Notch Signaling Controls Oligodendrocyte Regeneration in the Injured Telencephalon of Adult Zebrafish.

The myelination of axons in the vertebrate nervous system through oligodendrocytes promotes efficient axonal conduction, which is required for the normal function of neurons. The central nervous system (CNS) can regenerate damaged myelin sheaths through the process of remyelination, but the failure of remyelination causes neurological disorders such as multiple sclerosis. In mammals, parenchymal oligodendrocyte progenitor cells (OPCs) are known to be the principal cell type responsible for remyelination in demyelinating diseases and traumatic injuries to the adult CNS. However, growing evidence suggests that neural stem cells (NSCs) are implicated in remyelination in animal models of demyelination. We have previously shown that olig2+ radial glia (RG) have the potential to function as NSCs to produce oligodendrocytes in adult zebrafish. In this study, we developed a zebrafish model of adult telencephalic injury to investigate cellular and molecular mechanisms underlying the regeneration of oligodendrocytes. Using this model, we showed that telencephalic injury induced the proliferation of olig2+ RG and parenchymal OPCs shortly after injury, which was followed by the regeneration of new oligodendrocytes in the adult zebrafish. We also showed that blocking Notch signaling promoted the proliferation of olig2+ RG and OPCs in the normal and injured telencephalon of adult zebrafish. Taken together, our data suggest that Notch-regulated proliferation of olig2+ RG and parenchymal OPCs is responsible for the regeneration of oligodendrocytes in the injured telencephalon of adult zebrafish.

Schwann cells selectively myelinate primary motor axons via neuregulin-ErbB signaling.

Spinal motor neurons project their axons out of the spinal cord via the motor exit point (MEP) and regulate their target muscle fibers for diverse behaviors. Several populations of glial cells including Schwann cells, MEP glia, and perineurial glia are tightly associated with spinal motor axons in nerve fascicles. Zebrafish have two types of spinal motor neurons, primary motor neurons (PMNs) and secondary motor neurons (SMNs). PMNs are implicated in the rapid response, whereas SMNs are implicated in normal and slow movements. However, the precise mechanisms mediating the distinct functions of PMNs and SMNs in zebrafish are unclear. In this study, we found that PMNs were myelinated by MEP glia and Schwann cells, whereas SMNs remained unmyelinated at the examined stages. Immunohistochemical analysis revealed that myelinated PMNs solely innervated fast muscle through a distributed neuromuscular junction (NMJ), whereas unmyelinated SMNs innervated both fast and slow muscle through distributed and myoseptal NMJs, respectively, indicating that myelinated PMNs could provide rapid responses for startle and escape movements, while unmyelinated SMNs regulated normal, slow movement. Further, we demonstrate that neuregulin 1 (Nrg1) type III-ErbB signaling provides a key instructive signal that determines the myelination of primary motor axons by MEP glia and Schwann cells. Perineurial glia ensheathed unmyelinated secondary motor axons and myelinated primary motor nerves. Ensheathment required interaction with both MEP glia and Schwann cells. Collectively, these data suggest that primary and secondary motor neurons contribute to the regulation of movement in zebrafish with distinct patterns of myelination.

mRNA expression and metabolic regulation of npy and agrp1/2 in the zebrafish brain.

Neuropeptide Y (NPY) is an evolutionarily conserved neuropeptide implicated in feeding regulation in vertebrates. In mammals, NPY neurons coexpress Agouti-related protein (AgRP) in the arcuate nucleus of the hypothalamus, and NPY/AgRP neurons activate orexigenic signaling to increase food intake. Zebrafish express npy and two agrp genes, agrp1 and agrp2, in the brain. Similar to mammals, NPY and AgRP1 act as orexigenic factors in zebrafish, but the exact distribution of NPY and AgRP neurons in the zebrafish brain and the regulation of these genes by metabolic states remain unclear. In this study, we analyzed the tissue distribution of npy, agrp1, and agrp2 mRNA in the brain of larval and adult zebrafish. We detected the expression of agrp1, but not npy, in the hypothalamus of larval zebrafish. In the adult zebrafish brain, npy mRNA expression was detected in the dorsal area of the periventricular and lateral hypothalamus, but fasting induced upregulation of npy only in the lateral hypothalamus, indicating that NPY neurons in this area are implicated in feeding regulation. However, consistent with the findings in larval zebrafish, NPY neurons in the hypothalamus did not coexpress AgRP1. In contrast, fasting resulted in a dramatic increase in AgRP1 neurons in the ventral periventricular hypothalamus, which do not coexpress NPY. In addition, we found for the first time that npy- and agrp1-expressing neurons function as GABAergic inhibitory neurons in zebrafish, as they do in mammals. Taken together, our results show that the zebrafish NPY/AgRP system is involved in appetite regulation. In addition, our data suggest that although npy and agrp1 were initially expressed in distinct neurons, evolution has resulted in their coexpression in mammalian hypothalamic neurons.

ISL1-based LIM complexes control Slit2 transcription in developing cranial motor neurons.

LIM-homeodomain (HD) transcription factors form a multimeric complex and assign neuronal subtype identities, as demonstrated by the hexameric ISL1-LHX3 complex which gives rise to somatic motor (SM) neurons. However, the roles of combinatorial LIM code in motor neuron diversification and their subsequent differentiation is much less well understood. In the present study, we demonstrate that the ISL1 controls postmitotic cranial branchiomotor (BM) neurons including the positioning of the cell bodies and peripheral axon pathfinding. Unlike SM neurons, which transform into interneurons, BM neurons are normal in number and in marker expression in Isl1 mutant mice. Nevertheless, the movement of trigeminal and facial BM somata is stalled, and their peripheral axons are fewer or misrouted, with ectopic branches. Among genes whose expression level changes in previous ChIP-seq and microarray analyses in Isl1-deficient cell lines, we found that Slit2 transcript was almost absent from BM neurons of Isl1 mutants. Both ISL1-LHX3 and ISL1-LHX4 bound to the Slit2 enhancer and drove endogenous Slit2 expression in SM and BM neurons. Our findings suggest that combinations of ISL1 and LHX factors establish cell-type specificity and functional diversity in terms of motor neuron identities and/or axon development.

Distribution of galanin receptor 2b neurons and interaction with galanin in the zebrafish central nervous system.

Galanin is a multifunctional neuropeptide that is implicated in the modulation of physiological processes, including nociception, cognition, feeding behavior, neuronal growth, and reproduction. The physiological effects of galanin are mediated through its interaction with three different G protein-coupled receptors, i.e., GALR1, GALR2, and GALR3. Unlike mammals, zebrafish have four different receptors for galanin, diversified from GALR1 (GAL1a and GALR1b) and GALR2 (GALR2a and GALR2b). Despite the importance of galanin in the central nervous system (CNS), no information has been reported regarding GalR2 in zebrafish CNS. In this study, we found that galr2a is expressed at low levels in restricted areas of the brain; however, galr2b was widely expressed in CNS including olfactory bulb, midbrain tegmentum, preoptic region, dorsal thalamus, posterior tuberculum, postoptic commissure, hindbrain, and spinal cord. To further analyze the distribution of GALR2b neurons and their interaction with GAL, we generated Tg(galr2b:egfp) zebrafish, which express enhanced green fluorescent protein (EGFP) under the control of a galr2b promoter. Investigation of the CNS of transgenic reporter zebrafish revealed that galr2b:EGFP(+) neurons are distributed and interact with galanin-immunoreactive (galanin-IR) cells in various regions of the brain and spinal cord. We found that in some regions of the brain and spinal cord, galanin-IR nerve cells were not observed near galr2b:EGFP neurons, suggesting that GALR2b may have the potential to interact with other ligands instead of galanin in these regions.

Generation of demyelination models by targeted ablation of oligodendrocytes in the zebrafish CNS.

Demyelination is the pathological process by which myelin sheaths are lost from around axons, and is usually caused by a direct insult targeted at the oligodendrocytes in the vertebrate central nervous system (CNS). A demyelinated CNS is usually remyelinated by a population of oligodendrocyte progenitor cells, which are widely distributed throughout the adult CNS. However, myelin disruption and remyelination failure affect the normal function of the nervous system, causing human diseases such as multiple sclerosis. In spite of numerous studies aimed at understanding the remyelination process, many questions still remain unanswered. Therefore, to study remyelination mechanisms in vivo, a demyelination animal model was generated using a transgenic zebrafish system in which oligodendrocytes are conditionally ablated in the larval and adult CNS. In this transgenic system, bacterial nitroreductase enzyme (NTR), which converts the prodrug metronidazole (Mtz) into a cytotoxic DNA cross-linking agent, is expressed in oligodendrocyte lineage cells under the control of the mbp and sox10 promoter. Exposure of transgenic zebrafish to Mtz-containing media resulted in rapid ablation of oligodendrocytes and CNS demyelination within 48 h, but removal of Mtz medium led to efficient remyelination of the demyelinated CNS within 7 days. In addition, the demyelination and remyelination processes could be easily observed in living transgenic zebrafish by detecting the fluorescent protein, mCherry, indicating that this transgenic system can be used as a valuable animal model to study the remyelination process in vivo, and to conduct high-throughput primary screens for new drugs that facilitate remyelination.