Molecular Components of the Hair Cell Synaptic Vesicle Cycle

Hair cells (HCs) of the zebrafish inner ear act as sensory receptors for both auditory and vestibular stimuli, and have glutamatergic ribbon synapses.We have identified and positionally cloned two mutants that interfere with HC synaptic activity from an ENU mutagenesis screen for vestibular dysfunction in zebrafish (Nicolson et al., 1998 and unpublished results). We find that the comet gene encodes the lipid phosphatase synaptojanin 1 (synj1). comet mutant larvae display a balance defect that increases in severity when challenged, suggesting fatigability, perhaps due to insufficient SV recycling.We have sequenced three alleles of comet/synj1, all of which encode severe truncations that presumably lead to functional null phenotypes. We confirm the expression of synj1 in the CNS and, in addition, show expression in HCs of the ear by in situ hybridization. Morpholino-mediated suppression of the short splice variant of synj1, synj1-145 inefficiently generates a balance defect that is dissimilar to that of the comet mutant. Morpholino knockdown of Synj1 produces a phenocopy. At the electron microscopy level, comet/synj1 mutants show a decrease in synaptic ribbon diameter that accompanies a reduced number of ribbon-associated synaptic vesicles. This can be interpreted as evidence for altered release kinetics and ribbon maintenance in comet mutants. We also describe the completely novel phenotype for loss of synj1, basal membrane blebbing. Basal blebbing is stimulation-dependent in comet/synj1 mutants and the absence of synaptic exocytosis in comet/gemini double mutants that lack the synaptic Ca2+ channel cav1.3 (Sidi et al., 2004) abolishes blebbing. In addition, interference with endocytosis by exposing larvae to Latrunculin A phenocopies blebbing in wild-type HCs. Furthermore, we have identified vesicular glutamate transporter 3 (vglut3) as critical for HC function from the asteroid mutant strain. asteroid mutant larvae are deaf and display a profound balance defect, while hair-bundle morphology and FM 1-43 dye uptake appear normal. This phenotype suggests a transmission failure downstream of mechanotransduction. By inducing an early frameshift, the genomic lesion of the asteroid mutant results in a protein null phenotype. We can replicate this phenotype by morpholino-mediated knockdown of vglut3 in the wild-type. Unlike in mammals, vglut3 appears to be exclusively expressed in HCs of the ear and lateral line organ in the zebrafish. Restriction of Vglut3 to HCs and absence in the asteroid mutant were confirmed by immunohistochemistry. asteroid mutants show a 60% decrease in the number of ribbon-associated synaptic vesicles at the ultrastructural level, while ribbon diameters are comparable to the wild-type. This indicates a role for Vglut3 in synaptic vesicle biogenesis and/or trafficking, but Vglut3 is not the sole component required for these processes. Using in situ hybridization, we also detect vglut1 transcript in HCs, but the presence of vglut1 does not compensate for loss of vglut3 under the conditions tested, and qPCR data indicates that neither vglut1 nor vglut3 are significantly upregulated in asteroid/vglut3 mutants. In conclusion, we have identified, cloned and characterized two “novel” genes that are required for proper HC synaptic transmission. Both synj1 and vglut3 are involved in SV generation and recycling and their investigation should aid further elucidation of these mechanisms in future studies

synaptojanin1 Is Required for Temporal Fidelity of Synaptic Transmission in Hair Cells

To faithfully encode mechanosensory information, auditory/vestibular hair cells utilize graded synaptic vesicle (SV) release at specialized ribbon synapses. The molecular basis of SV release and consequent recycling of membrane in hair cells has not been fully explored. Here, we report that comet, a gene identified in an ENU mutagenesis screen for zebrafish larvae with vestibular defects, encodes the lipid phosphatase Synaptojanin 1 (Synj1). Examination of mutant synj1 hair cells revealed basal blebbing near ribbons that was dependent on Cav1.3 calcium channel activity but not mechanotransduction. Synaptojanin has been previously implicated in SV recycling; therefore, we tested synaptic transmission at hair-cell synapses. Recordings of post-synaptic activity in synj1 mutants showed relatively normal spike rates when hair cells were mechanically stimulated for a short period of time at 20 Hz. In contrast, a sharp decline in the rate of firing occurred during prolonged stimulation at 20 Hz or stimulation at a higher frequency of 60 Hz. The decline in spike rate suggested that fewer vesicles were available for release. Consistent with this result, we observed that stimulated mutant hair cells had decreased numbers of tethered and reserve-pool vesicles in comparison to wild-type hair cells. Furthermore, stimulation at 60 Hz impaired phase locking of the postsynaptic activity to the mechanical stimulus. Following prolonged stimulation at 60 Hz, we also found that mutant synj1 hair cells displayed a striking delay in the recovery of spontaneous activity. Collectively, the data suggest that Synj1 is critical for retrieval of membrane in order to maintain the quantity, timing of fusion, and spontaneous release properties of SVs at hair-cell ribbon synapses

Type I interferons and microbial metabolites of tryptophan modulate astrocyte activity and CNS inflammation via the aryl hydrocarbon receptor

Astrocytes play important roles in the central nervous system (CNS) during health and disease. Through genome-wide analyses we detected a transcriptional response to type I interferons (IFN-I) in astrocytes during experimental CNS autoimmunity and also in CNS lesions from multiple sclerosis (MS) patients. IFN-I signaling in astrocytes reduces inflammation and experimental autoimmune encephalomyelitis (EAE) disease scores via the ligand-activated transcription factor aryl hydrocarbon receptor (AhR) and suppressor of cytokine signaling 2 (SOCS2). The anti-inflammatory effects of nasally administered IFN-β are partly mediated by AhR. Dietary tryptophan is metabolized by the gut microbiota into AhR agonists that act on astrocytes to limit CNS inflammation. EAE scores were increased following ampicillin treatment during the recovery phase, and CNS inflammation was reduced in antibiotic-treated mice by supplementation with the tryptophan metabolites indole, indoxyl-3-sulfate (I3S), indole-3-propionic acid (IPA) and indole-3-aldehyde (IAld), or the bacterial enzyme tryptophanase. In individuals with MS, the circulating levels of AhR agonists were decreased. These findings suggest that IFN-I produced in the CNS act in combination with metabolites derived from dietary tryptophan by the gut flora to activate AhR signaling in astrocytes and suppress CNS inflammation

Multibow: Digital Spectral Barcodes for Cell Tracing

We introduce a multicolor labeling strategy (Multibow) for cell tracing experiments in developmental and regenerative processes. Building on Brainbow-based approaches that produce colors by differential expression levels of different fluorescent proteins, Multibow adds a layer of label diversity by introducing a binary code in which reporters are initially OFF and then probabilistically ON or OFF following Cre recombination. We have developed a library of constructs that contains seven different colors and three different subcellular localizations. Combining constructs from this library in the presence of Cre generates cells labeled with multiple independently expressed colors based on if each construct is ON or OFF following recombination. These labels form a unique "barcode" that allows the tracking of the cell and its clonal progenies in addition to expression level differences of each color. We tested Multibow in zebrafish which validates its design concept and suggests its utility for cell tracing applications in development and regeneration

Spatial temporal coverage and stability of Multibow labeling.

<p><b>a.</b> Spatial and cell type coverage of Multibow. The embryo was injected with 6 Multibow colors (mR/mG/nR/nG/R/G) at single cell stage and heat-shocked at 1 day-post-fertilization (dpf) for 2 hours. The whole 4dpf larva was imaged in 2 channels (G/R). Positive cells can be seen distributed from head to tail throughout the larva, indicating high spatial coverage. In inserts 1 and 2, distinctly shaped skin, muscle, mesenchymal and neural cells can be observed by cytoplasmic or membrane Multibow labeling. Scale bars: 100μm. <b>b.</b> Temporal stability of labeling. The embryo was injected with 6 Multibow colors (mR/mG/nR/nG/R/G) at single cell stage and heat-shocked at 1 day-post-fertilization (dpf) for 2 hours. The same embryo was imaged once per day to 11dpf. The persistence of labeling indicates genomic insertion of Multibow cassettes. Red patches around the eye and along the gut are auto-fluorescence. Enlarged views of white boxed areas show that the area is stably fluorescent. Scale bar in enlarged views: 100μm. <b>c.</b> Label stability of color codes over time. The embryo was injected with 12 (B/G/Y/R) Multibow constructs at one cell stage. Heat-shock of this tg(<i>hsp70</i>:<i>cerulean-cre</i>) individual was at 30hpf (duration: 2 hours). Its developing larval tail fin was imaged every 24 hours starting at 54hpf using four channels (B/G/Y/R). The color codes of the cells remain unchanged despite fluorescent intensity differences at different days, allowing identification of the same cells/clones(e.g., α and β, shown in enlarged regions marked by white boxes). Color codes: α: nG/nY; β: mB. Scale bar: 100μm. See also Fig d in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127822#pone.0127822.s002" target="_blank">S2 Fig</a>.</p

Design and test of Multibow in zebrafish.

<p><b>a.</b> Modified “Brainbow [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127822#pone.0127822.ref001" target="_blank">1</a>]” cassette that allows a binary ON/OFF switch. <b>b.</b> Multibow Strategy. Each cell harbors multiple different ON/OFF cassettes to generate random color “digital” barcodes upon Cre-mediated recombination. <b>c.</b> Table of Multibow Tags and Fluorescent Proteins (FPs). <b>d.</b> Diversity of color codes. Image is a densely labeled region along the trunk of a 40hpf <i>hsp70</i>:<i>cerulean-cre</i> embryo injected with all 21 Multibow constructs and heat-shocked at 10hpf for 1 hour. The color and tag diversity generates barcodes for cell clones that appear random and diverse. Intensity differences further help distinguish cells from neighbors visually. The Composite image is made from the green, yellow (turned to blue) and red panels. 3 different clones are highlighted by α, β, γ and corresponding arrows. Scale bar: 10μm. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127822#pone.0127822.s006" target="_blank">S3 Table</a>. <b>e.</b> Partial table of clones of different color codes found in <b>d.</b>. The colored square labels of the top row indicate nuclear, membrane and cytoplasmic, respectively. A black square in the table indicates this clone being positive for the corresponding color. Distinct "barcodes" form for different clones. The α, β, γ clones are indicated by arrows. The number of annotated cells labels (~30) represents a large fraction of cells found in the image in <b>d</b>, which contains ~50 cells. The fact that most of these cells have a color code distinct from any other cell (except clones that have the same color) show that Multibow label is highly random.</p

Examples of Multibow Cell Tracing in Development and Regeneration.

<p><b>a.</b> Cranial facial development mapped by Multibow. The embryo was heat-shocked at 6hpf. 4 channels (B/G/Y/R) were used. The left face of the larva was imaged. Red boxes: regions highlighted in <b>b.</b> and <b>c.</b>. Scale bars: 50μm. <b>b.</b> Lineage relationship between neuromast hair cells. Dashed line circle indicates the hair bundle. Multibow labeled hair cell color codes: 1(mB/nY/R), 2(mB/mG/nR), 3(mB), 4(nG), 5(mB/nR), 6(R). The same pattern was already observed at 30hpf. Scale bars: 10μm. <b>c.</b> Identification of cells that undergo remarkable morphological changes during semicircular canal formation. Arrows: initial locations of the two mesenchymal cells that span the projection later. Grey circle: posterior otolith. Scale bars: 50μm. <b>d.</b> Clonal expansion near the eye over long time periods. The embryo was injected with 12 constructs (B/G/Y/R) and heat-shocked at 10hpf. Arrows indicate locations of identified clones α (nG), β (nG/R), γ (nY/mR). These clones can be seen amplified in number at 54hpf or 129hpf (α: 2 to 4; β: 2 to 4; γ: 2 to 3). Scale bar: 100μm. <b>e.</b> Multibow analysis of regeneration in the larval tail. Heat-shock labeling (1 hour), amputation and imaging were performed as labeled in the timeline. Immediately after amputation, the tissue shrank and cells near the wound converged (the images overlay may appear to be slightly out of register due to the changes of the live tissue during the acquisition of different channels, cell identification is not affected as these changes are small and predictable). By 2 days after amputation, most cells that had converged at the frontier of the wound were gone (their unique color codes disappeared, red arrowheads). The regenerated tissue came from clonal expansion of cells away from the frontier (highlighted examples in enlarged view from the white boxes 1 and 2). These clones show lineage restriction to the original cell type (the morphology of cells in the same clone remains similar, e.g., the blue cells in box 1 increased in number while size and shape do not have major changes.). Scale bars: 50μm.</p

Myc and Fgf Are Required for Zebrafish Neuromast Hair Cell Regeneration

Unlike mammals, the non-mammalian vertebrate inner ear can regenerate the sensory cells, hair cells, either spontaneously or through induction after hair cell loss, leading to hearing recovery. The mechanisms underlying the regeneration are poorly understood. By microarray analysis on a chick model, we show that chick hair cell regeneration involves the activation of proliferation genes and downregulation of differentiation genes. Both MYC and FGF are activated in chick hair cell regeneration. Using a zebrafish lateral line neuromast hair cell regeneration model, we show that the specific inhibition of Myc or Fgf suppresses hair cell regeneration, demonstrating that both pathways are essential to the process. Rapid upregulation of Myc and delayed Fgf activation during regeneration suggest a role of Myc in proliferation and Fgf in differentiation. The dorsal-ventral pattern of fgfr1a in the neuromasts overlaps with the distribution of hair cell precursors. By laser ablation, we show that the fgfr1a-positive supporting cells are likely the hair cell precursors that directly give rise to new hair cells; whereas the anterior-posterior fgfr1a-negative supporting cells have heightened proliferation capacity, likely to serve as more primitive progenitor cells to replenish lost precursors after hair cell loss. Thus fgfr1a is likely to mark compartmentalized supporting cell subtypes with different capacities in renewal proliferation and hair cell regeneration. Manipulation of c-MYC and FGF pathways could be explored for mammalian hair cell regeneration

Specified neural progenitors sort to form sharp domains after noisy Shh signaling

Sharply delineated domains of cell types arise in developing tissues under instruction of inductive signal (morphogen) gradients, which specify distinct cell fates at different signal levels. The translation of a morphogen gradient into discrete spatial domains relies on precise signal responses at stable cell positions. However, cells in developing tissues undergoing morphogenesis and proliferation often experience complex movements, which may affect their morphogen exposure, specification, and positioning. How is a clear pattern achieved with cells moving around? Using in toto imaging of the zebrafish neural tube, we analyzed specification patterns and movement trajectories of neural progenitors. We found that specified progenitors of different fates are spatially mixed following heterogeneous Sonic Hedgehog signaling responses. Cell sorting then rearranges them into sharply bordered domains. Ectopically induced motor neuron progenitors also robustly sort to correct locations. Our results reveal that cell sorting acts to correct imprecision of spatial patterning by noisy inductive signals