Larval zebrafish rapidly sense the water flow of a predator's strike

Larval fishes have a remarkable ability to sense and evade the feeding strike of a predator fish with a rapid escape manoeuvre. Although the neuromuscular control of this behaviour is well studied, it is not clear what stimulus allows a larva to sense a predator. Here we show that this escape response is triggered by the water flow created during a predator''s strike. Using a novel device, the impulse chamber, zebrafish (Danio rerio) larvae were exposed to this accelerating flow with high repeatability. Larvae responded to this stimulus with an escape response having a latency (mode=13–15 ms) that was fast enough to respond to predators. This flow was detected by the lateral line system, which includes mechanosensory hair cells within the skin. Pharmacologically ablating these cells caused the escape response to diminish, but then recover as the hair cells regenerated. These findings demonstrate that the lateral line system plays a role in predator evasion at this vulnerable stage of growth in fishes.     

Regeneration in zebrafish lateral line neuromasts: expression of the neural progenitor cell marker sox2 and proliferation-dependent and-independent mechanisms of hair cell renewal

Mechanosensory hair cells are essential for audition in vertebrates, and in many species, have the capacity for regeneration when damaged. Regeneration is robust in the fish lateral line system as new hair cells can reappear after damage induced by waterborne aminoglycoside antibiotics, platinum-based drugs, and heavy metals. Here, we characterize the loss and reappearance of lateral line hair cells induced in zebrafish larvae treated with copper sulfate using diverse molecular markers. Transgenic fish that express green fluorescent protein in different cell types in the lateral line system have allowed us to follow the regeneration of hair cells after different damage protocols. We show that conditions that damage only differentiated hair cells lead to reappearance of new hair cells within 24 h from nondividing precursors, whereas harsher conditions are followed by a longer recovery period that is accompanied by extensive cell division. In order to characterize the cell population that gives rise to new hair cells, we describe the expression of a neural stem cell marker in neuromasts. The zebrafish sox2 gene is strongly expressed in neuromast progenitor cells, including those of the migrating lateral line primordium, the accessory cells that underlie the hair cells in neuromasts, and in interneuromastic cells that give rise to new neuromasts. Moreover, we find that most of the cells that proliferate within the neuromast during regeneration express this marker. Thus, our results describe the dynamics of hair cell regeneration in zebrafish and suggest the existence of at least two mechanisms for recovery of these cells in neuromasts.     

The vesicular integral protein-like gene is essential for development of a mechanosensory system in zebrafish

The zebrafish hi472 mutation is caused by a retroviral insertion into the vesicular integral protein-like gene, or zVIPL, a poorly studied lectin implicated in endoplasmic reticulum (ER)-Golgi trafficking. A mutation in the shorter isoform of zVIPL (zVIPL-s) results in a reduction of mechanosensitivity and consequent loss of escape behavior. Here we show that motoneurons and hindbrain reticulospinal neurons, which normally integrate mechanosensory inputs, failed to fire in response to tactile stimuli in hi472 larvae, suggesting a perturbation in sensory function. The hi472 mutant larvae in fact suffered from a severe loss of functional neuromasts of the lateral line mechanosensory system, a reduction of zVIPL labeling in support cells, and a reduction or even a complete loss of hair cells in neuromasts. The Delta-Notch signaling pathway is implicated in cellular differentiation of neuromasts, and we observed an increase in Notch expression in neuromasts of hi472 mutant larvae. Treatment of hi472 mutant larvae with DAPT, an inhibitor of Notch signaling, or overexpression of the Notch ligand deltaB in hi472 mutant blastocysts produced partial rescue of the morphological defects and of the startle response behavior. We conclude that zVIPL-s is a necessary component of Delta-Notch signaling during neuromast development in the lateral line mechanosensory system.     

Organization and physiology of posterior lateral line afferent neurons in larval zebrafish

The lateral line system of larval zebrafish can translate hydrodynamic signals from the environment to guide body movements. Here, I demonstrate a spatial relationship between the organization of afferent neurons in the lateral line ganglion and the innervation of neuromasts along the body. I developed a whole cell patch clamp recording technique to show that afferents innervate multiple direction-sensitive neuromasts, which are sensitive to low fluid velocities. This work lays the foundation to integrate sensory neuroscience and the hydrodynamics of locomotion in a model genetic system.     

Single-cell transcriptome analysis reveals three sequential phases of gene expression during zebrafish sensory hair cell regeneration

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A neuronal circuit that generates the temporal motor sequence for the defensive response in zebrafish larvae

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An Assay for Lateral Line Regeneration in Adult Zebrafish

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al.¹⁷ that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.     

Gene miles-apart is required for formation of otic vesicle and hair cells in zebrafish

Hearing loss is a serious burden to physical and mental health worldwide. Aberrant development and damage of hearing organs are recognized as the causes of hearing loss, the molecular mechanisms underlining these pathological processes remain elusive. Investigation of new molecular mechanisms involved in proliferation, differentiation, migration and maintenance of neuromast primordium and hair cells will contribute to better understanding of hearing loss pathology. This knowledge will enable the development of protective agents and mechanism study of drug ototoxicity. In this study, we demonstrate that the zebrafish gene miles-apart, a homolog of sphingosine-1-phosphate receptor 2 (s1pr2) in mammals, has an important role in the development of otic vesicle, neuromasts and survival of hair cells. Whole-mount in situ hybridization of embryos showed that miles-apart expression occurred mainly in the encephalic region and the somites at 24 h.p.f. (hour post fertilization), in the midbrain/hindbrain boundary, the brainstem and the pre-neuromast of lateral line at 48 h.p.f. in a strict spatiotemporal regulation. Both up- and downregulation of miles-apart led to abnormal otoliths and semicircular canals, excess or few hair cells and neuromasts, and their disarranged depositions in the lateral lines. Miles-apart (Mil) dysregulation also caused abnormal expression of hearing-associated genes, including hmx2, fgf3, fgf8a, foxi1, otop1, pax2.1 and tmieb during zebrafish organogenesis. Moreover, in larvae miles-apart gene knockdown significantly upregulated proapoptotic gene zBax2 and downregulated prosurvival gene zMcl1b; in contrast, the level of zBax2 was decreased and of zMcl1b enhanced by miles-apart overexpression. Collectively, Mil activity is linked to organization and number decision of hair cells within a neuromast, also to deposition of neuromasts and formation of otic vesicle during zebrafish organogenesis. At the larva stage, Mil as an upstream regulator of bcl-2 gene family has a role in protection of hair cells against apoptosis by promoting expression of prosurvival gene zMcl1b and suppressing proapoptotic gene zBax2.     

A zebrafish SKIV2L2-enhancer trap line provides a useful tool for the study of peripheral sensory circuit development

The zebrafish is an ideal model for elucidating the cellular and molecular mechanisms that underlie development of the peripheral nervous system. A transgenic line that selectively labels all the sensory circuits would be a valuable tool for such investigations. In this study, we describe such a line: the enhancer trap zebrafish line Tg(SKIV2L2:gfp)^j1775 which expresses green fluorescent protein (gfp) in the peripheral sensory ganglia. We show that this transgene marks all peripheral ganglia and sensory nerves, beginning at the time when the neurons are first extending their processes, but does not label the efferent nerves. The trapped reporter is inserted just upstream of a previously poorly described gene: lhfpl4 on LG6. The expression pattern of this gene by in situ hybridization reveals a different, but overlapping, pattern of expression compared to that of the transgene. This pattern also does not mimic that of the gene (skiv2l2), which provided the promoter element in the construct. These findings indicate that reporter expression is not dictated by an endogenous enhancer element, but instead arises through an unknown mechanism. Regardless, this reporter line should prove to be a valuable tool in the investigation of peripheral nervous system formation in the zebrafish.     

Therapeutic potential of neurotrophins for treatment of hearing loss

Degeneration of spiral ganglion neurons (SGNs) and hair cells in the cochlea induced by aging, injury, ototoxic drugs, acoustic trauma, and various diseases is the major cause of hearing loss. Discovery of growth factors that can either prevent SGN and hair-cell death or stimulate hair-cell regeneration would be of great interest. Studies over the past several years have provided evidence that specific neurotrophins are potent survival factors for SGNs and protect these neurons from ototoxic drugs in vitro and in vivo. Current research focuses more on understanding the mechanism of hair-cell regeneration/differentiation and identification of growth factors that can stimulate hair-cell regeneration. SGNs are required to relay the signal to the central nervous system even when a cochlear implant is used to replace hair-cell function or in the case that cochlear sensory epithelium can be stimulated to regenerate new hair cells successfully. Therefore, neurotrophins may have their therapeutic value in prevention and treatment of hearing impairment.     

A role for TREM2 ligands in the phagocytosis of apoptotic neuronal cells by microglia

Following neuronal injury, microglia initiate repair by phagocytosing dead neurons without eliciting inflammation. Prior evidence indicates triggering receptor expressed by myeloid cells-2 (TREM2) promotes phagocytosis and retards inflammation. However, evidence that microglia and neurons directly interact through TREM2 to orchestrate microglial function is lacking. We here demonstrate that TREM2 interacts with endogenous ligands on neurons. Staining with TREM2-Fc identified TREM2 ligands (TREM2-L) on Neuro2A cells and on cultured cortical and dopamine neurons. Apoptosis greatly increased the expression of TREM2-L. Furthermore, apoptotic neurons stimulated TREM2 signaling, and an anti-TREM2 mAb blocked stimulation. To examine the interaction between TREM2 and TREM2-L in phagocytosis, we studied BV2 microglial cells and their engulfment of apoptotic Neuro2A. One of our anti-TREM2 mAb, but not others, reduced engulfment, suggesting the presence of a functional site on TREM2 interacting with neurons. Further, Chinese hamster ovary cells transfected with TREM2 conferred phagocytic activity of neuronal cells demonstrating that TREM2 is both required and sufficient for competent uptake of apoptotic neuronal cells. Finally, while TREM2-L are expressed on neurons, TREM2 is not; in the brain, it is found on microglia. TREM2 and TREM2-L form a receptor–ligand pair connecting microglia with apoptotic neurons, directing removal of damaged cells to allow repair.     

Proneural gene requirement for hair cell differentiation in the zebrafish lateral line

The lateral line system comprises an array of mechanosensory organs, the neuromasts, distributed over the body surface. Each neuromast consists of a patch of mechanosensory hair cells surrounded by support cells. We show that, in the zebrafish, two proneural genes are essential for differentiation of the hair cells, neuroD (nrd) and atonal homolog 1 (ath1). Gene knockdown experiments demonstrate that loss of function of either gene, but not of the related proneural gene neurogenin1 (ngn1), abrogate the appearance of hair cell markers. This is in contrast to other sensory systems, such as the neurons of the lateral line ganglion, where nrd is regulated by ngn1 and not by ath1. Overexpression of ath1 can induce nrd, and the phenotype produced by loss of ath1 function can be partially rescued by injection of nrd mRNA. This supports the conclusion that the activation of nrd probably requires ath1 in the hair cell lineage, whereas in sensory neurons nrd activation requires ngn1. We propose that the emergence of two atonal homologs, ath1 and ngn1, allowed the cellular segregation of mechanoreception and signal transmission that were originally performed by a single cell type as found in insects.     

Adaptive cell invasion maintains lateral line organ homeostasis in response to environmental changes

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A screen for mutations in zebrafish that affect myelin gene expression in Schwann cells and oligodendrocytes

Myelin is the multi-layered glial sheath around axons in the vertebrate nervous system. Myelinating glia develop and function in intimate association with neurons and neuron-glial interactions control much of the life history of these cells. However, many of the factors that regulate key aspects of myelin development and maintenance remain unknown. To discover new molecules that are important for glial development and myelination, we undertook a screen of zebrafish mutants with previously characterized neural defects. We screened for myelin basic protein (mbp) mRNA by in situ hybridization and identified four mutants (neckless, motionless, iguana and doc) that lacked mbp expression in parts of the peripheral and central nervous systems (PNS or CNS), despite the presence of axons. In all four mutants electron microscopy revealed that myelin-forming glia were present and had formed loose wraps around axons but did not form compact myelin. We found that addition of exogenous retinoic acid (RA) rescued mbp expression in neckless mutant embryos, which lack endogenous RA synthesis. Timed application of the RA synthesis inhibitor DEAB to wild type embryos showed that RA signalling is required at least 48 h before the onset of myelin protein synthesis in both CNS and PNS.     

A Pdgf‐cCreERT2 knock‐in mouse model for tracing PDGF‐C cell lineages during development

PDGF‐C, a member of the platelet‐derived growth factor (PDGF) family, plays important roles in the development of craniofacial structures, the neural system, the vascular system, and tumors. PDGF‐C could also be required for the regulation of certain types of stem or progenitor cells as suggested by its expression in the regions where these cells are located. To further characterize the role of PDGF‐C in development, we generated a Pdgf‐c^CreERT2 mouse strain, in which a tamoxifen‐inducible Cre (CreERT2) cDNA was specifically targeted into the Pdgf‐c genomic locus and controlled by the endogenous Pdgf‐c regulatory elements. We also showed that Cre activity in this mouse strain could be specifically induced by tamoxifen, which allowed the fate of PDGF‐C‐expressing cells to be traced at various stages of development. Using this model system, we demonstrated for the first time that PDGF‐C‐expressing cells could be multipotent, generating multiple cell lineages required for the formation of the cerebellum. Therefore, the Pdgf‐c^CreERT2 mouse strain generated in this study will be a valuable transgenic tool for exploring the function of PDGF‐C in development and stem cell biology.     

A role for the Rab6B Bicaudal-D1 interaction in retrograde transport in neuronal cells

The Rab6 subfamily of small GTPases consists of three different isoforms: Rab6A, Rab6A' and Rab6B. Both Rab6A and Rab6A' are ubiquitously expressed whereas Rab6B is predominantly expressed in brain. Recent studies have shown that Rab6A' is the isoform regulating the retrograde transport from late endosomes via the Golgi to the ER and in the transition from anaphase to metaphase during mitosis. Since the role of Rab6B is still ill defined, we set out to characterize its intracellular environment and dynamic behavior. In a Y-2H search for novel Rab6 interacting proteins, we identified Bicaudal-D1, a large coiled-coil protein known to bind to the dynein/dynactin complex and previously shown to be a binding partner for Rab6A/Rab6A'. Co-immunoprecipitation studies and pull down assays confirmed that Bicaudal-D1 also interacts with Rab6B in its active form. Using confocal laser scanning microscopy it was established that Rab6B and Bicaudal-D1 co-localize at the Golgi and vesicles that align along microtubules. Furthermore, both proteins co-localized with dynein in neurites of SK-N-SH cells. Live cell imaging revealed bi-directional movement of EGFP-Rab6B structures in SK-N-SH neurites. We conclude from our data that the brain-specific Rab6B via Bicaudal-D1 is linked to the dynein/dynactin complex, suggesting a regulatory role for Rab6B in the retrograde transport of cargo in neuronal cells.