Identification of compounds that rescue otic and myelination defects in the zebrafish adgrg6 (gpr126) mutant
- University of Sheffield, United Kingdom
- Sosei Heptares, United Kingdom
Figures
Figure 1 with 1 supplement
Comparison of adgrg6 mutant allele phenotypes in the inner ear and peripheral nervous system.
(A) (i–iii) Live images of 4 dpf otic vesicles, lateral view. (i) Wild-type sibling, (ii) adgrg6tb233c, (iii) adgrg6fr24 showing the swollen, unfused projections in the homozygous mutant otic …
Figure 1—figure supplement 1
Quantification of mbp expression around the posterior lateral line ganglion in adgrg6tb233c mutants and wild-type sibling embryos.
(A) Top, wild-type (WT) sibling: bright-field image of 4 dpf embryo stained with mbp. Dorsal view, anterior to the left. Blue box indicates the region of interest (ROI) used for the quantification …
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Figure 1—figure supplement 1—source data 1
Source data for the percentage area of mbp expression shown in Figure 1—figure supplement 1.
- https://doi.org/10.7554/eLife.44889.004
Figure 2
Overview of the screening assay protocol and strategy.
(A) Schematic of the screening assay protocol. Homozygous adult adgrg6tb233c mutant fish were paired to raise large numbers of adgrg6tb233c mutant embryos. Embryos were grown until 60 hpf, when the …
Figure 3 with 1 supplement
A primary drug screen identified 92 (Tocris) and 205 (Spectrum) putative hit compounds able to down-regulate vcanb mRNA expression in adgrg6tb233c mutants.
(A) Scoring system used to assess vcanb mRNA expression levels in the inner ear of adgrg6tb233c embryos after treatment. (Ai) vcanb mRNA expression in the untreated/DMSO-treated adgrg6tb233c mutant …
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Figure 3—source data 1
Source data for Figure 3D.
Dendrogram representing structural similarity between library compounds (Tocris). Dendrogram of the Tocriscreen Total library compounds based on the similarity matrix between all pairs of compounds (Ward’s method of hierarchical agglomerative clustering—see Materials and methods). Compounds are named by their plate and well ID.
- https://doi.org/10.7554/eLife.44889.011
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Figure 3—source data 2
Source data for Figure 3F.
Dendrogram representing structural similarity between library compounds (Spectrum). Dendrogram of the Spectrum library compounds based on the similarity matrix between all pairs of compounds. Compounds are named by their plate and well ID.
- https://doi.org/10.7554/eLife.44889.012
Figure 3—figure supplement 1
Scaffold analysis of compound structures in the Tocriscreen Total and Spectrum libraries.
Two different methods were used to remove side chains and determine the core structures of each compound. Scaffolds were then compared and a histogram produced with the number of molecules per …
Figure 4
Retesting and counter screen for mbp expression reveals chemical clustering of hit compounds.
(A) Scoring system used to assess mbp mRNA expression levels around the PLLg of adgrg6tb233c embryos after treatment. (Ai) A score of 3 was given to embryos where mbp mRNA expression was similar to …
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Figure 4—source data 1
Source data for Figure 4D.
Dendrogram representing structural similarity between library compounds (Combined). Dendrogram of the combined Spectrum and Tocriscreen Total library compounds based on the similarity matrix between all pairs of compounds. Compounds are named by their plate and well ID.
- https://doi.org/10.7554/eLife.44889.014
Figure 5
Heatmap of the assay results and network analysis for 68 compounds identified in the vcanb screen.
(A) Heatmap of the assay results for each of the 68 hit compounds. Each box represents an embryo screened in each of the three assays (vcanb, mbp and fr24) as listed at the bottom of the heatmap. …
Figure 6
Hit compounds from the vcanb screen vary in their ability to restore mbp expression in adgrg6tb233c mutant embryos.
(A) Section of the heatmap in Figure 5A showing the results for nifedipine, cilnidipine, tracazolate hydrochloride and FPL 64176. (B) Enlargement of the dihydropyridine cluster (cluster 1 in Figures …
Figure 7 with 3 supplements
Selected hit compounds rescue the adgrg6tb233c mutant ear phenotype in a dose-dependent manner.
adgrg6tb233c homozygous embryos were exposed to a 1.5-fold dilution series of concentrations (ranging from 0.3 μM to 33.7 μM), tailored to the toxicity of nifedipine, cilnidipine, tracazolate …
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Figure 7—source data 1
Source data for the dose-response experiments shown in Figure 7D.
- https://doi.org/10.7554/eLife.44889.023
Figure 7—figure supplement 1
Additional dihydropyridines are able to downregulate otic vcanb expression in adgrg6tb233c mutant embryos.
(A) Adapted dihydropyridine cluster including compounds not represented in the Tocris or Spectrum collections. The new compounds tested are shown as green circles. Nemadipine-A falls just below the …
Figure 7—figure supplement 2
Normalisation of ear width with respect to size of the head.
(A) Live DIC image of an adgrg6tb233c mutant embryo at 110 hpf, mounted dorsally with anterior to the left, showing the parameters A, B and C (as defined in the figure) used to calculate the …
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Figure 7—figure supplement 2—source data 1
Source data for the SSMD calculations shown in Figure 7—figure supplement 2B.
- https://doi.org/10.7554/eLife.44889.024
Figure 7—figure supplement 3
LD50 curves from the treatment of wild-type embryos from 60 to 110 hpf.
Sixteen LWT wild-type embryos, each kept in a separate well of a 96-well plate, were treated with each of the following concentrations: 5, 10, 20, 40, 60, 80 and 100 μM, from 60 to 110 hpf. At the …
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Figure 7—figure supplement 3—source data 2
Source data for the mortality counts shown in Figure 7—figure supplement 3.
- https://doi.org/10.7554/eLife.44889.025
Figure 8
Assay for rescue of the fr24 strong allele distinguishes compounds likely to rescue downstream, or at the level of, the Adgrg6 receptor.
(A) Section of the heatmap in Figure 5A showing the results for colforsin, dihydrofissinolide, deoxygedunin and carapin-8(9)-ene. (B) Enlargement of the cluster containing gedunin-related compounds …
Videos
Video 1
Light-sheet microscope time-lapse video of the ear shown in Figure 1Ci-iii.
Dorsal view (anterior to top) of the left inner ear of a phenotypically wild-type sibling embryo showing the anterior, lateral and posterior projections (the anterior projection is partially out of …
Video 2
Light-sheet microscope time-lapse video of the ear shown in Figure 1Civ-vi.
Dorsal view of the right inner ear of an adgrg6fr24 mutant embryo showing anterior, lateral and posterior projections (the posterior projection is partially out of view). In the video, the anterior …
Tables
Table 1
List of the 41 hit compounds that rescued the expression of both vcanb and mbp in adgrg6tb233c mutants, thus representing putative Adgrg6 pathway modulators.
The table includes the plate and well ID, along with known activities and the average score from nine adgrg6tb233c embryos in the vcanb assay, from six adgrg6tb233c embryos in the mbp assay and from …
| # | Plate | Well | Compound name | Known activity | vcanb score | mbp score | fr24 score |
|---|---|---|---|---|---|---|---|
| 1 | S18 | C09 | CARAPIN-8(9)-ENE | undetermined | 0.00 | 8.50 | 9.00 |
| 2 | S25 | D08 | 3-ISOBUTYL-1-METHYLXANTHINE (IBMX) | phosphodiesterase inhibitor, non-selective adenosine receptor antagonist | 2.00 | 8.50 | 9.00 |
| 3 | S17 | F05 | DEOXYGEDUNIN | neuroprotective | 2.00 | 8.00 | 9.00 |
| 4 | S23 | F10 | DIHYDROFISSINOLIDE | undetermined | 2.67 | 7.50 | 9.00 |
| 5 | S04 | B02 | IVERMECTIN | antiparasitic | 2.33 | 7.00 | 9.00 |
| 6 | T01 | F06 | SC-10 | protein kinase C activator, NMDA receptor activator | 5.67 | 6.50 | 9.00 |
| 7 | T01 | H11 | 1,3-Dipropyl-8-phenylxanthine | Selective adenosine A1 receptor antagonist | 3.33 | 6.50 | 9.00 |
| 8 | S17 | E02 | 3-DEOXO-3beta-ACETOXYDEOXYDIHYDROGEDUNIN | undetermined | 0.00 | 6.50 | 9.00 |
| 9 | T11 | F07 | Cilnidipine* | dihydropyridine N- and L-type Ca2+ channel blocker | 2.00 | 6.50 | 9.00 |
| 10 | S13 | F03 | AMIODARONE HYDROCHLORIDE | coronary vasodilator, Ca2+ channel blocker | 5.00 | 6.50 | 9.00 |
| 11 | S06 | E02 | HYDROCORTISONE HEMISUCCINATE | glucocorticoid | 3.67 | 6.00 | 9.00 |
| 12 | T01 | C04 | (RS)-(Tetrazol-5-yl)glycine | highly potent NMDA receptor agonist | 3.00 | 5.00 | 9.00 |
| 13 | S02 | E05 | LOMEFLOXACIN HYDROCHLORIDE | antibacterial | 5.33 | 5.00 | 9.00 |
| 14 | S13 | E04 | ETHAMIVAN | CNS & respiratory stimulant | 4.67 | 5.00 | 9.00 |
| 15 | T08 | B04 | CGS 15943 | potent adenosine receptor antagonist | 5.33 | 4.50 | 9.00 |
| 16 | S13 | E09 | ASTEMIZOLE | H1 antihistamine (nonsedating) | 4.67 | 4.50 | 9.00 |
| 17 | T02 | A09 | SKF 91488 dihydrochloride | histamine N-methyltransferase inhibitor | 3.00 | 4.00 | 9.00 |
| 18 | S25 | F05 | 11alpha-HYDROXYPROGESTERONE HEMISUCCINATE | glucocorticoid | 2.67 | 4.00 | 9.00 |
| 19 | T14 | A07 | Efonidipine hydrochloride monoethanolate | dihydropyridine L-type and T-type Ca2+ channel blocker | 3.67 | 4.00 | 9.00 |
| 20 | T05 | C09 | Nifedipine | dihydropyridine L-type Ca2+ channel blocker | 4.33 | 7.00 | 8.00 |
| 21 | T05 | E08 | CGP 37157 | antagonist of mitochondrial Na+/Ca2+ exchange | 3.67 | 6.50 | 8.00 |
| 22 | S05 | D03 | DANAZOL | anterior pituitary suppressant, anti-estrogenic | 1.00 | 5.00 | 8.00 |
| 23 | S18 | H09 | XANTHYLETIN | undetermined | 1.00 | 4.50 | 8.00 |
| 24 | S18 | A06 | FERULIC ACID | antineoplastic, choleretic, food preservative | 3.67 | 4.00 | 8.00 |
| 25 | S18 | F02 | alpha-DIHYDROGEDUNOL | undetermined | 2.33 | 4.00 | 8.00 |
| 26 | T05 | F04 | (S)-(+)-Niguldipine hydrochloride | dihydropyridine L-type Ca2+ channel blocker, α1 antagonist | 3.67 | 5.00 | 7.00 |
| 27 | T07 | F02 | Tracazolate hydrochloride | subtype-selective GABAAallosteric modulator | 2.33 | 4.50 | 7.00 |
| 28 | S10 | E02 | NIMODIPINE | dihydropyridine L-type Ca2+ channel blocker | 0.33 | 7.00 | 6.00 |
| 29 | S17 | E06 | 3beta-ACETOXYDEOXODIHYDROGEDUNIN | undetermined | 2.00 | 4.50 | 5.00 |
| 30 | S17 | F02 | DIHYDROGEDUNIN | undetermined | 1.67 | 5.00 | 2.00 |
| 31 | S22 | F09 | TANGERITIN | undetermined | 1.33 | 5.50 | 1.00 |
| 32 | S10 | F07 | COLFORSIN | adenylate cyclase activator, antiglaucoma, hypotensive, vasodilator | 0.00 | 9.00 | 0.00 |
| 33 | T04 | G02 | Imiloxan hydrochloride | selective α2B-adrenoceptor antagonist | 0.67 | 9.00 | ND |
| 34 | S24 | C03 | 3alpha-ACETOXYDIHYDRODEOXYGEDUNIN | undetermined | 0.33 | 8.50 | DE |
| 35 | S11 | E02 | EZETIMIBE | antihyperlipidemic (sterol absorption inhibitor) | 2.00 | 7.50 | 0.00 |
| 36 | S10 | E06 | NITRENDIPINE | dihydropyridine L-type Ca2+ channel blocker | 1.33 | 7.00 | ND |
| 37 | S11 | E08 | ROSUVASTATIN CALCIUM | antihyperlipidemic | 0.00 | 6.00 | 0.00 |
| 38 | S22 | C07 | DEMETHYLNOBILETIN | undetermined | 0.00 | 6.00 | 0.00 |
| 39 | S22 | G11 | HEXAMETHYLQUERCETAGETIN | undetermined | 0.00 | 5.50 | DE |
| 40 | S22 | F08 | NOBILETIN | matrix metaloproteinase inhibitor, antineoplastic, anti-ERK, NF-κB suppressor | 0.00 | 5.00 | DE |
| 41 | S12 | H07 | PREGNENOLONE SUCCINATE | glucocortcoid, antiinflammatory | 4.67 | 4.00 | DE |
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Table 1—source data 1
Source data for Table 1.
- https://doi.org/10.7554/eLife.44889.017
Additional files
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Supplementary file 1
List of the 89 hit compounds that rescued the expression of vcanb in adgrg6tb233c mutants and were followed up by mbp counter screens.
The table includes the plate and well position of each compound, along with known activities and the raw data scores from nine adgrg6tb233c embryos in the vcanb assay (v1–v9), from six adgrg6tb233c embryos in the mbp assay (m1–m6) and from three adgrg6fr24 embryos in the fr24 (fr1–3) assay. Abbreviations: DE, dead embryo; ND, no data; S, Spectrum; T, Tocris. *Deoxygedunin: (Jang et al., 2010); Nobiletin: (Cheng et al., 2016); Angolensin (R): (Weisman et al., 2006); Sinensetin: (Kang et al., 2015); Larixol acetate: (Urban et al., 2016); Gedunin: (Hieronymus et al., 2006; Subramani et al., 2017).
- https://doi.org/10.7554/eLife.44889.027
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Transparent reporting form
- https://doi.org/10.7554/eLife.44889.028
