Adult zebrafish, in contrast to mammals, regrow axons descending from the brainstem after spinal cord transection. L1.1, a homolog of the mammalian recognition molecule L1, is upregulated by brainstem neurons during axon regrowth. However, its functional relevance for regeneration is unclear. Here, we show with a novel morpholino-based approach that reducing L1.1 protein expression leads to impaired locomotor recovery as well as reduced regrowth and synapse formation of axons of supraspinal origin after spinal cord transection. This indicates that L1.1 contributes to successful regrowth of axons from the brainstem and locomotor recovery after spinal cord transection in adult zebrafish.

Axon guidance by molecular gradients plays a crucial role in wiring up the nervous system. However, the mechanisms axons use to detect gradients are largely unknown. We first develop a Bayesian ''ideal observer'' analysis of gradient detection by axons, based on the hypothesis that a principal constraint on gradient detection is intrinsic receptor binding noise. Second, from this model, we derive an equation predicting how the degree of response of an axon to a gradient should vary with gradient steepness and absolute concentration. Third, we confirm this prediction quantitatively by performing the first systematic experimental analysis of how axonal response varies with both these quantities. These experiments demonstrate a degree of sensitivity much higher than previously reported for any chemotacting system. Together, these results reveal both the quantitative constraints that must be satisfied for effective axonal guidance and the computational principles that may be used by the underlying signal transduction pathways, and allow predictions for the degree of response of axons to gradients in a wide variety of in vivo and in vitro settings.axon guidance ͉ chemotaxis ͉ growth cone ͉ nerve growth factor ͉ nerve regeneration E ndogenous chemical gradients are a key source of information used by developing axons when wiring up the nervous system. Furthermore, artificially generated gradients are a potential therapy for restoring connectivity after neural injury. Many of the molecular gradients that direct axons in the developing nervous system have recently been identified, together with some of the signaling pathways through which they operate (1-8). However, our understanding of the mechanisms by which axons actually detect gradients remains qualitative. This limits our ability to predict both the response of axons when gradients are perturbed and the optimal parameters for promoting regrowth after injury.To be guided by a gradient, axons must be able to detect small spatial variations in receptor binding. This requires integrating signals from spatially distributed receptors to make a decision as to the direction of the gradient. Resources within the growth cone can then be marshaled appropriately by this information, for instance, via the production of steep gradients of intracellular signaling molecules (8). Although there is evidence for a role for gradients of molecules such as Ca 2ϩ in this latter step (9, 10), very little is known about the computations required to accurately make the initial decision.What constrains the ability of an axon to make a decision regarding gradient direction? Both experimental and computational work addressing chemotaxis in related systems such as bacteria, leukocytes, and Dictyostelium has identified the fundamental role of noise in limiting gradient perception. Noise can arise from low numbers of ligand molecules, from the stochastic nature of receptor binding, and from intracellular signaling pathways (11-16). Such constraints must also apply to axonal gradient sensing (17...

Guidance of axons by molecular gradients is crucial for wiring up the developing nervous system. It often is assumed that the unique signature of such guidance is immediate and biased turning of the axon tip toward or away from the gradient. However, here we show that such turning is not required for guidance. Rather, by a combination of experimental and computational analyses, we demonstrate that growth-rate modulation is an alternative mechanism for guidance. Furthermore we show that, although both mechanisms may operate simultaneously, biased turning dominates in steep gradients, whereas growth-rate modulation may dominate in shallow gradients. These results suggest that biased axon turning is not the only method by which guidance can occur.chemotaxis | growth cone | nerve growth factor | neural development | computational neuroscience F or the brain to function correctly, its neurons must be connected correctly, and wiring problems are known to underlie a number of nervous system disorders (1, 2). Some of the most important cues guiding the formation of connections between neurons in the developing nervous system are molecular gradients (3-10). To respond to a gradient cue, an axon must be able both to make a decision regarding gradient direction and then to convert that decision into directed motion (8). Although recent research has addressed the mechanisms involved in the decision-making step (11), how a decision regarding gradient direction is subsequently converted into a change in behavior of the growth cone is largely unknown.The most obvious mechanism in this regard is immediate and biased turning, whereby the axon tip is more likely to turn up the gradient (or down in the case of repulsive factors) each time the gradient direction is assessed. Such behavior is observed in in vitro assays that examine the response of axons to steep gradients of tropic factors in two dimensions (12-17). However, 3D in vitro assays, which reproduce more closely the conditions of axon growth in vivo, often fail to show consistent turning of axons. Rather, in these assays it tends to be the collective growth of a population of axons that is biased by the gradient (e.g., refs.18-20). Whether immediate and biased turning of axons in response to gradient signals occurs in vivo can be hard to assess, because in any specific situation axons are likely to respond to a combination of many different types of guidance cue. Here, we show that asymmetric growth of axons in response to a gradient signal does not require immediate and biased turning, and we identify an alternative mechanism for axonal chemotaxis based on growth-rate modulation. Our data suggest that, although immediate and biased turning and growthrate modulation may operate together, the former dominates in steep gradients, whereas the latter dominates in shallow gradients.

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Neuropilin-1, a receptor for axon-repellent semaphorins and vascular endothelial growth factor (VEGF), functions both in angiogenesis and axon growth. Here, we show strong expression of neuropilin-1a in primary motor neurons in the trunk of embryonic zebrafish. Reducing the expression of neuropilin-1a using antisense morpholino oligonucleotides induced aberrant branching of motor nerves, additional exit points of motor nerves from the spinal cord, and migration of neurons out of the spinal cord along the motor axon pathway in a dose-dependent manner. These phenotypes could be partially rescued by coinjecting neuropilin-1a mRNA. Other axons in the spinal cord and head appeared unaffected by the morpholino treatment. In addition, neuropilin-1a morpholino treatment disturbed normal formation of blood vessels in the trunk of 24 hours postfertilization embryos, as shown by microangiography. Morpholinos to VEGF also disturbed formation of blood vessels but did not affect motor axons, indicating that correct formation of blood vessels is not needed for the growth of primary motor axons. Morpholinos to the semaphorin 3A homologs semaphorin 3A1 and semaphorin 3A2 also had no effect on motor axon growth. However, combined injections of neuropilin-1a morpholino, at a concentration that did not elicit axonal aberrations when injected alone, with VEGF, semaphorin 3A1, or semaphorin 3A2 morpholinos synergistically increased the proportion of embryos showing aberrant motor axon growth. Thus, neuropilin-1a in primary motor neurons may integrate signals from several ligands and is needed for proper segmental growth of primary motor nerves in zebrafish. Developmental Dynamics 234:535-549, 2005.

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The pioneering primary motor axons in the zebrafish trunk are guided by multiple cues along their pathways. Plexins are receptor components for semaphorins that influence motor axon growth and path finding. We cloned plexinA3 in zebrafish and localized plexinA3 mRNA in primary motor neurons during axon outgrowth. Antisense morpholino knock-down led to substantial errors in motor axon growth. Errors comprised aberrant branching of primary motor nerves as well as additional exit points of axons from the spinal cord. Excessively branched and supernumerary nerves were found in both ventral and dorsal pathways of motor axons. The trunk environment and several other types of axons, including trigeminal axons, were not detectably affected by plexinA3 knock-down. RNA overexpression rescued all morpholino effects. Synergistic effects of combined morpholino injections indicate interactions of plexinA3 with semaphorin3A homologs. Thus, plexinA3 is a crucial receptor for axon guidance cues in primary motor neurons.

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