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 Figure 1 - video 1) (Blard et al., 2026). 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 (Degnan and Degnan, 2010; Say and Degnan, 2020). 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 enriched KEGG pathways (FDR < 0.05) based on significantly upregulated genes at each developmental stage; GO-MWU analysis of upregulated genes reveal stage-specific enrichments largely consistent with the KEGG analysis (Figure 1 – figure supplement 1; Supplementary file 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 Figure 1G.

Dynamic and high-level expression of transcription factor genes during larval development and early metamorphosis.

A, Scaled heatmap of 159 TF genes expressed during larval and postlarval development, of which 127 are significantly differentially expressed between at least two successive stages. The 40 most highly expressed TFs, all of which are among the top 5% of the most-highly expressed genes, are annotated to the right of the heatmap. TF genes names are followed by their gene family name, which is colour-coded into families; this colour- coding is used in all figures. B, Lists of TF genes in the order of their level of expression for all larval and postlarval stages. TFs in the pink and green boxes are amongst the top 1 and 5% of all expressed genes, respectively (Supplementary file 6). C, Scaled heatmap of the 40 most highly expressed TFs in larval cell types (Sebé-Pedrós et al., 2018). External epithelial cells are to the left - cell types ordered left-to-right based on their enrichment level along the anterior-posterior (A-P) axis; internal archaeocyte-like cells are to the right (Supplementary file 7).

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. On average, there are 16,469 and 26,177 genes expressed and not expressed at these four stages, respectively (Supplementary file 10). 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 first 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 Figure 2A; 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 file 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 file 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 file 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 file 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 (Figure 1D), and larvae exposed to natural and constant light. B, Ten top KEGG categories in larvae exposed to natural and constant light (Supplementary file 18). C, Volcano plot of TFs up- and downregulated in larvae exposed to constant light; bolded TFs are expressed in the top 5% of all genes. 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 (bottom). The TFs that are within the top 5% are in bold. E, Proximal CLOCK OCRs do not change in light-exposed larvae (green box; see Figure 4A), 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 Figure 4.

Environmental cues, genomic regulatory processes and signalling events underlying larval competence and early metamorphosis in A. queenslandica.

This model combines results from this and previous studies (Nakanishi et al., 2015; Say and Degnan, 2020; Ueda et al., 2016). A, Pre-competent larvae emerge from adults early in the afternoon, then swim in the water column for several hours before becoming competent to settle and initiate metamorphosis immediately after sunset (Degnan and Degnan, 2010; Say and Degnan, 2020). With the acquisition of competence is: (i) the upregulation of genes encoding CLOCK and other bHLH–PAS TFs, and other TFs, and the downregulation of AP-1 and other bZIPs; and (ii) a change in chromatin accessability in the vicinity of genes that are differentially expressed at competence and in the first hour of metamorphosis. B, Larvae prevented from experiencing sunset do not settle in the presense of inductive algae nor initiate metamorphosis (Say and Degnan, 2020). Chromatin accessibility in light-exposed larvae decreases markedly and larvae move into a alternative state that allow for prolonged periods in the plankton. This includes the repression of over 40 TF genes, including CLOCK and HIF. C, A cue on the surface of the alga A. fragilissima induces an internal signalling cascade that includes activation of calcium (Ca2+), nitric oxide (NO-cGMP) and MAPK pathways; larvae exposed to pharmacological agents that inhibit these pathways do not settle and metamorphose (Nakanishi et al., 2015; Song et al., 2020; Ueda et al., 2016). Newly settled postlarvae undergo large changes in chromatin accessability and gene expression within the first hour of settling, repressing larval genes and activating multiple developmental signallng pathways and over 60 TFs, including AP-1 and other potential partner bZIP TFs, and other conserved Immediate-early (IE) TFs. During this first hour there are rapid and extensive morphogenetic changes with multiple cell types being reprogrammed (Blard et al., 2026). D, 5-6 hour later, most developmental TF and signalling genes are downregulated, and diverse metabolic genes are activated, indicative of an overall shift in postlarval developmental and physiological state.