Afferent Neurons of the Zebrafish Lateral Line Are Strict Selectors of Hair-Cell Orientation

Hair cells in the inner ear display a characteristic polarization of their apical stereocilia across the plane of the sensory epithelium. This planar orientation allows coherent transduction of mechanical stimuli because the axis of morphological polarity of the stereocilia corresponds to the direction of excitability of the hair cells. Neuromasts of the lateral line in fishes and amphibians form two intermingled populations of hair cells oriented at 180° relative to each other, however, creating a stimulus-polarity ambiguity. Therefore, it is unknown how these animals resolve the vectorial component of a mechanical stimulus. Using genetic mosaics and live imaging in transgenic zebrafish to visualize hair cells and neurons at single-cell resolution, we show that lateral-line afferents can recognize the planar polarization of hair cells. Each neuron forms synapses with hair cells of identical orientation to divide the neuromast into functional planar-polarity compartments. We also show that afferent neurons are strict selectors of polarity that can re-establish synapses with identically oriented targets during hair-cell regeneration. Our results provide the anatomical bases for the physiological models of signal-polarity resolution by the lateral line

OptoGluNAM4.1, a Photoswitchable allosteric antagonist for real-time control of mGlu4 receptor activity

OptoGluNAM4.1, a negative allosteric modulator (NAM) of metabotropic glutamate receptor 4 (mGlu4) contains a reactive group that covalently binds to the receptor and a blue-light-activated, fast-relaxing azobenzene group that allows reversible receptor activity photocontrol in vitro and in vivo. OptoGluNAM4.1 induces light-dependent behavior in zebrafish and reverses the activity of the mGlu4 agonist LSP4-2022 in a mice model of chronic pain, defining a photopharmacological tool to better elucidate the physiological roles of the mGlu4 receptor in the nervous system

Illuminating phenylazopyridines to photoswitch metabotropic glutamate receptors: from the flask to the animals

Phenylazopyridines are photoisomerizable compounds with high potential to control biological functions with light. We have obtained a series of phenylazopyridines with light dependent activity as negative allosteric modulators (NAM) of metabotropic glutamate receptor subtype 5 (mGlu5). Here we describe the factors needed to achieve an operational molecular photoisomerization and its effective translation into in vitro and in vivo receptor photoswitching, which includes zebrafish larva motility and the regulation of the antinociceptive effects in mice. The combination of light and some specific phenylazopyridine ligands displays atypical pharmacological profiles, including light-dependent receptor overactivation, which can be observed both in vitro and in vivo. Remarkably, the localized administration of light and a photoswitchable compound in the peripheral tissues of rodents or in the brain amygdalae results in an illumination-dependent analgesic effect. The results reveal a robust translation of the phenylazopyridine photoisomerization to a precise photoregulation of biological activity

Synaptojanine2 et Ocrl1, protéine du syndrome de Lowe (étude de deux nouvelles connexions directes entre les phosphoinositides et la GTPase Rac)

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Delaying Gal4-driven gene expression in the zebrafish with morpholinos and Gal80.

The modular Gal4/UAS gene expression system has become an indispensable tool in modern biology. Several large-scale gene- and enhancer-trap screens in the zebrafish have generated hundreds of transgenic lines expressing Gal4 in unique patterns. However, the early embryonic expression of the Gal4 severely limits their use for studies on regeneration or behavior because UAS-driven effectors could disrupt normal organogenesis. To overcome this limitation, we explored the use of the Gal4 repressor Gal80 in transient assays and with stable transgenes to temporally control Gal4 activity. We also validated a strategy to delay Gal4-driven gene expression using a morpholino targeted to Gal4. The first approach is limited to transgenes expressing the native Gal4. The morphant approach can also be applied to transgenic lines expressing the Gal4-VP16 fusion protein. It promises to become a standard approach to delay Gal4-driven transgene expression and enhance the genetic toolkit for the zebrafish

Temporal control of Gal80 expression.

<p>(<b>A</b>) Schematic representation of the construct hsp70:Gal80-myc. (<b>B</b>) Anti-myc western blotting of 48 hpf fish resulting from a cross of <i>Tg[hsp70:Gal80-myc]</i> fish after 0, 30 and 60 minutes of heat-shock. (<b>C</b>) Embryos resulting from a cross between <i>Tg[hsp70:Gal80-Myc]</i> and <i>Tg[UAS:Kaede]</i> fish were not submitted (a–c) or submitted to a one-hour heat-shock (d–f). Representative specimens are depicted for anti-Gal80 <i>in situ</i> hybridization at 30 hpf (a, d) or for Kaede expression at 2 dpf (b–c, d–e). (Scale bars: 150 µm). (<b>D</b>) Embryos resulting from a cross between <i>Tg[hsp70:Gal80-Myc]</i> and <i>Tg[UAS:Kaede]</i> fish were injected with a DNA encoding full-length Gal4 under the control of the β-actin promoter. At 24 hpf, the embryos were subjected to a 30-minute heat-shock at 39 degrees and immediately photo-converted. The following day, immunostaining anti-myc (c, g) was performed on fish expressing Gal80 (a–d) or not expressing Gal80 (e–h). The figure depicts muscle fibers for the expression of Kaede^red (a, e) and Kaede^green (b, f). (Scale bars: 20 µm).</p

Gal80 expression inhibits Gal4 activity.

<p>(<b>A–H</b>) Embryos resulting from a cross between <i>Tg[hsp70:Gal4]</i> and <i>Tg[UAS:Kaede]</i> fish were either non-injected (NI, A–D) or injected with 100 pg of mRNA encoding full-length Gal80 (E–H). Representative specimens are depicted at 24 hpf (A–B, N = 18 GFP<b>⁺</b>/72 fish; E–F, N = 0 GFP<b>⁺</b>/75 fish) and 5 dpf (C–D, N = 18 GFP<b>⁺</b>/77 fish; G–H, N = 19 GFP<b>⁺</b>/82 fish). In H, asterisks indicate the fish displaying GFP. (<b>I–N</b>) Embryos resulting from a cross of <i>Tg[UAS:Kaede]</i> fish were injected with a DNA encoding full-length Gal4 under the control of the HuC promoter either alone (NI, I–K, N = 32) or with 100 pg of mRNA encoding full-length Gal80 (L–N, N = 37). Representative specimens are depicted at 48 hpf, 3 and 7 dpf.</p

Gal4 morpholino temporarily inhibits Gal4 expression in a dose-dependant manner.

<p>Embryos resulting from a cross of <i>Tg[hspGFF53A;UAS:EGFP]</i> double transgenic animals were either non-injected (A–E) or injected with 1 ng (F–J), 3 ng (K–O) or 5 ng (P–T) of Gal4 MO (N = 94). Representative specimens are depicted at 2, 4 and 6 dpf. White arrows indicate green fluorescence at the level of the posterior afferent lateralis ganglion. (Scale bars: 150 µm).</p

Gal4 morpholino temporarily inhibits Gal4 expression in Gal4-VP16 lines.

<p>(<b>A</b>) Embryos resulting from a cross between <i>Tg[hsp70:Gal4]</i> and <i>Tg[UAS:mem-TdTomato]</i> fish were either non-injected (NI, Aa-b, N = 27) or injected with 3 ng of Gal4 MO (Ac-d, N = 37). Representative specimens are depicted at 2 dpf. (<b>B</b>) Embryos resulting from a cross of <i>Tg[ET(hsp:Gal4VP16^s1006t);UAS:Kaede]</i> double transgenic animals were either non-injected (NI, Ba-d, N = 47) or injected with 3 ng of Gal4 MO (Be-h, N = 52). Representative specimens are depicted at 24 hpf (Ba, e), 4 dpf (Bb, f) and 6 dpf (Bc-d, Bg-h). (<b>C</b>) Embryos resulting from a cross of <i>Tg[hspGFF53A;UAS:EGFP]</i> double transgenic animals were either non-injected (NI, Ca-b, N = 64) or injected with 3 ng of Gal4 MO (Cc-d, N = 58). Representative specimens are depicted at 48 hpf (Ca, c) and 4 dpf (Cb, d). (<b>D</b>). Embryos resulting from a cross of <i>Tg[hspGFF53A;UAS:EGFP]</i> double transgenic animals were either non-injected (NI, Da), injected with 5 ng of Gal4 MO (Db) or injected with 5 ng of a control MO (Dc). Representative specimens are depicted at 48 hpf. (Scale bars: 600 µm for A; 300 µm for B and C, 150 µm for D).</p