Ellen A. Schmitt

Early retinal development in the zebrafish, Danio rerio: Light and electron microscopic analyses

The morphological differentiation of the zebrafish retina was analyzed by using light (LM) and transmission electron (TEM) microscopy between the time of initial ganglion cell differentiation (≈32 hours postfertilization; hpf) and shortly after the point when the retina appears functional (≈74 hpf), i.e., when all major cell types and basic synaptic connections are in place. The results show that the inner retinal neurons, like the photoreceptor and ganglion cells, differentiate first within the ventronasal region, and differentiation subsequently spreads asymmetrically into the nasal and dorsal regions before reaching the ventrotemporal retina. In addition, we show that the attenuation of the optic stalk occurs in parallel with ganglion cell differentiation between 32 and 40 hpf. The first conventional synapses appear within the inner plexiform layer simultaneously with the first photoreceptor outer segment discs at 60 hpf; functional ribbon triads arise within photoreceptor synaptic terminals at 65 hpf; and synaptic ribbons occur within bipolar cell axon terminals at the time larvae exhibit their first visual responses (≈70 hpf). Although development is initially more advanced within the ventronasal region between 50 and 60 hpf, development across the retina rapidly equilibrates such that it is relatively comparable within all quadrants of the central retina by 70 hpf. An area within the temporal retina characterized by tightly packed and highly tiered cones emerges with subsequent development. Retinal differentiation in the zebrafish corresponds with that generally described in other vertebrates and can be correlated with the development of visual and electroretinographic responses in the animal. J. Comp. Neurol. 404:515–536, 1999.

Comparison of topographical patterns of ganglion and photoreceptor cell differentiation in the retina of the zebrafish, Danio rerio

Earlier studies suggested retinal differentiation in the zebrafish commences ventrally rather than centrally as is the case in other vertebrates. Here we describe the topographical spread of cell differentiation for ganglion cells, double cones and rods in the zebrafish retina between 36 and 72 hours postfertilization (hpf), by using immunohistochemical markers in retinal wholemounts. Staining for all three cell types commenced within the ventral retina on the nasal side of the optic nerve and choroid fissure, at 38 hpf for ganglion cells and 50 hpf for double cones and rods. Within 3 to 4 hours, the staining of ganglion cells and double cones spread in a continuous wave‐like fashion into the nasal region of the ventral retina. After this time, the staining patterns for ganglion cells and double cones progressed dorsally into the central and temporal retina. Finally, stained somata of ganglion cells were observed within the temporal‐ventral region by ∼48 hpf, more than 8 hours later than the first ganglion cells within the nasal retina. The topographical spread of double cone staining was slightly less orderly. After staining had extended into the nasal retina between 50 and 54 hpf, a small group of stained double cones often appeared at the temporal edge of the choroid fissure by 56 hpf, simultaneously with initial staining observed dorsal and temporal to the optic nerve.

Temporal and spatial patterns of opsin gene expression in zebrafish (Danio rerio)

In zebrafish, the first class of cone photoreceptor to become morphologically distinct is the ultraviolet-sensitive short single cone, at 4 days postfertilization, whereas the last class, the red- and green-sensitive double cone, becomes distinct at 10 days postfertilization. We have examined the time course of visual pigment gene expression in zebrafish using whole-mount in situ hybridization. Within the retina, opsins may be detected as early as 40 h postfertilization with the ultraviolet and rod visual pigments being expressed before the blue- (48 h) and red- (60 h) sensitive pigments. In the pineal, red-sensitive opsin is expressed at 48 h postfertilization. Visual pigment expression provides a useful tool for investigations of early cell fate in zebrafish.

Erratum: Temporal and spatial patterns of opsin gene expression in the zebrafish ( Danio rerio ): Corrections with additions

We report here a reexamination of the developmental expression of cone opsins in the zebrafish retina. The red- and blue-sensitive opsins appear at 51 h postfertilization (hpf) whereas ultraviolet (UV) opsin is not seen until after 55 hpf. More cells show red cone opsin expression than blue at 51 and 55 hpf, indicating the sequence of cone opsin expression in zebrafish is first red, then blue, and finally UV. Curiously, morphological development of the cones is in reverse order; UV cones appear quite mature by day 6-7 postfertilization (pf), but morphologically, red cones do not appear adult-like until 15-20 days pf.

Retinal patterning in the zebrafish mutant cyclops

Determination of cell fate in the vertebrate retina has been shown to be largely independent of lineage. After cell fates are determined, retinal neurons become organized in a precise laminar pattern. The mechanisms for this patterning could involve morphogens distributed in gradients or, alternatively, direct cell‐cell interactions. In the zebrafish mutant cyclops (cycb16), most embryos have two partial retinas joined in the ventral midline. This presents developing retinal cells near the midline with abnormal cellular environments, whereas laterally the pattern of developing cells is normal. We examined the consequences of this for patterning in the mutants retina. We found that the retinas are joined in the midline at the apical surfaces of the photoreceptor layers. A laminar pattern emerges in the midline that preserves normal positional relationships between retinal cell types locally but is abnormal with respect to patterning over the entire retina. Lateral to the midline, retinal patterning appears normal. Metabolic labeling experiments showed that late rounds of DNA synthesis precede the emergence of the novel pattern in this midline region. We conclude that these observations in the cyclops mutant are compatible with mechanisms of pattern formation in the retina involving local cell interactions. J. Comp. Neurol. 381:449‐460, 1997.