Assembly of lamina-specific neuronal connections by slit bound to type IV collagen.

The mechanisms that generate specific neuronal connections in the brain are under intense investigation. In zebrafish, retinal ganglion cells project their axons into at least six layers within the neuropil of the midbrain tectum. Each axon elaborates a single, planar arbor in one of the target layers and forms synapses onto the dendrites of tectal neurons. We show that the laminar specificity of retinotectal connections does not depend on self-sorting interactions among RGC axons. Rather, tectum-derived Slit1, signaling through axonal Robo2, guides neurites to their target layer. Genetic and biochemical studies indicate that Slit binds to Dragnet (Col4a5), a type IV Collagen, which forms the basement membrane on the surface of the tectum. We further show that radial glial endfeet are required for the basement-membrane anchoring of Slit. We propose that Slit1 signaling, perhaps in the form of a superficial-to-deep gradient, presents laminar positional cues to ingrowing retinal axons.

An essential role for Radar (Gdf6a) in inducing dorsal fate in the zebrafish retina.

Retinal ganglion cells form orderly topographic connections with the tectum, establishing a continuous neural representation of visual space. Mapping along the dorsal-ventral axis requires interactions between EphB and ephrin-B cell-surface molecules expressed as countergradients in both retina and tectum. We have discovered that the diffusible TGFss-related factor Radar (Gdf6a) is necessary and sufficient for activation of dorsal markers, such as Bmp4, Tbx5, Tbx2b, and Ephrin-B2, and suppression of the ventral marker Vax2 in the zebrafish retina. Radar mutant axons innervate only the dorsal half of the tectum, where they form a compressed retinotectal map. Wild-type cells transplanted into the dorsal retina are able to rescue the dorsal identity of nearby mutant cells. Moreover, Radar overexpression "dorsalizes" retinal ganglion cell identity in the ventral retina. We conclude that Radar is near the top of a signaling cascade that establishes dorsal-ventral positional information in the retina and controls the formation of the retinotectal map.

Retinotopic order in the absence of axon competition.

The retinotectal projection has long been studied experimentally and theoretically, as a model for the formation of topographic brain maps. Neighbouring retinal ganglion cells (RGCs) project their axons to neighbouring positions in the optic tectum, thus re-establishing a continuous neural representation of visual space. Mapping along this axis requires chemorepellent signalling from tectal cells, expressing ephrin-A ligands, to retinal growth cones, expressing EphA receptors. High concentrations of ephrin A, increasing from anterior to posterior, prevent temporal axons from invading the posterior tectum. However, the force that drives nasal axons to extend past the anterior tectum and terminate in posterior regions remains to be identified. We tested whether axon-axon interactions, such as competition, are required for posterior tectum innervation. By transplanting blastomeres from a wild-type (WT) zebrafish into a lakritz (lak) mutant, which lacks all RGCs, we created chimaeras with eyes that contained single RGCs. These solitary RGCs often extended axons into the tectum, where they branched to form a terminal arbor. Here we show that the distal tips of these arbors were positioned at retinotopically appropriate positions, ruling out an essential role for competition in innervation of the ephrin-A-rich posterior tectum. However, solitary arbors were larger and more complex than under normal, crowded conditions, owing to a lack of pruning of proximal branches during refinement of the retinotectal projection. We conclude that dense innervation is not required for targeting of retinal axons within the zebrafish tectum but serves to restrict arbor size and shape.

Targeting neural circuitry in zebrafish using GAL4 enhancer trapping.

We present a pilot enhancer trap screen using GAL4 to drive expression of upstream activator sequence (UAS)-linked transgenes in expression patterns dictated by endogenous enhancers in zebrafish. The patterns presented include expression in small subsets of neurons throughout the larval brain, which in some cases persist into adult. Through targeted photoconversion of UAS-driven Kaede and variegated expression of UAS-driven GFP in single cells, we begin to characterize the cellular components of labeled circuits.

Vesicular glutamate transport at a central synapse limits the acuity of visual perception in zebrafish.

The neural circuitry that constrains visual acuity in the CNS has not been experimentally identified. We show here that zebrafish blumenkohl (blu) mutants are impaired in resolving rapid movements and fine spatial detail. The blu gene encodes a vesicular glutamate transporter expressed by retinal ganglion cells. Mutant retinotectal synapses release less glutamate, per vesicle and per terminal, and fatigue more quickly than wild-type in response to high-frequency stimulation. In addition, mutant axons arborize more extensively, thus increasing the number of synaptic terminals and effectively normalizing the combined input to postsynaptic cells in the tectum. This presumably homeostatic response results in larger receptive fields of tectal cells and a degradation of the retinotopic map. As predicted, mutants have a selective deficit in the capture of small prey objects, a behavior dependent on the tectum. Our studies successfully link the disruption of a synaptic protein to complex changes in neural circuitry and behavior.

Forward genetic analysis of visual behavior in zebrafish.

The visual system converts the distribution and wavelengths of photons entering the eye into patterns of neuronal activity, which then drive motor and endocrine behavioral responses. The gene products important for visual processing by a living and behaving vertebrate animal have not been identified in an unbiased fashion. Likewise, the genes that affect development of the nervous system to shape visual function later in life are largely unknown. Here we have set out to close this gap in our understanding by using a forward genetic approach in zebrafish. Moving stimuli evoke two innate reflexes in zebrafish larvae, the optomotor and the optokinetic response, providing two rapid and quantitative tests to assess visual function in wild-type (WT) and mutant animals. These behavioral assays were used in a high-throughput screen, encompassing over half a million fish. In almost 2,000 F2 families mutagenized with ethylnitrosourea, we discovered 53 recessive mutations in 41 genes. These new mutations have generated a broad spectrum of phenotypes, which vary in specificity and severity, but can be placed into only a handful of classes. Developmental phenotypes include complete absence or abnormal morphogenesis of photoreceptors, and deficits in ganglion cell differentiation or axon targeting. Other mutations evidently leave neuronal circuits intact, but disrupt phototransduction, light adaptation, or behavior-specific responses. Almost all of the mutants are morphologically indistinguishable from WT, and many survive to adulthood. Genetic linkage mapping and initial molecular analyses show that our approach was effective in identifying genes with functions specific to the visual system. This collection of zebrafish behavioral mutants provides a novel resource for the study of normal vision and its genetic disorders.