David A. Cameron

Responses of Müller glia to retinal injury in adult zebrafish

In an effort to identify the cellular events that enable neuronal regeneration in the vertebrate retina, the identity and characteristics of mitotic and apoptotic cells were examined in lesioned retinas of adult zebrafish. Following lesion a complex spatiotemporal pattern of mitosis was observed, including a delayed entry of Müller glia into the cell cycle. Characteristics of these proliferative Müller glia indicated they might serve as a stem/precursor cell of regenerated retina. The results suggested a model of retinal regeneration in which lesions are filled, in part, by a localized en place cytogenesis within intact retina surrounding the lesion site.

A molecular phenotype atlas of the zebrafish retina.

The rasborine cyprinid Danio rerio (the zebrafish) has become a popular model of retinal function and development. Its value depends, in part, on validation of homologies with retinal cell populations of cyprinine cyprinids. This atlas provides raw and interpreted molecular phenotype data derived from computationally classified sets of small molecule signals from different cell types in the zebrafish retina: L-alanine, L-aspartate, L-glutamine, L-glutamate, glutathione, glycine, taurine and γ-aminobutyrate. This basis set yields an 8-dimensional signature for every retinal cell and formally establishes molecular signature homologies with retinal neurons, glia, epithelia and endothelia of other cyprinids. Zebrafish photoreceptor classes have been characterized previously: we now show their metabolic profiles to be identical to those of the corresponding photoreceptors in goldfish. The inner nuclear layer is partitioned into precise horizontal, bipolar and amacrine cell layers. The horizontal cell layer contains at least three and perhaps all four known classes of cyprinine horizontal cells. Homologues of cyprinid glutamatergic ON-center and OFF-center mixed rod-cone bipolar cells are present and it appears likely that all five classes are present in zebrafish. The cone bipolar cells defy simple analysis but comprise the largest fraction of bipolar cells, as in all cyprinids. Signature analysis reveals six molecular phenotypes in the bipolar cell cohort: most are superclasses. The amacrine cell layer is composed of ≈64% GABA+ and 35% glycine+ amacrine cells, with the remainder being sparse dopaminergic interplexiform cells and other rare unidentified neurons. These different amacrine cell types are completely distinct in the dark adapted retina, but light adapted retinas display weak leakage of GABA signals into many glycinergic amacrine cells, suggesting widespread heterocellular coupling. The composition of the zebrafish ganglion cell layer is metabolically indistinguishable from that in other cyprinids, and the signatures of glial and non-neuronal cells display strong homologies with those in mammals. As in most vertebrates, zebrafish Müller cells possess a high glutamine, low glutamate signature and contain the dominant pool of glutathione in the neural retina. The retinal pigmented epithelium shows a general mammalian signature but also has exceptional glutathione content (5–10 mM), perhaps required by the unusually high oxygen tensions of teleost retinas. The optic nerve and the marginal zone of the retina reveal characteristic metabolic specializations. The marginal zone is strongly laminated and its nascent neurons display their characteristic signatures before taking their place in the retina proper.

A Visual Pigment Expressed in Both Rod and Cone Photoreceptors

Rods and cones contain closely related but distinct G protein-coupled receptors, opsins, which have diverged to meet the differing requirements of night and day vision. Here, we provide evidence for an exception to that rule. Results from immunohistochemistry, spectrophotometry, and single-cell RT-PCR demonstrate that, in the tiger salamander, the green rods and blue-sensitive cones contain the same opsin. In contrast, the two cells express distinct G protein transducin alpha subunits: rod alpha transducin in green rods and cone alpha transducin in blue-sensitive cones. The different transducins do not appear to markedly affect photon sensitivity or response kinetics in the green rod and blue-sensitive cone. This suggests that neither the cell topology or the transducin is sufficient to differentiate the rod and the cone response.

Cellular proliferation and neurogenesis in the injured retina of adult zebrafish

The retinas of adult teleost fish can regenerate neurons following a chemical or mechanical injury. Previous studies have demonstrated that mechanical excision of fish retina induces a hyperplasia within the retinal sheet, including the formation of a proliferative blastema from whence new retinal cells are produced to fill the excision site. The current study was designed to address two issues regarding injury-induced retinal hyperplasia: (1) Retinas of adult zebrafish can regenerate following a surgical excision, but compared to other fish they contain very few proliferative cells: Might retinal injury in adult zebrafish therefore induce minimal, or perhaps no, hyperplasia? (2) The fate of injury-induced, proliferative retinal cells outside surgical excision sites has yet to be determined. Do such cells produce retinal neurons? Evidence is presented that mechanical injury to the adult zebrafish retina induces a dramatic increase in the number of proliferative cells both within and external to the lesion site, and some of these cells apparently migrate within the radial dimension of the retina. Evidence is also presented that injury-induced proliferative cells outside a lesion site can produce retinal neurons--including cone photoreceptors, interplexiform cells, and amacrine cells--that are incorporated into the extant retina. The results suggest that the adult zebrafish retina contains a latent population of cells that is induced to proliferate following retinal injury, and that these cells might represent a novel avenue for pluripotent neurogenesis within the intact adult teleost retina.

Evidence for two distinct mechanisms of neurogenesis and cellular pattern formation in regenerated goldfish retinas

After its destruction by intraocular injection of ouabain, the goldfish retina regenerates, but little is known about the histogenesis of the new tissue, including the structure and formation of regenerated cell mosaic patterns. In an effort to determine how retinal cells are generated and spatially organized within retina regenerated after ouabain injection, in situ hybridization and immunocytochemical techniques were combined with computational analyses of two‐dimensional spatial patterns of identified neurons. Labeling with specific opsin riboprobes revealed two distinct cone patterns in the ouabain‐injected eyes, each of which was different from the relatively orderly cone patterns of native retina. Central, regenerated regions had sparse aggregates of cones, and a relatively lower density of each cone type. Peripheral regions of experimental retina, likely derived from the circumferential germinal zone, had high densities of all cone types, each of which tended to be distributed randomly. The spatial patterns of inner retinal neurons in experimental eyes were also disorganized with respect to native retina. These results indicate that although some aspects of retinal regeneration resemble normal retinal development and growth, ouabain‐induced regeneration does not produce well‐organized mosaics of neurons, indicating a failure of the developmental interactions needed for proper pattern formation, which in turn could compromise visual recovery. Furthermore, the distinct cone patterns in different regions of experimental retina support the hypothesis that new goldfish retina arises via two spatially and cellularly distinct mechanisms after exposure to ouabain. J. Comp. Neurol. 431:363–381, 2001.

Cell mosaic patterns in the native and regenerated inner retina of zebrafish: implications for retinal assembly

In part because of its laminar organization and morphologically distinct cell populations, the vertebrate retina has often been used as a system for investigating the assembly of neural structures. The retinas of adult teleost fish, because they grow throughout life and can regenerate following an injury, provide an especially attractive model system for such investigations. In an effort to provide a quantitative foundation for testing hypotheses regarding the mechanisms of pattern formation during growth and regeneration of the vertebrate retina, nearest neighbor and auto‐correlation analyses were used to examine the mosaic patterns of eight inner retinal cell groups in the native and regenerated retina of adult zebrafish. In both native and regenerated retina, the mosaic patterns of most inner retinal cells are non‐random. However, regenerated mosaics tend toward significantly lower nearest neighbor distances, less orderly patterns, and more variable radial locations than their native retina counterparts. The individual cell groups in both native and regenerated inner retina are likely to be spatially distributed independently. The results support the hypotheses that, in the adult zebrafish: 1) distinct inner retinal cell groups of native retina are also present in regenerated retina; 2) the assembly of inner retinal cell mosaics is controlled by non‐random spatial organizing mechanisms during development, growth, and regeneration; and 3) the spatial organization of cell mosaics is disrupted during regeneration. The results suggest that retinal regeneration may represent a spatially disrupted recapitulation of retinal developmental mechanisms. J. Comp. Neurol. 416:356‐367, 2000.

Mapping absorbance spectra, cone fractions, and neuronal mechanisms to photopic spectral sensitivity in the zebrafish.

The four spectral cone types in the zebrafish retina each contribute to photopic visual sensitivity as measured by the b-wave of the electroretinogram (ERG). The goal of the current study was to evaluate a model of photopic b-wave spectral sensitivity in the zebrafish that mapped first-order cellular and biophysical aspects of cone photoreceptors (visual pigment absorbance spectra and cone fractions) onto a second-order physiological aspect of cone-derived neural activity in the retina. Good correspondence between the model and photopic ERG data was attained using new visual pigment absorbance data for zebrafish cones (lambda(max) of the L, M, and S cones were 564, 473, and 407 nm, respectively), visual pigment templates, and linearly gained cone fractions. The model inferred four distinct cone processing channels that contribute to the photopic b-wave, two of which are antagonistic combinations of cone-derived signals (L-M and M-S), and two of which are noncombinatorial signals from S and U cones. The nature of the gains and the processing channels suggested general rules of cone-specific inputs to second-order neurons. The model further suggested that the zebrafish retina utilizes neuronal mechanisms for enhancing sensitivity to luminance contrast at short wavelengths and chromatic contrast at middle and long wavelengths. The results indicated that first-order cellular and biophysical aspects of cone photoreceptors can successfully explain physiological aspects of cone-derived neuronal activity in the zebrafish retina.

Photoreceptor Differentiation during Retinal Development, Growth, and Regeneration in a Metamorphic Vertebrate

To test the hypothesis that growth and regeneration of the adult retina involves a mechanistic recapitulation of retinal development, the patterns of photoreceptor differentiation were investigated in the developing retina, as well as growing and regenerating adult retina, of a metamorphic vertebrate, the winter flounder. Only one opsin, of type RH2 (a “green” cone opsin), was expressed in premetamorphic (developing) retina, and a corresponding middle-wavelength visual pigment was observed. In premetamorphic retinas there was no evidence for any other cone opsins or pigments, rods, rod opsin expression, or rod visual pigment. In contrast, a rod opsin (RH1) and three cone opsins (SWS2, RH2, and LWS) were expressed in postmetamorphic (adult) retina, and these opsins were consistent with the observed repertoire of visual pigments. During postmetamorphic retinal growth and regeneration, cones were always produced before rods, but the different cone types were apparently produced simultaneously, suggesting that cone differentiation mechanisms might change after metamorphosis. The results support the hypothesis that photoreceptor differentiation during growth and regeneration of the adult retina involves a recapitulation of mechanisms that control the sequence of photoreceptor production during retinal development.

Layer 6 cortical neurons require Reelin-Dab1 signaling for cellular orientation, Golgi deployment, and directed neurite growth into the marginal zone

BackgroundThe secreted ligand Reelin is believed to regulate the translocation of prospective layer 6 (L6) neocortical neurons into the preplate, a loose layer of pioneer neurons that overlies the ventricular zone. Recent studies have also suggested that Reelin controls neuronal orientation and polarized dendritic growth during this period of early cortical development. To explicitly characterize and quantify how Reelin controls this critical aspect of neurite initiation and growth we used a new ex utero explant model of early cortical development to selectively label a subset of L6 cortical neurons for complete 3-D reconstruction.ResultsThe total neurite arbor sizes of neurons in Reelin-deficient (reeler mutant) and Dab1-deficient (Reelin-non-responsive scrambler mutant) cortices were quantified and unexpectedly were not different than control arbor lengths (p = 0.51). For each mutant, however, arbor organization was markedly different: mutant neurons manifested more primary processes (neurites emitted directly from the soma) than wild type, and these neurites were longer and displayed less branching. Reeler and scrambler mutant neurites extended tangentially rather than radially, and the Golgi apparatus that normally invests the apical neurite was compact in both reeler and scrambler mutants. Mutant cortices also exhibited a neurite “exclusion zone” which was relatively devoid of L6 neuron neurites and extended at least 15 μm beneath the pial surface, an area corresponding to the marginal zone (MZ) in the wild type explants. The presence of an exclusion zone was also indicated in the orientation of mutant primary neurite and neuronal somata, which failed to adopt angles within ~20˚ of the radial line to the pial surface. Injection of recombinant Reelin to reeler, but not scrambler, mutant cortices fully rescued soma orientation, Golgi organization, and dendritic projection defects within four hrs.ConclusionsThese findings indicate Reelin promotes directional dendritic growth into the MZ, an otherwise exclusionary zone for L6 neurites.

Plasticity of photoreceptor-generating retinal progenitors revealed by prolonged retinoic acid exposure

BackgroundRetinoic acid (RA) is important for vertebrate eye morphogenesis and is a regulator of photoreceptor development in the retina. In the zebrafish, RA treatment of postmitotic photoreceptor precursors has been shown to promote the differentiation of rods and red-sensitive cones while inhibiting the differentiation of blue- and UV-sensitive cones. The roles played by RA and its receptors in modifying photoreceptor fate remain to be determined.ResultsTreatment of zebrafish embryos with RA, beginning at the time of retinal progenitor cell proliferation and prior to photoreceptor terminal mitosis, resulted in a significant alteration of rod and cone mosaic patterns, suggesting an increase in the production of rods at the expense of red cones. Quantitative pattern analyses documented increased density of rod photoreceptors and reduced local spacing between rod cells, suggesting rods were appearing in locations normally occupied by cone photoreceptors. Cone densities were correspondingly reduced and cone photoreceptor mosaics displayed expanded and less regular spacing. These results were consistent with replacement of approximately 25% of positions normally occupied by red-sensitive cones, with additional rods. Analysis of embryos from a RA-signaling reporter line determined that multiple retinal cell types, including mitotic cells and differentiating rods and cones, are capable of directly responding to RA. The RA receptors RXRγ and RARαb are expressed in patterns consistent with mediating the effects of RA on photoreceptors. Selective knockdown of RARαb expression resulted in a reduction in endogenous RA signaling in the retina. Knockdown of RARαb also caused a reduced production of rods that was not restored by simultaneous treatments with RA.ConclusionsThese data suggest that developing retinal cells have a dynamic sensitivity to RA during retinal neurogenesis. In zebrafish RA may influence the rod vs. cone cell fate decision. The RARαb receptor mediates the effects of endogenous, as well as exogenous RA, on rod development.