Teleost Retina Research Articles

Rod precursors in the adult retina of the Austrolebias charrua annual fish

Rod photoreceptors in the adult teleost retina are produced by rod precursors located in the outer nuclear layer (ONL). Annual fishes of the genus Austrolebias exhibit extensive adult retinal cell proliferation and neurogenesis, as well as surprising adaptive strategies to their extreme and changing environment, including adult retinal plasticity. Thus, here we identify and characterize rod precursors in the ONL of the Austrolebias charrua retina. For this aim we used classical histological techniques, transmission electron microscopy, detection of cell proliferation, and immunohistochemistry. Through these complementary approaches, we describe a cell population clearly distinguishable from photoreceptors in the ONL of the adult retina of A. charrua, which we propose corresponds to the rod precursor population. These cells exhibited particular morphological and ultrastructural characteristics, uptake of cell proliferation markers (BrdU+) and expression of stem cell markers (Sox2+). Determining the existence of the population of rod precursors is crucial to understand the sequence of events related to retinal plasticity and regeneration.

Decline in Constitutive Proliferative Activity in the Zebrafish Retina with Ageing.

It is largely assumed that the teleost retina shows continuous and active proliferative and neurogenic activity throughout life. However, when delving into the teleost literature, one finds that assumptions about a highly active and continuous proliferation in the adult retina are based on studies in which proliferation was not quantified in a comparative way at the different life stages or was mainly studied in juveniles/young adults. Here, we performed a systematic and comparative study of the constitutive proliferative activity of the retina from early developing (2 days post-fertilisation) to aged (up to 3–4 years post-fertilisation) zebrafish. The mitotic activity and cell cycle progression were analysed by using immunofluorescence against pH3 and PCNA, respectively. We observed a decline in the cell proliferation in the retina with ageing despite the occurrence of a wave of secondary proliferation during sexual maturation. During this wave of secondary proliferation, the distribution of proliferating and mitotic cells changes from the inner to the outer nuclear layer in the central retina. Importantly, in aged zebrafish, there is a virtual disappearance of mitotic activity. Our results showing a decline in the proliferative activity of the zebrafish retina with ageing are of crucial importance since it is generally assumed that the fish retina has continuous proliferative activity throughout life.

Active Blood Acidification Greatly Enhances Oxygen Supply to the Teleost Retina

Most ray-finned fishes possess a unique system for retinal oxygen delivery, where intracellular acidification of red blood cells (RBCs) in the retinal capillaries induces oxygen offloading from pH-sensitive hemoglobin. A countercurrent vascular network, the choroid rete mirabile, posterior to the retina augments this “oxygen-secretory” mechanism and confers retinal PO2 values that can exceed 1300 mmHg in some species. We hypothesized that the choroid rete mirabile exerts an active role in intracellular RBC acidification to facilitate retinal oxygen secretion. We show the presence and enzymatic activities of extracellular carbonic anhydrase (CA) and vacuolar-type H+-ATPase (VHA) in the lumen of the choroid rete mirabile. These data are consistent with a pathway, where VHA secretes H+ into the choroid rete mirabile lumen, which combines with HCO3– to form CO2 (catalyzed by extracellular CA) that rapidly diffuses into the RBC facilitating hemoglobin oxygen offloading. Next, we validated the model in vivo by injecting an extracellular CA inhibitor (C18) into the dorsal aorta of anesthetized rainbow trout with a PO2 electrode implanted into the retina. Here, the inhibition of extracellular CA resulted in a rapid reduction in retinal PO2 from 517 to 226 mmHg within minutes. These data strongly support the proposed model for RBC acidification in the choroid rete mirabile. Further, in vivo data show that this mechanism primarily augments oxygen diffusion to the inner retinal segments involved in signal-processing and that the evolution of this pathway was tightly associated with the morphological expansion of this part of the retina. Together, these data suggest that the evolutionary origin of retinal oxygen secretion in the early history of the ray-finned fishes was associated with a greatly enhanced capacity for visual processing, potentially permitting improved visual prey detection and the colonization of novel environments.

The lasting after-effects of an ancient polyploidy on the genomes of teleosts.

The ancestor of most teleost fishes underwent a whole-genome duplication event three hundred million years ago. Despite its antiquity, the effects of this event are evident both in the structure of teleost genomes and in how the surviving duplicated genes still operate to drive form and function. I inferred a set of shared syntenic regions that survive from the teleost genome duplication (TGD) using eight teleost genomes and the outgroup gar genome (which lacks the TGD). I then phylogenetically modeled the TGD’s resolution via shared and independent gene losses and applied a new simulation-based statistical test for the presence of bias toward the preservation of genes from one parental subgenome. On the basis of that test, I argue that the TGD was likely an allopolyploidy. I find that duplicate genes surviving from this duplication in zebrafish are less likely to function in early embryo development than are genes that have returned to single copy at some point in this species’ history. The tissues these ohnologs are expressed in, as well as their biological functions, lend support to recent suggestions that the TGD was the source of a morphological innovation in the structure of the teleost retina. Surviving duplicates also appear less likely to be essential than singletons, despite the fact that their single-copy orthologs in mouse are no less essential than other genes.

Evolutionary history of teleost intron-containing and intron-less rhodopsin genes

Recent progress in whole genome sequencing has revealed that animals have various kinds of opsin genes for photoreception. Among them, most opsin genes have introns in their coding regions. However, it has been known for a long time that teleost retinas express intron-less rhodopsin genes, which are presumed to have been formed by retroduplication from an ancestral intron-containing rhodopsin gene. In addition, teleosts have an intron-containing rhodopsin gene (exo-rhodopsin) exclusively for pineal photoreception. In this study, to unravel the evolutionary origin of the two teleost rhodopsin genes, we analyzed the rhodopsin genes of non-teleost fishes in the Actinopterygii. The phylogenetic analysis of full-length sequences of bichir, sturgeon and gar rhodopsins revealed that retroduplication of the rhodopsin gene occurred after branching of the bichir lineage. In addition, analysis of the tissue distribution and the molecular properties of bichir, sturgeon and gar rhodopsins showed that the abundant and exclusive expression of intron-containing rhodopsin in the pineal gland and the short lifetime of its meta II intermediate, which leads to optimization for pineal photoreception, were achieved after branching of the gar lineage. Based on these results, we propose a stepwise evolutionary model of teleost intron-containing and intron-less rhodopsin genes.

Distribution and density of mixed-input ON bipolar cells of the goldfish (Carassius auratus) during growth.

Neurons are continuously produced at different rates and locations in the teleost retina. Goldfish rods are homogeneously distributed and maintain a stable density throughout growth, whereas little is known about their postsynaptic partners. We examined the distribution and density of mixed-input ON bipolar cells (ON mBCs) in 57 goldfish of various sizes by immunolabeling their retinas with an antibody against PKCα and counting PKCα-positive neurons in wholemounts. Cell densities were correlated with morphometric data for the same animals, and the spatial resolution of the ON mBC mosaic was calculated in each case. The distribution of ON mBCs is homogeneous throughout growth. For a 10-fold change in body size (i.e., from 20 to 200 mm), the total number of ON mBCs increases 2.8 times, while retinal area expands around 10 times. As a consequence, the density of ON mBCs in large fish falls to ∼1/3 of that of small animals, and intercellular spacing doubles. The eye and the lens become around three times larger from small to large fish. This causes the retinal magnification factor (and thereby the image projected onto retina) to augment by the same amount. Because the retinal magnification factor rises more than the intercellular spacing in the same animals, the spatial resolution of the ON mBC mosaic improves from 0.8 to 1.4 cycles/degree as the body size increases from 20 to 200 mm. As ON mBCs are mostly rod-driven, our results suggest that the scotopic acuity of the goldfish may improve as the animal grows.

Photo-regulation of rod precursor cell proliferation

Teleosts are unique in their ability to undergo persistent neurogenesis and to regenerate damaged and lost retinal neurons in adults. This contrasts with the human retina, which is incapable of replacing lost retinal neurons causing vision loss/blindness in the affected individuals. Two cell populations within the adult teleost retina generate new retinal neurons throughout life. Stem cells within the ciliary marginal zone give rise to all retinal cell types except for rod photoreceptors, which are produced by the resident Müller glia that are located within the inner nuclear layer of the entire retina. Understanding the mechanisms that regulate the generation of photoreceptors in the adult teleost retina may ultimately aid developing strategies to overcome vision loss in diseases such as retinitis pigmentosa. Here, we investigated whether photic deprivation alters the proliferative capacity of rod precursor cells, which are generated from Müller glia. In dark-adapted retinas, rod precursor cell proliferation increased, while the number of proliferating Müller glia and their derived olig2:EGFP-positive neuronal progenitor cells was not significantly changed. Cell death of rod photoreceptors was excluded as the inducer of rod precursor cell proliferation, as the number of TUNEL-positive cells and l-plastin-positive microglia in both the outer (ONL) and inner nuclear layer (INL) remained at a similar level throughout the dark-adaptation timecourse. Rod precursor cell proliferation in response to dark-adaptation was characterized by an increased number of EdU-positive cells, i.e. cells that were undergoing DNA replication. These proliferating rod precursor cells in dark-adapted zebrafish differentiated into rod photoreceptors at a comparable percentage and in a similar time frame as those maintained under standard light conditions suggesting that the cell cycle did not stall in dark-adapted retinas. Inhibition of IGF1-receptor signaling reduced the dark-adaptation-mediated proliferation response; however, caloric restriction which has been suggested to be integrated by the IGF1/growth hormone signaling axis did not influence rod precursor cell proliferation in dark-adapted retinas, as similar numbers were observed in starved and normal fed zebrafish. In summary, photic deprivation induces cell cycle entry of rod precursor cells via IGF1-receptor signaling independent of Müller glia proliferation.

Müller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish.

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after severe damage. Investigating the regeneration pathways in zebrafish might bring new insights to develop innovative strategies for the treatment of retinal degenerative diseases in mammals. Herein, we focused on the induction of a focal lesion to the outer retina in adult zebrafish by means of a 532 nm diode laser. A localized injury allows investigating biological processes that take place during retinal degeneration and regeneration directly at the area of damage. Using non-invasive optical coherence tomography (OCT), we were able to define the location of the damaged area and monitor subsequent regeneration in vivo. Indeed, OCT imaging produces high-resolution, cross-sectional images of the zebrafish retina, providing information which was previously only available with histological analyses. In order to confirm the data from real-time OCT, histological sections were performed and regenerative response after the induction of the retinal injury was investigated by immunohistochemistry.

Müller glia and phagocytosis of cell debris in retinal tissue.

Müller cells are the predominant glial cell type in the retina of vertebrates. They play a wide variety of roles in both the developing and the mature retina that have been widely reported in the literature. However, less attention has been paid to their role in phagocytosis of cell debris under physiological, pathological or experimental conditions. Müller glia have been shown to phagocytose apoptotic cell bodies originated during development of the visual system. They also engulf foreign molecules that are injected into the eye, cone outer segments and injured photoreceptors. Phagocytosis of photoreceptor cell debris in the light-damaged teleost retina is primarily carried out by Müller cells. Once the microglial cells become activated and migrate to the photoreceptor cell layer, the phagocytic activity of Müller cells progressively decreases, suggesting a possible mechanism of communication between Müller cells and neighbouring microglia and photoreceptors. Additionally, it has been shown that phagocytic Müller cells acquire proliferating activity in the damaged teleost retina, suggesting that engulfment of apoptotic photoreceptor debris might stimulate the Müller glia to proliferate during the regenerative response. These findings highlight Müller glia phagocytosis as an underlying mechanism contributing to degeneration and regeneration under pathological conditions.

Rhythmic expressed clock regulates the transcription of proliferating cellular nuclear antigen in teleost retina

Teleost fish continues to grow their eyes throughout life with the body size. In Astatotilapia burtoni, the fish retina increases by adding new retinal cells at the ciliary marginal zone (CMZ) and in the outer nuclear layer (ONL). Cell proliferation at both sites exhibits a daily rhythm in number of dividing cells. To understand how this diurnal rhythm of new cell production is controlled in retinal progenitor cells, we studied the transcription pattern of clock genes in retina, including clock1a, clock1b, bmal1a (brain and muscle ARNT-Like), and per1b (period1b). We found that these genes have a strong diurnal rhythmic transcription during light-dark cycles but not in constant darkness. An oscillation in pcna transcription was also observed during light-dark cycles, but again not in constant darkness. Our results also indicate an association between Clock proteins and the upstream region of pcna (proliferating cellular nuclear antigen) gene. A luciferase reporter assay conducted in an inducible clock knockdown cell line further demonstrated that the mutation on predicted E-Boxes in pcna promoter region significantly attenuated the transcriptional activation induced by Clock protein. These results suggested that the diurnal rhythmic expression of clock genes in A. burtoni retina could be light dependent and might contribute to the daily regulation of the proliferation of the retina progenitors through key components of cell cycle machinery, for instance, pcna.

Opposing Actions of Fgf8a on Notch Signaling Distinguish Two Muller Glial Cell Populations that Contribute to Retina Growth and Regeneration

The teleost retina grows throughout life and exhibits a robust regenerative response following injury. Critical to both these events are Muller glia (or, Muller glial cells; MGs), which produce progenitors for retinal growth and repair. We report that Fgf8a may be an MG niche factor that acts through Notch signaling to regulate spontaneous and injury-dependent MG proliferation. Remarkably, forced Fgf8a expression inhibits Notch signaling and stimulates MG proliferation in young tissue but increases Notch signaling and suppresses MG proliferation in older tissue. Furthermore, cessation of Fgf8a signaling enhances MG proliferation in both young and old retinal tissue. Our study suggests that multiple MG populations contribute to retinal growth and regeneration, and it reveals a previously unappreciated role for Fgf8a and Notch signaling in regulating MG quiescence, activation, and proliferation.

Characterization and Evolution of the Spotted Gar Retina.

In this study, we characterize the retina of the spotted gar, Lepisosteus oculatus, a ray-finned fish. Gar did not undergo the whole genome duplication event that occurred at the base of the teleost fish lineage, which includes the model species zebrafish and medaka. The divergence of gars from the teleost lineage and the availability of a high-quality genome sequence make it a uniquely useful species to understand how genome duplication sculpted features of the teleost visual system, including photoreceptor diversity. We developed reagents to characterize the cellular organization of the spotted gar retina, including representative markers for all major classes of retinal neurons and Müller glia. We report that the gar has a preponderance of predicted short-wavelength shifted (SWS) opsin genes, including a duplicated set of SWS1 (ultraviolet) sensitive opsin encoding genes, a SWS2 (blue) opsin encoding gene, and two rod opsin encoding genes, all of which were expressed in retinal photoreceptors. We also report that gar SWS1 cones lack the geometric organization of photoreceptors observed in teleost fish species, consistent with the crystalline photoreceptor mosaic being a teleost innovation. Of note the spotted gar expresses both exo-rhodopsin (RH1-1) and rhodopsin (RH1-2) in rods. Exo-rhodopsin is an opsin that is not expressed in the retina of zebrafish and other teleosts, but rather is expressed in regions of the brain. This study suggests that exo-rhodopsin is an ancestral actinopterygian (ray finned fish) retinal opsin, and in teleosts its expression has possibly been subfunctionalized to the pineal gland.

Exclusive multipotency and preferential asymmetric divisions in post-embryonic neural stem cells of the fish retina.

The potency of post-embryonic stem cells can only be addressed in the living organism, by labeling single cells after embryonic development and following their descendants. Recently, transplantation experiments involving permanently labeled cells revealed multipotent neural stem cells (NSCs) of embryonic origin in the medaka retina. To analyze whether NSC potency is affected by developmental progression, as reported for the mammalian brain, we developed an inducible toolkit for clonal labeling and non-invasive fate tracking. We used this toolkit to address post-embryonic stem cells in different tissues and to functionally differentiate transient progenitor cells from permanent, bona fide stem cells in the retina. Using temporally controlled clonal induction, we showed that post-embryonic retinal NSCs are exclusively multipotent and give rise to the complete spectrum of cell types in the neural retina. Intriguingly, and in contrast to any other vertebrate stem cell system described so far, long-term analysis of clones indicates a preferential mode of asymmetric cell division. Moreover, following the behavior of clones before and after external stimuli, such as injuries, shows that NSCs in the retina maintained the preference for asymmetric cell division during regenerative responses. We present a comprehensive analysis of individual post-embryonic NSCs in their physiological environment and establish the teleost retina as an ideal model for studying adult stem cell biology at single cell resolution.

Functional diversity of melanopsins and their global expression in the teleost retina.

Melanopsin (OPN4) is an opsin photopigment that, in mammals, confers photosensitivity to retinal ganglion cells and regulates circadian entrainment and pupil constriction. In non-mammalian species, two forms of opn4 exist, and are classified into mammalian-like (m) and non-mammalian-like (x) clades. However, far less is understood of the function of this photopigment family. Here we identify in zebrafish five melanopsins (opn4m-1, opn4m-2, opn4m-3, opn4x-1 and opn4x-2), each encoding a full-length opsin G protein. All five genes are expressed in the adult retina in a largely non-overlapping pattern, as revealed by RNA in situ hybridisation and immunocytochemistry, with at least one melanopsin form present in all neuronal cell types, including cone photoreceptors. This raises the possibility that the teleost retina is globally light sensitive. Electrophysiological and spectrophotometric studies demonstrate that all five zebrafish melanopsins encode a functional photopigment with peak spectral sensitivities that range from 470 to 484nm, with opn4m-1 and opn4m-3 displaying invertebrate-like bistability, where the retinal chromophore interchanges between cis- and trans-isomers in a light-dependent manner and remains within the opsin binding pocket. In contrast, opn4m-2, opn4x-1 and opn4x-2 are monostable and function more like classical vertebrate-like photopigments, where the chromophore is converted from 11-cis to all-trans retinal upon absorption of a photon, hydrolysed and exits from the binding pocket of the opsin. It is thought that all melanopsins exhibit an invertebrate-like bistability biochemistry. Our novel findings, however, reveal the presence of both invertebrate-like and vertebrate-like forms of melanopsin in the teleost retina, and indicate that photopigment bistability is not a universal property of the melanopsin family. The functional diversity of these teleost melanopsins, together with their widespread expression pattern within the retina, suggests that melanopsins confer global photosensitivity to the teleost retina and might allow for direct "fine-tuning" of retinal circuitry and physiology in the dynamic light environments found in aquatic habitats.

Purinergic Signals Regulate Daily S-Phase Cell Activity in the Ciliary Marginal Zone of the Zebrafish Retina

Regeneration and growth that occur in the adult teleost retina have been helpful in identifying molecular and cellular mechanisms underlying cell proliferation and differentiation. Here, it is reported that S-phase cell number, in the ciliary marginal zone (CMZ) of the adult zebrafish retina, exhibits day-night variations with a mid-light phase peak. Oscillations persist for 24 h in constant darkness (DD), suggesting control by a circadian component. However, variations in the S-phase nuclei number were rapidly dampened and not present during and after a second day in DD. An ADPβS treatment significantly enhanced S-phase activity at night to mid-light levels, as assessed by in vivo BrdU incorporation in a 2-h interval. Moreover, daylight increase in S-phase cell number was completely abolished when extracellular nucleotide levels or their extracellular hydrolysis by ectonucleoside triphosphate diphosphohydrolases (NTPDases) were significantly disrupted or when a selective antagonist of purinergic P2Y1 receptors was intraocularly injected before BrdU exposure. Extracellular nucleotides and NTPDase action were also important for maintaining nocturnal low levels of S-phase activity in the CMZ. Finally, we showed that mRNAs of NTPDases 1, 2 (3 isoforms), and 3 as well as of P2Y1 receptor are present in the neural retina of zebrafish. NTPDase mRNA expression exhibited a 2-fold increment in light versus dark conditions as assessed by quantitative RT-PCR, whereas P2Y1 receptor mRNA levels did not show significant day-night variations. This study demonstrates a key role for nucleotides, principally ADP as a paracrine signal, as well as for NTPDases, the plasma membrane-bound enzymes that control extracellular nucleotide concentration, for inducing S-phase cell entry in the CMZ-normally associated with retinal growth-throughout the light-dark cycle.

Genetics of photoreceptor degeneration and regeneration in zebrafish

Zebrafish are unique in that they provide a useful model system for studying two critically important problems in retinal neurobiology, the mechanisms responsible for triggering photoreceptor cell death and the innate stem cell–mediated regenerative response elicited by this death. In this review we highlight recent seminal findings in these two fields. We first focus on zebrafish as a model for studying photoreceptor degeneration. We summarize the genes currently known to cause photoreceptor degeneration, and we describe the phenotype of a few zebrafish mutants in detail, highlighting the usefulness of this model for studying this process. In the second section, we discuss the several different experimental paradigms that are available to study regeneration in the teleost retina. A model outlining the sequence of gene expression starting from the dedifferentiation of Müller glia to the formation of rod and cone precursors is presented.

Extracellular ADP regulates lesion‐induced in vivo cell proliferation and death in the zebrafish retina

Regeneration and growth that occur in the adult teleost retina by neurogenesis have been helpful in identifying molecular and cellular mechanisms underlying cell proliferation and differentiation. In this report, we demonstrate that endogenous purinergic signals regulate cell proliferation induced by a cytotoxic injury of the adult zebrafish retina which mainly damages inner retinal layers. Particularly, we found that ADP but not ATP or adenosine significantly enhanced cell division as assessed by 5-bromo-2'-deoxyuridine incorporation following injury, during the degenerative and proliferative phase of the regeneration process. This effect of ADP occurs via P2Y1 metabotropic receptors as shown by intra-ocular injection of selective antagonists. Additionally, we describe a role for purinergic signals in regulating cell death induced by injury. Scavenging of extracellular nucleotides significantly increased cell death principally seen in the inner retinal layers. This effect is partially reproduced by blocking P2Y1 receptors suggesting a neuroprotective function for ADP, which is derived from extracellular ATP probably released by dying cells as a consequence of the ouabain treatment. This study demonstrates a crucial role for ADP as a paracrine signal in the repair of retinal tissue following injury.

Discovery of a novel prolactin in non-mammalian vertebrates: evolutionary perspectives and its involvement in teleost retina development.

BackgroundThe three pituitary hormones, viz. prolactin (PRL), growth hormone (GH) and somatolactin (SL), together with the mammalian placental lactogen (PL), constitute a gene family of hormones with similar gene structure and encoded protein sequences. These hormones are believed to have evolved from a common ancestral gene through several rounds of gene duplication and subsequent divergence.Principal FindingsIn this study, we have identified a new PRL-like gene in non-mammalian vertebrates through bioinformatics and molecular cloning means. Phylogenetic analyses showed that this novel protein is homologous to the previously identified PRL. A receptor transactivation assay further showed that this novel protein could bind to PRL receptor to trigger the downstream post-receptor event, indicating that it is biologically active. In view of its close phylogenetic relationship with PRL and also its ability to activate PRL receptor, we name it as PRL2 and the previously identified PRL as PRL1. All the newly discovered PRL2 sequences possess three conserved disulfide linkages with the exception of the shark PRL2 which has only two. In sharp contrast to the classical PRL1 which is predominantly expressed in the pituitary, PRL2 was found to be mainly expressed in the eye and brain of the zebrafish but not in the pituitary. A largely reduced inner nuclear layer of the retina was observed after morpholino knockdown of zebrafish PRL2, indicating its role on retina development in teleost.SignificanceThe discovery of this novel PRL has revitalized our understanding on the evolution of the GH/PRL/SL/PL gene family. Its unique expression and functions in the zebrafish eye also provide a new avenue of research on the neuroendocrine control of retina development in vertebrates.

Cellular expression of midkine‐a and midkine‐b during retinal development and photoreceptor regeneration in zebrafish

AbstractIn the retina of adult teleosts, stem cells are sustained in two specialized niches: the ciliary marginal zone (CMZ) and the microenvironment surrounding adult Müller glia. Recently, Müller glia were identified as the regenerative stem cells in the teleost retina. Secreted signaling molecules that regulate neuronal regeneration in the retina are largely unknown. In a microarray screen to discover such factors, we identified midkine‐b (mdkb). Midkine is a highly conserved heparin‐binding growth factor with numerous biological functions. The zebrafish genome encodes two distinct midkine genes: mdka and mdkb. Here we describe the cellular expression of mdka and mdkb during retinal development and the initial, proliferative phase of photoreceptor regeneration. The results show that in the embryonic and larval retina mdka and mdkb are expressed in stem cells, retinal progenitors, and neurons in distinct patterns that suggest different functions for the two molecules. Following the selective death of photoreceptors in the adult, mdka and mdkb are coexpressed in horizontal cells and proliferating Müller glia and their neurogenic progeny. These data reveal that Mdka and Mdkb are signaling factors present in the retinal stem cell niches in both embryonic and mature retinas, and that their cellular expression is actively modulated during retinal development and regeneration. J. Comp. Neurol. 514:1–10, 2009. © 2009 Wiley‐Liss, Inc.

Cellular expression of Midkine‐a and Midkine‐b during retinal development and photoreceptor regeneration in zebrafish

In the retina of adult teleosts, stem cells are sustained in two specialized niches: the ciliary marginal zone (CMZ) and the microenvironment surrounding adult Müller glia. Recently, Müller glia were identified as the regenerative stem cells in the teleost retina. Secreted signaling molecules that regulate neuronal regeneration in the retina are largely unknown. In a microarray screen to discover such factors, we identified midkine-b (mdkb). Midkine is a highly conserved heparin-binding growth factor with numerous biological functions. The zebrafish genome encodes two distinct midkine genes: mdka and mdkb. Here we describe the cellular expression of mdka and mdkb during retinal development and the initial, proliferative phase of photoreceptor regeneration. The results show that in the embryonic and larval retina mdka and mdkb are expressed in stem cells, retinal progenitors, and neurons in distinct patterns that suggest different functions for the two molecules. Following the selective death of photoreceptors in the adult, mdka and mdkb are coexpressed in horizontal cells and proliferating Müller glia and their neurogenic progeny. These data reveal that Mdka and Mdkb are signaling factors present in the retinal stem cell niches in both embryonic and mature retinas, and that their cellular expression is actively modulated during retinal development and regeneration.