Neurogenesis in the inner ear: the zebrafish statoacoustic ganglion provides new neurons from a Neurod/Nestin-positive progenitor pool well into adulthood.

The vertebrate inner ear employs sensory hair cells and neurons to mediate hearing and balance. In mammals, damaged hair cells and neurons are not regenerated. In contrast, hair cells in the inner ear of zebrafish are produced throughout life and regenerate after trauma. However, it is unknown whether new sensory neurons are also formed in the adult zebrafish statoacoustic ganglion (SAG), the sensory ganglion connecting the inner ear to the brain. Using transgenic lines and marker analysis, we identify distinct cell populations and anatomical landmarks in the juvenile and adult SAG. In particular, we analyze a Neurod/Nestin-positive progenitor pool that produces large amounts of new neurons at juvenile stages, which transitions to a quiescent state in the adult SAG. Moreover, BrdU pulse chase experiments reveal the existence of a proliferative but otherwise marker-negative cell population that replenishes the Neurod/Nestin-positive progenitor pool at adult stages. Taken together, our study represents the first comprehensive characterization of the adult zebrafish SAG showing that zebrafish, in sharp contrast to mammals, display continued neurogenesis in the SAG well beyond embryonic and larval stages.

Single cell sequencing of radial glia progeny reveals the diversity of newborn neurons in the adult zebrafish brain.

Zebrafish display widespread and pronounced adult neurogenesis, which is fundamental for their regeneration capability after central nervous system injury. However, the cellular identity and the biological properties of adult newborn neurons are elusive for most brain areas. Here, we have used short-term lineage tracing of radial glia progeny to prospectively isolate newborn neurons from the her4.1+ radial glia lineage in the homeostatic adult forebrain. Transcriptome analysis of radial glia, newborn neurons and mature neurons using single cell sequencing identified distinct transcriptional profiles, including novel markers for each population. Specifically, we detected two separate newborn neuron types, which showed diversity of cell fate commitment and location. Further analyses showed that these cell types are homologous to neurogenic cells in the mammalian brain, identified neurogenic commitment in proliferating radial glia and indicated that glutamatergic projection neurons are generated in the adult zebrafish telencephalon. Thus, we prospectively isolated adult newborn neurons from the adult zebrafish forebrain, identified markers for newborn and mature neurons in the adult brain, and revealed intrinsic heterogeneity among adult newborn neurons and their homology with mammalian adult neurogenic cell types.

Zebrafish Spinal Cord Repair Is Accompanied by Transient Tissue Stiffening.

Severe injury to the mammalian spinal cord results in permanent loss of function due to the formation of a glial-fibrotic scar. Both the chemical composition and the mechanical properties of the scar tissue have been implicated to inhibit neuronal regrowth and functional recovery. By contrast, adult zebrafish are able to repair spinal cord tissue and restore motor function after complete spinal cord transection owing to a complex cellular response that includes axon regrowth and is accompanied by neurogenesis. The mechanical mechanisms contributing to successful spinal cord repair in adult zebrafish are, however, currently unknown. Here, we employ atomic force microscopy-enabled nanoindentation to determine the spatial distributions of apparent elastic moduli of living spinal cord tissue sections obtained from uninjured zebrafish and at distinct time points after complete spinal cord transection. In uninjured specimens, spinal gray matter regions were stiffer than white matter regions. During regeneration after transection, the spinal cord tissues displayed a significant increase of the respective apparent elastic moduli that transiently obliterated the mechanical difference between the two types of matter before returning to baseline values after the completion of repair. Tissue stiffness correlated variably with cell number density, oligodendrocyte interconnectivity, axonal orientation, and vascularization. This work constitutes the first quantitative mapping of the spatiotemporal changes of spinal cord tissue stiffness in regenerating adult zebrafish and provides the tissue mechanical basis for future studies into the role of mechanosensing in spinal cord repair.

Ligand-Controlled Site-Specific Recombination in Zebrafish.

Cre-mediated site-specific recombination has emerged as an indispensable tool for the precise manipulation of genomes allowing lineage-tracing studies, temporal and spatial misexpressions, and in particular the generation of conditional knockout alleles. Previously, we and others showed that Cre and its ligand-inducible variant CreERT2 are also highly efficient in the developing and adult zebrafish. The number of Cre driver and effector lines is currently still limited in zebrafish. However, the recent advent of novel genome editing tools such as TALEN and CRISPR/Cas will significantly increase interest in the conditional Cre/lox-technology in this organism. The considerations of basic transgene design and subsequent transgenesis have been addressed elsewhere. Here we outline practical experimental steps for transient functionality tests of CreERT2 driver and effector constructs. In addition, we introduce detailed protocols to elicit CreERT2-mediated recombination in vivo at embryonic as well as adult stages.

Characterization of light lesion paradigms and optical coherence tomography as tools to study adult retina regeneration in zebrafish.

Light-induced lesions are a powerful tool to study the amazing ability of photoreceptors to regenerate in the adult zebrafish retina. However, the specificity of the lesion towards photoreceptors or regional differences within the retina are still incompletely understood. We therefore characterized the process of degeneration and regeneration in an established paradigm, using intense white light from a fluorescence lamp on swimming fish (diffuse light lesion). We also designed a new light lesion paradigm where light is focused through a microscope onto the retina of an immobilized fish (focused light lesion). Focused light lesion has the advantage of creating a locally restricted area of damage, with the additional benefit of an untreated control eye in the same animal. In both paradigms, cell death is observed as an immediate early response, and proliferation is initiated around 2 days post lesion (dpl), peaking at 3 dpl. We furthermore find that two photoreceptor subtypes (UV and blue sensitive cones) are more susceptible towards intense white light than red/green double cones and rods. We also observed specific differences within light lesioned areas with respect to the process of photoreceptor degeneration: UV cone debris is removed later than any other type of photoreceptor in light lesions. Unspecific damage to retinal neurons occurs at the center of a focused light lesion territory, but not in the diffuse light lesion areas. We simulated the fish eye optical properties using software simulation, and show that the optical properties may explain the light lesion patterns that we observe. Furthermore, as a new tool to study retinal degeneration and regeneration in individual fish in vivo, we use spectral domain optical coherence tomography. Collectively, the light lesion and imaging assays described here represent powerful tools for studying degeneration and regeneration processes in the adult zebrafish retina.

Dopamine from the brain promotes spinal motor neuron generation during development and adult regeneration.

Coordinated development of brain stem and spinal target neurons is pivotal for the emergence of a precisely functioning locomotor system. Signals that match the development of these far-apart regions of the central nervous system may be redeployed during spinal cord regeneration. Here we show that descending dopaminergic projections from the brain promote motor neuron generation at the expense of V2 interneurons in the developing zebrafish spinal cord by activating the D4a receptor, which acts on the hedgehog pathway. Inhibiting this essential signal during early neurogenesis leads to a long-lasting reduction of motor neuron numbers and impaired motor responses of free-swimming larvae. Importantly, during successful spinal cord regeneration in adult zebrafish, endogenous dopamine promotes generation of spinal motor neurons, and dopamine agonists augment this process. Hence, we describe a supraspinal control mechanism for the development and regeneration of specific spinal cell types that uses dopamine as a signal.

Lesion-induced generation of interneuron cell types in specific dorsoventral domains in the spinal cord of adult zebrafish.

In contrast to mammals, adult zebrafish regenerate neurons in the lesioned spinal cord. For example, motor neurons are generated from an olig2-expressing population of pMN-like ependymoradial glial cells in a ventrolateral position at the central canal. However, the extent of neuronal regeneration is unclear. Here we show, using a transgenic fish in which V2 interneurons are labeled by green fluorescent protein (GFP) under the control of the vsx1 promoter, that after a complete spinal cord transection, large numbers of V2 interneurons are generated in the vicinity of the lesion site. Tg(vsx1:GFP)⁺ cells are not present in the unlesioned spinal cord and label with the proliferation marker bromodeoxyuridine (BrdU) after a lesion. Some mediolaterally elongated Tg(vsx1:GFP)⁺ cells contact the central canal in a medial position. These cells likely arise from a p2-like domain of ependymoradial glial progenitor cells, indicated by coexpression of Pax6 and Nkx6.1, but not DsRed driven by the olig2 promoter in these cells. We also present evidence that Pax2⁺ interneurons are newly generated after a spinal lesion, whereas the generation rate for a dorsal population of parvalbuminergic interneurons is comparatively low. Our results identify the regenerative potential of different interneuron types for the first time and support a model in which different progenitor cell domains in distinct dorsoventral positions around the central canal are activated by a lesion to give rise to diverse neuronal cell types in the adult zebrafish spinal cord.

Plasticity of tyrosine hydroxylase and serotonergic systems in the regenerating spinal cord of adult zebrafish.

Monoaminergic innervation of the spinal cord has important modulatory functions for locomotion. Here we performed a quantitative study to determine the plastic changes of tyrosine hydroxylase-positive (TH1(+); mainly dopaminergic), and serotonergic (5-HT(+)) terminals and cells during successful spinal cord regeneration in adult zebrafish. TH1(+) innervation in the spinal cord is derived from the brain. After spinal cord transection, TH1(+) immunoreactivity is completely lost from the caudal spinal cord. Terminal varicosities increase in density rostral to the lesion site compared with unlesioned controls and are re-established in the caudal spinal cord at 6 weeks post lesion. Interestingly, axons mostly fail to re-innervate more caudal levels of the spinal cord even after prolonged survival times. However, densities of terminal varicosities correlate with recovery of swimming behavior, which is completely lost again after re-lesion of the spinal cord. Similar observations were made for terminals derived from descending 5-HT(+) axons from the brain. In addition, spinal 5-HT(+) neurons were newly generated after a lesion and transiently increased in number up to fivefold, which depended in part on hedgehog signaling. Overall, TH1(+) and 5-HT(+) innervation is massively altered in the successfully regenerated spinal cord of adult zebrafish. Despite these changes in TH and 5-HT systems, a remarkable recovery of swimming capability is achieved, suggesting significant plasticity of the adult spinal network during regeneration.

Claudin k is specifically expressed in cells that form myelin during development of the nervous system and regeneration of the optic nerve in adult zebrafish.

The zebrafish has become an important model organism to study myelination during development and after a lesion of the adult central nervous system (CNS). Here, we identify Claudin k as a myelin-associated protein in zebrafish and determine its localization during development and adult optic nerve regeneration. We find Claudin k in subcellular compartments consistent with location in autotypic tight junctions of oligodendrocytes and myelinating Schwann cells. Expression starts in the hindbrain at 2 days (mRNA) and 3 days (protein) postfertilization and is maintained in adults. A newly generated claudin k:green fluorescent protein (GFP) reporter line allowed us to characterize oligodendrocytes in the adult retina that express Claudin k and olig2, but not P0 and uniquely only form loose wraps of membrane around axons. After a crush of the adult optic nerve, Claudin k protein levels were first reduced and then recovered within 4 weeks postlesion, concomitant with optic nerve myelin de- and regeneration. During optic nerve regeneration, oligodendrocytes, many of which were newly generated, repopulated the lesion site and exhibited increasing morphological complexity over time. Thus, Claudin k is a novel myelin-associated protein expressed by oligodendrocytes and Schwann cells from early stages of wrapping and myelin formation in zebrafish development and adult regeneration, suggesting important functions of the gene for myelin formation and maintenance. Our Claudin k antibodies and claudin k:GFP reporter line represent excellent ways to visualize oligodendrocyte and Schwann cell differentiation in vivo.

Sonic hedgehog is a polarized signal for motor neuron regeneration in adult zebrafish.

In contrast to mammals, the spinal cord of adult zebrafish has the capacity to reinitiate generation of motor neurons after a lesion. Here we show that genes involved in motor neuron development, i.e., the ventral morphogen sonic hedgehog a (shha), as well as the transcription factors nkx6.1 and pax6, together with a Tg(olig2:egfp) transgene, are expressed in the unlesioned spinal cord of adult zebrafish. Expression is found in ependymoradial glial cells lining the central canal in ventrodorsal positions that match expression domains of these genes in the developing neural tube. Specifically, Tg(olig2:egfp)(+) ependymoradial glial cells, the adult motor neuron progenitors (pMNs), coexpress Nkx6.1 and Pax6, thus defining an adult pMN-like zone. shha is expressed in distinct ventral ependymoradial glial cells. After a lesion, expression of all these genes is strongly increased, while relative spatial expression domains are maintained. In addition, expression of the hedgehog (hh) receptors patched1 and smoothened becomes detectable in ependymoradial glial cells including those of the pMN-like zone. Cyclopamine-induced knock down of hh signaling significantly reduces ventricular proliferation and motor neuron regeneration. Expression of indicator genes for the FGF and retinoic acid signaling pathways was also increased in the lesioned spinal cord. This suggests that a subclass of ependymoradial glial cells retain their identity as motor neuron progenitors into adulthood and are capable of reacting to a sonic hedgehog signal and potentially other developmental signals with motor neuron regeneration after a spinal lesion.

Motor neuron regeneration in adult zebrafish.

The mammalian spinal cord does not regenerate motor neurons that are lost as a result of injury or disease. Here we demonstrate that adult zebrafish, which show functional spinal cord regeneration, are capable of motor neuron regeneration. After a spinal lesion, the ventricular zone shows a widespread increase in proliferation, including slowly proliferating olig2-positive (olig2+) ependymo-radial glial progenitor cells. Lineage tracing in olig2:green fluorescent protein transgenic fish indicates that these cells switch from a gliogenic phenotype to motor neuron production. Numbers of undifferentiated small HB9+ and islet-1+ motor neurons, which are double labeled with the proliferation marker 5-bromo-2-deoxyuridine (BrdU), are transiently strongly increased in the lesioned spinal cord. Large differentiated motor neurons, which are lost after a lesion, reappear at 6-8 weeks after lesion, and we detected ChAT+/BrdU+ motor neurons that were covered by contacts immunopositive for the synaptic marker SV2. These observations suggest that, after a lesion, plasticity of olig2+ progenitor cells may allow them to generate motor neurons, some of which exhibit markers for terminal differentiation and integration into the existing adult spinal circuitry.