DeTCT analysis identifies three groups of differentially expressed genes. (A) Diagram of the experiment. Embryos from a slc39a14^+/− incross were either left unexposed or exposed to 50 µM MnCl₂ from 2 to 5 dpf. (B) Principal component analysis of the samples. Principal component (PC) 1 is plotted on the x-axis and PC2 on the y-axis. Samples belonging to the same condition are grouped together. Circles represent wild-type embryos, diamonds represent heterozygotes and squares represent homozygote mutants. Unexposed sibling embryos are indicated in light blue and MnCl₂-exposed embryos are in dark blue. Unexposed mutants are coloured light red and exposed mutants are dark red. (C) Group 1 (Mn toxicity) genes were defined as those with a significant difference between exposed and unexposed siblings (red bar with asterisk). An example plot of normalised counts for the soul5 gene is shown. The colour scheme for C-E is the same as in B. (D) Group 2 (Increased sensitivity) genes were defined as those with a significant difference between exposed mutants and unexposed siblings (red bar with asterisk), but without significant differences between either unexposed mutants or exposed siblings when compared to unexposed siblings (black bars labelled NS, not significant). An example plot of normalised counts for the opn1mw2 gene is shown. (E) Group3 (Mutant effect) was defined as genes with a significant difference between unexposed mutants and unexposed siblings (red bar with asterisk). Example plot of normalised counts for the cdh24b gene. *P<0.05 determined using the Wald test (see Materials and Methods).

Effect of Mn treatment in slc39a14^−/− mutants. (A) Heatmap of the expression of all 613 genes with a significant difference between exposed mutant and unexposed sibling embryos, but without without significant effects of treatment or genotype. The heatmaps are split into genes that show either synergistic or additive effects of the individual genotype and treatment effects. Each row represents a different gene and each column is a sample. The normalised counts for each gene have been mean centred and scaled by dividing by the standard deviation. (B) Example of a gene (hspa5) with a synergistic effect of treatment and genotype. The difference between the exposed mutants and unexposed siblings cannot be explained by adding together the separate effects of Mn treatment and the slc39a14^−/− mutation. Unexposed sibling embryos are indicated in light blue and MnCl₂-exposed embryos are in dark blue. Unexposed mutants are coloured light red and exposed mutants are dark red. (C) Example of a gene (pde6c) that has an additive effect of treatment and genotype. The difference between exposed mutants and unexposed siblings is consistent with adding together the two sub-threshold effects of treatment and genotype produce. Colour scheme is as in B. FC, fold change. Wald test was used to determine significance in B,C. (D) qRT-PCR showed comparable gene expression changes for hspa5 as for the single-embryo sequencing dataset. The individual samples are displayed as fold change relative to the mean value for unexposed siblings, and the mean and 95% confidence intervals for each condition indicated are in orange. Compare with B. (E) Enrichment Map network of the Zebrafish Anatomy Ontology (ZFA) enrichment results. Each node represents an enriched ZFA term and the edges join nodes that have overlapping genes annotated to them. The width of each edge is proportional to the amount of overlap; nodes are coloured by −log₁₀[adjusted P-value] (Wald test); and the size represents the number of significant genes annotated to the term. (F) ClueGO network diagram of the enrichment of GO terms. Nodes represent enriched GO terms and edges connect nodes that share annotations to the significant genes. Different components of the network are coloured according to the categories as labelled on the diagram. The sizes of the circles represent the adjusted P-values for each GO term as indicated below (Wald test).

MnCl₂ treatment alters neuronal activity in both wild-type and slc39a14^−/− larvae. (A-D) Z-projection of cfos mRNA expression in the brain of wild-type (A) and homozygous mutant larvae (C) at 6 dpf following treatment with 50 µM MnCl₂ from 2 dpf. B and D list the brain regions with enhanced (magenta) and suppressed (green) neuronal activity by genotype. A, anterior; P, posterior; D, dorsal; V, ventral; L, lateral; M, medial. Scale bars: 100 µm.

MnCl₂ treatment causes locomotor abnormalities and Ca²⁺dyshomeostasis. (A) Average locomotor activity of wild-type (blue) and slc39a14^−/− larvae (red) during the day and night in response to increasing concentrations of MnCl₂. Data are presented as mean±s.e.m. (two-way ANOVA with Tukey's posthoc test; *P<0.05), n=24 larvae per group. The y-axes represent the average activity of larval zebrafish in seconds per 10 minutes. (B) Frame-by-frame analysis of the locomotor activity of wild-type and slc39a14^−/− larvae at 6 dpf that were unexposed and exposed to 50 µM MnCl₂. White shading representing the day (lights ON) and grey shading represents the night (lights OFF). The arrow indicates the ‘lights OFF’ switch. Summed and smoothed Δ pixels traces (Delta Px) are shown as mean±s.e.m. (bold lines and shaded surrounding areas). n=24 larvae per group. (C) Calcium, magnesium and manganese concentrations determined by ICP-MS in untreated and MnCl₂ (50 µM)-treated wild-type and slc39a14^−/− larvae. Data are presented as mean±s.d. (one-way ANOVA with Tukey's post hoc test; *P<0.05; **P<0.01; ***P<0.001). (D) Apoptotic cell death upon MnCl₂ exposure in both wild-type (blue) and mutant (red) larvae at 5 dpf detected by TUNEL staining in the telencephalon, diencephalon and hindbrain. Data are presented as mean±s.d.

Exogenous Mn restores normal expression of genes that are differentially expressed in unexposed slc39a14^−/− mutants. (A) Heatmap of the expression of 266 genes with a significant difference between unexposed mutants and unexposed siblings. Each row represents a different gene and each column is a sample. The normalised counts for each gene have been mean centred and scaled by dividing by the standard deviation. (B) Plot of normalised counts for the add2 gene. Gene expression is decreased in both unexposed and MnCl₂-exposed mutant embryos. Unexposed sibling embryos are indicated in light blue and Mn-exposed embryos are in dark blue. Unexposed mutants are coloured light red and exposed mutants are dark red. (C) Plot of normalised counts for the pcdh7b gene. There were decreased counts in the unexposed mutant embryos that were rescued back to wild-type levels upon 50 µM MnCl₂ treatment. Colour scheme is as in B. FC, fold change. Wald test was used to determine significance in B,C. (D) Enrichment Map diagram of the enrichment of ZFA terms for the genes differentially expressed in unexposed mutants that are rescued by Mn treatment. Nodes represent enriched ZFA terms and edges connect nodes that share annotations to the significant genes. The width of each edge is proportional to amount of overlap, nodes are coloured by −log₁₀[adjusted P-value] (Wald test) and the size represents the number of significant genes annotated to the term. (E) ClueGO network diagram of the enrichment of GO terms associated with the genes that are rescued by Mn treatment. Nodes represent enriched GO terms and edges connect nodes that share annotations to the significant genes. Different components of the network are coloured according to the categories as labelled on the diagram. The sizes of the circles represent the adjusted P-values for each GO term as indicated on the right (Wald test).