Figure 4—source data 1.

(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.