Introduction
In the teleost retina, extensive neuronal death [
20,
30] or the selective death of photoreceptors [
35,
38] stimulates intrinsic stem cells to proliferate and give rise to regenerated neurons, which then integrate into existing neural circuits (see [
18]). Consequently, the teleost retina is an ideal system to study the intrinsic cellular and molecular mechanisms that allow stem cells to regenerate neurons in the vertebrate central nervous system (CNS). Identifying and testing the molecular determinants of neuronal regeneration in the teleost retina should yield results that will have implications for understanding the regenerative potential of stem cells in the human retina and brain and the potential use of stem-cell-based therapies to treat CNS injuries or disease.
Significant progress has been made in characterizing the cells that are involved in mediating neuronal regeneration of the teleost retina. Injury to photoreceptors was long considered necessary to elicit regeneration [
30], although a recent study showed that cell death within the inner nuclear layer (INL) that spares photoreceptors is sufficient [
11]. Irrespective of the locus of cell death, Müller glia are the regenerative stem cells [
3,
9], and re-expression of developmental regulatory genes and proliferation by Müller glia are required for neuronal regeneration [
10,
33]. Although dying neurons and Müller glia are obligatory components of this regenerative neurogenesis, microglia resident within the retina migrate to the site of injury [
31,
38] and are also likely critical for neuronal regeneration. Finally, in teleosts, photoreceptors are regenerated in their normal birth order, cones before rods [
23,
29,
38]. Following photoreceptor-only death, injury-induced progenitors serve exclusively as “cone progenitors.” Therefore, the teleost retina also provides a model in which to elucidate the molecular mechanisms that govern the selective regeneration of cones.
Since none of the cell types described above is unique to teleost fish [
12,
25,
26], a fundamental difference between animals capable of neuronal regeneration and those that are not may be the molecules elicited in response to injury. Previous studies have described the transcriptional changes that occur in the teleost retina in response to either global [
6] or photoreceptor-specific injuries [
21], and these studies have provided insight into the molecular basis for neuronal regeneration. Particularly revealing are changes in the molecular signature of Müller glia as they adopt the features of neural stem cells [
21]. However, knowledge about signaling molecules in the local microenvironment of a retinal injury and the transcriptional events they activate during neuronal death and regeneration is still lacking.
As a means to identify genes necessary for photoreceptor regeneration, we evaluated transcriptional changes for cells in the outer nuclear layer (ONL) as photoreceptors die and are regenerated. To accomplish this, we combined light-induced photoreceptor lesions, laser-capture microdissection (LCM) of the ONL and analysis of gene expression using oligonucleotide arrays. By selectively harvesting cells from the ONL, we were able to limit our analysis to transcriptional changes among cells within the site of injury. By this approach, we hope further to characterize the molecular signatures of injured and dying photoreceptors, cone progenitors, and activated microglia. Further, we are particularly interested in identifying novel extracellular signaling molecules involved in these regenerative events, and here we describe the cellular expression of three such factors.
Materials and methods
Histology
Fish were anesthetized in 0.1% methane sulfonate salt (MS222; Sigma Aldridge, St. Louis, MO, USA). Eyecups were then dissected, fixed overnight in 4% paraformaldehyde, cryoprotected by infiltration in 20% sucrose in phosphate buffer, and frozen in OCT (Sakura Finetek, Torrance, CA, USA). Radial sections (8 μm) were cut with a cryostat and mounted on glass slides.
Immunohistochemistry was performed using standard procedures. Briefly, sections were rinsed in phosphate-buffered saline and 0.5% triton X-100 (PBST), incubated with 20% normal goat serum (NGS) in PBST, followed by overnight incubation at 4°C in primary antibody (anti-4C4, gift from Dr. Pamela Raymond; anti-zebra-fish galectin-1-like-2, gift from Geraldo Vasta; rabbit anti-GFP, Invitrogen Corp., Carlsbad, CA, USA) at a concentration of 1:200 diluted in 2% NGS–PBST. After washing with PBST, sections were incubated in fluorescently labeled Alexafluor 555 or Alexa 488 secondary antibodies diluted 1:500 in 2% NGS–PBST (Molecular Probes, Eugene, OR) for 1.5 h at room temperature, washed extensively in PBST, and sealed with mounting media and glass coverslips. Sections were counterstained with 1:1,000 dilution of bisbenzimide to label nuclei. For immunostaining with antibodies against proliferating cell nuclear antigen (PCNA; Sigma clone PC-10, St. Louis, MO, USA), slides were first boiled for 20 min in 10 mM sodium citrate, pH 6.0, with 0.05% Tween 20 prior to the first rinse in PBST.
To identify dying cells in cryosections, terminal deoxynucleotide end labeling (TUNEL) with TUNEL kit (Roche Diagnostics, Indianapolis, IN, USA) was used according to the manufacturer’s protocol.
In situ hybridization was performed on cryosections as previously described [
19]. Briefly, sense and antisense riboprobes were synthesized from linearized plasmids, and digoxigenin (DIG)-labeled probes were generated by in vitro transcription using the DIG RNA labeling kit (Roche Diagnostics, Indianapolis, IN, USA). Following prehybridization, 200 ng of probe in 80 μl of hybridization solution was pipetted onto each slide, coverslipped, and hybridized overnight at 55°C. The next day, the sections were washed and digoxigenin was immunolabeled using an alkaline-phosphatase-conjugated antibody. NBT–BCIP or Fast Red (Roche Diagnostics, Indianapolis, IN, USA) served as the enzymatic substrate. After the color reaction, sections were fixed in 4% paraformaldehyde for 15 min before proceeding with immunohistochemistry.
The cDNA encoding galectin-9-like 1 (Accession number BC059573, Open Biosystems, Huntsville, AL, USA) was linearized with EcoRI, and riboprobes were synthesized with T7 polymerase. Similarly, the cDNA encoding progranulin-a (Accession number NM001001949; a gift from Dr. Hugh Bennett) was linearized with SmaI, and riboprobes were synthesized with T3 polymerase. Negative controls were riboprobes encoding the sense strand of the respective cDNAs, which failed to hybridize to retinal sections (data not shown).
Sections were photographed with using a Nikon E300 photomicroscope and a Nikon DMX 1200 digital camera. Images were compiled in Adobe Photoshop CS2 (Adobe, San Jose, CA, USA). Images were resized and occasionally modified for contrast and brightness using the Image-Adjustments-Contrast–Brightness setting. All images within an experiment were manipulated in exactly the same manner.
Discussion
The dataset described in this report provides the vision community with a resource to mine for transcriptional changes that regulate the first stages of photoreceptor regeneration, from death to the specification of photoreceptor progenitors. Early transcriptional changes were observed in genes likely expressed by injured photoreceptors. Later transcriptional changes allowed us to identify novel genes expressed in Müller glia and cone progenitors, which give rise to regenerated photoreceptors, and microglia, which may also play a crucial role in photoreceptor regeneration. More broadly, this dataset provides information about the molecular mechanisms that regulate injury-induced neurogenesis in an adult nervous system and thus has utility for neuroscience in general.
By combining LCM of the ONL with a time course of injury-induced gene expression, we were able nominally to assign genes to each of the three cell types the cellular analysis identified within the lesioned ONL. We predicted that changes in gene expression that occur during the first 24 h are largely attributable to the injured photoreceptors. Thus, as anticipated, we observed transcriptional changes in known injury-induced genes at the early time points, including the heat shock proteins. Similarly, we detected a decrease in photoreceptor-specific genes, including genes encoding opsins and phototransduction proteins. The decrease in the expression of these genes is most easily explained by the apoptotic loss of photoreceptors. Nonetheless, the observed decrease in photoreceptor-specific genes serves to validate the logic behind our approach to assign early gene changes to photoreceptors. In the mammalian retina, injury to photoreceptors results in increased transcription of
endothelin2, which encodes a secreted protein that signals through endothelin B receptors on Müller glia [
28]. We anticipate that similar signaling events occur in the regenerating retina of teleosts (although no changes in endothelin 1 or 2 were observed), and genes encoding molecules that signal from photoreceptors to Müller glia are represented in the dataset. One category of genes worth investigating further are the stress response genes. Recent studies have suggested that heat shock proteins may be secreted [
8] and thus may serve as extracellular signaling molecules during the early events of photoreceptor regeneration.
In addition, we anticipated identifying known and novel genes expressed by cone progenitors in the ONL at the 48-h time point. It should be noted, however, that the changes in the expression of these genes, and in contrast to the loss of photoreceptors, are most easily explained by the accretion of cone progenitors migrating from the INL to the ONL. Nonetheless, our approach allowed us to identify putative cone-progenitor-specific genes, and these data will provide insights into the molecular mechanisms that govern the terminal steps of cone specification and differentiation. In support of this hypothesis, we detected an increase in the expression of lgals1l2, which encodes a secreted protein, and demonstrated that Lgals1l2 is expressed by putative cone progenitors (and microglia and Müller glia).
Based on our cellular analysis, we also anticipated identifying genes expressed by reactive microglia that migrate to the damaged ONL. Microglia are a little studied cell type but one whose importance in various developmental and disease states is becoming recognized [
14,
24]. Of note, each of the genes encoding the secreted proteins
lgals1l2,
lgals9l1, and
prgna is expressed by resident microglia after injury to photoreceptors. Microglia are phagocytic cells that can secrete both proinflammatory and anti-inflammatory signals to either inhibit or promote neuronal repair and regeneration [
14]. Therefore, microglial-derived secreted signals are likely important regulators of cone photoreceptor regeneration. Functional evaluation of microglial-specific genes should provide further insights into the mechanisms that regulate photoreceptor (and neuronal) regeneration.
Gene array technologies have been used previously to profile transcriptional changes in zebra fish during regeneration of the fin [
32], heart [
22], and retina [
6,
21]. Recently, LCM and gene arrays were combined to identify transcriptional changes in retinal ganglion cells during axonal regeneration [
34]. Of note, some of the gene coding for secreted factors identified in our dataset are also upregulated during heart and fin regeneration [
22,
32]. The injury-induced upregulation of these genes suggest that in zebra fish common molecular mechanisms may regulate all regenerative processes. However, further comparisons of datasets from zebra fish arrays should also reveal molecular signatures that are specific to retinal stem cells and the regeneration of cone photoreceptors. Finally, although the annotation of the zebra fish genome does not yet match that for mammals, comparing transcriptional changes in the injured and regenerating retina of zebra fish with similar studies conducted in mammals [
7,
28] may begin to identify molecular mechanisms that are essential to control the fates of stem cells in the mammalian retina and brain.
Acknowledgements
We thank Drs. Pamela Raymond for the transgenic (gfap:GFP)mi2001 reporter fish and the 4C4 antibody, Geraldo Vasta for the anti-zebra-fish Galectin-1-like-2 antibody and Hugh Bennett for the progranulin-a cDNA. We also thank Dr. David Reed, Matthew Brooks, and Ritu Khanna for their help in conducting and analyzing the microarray experiments and Erica Dawsey and Laura Kakuk-Atkins for technical assistance. This work was supported by the National Institutes of Health Grants R01-EY007060 and P30-EY07003 (PFH) and T32-EY13934 (S.E.L.C.) and Research to Prevent Blindness. S.E.L.C. was also supported by a postdoctoral fellowship from the Foundation Fighting Blindness-Canada.