Cell fate specification modes shape transcriptome evolution in the highly conserved spiral cleavage

Abstract

Early animal development can be remarkably variable, influenced by lineage-specific reproductive strategies and adaptations. Yet, early embryogenesis is also strikingly conserved in certain groups, such as Spiralia. In this clade, a shared cleavage program (i.e., spiral cleavage) and similar cell lineages are ancestral to at least seven phyla. Why early development is so conserved in specific groups and plastic in others is not fully understood. Here, we investigated two annelid species (Owenia fusiformis and Capitella teleta) with spiral cleavage but different modes of specifying their primary progenitor cells. By generating high-resolution transcriptomic time courses from the oocyte to gastrulation, we demonstrate that transcriptional dynamics differ markedly between these species during spiral cleavage and instead reflect their distinct timings of embryonic organiser specification. However, the end of cleavage and gastrulation exhibit high transcriptomic similarity, when orthologous transcription factors share gene expression domains, suggesting this period is a previously overlooked mid-developmental transition in annelid embryogenesis. Together, our data reveal hidden transcriptomic plasticity during spiral cleavage, indicating an evolutionary decoupling of morphological and transcriptomic conservation during early embryogenesis.

Keywords: Annelida; Maternal-to-Zygotic Transition; Phylotypic Stage; Spiral Cleavage; Zygotic Genome Activation.

Figure 1. Equal and unequal spiral cleavage in two annelid species.

(A, D) Schematic drawings of equal (or conditional) (A) and unequal (or autonomous) (D) spiral cleavage as exemplified by the early development of the annelids O. fusiformis (B) and C. teleta (E). The drawings highlight the two different types of first zygotic divisions, the idiosyncratic spiral arrangement of the animal and vegetal blastomeres at the 8-cell stage, the different timings of embryonic organiser specification, and distinct gastrula morphologies in embryos that will develop into planktotrophic (O. fusiformis) and lecithotrophic (C. teleta) larvae. (C, F) Z-projections of confocal stacks of oocytes and embryos at the exact stages collected for transcriptomic profiling in O. fusiformis (C) and C. teleta (F). Samples are stained against tubulin and actin (both in grey) to reveal cell membranes and contours. In C. teleta (F), the blastomere lineages at the 2-, 4- and 8-cell stages are highlighted. The cell acting as an embryonic organiser (the 4d micromere in O. fusiformis and the 2d blastomere in C. teleta) is false-coloured in red at the 5 hpf and 16-cell stage, respectively. Drawings are not to scale, and asterisks indicate the animal/anterior pole. bc blastocoele, bp blastopore, hpf hours post-fertilisation, pb polar bodies. In (C, F), scale bars are 50 μm. (C, F) All images come from representative specimens of at least two biological replicates. (E) Copyright 2025 by François Michonneau (CC-BY 4.0). Source data are available online for this figure.

Figure 2. Global transcriptional dynamics during conditional and autonomous spiral cleavage.

(A, B) Principal component analyses support that developmental time accounts for the most considerable fraction of the variance in the transcriptomic time courses during spiral cleavage in O. fusiformis and C. teleta. (C) Similarity clustering reveals three primary sample groups during spiral cleavage in both species. (D) O. fusiformis and C. teleta show different dynamics in the total number of expressed genes during spiral cleavage, which align with the different timings of organiser specification in these species (highlighted by a yellow and green bar, respectively). (E, F) Bar plots depicting the number of differentially expressed genes (upregulated in red and downregulated in blue) during spiral cleavage in O. fusiformis (E) and C. teleta (F). The earliest signs of zygotic genome activation are highlighted with a yellow and green bar, respectively.

Figure 3. The zygotic genome activation in O. fusiformis and C. teleta.

(A, B) Z-projections of confocal stacks showing the morphological phenotype of 24 h post-fertilisation O. fusiformis larvae (A) and stage 5 C. teleta larvae (B) after windows of actinomycin D treatment during early cleavage. Inhibition of zygotic transcription from the 16-cell stage on compromises normal embryogenesis in the two annelids. However, in O. fusiformis, there is cellular differentiation between 3 to 5 hpf (white arrows), and in both annelids, the most severe phenotypes appear when inhibiting zygotic genome activation during the specification of the embryonic organiser and major wave of differential gene expression (grey background). (C, D) Volcano plots indicating up- and downregulated genes in the 8-cell to 16-cell transition in O. fusiformis (C) and C. teleta (D). Only two one-to-one orthologs (fjx1 and jub) are differentially expressed at this stage in both species. LFC stands for log-fold change. P-values in (C, D) were derived from the described DESeq2 pipeline (Wald test) and Benjamini–Hochberg-adjusted. (E, F) Whole-mount in situ hybridisation of jbx1 from the 16-cell stage (or 3 hpf in O. fusiformis) until gastrulation in O. fusiformis (E) and C. teleta (F). In O. fusiformis, jbx1 is asymmetrically expressed in the anterior ectoderm. In C. teleta, jbx1 is broadly expressed during cleavage and asymmetrically localised in the anterior neuroectoderm and endoderm in the gastrula. All images come from representative specimens of at least two biological replicates. Asterisks in (E, F) indicate the animal pole. ao apical organ, bc blastocoele, bp blastopore, br brain, cb ciliary band, gp gastral plate, mo mouth. Source data are available online for this figure.

Figure 4. Distinct transcriptomic routes during spiral cleavage in the annelids O. fusiformis and C. teleta.

(A) Jensen-Shannon distance represents the transcriptomic divergence during the spiral cleavage of O. fusiformis and C. teleta. The point of maximal transcriptomic similarity between these annelids occurs at the late cleavage and gastrulation. (B, C) Soft k-means clustering of temporally coexpressed genes in O. fusiformis (B) and C. teleta (C). In both species, the first two clusters likely represent maternal transcripts that decay early, while the rest largely or entirely comprise zygotically expressed genes. Unlike O. fusiformis, C. teleta has cleavage-specific clusters at the 16-cell, 32-cell and 64-cell stages. (D) Comparison of gene family cluster composition between O. fusiformis and C. teleta. Although gene-specific transcriptional dynamics are dissimilar, the deployment of gene families in maternal, early zygotic, and embryonic clusters is similar between these annelids. P values were derived from upper-tail hypergeometric tests and Benjamini–Hochberg-adjusted (adj. P value). (E) Alluvial plot depicting the comparative deployment of one-to-one orthologs (n = 7607) in clusters of temporally coexpressed genes during spiral cleavage in O. fusiformis and C. teleta. The embryonic clusters exhibit the most extensive conservation of gene composition between species. (F) Gene Ontology (GO) enrichment of shared orthologs between oocyte/zygote, maternal decay, early zygotic, and embryonic clusters, according to the GO annotation of O. fusiformis. While oocyte/zygotic genes are involved in metabolism, shared orthologs of maternal decay are involved in the cell cycle and cellular organisation, and early zygotic and embryonic genes are involved in transcriptional control and development. P values were computed from upper-tail Fisher’s exact tests to detect overrepresented terms.

Figure 5. The comparative dynamics of transcription factor activation in conditional and autonomous annelids.

(A, B) Nightingale rose charts of transcription factor (TF) distribution according to developmental time points (A) and clusters of coexpressed genes (B). (C) Comparison of gene family cluster composition between O. fusiformis and C. teleta. TF similarity in clusters of genes showing maternal decay and embryonic expression is high. However, there are prominent shifts in TF gene families allocated to the embryonic cluster in one species to earlier clusters in the other. (D) Alluvial plot depicting the comparative deployment of one-to-one orthologs (n = 306) exhibiting shifts in temporal activation (TPM >2) during spiral cleavage in O. fusiformis and C. teleta. (E) Pie chart showing that most genes with a temporal shift between O. fusiformis and C. teleta are expressed earlier in the latter. (F) The bar plots indicate the allocation of genes with a temporal shift to the clusters of coregulated genes in O. fusiformis and C. teleta. Most genes exhibiting temporal shifts between these species belong to late clusters in O. fusiformis and shift towards earlier clusters in C. teleta. (G) Gene Ontology (GO) enrichment of one-to-one orthologs (n = 306) exhibiting temporal shifts in transcriptional activation between O. fusiformis and C. teleta, according to the GO annotation of C. teleta. P values were derived from upper-tail Fisher’s exact tests.

Figure 6. Spatial expression dynamics of orthologous transcription factors in O. fusiformis and C. teleta.

(A–N) Whole-mount in situ hybridisation of seven orthologous transcription factors (pax2/5/8, tbx2/3, vsx2, uncx, AP2, HNF4, and prop1) during mid and late cleavage and at the gastrula stage in O. fusiformis (A–G) and C. teleta (H–N). In O. fusiformis, pax2/5/8 (A), tbx2/3 (B), vsx2 (C) and uncx (D) are expressed in the anterior neuroectoderm (arrowheads), as well as in the posterior blastoporal rim (pax2/5/8 and uncx, arrows) and endoderm (tbx2/3 and vsx2). AP2 (E) is expressed in the posterior ectoderm (arrowhead), and HNF4 (F) and prop1 (G) are in the gastral plate before gastrulation and in seven equatorial clusters of two ectodermal cells in the gastrula (prop1, arrowheads). No expression is detected before 5 hpf for any of these genes. In C. teleta, pax2/5/8 (H), tbx2/3 (I), vsx2 (J), uncx (K) and AP2 (L) are broadly expressed during mid and late cleavage. In the gastrula, pax2/5/8, tbx2/3, vsx2 and uncx are detected in the anterior neuroectoderm (arrowheads), four cell clusters posterior to the foregut (pax2/5/8, arrows), the endoderm (tbx2/3 and uncx) and two lateral mesodermal clusters (uncx, arrows). HNF4 (M) is broadly detected at 32- and 64-cells and in the endoderm of the gastrula, and prop1 (N) expression is only apparent at the 64-cell stage. Insets are animal/vegetal views, except for the gastrula stage in (H–N), which are lateral views. All images come from representative specimens of at least two biological replicates. Asterisks indicate the animal pole. bc blastocoele, bp blastopore, en endoderm, gp gastral plate.

Figure 7. A mid-developmental transition in spiral cleavage.

(A, B) Jensen-Shannon transcriptomic divergence between all possible inter-species pairwise comparisons during the entire life cycle, from oocyte to juvenile or competent larva of O. fusiformis and C. teleta (A) and O. fusiformis and C. gigas (B). The point of maximal transcriptomic similarity between these annelids occurs at the late cleavage and gastrulation, while it happens at the ciliated larval stage between O. fusiformis and C. gigas. (C, D) Bar plots indicate the distribution of expressed genes according to their age or phylostratum at each cluster of temporally coregulated genes in O. fusiformis (C) and C. teleta (D). More ancestral genes comprise around half of the transcriptome during spiral cleavage in both species, except in the stage-specific clusters of C. teleta at the 16-cell, 32-cell, and 64-cell stages. (E, F) The heatmaps display the stages with the highest gene expression, categorised by age. In O. fusiformis (E), metazoan, protostomian, and lophotrochozoan genes are more expressed at the 5 hpf and gastrula, while in C. teleta (F), metazoan and bilaterian genes are highly expressed at the gastrula stage.

Figure 8. Transcriptional and morphological dynamics are decoupled in spiral cleavage.

(A) Schematic summary of the transcriptomic dynamics during spiral cleavage in O. fusiformis and C. teleta. Two waves of mRNA decay (one that is oocyte/zygote-specific and another that extends to about the 8-cell stage) occur during early cleavage. Zygotic genome activation and the maternal-to-zygotic transition (MZT) likely occur between the second (4-cell stage) and fourth cell division (16-cell stage) and more intensely in C. teleta, as it coincides with the specification of the embryonic organiser (schematic drawing and dotted vertical line). The large increase in zygotic transcriptional activity occurs at the sixth cell division (64-cell stage) in O. fusiformis, when the embryonic organiser is established in this species (dotted line and schematic drawing). (B) Schematic comparison of broad areas of developmental competence at the gastrula stage between O. fusiformis and C. teleta (see main text for details). (C) Differently from other animal phyla, morphological and transcriptomic similarities are uncoupled during development in Annelida. The early embryonic phase of spiral cleavage is morphologically stereotypical but transcriptionally divergent. However, annelid embryos converge into a broad transcriptomic and molecular patterning similarity phase by the gastrula stage, which acts as a mid-developmental transitional period. Annelid embryos diverge (morphologically and transcriptionally) upon gastrulation as they develop into lineage-specific larval forms. Drawings are not to scale.

Figure EV1. Dynamics of RNA polymerase II nuclearisation during spiral cleavage.

(A, C) Z-projections of confocal stacks of embryos of O. fusiformis (A) and C. teleta (C) from the 2-cell stage to 4 h post-fertilisation (hpf) or the 32-cell stage. RNA polymerase II localises to the nuclei from the 4-cell stage onwards in both annelids. In C. teleta, the nuclearisation is more intense in the C and D blastomeres than in the A and B cells. (B, D) Expression dynamics of the RPB1 gene (largest subunit of the RNA polymerase II, recognised by the antibody used in A and C) in O. fusiformis (B) and C. teleta (D). In the two annelids, RPB1 is a highly abundant maternal gene. Gene expression values are the average of two biological replicates.

Figure EV2. Transcriptomic dynamics during spiral cleavage in Annelida.

(A, B) Jensen-Shannon transcriptomic divergence during the spiral cleavage between O. fusiformis (A) and C. teleta (B) and four other annelid species with publicly available transcriptomic resources covering at least one cleavage stage and the gastrula stage. In all cases, the point of maximal transcriptomic similarity occurs at the late cleavage and gastrulation (grey horizontal bar).

Figure EV3. The comparison of clusters of temporally coregulated genes between conditional and autonomous spiral cleavage.

(A) Upset plot indicating the number of shared one-to-one orthologs between clusters of temporally coregulated genes. The inset indicates the total number of genes in each cluster. (B–F) Bar plots indicating the top ten Gene Ontology (GO) categories amongst shared orthologous genes in the oocyte/zygote, maternal decay, early zygotic, and embryonic clusters, according to the GO annotation for the C. teleta ortholog. P values were computed from upper-tail Fisher’s exact tests.

Figure EV4. Heterochronic shifts in gene expression between selected molluscan and annelid species.

(A–F) Alluvial plots depicting the comparative deployment of one-to-one orthologs exhibiting shifts in temporal activation during spiral cleavage between O. fusiformis and P. dumerilii (A), O. fusiformis and U. unicinctus (B), C. teleta and P. dumerilii (C), C. teleta and U. unicinctus (D), O. fusiformis and C. gigas (E), and C. teleta and C. gigas (F). Generally, more genes shift from late cleavage stages in O. fusiformis to early stages in other species than when C. teleta is included in equivalent comparisons.

Figure EV5. Transcriptional similarity between Molluscan and Annelid development.

(A–F) Jensen-Shannon transcriptomic divergence between all possible inter-species pairwise comparisons during the entire life cycle, from oocyte or cleavage to juvenile or competent larva, between molluscan and annelid species with a high-resolution time course. In intra-phylum comparisons, the stages of maximal similarity are at or around gastrulation. In contrast, in inter-phylum comparisons, the larval stages are more transcriptionally similar (except for the O. fusiformis versus Haliotis discus hannai comparisons, which might be due to the poor quality of the molluscan dataset).