Defect patterns on the curved surface of fish retinae suggest a mechanism of cone mosaic formation.

The outer epithelial layer of zebrafish retinae contains a crystalline array of cone photoreceptors, called the cone mosaic. As this mosaic grows by mitotic addition of new photoreceptors at the rim of the hemispheric retina, topological defects, called "Y-Junctions", form to maintain approximately constant cell spacing. The generation of topological defects due to growth on a curved surface is a distinct feature of the cone mosaic not seen in other well-studied biological patterns like the R8 photoreceptor array in the Drosophila compound eye.

Midkine-a Is Required for Cell Cycle Progression of Müller Glia during Neuronal Regeneration in the Vertebrate Retina.

In the retina of zebrafish, Müller glia have the ability to reprogram into stem cells capable of regenerating all classes of retinal neurons and restoring visual function. Understanding the cellular and molecular mechanisms controlling the stem cell properties of Müller glia in zebrafish may provide cues to unlock the regenerative potential in the mammalian nervous system. Midkine is a cytokine/growth factor with multiple roles in neural development, tissue repair, and disease.

Novel Animal Model of Crumbs-Dependent Progressive Retinal Degeneration That Targets Specific Cone Subtypes.

Purpose: Human Crb1 is implicated in some forms of retinal degeneration, suggesting a role in photoreceptor maintenance. Multiple Crumbs (Crb) polarity genes are expressed in vertebrate retina, although their functional roles are not well understood. To gain further insight into Crb and photoreceptor maintenance, we compared retinal cell densities between wild-type and Tg(RH2-2:Crb2b-sfEX/RH2-2:GFP)pt108b transgenic zebrafish, in which the extracellular domain of Crb2b-short form (Crb2b-sfEX) is expressed in the retina as a secreted protein, which disrupts the planar organization of RGB cones (red, green, and blue) by interfering with Crb2a/2b-based cone-cone adhesion.

Anisotropic Müller glial scaffolding supports a multiplex lattice mosaic of photoreceptors in zebrafish retina.

Background: The multiplex, lattice mosaic of cone photoreceptors in the adult fish retina is a compelling example of a highly ordered epithelial cell pattern, with single cell width rows and columns of cones and precisely defined neighbor relationships among different cone types. Cellular mechanisms patterning this multiplex mosaic are not understood. Physical models can provide new insights into fundamental mechanisms of biological patterning.

Rapid, Dynamic Activation of Müller Glial Stem Cell Responses in Zebrafish.

Purpose: Zebrafish neurons regenerate from Müller glia following retinal lesions. Genes and signaling pathways important for retinal regeneration in zebrafish have been described, but our understanding of how Müller glial stem cell properties are regulated is incomplete. Mammalian Müller glia possess a latent neurogenic capacity that might be enhanced in regenerative therapies to treat degenerative retinal diseases.

Regeneration: New Neurons Wire Up.

Functional repair of damage in the nervous system requires re-establishment of precise patterns of synaptic connectivity. A new study shows that after selective ablation, zebrafish retinal neurons regenerate and reconstruct some, although not all, of their stereotypic wiring.

Transmission from the dominant input shapes the stereotypic ratio of photoreceptor inputs onto horizontal cells.

Many neurons receive synapses in stereotypic proportions from converging but functionally distinct afferents. However, developmental mechanisms regulating synaptic convergence are not well understood. Here we describe a heterotypic mechanism by which one afferent controls synaptogenesis of another afferent, but not vice versa.

Patterning the cone mosaic array in zebrafish retina requires specification of ultraviolet-sensitive cones.

Cone photoreceptors in teleost fish are organized in precise, crystalline arrays in the epithelial plane of the retina. In zebrafish, four distinct morphological/spectral cone types occupy specific, invariant positions within a regular lattice. The cone lattice is aligned orthogonal and parallel to circumference of the retinal hemisphere: it emerges as cones generated in a germinal zone at the retinal periphery are incorporated as single-cell columns into the cone lattice.

Müller glia: Stem cells for generation and regeneration of retinal neurons in teleost fish.

Adult zebrafish generate new neurons in the brain and retina throughout life. Growth-related neurogenesis allows a vigorous regenerative response to damage, and fish can regenerate retinal neurons, including photoreceptors, and restore functional vision following photic, chemical, or mechanical destruction of the retina. Müller glial cells in fish function as radial-glial-like neural stem cells.

A self-renewing division of zebrafish Müller glial cells generates neuronal progenitors that require N-cadherin to regenerate retinal neurons.

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.

Retinal regeneration in adult zebrafish requires regulation of TGFβ signaling.

Müller glia are the resident radial glia in the vertebrate retina. The response of mammalian Müller glia to retinal damage often results in a glial scar and no functional replacement of lost neurons. Adult zebrafish Müller glia, in contrast, are considered tissue-specific stem cells that can self-renew and generate neurogenic progenitors to regenerate all retinal neurons after damage.

Coupling mechanical deformations and planar cell polarity to create regular patterns in the zebrafish retina.

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.

β-catenin/Wnt signaling controls progenitor fate in the developing and regenerating zebrafish retina.

Background: The zebrafish retina maintains two populations of stem cells: first, the germinal zone or ciliary marginal zone (CMZ) contains multipotent retinal progenitors that add cells to the retinal periphery as the fish continue to grow; second, radial glia (Müller cells) occasionally divide asymmetrically to generate committed progenitors that differentiate into rod photoreceptors, which are added interstitially throughout the retina with growth. Retinal injury stimulates Müller glia to dedifferentiate, re-enter the cell cycle, and generate multipotent retinal progenitors similar to those in the CMZ to replace missing neurons. The specific signals that maintain these two distinct populations of endogenous retinal stem cells are not understood.

Microarray-based gene profiling analysis of Müller glia-derived retinal stem cells in light-damaged retinas from adult zebrafish.

Microarray-based gene profiling has become an important technique to measure changes in gene expression on a genome-wide scale. Recently, cell-specific microarrays have been reported to study changes in gene expression of a particular cell type in several model organisms. Here, we describe a protocol to prepare RNA samples for microarray analysis of isolated Müller glia-derived retinal stem cells from light-damaged adult zebrafish expressing a fluorescent marker in Müller cells using enzymatic retinal dissociation followed by fluorescence-activated cell sorting (FACS).

FGF signaling regulates rod photoreceptor cell maintenance and regeneration in zebrafish.

Fgf signaling is required for many biological processes involving the regulation of cell proliferation and maintenance, including embryonic patterning, tissue homeostasis, wound healing, and cancer progression. Although the function of Fgf signaling is suggested in several different regeneration models, including appendage regeneration in amphibians and fin and heart regeneration in zebrafish, it has not yet been studied during zebrafish photoreceptor cell regeneration. Here we demonstrate that intravitreal injections of FGF-2 induced rod precursor cell proliferation and photoreceptor cell neuroprotection during intense light damage.

Ontogeny of cone photoreceptor mosaics in zebrafish.

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.

Midkine expression is regulated by the circadian clock in the retina of the zebrafish.

The retina displays numerous processes that follow a circadian rhythm. These processes are coordinated through the direct action of light on photoreceptive molecules and, in the absence of light, through autocrine/paracrine actions of extracellular neuromodulators. We previously described the expression of the genes encoding the secreted heparin-binding growth factors, midkine-a (mdka) and midkine-b (mdkb), in the retina of the zebrafish.

Genetic evidence for shared mechanisms of epimorphic regeneration in zebrafish.

In a microarray-based gene profiling analysis of Müller 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 Müller 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 Müller glia-based cone photoreceptor regeneration in adult zebrafish retina.

The teleost retina as a model for developmental and regeneration biology.

Retinal development in teleosts can broadly be divided into three epochs. The first is the specification of cellular domains in the larval forebrain that give rise to the retinal primordia and undergo early morphogenetic movements. The second is the neurogenic events within the retina proper-proliferation, cell fate determination, and pattern formation-that establish neuronal identities and form retinal laminae and cellular mosaics.

pak2a mutations cause cerebral hemorrhage in redhead zebrafish.

The zebrafish is a powerful model for studying vascular development, demonstrating remarkable conservation of this process with mammals. Here, we identify a zebrafish mutant, redhead (rhd(mi149)), that exhibits embryonic CNS hemorrhage with intact gross development of the vasculature and normal hemostatic function. We show that the rhd phenotype is caused by a hypomorphic mutation in p21-activated kinase 2a (pak2a).

Late-stage neuronal progenitors in the retina are radial Müller glia that function as retinal stem cells.

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.

Have we achieved a unified model of photoreceptor cell fate specification in vertebrates?

How does a retinal progenitor choose to differentiate as a rod or a cone and, if it becomes a cone, which one of their different subtypes? The mechanisms of photoreceptor cell fate specification and differentiation have been extensively investigated in a variety of animal model systems, including human and non-human primates, rodents (mice and rats), chickens, frogs (Xenopus) and fish. It appears timely to discuss whether it is possible to synthesize the resulting information into a unified model applicable to all vertebrates. In this review we focus on several widely used experimental animal model systems to highlight differences in photoreceptor properties among species, the diversity of developmental strategies and solutions that vertebrates use to create retinas with photoreceptors that are adapted to the visual needs of their species, and the limitations of the methods currently available for the investigation of photoreceptor cell fate specification.

Zebrafish dou yan mutation causes patterning defects and extensive cell death in the retina.

The size of an organ is largely determined by the number of cells it contains, which in turn is regulated by two opposing processes, cell proliferation and cell death, however, it is generally not clear how cell proliferation and cell death are coordinated during development. Here, we characterize the zebrafish dou yan(mi234) mutation that results in a dramatic reduction of retinal size and a disruption of retinal differentiation and lamination. The retinal size reduction is caused by increased retinal cell death in a non-cell-autonomous manner during early development.

Molecular characterization of retinal stem cells and their niches in adult zebrafish.

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.

Results: 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 (CMZ), a continuous annulus around the retinal circumference, and 2) the microenvironment around some Müller glia in the differentiated retina.

GFAP transgenic zebrafish.

We have generated transgenic zebrafish that express green fluorescent protein (GFP) in glial cells driven by the zebrafish glial fibrillary acidic protein (GFAP) regulatory elements. Transgenic lines Tg(gfap:GFP) were generated from three founders; the results presented here are from the mi2001 line. GFP expression was first visible in the living embryo at the tail bud-stage, then in the developing brain by the 5-somite-stage ( approximately 12 h post-fertilization, hpf) and then spreading posteriorly along the developing spinal cord by the 12-somite stage (approximately 15 hpf).