ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

(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 of the left and right posterior lateral line ganglion region. Below, three sets of images of the left and right ROI for each of 10 embryos (total of 20 areas), including the bright-field images, the threshold area (red) levels set with HSB on Fiji (Hue: 166–236, Saturation: 43–98, Brightness: 31–184), and the binary image used to determine the area of staining. (B) adgrg6^tb233c mutant embryo images comparable to those shown in A with identical threshold settings used in Fiji. (C) Comparison of the area of mbp staining between the adgrg6^tb233c mutant and wild-type sibling embryos using the quantification obtained in A and B. ****p<0.0001, Student’s t-test. Error bars represent the mean ±95% CI.

10.7554/eLife.44889.004Figure 1—figure supplement 1—source data 1.

(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 of the left and right posterior lateral line ganglion region. Below, three sets of images of the left and right ROI for each of 10 embryos (total of 20 areas), including the bright-field images, the threshold area (red) levels set with HSB on Fiji (Hue: 166–236, Saturation: 43–98, Brightness: 31–184), and the binary image used to determine the area of staining. (B) adgrg6^tb233c mutant embryo images comparable to those shown in A with identical threshold settings used in Fiji. (C) Comparison of the area of mbp staining between the adgrg6^tb233c mutant and wild-type sibling embryos using the quantification obtained in A and B. ****p<0.0001, Student’s t-test. Error bars represent the mean ±95% CI.

10.7554/eLife.44889.004Figure 1—figure supplement 1—source data 1.

(A) Schematic of the screening assay protocol. Homozygous adult adgrg6^tb233c mutant fish were paired to raise large numbers of adgrg6^tb233c mutant embryos. Embryos were grown until 60 hpf, when the lateral, anterior and posterior epithelial projections in the inner ear are evident. Three embryos were aliquoted into each well of a mesh-bottomed multiwell plate in E3 medium. The mesh-bottomed plate was then transferred to the drug plate containing control compounds as shown in the plate layout and library compounds at 25 µM in 250 µL of E3 embryo medium. Plates were incubated at 28°C until 90 hpf. The mesh-bottomed plate and embryos were then transferred to 4% PFA for fixation (4°C, overnight) and then processed for ISH to vcanb. Micrographs show a selection of typical results. Treatment with 100 µM IBMX (positive control, top) results in loss (rescue) of otic vcanb expression (white arrowhead). Strong otic vcanb expression (black arrowhead) is evident in embryos where the compound had no effect (non-hit) and in negative control wells (not shown). Note the spot of stain in each embryo, marking expression in the otic vesicle. Three examples are shown of wells where compounds were scored as a hit; one of these (Hit 2) was IBMX, represented in the Spectrum collection. (B). Pipeline of the compound screening strategy and chemoinformatics analysis. The left hand side describes the flow of experimental work and the right hand side describes the complementary chemoinformatics processes. For details, see the text.

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 scaffold. The histograms on the left use Bemis and Murcko scaffolds (Bemis and Murcko, 1996), an example of which shown at the top. The histograms on the right were generated using CSK scaffolds. The number of scaffolds for each library is shown in the top right of each graph. An example BMS scaffold and CSK scaffold (obtained from the same compound) are shown above the histograms.

(A) Scoring system used to assess mbp mRNA expression levels around the PLLg of adgrg6^tb233c embryos after treatment. (Ai) A score of 3 was given to embryos where mbp mRNA expression was similar to wild-type levels. Black arrowhead: mbp expression around the PLLg. (Aii) A score of 2 was given to embryos that showed weak mbp expression around the PLLg. (Aiii) A score of 1 was given to embryos with mbp expression identical to that in untreated adgrg6^tb233c mutants (absence of mbp expression around the PLLg (white arrowhead), with weak expression elsewhere). (Aiv) A score of 0 was used to indicate embryos where mbp mRNA expression was absent throughout the PNS. Asterisks mark expression near the three cristae of the ear. Scale bar: 50 μm. (B, C) mbp scoring system and classification of the compounds. (B) Compounds were categorised according to the mbp score sum from three embryos (average from two experiments; six embryos total) and grouped into compounds able to rescue mbp expression (score >3.5–9) and unable to rescue mbp expression (>1.5–3.5). A third class of compounds down-regulated both vcanb and mbp (score 0–1.5) and were not followed further. (C) Distribution of the compounds in the different rescue categories after the mbp counter screen. (D) Compounds from both libraries are represented as individual dots in a combined polar scatterplot (3120 compounds in total; https://adlvdl.github.io/visualizations/polar_scatterplot_whitfield_vcanb.html). Compounds were ordered according to similarities in their chemical structure and placed in concentric circles according to the category A–G they were assigned to after the primary screen, with jitter (noise) introduced to improve visualisation. (E) Polar scatter plot of the 91 hit compounds that passed the first retest and were followed up with mbp counter screens; previous scores for the compounds not followed are faded. (F) Polar scatter plot of the final 68 hit compounds (non-faded) after mbp counter screens. Bigger dots represent compounds that rescued mbp expression, whereas smaller dots correspond to the compounds that did not rescue mbp expression; compounds that downregulated mbp expression, or were not followed, are faded. Wedges on the scatter plot delineate the two clusters of compounds with similar structures for which some hits were followed up in further analysis (see text). The positions of IBMX (I) and colforsin (C) are indicated (red arrowheads). (G) Overview of the hit selection process. The length of the horizontal bars is proportional to the number of hit compounds taken through to each stage. Data for the Tocris library are on the left-hand side; data for the Spectrum library are on the right-hand side. The proportion of compounds in hit categories A, B and C are shown using the same colour scheme as in Figure 3, with the top bar representing the number of hits from the primary screen listed in Figure 3B. The second bar shows the number of compounds that were cherry-picked. The average scores from nine embryos (after retests) is shown in the third bar. Note that some compounds will change category after the retests and the number of category C compounds is increased. Any compounds that failed to rescue in any single retest were also not taken forward (fourth bar). The mbp data (E) are represented in the fifth and sixth bars. The final bar represents the compounds that were unable to rescue the strong fr24 allele. The total number of compounds at each stage is shown in the centre. Asterisks denote numbers that do not include duplicate compounds.

10.7554/eLife.44889.014Figure 4—source data 1.

(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 4G and 5B), highlighting cilnidipine and nifedipine. Compounds that rescued mbp expression are shown as larger nodes, whereas compounds that did not rescue mbp expression are shown as smaller nodes. The relationship of nilvadipine (green circle) to the other compounds in this cluster is also illustrated. (C) (i–vii) Lateral images of the inner ear at 4 dpf stained for vcanb by ISH. (i) Wild-type, (ii) adgrg6^tb233c mutant treated with DMSO as a control, (iii–vii) treatment of adgrg6^tb233c mutants with test compounds at 25 µM, with the exception of nilvadipine, which was tested at 22.5 µM. (viii–xiv) mbp mRNA expression of embryos treated as indicated above. Black arrowheads indicate mbp expression around the PLLg; white arrowhead in (ix) indicates the position of the PLLg in the untreated mutant, lacking mbp expression. Nifedipine, cilnidipine, tracazolate hydrochloride and nilvadipine all rescued mbp expression around the PLLg, whereas FPL 64176 did not rescue mbp expression around the PLLg so efficiently. (xv–xix) Representation of the chemical structures of the five compounds tested. Scale bar in (i), 50 µm (applies to i-vii); scale bar in viii, 50 µm (applies to viii–xiv).

(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 threshold of the network analysis performed, illustrated by the dotted line connecting to its closest related compound. (B) Dose-responsive activity of the dihydropyridines in the vcanb assay. (i-xii) Lateral views of the inner ear at four dpf stained for vcanb by ISH; anterior to the left. Controls: (i) wild-type, untreated; (vii) adgrg6^tb233c mutant treated with DMSO as a negative control; (xii) adgrg6^tb233c mutant treated with 100 µM IBMX as a positive control. (ii-vi) Treatment of adgrg6^tb233c mutants with test compounds at a low concentration (25–33.8 µM), (viii-xi) treatment of adgrg6^tb233c mutants with test compounds at a high concentration (40–50.6 µM), (xiii-xvii) Representation of the chemical structure of the five compounds tested. Scale bar in (i), 50 µm (applies to i-xii).

(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 threshold of the network analysis performed, illustrated by the dotted line connecting to its closest related compound. (B) Dose-responsive activity of the dihydropyridines in the vcanb assay. (i-xii) Lateral views of the inner ear at four dpf stained for vcanb by ISH; anterior to the left. Controls: (i) wild-type, untreated; (vii) adgrg6^tb233c mutant treated with DMSO as a negative control; (xii) adgrg6^tb233c mutant treated with 100 µM IBMX as a positive control. (ii-vi) Treatment of adgrg6^tb233c mutants with test compounds at a low concentration (25–33.8 µM), (viii-xi) treatment of adgrg6^tb233c mutants with test compounds at a high concentration (40–50.6 µM), (xiii-xvii) Representation of the chemical structure of the five compounds tested. Scale bar in (i), 50 µm (applies to i-xii).

(A) Live DIC image of an adgrg6^tb233c 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 normalised ear width. This value was used to assess how the ear swelling is affected after treatment with different compounds. (B) Table showing the strictly standardised mean difference (SSMD) values for each treatment group in respect to the vehicle control (adgrg6^tb233c, 1% DMSO), as a means of assessing the size of compound effect at different concentrations. SSMD scores > 2 indicate a strong effect (pale orange); SSMD scores > 3 indicate a very strong effect (dark orange).

10.7554/eLife.44889.024Figure 7—figure supplement 2—source data 1.

(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 (cluster two in Figures 4G and 5B), highlighting deoxygedunin, dihydrofissinolide and carapin-8(9)-ene. Compounds that rescued mbp expression are shown as larger nodes; compounds that did not rescue mbp expression are shown as smaller nodes. (C) (i–x) The inner ear at 4 dpf stained for vcanb. Lateral views; anterior to the left. Scale bar (applies to panels i–x): 50 µm. (i) adgrg6^tb233c/DMSO mutant control. (ii – v) Treatment of adgrg6^tb233c mutants with the compounds at 25 µM indicated was able to rescue the tb233c mutant ear phenotype to variable degrees. (vi) adgrg6^fr24/DMSO mutant control. (vii–x) Treatment of adgrg6^fr24 mutants with colforsin rescued otic vcanb expression in the fr24 allele, whereas treatment with dihydrofissinolide, deoxygedunin and carapin-8(9)-ene was unable to rescue the fr24 ear phenotype. (xi–xiv) Representation of the chemical structure of the four compounds tested. Note the structural similarity between deoxygedunin, dihydrofissinolide and carapin-8(9)-ene.