focused at the level of the basal processes of Müller glia (green) in the inner plexiform layer. (C) Müller glia in a lightlesioned mi2004 fish with inducible nGFP (green) in at 42 hpl. (D) Planimetric density of GFP+ Müller glial cells. Scales: 20 μm A, B, C. * p < 0.005.

Basal processes of Muller glia collapse and markers of differentiation are downregulated after intraocular ouabain injection.
(A, B) Immunocytochemistry for the Müller glial marker glutamine synthetase (GS) (white/magenta) in unlesioned (A) and 2 dpi (B) gfap:GFP retinas with Müller glial reporter (green). GS+/GFP+ Müller glial processes in the inner retina are disrupted, whereas the organization of the outer retina, including the adherens junctions at the outer limiting membrane, remain intact at 2 dpi. (C) PCNA+ (green) activated microglia (4C4+, magenta) are abundant in the damaged inner retina at 2 dpi. (D, E) In situ hybridization for another Müller glia marker, rlbp1a (white/magenta, D), shows expression is gone at 1 dpi (E). (F) Weak Alcama immunoreactivity (magenta/ white) appears in the basal process (arrow) of a Müller glia with the inducible nGFP reporter (green) at 3 dpi. Scales: 20 μm, A-E; 10 μm, F.

Regeneration of cone photoreceptors in Tg(sws1:GFP;sws2:mCherry); cdh2+/m117 heterozygote retinas.
(A, B) Immunocytochemistry for red/green double cone marker, zpr1 (magenta); UV cones (green), blue cones (red). In unlesioned retinas, all cone subtypes are present and appear morphologically normal in the cdh2+/m117 het retinas (B) similar to the wild-type sibs (A). (C-F) Immunocytochemistry for zpr1 (magenta) in unlesioned, flat-mounted retinas illustrates the organized cone mosaic pattern in wild-type sib (C) and cdh2+/m117 het retinas (E). At 14 dpl, all cone subtypes regenerate, but the mosaic pattern is not restored in sib (D) or cdh2+/m117 (F). Scales: 20 μm, A-F.

Injury-induced Müller glia produce proliferating retinal progenitors, but in cdh2+/m117 heterozygotes their morphology is abnormal after destruction of inner retinal neurons.
(A,B) Immunocytochemistry for PCNA (magenta) in wild-type sib and cdh2+/m117 het retinas after intraocular injection of ouabain. (A) In the wild-type sib, PCNA is expressed in Müller glial nuclei that migrate apically at 3 dpi, and PCNA+ progenitors form elongated, spindle-shaped neurogenic clusters at 5 dpi. The number of PCNA+ cells decreases at 7 dpi. (B) In the cdh2+/m117 hets, at 5 dpi PCNA+ progenitors fail to form cohesive, neurogenic clusters, and their nuclei are rounded, rather than spindle-shaped. The number of PCNA+ cells is reduced at 7 dpi, as in wild-type sibs. Scales: 20 μm, A,B.