Retina development in zebrafish requires the heparan sulfate proteoglycan agrin

Recent studies from our laboratory have begun to elucidate the role of agrin in zebrafish development. One agrin morphant phenotype that results from agrin knockdown is microphthalmia (reduced eye size). To begin to understand the mechanisms underlying the role of agrin in eye development, we have analyzed retina development in agrin morphants. Retinal differentiation is impaired in agrin morphants, with retinal lamination being disrupted following agrin morpholino treatment. Pax 6.1 and Mbx1 gene expression, markers of eye development, are markedly reduced in agrin morphants. Formation of the optic fiber layer of the zebrafish retina is also impaired, exhibited as both reduced size of the optic fiber layer, and disruption of retinal ganglion cell axon growth to the optic tectum. The retinotectal topographic projection to the optic tectum is perturbed in agrin morphants in association with a marked loss of heparan sulfate expression in the retinotectal pathway, with this phenotype resembling retinotectal phenotypes observed in mutant zebrafish lacking enzymes for heparan sulfate synthesis. Treatment of agrin morphants with a fibroblast growth factor (Fgf) receptor inhibitor, rescue of the retinal lamination phenotype by transplantation of Fgf8-coated beads, and disruption of both the expression of Fgf-dependent genes and activation of ERK in agrin morphants provides evidence that agrin modulation of Fgf function contributes to retina development. Collectively, these agrin morphant phenotypes provide support for a crucial role of agrin in retina development and formation of an ordered retinotectal topographic map in the optic tectum of zebrafish.     

Two homeobox genes define the domain of EphA3 expression in the developing chick retina

Graded expression of the Eph receptor EphA3 in the retina and its two ligands, ephrin A2 and ephrin A5 in the optic tectum, the primary target of retinal axons, have been implicated in the formation of the retinotectal projection map. Two homeobox containing genes, SOHo1 and GH6, are expressed in a nasal-high, temporal-low pattern during early retinal development, and thus in opposing gradients to EphA3. Retroviral misexpression of SOHo1 or GH6 completely and specifically repressed EphA3 expression in the neural retina, but not in other parts of the central nervous system, such as the optic tectum. Under these conditions, some temporal ganglion cell axons overshot their expected termination zones in the rostral optic tectum, terminating aberrantly at more posterior locations. However, the majority of ganglion cell axons mapped to the appropriate rostrocaudal locations, although they formed somewhat more diffuse termination zones. These findings indicate that other mechanisms, in addition to differential EphA3 expression in the neural retina, are required for retinal ganglion axons to map to the appropriate rostrocaudal locations in the optic tectum. They further suggest that the control of topographic specificity along the retinal nasal-temporal axis is split into several independent pathways already at a very early time in development.     

Eph Family Receptors and Their Ligands Distribute in Opposing Gradients in the Developing Mouse Retina

The Eph family of receptor tyrosine kinases and their ligands can be divided into two specificity subclasses: the Eck-related receptors and their GPI-anchored ligands, and the Elk-related receptors and their transmembrane ligands. Previous reports demonstrated that Eck- and Elk-related receptors in the retina distribute in high temporal–low nasal and high ventral–low dorsal gradients, respectively. While others have focused on complementary ligand gradients in the retinal axon target, the tectum, we report that ligands from each subclass also distribute in gradients opposing those of their corresponding receptors within the retina itself. Moreover, ligand gradients in the retina precede ganglion cell genesis. These results support an intraretinal role for Eph family members in addition to their previously proposed role in the development of retinotectal topography. The distinct distributions of Eph family members suggest that each subclass specifies positional information along independent retinal axes.     

Regulation of axial patterning of the retina and its topographic mapping in the brain

Topographic maps are a fundamental organizational feature of axonal connections in the brain. A prominent model for studying axial polarity and topographic map development is the vertebrate retina and its projection to the optic tectum (or superior colliculus). Linked processes are controlled by molecules that are graded along the axes of the retina and its target fields. Recent studies indicate that ephrin-As control the temporal-nasal mapping of the retina in the optic tectum/superior colliculus by regulating the topographically-specific interstitial branching of retinal axons along the anterior-posterior tectal axis. This branching is mediated by relative levels of EphA receptor repellent signaling. A major recent advance is the demonstration that EphB receptor forward signaling and ephrin-B reverse signaling mediate axon attraction to control dorsal-ventral retinal mapping along the lateral-medial tectal axis. In addition, several classes of regulatory proteins have been implicated in the control of the axial patterning of the retina, and its ultimate readout of topographic mapping.     

A simple model can unify a broad range of phenomena in retinotectal map development

A paradigm model system for studying the development of patterned connections in the nervous system is the topographic map formed by retinal axons in the optic tectum/superior colliculus. Starting in the 1970s, a series of computational models have been proposed to explain map development in both normal conditions, and perturbed conditions where the retina and/or tectum/superior colliculus are altered. This stands in contrast to more recent models that have often been simpler than older ones, and tend to address more limited data sets, but include more recent genetic manipulations. The original exploration of many of the early models was one-dimensional and limited by the computational resources of the time. This leaves open the ability of these early models to explain both map development in two dimensions, and the genetic manipulation data that have only appeared more recently. In this article, we show that a two-dimensional and updated version of the XBAM model (eXtended Branch Arrow Model), first proposed in 1982, reproduces a range of surgical map manipulations not yet demonstrated by more modern models. A systematic exploration of the parameter space of this model in two dimensions also reveals richer behavior than that apparent from the original one-dimensional versions. Furthermore, we show that including a specific type of axon?Caxon interaction can account for the map collapse recently observed when particular receptor levels are genetically manipulated in a subset of retinal ganglion cells. Together these results demonstrate that balancing multiple influences on map development seems to be necessary to explain many biological phenomena in retinotectal map formation, and suggest important constraints on the underlying biological variables.     

Shared and distinct functions of RAGS and ELF-1 in guiding retinal axons.

Two ligands for Eph-related receptor tyrosine kinases, RAGS and ELF-1, have been implicated in the control of development of the retinotectal projection. Both molecules are expressed in overlapping gradients in the tectum, the target area of retinal ganglion cell axons. In two in vitro assays ELF-1 is shown to have a repellent axon guidance function for temporal, but apparently not for nasal axons. RAGS on the other hand is repellent for both types of axons, though to different degrees. Thus, RAGS and ELF-1 share some and differ in other properties. The biological activities of these molecules correlate with the strength of interaction with their receptors expressed on RGC axons. The meaning of these findings for guidance of retinal axons in the tectum is discussed.     

The receptor tyrosine phosphatase CRYPalpha affects growth cone morphology

During development of the nervous system receptor tyrosine kinases and receptor protein tyrosine phosphatases act in a coordinate way during axon growth and guidance. In the developing avian retinotectal system, many different receptor protein tyrosine phosphatases are expressed. Most of them have unknown functions. Retinal ganglion cells express at least three different members of this receptor family on their axons and growth cones: CRYPalpha, CRYP-2 and PTPmu. CRYPalpha interacts heterophilically with at least two different ligands found in the basal membranes of the retina and the optic tectum. To analyze the role of the CRYPalpha-ligand interaction, retinal ganglion cell axons were grown on retinal basal membranes (inner limiting membrane) and the receptor-ligand interaction was blocked from both the receptor side (by receptor specific antibodies) and from the ligand side by using a receptor-alkaline phosphatase fusion protein. Both of these treatments reduced average retinal axon length and induced a dramatic change in morphology of retinal ganglion cell growth cones on basal membranes, but not on other substrates like laminin, N-cadherin, matrigel- and detergent-treated basal membranes. These results suggest that CRYPalpha and its ligand act as growth-promoting molecules during intraretinal axon growth.     

Topographic mapping from the retina to the midbrain is controlled by relative but not absolute levels of EphA receptor signaling

Topographic maps are a fundamental feature of sensory representations in nervous systems. The formation of one such map, defined by the connection of ganglion cells in the retina to their targets in the superior colliculus of the midbrain, is thought to depend upon an interaction between complementary gradients of retinal EphA receptors and collicular ephrin-A ligands. We have tested this hypothesis by using gene targeting to elevate EphA receptor expression in a subset of mouse ganglion cells, thereby producing two intermingled ganglion cell populations that express distinct EphA receptor gradients. We find that these two populations form separate maps in the colliculus, which can be predicted as a function of the net EphA receptor level that a given ganglion cell expresses relative to its neighbors.     

Expression of receptor tyrosine phosphatases during development of the retinotectal projection of the chick

Receptor tyrosine kinases and receptor protein tyrosine phosphatases (RPTPs) appear to coordinate many aspects of neural development, including axon growth and guidance. Here, we focus on the possible roles of RPTPs in the developing avian retinotectal system. Using both in situ hybridization analysis and immunohistochemistry, we show for the first time that five RPTP genes--CRYPalpha, CRYP-2, PTPmu, PTPgamma, and PTPalpha--have different but overlapping expression patterns throughout the retina and the tectum. PTPalpha is restricted to Muller glia cells and radial glia of the tectum, indicating a possible function in controlling neuronal migration. PTPgamma expression is restricted to amacrine neurons. CRYPalpha and CRYP-2 mRNAs in contrast are expressed throughout the retinal ganglion cell layer from where axons grow out to their tectal targets. PTPmu is expressed in a subset of these ganglion cells. CRYPalpha, CRYP-2, and PTPmu proteins are also localized in growth cones of retinal ganglion cell axons and are present in defined laminae of the tectum. Thus, the spatial and temporal expression of three distinct RPTP subtypes--CRYPalpha, CRYP-2, and PTPmu--are consistent with the possibility of their involvement in axon growth and guidance of the retinotectal projection.     

Response of retinal ganglion cell axons to striped linear gradients of repellent guidance molecules

Although molecular gradients have long been postulated to play a role in the development of topographic projections in the nervous system, relatively little is known about how axons evaluate gradients. Do growth cones respond to concentration or to slope? Do they react suddenly or gradually? Is there adaptation? In the developing retinotectal system, temporal retinal ganglion cell axons have previously been shown to avoid repellent cell-surface activities distributed in gradients across the optic tectum. We confronted temporal retinal axons with precisely formed striped linear gradients of repellent tectal membranes and of two candidate repellent molecules, ephrin-A2 and -A5. Axons entered gradient stripes independently of their slope and extended unhindered in the uphill direction until they suddenly avoided an apparent threshold concentration of repellent material that was independent of slope. This critical concentration was similar in both linear and nonlinear gradients, and hence independent of gradient shape. When gradients of identical slope were formed on different basal levels of repellent material, axons grew uphill for a fixed increment of concentration, possibly measured from the lowest point of the gradient, rather than up to a fixed absolute concentration. The speed of growth cones was not affected by repellent unstriped gradients below the critical concentration level. Similar results were found with membranes from cell lines stably transfected with either ephrin-A5 or ephrin-A2, two previously identified growth cone repellent cell-surface proteins. These data suggest that growth cones or axons can integrate guidance information over large distances, probably by a combined memory and adaptation mechanism. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 541–562, 1998     

Genetic analysis of ephrin-A2 and ephrin-A5 shows their requirement in multiple aspects of retinocollicular mapping

Ephrin-A2 and -A5 are thought to be anteroposterior mapping labels for the retinotectal/retinocollicular projection. Here, gene disruptions of both these ephrins are characterized. Focal retinal labeling reveals moderate map abnormalities when either gene is disrupted. Double heterozygotes also have a phenotype, showing an influence of absolute levels. In vitro assays indicate these ephrins are required for repellent activity in the target and also normal responsiveness in the retina. In double homozygotes, anteroposterior order is almost though not completely lost. Temporal or nasal retinal labelings reveal quantitatively similar but opposite shifts, with multiple terminations scattered widely over the target. These results indicate an axon competition mechanism for mapping, with a critical role for ephrins as anteroposterior topographic labels. Dorsoventral topography is also impaired, showing these ephrins are required in mapping both axes.     

The Caenorhabditis elegans Six/sine oculis class homeobox gene ceh-32 is required for head morphogenesis

Repulsion plays a fundamental role in the establishment of a topographic map of the chick retinotectal projections. This has been highlighted by studies demonstrating the role of opposing gradients of the EphA3 receptor tyrosine kinase on retinal axons and two of its ligands, ephrin-A2 and ephrin-A5, in the tectum. We have analyzed the distribution of these two ephrins in other retinorecipient structures in the chick diencephalon and mesencephalon during the period when visual connections are being established. We have found that both ephrin-A2 and ephrin-A5 and their receptors EphA4 and EphA7 are expressed in gradients whose orientation is consistent with the topography of the nasotemporal axis of the respective retinofugal projections. In addition, their distribution suggests that receptor-ligand interactions may be involved in the organization of connections between the different primary visual centers and, thus, in the topographic organization of secondary visual projections. Interestingly, where projections lack a clear topographic representation, a uniform expression of the Eph-ephrin molecules was observed. Finally, we also show that a similar patterning mechanism may be implicated in the transfer of visual information to the telencephalon. These results suggest a conserved function for EphA receptors and their ligands in the elaboration of topographic maps at multiple levels of the visual pathway.     

Pathways of Retinotectal Projection in Developing Xenopus Tadpoles Revealed by Selectively Labeling of Retinal Axons with Horseradish Peroxidase (HRP)

Anatomical mapping was made of the retinal central pathways from the chiasm to the targets within the tectum in the developing Xenopus tadpoles, after labeling a specific regional population of retinal axons with horseradish peroxidase (HRP). In the tadpoles at stage 50, pathway sorting of retinal axons within the optic tract was clear for the dorsoventral axis of the retina, but not for the nasotemporal axis. Most nasal retinal axons and some dorsal and ventral retinal axons invaded the tectum directly at the diencephalotectal junction, and arrived at their correct sites of innervation after running through ectopic parts of the tectum. These findings indicate that the pathway orientation before targets is not a prerequisite factor for establishment of the orderly map of the retinotectal projection. Rather, a direct interaction between ingrowing retinal axons and tectal cells seems to be a predominant factor for specification of retinal central connections.     

Getting into shape: optimal ligand gradients for axonal guidance

During neural development, neurons from downstream, presynaptic regions of the nervous system (such as the retina) send spatially patterned axonal projections to upstream, target regions (the tectum or superior colliculus). A servomechanism model has been proposed to explain the pattern and time-course of axonal growth between these two regions [Honda, H., 1998. Topographic mapping in the retinotectal projection by means of complementary ligand and receptor gradients: a computer simulation study. J. Theor. Biol., 192, 235-246]. Here, we show that a modification of this model incorporating a different criterion for axonal decision-making, called the local optimum rule, is guaranteed to converge to a topographic map under a wide range of conditions encountered during neural development. A theoretical investigation of these conditions leads to new hypotheses regarding map formation.