Neuronal progenitors in the mammalian brain derive from radial glia or specialized astrocytes. In developing neural retina, radial glia-like Müller cells are generated late in neurogenesis and are not considered to be neuronal progenitors, but they do proliferate after injury and can express neuronal markers, suggesting a latent neurogenic capacity. To examine the neurogenic capacity of retinal glial cells, we used lineage tracing in transgenic zebrafish with a glial-specific promoter (gfap, for glial fibrillary acid protein) driving green fluorescent protein in differentiated Müller glia. We found that all Müller glia in the zebrafish retina express low levels of the multipotent progenitor marker Pax6 (paired box gene 6), and they proliferate at a low frequency in the intact, uninjured retina. Müller glia-derived progenitors express Crx (cone rod homeobox) and are late retinal progenitors that generate the rod photoreceptor lineage in the postembryonic retina. These Müller glia-derived progenitors also remain competent to produce earlier neuronal lineages, in that they respond to loss of cone photoreceptors by specifically regenerating the missing neurons. We conclude that zebrafish Müller glia function as multipotent retinal stem cells that generate retinal neurons by homeostatic and regenerative developmental mechanisms.

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

Cone photoreceptors in the zebrafish retina are arranged in a crystalline lattice, with each spectral subtype at a specific position in the array; rod photoreceptors are inserted around the cones. Patterning events and developmental mechanisms that lead to the formation of the cone mosaic are not known. To begin investigating this issue, we examined the initial stages of opsin expression in zebrafish embryos by in situ hybridization with goldfish opsin cRNA probes to determine how and when the cone mosaic pattern arises. We found both differences and similarities in the spatiotemporal patterns of rod and cone development, which suggest the following: 1) Expression of opsin message (including rod opsin, blue and red cone opsins) was found in a ventral patch of retina located nasal to the choroid fissure. 2) The cone mosaic pattern was generated by a crystallization-like process initiated in the precocial ventral patch and secondarily in nasal retina, which then swept like a wave into dorsotemporal retina. 3) The remainder of the retina, suggesting that these precocial rods might differ from typical rods. 4) Developmental maturation of rods in zebrafish, as reflected by expression of opsin, may be accelerated compared to cones, which are thought to become postmitotic before rods. These data are consistent with a model in which lateral inductive interactions among differentiating photoreceptors lead to patterning of the array.

Müller glia function as retinal stem cells in adult zebrafish. In response to loss of retinal neurons, Müller glia partially dedifferentiate, re-express neuroepithelial markers and re-enter the cell cycle. We show that the immunoglobulin superfamily adhesion molecule Alcama is a novel marker of multipotent retinal stem cells, including injury-induced Müller glia, and that each Müller glial cell divides asymmetrically only once to produce an Alcama-negative, proliferating retinal progenitor. The initial mitotic division of Müller glia involves interkinetic nuclear migration, but mitosis of retinal progenitors occurs in situ. Rapidly dividing retinal progenitors form neurogenic clusters tightly associated with Alcama/N-cadherinlabeled Müller glial radial processes. Genetic suppression of Ncadherin function interferes with basal migration of retinal progenitors and subsequent regeneration of HuC/D + inner retinal neurons.

The following describes a modified technique for obtaining 3-sm sections for light microscopic level immunocytochemistry. By combining 20% sucrose with Tissue-Tek OCT embedding compound in a ratio of21, we produced a block that was suitable for cutting 3-tm sections on a conventional cryostat. The 3-sm sections were dramatically improved cornpared with 10-srn sections cut frorn tissue ernbedded in OCT RAYMOND Literature Cited ' Johnson GD, Araujo GM: A simple method of reducing the fading of immunofluorescence during microscopy. J Immunol Methods 43:349, is Bignami A: Glial fibnillary acidic (GFA) protein in Mullen glia. Immu-1981 nofluorescence study of the goldfish retina. Brain Res 300:175, 1984 5. Raymond PA, Banthel LK: Retinal regeneration in goldfish: use of cell 2. Campbell AM: Monoclonal antibody technology. In Burdon RH, van specific monoclonal antibodies to assess the reestablishment of normal Knippenbeng PH, eds. Laboratory techniques in biochemistry and mo-cytoanchitecture.

In a microarray-based gene profiling analysis of Mü ller glia-derived retinal stem cells in light-damaged retinas from adult zebrafish, we found that 2 genes required for regeneration of fin and heart tissues in zebrafish, hspd1 (heat shock 60-kDa protein 1) and mps1 (monopolar spindle 1), were up-regulated. Expression of both genes in the neurogenic Mü ller glia and progenitors was independently verified by quantitative reverse transcriptase PCR and in situ hybridization. Functional analysis of temperature-sensitive mutants of hspd1 and mps1 revealed that both are necessary for Mü ller glia-based cone photoreceptor regeneration in adult zebrafish retina. In the amputated fin, hspd1 is required for the induction of mesenchymal stem cells and blastema formation, whereas mps1 is required at a later step for rapid cell proliferation and outgrowth. This temporal sequence of hspd1 and mps1 function is conserved in the regenerating retina. Comparison of gene expression profiles from regenerating zebrafish retina, caudal fin, and heart muscle revealed additional candidate genes potentially implicated in injury-induced epimorphic regeneration in diverse zebrafish tissues.T he study of regeneration has long fascinated biologists and has lately experienced a renaissance associated with growing interest in regenerative medicine and the therapeutic potential of stem cells. Zebrafish (Danio rerio) are an ideal genetic model for studying regeneration in vertebrates (1) because they have remarkable capabilities to regenerate fins (2), heart muscle (3), and nervous tissues (4-7) following injury. A forward mutagenesis screen for temperature-sensitive mutations that interfere with regeneration of amputated caudal fin identified several genes whose functions are critical for specific steps in fin regeneration, including mps1 (monopolar spindle 1, also called ttk, a kinase required for mitotic checkpoint regulation), hspd1 (heat shock 60-kDa protein 1, a mitochondrial chaperone), and fgf20 (fibroblast growth factor 20) (8-10). In addition, gene profiling analysis of regenerating tissues has provided lists of candidate genes associated with regeneration in fin (11), heart (12), and neural retina (13-15).The regeneration of retinal neurons in adult zebrafish is an especially powerful model for studying regeneration of neuronal tissues; laminar retinal architecture and visual function are restored following damage inflicted by surgical lesions, neurotoxins, and laser or photic lesions of retina (16). The neural stem cells in the retina arise from differentiated Müller glia, which respond to local retinal injuries by dedifferentiation, proliferation, and production of multipotent neuronal progenitors (retinal stem cells) that can regenerate all types of retinal neurons (17-19). To discover genes expressed in injury-activated neurogenic Müller glial cells that activate stem cell properties and trigger a neurogenic program, we generated transcriptional profiles of isolated fluorescent-tagged Müller glial cells from light-lesioned adult ...

Cone photoreceptors in fish are typically arranged into a precise, reiterated pattern known as a 'cone mosaic'. Cone mosaic patterns can vary in different fish species and in response to changes in habitat, yet their function and the mechanisms of their development remain speculative. Zebrafish (Danio rerio) have four cone subtypes arranged into precise rows in the adult retina.Here we describe larval zebrafish cone patterns and investigate a previously unrecognized transition between larval and adult cone mosaic patterns. Cone positions were determined in transgenic zebrafish, expressing green fluorescent protein (GFP) in their UV-sensitive cones, by the use of multiplex in situ hybridization labelling of various cone opsins. We developed a 'mosaic metric' statistical tool to measure local cone order. We found that ratios of the various cone subtypes in larval and adult zebrafish were statistically different. The cone photoreceptors in larvae form a regular heterotypic mosaic array, i.e. the position of any one cone spectral subtype relative to the other cone subtypes is statistically different from random. However, the cone spectral subtypes in larval zebrafish are not arranged in continuous rows as in the adult. We used cell birthdating to show that the larval cone mosaic pattern remains relatively disorganized, or perhaps is somewhat remodeled, within the adult retina. The abundance of cone subtypes relative to other subtypes is different in this larval remnant compared to that of larvae or canonical adult zebrafish retina. These observations provide baseline data for understanding the development of cone mosaics via comparative analysis of larval and adult cone development in a model species.

The orderly packing and precise arrangement of epithelial cells is essential to the functioning of many tissues, and refinement of this packing during development is a central theme in animal morphogenesis. The mechanisms that determine epithelial cell shape and position, however, remain incompletely understood. Here, we investigate these mechanisms in a striking example of planar order in a vertebrate epithelium: The periodic, almost crystalline distribution of cone photoreceptors in the adult teleost fish retina. Based on observations of the emergence of photoreceptor packing near the retinal margin, we propose a mathematical model in which ordered columns of cells form as a result of coupling between planar cell polarity (PCP) and anisotropic tissue-scale mechanical stresses. This model recapitulates many observed features of cone photoreceptor organization during retinal growth and regeneration. Consistent with the model's predictions, we report a planar-polarized distribution of Crumbs2a protein in cone photoreceptors in both unperturbed and regenerated tissue. We further show that the pattern perturbations predicted by the model to occur if the imposed stresses become isotropic closely resemble defects in the cone pattern in zebrafish lrp2 mutants, in which intraocular pressure is increased, resulting in altered mechanical stress and ocular enlargement. Evidence of interactions linking PCP, cell shape, and mechanical stresses has recently emerged in a number of systems, several of which show signs of columnar cell packing akin to that described here. Our results may hence have broader relevance for the organization of cells in epithelia. Whereas earlier models have allowed only for unidirectional influences between PCP and cell mechanics, the simple, phenomenological framework that we introduce here can encompass a broad range of bidirectional feedback interactions among planar polarity, shape, and stresses; our model thus represents a conceptual framework that can address many questions of importance to morphogenesis.