BACKGROUND
the identification and characterization of neural progenitors that produce neurons and glia in the central nervous system is a subject of intense investigation.
it is now widely recognized that neural stem cells persist in specialized 'niches' in the adult mammalian forebrain where they generate large numbers of selected types of neurons  <cit> .
one of the most intriguing recent discoveries is that these adult neural stem cells exhibit some properties of glial cells  <cit>  and that neurons in certain regions of the developing embryonic mammalian and avian brains also derive from radial glia  <cit> .
in the adult brain, the microenvironmental compartments called 'niches' provide an embryonic-like milieu to support the maintenance of neural stem cells with the essential properties of self-renewal and pluripotency, i.e.
capacity for multi-lineage differentiation  <cit> .
although still poorly understood, some defining characteristics of adult stem cell niches in the brain and elsewhere are beginning to emerge  <cit> .
some common features of neural stem cells and their niches include: prominent cadherin-mediated adhesive junctions, a rich extracellular matrix and contact with a specialized basal lamina via integrin-mediated junctions, close association with blood vessels, cell-surface carbohydrate markers , expression of blbp , expression of selected classes of intermediate filament proteins , responsiveness to extrinsic signals such as igf , tgfβ/bmp  family, wnts, shh , notch, and lif .
probably not coincidentally, these extrinsic regulators represent all of the major families of signaling pathways that are essential for early embryonic development  <cit> .
the neural retina is an embryonic derivative of the forebrain, but unlike the cerebral cortex, adult neural stem cells have not been described in mammalian retina in vivo.
with the exception of fish and larval amphibians, retinal neurogenesis in vertebrates is completed during embryonic or early postembryonic development  <cit> .
neural progenitors with the capacity to generate retinal neurons can be recognized from the earliest stages of neural induction by regionalized expression of a series of transcriptional regulators that specify retinal identity  <cit> .
these early 'eye field transcription factors' include the paired-class homeobox genes pax <dig> and rx, a member of the sine oculis homeobox family, six <dig>  and a lim-homeobox gene, lhx.
additional members of the paired-class homeobox family, chx <dig>  and crx, appear at later stages in the developing optic cup  <cit> .
continued co-expression of these homeobox transcription factors is a distinguishing feature of multipotent retinal progenitor cells  <cit> .
in larval amphibians and teleost fish, retinal neurogenesis continues postembryonically at the interface between neural retina and ciliary epithelium, a region called the ciliary marginal zone  or circumferential germinal zone  <cit> .
in these species the cmz generates the majority of retinal tissue found in the adult eye  <cit> , but the specialized cellular and molecular characteristics of the cmz niche that support the maintenance and regulate the activity of retinal stem cells in the adult fish eye have not yet been elucidated.
a reduced and transient cmz has been described recently in postnatal chicks and marsupial mammals  <cit>  and a residual cmz is seen in mice with a genetic lesion that increases activity of sonic hedgehog signaling  <cit> .
although the ciliary epithelium of the adult mammalian retina may retain some neurogenic potential in vivo  <cit>  and in vitro  <cit> , any endogenous capacity for neurogenesis in these species remains latent and without functional consequence.
in addition to the cmz, another source of retinal progenitor cells persists in the adult teleost fish retina, although the identity and characteristics of these cells are not well understood.
in the uninjured, differentiated retina, rod photoreceptors accumulate in central retina as the fish eye grows, and they are generated by rapidly dividing, lineage-restricted rod precursor cells located in the outer nuclear layer , among the nuclei of differentiated rods  <cit> .
the rod precursors are derived from more slowly proliferating progenitors in the inner nuclear layer  that give rise to clusters of rapidly proliferating progenitors , which migrate into the outer nuclear layer along the radial fibers of the müller glial cells in larval  <cit>  and adult  <cit>  fish.
not all of the müller glia are associated with neurogenesis and the spatial distribution of rod precursors and clusters of inl progenitors varies in that they are present in greater density near the cmz, i.e., in the most recently generated retina  <cit> .
the slowly dividing progenitors in the inl are immunoreactive for pax <dig>  <cit> , but it is not known whether they express other retinal progenitor transcription factors found in the cmz.
the rod precursors in the onl do not express pax <dig>  but they do express the proneural bhlh transcription factor, neurod, suggesting that these onl progenitors may be committed to the photoreceptor lineage  <cit> .
although their identity has not yet been established, it is clear that multipotent retinal stem cells  persist not only in the germinal zone of the cmz but also in central retina.
adult teleost fish have a robust capacity to regenerate retinal neurons and to restore the appropriate laminar retinal architecture following damage inflicted by surgical lesions, neurotoxins, laser or photic lesions  <cit> .
all of these studies concluded that the source of regenerated neurons in central retina is an intrinsic progenitor population and not the cmz.
in the intact retina, the progeny of the inl progenitors differentiate only as rod photoreceptors, but several authors have speculated that these progenitors may retain the capacity to generate multiple lineages  <cit> .
alternatively, it has been suggested that müller glial cells, which proliferate rapidly in response to retinal damage, may retain a latent capacity to generate neurons in the fish retina  <cit> .
in the present study we define the molecular profiles of retinal progenitor cells in the growing and regenerating adult zebrafish retina and we examine the properties of the cellular niches in which they reside.
we introduce a simple heat lesion paradigm for analysis of retinal regeneration, we describe the time course of regeneration of cone photoreceptors, and we examine the molecular profile of the injury-induced proliferating progenitor cells that give rise to regenerated cones.
our data show that the adult fish retina has two types of specialized microenvironmental niches that sustain multipotent, self-renewing retinal stem cells: the peripheral cmz and the microenvironment created by some müller glia in differentiated retina.
we further demonstrate that the retinal stem cell niches in the cmz germinal zone and the injury-induced stem cell niche in the regenerating retina share a common molecular signature that includes activated notch signaling, diffuse distribution of n-cadherin, expression of blbp, and expression of the retinal homeobox genes rx <dig>  pax6a and vsx2/chx <dig>  müller glia form the retinal stem cell niche and may generate retinal stem cells in the regenerating retina.
like the neuroepithelial cells  of the cmz they are associated with the specialized basal lamina of blood vessels at the vitreal surface of the retina and their nuclei divide at the apical surface in contact with the subretinal space .
RESULTS
molecular profile of retinal stem cells and progenitor cells in the zebrafish cmz
the initial differentiation of retinal neurons and formation of retinal laminae in zebrafish is completed by the end of embryonic development at  <dig> days post-fertilization , after which proliferating retinal progenitors are concentrated in a germinal zone at the peripheral margins of the retina in a wedge of cells formed by the converging retinal laminae, called the ciliary marginal zone, cmz .
a subset of the proliferating retinal progenitors in the zebrafish cmz express paired-class homeobox transcription factors associated with retinal specification, including rx <dig>  which is also expressed in differentiating  cone photoreceptors in the onl , vsx2/chx <dig>  and pax6a .
in the most peripheral region of the germinal zone adjacent to the ciliary epithelium, pax6a is co-expressed with rx <dig> , which is indicative of a multi-lineage capacity.
in the peripheral region of the germinal zone rx <dig>  vsx2/chx <dig>  and pax6a show the highest signal levels of all the probes we examined .
expression of pax6a is moderately high in the peripheral germinal zone and remains strong in middle and central-inner regions, consistent with the continued expression of this gene in differentiated amacrine cells.
the level of rx <dig> signal is moderate in the peripheral germinal zone, and rx <dig> is slightly weaker.
both rx genes are sharply down-regulated in the middle germinal zone, but reappear in the central-outer region in differentiating cone photoreceptors.
the profile of vsx2/chx <dig> is similar to rx1/rx <dig> in that expression levels are highest in the peripheral cmz and are sharply down-regulated in the middle cmz.
expression of vsx2/chx <dig> reappears in the differentiated inl, presumably in bipolar cells, but outside the cmz  <cit> .
the expression domain of these transcription factors extends from the cmz into the adjacent ciliary epithelium .
other transcriptional regulators associated with retinal progenitors in several vertebrate species are members of the basic helix-loop-helix  family of proneural genes  <cit> .
a zebrafish ortholog of mammalian/chick mash1/cash <dig>  called ascl1a  has a moderate signal level in the peripheral cmz, is reduced in the middle and is absent from central regions .
proneural genes related to the atonal family, such as neurod, are typically activated at later stages in the neuronal differentiation cascade and are associated with determination of specific neuronal cell types  <cit> .
in the retina, neurod specifies photoreceptors in zebrafish  <cit> .
the neurod probe produces only a very weak signal in the peripheral and middle cmz, but increases sharply in the central regions, especially the outer part, where photoreceptors are differentiating .
all of these results are consistent with the idea that the most primitive  retinal stem cells are located in the most peripheral region of the cmz adjacent to the ciliary epithelium and more restricted retinal progenitors lie closer to the differentiated retina.
the notch-delta signaling pathway plays an important role in cell fate choice in the retina and in particular the decision of whether to differentiate as a neuron or a müller glia or to remain undifferentiated  <cit> .
several constituents of this bidirectional signaling pathway are expressed in the cmz in larval and juvenile zebrafish, including the notch ligands deltaa, deltab, deltac, deltad, several notch family members  and downstream mediators of activated notch, her <dig>  the zebrafish ortholog of mammalian hes <dig> and her2/hes <dig> .
analysis of relative expression levels shows that in the most peripheral region of the cmz components of the notch-delta signaling cascade give moderate signals .
expression levels are lower in the middle and central regions of the cmz.
the signals for notch <dig> are similar to the notch <dig> paralogs .
these data indicate that the notch-delta signaling pathway is activated in retinal progenitors including multipotent retinal stem cells that co-express a set of transcriptional regulators implicated in maintenance of multipotency and specification of retinal cell fate.
the microenvironment of the zebrafish cmz has features typical of neural stem cell niches
the radial glia/astrocyte marker blbp has been associated with adult neural stem cell niches in mammalian brain  <cit> .
we used a polyclonal antibody against a synthetic peptide of human blbp that is 90% identical to the predicted amino acid sequence of the orthologous zebrafish gene, fabp7a  <cit> .
we show that retinal stem cells and progenitors in the cmz and immature müller glial cells are immunoreactive for blbp .
blbp immunoreactivity extends throughout the cmz and into the adjacent ciliary epithelium.
in larval zebrafish retina , the cmz and immature müller glia distributed across the retina are blbp+ , but in 2-month-old juvenile fish only the recently generated, immature müller glial cells closest to the cmz are labeled .
thus, the blbp protein is down-regulated with maturation of zebrafish müller glia.
in mammals, the extracellular matrix-associated carbohydrate epitope ssea-1/lex/cd <dig> is a surface marker for embryonic stem cells and adult neural stem cells  <cit>  as well as subpopulations of embryonic retinal progenitor cells  <cit> .
the antigen is the trisaccharide, 3-fucosyl-n-acetyl-lactosamine, which is a component of membrane glycolipids and glycoproteins from a variety of tissues.
in zebrafish, the ssea- <dig> epitope is present at high levels around the circumferential blood vessel  <cit>  that lies on the vitreal surface at the border between neural retina and ciliary epithelium overlying the cmz  and around other blood vessels that line the vitreal surface .
a second site of high expression is a discrete laminar zone of undetermined identity in the inner plexiform layer , potentially on amacrine cell processes  <cit> .
the retinal pigmented epithelium  also expresses ssea- <dig> .
the calcium-dependent, homophilic cell adhesion protein n-cadherin  is diffusely distributed on the basolateral plasma membranes of retinal stem cells and progenitors in the cmz and adjacent ciliary epithelium , similar to the distribution of n-cadherin on undifferentiated neuroepithelial progenitors in the embryonic retinal primordium  <cit> .
proliferating retinal progenitor cells in the cmz are ensheathed in a network of cadherin-mediated adhesive interactions with neighboring cells.
in contrast, in the differentiated retina, n-cadherin is targeted to the zonula adherens that form the outer limiting membrane , to the synaptic  layers and to axons of retinal ganglion cells .
progenitors with the molecular profile of retinal stem cells are activated locally in differentiated retina by thermal lesions
to determine whether retinal stem cells responsible for retinal regeneration following injury have molecular profiles similar to those in the cmz, we created local thermal lesions largely limited to the rpe and underlying photoreceptors in the retina of adult zebrafish .
the diameter of the lesioned area ranges from ~ <dig> to  <dig> μm.
loss of photoreceptors is apparent within  <dig> day post-lesion  and pyknotic nuclei fill the onl and subretinal space between neural retina and rpe .
destruction of cone photoreceptors within the lesion is revealed by the absence of immunoreactivity with zpr <dig> , a highly specific cell-surface marker for zebrafish double cones .
thermal lesions trigger a local increase in mitotic activity in the retina, which begins within  <dig> dpl .
in lesioned areas elongated clusters of cells associated with radial fibers of müller glia appear by  <dig> dpl .
cells in these clusters incorporate brdu .
we confirmed that incorporation of brdu reflects mitotic cell division and not dna repair in response to damage  <cit>  by immunostaining for proliferating cell nuclear antigen, pcna .
these mitotic cells are closely associated with radial fibers of müller glia, all of which are strongly immunoreactive with antibodies against glial fibrillary acidic protein, gfap .
nuclei of the activated müller glia also reenter the cell cycle and migrate from the inl into the onl , as we showed previously in regenerating goldfish retina  <cit> .
at  <dig> and  <dig> dpl the injury-induced proliferating cells associated with gfap+ radial müller fibers co-express at high levels the retinal stem cell markers rx <dig>  vsx2/chx <dig>  and pax6a .
brdu+ mitotic clusters show strong rx <dig>  and vsx2/chx <dig> signals .
the expression of these transcription factors within the lesion extends vertically beyond the laminar boundaries of their respective expression domains in the differentiated retina .
constituents of the notch-delta signaling pathway are also strongly upregulated in the injured retina after thermal lesions .
between  <dig> and  <dig> dpl, deltac, notch1b and notch <dig> probes produce signals in the lesion area comparable in strength to those in the cmz.
some  brdu+ cells in the lesion area at  <dig> dpl co-label with deltac .
the deltac and notch signals are largely in adjacent cells and are not co-localized .
the deltaa, deltab, deltad, and her6/hes <dig> probes similarly produce strong signals in the lesion area .
in summary, injury-induced proliferating cells closely associated with radial fibers of müller glia express retinal stem cell transcription factors and activate notch signaling.
injury induces a retinal stem cell niche that recreates the microenvironment of the cmz
müller glial cells activated by the lesion acquire several features characteristic of retinal stem/progenitor cells in the cmz and in the embryonic retina.
activated müller glial cells up-regulate expression of n-cadherin and the protein becomes distributed diffusely throughout the plasma membrane as it is in retinal stem cells and progenitors in the cmz .
the inset in fig.
4e shows radial fibers of activated müller glia co-labeled with a monoclonal antibody specific for zebrafish gfap  and polyclonal antibodies against zebrafish n-cadherin.
in contrast, in the uninjured differentiated zebrafish retina, n-cadherin immunoreactivity primarily localizes to the adherens junctions formed by müller glia at the apical surface of the retina , to the synaptic neuropil of the inner and outer plexiform layers, and to retinal ganglion cell axons  <cit> .
the injury-induced clusters of brdu+ nuclei are tightly associated with radial müller fibers that are immunoreactive for n-cadherin .
activated müller glia in the region of the lesion also up-regulate expression of blbp , which is characteristic of retinal stem/progenitor cells in the cmz and immature müller glia .
the boundary between blbp+ and blbp- müller glia is very abrupt , which suggests that only the müller glia directly impacted by the retinal injury dedifferentiate and reacquire an immature molecular profile.
cone photoreceptors regenerate within a week
some of the mitotically active cells induced by the thermal lesion are retinal stem/progenitor cells that differentiate into retinal neurons, thus repairing the retinal damage.
to identify the retinal stem cells responsible for regenerating retinal neurons, we first needed to know when the regenerated neurons are born so we could focus on cells proliferating during and/or before that time.
not all of the mitotic cells at every interval following the lesion are retinal stem cells; for example, microglia proliferate rapidly in response to retinal injury  <cit>  and phagocytize the dead cells and debris.
differentiated müller glia proliferate after injury, even in mammalian retinas that fail to regenerate neurons  <cit> .
in this study, we confined our analysis of the time course of neuronal regeneration in the retina to cone photoreceptors because they have distinctive and highly specialized morphology and several unique cell-specific markers.
to document regeneration of cones, we first labeled proliferating cells with brdu at  <dig> to  <dig> dpl and then waited for several days up to one month to allow the progeny of the mitotic cells to differentiate.
we then identified double-cones with the zpr <dig> antibody .
when the retina is examined  <dig> hours after brdu exposure at  <dig> dpl, mitotic cells are labeled in the region of the lesion .
at this stage, microglia  are in the subretinal space between the outer limiting membrane and the rpe and are not part of the pool of proliferating cells within the retina .
when fish survived for  <dig> days after the brdu , a few progeny of the brdu+ cells had begun to differentiate and express the double cone marker zpr <dig>  and they are small and immature compared with the intact cones adjacent to the lesion .
by  <dig> dpl the regenerated cones are fully differentiated and indistinguishable from the surrounding photoreceptors, with the exception that their nuclei retain the brdu-label .
in all lesions at long survival intervals, persistent brdu+ nuclei are seen in the inl .
we do not know the identity of these cells, although they do not have the morphological characteristics of müller glia and they are not immunoreactive with the microglia-specific antibody, 4c <dig> .
we presume that most of them are retinal neurons, which were regenerated following loss due to the lesion, as has been well documented in several other models of retinal regeneration in teleost fish  <cit> .
to determine the time course of cone photoreceptor regeneration, we labeled proliferating cells with brdu at  <dig>   <dig>   <dig> or  <dig> dpl, and counted double-labeled cone photoreceptors at  <dig> dpl.
because the zpr <dig> antibody labels only the red-green double cones and zebrafish have two additional single cone types that express either blue or ultraviolet-sensitive opsin, we used a cocktail of rna probes to the four cone opsin classes – red , green , blue  and ultraviolet  – to label all cone types .
we also counted brdu+ rod photoreceptors in the onl in the area of the lesion .
cell counts  show that regenerated cones in zebrafish are born between  <dig> and  <dig> dpl whereas most rods are born at  <dig> dpl or later .
in the developing zebrafish retina, cones are born before rods as they are in the embryonic mammalian retina  <cit> .
in our previous studies of retinal regeneration in goldfish we found that the ongoing production of rod photoreceptors from mitotic rod precursors largely ceases while the complement of cones is restored  <cit>  and here we show that a similar process occurs in zebrafish.
this temporal shift in the distribution of photoreceptor cell types generated by the retinal stem cells suggests that they respond to changing microenvironmental factors similar to those found in the developing embryonic retina, which regulate the sequential production of retinal neurons.
identity of the injury-activated retinal stem cells/progenitors
the longitudinal birthdating analysis identified cells proliferating between  <dig> and  <dig> dpl as retinal progenitors whose progeny differentiate into cone photoreceptors.
since cone photoreceptors are not normally produced in the differentiated regions of the intact retina, these data demonstrate that multipotent retinal stem cells persist in central retina with the capacity to respond to injury by altering their lineage profile to compensate for loss of specific neuronal subtypes.
as described above, most of the mitotic activity in the lesion area at this time is in elongated clusters of proliferating cells in the inl closely associated with the radial fibers of müller glia .
these proliferating cell clusters have been observed in all previous studies of retinal regeneration in teleost fish, and their similarity to the progenitors in the rod photoreceptor lineage of the uninjured retina has been noted  <cit> .
another potential source of latent retinal stem cells are the müller glia themselves  <cit> .
this question has been difficult to resolve because of the tight association of retinal progenitors in the rod lineage with the radial fibers of müller glia in both the uninjured retina  <cit>  and the regenerating retina , and the complexity of müller processes that enwrap neighboring cells  <cit> .
these intimate and complex anatomical relationships make it difficult to determine with fluorescent microscopy and the standard, cytoplasmic müller-specific markers, e.g.
gfap, cellular retinaldehyde binding protein  and glutamine synthetase, whether a given brdu+ nucleus belongs to the müller cell or to an associated progenitor.
in an attempt to examine this question with better techniques, we sought a müller-specific marker that is confined to the cell body.
apolipoprotein e  is a small protein component of several different lipoproteins that is secreted by many cells including glia  <cit> .
to determine whether apoe is expressed by müller cells, we used transgenic zebrafish  <cit>  in which enhanced green fluorescent protein  is controlled by transcriptional regulatory elements of the zebrafish gfap gene.
the gfp reporter in this transgenic line is cytoplasmic and fills the cell body and all the processes of müller glia.
we found that zebrafish apoe is expressed selectively and at high levels in gfap: gfp+ differentiated müller glia, and the signal is largely confined to the cell bodies in the inl .
the apoe probe also co-localizes with a probe that hybridizes to rlbp1-l  the zebrafish orthologue of the müller glial marker cralbp   <cit>  .
it is therefore a useful marker to identify brdu+ proliferating müller glia.
if müller glia function as retinal progenitors/stem cells in the injured retina, they should be proliferating before and/or during the interval when regenerated cone photoreceptors are being born , which we found is between  <dig> and  <dig> dpl.
very few cells incorporate brdu at  <dig> dpl , but at  <dig> dpl approximately 80% of the proliferating cells are apoe+/brdu+ müller glia .
the fraction of apoe+/brdu+ müller glia in the population of proliferating cells dropped to ~50% by  <dig> and  <dig> dpl, while the total number of brdu+ nuclei increased from a mean of  <dig> per section at  <dig> dpl, to  <dig> per section at  <dig> dpl, to  <dig> per section at  <dig> dpl .
these data are consistent with several interpretations.
müller glia that express apoe constitute the vast majority  of the proliferating cells at  <dig> dpl when the size of the brdu+ population is smallest.
the remaining 20% of the brdu+ cells that are apoe- at this time could be progenitors in the rod lineage or dedifferentiated müller glia or progeny of müller glia that have down-regulated apoe expression.
a closer examination of the rare brdu+ cells at early intervals prior to  <dig> dpl with specific markers for retinal stem cells and progenitors  might answer this question.
the decline in the müller glia fraction of the expanding brdu+ population at  <dig> and  <dig> dpl could be explained by dedifferentiation of proliferating müller cells and/or a more rapid rate of proliferation of non-müller retinal progenitors.
finally, these data are also consistent with the hypothesis that injury-induced retinal stem cells might derive from müller glia that begin to proliferate at or before  <dig> dpl, dedifferentiate and give rise to retinal stem cells.
discussion
we have characterized the molecular microenvironment of two kinds of specialized niches that sustain retinal stem cells in zebrafish: 1) the ciliary marginal zone  at the peripheral retinal margin and 2) some müller glia in the differentiated retina .
the retinal stem cell niche in the zebrafish cmz has several features in common with the neurogenic niches in the adult mammalian brain, including the subventricular zone  and the dentate gyrus subgranular zone , which have been described as 'displaced neuroepithelium'.
adult neural stem cells are specialized astrocytes that serve as both neural stem cells and niche cells; they extend processes to form apical junctions at the ventricular surface and also have extensive contacts with the basal lamina surrounding blood vessels  <cit> .
the retinal stem cells in the zebrafish cmz that co-express rx <dig>  pax6a and vsx2/chx <dig> contact both the apical surface and the basal lamina and are in close proximity to a large blood vessel  that is ssea-1+ .
other characteristics that mimic neuroepithelial cells of the developing neural tube and retina include activation of the notch signaling cascade  and diffuse distribution of n-cadherin on the plasma membrane  <cit> .
the radial glial marker blbp is a downstream target of notch signaling  <cit>  and of pax <dig>  <cit> , both of which are implicated in the maintenance of neural stem cell fate.
the cmz cells in zebrafish express the radial glial marker blbp, as do the cells of the ciliary epithelium and retinal pigmented epithelium, and immature müller glia.
mitotic activity in the cmz is stimulated by insulin-like growth factor  in goldfish  <cit> , and by wnt or sonic hedgehog  in chick, mouse and xenopus retinas  <cit> .
the ssea- <dig> carbohydrate binds wnts  <cit> , and its association with the ora collection vessel in zebrafish may contribute to the special microenvironment that maintains a neuroepithelium at the cmz.
the proximity of the rpe on the apical side is another potential source of secreted signals that might promote neuroepithelial properties in the cmz.
the rpe in zebrafish expresses blbp and is a source of shh  <cit> .
we have not yet looked for pedf  in zebrafish, but this growth factor is secreted by components of the svz and supports self-renewal of adult neural stem cells in mice  <cit> .
a separate type of retinal stem cell niche is created within the differentiated retina by some of the müller glia.
retinal müller glia are often compared with radial glia of the developing brain  <cit> .
like radial glia, processes of müller glia span the width of the retinal epithelium from the apical epithelial surface  to the basal lamina at the vitreal surface and they express structural and functional markers of astrocytes .
in the intact retina of teleost fish, rapidly proliferating inl progenitors  are clustered around radial müller cell fibers and they use these radial processes as guides along which to migrate into the onl  <cit> , as we showed previously by serial reconstruction electron microscopy  <cit> .
not all müller glia are associated with inl progenitors, and the frequency is highest near the retinal periphery   <cit> .
we do not yet know whether these neurogenic niches are associated with a specialized subpopulation of müller glial cells.
in the intact retina, the retinal progenitors in these putative müller cell niches are directed exclusively into the rod lineage, but following injury, the frequency of neurogenic clusters increases  <cit> , the fate of the retinal progenitors is altered  <cit> , and the structural and molecular characteristics of the müller glia change  <cit>  .
müller glia in the injured fish retina exhibit many phenotypic changes associated with reactive gliosis, such as up-regulation of gfap  <cit> , which is often considered an indicator of stress and pathological processes  <cit>  and reentry into the cell cycle  <cit> .
here we show that they also exhibit phenotypic changes suggestive of dedifferentiation, such as re-expression of blbp and migration of their nuclei toward the apical surface, both of which are characteristic of immature müller glia in the larval zebrafish retina .
the injury-induced retinal stem cells/multipotent progenitors are entwined in müller processes, which in response to injury up-regulate components of the notch-delta signaling pathways and n-cadherin   <cit> .
the n-cadherin protein becomes distributed to the basolateral membranes of the activated müller glia, similar to its distribution on neuroepithelial cells.
radial glia in the embryonic mammalian brain are neurogenic progenitors  <cit>  and specialized astrocytes in the adult mammalian brain function as neural stem cells  <cit> , so it is not surprising that we and others have suggested that müller glia might generate the injury-induced retinal stem cells in teleost fish  <cit> , although direct evidence that employs a selective and permanent lineage tracer for müller glial fate mapping has not yet been accomplished.
fischer and reh  <cit>  have proposed that a latent neurogenic capacity persists in müller glia of early postnatal chick based on observations of brdu incorporation and expression of neuronal markers in immature müller cells following intraocular injection of neurotoxins  or growth factors , and similar observations were made in rat retina  <cit> .
however, physiologically relevant, regenerative repair of retinal neuron loss has only been demonstrated in teleost fish  <cit>  and some amphibians  <cit> .
one of the most striking morphological changes in the injured fish retina is the migration of proliferating retinal progenitor cells and müller nuclei to the apical surface and the subsequent reestablishment of a neuroepithelium; this anatomical organization is a necessary condition for regeneration of retinal neurons independent of the mechanism of injury  <cit> .
for example, neurotoxins that selectively destroy retinal neurons in the inner layers of adult goldfish fail to elicit a retinal stem cell response and the missing neurons are not replaced.
only when photoreceptors are destroyed is the regenerative process triggered and the retina repaired including inner neurons as well as photoreceptors.
one possible explanation for this differential response to injury is that damaged photoreceptors release factors that stimulate retinal stem cells and create a 'regeneration niche'.
another possibility, not mutually exclusive, is that in the absence of photoreceptors, the proliferating retinal progenitors reestablish contact with the apical surface thus recreating a neuroepithelial organization similar to that found in the cmz.
migration of müller nuclei to the apical surface following loss of photoreceptors is clearly not a sufficient trigger to elicit a regenerative stem cell response, since müller glia in the mammalian retina undergo a similar behavior in the injured or dystrophic mammalian retina but do not generate neurons  <cit> .
the key unanswered question is what extrinsic signals are necessary and sufficient to provoke the formation of a regenerative retinal stem cell niche?
several extracellular factors associated with maintenance of the undifferentiated, multipotent state in neural progenitors in early development, promote glial differentiation at later stages, including activation of lif and notch signaling  <cit> .
in the developing retina, activation of notch signaling either maintains undifferentiated progenitors or promotes müller glia differentiation, depending on stage-dependent contextual cues  <cit> .
the lif pathway may also be implicated: müller glia isolated from the neonatal mouse retina and induced to proliferate by exposure to growth factors express lif  <cit> , and lif is necessary for injury-induced neurogenesis in mouse olfactory epithelium  <cit> .
CONCLUSIONS
our data indicate that müller glia in the adult fish retina have a complex response to local injury that includes some features of reactive gliosis  together with dedifferentiation and re-acquisition of phenotypic and molecular characteristics of multipotent retinal progenitors .
the outcome of this injury-induced glial activation is not generation of more müller glia and formation of a glial scar, as is typical in mammalian retina  <cit> , but instead a retinal stem cell niche is recreated with molecular and morphological features that mirror the neurogenic cmz.
our data suggest that in the injured retina activated müller glia cells define the retinal stem cell niche and our results are consistent with the proposal that retinal stem cells derive from proliferating müller glia.
this idea fits with the emerging view that adult neural stem cells in vivo are a subpopulation of radial glial-derived cells that retain a neurogenic potential.

 the identification and characterization of neural progenitors that produce neurons and glia in the central nervous system is a subject of intense investigation. it is now widely recognized that neural stem cells persist in specialized'niches' in the adult mammalian forebrain where they generate large numbers of selected types of neurons. one of the most intriguing recent discoveries is that these adult neural stem cells exhibit some properties of glial cells and that neurons in certain regions of the developing embryonic mammalian and avian brains also derive from radial glia. in the adult brain, the microenvironmental compartments called'niches' provide an embryonic-like milieu to support the maintenance of neural stem cells with the essential properties of self-renewal and pluripotency, i.e. capacity for multi-lineage differentiation. although still poorly understood, some defining characteristics of adult stem cell niches in the brain and elsewhere are beginning to emerge. some common features of neural stem cells and their niches include: prominent cadherin-mediated adhesive junctions, a rich extracellular matrix and contact with a specialized basal lamina via integrin-mediated junctions, close association with blood vessels, cell-surface carbohydrate markers, expression of blbp, expression of selected classes of intermediate filament proteins, responsiveness to extrinsic signals such as igf, tgfβ/bmp family, wnts, shh, notch, and lif. probably not coincidentally, these extrinsic regulators represent all of the major families of signaling pathways that are essential for early embryonic development. the neural retina is an embryonic derivative of the forebrain, but unlike the cerebral cortex, adult neural stem cells have not been described in mammalian retina in vivo. with the exception of fish and larval amphibians, retinal neurogenesis in vertebrates is completed during embryonic or early postembryonic development. neural progenitors with the capacity to generate retinal neurons can be recognized from the earliest stages of neural induction by regionalized expression of a series of transcriptional regulators that specify retinal identity. these early'eye field transcription factors' include the paired-class homeobox genes pax and rx, a member of the sine oculis homeobox family, six and a lim-homeobox gene, lhx. additional members of the paired-class homeobox family, chx and crx, appear at later stages in the developing optic cup. continued co-expression of these homeobox transcription factors is a distinguishing feature of multipotent retinal progenitor cells. in larval amphibians and teleost fish, retinal neurogenesis continues postembryonically at the interface between neural retina and ciliary epithelium, a region called the ciliary marginal zone or circumferential germinal zone. in these species the cmz generates the majority of retinal tissue found in the adult eye, but the specialized cellular and molecular characteristics of the cmz niche that support the maintenance and regulate the activity of retinal stem cells in the adult fish eye have not yet been elucidated. a reduced and transient cmz has been described recently in postnatal chicks and marsupial mammals and a residual cmz is seen in mice with a genetic lesion that increases activity of sonic hedgehog signaling. although the ciliary epithelium of the adult mammalian retina may retain some neurogenic potential in vivo and in vitro, any endogenous capacity for neurogenesis in these species remains latent and without functional consequence. in addition to the cmz, another source of retinal progenitor cells persists in the adult teleost fish retina, although the identity and characteristics of these cells are not well understood. in the uninjured, differentiated retina, rod photoreceptors accumulate in central retina as the fish eye grows, and they are generated by rapidly dividing, lineage-restricted rod precursor cells located in the outer nuclear layer, among the nuclei of differentiated rods. the rod precursors are derived from more slowly proliferating progenitors in the inner nuclear layer that give rise to clusters of rapidly proliferating progenitors, which migrate into the outer nuclear layer along the radial fibers of the müller glial cells in larval and adult fish. not all of the müller glia are associated with neurogenesis and the spatial distribution of rod precursors and clusters of inl progenitors varies in that they are present in greater density near the cmz, i.e., in the most recently generated retina. the slowly dividing progenitors in the inl are immunoreactive for pax, but it is not known whether they express other retinal progenitor transcription factors found in the cmz. the rod precursors in the onl do not express pax but they do express the proneural bhlh transcription factor, neurod, suggesting that these onl progenitors may be committed to the photoreceptor lineage. although their identity has not yet been established, it is clear that multipotent retinal stem cells persist not only in the germinal zone of the cmz but also in central retina. adult teleost fish have a robust capacity to regenerate retinal neurons and to restore the appropriate laminar retinal architecture following damage inflicted by surgical lesions, neurotoxins, laser or photic lesions. all of these studies concluded that the source of regenerated neurons in central retina is an intrinsic progenitor population and not the cmz. in the intact retina, the progeny of the inl progenitors differentiate only as rod photoreceptors, but several authors have speculated that these progenitors may retain the capacity to generate multiple lineages. alternatively, it has been suggested that müller glial cells, which proliferate rapidly in response to retinal damage, may retain a latent capacity to generate neurons in the fish retina. in the present study we define the molecular profiles of retinal progenitor cells in the growing and regenerating adult zebrafish retina and we examine the properties of the cellular niches in which they reside. we introduce a simple heat lesion paradigm for analysis of retinal regeneration, we describe the time course of regeneration of cone photoreceptors, and we examine the molecular profile of the injury-induced proliferating progenitor cells that give rise to regenerated cones. our data show that the adult fish retina has two types of specialized microenvironmental niches that sustain multipotent, self-renewing retinal stem cells: the peripheral cmz and the microenvironment created by some müller glia in differentiated retina. we further demonstrate that the retinal stem cell niches in the cmz germinal zone and the injury-induced stem cell niche in the regenerating retina share a common molecular signature that includes activated notch signaling, diffuse distribution of n-cadherin, expression of blbp, and expression of the retinal homeobox genes rx pax6a and vsx2/chx müller glia form the retinal stem cell niche and may generate retinal stem cells in the regenerating retina. like the neuroepithelial cells of the cmz they are associated with the specialized basal lamina of blood vessels at the vitreal surface of the retina and their nuclei divide at the apical surface in contact with the subretinal space.  molecular profile of retinal stem cells and progenitor cells in the zebrafish cmz the initial differentiation of retinal neurons and formation of retinal laminae in zebrafish is completed by the end of embryonic development at days post-fertilization, after which proliferating retinal progenitors are concentrated in a germinal zone at the peripheral margins of the retina in a wedge of cells formed by the converging retinal laminae, called the ciliary marginal zone, cmz. a subset of the proliferating retinal progenitors in the zebrafish cmz express paired-class homeobox transcription factors associated with retinal specification, including rx which is also expressed in differentiating cone photoreceptors in the onl, vsx2/chx and pax6a. in the most peripheral region of the germinal zone adjacent to the ciliary epithelium, pax6a is co-expressed with rx, which is indicative of a multi-lineage capacity. in the peripheral region of the germinal zone rx vsx2/chx and pax6a show the highest signal levels of all the probes we examined. expression of pax6a is moderately high in the peripheral germinal zone and remains strong in middle and central-inner regions, consistent with the continued expression of this gene in differentiated amacrine cells. the level of rx signal is moderate in the peripheral germinal zone, and rx is slightly weaker. both rx genes are sharply down-regulated in the middle germinal zone, but reappear in the central-outer region in differentiating cone photoreceptors. the profile of vsx2/chx is similar to rx1/rx in that expression levels are highest in the peripheral cmz and are sharply down-regulated in the middle cmz. expression of vsx2/chx reappears in the differentiated inl, presumably in bipolar cells, but outside the cmz. the expression domain of these transcription factors extends from the cmz into the adjacent ciliary epithelium. other transcriptional regulators associated with retinal progenitors in several vertebrate species are members of the basic helix-loop-helix family of proneural genes. a zebrafish ortholog of mammalian/chick mash1/cash called ascl1a has a moderate signal level in the peripheral cmz, is reduced in the middle and is absent from central regions. proneural genes related to the atonal family, such as neurod, are typically activated at later stages in the neuronal differentiation cascade and are associated with determination of specific neuronal cell types. in the retina, neurod specifies photoreceptors in zebrafish. the neurod probe produces only a very weak signal in the peripheral and middle cmz, but increases sharply in the central regions, especially the outer part, where photoreceptors are differentiating. all of these results are consistent with the idea that the most primitive retinal stem cells are located in the most peripheral region of the cmz adjacent to the ciliary epithelium and more restricted retinal progenitors lie closer to the differentiated retina. the notch-delta signaling pathway plays an important role in cell fate choice in the retina and in particular the decision of whether to differentiate as a neuron or a müller glia or to remain undifferentiated. several constituents of this bidirectional signaling pathway are expressed in the cmz in larval and juvenile zebrafish, including the notch ligands deltaa, deltab, deltac, deltad, several notch family members and downstream mediators of activated notch, her the zebrafish ortholog of mammalian hes and her2/hes. analysis of relative expression levels shows that in the most peripheral region of the cmz components of the notch-delta signaling cascade give moderate signals. expression levels are lower in the middle and central regions of the cmz. the signals for notch are similar to the notch paralogs. these data indicate that the notch-delta signaling pathway is activated in retinal progenitors including multipotent retinal stem cells that co-express a set of transcriptional regulators implicated in maintenance of multipotency and specification of retinal cell fate. the microenvironment of the zebrafish cmz has features typical of neural stem cell niches the radial glia/astrocyte marker blbp has been associated with adult neural stem cell niches in mammalian brain. we used a polyclonal antibody against a synthetic peptide of human blbp that is 90% identical to the predicted amino acid sequence of the orthologous zebrafish gene, fabp7a. we show that retinal stem cells and progenitors in the cmz and immature müller glial cells are immunoreactive for blbp. blbp immunoreactivity extends throughout the cmz and into the adjacent ciliary epithelium. in larval zebrafish retina, the cmz and immature müller glia distributed across the retina are blbp+, but in 2-month-old juvenile fish only the recently generated, immature müller glial cells closest to the cmz are labeled. thus, the blbp protein is down-regulated with maturation of zebrafish müller glia. in mammals, the extracellular matrix-associated carbohydrate epitope ssea-1/lex/cd is a surface marker for embryonic stem cells and adult neural stem cells as well as subpopulations of embryonic retinal progenitor cells. the antigen is the trisaccharide, 3-fucosyl-n-acetyl-lactosamine, which is a component of membrane glycolipids and glycoproteins from a variety of tissues. in zebrafish, the ssea- epitope is present at high levels around the circumferential blood vessel that lies on the vitreal surface at the border between neural retina and ciliary epithelium overlying the cmz and around other blood vessels that line the vitreal surface. a second site of high expression is a discrete laminar zone of undetermined identity in the inner plexiform layer, potentially on amacrine cell processes. the retinal pigmented epithelium also expresses ssea-. the calcium-dependent, homophilic cell adhesion protein n-cadherin is diffusely distributed on the basolateral plasma membranes of retinal stem cells and progenitors in the cmz and adjacent ciliary epithelium, similar to the distribution of n-cadherin on undifferentiated neuroepithelial progenitors in the embryonic retinal primordium. proliferating retinal progenitor cells in the cmz are ensheathed in a network of cadherin-mediated adhesive interactions with neighboring cells. in contrast, in the differentiated retina, n-cadherin is targeted to the zonula adherens that form the outer limiting membrane, to the synaptic layers and to axons of retinal ganglion cells. progenitors with the molecular profile of retinal stem cells are activated locally in differentiated retina by thermal lesions to determine whether retinal stem cells responsible for retinal regeneration following injury have molecular profiles similar to those in the cmz, we created local thermal lesions largely limited to the rpe and underlying photoreceptors in the retina of adult zebrafish. the diameter of the lesioned area ranges from ~ to μm. loss of photoreceptors is apparent within day post-lesion and pyknotic nuclei fill the onl and subretinal space between neural retina and rpe. destruction of cone photoreceptors within the lesion is revealed by the absence of immunoreactivity with zpr, a highly specific cell-surface marker for zebrafish double cones. thermal lesions trigger a local increase in mitotic activity in the retina, which begins within dpl. in lesioned areas elongated clusters of cells associated with radial fibers of müller glia appear by dpl. cells in these clusters incorporate brdu. we confirmed that incorporation of brdu reflects mitotic cell division and not dna repair in response to damage by immunostaining for proliferating cell nuclear antigen, pcna. these mitotic cells are closely associated with radial fibers of müller glia, all of which are strongly immunoreactive with antibodies against glial fibrillary acidic protein, gfap. nuclei of the activated müller glia also reenter the cell cycle and migrate from the inl into the onl, as we showed previously in regenerating goldfish retina. at and dpl the injury-induced proliferating cells associated with gfap+ radial müller fibers co-express at high levels the retinal stem cell markers rx vsx2/chx and pax6a. brdu+ mitotic clusters show strong rx and vsx2/chx signals. the expression of these transcription factors within the lesion extends vertically beyond the laminar boundaries of their respective expression domains in the differentiated retina. constituents of the notch-delta signaling pathway are also strongly upregulated in the injured retina after thermal lesions. between and dpl, deltac, notch1b and notch probes produce signals in the lesion area comparable in strength to those in the cmz. some brdu+ cells in the lesion area at dpl co-label with deltac. the deltac and notch signals are largely in adjacent cells and are not co-localized. the deltaa, deltab, deltad, and her6/hes probes similarly produce strong signals in the lesion area. in summary, injury-induced proliferating cells closely associated with radial fibers of müller glia express retinal stem cell transcription factors and activate notch signaling. injury induces a retinal stem cell niche that recreates the microenvironment of the cmz müller glial cells activated by the lesion acquire several features characteristic of retinal stem/progenitor cells in the cmz and in the embryonic retina. activated müller glial cells up-regulate expression of n-cadherin and the protein becomes distributed diffusely throughout the plasma membrane as it is in retinal stem cells and progenitors in the cmz. the inset in fig. 4e shows radial fibers of activated müller glia co-labeled with a monoclonal antibody specific for zebrafish gfap and polyclonal antibodies against zebrafish n-cadherin. in contrast, in the uninjured differentiated zebrafish retina, n-cadherin immunoreactivity primarily localizes to the adherens junctions formed by müller glia at the apical surface of the retina, to the synaptic neuropil of the inner and outer plexiform layers, and to retinal ganglion cell axons. the injury-induced clusters of brdu+ nuclei are tightly associated with radial müller fibers that are immunoreactive for n-cadherin. activated müller glia in the region of the lesion also up-regulate expression of blbp, which is characteristic of retinal stem/progenitor cells in the cmz and immature müller glia. the boundary between blbp+ and blbp- müller glia is very abrupt, which suggests that only the müller glia directly impacted by the retinal injury dedifferentiate and reacquire an immature molecular profile. cone photoreceptors regenerate within a week some of the mitotically active cells induced by the thermal lesion are retinal stem/progenitor cells that differentiate into retinal neurons, thus repairing the retinal damage. to identify the retinal stem cells responsible for regenerating retinal neurons, we first needed to know when the regenerated neurons are born so we could focus on cells proliferating during and/or before that time. not all of the mitotic cells at every interval following the lesion are retinal stem cells; for example, microglia proliferate rapidly in response to retinal injury and phagocytize the dead cells and debris. differentiated müller glia proliferate after injury, even in mammalian retinas that fail to regenerate neurons. in this study, we confined our analysis of the time course of neuronal regeneration in the retina to cone photoreceptors because they have distinctive and highly specialized morphology and several unique cell-specific markers. to document regeneration of cones, we first labeled proliferating cells with brdu at to dpl and then waited for several days up to one month to allow the progeny of the mitotic cells to differentiate. we then identified double-cones with the zpr antibody. when the retina is examined hours after brdu exposure at dpl, mitotic cells are labeled in the region of the lesion. at this stage, microglia are in the subretinal space between the outer limiting membrane and the rpe and are not part of the pool of proliferating cells within the retina. when fish survived for days after the brdu, a few progeny of the brdu+ cells had begun to differentiate and express the double cone marker zpr and they are small and immature compared with the intact cones adjacent to the lesion. by dpl the regenerated cones are fully differentiated and indistinguishable from the surrounding photoreceptors, with the exception that their nuclei retain the brdu-label. in all lesions at long survival intervals, persistent brdu+ nuclei are seen in the inl. we do not know the identity of these cells, although they do not have the morphological characteristics of müller glia and they are not immunoreactive with the microglia-specific antibody, 4c. we presume that most of them are retinal neurons, which were regenerated following loss due to the lesion, as has been well documented in several other models of retinal regeneration in teleost fish. to determine the time course of cone photoreceptor regeneration, we labeled proliferating cells with brdu at or dpl, and counted double-labeled cone photoreceptors at dpl. because the zpr antibody labels only the red-green double cones and zebrafish have two additional single cone types that express either blue or ultraviolet-sensitive opsin, we used a cocktail of rna probes to the four cone opsin classes – red, green, blue and ultraviolet – to label all cone types. we also counted brdu+ rod photoreceptors in the onl in the area of the lesion. cell counts show that regenerated cones in zebrafish are born between and dpl whereas most rods are born at dpl or later. in the developing zebrafish retina, cones are born before rods as they are in the embryonic mammalian retina. in our previous studies of retinal regeneration in goldfish we found that the ongoing production of rod photoreceptors from mitotic rod precursors largely ceases while the complement of cones is restored and here we show that a similar process occurs in zebrafish. this temporal shift in the distribution of photoreceptor cell types generated by the retinal stem cells suggests that they respond to changing microenvironmental factors similar to those found in the developing embryonic retina, which regulate the sequential production of retinal neurons. identity of the injury-activated retinal stem cells/progenitors the longitudinal birthdating analysis identified cells proliferating between and dpl as retinal progenitors whose progeny differentiate into cone photoreceptors. since cone photoreceptors are not normally produced in the differentiated regions of the intact retina, these data demonstrate that multipotent retinal stem cells persist in central retina with the capacity to respond to injury by altering their lineage profile to compensate for loss of specific neuronal subtypes. as described above, most of the mitotic activity in the lesion area at this time is in elongated clusters of proliferating cells in the inl closely associated with the radial fibers of müller glia. these proliferating cell clusters have been observed in all previous studies of retinal regeneration in teleost fish, and their similarity to the progenitors in the rod photoreceptor lineage of the uninjured retina has been noted. another potential source of latent retinal stem cells are the müller glia themselves. this question has been difficult to resolve because of the tight association of retinal progenitors in the rod lineage with the radial fibers of müller glia in both the uninjured retina and the regenerating retina, and the complexity of müller processes that enwrap neighboring cells. these intimate and complex anatomical relationships make it difficult to determine with fluorescent microscopy and the standard, cytoplasmic müller-specific markers, e.g. gfap, cellular retinaldehyde binding protein and glutamine synthetase, whether a given brdu+ nucleus belongs to the müller cell or to an associated progenitor. in an attempt to examine this question with better techniques, we sought a müller-specific marker that is confined to the cell body. apolipoprotein e is a small protein component of several different lipoproteins that is secreted by many cells including glia. to determine whether apoe is expressed by müller cells, we used transgenic zebrafish in which enhanced green fluorescent protein is controlled by transcriptional regulatory elements of the zebrafish gfap gene. the gfp reporter in this transgenic line is cytoplasmic and fills the cell body and all the processes of müller glia. we found that zebrafish apoe is expressed selectively and at high levels in gfap: gfp+ differentiated müller glia, and the signal is largely confined to the cell bodies in the inl. the apoe probe also co-localizes with a probe that hybridizes to rlbp1-l the zebrafish orthologue of the müller glial marker cralbp. it is therefore a useful marker to identify brdu+ proliferating müller glia. if müller glia function as retinal progenitors/stem cells in the injured retina, they should be proliferating before and/or during the interval when regenerated cone photoreceptors are being born, which we found is between and dpl. very few cells incorporate brdu at dpl, but at dpl approximately 80% of the proliferating cells are apoe+/brdu+ müller glia. the fraction of apoe+/brdu+ müller glia in the population of proliferating cells dropped to ~50% by and dpl, while the total number of brdu+ nuclei increased from a mean of per section at dpl, to per section at dpl, to per section at dpl. these data are consistent with several interpretations. müller glia that express apoe constitute the vast majority of the proliferating cells at dpl when the size of the brdu+ population is smallest. the remaining 20% of the brdu+ cells that are apoe- at this time could be progenitors in the rod lineage or dedifferentiated müller glia or progeny of müller glia that have down-regulated apoe expression. a closer examination of the rare brdu+ cells at early intervals prior to dpl with specific markers for retinal stem cells and progenitors might answer this question. the decline in the müller glia fraction of the expanding brdu+ population at and dpl could be explained by dedifferentiation of proliferating müller cells and/or a more rapid rate of proliferation of non-müller retinal progenitors. finally, these data are also consistent with the hypothesis that injury-induced retinal stem cells might derive from müller glia that begin to proliferate at or before dpl, dedifferentiate and give rise to retinal stem cells. discussion we have characterized the molecular microenvironment of two kinds of specialized niches that sustain retinal stem cells in zebrafish: 1) the ciliary marginal zone at the peripheral retinal margin and 2) some müller glia in the differentiated retina. the retinal stem cell niche in the zebrafish cmz has several features in common with the neurogenic niches in the adult mammalian brain, including the subventricular zone and the dentate gyrus subgranular zone, which have been described as'displaced neuroepithelium'. adult neural stem cells are specialized astrocytes that serve as both neural stem cells and niche cells; they extend processes to form apical junctions at the ventricular surface and also have extensive contacts with the basal lamina surrounding blood vessels. the retinal stem cells in the zebrafish cmz that co-express rx pax6a and vsx2/chx contact both the apical surface and the basal lamina and are in close proximity to a large blood vessel that is ssea-1+. other characteristics that mimic neuroepithelial cells of the developing neural tube and retina include activation of the notch signaling cascade and diffuse distribution of n-cadherin on the plasma membrane. the radial glial marker blbp is a downstream target of notch signaling and of pax, both of which are implicated in the maintenance of neural stem cell fate. the cmz cells in zebrafish express the radial glial marker blbp, as do the cells of the ciliary epithelium and retinal pigmented epithelium, and immature müller glia. mitotic activity in the cmz is stimulated by insulin-like growth factor in goldfish, and by wnt or sonic hedgehog in chick, mouse and xenopus retinas. the ssea- carbohydrate binds wnts, and its association with the ora collection vessel in zebrafish may contribute to the special microenvironment that maintains a neuroepithelium at the cmz. the proximity of the rpe on the apical side is another potential source of secreted signals that might promote neuroepithelial properties in the cmz. the rpe in zebrafish expresses blbp and is a source of shh. we have not yet looked for pedf in zebrafish, but this growth factor is secreted by components of the svz and supports self-renewal of adult neural stem cells in mice. a separate type of retinal stem cell niche is created within the differentiated retina by some of the müller glia. retinal müller glia are often compared with radial glia of the developing brain. like radial glia, processes of müller glia span the width of the retinal epithelium from the apical epithelial surface to the basal lamina at the vitreal surface and they express structural and functional markers of astrocytes. in the intact retina of teleost fish, rapidly proliferating inl progenitors are clustered around radial müller cell fibers and they use these radial processes as guides along which to migrate into the onl, as we showed previously by serial reconstruction electron microscopy. not all müller glia are associated with inl progenitors, and the frequency is highest near the retinal periphery. we do not yet know whether these neurogenic niches are associated with a specialized subpopulation of müller glial cells. in the intact retina, the retinal progenitors in these putative müller cell niches are directed exclusively into the rod lineage, but following injury, the frequency of neurogenic clusters increases, the fate of the retinal progenitors is altered, and the structural and molecular characteristics of the müller glia change. müller glia in the injured fish retina exhibit many phenotypic changes associated with reactive gliosis, such as up-regulation of gfap, which is often considered an indicator of stress and pathological processes and reentry into the cell cycle. here we show that they also exhibit phenotypic changes suggestive of dedifferentiation, such as re-expression of blbp and migration of their nuclei toward the apical surface, both of which are characteristic of immature müller glia in the larval zebrafish retina. the injury-induced retinal stem cells/multipotent progenitors are entwined in müller processes, which in response to injury up-regulate components of the notch-delta signaling pathways and n-cadherin. the n-cadherin protein becomes distributed to the basolateral membranes of the activated müller glia, similar to its distribution on neuroepithelial cells. radial glia in the embryonic mammalian brain are neurogenic progenitors and specialized astrocytes in the adult mammalian brain function as neural stem cells, so it is not surprising that we and others have suggested that müller glia might generate the injury-induced retinal stem cells in teleost fish, although direct evidence that employs a selective and permanent lineage tracer for müller glial fate mapping has not yet been accomplished. fischer and reh have proposed that a latent neurogenic capacity persists in müller glia of early postnatal chick based on observations of brdu incorporation and expression of neuronal markers in immature müller cells following intraocular injection of neurotoxins or growth factors, and similar observations were made in rat retina. however, physiologically relevant, regenerative repair of retinal neuron loss has only been demonstrated in teleost fish and some amphibians. one of the most striking morphological changes in the injured fish retina is the migration of proliferating retinal progenitor cells and müller nuclei to the apical surface and the subsequent reestablishment of a neuroepithelium; this anatomical organization is a necessary condition for regeneration of retinal neurons independent of the mechanism of injury. for example, neurotoxins that selectively destroy retinal neurons in the inner layers of adult goldfish fail to elicit a retinal stem cell response and the missing neurons are not replaced. only when photoreceptors are destroyed is the regenerative process triggered and the retina repaired including inner neurons as well as photoreceptors. one possible explanation for this differential response to injury is that damaged photoreceptors release factors that stimulate retinal stem cells and create a'regeneration niche'. another possibility, not mutually exclusive, is that in the absence of photoreceptors, the proliferating retinal progenitors reestablish contact with the apical surface thus recreating a neuroepithelial organization similar to that found in the cmz. migration of müller nuclei to the apical surface following loss of photoreceptors is clearly not a sufficient trigger to elicit a regenerative stem cell response, since müller glia in the mammalian retina undergo a similar behavior in the injured or dystrophic mammalian retina but do not generate neurons. the key unanswered question is what extrinsic signals are necessary and sufficient to provoke the formation of a regenerative retinal stem cell niche? several extracellular factors associated with maintenance of the undifferentiated, multipotent state in neural progenitors in early development, promote glial differentiation at later stages, including activation of lif and notch signaling. in the developing retina, activation of notch signaling either maintains undifferentiated progenitors or promotes müller glia differentiation, depending on stage-dependent contextual cues. the lif pathway may also be implicated: müller glia isolated from the neonatal mouse retina and induced to proliferate by exposure to growth factors express lif, and lif is necessary for injury-induced neurogenesis in mouse olfactory epithelium.  our data indicate that müller glia in the adult fish retina have a complex response to local injury that includes some features of reactive gliosis together with dedifferentiation and re-acquisition of phenotypic and molecular characteristics of multipotent retinal progenitors. the outcome of this injury-induced glial activation is not generation of more müller glia and formation of a glial scar, as is typical in mammalian retina, but instead a retinal stem cell niche is recreated with molecular and morphological features that mirror the neurogenic cmz. our data suggest that in the injured retina activated müller glia cells define the retinal stem cell niche and our results are consistent with the proposal that retinal stem cells derive from proliferating müller glia. this idea fits with the emerging view that adult neural stem cells in vivo are a subpopulation of radial glial-derived cells that retain a neurogenic potential. 
müller glia in the teleost retina have a complex response to local injury that includes some features of reactive gliosis together with dedifferentiation and re-acquisition of phenotypic and molecular characteristics of multipotent retinal progenitors in the cmz along with characteristics associated with radial glia. 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. 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. 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, 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 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. we also describe a novel specific marker for müller glia, apoe.
 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.  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, 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 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 together with dedifferentiation and re-acquisition of phenotypic and molecular characteristics of multipotent retinal progenitors in the cmz along with characteristics associated with radial glia. we also describe a novel specific marker for müller glia, apoe.  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. 