Light-entrained chromatin priming poises rapid metamorphosis in a marine sponge
- Centre for Marine Science and School of the Environment, The University of Queensland, Australia
Figures
Figure 1 with 2 supplements
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, 5–6 hr 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 8029 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) Weighted gene co-expression network analysis (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 transcription factors (TFs) are shown. (J) Alluvial plot showing dynamics of 127 significantly differentially expressed TF genes as per G.
Figure 1—figure supplement 1
Larval and postlarval gene expression.
(A) Weighted gene co-expression network analysis (WGCNA) co-expression modules of genes expressed through larval and postlarval development. Number of coding genes and transcription factors (TFs) are shown. KEGG (B) and GO-MWU (C) analysis of WGCNA co-expression modules that comprise genes that are down- (blue, pink) and up- (red, yellow-green, tan) regulated at the start of metamorphosis. BP, biological process; CC, cellular component; and MF, molecular function. (D) Developmentally enriched GO-MWU terms (−log p-value; maximum of 10 shown) based on significantly upregulated genes at each developmental stage. (E, F) Scaled heatmaps of expression of TFs in larval cell types previously defined using scRNA-seq (Sebé-Pedrós et al., 2018). (E) TF genes significantly differentially expressed between precompetent and competent larval stages. (F) TF genes significantly differentially expressed between competent larval and 1 hps postlarval stages. Highly expressed TFs are in bold, and arrows in E and F show whether the TF is up- or downregulated between successive stages.
Figure 1—video 1
Time-lapse video of the first 1–2 hr of metamorphosis, beginning within 10 min of an A. queenslandica larva attaching to a fragment of the coralline alga A. fragilissima.
Images were obtained every 3 min for approximately 2 hr with each time point consisting of a z-stack of 14 images at an interval of 25 µm. The movie is 180x normal speed.
Figure 2 with 1 supplement
Dynamic and high-level expression of transcription factor (TF) 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 gene 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 among 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).
Figure 2—figure supplement 1
ATAC-seq analysis of A. queenslandica larval and early postlarval chromatin.
Analyses were performed on cells dissociated from three individual precompetent and competent larvae, and 1 and 6–7 hps postlarvae. (A) Insert size distribution of ATAC-seq libraries. Three biological replicates for each stage shown. (B) Bar graph of number of ATAC-seq peaks in each replicate. Developmental stages are colour-coded as per Figure 1. (C) Bar graph of the total number of open chromatin regions (OCRs) identified per developmental stage. (D) Bar graph of the number of ATAC-seq peaks shared between larval and postlarval stages. Most peaks are present in all four stages. (E) Bar graph of the number of ATAC-seq peaks shared between stages. (F) ATAC-seq FRiP scores for all replicates. Dotted lines, ENCODE standards (https://www.encodeproject.org/atac-seq/). (G) Boxplot of ATAC-seq peak insert size, calculated using the arithmetic mean across replicates for each stage. (H) Boxplot of ATAC-seq peak width size, calculated as per H. (I) Heatmaps of total ATAC-seq reads for each developmental stage within 1 kb up- and downstream of TSSs of all genes. (J) Normalised ATAC-seq read density from TSS to transcription end site (TES) and 5 kb up- and downstream for all genes in each replicate and stage, showing enrichment and depletion at TSSs and TESs, respectively. (K) Chromatin accessibility read density 1 kb up- and downstream of the TSS, by stage. Top panel plots the read density for three replicate ATAC-seq analyses for each larval and postlarval stage. Bottom panel shows the consensus plot for each stage. (L) The percentage of ATAC-seq peaks mapping to genomic features for larval and postlarval replicates. Promoter region is defined as a region within 500 bp of the TSS. Downstream is defined as ≤300 bp of the end of a gene. (M) The percentage of ATAC-seq peaks up- and downstream of the TSS for larval and postlarval replicates, partitioned into classes based on distance from the TSS.
Figure 3 with 1 supplement
Open chromatin regions (OCRs) associated with expressed genes are enriched for binding sites of highly expressed transcription factors (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. JASPAR 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 differentially accessible chromatin regions (DACRs) across larval development and early metamorphosis. Open and close, chromatin accessibility significantly increases and decreases, respectively.
Figure 3—figure supplement 1
Chromatin dynamics through larval and early postlarval development.
(A) PCA of the 3482 differentially accessible chromatin regions (DACRs) that significantly change in at least one larval or postlarval stage; n = 3 for all stages. (B) Hierarchical clustered heatmap of Pearson correlation of replicated larval and postlarval stages based on log10-transformed read counts of the 3482 DACRs. (C) Examples of the location and frequency of ATAC-seq reads mapped to the genome are shown and the regions of significant change are highlighted in grey. Scaffold locations of open chromatin regions (OCRs) are shown above and gene names below. Direction of transcription is depicted by chevrons and exons are depicted by black blocks. Below gene name is stage of development in which the gene is up- or downregulated, and the p-adjusted significance values of the OCRs and expression levels. PHAF1, Phagosome Assembly Factor 1; ACBD5, Acyl-CoA binding domain-containing 5; UVSSA, UV stimulated scaffold protein A; WASHC2C, WASH complex subunit 2C; PTPRK, Protein tyrosine phosphatase receptor type K. (D) Venn diagram of the OCRs unique to and shared between precompetent and competent larvae, and 1 and 6–7 hps postlarvae (Supplementary file 10). (E) Venn diagram of the conserved TFBMs unique to and shared between precompetent and competent larvae, and 1 and 6–7 hps postlarvae as determined by Homer (Supplementary file 10). (F–H) Analysis of TFBMs present in DACRs associated with the five weighted gene co-expression network analysis (WGCNA) modules whose genes change across the initiation of metamorphosis (Figure 1I and Supplementary file 11); module colours shown. (F) Upset plot of TFBMs unique to and shared between WGCNA modules. (G) Heatmap of the most common TFBMs shared across all modules. TFBMs that potentially interact with highly expressed transcription factors (TFs) is colour-coded by TF class as per Figure 2A; black, TFBMs of other expressed TFs. JASPAR code follows TF class/family name to the right. (H) Top, the two TFBMs that are significantly enriched in OCRs of all five WGCNA modules, with Homer p-values to the right. Bottom, three TFBMs enriched in OCRs associated with genes comprising blue and pink modules.
Figure 4
TFBMs enriched in dynamic open chromatin regions (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 transcription factor (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) TFBM 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.
Figure 5
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 transcription factors (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 open chromatin regions (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.
Figure 6
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 (1) the upregulation of genes encoding CLOCK and other bHLH–PAS transcription factors (TFs), and other TFs, and the downregulation of AP-1 and other bZIPs; and (2) a change in chromatin accessibility 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 presence of inductive algae nor initiate metamorphosis (Say and Degnan, 2020). Chromatin accessibility in light-exposed larvae decreases markedly and larvae move into an alternative physiological state and remain 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., 2021; Ueda et al., 2016). Newly settled postlarvae undergo large changes in chromatin accessibility and gene expression within the first hour of settling, repressing larval genes and activating multiple developmental signalling 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 hr 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.
Additional files
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Supplementary file 1
Information on CEL-seq2 libraries.
- https://cdn.elifesciences.org/articles/111060/elife-111060-supp1-v1.xlsx
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Supplementary file 2
Normalised and average CEL-seq2 counts for all expressed genes using DESeq2.
- https://cdn.elifesciences.org/articles/111060/elife-111060-supp2-v1.xlsx
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Supplementary file 3
Genes that are differentially expressed during larval development and early metamorphosis.
- https://cdn.elifesciences.org/articles/111060/elife-111060-supp3-v1.xlsx
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Supplementary file 4
KEGG enrichments for genes that are upregulated at each larval and early postlarval stage and GO-MWU enrichments of differentially expressed genes between successive stages.
- https://cdn.elifesciences.org/articles/111060/elife-111060-supp4-v1.xlsx
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Supplementary file 5
KEGG enrichments for genes in each WGCNA modules.
- https://cdn.elifesciences.org/articles/111060/elife-111060-supp5-v1.xlsx
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Supplementary file 6
TF genes that are expressed and differentially expressed during larval development and early metamorphosis.
- https://cdn.elifesciences.org/articles/111060/elife-111060-supp6-v1.xlsx
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Supplementary file 7
Raw and normalised MARS-seq counts (Sebé-Pedrós et al., 2018) of differentially expressed TF genes in larval cell types.
- https://cdn.elifesciences.org/articles/111060/elife-111060-supp7-v1.xlsx
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Supplementary file 8
Information on ATAC-seq libraries: sequencing reads, mapping ratios, peaks, and fractions of reads in called peak regions.
- https://cdn.elifesciences.org/articles/111060/elife-111060-supp8-v1.xlsx
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Supplementary file 9
Genomic location and TFBMs enrichment in OCRs present during larval development and early metamorphosis.
Larval enhancers and promoters inferred by ChIP-seq (Gaiti et al., 2017) overlapped with OCRs identified in precompetent larvae in the present study.
- https://cdn.elifesciences.org/articles/111060/elife-111060-supp9-v1.xlsx
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Supplementary file 10
OCRs and their TFBMs associated with expressed genes at all four stages.
- https://cdn.elifesciences.org/articles/111060/elife-111060-supp10-v1.xlsx
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Supplementary file 11
OCRs and their TFBMs associated with WGCNA modules.
- https://cdn.elifesciences.org/articles/111060/elife-111060-supp11-v1.xlsx
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Supplementary file 12
OCRs and their TFBMs associated with DEGs between precompetent and competent and competent and 1 hps.
- https://cdn.elifesciences.org/articles/111060/elife-111060-supp12-v1.xlsx
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Supplementary file 13
OCRs and their PAS related (bHLH) TFBMs associated with differentially expressed genes during larval development and early metamorphosis.
- https://cdn.elifesciences.org/articles/111060/elife-111060-supp13-v1.xlsx
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Supplementary file 14
OCRs and their Fos/Jun-related (bZIP) TFBMs associated with differentially expressed genes during larval development and early metamorphosis.
- https://cdn.elifesciences.org/articles/111060/elife-111060-supp14-v1.xlsx
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Supplementary file 15
OCRs and TFBMs found in and around CLOCK during larval development and early metamorphosis.
- https://cdn.elifesciences.org/articles/111060/elife-111060-supp15-v1.xlsx
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Supplementary file 16
OCRs and TFBMs found in and around Jun during larval development and early metamorphosis.
- https://cdn.elifesciences.org/articles/111060/elife-111060-supp16-v1.xlsx
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Supplementary file 17
OCRs and TFBMs found in and around Fos during larval development and early metamorphosis.
- https://cdn.elifesciences.org/articles/111060/elife-111060-supp17-v1.xlsx
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Supplementary file 18
Comparison of gene expression, chromatin state and accessible TFBMs in larvae exposed to natural and constant light.
- https://cdn.elifesciences.org/articles/111060/elife-111060-supp18-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/111060/elife-111060-mdarchecklist1-v1.docx
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Light-entrained chromatin priming poises rapid metamorphosis in a marine sponge
eLife 15:RP111060.
https://doi.org/10.7554/eLife.111060.3
