Single-cell atlases of two lophotrochozoan larvae highlight their complex evolutionary histories

Abstract

Pelagic larval stages are widespread across animals, yet it is unclear whether larvae were present in the last common ancestor of animals or whether they evolved multiple times due to common selective pressures. Many marine larvae are at least superficially similar; they are small, swim through the beating of bands of cilia, and sense the environment with an apical organ. To understand these similarities, we have generated single-cell atlases for marine larvae from two animal phyla and have compared their cell types. We found clear similarities among ciliary band cells and between neurons of the apical organ in the two larvae pointing to possible homology of these structures, suggesting a single origin of larvae within Spiralia. We also find several clade-specific innovations in each larva, including distinct myocytes and shell gland cells in the oyster larva. Oyster shell gland cells express many recently evolved genes that have made previous gene age estimates for the origin of trochophore larvae too young.

Fig. 1.. Larvae are a common feature of Metazoa.

(A) A phylogeny of animals shows the widespread presence of ciliated larvae, especially among the superphylum Lophotrochozoa (Loph.). Black dots, character presence; white dots, absence; gray dots, unconfirmed. (B and C) Schematics of larvae presented in this study, (B) in blue box the trochophore larva of the Pacific oyster Crassostrea gigas, represented as both ventral (V) and lateral (L) views. C. gigas larvae at the trochophore stage lack an apical tuft of sensory cilia and paired protonephridia. (C) In yellow, schematics of the Müller’s larva of the polyclad flatworm Prostheceraeus crozieri depicted as ventral and lateral views.

Fig. 2.. Cell atlas of the trochophore larva of C. gigas.

(A) Two-dimensional uniform manifold approximation and projection (UMAP) showing cell clusters for the oyster larva. (B) Dotplot of marker gene expression for different cell clusters: yellow highlights the shell gland clusters, pink highlights the neuronal clusters, red highlights the myocyte clusters, and blue highlights the ciliary clusters. Dotplots show expression of genes (x axis) in each cell cluster (y axis) of the oyster scRNA-seq. Shades of blue indicate average expression, and the size of dots indicates the percentage of cells expressing the gene. (C) ISH of the marker genes shown in (B) with schematic of expression in the larva (color of the square indicates which gene/s was/were used for each schematic). A, apical view; P, posterior view; V, ventral view; D, dorsal view; L, lateral view with mouth on the right. HCR expression without 4′,6-diamidino-2-phenylindole in a larger format is available in fig S1. Scale bar, 50 μm.

Fig. 3.. Gene age analyses in different cell types of the oyster larva show that shell gland cells have a young gene signature.

(A) Transcriptome age indices (TAI) for different cell types; smaller TAI values correspond to “older” gene ages. Gene age is inferred using a phylostratigraphy approach; the TAI is then calculated on the log-transformed gene average expression per cluster. (B) Heatmap showing enrichment test −log₁₀(P value) for marker genes phylostrata per cell type in the oyster. Enrichment was computed using a hypergeometric test applied to the number of marker genes in each cluster per phylostrata compared to the global set of expressed genes.

Fig. 4.. Single-cell atlas of the Müller’s larva of P. crozieri.

(A) UMAP showing cell clusters for the polyclad flatworm larva. (B) Dotplot of marker gene expression for cell clusters: yellow highlights the gut clusters, pink highlights the neuronal clusters, red highlights the myocyte clusters, and blue highlights the ciliary clusters. Dotplot graphs show expression of genes (x axis) in each cell cluster (y axis) of the flatworm scRNA-seq: Shades of blue indicate average expression, and the size of the dots indicates the percentage of cells expressing the gene. (C) HCR of the marker genes shown in (B) with schematics of their expression in the larva (color of the square indicates which gene/s was/were used for schematic). A, apical view; P, posterior view; V, ventral view; D, dorsal view; L, lateral view with mouth on the left. Images of HCR expression without 4′,6-diamidino-2-phenylindole and in larger format is available in fig S9. Scale bar is 50 μm.

Fig. 5.. The complexity of the nervous system of the polyclad flatworm larva.

(A) HCR staining of cluster markers and neuropeptides (NPs). (B) HCRs with a focus on apical organ neurons and (C) schematic drawing of the Müller’s larva apical organ. All HCR stainings are maximum projections. DAPI, 4′,6-diamidino-2-phenylindole; A, apical view; P, posterior view; V, ventral view; D, dorsal view; L, lateral view with mouth on the left; ee, epidermal eye; ce, cerebral eye. Scale bars, 50 μm.

Fig. 6.. SAMap cell clusters alignment scores between invertebrate larvae.

(A) Mapping of cell clusters between the trochophore larva of the Pacific oyster (Cg) and the Müller larva of polyclad flatworm (Pc). (B) Mapping of cell clusters between the trochophore larva of the Pacific oyster (Cg) and the pluteus larva of the sea urchin Strongylocentrotus purpuratus (Sp). (C) Mapping of cell types between the Müller larva of polyclad flatworm (Pc) and the pluteus larva of sea urchin (Sp). Alignment scores are defined as the average number of mutual nearest cross-species neighbors of each cell relative to the maximum possible number of neighbors (13).