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Front Cell Dev Biol
2022 Sep 15;10:991664. doi: 10.3389/fcell.2022.991664.
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Integrating single cell transcriptomics and volume electron microscopy confirms the presence of pancreatic acinar-like cells in sea urchins.
Paganos P
,
Ronchi P
,
Carl J
,
Mizzon G
,
Martinez P
,
Benvenuto G
,
Arnone MI
.
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The identity and function of a given cell type relies on the differential expression of gene batteries that promote diverse phenotypes and functional specificities. Therefore, the identification of the molecular and morphological fingerprints of cell types across taxa is essential for untangling their evolution. Here we use a multidisciplinary approach to identify the molecular and morphological features of an exocrine, pancreas-like cell type harbored within the sea urchin larval gut. Using single cell transcriptomics, we identify various cell populations with a pancreatic-like molecular fingerprint that are enriched within the S. purpuratus larva digestive tract. Among these, in the region where they reside, the midgut/stomach domain, we find that populations of exocrine pancreas-like cells have a unique regulatory wiring distinct from the rest the of the cell types of the same region. Furthermore, Serial Block-face scanning Electron Microscopy (SBEM) of the exocrine cells shows that this reported molecular diversity is associated to distinct morphological features that reflect the physiological and functional properties of this cell type. Therefore, we propose that these sea urchin exocrine cells are homologous to the well-known mammalian pancreatic acinar cells and thus we trace the origin of this particular cell type to the time of deuterostome diversification. Overall, our approach allows a thorough characterization of a complex cell type and shows how both the transcriptomic and morphological information contribute to disentangling the evolution of cell types and organs such as the pancreatic cells and pancreas.
FIGURE 1. Pancreatic-like molecular signature of the S. purpuratus larva cell type families. (A) Uniform Manifold Approximation and Projection (UMAP) showing the 3 dpf S. purpuratus cell type families (modified from Paganos et al., 2021). (B) X-ray Microtomography (MicroCT) of the 3 dpf S. purpuratus pluteus larva, placed in lateral view, in which the different cell type families are labelled with pseudo-coloring. Color code is the same as the one used in (A). (C) Simplified schematic representation of pancreatic gene markers known to be present in specific stages of pancreatic development and distinct pancreatic lineages. (D) Dotplot showing the average score of the sea urchin pancreatic marker orthologs shown in (C) across the larval cell type families. (E) Dotplot showing the average expression of the pancreatic markers across the larval cell type families. Color code in (E) is the same as the one used in (C).
FIGURE 2. Distribution of endocrine and exocrine pancreas-like markers across the digestive tract. (A) Summary of the pipeline used to increase the resolution of the digestive tract cell type composition. Digestive tract related cell type families are highlighted in yellow (left), subsetted and subclustered resulting in the generation of 15 clusters (right). (B) Dotplot showing the average expression of pancreatic gene markers across the digestive tract subclustered dataset. Colored boxes are used to highlight endocrine-like (green) and exocrine-like (magenta) molecular signatures. (C–J) FISH using antisense probes designed against several of the pancreatic markers used in (B). Color code of the signal is the same as in (B). DAPI was used to label the nuclei (gray). (K) MicroCT of the 3 dpf S. purpuratus pluteus larva, placed in lateral view, in which the different pancreatic-like molecular fingerprints found in the digestive tract are labelled with pseudo-coloring. Color code is the same as the one using in (B). A, Anus; Es, Esophagus; Cs, Cardiac sphincter; Epl: Exocrine pancreas-like; In, Intestine; Ps, Pyloric sphincter; St, stomach.
FIGURE 3. Molecular characterization of the S. purpuratus larva exocrine pancreas-like cells. (A) FISH using antisense probes against Sp-Ptf1a (A1) and Sp-Cpa2L (A2–4) at gastrula and various larval stages, respectively. Contour of the digestive tract compartment (gray) and the double belt row structure (magenta) are shown in dotted lines. Arrowheads indicate the position of the presumptive (A1) and the actual cardiac sphincter (A2–4). Specimens in A1, A2, and A4 are oriented in lateral view and the larva in A3 is viewed from the cardiac sphincter. (B) Dotplot showing the average expression of the top 25 exocrine pancreas-like marker genes. (C) Dotplot showing the average expression of sea urchin orthologous genes encoding for proteins found in mammalian acinar cells. (D) FISH validations of gene predictions shown in (C) using antisense mRNA probes for Sp-Ptf1a (D1), Sp-Serp2/3 (D2, D4), Sp-Trypsin2 (D3) and Cpa2L (D4). FISH shown in D4 was paired with IHC for the midgut and posterior gut marker Endo1. DAPI was used to label the nuclei (gray). Epl, Exocrine pancreas-like; M, Mouth.
FIGURE 4. Morphological characterization of the S. purpuratus larva exocrine pancreas-like cells. (A) Nuclei segmentation of the entire midgut region in which cell membranes are visible (semitransparent). Midgut (blue), differentiating (red) and exocrine pancreas-like nuclei (yellow) are shown. The nuclei shown with lower transparency are the ones segmented and used for the analysis. Cell membranes are semi-transparent. (B) Overlay and stitching of the FISH for Sp-Cpa2L (magenta), IMH for endo1 and the vEM dataset. (C–E) Isolated slices showing an example of the vesicle relative number and polarization per cell type identified. 3D reconstruction of the cells located in the upper part of the midgut region. Plasma membranes of cell types analyzed were segmented and shown projected on the stitched EM dataset. (F–J) Individual 3D reconstructions of the segmented cell types of interest. The plasma membranes visible in a semi-transparent manner, revealing the nuclei and vesicle number and position. Yellow and lime, exocrine cell; Red, differentiating cell; cyan; main stomach cell. c, cilium; g, golgi; n, nucleus; l, lumen; lv, larger vesicles; m, microvilli.
FIGURE 5. Sea urchin acinar-like cells. Schematic representation summarizing the molecular and morphological conserved features of vertebrate acinar and invertebrate acinar-like cells as shown in this study. Cartoons show the distribution and organization of pancreatic cells in mammals (top row) and sea urchin (bottom row) and (on the right) an overview of the evolutionary conserved gene toolkit shared between sea urchin and mammalian exocrine pancreatic cells. In mammals, exocrine (magenta) and endocrine (green) cells coalesce into a single organ; the pancreas. In sea urchins, acinar-like (magenta) and endocrine pancreas–like (green) cells are scattered within the digestive tract. The cartoon on the right represents an overview of the evolutionary conserved regulatory and terminal differentiation genes found both in mammalian and sea urchin exocrine cells. A full gene list of the sea urchin transcripts found in the acinar-like cell type family can be found in Supplementary Files 2 and 3.
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