Dynamics of axonal regeneration in adult and aging zebrafish reveal the promoting effect of a first lesion

Axonal regeneration is a major issue in the maintenance of adult nervous systems, both after nerve injuries and in neurodegenerative diseases. However, studying this process in vivo is difficult or even impossible in most vertebrates. Here we show that the posterior lateral line (PLL) of zebrafish is a suitable system to study axonal regeneration in vivo because of both the superficial location and reproducible spatial arrangement of neurons and targets, and the possibility of following reinnervation in live fish on a daily basis. Axonal regeneration after nerve cut has been demonstrated in this system during the first few days of life, leading to complete regeneration within 24 h. However, the potential for PLL nerve regeneration has not been tested yet beyond the early larval stage. We explore the regeneration potential and dynamics of the PLL nerve in adult zebrafish and report that regeneration occurs throughout adulthood. We observed that irregularities in the original branching pattern are faithfully reproduced after regeneration, suggesting that regenerating axons follow the path laid down by the original nerve branches. We quantified the extent of target reinnervation after a nerve cut and found that the latency before the nerve regenerates increases with age. This latency is reduced after a second nerve cut at all ages, suggesting that a regeneration-promoting factor induced by the first cut facilitates regeneration on a second cut. We provide evidence that this factor remains present at the site of the first lesion for several days and is intrinsic to the neurons.The potential of adult neurons to regenerate their axons and to reinnervate target organs after injury is not as well understood as early axonogenesis. Whether and how this capability is modified on aging is an even less explored area. In all vertebrates studied so far, neurons of the peripheral nervous system retain the ability to reextend peripheral axons and reestablish functional connections. This ability is thought to involve intrinsic mechanisms of repair as well as extrinsic signals from the local environment, mostly coming from Schwann cells and macrophages (1).The posterior lateral line (PLL) of fish is a convenient yet unexplored sensory system to address the issue of axonal regeneration throughout adulthood. The PLL comprises a set of superficial mechanosensory organs called neuromasts, which are distributed over the body and tail. Neuromasts are composed of a core of mechanosensory hair cells providing information about the local water flow, surrounded by accessory cells. The afferent neurons innervating neuromasts are clustered in a ganglion posterior to the otic vesicle, and their peripheral axons extend toward the tail, right under the skin. This sensory system is involved in a large repertoire of behaviors (2).The juvenile PLL of zebrafish comprises four lines of neuromasts that extend at different dorsoventral levels, totaling about 50 organs (3). This pattern is established around 1 mo postfertilization (mpf) and remains essentially unchanged throughout adulthood, except that each juvenile neuromast gives rise to a number of “accessory” neuromasts through a budding process (4, 5) that depends on innervation (6). Bud-neuromasts remain closely associated and form dorsoventrally arranged linear clusters, or “stitches” (7). Here we concentrate on the most extensive of the four lines, the ventral one. This line lies originally along the horizontal myoseptum of the embryo and comprises only five neuromasts. More neuromasts develop during larval life, such that the line eventually consists of one neuromast on every intersomitic border, or about 30 altogether (8). The line migrates ventrally to reach its final position at the juvenile stage, and an axonal branch follows each neuromast during this migration.Axonal regeneration after nerve cut has been demonstrated in the PLL during the first few days of life, leading to complete reinnervation of neuromasts within 24 h (9, 10). Regenerating axons follow either the Schwann cells that ensheathe the nerve (10) or the interneuromast cells that extend between consecutive neuromasts (11). PLL nerve regeneration has not yet been addressed beyond the early larval stage.Here we examine whether the PLL nerve is able to regenerate in adult and aging zebrafish. Our data clearly demonstrate the effectiveness and fidelity of regeneration at all ages studied, from 1 to 15 mpf, although the onset of reinnervation is increasingly delayed with age, thereby linking neuronal aging with a progressive decline in neuronal reactivity to axonal damage. Whenever a second cut is made after complete regeneration, the latency of reinnervation is reduced at all ages, provided the second cut is immediately distal to the first one. We show that although Schwann cells act as guidance cues to help the axons regrow along their original path, they are not involved in this regeneration-promoting effect. We conclude that the promoting effect of a first lesion is mostly caused by an intrinsic, local change occurring in the injured axons. Altogether, our results reveal that the zebrafish PLL is a convenient system to study axonal regeneration in vivo compared with mammalian systems, in which nerve wiring is more complex and less traceable.     

A Phosphorylation Epitope on MAP 1B that is Transiently Expressed in Growing Axons in the Developing Rat Nervous System

We have isolated a monoclonal antibody (150) that recognizes a phosphorylation epitope on the microtubule-associated protein (MAP) 1B. Immunoblot analysis of the developing rat central nervous system shows that monoclonal antibody 150 is directed against a protein of ∼325 kDa (MAP 1B) that copolymerizes with microtubules through successive cycles of temperature-dependent assembly and disassembly. Furthermore, immunoprecipitated MAP 1B contains the epitope recognized by monoclonal antibody 150. Removal of phosphate from blotted proteins using alkaline phosphatase abolishes the binding of monoclonal antibody 150 to MAP 1B, indicating that the epitiope is phosphorylated. In the developing rat nervous system, immunohistochemistry with monoclonal antibody 150 shows that the phosphorylation epitope on MAP 1B is transiently expressed in growing axons but not in dendrites. For instance, in the neonatal rat cerebellum, the parallel fibres of granule cells are stained only during elongation and not after synaptogenesis. The monoclonal antibody 150 epitope is also transiently expressed in radial glial fibres and in certain cell nuclei. All immunostaining of sections with monoclonal antibody 150 was completely abolished by alkaline phosphatase treatment. These observations and previous ones made by us in cell culture (Mansfield et al., J. Neurocytol. , 20 , 654 – 666, 1991) suggest that the phosphorylation epitope on MAP 1B recognized by monoclonal antibody 150, which has not been previously detected in vivo , may be important in axonogenesis.     

Vertebrate-specific glutaredoxin is essential for brain development

Cellular functions and survival are dependent on a tightly controlled redox potential. Currently, an increasing amount of data supports the concept of local changes in the redox environment and specific redox signaling events controlling cell function. Specific protein thiol groups are the major targets of redox signaling and regulation. Thioredoxins and glutaredoxins catalyze reversible thiol-disulfide exchange reactions and are primary regulators of the protein thiol redox state. Here, we demonstrate that embryonic brain development depends on the enzymatic activity of glutaredoxin 2. Zebrafish with silenced expression of glutaredoxin 2 lost virtually all types of neurons by apoptotic cell death and the ability to develop an axonal scaffold. As demonstrated in zebrafish and in a human cellular model for neuronal differentiation, glutaredoxin 2 controls axonal outgrowth via thiol redox regulation of collapsin response mediator protein 2, a central component of the semaphorin pathway. This study provides an example of a specific thiol redox regulation essential for vertebrate embryonic development.     

Mllt11 Regulates Migration and Neurite Outgrowth of Cortical Projection Neurons during Development

The formation of connections within the mammalian neocortex is highly regulated by both extracellular guidance mechanisms and intrinsic gene expression programs. There are two types of cortical projection neurons (CPNs): those that project locally and interhemispherically and those that project to subcerebral structures such as the thalamus, hindbrain, and spinal cord. The regulation of cortical projection morphologies is not yet fully understood at the molecular level. Here, we report a role for Mllt11 (Myeloid/lymphoid or mixed-lineage leukemia; translocated to chromosome 11/All1 Fused Gene From Chromosome 1q) in the migration and neurite outgrowth of callosal projection neurons during mouse brain formation. We show that Mllt11 expression is exclusive to developing neurons and is enriched in the developing cortical plate (CP) during the formation of the superficial cortical layers. In cultured primary cortical neurons, Mllt11 is detected in varicosities and growth cones as well as the soma. Using conditional loss-of-function and gain-of-function analysis we show that Mllt11 is required for neuritogenesis and proper migration of upper layer CPNs. Loss of Mllt11 in the superficial cortex of male and female neonates leads to a severe reduction in fibers crossing the corpus callosum (CC), a progressive loss in the maintenance of upper layer projection neuron gene expression, and reduced complexity of dendritic arborization. Proteomic analysis revealed that Mllt11 associates with stabilized microtubules, and Mllt11 loss affected microtubule staining in callosal axons. Taken together, our findings support a role for Mllt11 in promoting the formation of mature upper-layer neuron morphologies and connectivity in the cerebral cortex.SIGNIFICANCE STATEMENT The regulation of cortical projection neuron (CPN) morphologies is an area of active investigation since the time of Cajal. Yet the molecular mechanisms of how the complex dendritic and axonal morphologies of projection neurons are formed remains incompletely understood. Although conditional mutagenesis analysis in the mouse, coupled with overexpression assays in the developing fetal brain, we show that a novel protein called Mllt11 is sufficient and necessary to regulate the dendritic and axonal characteristics of callosal projection neurons in the developing mammalian neocortex. Furthermore, we show that Mllt11 interacts with microtubules, likely accounting for its role in neuritogenesis.     

Ciliary neurotrophic factor stimulates neurite outgrowth from spinal cord neurons

Ciliary neurotrophic factor (CNTF) has been shown to promote the survival of motoneurons, but its effects on axonal outgrowth have not been examined in detail. Since nerve growth factor (NGF) promotes the outgrowth of neurites within the same populations of neurons that depend on NGF for survival, we investigated whether CNTF would stimulate neurite outgrowth from motoneurons in addition to enhancing their survival. We found that CNTF is a powerful promoter of neurite outgrowth from cultured chick embryo ventral spinal cord neurons. An effect of CNTF on neurite outgrowth was detectable within 7 hours, and at a concentration of 10 ng/ml, CNTF enhanced neurite length by about 3- to 4-fold within 48 hours. The neurite growth-promoting effect of CNTF does not appear to be a consequence of its survival-promoting effect. To determine whether the effect of CNTF on spinal cord neurons was specific for motoneurons, we analyzed cell survival and neurite outgrowth for motoneurons labeled with diI, as well as for neurons taken from the dorsal half of the spinal cord, which lacks motoneurons. We found that the effect of CNTF was about the same for motoneurons as it was for neurons from the dorsal spinal cord. The responsiveness of a variety of spinal cord neurons to CNTF may broaden the appeal of CNTF as a candidate for the treatment of spinal cord injury or disease. © 1996 Wiley-Liss, Inc.     

Restricted expression of the neuronal intermediate filament protein plasticin during zebrafish development

In the adult goldfish visual pathway, expression of the neuronal intermediate filament (nIF) protein plasticin is restricted to differentiating retinal ganglion cells (RGCs) at the margin of the retina. Following optic nerve injury, plasticin expression is elevated transiently in all RGCs coincident with the early stages of axon regeneration. These results suggest that plasticin may be expressed throughout the nervous system during the early stages of axonogenesis. To test this hypothesis, we analyzed plasticin expression during zebrafish (Danio rerio) neuronal development. By using immunocytochemistry and in situ hybridization, we found that plasticin is expressed in restricted subsets of early zebrafish neurons. Expression coincides with axon outgrowth in projection neurons that pioneer distinct axon tracts in the embryo. Plasticin is expressed first in trigeminal, Rohon-Beard, and posterior lateral line ganglia neurons, which are among the earliest neurons to initiate axonogenesis in zebrafish. Plasticin is expressed also in reticulospinal neurons and in caudal primary motoneurons. Together, these neurons establish the first behavioral responses in the embryo. Plasticin expression also coincides with initial RGC axonogenesis and progressively decreases after RGC axons reach the tectum. At later developmental stages, plasticin is expressed in a subset of the cranial nerves. The majority of plasticin-positive neurons are within or project axons to the peripheral nervous system. Our results suggest that plasticin subserves the changing requirements for plasticity and stability during axonal outgrowth in neurons that project long axons. J. Comp. Neurol. 399:561–572, 1998. © 1998 Wiley-Liss, Inc.