Gene expression during larval development and the initiation of sponge metamorphosis.

A-C, Photomicrographs of the first hour of metamorphosis after competent A. queenslandica larvae settle on the coralline alga A. fragilissima (Alga). L Ant and L Pos, larval anterior-posterior axis; P Api and P Bas, postlarval apical-basal axis; Ppr, posterior pigment ring; mps, minutes post-settlement; scale bar, 100 µm (see Supplementary Video 1). D, Timeline of larval and early postlarval developmental stages analysed using CEL-seq2 and ATAC-seq (see Methods). Larvae become competent to respond to an inductive cue associated with A. fragilissima just after sunset, 4-6 h after emerging from the adult sponge 7,18. E, Principal component analysis (PCA) of CEL-seq2 transcriptomes with 95% confidence level ellipses shown; n = 6 for each stage. F, Hierarchical clustered heatmap of Pearson correlation coefficients of replicated larval and postlarval transcriptomes based on DESeq2-normalised counts of the 8,029 significantly differentially expressed genes (DESeq2; p-adj < 0.1). G, Alluvial plot showing dynamics of differentially expressed genes through larval development and early metamorphosis. H, Top 10 significantly (FDR < 0.05) enriched KEGG pathways based on significantly upregulated genes at each developmental stage (Supplementary Table 4). FDR, false discovery rate. I, WGCNA co-expression modules that comprise genes that are down (blue, pink) and up (red, yellow-green, tan) regulated at the start of metamorphosis. Number of coding genes and TFs are shown. J, Alluvial plot showing dynamics of 127 significantly differentially expressed TF genes as per Fig. 1G. K, Scaled heatmap of 159 TF genes expressed during larval and postlarval development. The TFs that are within the top 5% of the most-highly expressed genes are annotated above the heatmap (Supplementary Table 6).

Open chromatin regions associated with expressed genes are enriched for binding sites of highly-expressed TFs.

A, The number of OCRs associated with protein-coding genes expressed in larval and postlarval stages compared to genes not expressed at these stages. B, The number of significantly up (↑) and down (↓) regulated genes that have nearby OCRs. Colour coding within the bars show at which stage the OCRs appear. C, Upset plot showing the distribution of the 173 TFBMs shared and unique to each larval and postlarval stage. D, Heatmap of the 43 TFBMs that are significantly enriched in all larval and postlarval stages. TFBMs that potentially interact with highly expressed TFs are colour-coded by TF class as per Fig. 1K; black, TFBMs of other expressed TFs. Jasper code follows TF class/family name to the right. E, Venn diagram of the differential enhancement of TFBMs in OCRs associated with genes that are differentially expressed between competent larvae and 1 hps postlarvae, with the TFBM class and family name size scaled to prevalence (Supplementary Table 12). F, Alluvial plot showing DACRs across larval development and early metamorphosis. Open and close, chromatin accessibility significantly increases and decreases, respectively.

TFBMs enriched in dynamic OCRs and in CLOCK, Jun and Fos.

A, TFBM families mapped to CLOCK proximal OCRs (coloured bars under ATAC-seq peaks) in larval and postlarval stages (Supplementary Table 15). The locations of the putative TFBMs are demarcated by a black bar and annotated at the stage at which the OCR first appears. TFBMs colour-coded in bold correspond to highly expressed TF families. Log (odds) scores are positively correlated with motif match confidence. Protein-coding gene models at the bottom. Arrows, TSSs; chevrons, direction of transcription; exons, thick lines; UTRs, intermediate lines; introns, thin lines. The expression profiles are above the three genes (DESeq2 normalised counts). B, The TFBM families in CLOCK OCRs with the highest matches with defined motifs (higher log-odds ratio being more likely a functional binding site). C, TFMB families mapped to Jun proximal OCRs (Supplementary Table 16). See description in a for details. D, The TFBM families in Jun OCRs with the highest matches. See description in B for details. E, F, TFBM families mapped to Fos proximal OCRs (Supplementary Table 17). See descriptions in A, B for details.

The effect of constant light on larval gene expression and chromatin state.

A, PCA showing the relationship of normal transcriptomes of precompetent and competent larvae (Fig. 1D), and larvae exposed to natural and constant light. B, Ten top KEGG categories in larvae exposed to natural and constant light (Supplementary Table 18). C, Volcano plot of TFs up- and downregulated in larvae exposed to constant light. D, Scaled heatmap of 25 TF genes that are normally differentially expressed between precompetent and competent larvae, compared to their expression in natural and constant light. TFs that are normally markedly upregulated in competent larvae but repressed by constant light are boxed. The TFs that are within the top 5% are in bold. E, Proximal CLOCK OCRs do not change in light-exposed larvae (green box), but a downstream chromatin region in a QSOX intron becomes more accessible in larvae exposed to constant light (tan box). TFBMs present in this OCR are shown as per Fig. 3. F, Model of the genomic regulatory and signalling processes at the acquisition of competence and early metamorphosis in A. queenslandica, and the environmental cues controlling these processes, combining results from this and previous studies 18,19,22.