GAP-43 promoter elements in transgenic zebrafish reveal a difference in signals for axon growth during CNS development and regeneration

A pivotal event in neural development is the point at which differentiating neurons become competent to extend long axons. Initiation of axon growth is equally critical for regeneration. Yet we have a limited understanding of the signaling pathways that regulate the capacity for axon growth during either development or regeneration. Expression of a number of genes encoding growth associated proteins (GAPs) accompanies both developmental and regenerative axon growth and has led to the suggestion that the same signaling pathways regulate both modes of axon growth. We have tested this possibility by asking whether a promoter fragment from a well characterized GAP gene, GAP-43, is sufficient to activate expression in both developing and regenerating neurons. We generated stable lines of transgenic zebrafish that express green fluorescent protein (GFP) under regulation of a 1 kb fragment of the rat GAP-43 gene, a fragment that contains a number of evolutionarily conserved elements. Analysis of GFP expression in these lines confirms that the rat 1 kb region can direct growth-associated expression of the transgene in differentiating neurons that extend long axons. Furthermore, this region supports developmental down-regulation of transgene expression which, like the endogenous gene, coincides with neuronal maturation. Strikingly, these same sequences are insufficient for directing expression in regenerating neurons. This finding suggests that signaling pathways regulating axon growth during development and regeneration are not the same. While these results do not exclude the possibility that pathways involved in developmental axon growth are also active in regenerative growth, they do indicate that signaling pathway(s) controlling activation of the GAP-43 gene after CNS injury differ in at least one key component from the signals controlling essential features of developmental axon growth.     

The Asparaginyl Endopeptidase Legumain Is Essential for Functional Recovery after Spinal Cord Injury in Adult Zebrafish

Unlike mammals, adult zebrafish are capable of regenerating severed axons and regaining locomotor function after spinal cord injury. A key factor for this regenerative capacity is the innate ability of neurons to re-express growth-associated genes and regrow their axons after injury in a permissive environment. By microarray analysis, we have previously shown that the expression of legumain (also known as asparaginyl endopeptidase) is upregulated after complete transection of the spinal cord. In situ hybridization showed upregulation of legumain expression in neurons of regenerative nuclei during the phase of axon regrowth/sprouting after spinal cord injury. Upregulation of Legumain protein expression was confirmed by immunohistochemistry. Interestingly, upregulation of legumain expression was also observed in macrophages/microglia and neurons in the spinal cord caudal to the lesion site after injury. The role of legumain in locomotor function after spinal cord injury was tested by reducing Legumain expression by application of anti-sense morpholino oligonucleotides. Using two independent anti-sense morpholinos, locomotor recovery and axonal regrowth were impaired when compared with a standard control morpholino. We conclude that upregulation of legumain expression after spinal cord injury in the adult zebrafish is an essential component of the capacity of injured neurons to regrow their axons. Another feature contributing to functional recovery implicates upregulation of legumain expression in the spinal cord caudal to the injury site. In conclusion, we established for the first time a function for an unusual protease, the asparaginyl endopeptidase, in the nervous system. This study is also the first to demonstrate the importance of legumain for repair of an injured adult central nervous system of a spontaneously regenerating vertebrate and is expected to yield insights into its potential in nervous system regeneration in mammals.     

Growth and guidance cues for regenerating axons: where have they gone?

Both attractive and repellent cues are required to guide developing axons to their targets in the central nervous system. Critical guidance molecules in the developing brain include the semaphorins, netrins, slits, and ephrins. Current research indicates that many of these molecules and their receptors are expressed in the adult central nervous system (CNS), and that injury can alter the levels of these ligands/receptors. Recent studies have begun the process of elucidating the functions of these receptors in adult mammals, and the effects that they have on the regeneration of adult neurons. This review addresses our current knowledge with respect to the response of adult CNS neurons to axonal injury, interventions for enhancing the survival and regeneration of injured neurons, and the expression of developmental axon guidance cues in the injured mature CNS, with specific focus on the retino-tectal projection.     

Gene expression analysis of zebrafish retinal ganglion cells during optic nerve regeneration identifies KLF6a and KLF7a as important regulators of axon regeneration

Unlike mammals, teleost fish are able to mount an efficient and robust regenerative response following optic nerve injury. Although it is clear that changes in gene expression accompany axonal regeneration, the extent of this genomic response is not known. To identify genes involved in successful nerve regeneration, we analyzed gene expression in zebrafish retinal ganglion cells (RGCs) regenerating their axons following optic nerve injury. Microarray analysis of RNA isolated by laser capture microdissection from uninjured and 3-day post-optic nerve injured RGCs identified 347 up-regulated and 29 down-regulated genes. Quantitative RT-PCR and in situ hybridization were used to verify the change in expression of 19 genes in this set. Gene ontological analysis of the data set suggests regenerating neurons up-regulate genes associated with RGC development. However, not all regeneration-associated genes are expressed in differentiating RGCs indicating the regeneration is not simply a recapitulation of development. Knockdown of six highly induced regeneration-associated genes identified two, KLF6a and KLF7a, that together were necessary for robust RGC axon re-growth. These results implicate KLF6a and KLF7a as important mediators of optic nerve regeneration and suggest that not all induced genes are essential to mount a regenerative response.     

Histone deacetylase 1 controls cardiomyocyte proliferation during embryonic heart development and cardiac regeneration in zebrafish

In contrast to mammals, the zebrafish maintains its cardiomyocyte proliferation capacity throughout adulthood. However, neither the molecular mechanisms that orchestrate the proliferation of cardiomyocytes during developmental heart growth nor in the context of regeneration in the adult are sufficiently defined yet. We identified in a forward genetic N-ethyl-N-nitrosourea (ENU) mutagenesis screen the recessive, embryonic-lethal zebrafish mutant baldrian (bal), which shows severely impaired developmental heart growth due to diminished cardiomyocyte proliferation. By positional cloning, we identified a missense mutation in the zebrafish histone deacetylase 1 (hdac1) gene leading to severe protein instability and the loss of Hdac1 function in vivo. Hdac1 inhibition significantly reduces cardiomyocyte proliferation, indicating a role of Hdac1 during developmental heart growth in zebrafish. To evaluate whether developmental and regenerative Hdac1-associated mechanisms of cardiomyocyte proliferation are conserved, we analyzed regenerative cardiomyocyte proliferation after Hdac1 inhibition at the wound border zone in cryoinjured adult zebrafish hearts and we found that Hdac1 is also essential to orchestrate regenerative cardiomyocyte proliferation in the adult vertebrate heart. In summary, our findings suggest an important and conserved role of Histone deacetylase 1 (Hdac1) in developmental and adult regenerative cardiomyocyte proliferation in the vertebrate heart.     

Injury-associated induction of GAP-43 expression displays axon branch specificity in rat dorsal root ganglion neurons

Peripheral nerve injury results in the increased synthesis and axonal trasnport of the growth-associated protein GAP-43 in dorsal root ganglion (DRG) neurons, coincident with regenerative growth of the injured peripheral axon branches. To determine wheter the injury-associated signalling mechanism which leads to GAP-43 induction also operates through the central branches of DRG axons, we used immunocytochemistry to compare the expression of GAP-43 in adult rat DRG neurons 2 weeks after dorsal root crush lesions (central axotomy) or peripheral nerve crush lesions (peripheral axotomy). In uninjured ganglia, a subpopulation of smaller DRG neurons expresses moderate levels of GAP-43, whereas larger neurons generally do not. At 2 weeks following peripheral axotomy, virtually all axotomized neurons, large and small, express high levels of GAP-43. At 2 weeks following dorsal root lesions, no increase in GAP-43 expression is detected. Thus, the injury-associated up-regulation of GAP-43 expression in DRG neurons is triggered by a mechanism that is responsive to injury of only the peripheral, and not the central, axon branches. These findings support the hypothesis that GAP-43 induction in DRG neurons is caused by disconnection from peripheral target tissue, not by axon injury per se. © 1993 John Wiley & Sons, Inc.     

p38α MAPK regulates myocardial regeneration in zebrafish

Although adult mammals are unable to significantly regenerate their heart, this is not the case for a number of other vertebrate species. In particular, zebrafish are able to fully regenerate their heart following amputation of up to 20% of the ventricle. Soon after amputation, cardiomyocytes dedifferentiate and proliferate to regenerate the missing tissue. More recently, identical results have also been obtained in neonatal mice. Ventricular amputation of neonates leads to a robust regenerative response driven by the proliferation of existing cardiomyocytes in a similar manner to zebrafish. However, this ability is progressively lost during the first week of birth. The fact that adult zebrafish retain the capacity to regenerate their heart suggests that they either possess a unique regenerative mechanism, or that adult mammals lose/ inhibit this process. p38α ΜAPK has previously been shown to negatively regulate the proliferation of adult mammalian cardiomyocytes. We sought to determine whether a similar mechanism exists in adult zebrafish, and whether this needs to be overcome to allow regeneration to proceed. To determine whether p38α ΜAPK also regulates zebrafish cardiomyocytes in a similar manner, we generated conditional transgenic zebrafish in which either dominant-negative or active p38α ΜAPK are specifically expressed in cardiomyocytes. We found that active p38α ΜAPK but not dominantnegative p38α ΜAPK blocks proliferation of adult zebrafish cardiomyocytes and, consequently, heart regeneration as well. It appears that adult zebrafish cardiomyocytes share many characteristics with adult mammalian cardiomyocytes, including p38α MAPK-mediated cell cycle inhibition. These findings raise the possibility that zebrafish-like heart regeneration could be achieved in adult mammals.     

Axonally transported proteins associated with axon growth in rabbit central and peripheral nervous systems

In an effort to determine whether the “growth state” and the “mature state” of a neuron are differentiated by different programs of gene expression, we have compared the rapidly transported (group I) proteins in growing and nongrowing axons in rabbits. We observed two polypeptides (GAP-23 and GAP-43) which were of particular interest because of their apparent association with axon growth. GAP-43 was rapidly transported in the central nervous system (CNS) (retinal ganglion cell) axons of neonatal animals, but its relative amount declined precipitously with subsequent development. It could not be reinduced by axotomy of the adult optic nerves, which do not regenerate; however, it was induced after axotomy of an adult peripheral nervous system nerve (the hypoglossal nerve, which does regenerate) which transported only very low levels of GAP-43 before axotomy. The second polypeptide, GAP-23 followed the same pattern of growth-associated transport, except that it was transported at significant levels in uninjured adult hypoglossal nerves and not further induced by axotomy. These observations are consistent with the “GAP hypothesis” that the neuronal growth state can be defined as an altered program of gene expression exemplified in part by the expression of GAP genes whose products are involved in critical growth-specific functions. When interpreted in terms of GAP hypothesis, they lead to the following conclusions: (a) the growth state can be subdivided into a “synaptogenic state” characterized by the transport of GAP-23 but not GAP-43, and an “axon elongation state” requiring both GAPs; (b) with respect to the expression of GAP genes, regeneration involves a recapitulation of a neonatal state of the neuron; and (c) the failure of mammalian CNS neurons to express the GAP genes may underly the failure of CNS axons to regenerate after axon injury.     

Identification of Nogo-66 receptor (NgR) and homologous genes in fish

The Nogo-66 receptor NgR has been implicated in the mediation of inhibitory effects of central nervous system (CNS) myelin on axon growth in the adult mammalian CNS. NgR binds to several myelin-associated ligands (Nogo-66, myelin associated glycoprotein, and oligodendrocyte-myelin glycoprotein), which, among other inhibitory proteins, impair axonal regeneration in the CNS of adult mammals. In contrast to mammals, severed axons readily regenerate in the fish CNS. Nevertheless, fish axons are repelled by mammalian oligodendrocytes in vitro. Therefore, the identification of fish NgR homologs is a crucial step towards understanding NgR functions in vertebrate systems competent of CNS regeneration. Here, we report the discovery of four zebrafish (Danio rerio) and five fugu (Takifugu rubripes) NgR homologs. Synteny between fish and human, comparable intron-exon structures, and phylogenetic analyses provide convincing evidence that the true fish orthologs were identified. The topology of the phylogenetic trees shows that the extra fish genes were produced by duplication events that occurred in ray-finned fishes before the divergence of the zebrafish and pufferfish lineages. Expression of zebrafish NgR homologs was detected relatively early in development and prominently in the adult brain, suggesting functions in axon growth, guidance, or plasticity.     

Using zebrafish as the model organism to understand organ regeneration

The limited regenerative capacity of several organs, such as central nervous system(CNS), heart and limb in mammals makes related major diseases quite difficult to recover. Therefore, dissection of the cellular and molecular mechanisms underlying organ regeneration is of great scientific and clinical interests. Tremendous progression has already been made after extensive investigations using several model organisms for decades. Unfortunately, distance to the final achievement of the goal still remains. Recently, zebrafish became a popular model organism for the deep understanding of regeneration based on its powerful regenerative capacity, in particular the organs that are limitedly regenerated in mammals. Additionally, zebrafish are endowed with other advantages good for the study of organ regeneration. This review summarizes the recent progress in the study of zebrafish organ regeneration, in particular regeneration of fin, heart, CNS, and liver as the representatives. We also discuss reasons of the reduced regenerative capacity in higher vertebrate, the roles of inflammation during regeneration, and the difference between organogenesis and regeneration.     

Investigating regeneration and functional integration of CNS neurons: Lessons from zebrafish genetics and other fish species

Zebrafish possess a robust, innate CNS regenerative ability. Combined with their genetic tractability and vertebrate CNS architecture, this ability makes zebrafish an attractive model to gain requisite knowledge for clinical CNS regeneration. In treatment of neurological disorders, one can envisage replacing lost neurons through stem cell therapy or through activation of latent stem cells in the CNS. Here we review the evidence that radial glia are a major source of CNS stem cells in zebrafish and thus activation of radial glia is an attractive therapeutic target. We discuss the regenerative potential and the molecular mechanisms thereof, in the zebrafish spinal cord, retina, optic nerve and higher brain centres. We evaluate various cell ablation paradigms developed to induce regeneration, with particular emphasis on the need for (high throughput) indicators that neuronal regeneration has restored sensory or motor function. We also examine the potential confound that regeneration imposes as the community develops zebrafish models of neurodegeneration. We conclude that zebrafish combine several characters that make them a potent resource for testing hypotheses and discovering therapeutic targets in functional CNS regeneration. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases.     

Identification of Key Genes and Pathways Involved in the Heterogeneity of Intrinsic Growth Ability Between Neurons After Spinal Cord Injury in Adult Zebrafish

In the adult central nervous system (CNS), axon regeneration is a major hurdle for functional recovery after trauma. The intrinsic growth potential of an injured axon varies widely between neurons. The underlying molecular mechanisms of such heterogeneity are largely unclear. In the present study, the adult zebrafish dataset GSE56842 were downloaded. Differentially expressed genes (DEGs) were sorted and deeply analyzed by bioinformatics methods. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEGs were performed with the DAVID. A DEGs-associated protein–protein interaction network was constructed from the STRING database and visualized with Cytoscape software. In total, 621 DEGs were identified. GO analysis showed that the biological processes of DEGs focused mainly on the Notch signaling pathway, cell differentiation and positive regulation of neuron differentiation. The molecular functions mainly included calcium-transporting ATPase activity and calcium ion binding and structural constituents of the cytoskeleton. The cellular components included the plasma membrane, spectrin, and cytoplasmic and membrane-bound vesicles. KEGG pathway analysis showed that these DEGs were mainly involved in the metabolic pathway and Notch signaling pathway, and subnetworks revealed that genes within modules were involved in the metabolic pathway, Wnt signaling pathway, and calcium signaling pathway. This study identified DEG candidate genes and pathways involved in the heterogeneity of the intrinsic growth ability between neurons after spinal cord injury in adult zebrafish, which could facilitate our understanding of the molecular mechanisms underlying axon regeneration, and these candidate genes and pathways could be therapeutic targets for the treatment of CNS injury.

     

Why do mature CNS neurons of mammals fail to re-establish connections following injury--functions of bcl-2

Factors inside and outside neurons control the process of axonal growth and regeneration. Recently, it has become apparent that neurons are determined intrinsically for their ability to grow axons. In the mammalian CNS, the intrinsic machinery of neurons that triggers the growth of axons during early embryonic stages is shut down at a certain point in development; as a consequence, axon elongation and regeneration cannot occur in postnatal life. The proto-oncogene Bcl-2 has been recognized to act as a key regulator for the program of axon elongation inside neurons. However, expressing the gene Bcl-2 in CNS neurons is not sufficient to induce nerve regeneration in the adult CNS, eliminating the inhibitory mechanism in the mature CNS environment is still required. Recently, the formation of glia scar has been reported to be the major limiting factor in the CNS environment that blocks nerve regeneration. These new discoveries challenge the classical view of nerve regeneration in the mammalian CNS. It opens up a new dimension in the study of the cellular and molecular mechanisms underlying neurodevelopmental and neurodegenerative diseases.