Fig. S5

Transcriptional responses to elevated temperature tend to be cell-type-specific, revealing burdens on proteostasis in the notochord, related to Figure 4 (A) UMAP of all genes with significant temperature-dependent transcriptional changes (see STAR Methods). Each point represents an individual gene, and genes with similar responses (coefficient in differential expression model after accounting for stage) across cell types are grouped together. Each cluster is labeled with a different color, and a representative Gene Ontology (GO) term for the group of genes in each cluster is indicated. Clusters in which gene groups returned no significant GO terms are labeled “NA.” (B) Heatmap of expression levels for each gene module in (A) as an average for each cell type at elevated temperatures. Consistent with the heterogeneous grouping of temperature responses in (A), elevated module expression tends to be cell-type specific (examples boxed). Module 5, which contains genes related to translation and ER processing, has the broadest response across cell types, but the magnitude of the response is greatest within the notochord. (C) Scatterplot showing levels of UPR signature in the notochord cells of all individual embryos, colored by temperature. Embryos raised at higher temperature show higher levels of UPR signature at nearly all profiled stages. Lines and ribbons are derived from LOESS fits across pseudostage. (D) Boxplot showing marked reduction of notochord cells in embryos raised at high temperature, with raw counts in control and temperature-treated embryos shown as poi€. (E) Boxplot showing the effect of the UPR inhibitor ISRIB on whole-embryo stage at 28°C and 34°C; embryos treated with ISRIB showed significant increases over expected temperature-induced acceleration at 24 hpf, but this effect was not evident in 48 hpf embryos. (F) Hotspot analysis, as in Figure 4D, showing enriched transcriptional state of notochord sheath cells in embryos treated with ISRIB; note that ISRIB-treated cells do not occupy the high-temperature state identified in wild-type embryos. (G) TEM of sheath cells in wild-type and atf6 crispants showing effects in both untreated and temperature-treated embryos. ER structure is perturbed in atf6 crispants, and severe sheath defects are visible in the temperature-treated atf6 crispants. Scale bar, 200 nm.