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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 birth dating to show that the larval cone mosaic pattern remains as a distinct region within the adult retina and does not reorganize into the adult row pattern. In addition, the abundance of cone subtypes relative to other subtypes is different in this larval remnant compared with 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.

Cell adhesion molecules in the cadherin family have been implicated in histogenesis and maintenance of cellular structure and function in several organs. Zebrafish have emerged as an important new developmental model, but only three zebrafish cadherin molecules have been identified to date (N-cadherin, paraxial protocadherin, and VN-cadherin). We began a systematic study to identify other zebrafish cadherins by screening zebrafish cDNA libraries using an antibody raised to the cytoplasmic domain of mouse E-cadherin. Here, we report a partial cDNA with extensive sequence homology to R-cadherin. Spatial and temporal expression of this putative zebrafish R-cadherin was examined in embryos and adults by Northern analysis, RNase protection, and in situ hybridization. R-cadherin message increased during embryogenesis up to 80 hours postfertilization (hpf) and persisted in adults. In the embryonic brain, R-cadherin was first expressed in groups of cells in the diencephalon and pretectum. In adult zebrafish brain, R-cadherin continued to be expressed in several specific regions including primary visual targets. In the retina, R-cadherin was first detected at about 33 hours postfertilization in the retinal ganglion cell layer and the inner part of the inner nuclear layer. Expression levels were highest during periods of axon outgrowth and synaptogenesis. Retrograde labeling of the optic nerve with 1,1'-dioctadecyl-3,3,3',3', tetramethylindocarbocyanine perchlorate (DiI) followed by in situ hybridization confirmed that a subset of retinal ganglion cells in the embryo expressed R-cadherin message. In the adult, R-cadherin expression continued in a subpopulation of retinal ganglion cells. These results suggest that R-cadherin-mediated adhesion plays a role in development and maintenance of neuronal connections in zebrafish visual system.

Mechanisms underlying axonal pathfinding have been investigated for decades, and numerous molecules have been shown to play roles in this process, including members of the cadherin family of cell adhesion molecules. We showed in the companion paper that a member of the cadherin family (zebrafish R-cadherin) is expressed in retinal ganglion cells, and in presumptive visual structures in zebrafish brain, during periods when the axons were actively extending toward their targets. The present study extends the earlier work by using 1,1'-dioctadecyl-3,3,3',3', tetramethylindocarbocyanine perchlorate (DiI) anterograde tracing techniques to label retinal ganglion cell axons combined with R-cadherin in situ hybridization to explicitly examine the association ofretinal axons and brain regions expressing R-cadherin message. We found that in zebrafish embryos at 46-54 hours postfertilization, DiI-labeled retinal axons were closely associated with cells expressing R-cadherin message in the hypothalamus, the pretectum, and the anterolateral optic tectum. These results demonstrate that R-cadherin is appropriately distributed to play a role in regulating development of the zebrafish visual system, and in particular, pathfinding and synaptogenesis of retinal ganglion cell axons.

Vsx-1 and Vsx-2 are two homeobox genes that were cloned originally from an adult goldfish retinal library. They are members of the paired-like:CVC gene family, which is characterized by the presence of a paired homeodomain and an additional conserved region, termed the CVC domain. To analyze the possible roles for Vsx-1 and Vsx-2 in eye development, we used in situ hybridization to examine their expression patterns in zebrafish and goldfish embryos. Vsx-2 is initially expressed by proliferating neuroepithelial cells of the presumptive neural retina, then it is down-regulated as differentiation begins, and it is finally reexpressed at later stages of differentiation in a subset of cells, presumed to be bipolar cells, in the inner nuclear layer. In contrast, Vsx-1 is expressed only weakly in undifferentiated, presumptive neural retina and is then up-regulated selectively in presumptive bipolar cells at early stages of differentiation (when Vsx-2 is turned off), before decreasing to an intermediate level, which is maintained in the differentiated (adult) retina. The restricted expression patterns of Vsx-2 correspond to the observed phenotypes in mice with the ocular retardation mutation (orJ), further supporting the notion that Vsx-2 and Chx10 are homologues. The sequential complimentary and then corresponding expression patterns of Vsx-1 and Vsx-2 suggest that these similar transcription factors may be recruited for partially overlapping, but distinct, functions during the development of the retina.

In this study, we have compared spatial and temporal aspects of development of new rods and cones in the adult goldfish by using a combination of bromodeoxyuridine immunocytochemistry and opsin in situ hybridization to determine the intervals between terminal mitosis (cell "birth") and expression of opsin mRNA for each photoreceptor cell type. The goldfish opsins include rod opsin and four different cone opsins: red, green, blue, and ultraviolet. In a cohort of photoreceptors born at the same time, rods expressed opsin mRNA within 3 days of cell birth, while expression of cone opsin mRNA required at least 7 days. This temporal discrepancy in differentiation, coupled with a discordance in the site of cell genesis of rods and cones, allowed opsin expression to commence in both cell types in approximately the same retinal location. Commitment to the generic cone phenotype occurred within approximately 6 days throughout the cone cohort, as indicated by expression of interphotoreceptor retinoid-binding protein (IRBP) mRNA, but expression of a specific spectral phenotype was delayed until rods differentiated nearby. Onset of expression of cone opsin mRNA followed a phenotype-specific sequence: red, then green, then blue, and finally ultraviolet; in situ hybridization with two opsin probes confirmed that individual photoreceptors expressed only one type of opsin as they differentiated. This stepwise process of cone differentiation is consistent with the hypothesis that cell-cell interactions among developing photoreceptors may coordinate selection of specific photoreceptor phenotypes.

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.

This study describes the differentiation of photoreceptors in larval goldfish retina. The earliest photoreceptors to differentiate were cones; 3H-fucose labeled cone but not rod outer segments in larval as well as adult goldfish. All major cone types known to be present in the adult goldfish retina (double cones, long and short single cones) were found in the larval retina by 2 days after hatching. The cones matured rapidly; within a few days they had well-developed outer segments and synaptic pedicles that were smaller, but otherwise similar to those in adults. Rods were slower to mature. Their outer segments were at first short, wide, and misshapen; only as they grew longer and narrower did they become straight and properly aligned. Rod spherules were first seen in fish older than 1 month; immature rods contained perinuclear synaptic ribbons and invaginating processes penetrated the cell body. These results suggest that the influence of rods and cones on visual function in larval goldfish may be quite different from the adult.

Throughout a goldfish's life, new generations of ganglion cells are added on the retinal margin and their axons extend centrally to occupy predictable positions in the retinotectal pathway, adjacent to their predecessors and subjacent to the pia. The stacking of successive generations of axons defines the age-axis of the pathway. This study examined whether an ordered array of predecessor axons is a prerequisite for the patterned growth of new axons. One optic nerve was crushed intraorbitally and the fish was injected with 3H-thymidine to label the proliferating cells on the retinal margin. The ring of 3H-thymidine-labeled cells separated retina that was present at the time of nerve crush (inside the ring) from new retina added afterward (outside). After a period of 14-16 months postcrush, both tectal lobes received two punctate applications of horseradish peroxidase (HRP), one in the central and the other in peripheral tectum, to retrogradely label contralateral retinal ganglion cell bodies and their axons. The pattern of HRP labeling from the control tectum confirmed earlier work: axons on the central tectum had somata in the central retina, and axons on the peripheral tectum had somata in the peripheral retina. The labeled cells and axons were both in predictable patterns. The somata that were backfilled from applications to the center of the experimental tectum lay inside the radioactive ring and had therefore regenerated their axons. The patterns of their labeled axons in the optic pathway and of their somata in the retina were typical of the regenerated condition as described in earlier studies. The somata backfilled from the periphery of the experimental tectum were outside the radioactive ring and had been added after the optic nerve crush. The patterns of their labeled axons and somata were comparable to the normal pattern. These observations indicate that new axons do not depend on an ordered array of predecessors to reestablish normal order along the age-axis of the pathway.

The mechanisms that control cell proliferation in the developing nervous system are not well understood. In larval and adult goldfish addition of new retinal neurons continues as the eye grows, but the factors that modulate the rate of cell proliferation are unknown. The eyes of Black Moors grow excessively during postembryonic life, probably as a direct result of abnormally elevated intraocular pressure. Ocular growth must be partly autonomous in Black Moors because in some individuals the two eyes are very different in size. To determine whether cell proliferation and neuronal cell number in the retina were correlated with size of the eye, we counted dividing neuronal progenitor cells (rod precursors) and mature retinal neurons (ganglion cells) in the retinas of ocularly asymmetric fish. Rod precursors, which are scattered across the retina in the outer nuclear layer, were labeled with 3H-thymidine and counted on histological sections processed for autoradiography. Ganglion cells were counted in retinal whole mounts. We found that the total population of dividing rod precursors and the total number of ganglion cells were systematically greater in the large eye compared to the small eye of individual fish. We conclude that control of the rate of neuronal proliferation in the teleost retina is intrinsic to the eye and is probably regulated by the same factors that control ocular growth.

The retinotectal projection in larval goldfish was studied with the aid of anterograde filling of optic fibers with HRP applied to the retina. The results show that optic fibers have already reached the tectum and begun to form terminal arbors in newly hatched fish. The projection is topographic in that fibers from local regions of the retina project to discrete patches of tectum, with the smallest patch covering 3.5% of the total surface area of tectal neuropil. Many fibers in young larvae have numerous short side branches along their length and only some of them show evidence of terminal sprouting. The arbors are approximately elliptical in shape and average about 1,500 microns 2. Growth cones are seen frequently. In older larvae, terminal arbors are larger and more highly branched, and they have begun to resemble those in adult fish. Fibers terminate in two strata; those in the upper layer are smaller (1,800 microns 2 on average) than those in the deeper stratum (4,000 microns 2 on average). The fraction of tectal surface area covered by individual arbors (the "tectal coverage") ranges from 1.5% to 3% of the total surface area of the tectal neuropil. In contrast, the tectal coverage of individual arbors in young adult goldfish is much smaller, ranging from 0.02% to 0.42% of tectal surface area (Stuermer, '84, and unpublished). This apparent increase in precision of the map in older animals is not due to retraction of arbors, which are slightly larger in adults, but is accounted for by overall tectal growth: the tectal neuropil in goldfish increases in area by about 250-fold during this period (Raymond, '86).