Identification of 19 transcriptionally distinct cell types in the miracidium.

A) The miracidium is composed of ∼365 cells; i) DIC image of miracidium, ii) a Maximum Intensity Projection (MIP) of a confocal z-stack of DAPI-stained miracidium with nuclei segmented to enable counting (larval anterior pole at the top in all images). B) Experimental scheme describing the parasite’s life cycle (images of developmental stages not to scale), parasite dissociation, single-cell analysis of miracidium and validation pipeline. An average of 9975 miracidia per sample were dissociated; two samples were enriched for live cells (propidium iodide negative) using fluorescence-activated cell sorting (FACS), another two samples were unsorted. Cells were loaded according to the 10X Chromium single-cell 3’ protocol. Clustering was carried out to identify distinct populations and population-specific markers. Validation of population-specific markers was performed by in situ hybridisation (ISH). C) Uniform Manifold Approximation and Projection (UMAP) representation of 20,478 miracidium single cells. Cell clusters are coloured and distinctively labelled by cluster identity. D) Gene-expression profiles of the top population markers identified for each cell cluster (gene identifiers shown in parenthesis but with “Smp_” prefix removed for brevity). The colours represent the expression level from yellow (high expression) to dark blue (low expression). Gene expression has been log-normalised and scaled using Seurat(v. 4.3.0). The sizes of the circles represent the percentages of cells in those clusters that expressed a specific gene.

Orthogonal body wall muscles are transcriptionally distinct.

A) Dot plot depicting the expression profiles of specific- or enriched-marker genes in the two muscle clusters. Genes validated by in situ hybridisation (ISH) are marked in red. Gene identifiers shown in parenthesis but with “Smp_” prefix removed for brevity. Gene expression has been log-normalised and scaled using Seurat(v. 4.3.0). B) i-ii) Wholemount ISH (WISH) of paramyosin PRM+ and counterstaining with phalloidin reveals the nuclei of the circular body wall muscles (BWM), which form two distinct bilaterally symmetrical lines that run peripherally from the pole of the larva to the other (arrowheads in ii). The longitudinal BWM nuclei (ii - arrows) are spaced regularly between the lines of the circular BWM nuclei and their actin fibres run orthogonally to the circular muscles. These patterns were seen in 100% of individuals examined, n = 50. C) i) Segmentation of the PRM+ cells in one miracidium shows 74 muscle cells in total: ∼28 circular (segmented in orange), 33 longitudinal (yellow) and ii) 13 in an anterior unilateral cluster (inside dashed cyan line) adjacent to the apical gland cell (identifiable by its four nuclei, inside dashed red circle). D) Expression of markers for muscle 1 cluster; i) Kunitz-type protease inhibitor is expressed in circular BWM and wnt-11-1 is expressed in 7 Kunitz-type protease inhibitor+ cells at the posterior pole; ii) close-up of posterior end of another miracidium showing the co-expression of Kunitz-type protease inhibitor and wnt-11-1. In 100% of individuals examined, n = 30. E) i-iii) Notum, an inhibitor of wnt signalling, is expressed highly at the opposite pole to wnt-11-1 in the cluster of 13 muscle nuclei adjacent to the apical gland cell (nuclei in dashed red circle). In 100% of individuals examined, n = 30. F) Expression of markers for muscle 2 cluster; a calcium activated potassium channel (Smp_156150) and the transcription factor myoD (Smp_167400) show co-expression in longitudinal BWMs (arrowheads). In 100% of individuals examined, n = 30. Scale shown in B also applies to C, Di, E and Fi.

Neural complexity and ciliary plates in a simple larva.

A) Expression profiles of cell marker genes that are specific or enriched in the five neuronal clusters and ciliary plates. Genes validated by in situ hybridisation (ISH) are marked in red. Gene identifiers shown in parenthesis but with “Smp_” prefix removed for brevity. Gene expression has been log-normalised and scaled using Seurat(v. 4.3.0). Complexin (cpx) is expressed in all five nerve clusters and is a neural marker in the adult (Wendt et al., 2020). B) i) A dorso-ventral view of WISH of cpx shows expression in the brain, to the anterior - with projections around the three gland cells, and in two posterior clusters (100% of individuals examined, n = 50). ii) Segmentation of cpx+ cells (in magenta) in one miracidium (lateral view) reveals that at least 209 of the 365 cells that make up the larva are neural and that the clusters of cpx cells that are located posterior to the brain, and send projections to the posterior pole, sit perpendicular to the circular muscle cell nuclei (blue dashed lines). C) Multiplexed ISH for top markers for neuron 2 (Smp_201600) and neuron 3 (Smp_071050) shows expression in multiple cells of the brain, including cells adjacent to each other. Smp_071050 is also expressed in the apical gland cell (red dashed circle). 100% of individuals examined, n = 30. D) The top marker for neuron 4 Smp_319480 is expressed in four cells anteriorly (arrows) and ∼22 that sit lateral and just posterior to the brain; these later cells send projections into the brain and out to the body wall at the latitude between the 2nd and 3rd tier of ciliary plates (iii) seen here expressing the ciliary plate marker gene Smp_096390. 100% of individuals examined, n > 30. E) Neuron 5 marker Smp_343710, an EF-hand domain-containing protein, is expressed in (i) 10-20 cells whose nuclei sit outside of, and anterior to, the brain, in and around the paired lateral glands and their secretory ducts, and (Eii & iii, F) in a pair of bilaterally symmetrical bulbous protrusions. 100% of individuals examined, n = 30. F) Summary schematic of the in situ expression of marker genes for the neural cell clusters. G) i) A top marker for ciliary plates, a calcium binding protein (Smp_096390), shows transcripts expressed in all the ciliary plates of all four tiers (brackets) and in 6 cells towards the anterior pole. (ii & iii) Counterstaining with phalloidin shows that the nuclei of these anterior cells sit beneath the body wall muscle and send a projection externally between the 1st and 2nd tier of ciliary plates (arrowheads). 100% of individuals examined, n > 30. Scale shown in B also applies to C, D, Ei and Gi.

Identification of the tegument, protonephridia, parenchymal cells in the miracidium.

A) Expression profiles of cell marker genes specific or enriched in these cell type clusters. Genes validated by in situ hybridisation are in red. Gene identifiers shown in parenthesis but with “Smp_” prefix removed for brevity. Gene expression has been log-normalised and scaled using Seurat(v. 4.3.0). B Segmentation of nuclei of the cells expressing the marker genes for; i) protonephridia and tegument (multiplexed) show that 6 cells express Smp_335600 (ShKT-domain protein, a marker for protonephridia), 46 cells express Meg-6 (a marker for tegument) (segmentation of 1 larva), and ii) seven cells that express an uncharacterised gene, Smp_318890 (a marker for parenchymal cells)(segmentation of 1 larva). C & D) The tegument marker Meg-6 shows expression around nuclei in the posterior two-thirds of the larvae. The nuclei are below the body wall muscle, and cytoplasmic protrusions reach between muscle filaments (arrows) and form the epidermal ridges (arrowheads) between the ciliary plates (which are visible in Dii & iii expressing the ciliary plate marker Smp_096390) (asterisks). 100% of individuals examined, n > 30. E) The protonephridial marker Smp_335600 shows ‘S’-shaped expression with transcripts extending from the nucleus of the anterior flame cell (AFC) along the excretory tubule and its nucleus (ETN) to the posterior flame cell (PFC), Eii) it is expressed around the nuclei (arrow) rather than the barrels (arrowhead) of the flame cells. 100% of individuals examined, n > 30. Fi) The pan-parenchymal marker, Smp_318890, was expressed in 2 anterior cells (one on either side of the brain) and 5-7 cells posterior to the brain. Fii) These cells have long cytoplasmic protrusions that reach between all the other cells, Fiii) including the ago2-1+ stem cells. 100% of individuals examined, n > 30. Scale shown in B also applies to C, D, Ei and F.

Two defined populations of stem cells cluster by sex.

A) Expression profiles of cell marker genes that are specific or enriched in the stem cell clusters. Genes specific to the W (female-specific) sex chromosome are highlighted in blue. Genes validated by ISH are marked in red. Gene identifiers shown in parenthesis but with “Smp_” prefix removed for brevity. Gene expression has been log-normalised and scaled using Seurat(v. 4.3.0). B i) UMAP including all genes shows that there are two Delta/Phi and two Kappa stem clusters, ii) UMAP showing that removal of all genes specific to the W and Z sex chromosomes results in one Delta/Phi and one Kappa cluster, indicating that the stem cells are transcriptionally different in male and female miracidia due to the expression of sex-linked genes. C&D) Multiplexed ISH showing three stem cell markers simultaneously in the same individual: ago2-1 (Smp_179320) (pan-stem), p53 (Smp_139530) (Delta/Phi) and Uridine phosphorylase A (UPPA, Smp_308140) (Kappa). Ci) Ago2-1 expression reveals stem cells lateral and posterior to the brain (Optical section). 100% of individuals examined, n > 30. ii) MIP with segmentation of the 23 ago2-1+ cells. Di) P53 and UPPA expression shows the two stem cell populations intermingled in 100% of individuals examined, n > 30 ii) MIP and segmentation reveal there are more p53+ cells than UPPA+ cells (in this larva,15 are p53+ and 9 are UPPA+). Scale shown in C also applies to D.

RNA velocity analysis of stem and tegument cells from the miracidium and sporocyst show lineage-specific gene dynamics.

A) UMAP shows the life cycle stage origin of cells and cell cluster identity from Seurat analysis. B) RNA velocity analysis flow field show the generalised direction of RNA velocity. C) Latent time analysis shows an estimated temporal-relationship between cells. B) and C) are based on the expression of both spliced and unspliced transcripts, and their expression dynamics across cells and genes. The phase plot, velocity, and expression were calculated for D) p53-1, E) Zfp-1, F) p-53-2 and G) eled (no batch correction). Gene expression has been normalised (CPM) and log-transformed using scvelo(v. 0.2.4) For all of D) - G), the phase plot (left plot) shows the proportion of spliced and unspliced transcripts in each cell, where each point is a cell and is coloured by the clusters in B). The purple almond-shape overlaid represents the processes of transcription, splicing, and degradation, where this can be modelled. The dashed line shows the estimated steady state where RNA transcription is constant. The middle panel shows the RNA velocity, which for each gene is based on how the observations deviated from the estimated steady state towards induction or repression. The right panel shows gene expression. p53-1 and zfp-1 are predominantly expressed in the Delta/Phi-like miracidia stem cells and some miracidia tegument, and velocity indicates active expression of p53-1 but downregulation of zfp-1 in the stem cells. p53-2 is most highly expressed in the Kappa-like miracidia stem cells, and velocity indicates this gene is being actively transcribed. eled expression is very low and only spliced transcripts have been detected, but is generally restricted to the Kappa-like miracidia stem cells.

A model for the fate of the two stem populations in the miracidium.

Adding single-cell data for miracidia stem cells (this study) to existing stem cell scenarios on Schistosoma mansoni development (Wang et al., 2018; Li et al., 2021) shows the continuum of the Kappa (κ) population from the miracidium, through the intra-molluscan stages to the juvenile and adult stages inside the mammalian host. Wang et al. (2018) proposed that the κ cells give rise to epsilon (ε), eled+, cells in the juvenile primordial testes, ovaries, and vitellaria (germline), as well as in a gradient increasing toward the posterior growth zone (soma). They suggested that germ cells may be derived from ε-cells early in juvenile development, and eled is the earliest germline marker yet identified in schistosomes. This led to the idea that S. mansoni does not specify its germline until the juvenile stage (Wang et al., 2018) and a germline-specific regulatory program (including eled, oc-1, akkn, nanos-1, boule) was identified in intra-mammalian stages (Wang et al., 2018; Li et al., 2021). We show expression of these genes in κ stem cells in the miracidium. This suggests that after ∼6 days of embryogenesis, at hatching of the miracidium, the cells that may contribute to the germline might already be segregated into the κ population and the molecular regulatory program that differentiates somatic (delta/phi, δ/φ) and germ cell (κ) lineages is present. Furthermore, as p53-2 plays a genotoxic stress response role in adult reproductive cells (Wendt et al., 2022), its expression in κ cells in the miracidia is another line of evidence that indicates that this population may contain the pluripotent stem cells that likely give rise to the germline.

Tissue level segmentation of a miracidium reveals the contribution of each tissue to this simple larva.

In situ hybridisation using HCR (hybridisation chain reaction) enabled multiple tissue-level marker genes to be visualised simultaneously in the same larva. The nuclei of the cells of each tissue type were manually segmented using TrakEM2 in ImageJ (Cardona et al., 2012).