Sperry's experiment implied the existence of axon guidance cues but did not reveal where they were or how they worked. For a time, one prominent view was that recognition occurred mostly at or near the target and that mechanical forces or long-range chemotactic factors sufficed to get axons to the vicinity of the target.
We now know that axons reach distant targets in a series of discrete steps, making frequent decisions at closely spaced intervals along their route. To illustrate this point we shall trace in greater detail the path that Sperry was trying to understand, that of a retinal axon growing to the optic tectum.
Growth Cones Diverge at the Optic Chiasm
The first task of the axon of a retinal ganglion cell is to leave the retina. As it enters the optic fiber layer it extends along the basal lamina and glial end-feet positioned at the retina's edge. The growth of the axon is oriented from the outset, indicating that it can read directional cues in the environment. As it approaches the center of the retina it comes under the influence of attractants emanating from the optic nerve head (the junction of the optic nerve with the retina proper), which guide it into the optic stalk. It then follows the optic nerve toward the brain (Figure 54–11).
The axons of retinal ganglion cells grow to the optic tectum in discrete steps.
Two neurons that carry information from the nasal half of the retina are shown. The axon of one crosses the optic chiasm to reach the contralateral optic tectum. The axon of the other also crosses the optic chiasm but projects to the lateral geniculate nucleus. The numbers indicate important landmarks on the axon's journey. The growing axon is directed toward the optic nerve head with the retina (1), enters into the optic nerve (2), extends through the optic nerve (3), swerves to remain ipsilateral (not shown) or cross to the contralateral side at the optic chiasm (4), extends through the optic tract (5), enters into the optic tectum or lateral geniculate nucleus (6), navigates to an appropriate rostrocaudal and dorsoventral position on the tectum (7), descends from the tectal surface (8), stops at an appropriate layer where a rudimentary terminal arbor is formed (9), and finally is refined (10).
The first axons to travel this route follow the cells of the optic stalk, the rudiment of the neural tube that connects the retina to the diencephalon from which it arose. These "pioneer" axons then serve as scaffolds for later-arriving axons, which are able to extend accurately simply by following their predecessors (see strategy 3 in Figure 54–9). Once they reach the optic chiasm, however, the retinal axons must make a choice. Axons that arise from neurons in the nasal hemiretina of each eye cross the chiasm and proceed to the opposite side of the brain, whereas those from the temporal half are deflected as they reach the chiasm and so stay on the same side of the brain (Figure 54–12A).
Axons of retinal ganglion neurons diverge as they reach the optic chiasm.
A. A time lapse series shows axons approaching the midline. Axons that arise from the nasal hemiretina cross the optic chiasm and project to the contralateral tectum (left). In contrast axons from the temporal hemiretina reach the chiasm but fail to cross and thus project toward the ipsilateral tectum (right). (Reproduced, with permission, from Godemont, Wong, and Mason 1994.)
B. The axons of temporal hemiretina neurons, which express the tyrosine kinase receptor EphB1, encounter ephrin-B2 expressed by midline radial glial cells at the optic chiasm and so are prevented from crossing the midline. The axons of nasal hemiretina neurons, which lack EphB1 receptors, are unaffected by the presence of ephrin-B2 and cross to the contralateral side. (A, anterior; P, posterior.)
C. Higher-power view illustrating the trajectories of retinal ganglion cell axons at the chiasm.
This divergence in trajectory reflects the differential responses of axons from the nasal and temporal hemiretinas to guidance cues presented by midline chiasm cells. Some retinal axons contact and traverse chiasm cells, whereas others are inhibited by these cells and deflected away, thus remaining in the ipsilateral side. One of the key molecules presented by chiasm cells is a membrane-bound repellent of the ephrin-B family (Figure 54–12B), which also figures in later steps of retinal ganglion cell axon guidance.
The fraction of temporal retinal axons that project ipsilaterally varies among species: a few in lower vertebrates, some in rodents, and many in humans. These differences reflect placement of the eyes. In many animals the eyes point to the sides and monitor different parts of the visual world, so that information from the two eyes need not be combined. In humans both eyes look forward and sample largely overlapping regions of the visual world, so coordination of visual input is essential.
After crossing the optic chiasm, retinal axons assemble in the optic tract along the ventral surface of the diencephalon. Axons then leave the tract at different points. In most vertebrate species the tectum of the midbrain (called the superior colliculus in mammals) is the major target of retinal axons, but a small number of axons project to the lateral geniculate nucleus of the thalamus. In humans, however, most axons project to the lateral geniculate, a sizable number reach the colliculus, and small numbers project to the pulvinar, superchiasmatic nucleus, and pretectal nuclei. Within these targets different retinal axons project to different regions. As Sperry showed, the retinal axons form a precise retinotopic map on the tectal surface. Similar maps form in other areas innervated by retinal axons.
Having reached an appropriate position within the tectum, retinal axons need to find an appropriate synaptic partner. To achieve this last leg of their journey, retinal axons turn and dive into the tectal neuropil (Figure 54–11), descending along the surface of radial glial cells, which provide a scaffold for radial axonal growth. Although radial glial cells span the entire extent of the neuroepithelium, each retinal axon confines its synaptic terminals to a single layer. The dendrites of many postsynaptic cells extend through multiple layers and form synapses along their whole length, but retinal inputs are restricted to a small fraction of the target neuron's dendritic tree. These organizational features imply that layer-specific cues arrest axonal elongation and trigger arborization.
Axons therefore solve the problem of long-distance navigation by dividing their journey into short segments, and by recognizing and responding to intermediate targets along the path to their final targets. Some intermediate targets, such as the optic chiasm, are "decision" regions where axons need to diverge.
Reliance on intermediate targets is an effective solution to the problem of long-distance axonal navigation but is not the only one. In some cases the first axons reach their targets when the embryo is small and the distance to be covered is short. These "pioneer" axons respond to molecular cues embedded in cells or the extracellular matrix along their way. The first axons to exit the retina fall within this class. Axons that appear later, when distances are longer and obstacles more numerous, can reach their targets by following the pioneers. Yet another guidance mechanism is a molecular gradient. Indeed, as we will see, gradients of cell-surface molecules in the tectum inform axons about their proper termination zone.
Ephrins Provide Gradients of Inhibitory Signals in the Brain
So far we have seen how retinal axons reach the tectum by responding to a series of discrete directional cues. However, these choices during growth do not account for the smoothly graded connections implied by Sperry's analysis of the retinotopic map in the tectum. The quest for the hypothetical "graded map molecules" became a major focus for developmental neurobiologists, and so we describe it in some detail.
A key breakthrough in the quest for these molecules came with the development of bioassays in which explants from defined portions of the retina were laid on substrates of tectal membrane fragments. The membrane fragments were taken from defined anteroposterior portions of the tectum and arranged in alternating stripes. Axons from the temporal (posterior) hemiretina were found to grow preferentially on membranes from anterior tectum, a preference similar to that exhibited in vivo (Figure 54–13). This preference was found to result from the presence of inhibitory factors in posterior membranes rather than from attractive or adhesive substances in anterior membranes. This observation was one of the first to emphasize the role of inhibitory or repellent substances in axon guidance.
Repellant signals guide developing retinal axons in vitro.
A. Retinal ganglion axons from the posterior (temporal) hemiretina project into the anterior developing tectum. Conversely, axons from the anterior (nasal) hemiretina project into the posterior tectum.
B. Fragments of membrane were taken from specified anteroposterior portions of the tectum and arranged in alternating strips. Axons from explants of posterior retina grow selectively on the fragments from anterior tectum. The preferential growth of axons on anterior membrane results from an inhibitory cue in the posterior membrane. In contrast, axons from anterior retina grow on both anterior and posterior tectal membrane fragments. (A, anterior; P, posterior.) (Modified, with permission, from Walter, Henke-Fahle, and Bonhoeffer 1987.)
This stripe assay permitted the characterization of an inhibitory cue, present in membranes from the posterior but not the anterior tectum. Independently, molecular biologists identified a family of receptor tyrosine kinases, the Eph kinases, and a large family of membrane-associated ligands, the ephrins. Both receptors and ligands are divided into A and B subfamilies. The ephrin-A proteins bind and activate EphA kinases; conversely, ephrin-B proteins bind and activate EphB kinases (Figure 54–14).
The formation of retinotopic maps in vivo depends on ephrin-Eph kinase signaling.
A. In the retina EphA receptors are expressed in an anteroposterior (A-P) gradient, and ephrin-B is expressed in a dorsoventral (D-V) gradient. In the tectum ephrin-A receptors are distributed in an anteroposterior gradient and EphB in a dorsoventral gradient.
B. Expression of EphA in retinal axons that derive from neurons in the posterior (temporal) retina directs axon growth to the anterior tectum through avoidance of ephrin-A proteins. In EphA mutant mice posterior retinal axons are able to project to a more posterior domain within the tectum.
C. EphB signaling directs the projection of dorsal retinal axons to the ventral tectum. Blocking ephrin-B signaling with soluble EphB protein causes dorsal axons to project to an abnormally dorsal domain within the tectum.
The two lines of research converged when the tectal inhibitory cue was identified as ephrin-A5. We now know that the Eph kinases and ephrins serve many functions in neural and nonneural tissues and that each class of proteins can serve as ligands or receptors, depending on cellular context. In the developing nervous system these proteins comprise a major group of repellent signals.
Ephrin-Eph interactions account for formation of the retinotopic map in the tectum. In the tectum ephrin-A2 and ephrin-A5 levels are graded along the anteroposterior axis, and in the retina the levels of the Eph receptors are also graded along the anteroposterior axis. These gradients run in opposite directions in the tectum: ephrin-A grades from anterior-low to posterior-high and in the retina Eph-A kinase from posterior-high to anterior-low (Figure 54–14A). Such counter-gradients account, at least in part, for topographic mapping. Axons from posterior retinal ganglion cells with high levels of receptors are repelled most strongly by the high level of ephrin-A in the posterior tectum and thus are confined to the anterior tectum. The less sensitive axons from the anterior retina are able to penetrate further into the posterior domain of the tectum. Ephrin-A2 and -A5 are therefore strong candidates for chemospecificity factors of the type postulated by Sperry.
The crucial role of ephrins and Eph kinases in the formation of retinotopic maps has been confirmed in vivo. Overexpression of ephrin-A2 in the developing optic tectum of chick embryos generates small patches of cells in the rostral tectum that are abnormally rich in ephrin-A2. Temporal retinal axons, which normally avoid the ephrin-rich caudal tectum, also avoid these patches in the rostral tectum, and they terminate in abnormal positions. In contrast, nasal retinal axons, which normally grow toward the caudal tectum, are not perturbed by encounters with excess ephrin.
Conversely, in mice with targeted mutations in the ephrin-A2 and ephrin-A5 genes some posterior retinal axons terminate in inappropriately posterior tectal regions (Figure 54–14B). Anterior retinal axons, which naturally express low levels of EphA proteins, project normally in these mutants. In mice lacking both ephrin-A proteins these deficits are more severe than with either single mutant. Thus the interaction of ephrin-A with EphA receptors is crucial for the targeting of retinal axons in the tectum. These ephrin/EphA pairs possess the properties of the recognition molecules that Sperry predicted were necessary to direct topographic mapping along the anteroposterior axis of the tectum.
But the retinal map is two-dimensional, so what establishes order along the dorsoventral axis? The ephrin/EphB pairs are involved in establishing this axis. Just as ephrin-A and EphA are graded along the anteroposterior axis, ephrin-B and EphB are graded along the dorsoventral axis, and genetic manipulation of ephrin-B and EphB levels affects dorsoventral mapping (Figure 54–14C). Thus the retinotopic map is arranged along rectangular coordinates with ephrin/EphA and ephrin/EphB labeling the anteroposterior and dorsoventral axes, respectively.
This simple view is satisfying, but the reality is more complex. First, Eph kinases are expressed in the tectum as well as in the retina, and ephrins are expressed in the retina as well as in the tectum. Thus, so-called "cis" interactions (Eph and ephrin on the same cell) as well as "trans" interactions (Eph on growth cone, ephrin on target cell) may be involved. Second, both ligands and receptors are present at multiple points along the optic pathway and play multiple roles. As we have seen, ephrin/EphB interactions affect not only dorsoventral mapping but also the decision of an axon to cross to the contralateral side at the optic chiasm. Finally, in visual circuits precise axonal mapping is regulated by patterns of neural activity, as discussed in the next two chapters. Nonetheless, we now have the initial outline of a molecular strategy for the formation of specific topographic projections from the eye to the brain.