s pistillata cell atlas illuminates stony symbiosis, calcification and immunity Shani Levy, Anamaria Elek, Xavier Grau-Bové, Simón Menéndez-Bravo, Marta Iglesias, Amos Tanay, Tali Mass, Arnau Sebé-Pedrós

Scleractinia A multi-stage coral cell type atlas Calicoblasts have a metabolic and Ca skeleton producing Cross- cell type comparison Hexacorallia Actinaria Using single-cell RNA sequencing, we characterized over 40 cell types across the We show a strong conservation of broad cell types across Nematostella vectensis role in and more epidermal-like identity in adult coral Octocorallia three life stages of a stony coral Stylophora pistillata. three major cnidarian lineages that diverged from a common Xenia sp. Hydrozoa 30 ancestor 500 million years ago. Hydra vulgaris transmembrane channels Placozoa ion transporters At the level of specific cell types we observe many-to-many aquaporins Bilateria 20 similarities across species, suggestng a subsequent rapid Ctenophora

carbonic anhydrases Metazoa Porifera Ntox44 evolutionary diversification. 10 CREBL3, RFX4/6/8, Spis_gland % calicoblast cells thrombospondins Mucin 5AC, KSR1 kinase, Nvec_gland 0 fibronectin FKBP peptidylprolyl isomerase Xesp_gland cadherin Hvul_gland lectins GATA, ETV1/4/5, Pou4, Hvul_neuron Synapsin, NKCa channel, Nvec_neuron Repulsive guidance molecule, Spis_neuron TLE corepressor, CDC50,DAG kinase Figure 2. Fraction of callicoblast cells in each sampled life stage (left) and differential gene expression between primary polyp Xesp_neuron calicoblasts and adult colony calicoblasts (right). PTPRN Y_phosphatase, Nvec_muscle PRKAC kinaseadenylate cyclase, Xesp_muscle Gastrodermal cells hosting algal endosymbiont have a G-alpha, Propeptide convertase, Spis_gastrodermis ion glutamate transporter Nvec_gastrodermis distinct expression program enriched in metabolic functions Sox1/2/3-like, MLX, CACNB1, parvin, Spis_alga-hosting_cells ILK, ROR1, isocitrate dehydrogenase Hvul_gastrodermis Mitotic host cells 100 Xesp_gastrodermis Symbiodinium occupancy SIX4/5,NR2F1/2, NFIA/B/C, 80 Alga-hosting cells per coral metacell Xesp_alga-hosting_cells 60 Laminin C1/2/3, RBM46/47, Hvul_cnidocyte 40 Thrombospondin, Paxillin, Xesp_cnidocyte 20 cathepsin D, Kazal-domain protein Spis_cnidocyte 0 containing cells % Symbiodinium - Pax4/6, galectin, ApeC, Nvec_cnidocyte Symbiodinium UMI distribution ShK proteins, minicollagen, Nvec_precursors 10,000 per coral metacell FKBP peptidylprolyl isomerase Xesp_precursors 1,000 Spis_germline Peptidase M13, Peptidase S8, 100 Hvul_germline Gamma-glutamyl transpeptidase 10 Hvul_precursors 1 Symbiodinium UMIs/cell Sox14/21, NCL, Spis_digestive_filaments Metacells EIF4A1, ribosomal proteins Nvec_digestive_filaments Xesp_digestive_filaments lipid metabolisc process E2F7/8, cyclin B2, Tpx2, autophagy HAT1, HELLS helicase, Nvec_epidermis anion transport Spis_calicoblast carboxylic acid biosynt. and transport Growth-Arrest-Specific Protein 2 fatty acid metabolic process Spis_epidermis alpha−amino acid metabolic process TALE_MEIS, SSRP1, Xesp_epidermis steroid metabolic process p25-alpha, kinesin (x2), centrin_1 Hvul_epidermis Spis_immune PAX2/5/8, BACH/Nfe2, Figure 3. Percentage of coral cells containing a Symbiodinium cell in Spis_mitotic_host_cells each metacell, and Symbiodinium UMI count per cell in each coral Tetraspanin, DLG, Filamin, Mex3, Spis_unknown metacell (top left). Expression of a host cell-specific gene Cadherin, Fibronectin, Myosin18 (XP_022783975_1) detected by RNA ISH on adult tissue sections (top right). Scale bars: 50µm. Ap: Actinopharynx, Cn: Cnidocyte, Ep: Figure 5. Cnidarian species tree and cell type tree for the four species in this study. Cell type tree is based on hierarchical clustering of 1,227 variably Epidermis, GCv: Gastrovascular Cavity, Me: Mesoglea, MF: Mesenterial expressed one-to-one orthologs (max expression fold-change in any cell type > 1.8). Node values indicate percent jackknife support (with 75% downsampling). Filaments. Differential gene expression between alga-hosting cells and Selected genes supporting each node are indicated. gastrodermis (left).

Cellular immune system in implies existence of

0 5 conserved immune transcriptional response across Sterol dehydrogenase Normalized IRF Dynein expression IBR E3 ub-ligase Perforin NFIL3/TEF Wnt UPA Fibrinogen NFAT, IRF1/2 ETV6/7 Mucin Frizzled Olfactomedin IRF3/4/5/6/7/8/9 TGFbeta LectinC ATF3/JDP2, Homeobox unclass. Ependymin Tyrosinase ADAMTS Ldl receptor Trefoil factor Ephrin Collagen Secreted proteins: Mucin5A Cellulase perforin, LPS binding protein, prosaposin/granulysin Trypsin Astacin PAPSS1 Peptidase M10 secreted lectin, tyrosinase, ApeC antimicrobial Peptidase M13 Trypsin Nav-gated ion channel GABA receptor Peroxidasin Synaptotagmin MCTP1 ADAMTS PKD ion channel Astacin Peroxidasin GABA receptor transmembrane receptors: Zona pellucida prot. DERM Calmodulin interleukin1 receptor, TyrK, SEFIR, CD36 Aminotransferase5 PKD channel FibrinogenC Choline transp. lectin Ependymin AChR Hemopexin Transferrin GATA Laminin G3 Chymotrypsin Figure 6. The top 2% cross-species pairwise cell type similarities, based on Kullback–Leibler divergence (KLD), shown as arches connecting cell types. ShK protein Syntaxin intracellular signalling: Minicollagen The arch width is proportional to cell types' similarity. The normalized expression of the top shared orthologous genes (FC > 1.3) in two example cell types is LWamide NOD-like receptor, STING Spectrin shown, compared to the average expression of the same genes in all other cell types. SUPT6H NO synthase PRAS, RAB43 member RAS family Peptidase M13 BARX1/2 MORF4L1/2 ASC ion channel Sarcoglycan Synaptotagmin protein stability and lysosome: Transglutaminase GPCR RXFP1/2 Hsp70, Hsp40, E3 ubiquitin−protein ligase Trefoil factor CarboxypepditaseA More information can be Ephrin KCND1/2/3 cathepsins Trypsin GluR ion channel Summary found in our manuscript: Peptidase_A22B GABA receptor Astacin ATOH8 apoptosis: BCL2, caspase Levy et al. 2021 Cell 184 1–16 PhospholipaseC TRPA1 TAOK Acid protein https://doi.org/10.1016/j.cell Hemicentin Transferrin Stylophora pistillata cell atlas: GABA receptor GST TRPA1 ion channel vesicle traficking The data can be explored in Insulin DBH monooxygenase Nav-gated ion channel Cadherin an interactive database at Synaptotagmin MCTP1 TEK TyrK (1) provides new insights into molecular pathways and gene regulatory programs involved PKD ion channel Cyclin B2 sebe-lab.shinyapps.io/ CARP4 Hapless Na/HCO3 transporter ODT Stylophora_cell_atla Carbonic anhydrase Tubulin in coral reef formation, Zona pellucida prot. Synaptotagmin Peroxidasin Figure 4. Gene expression for selected immune-related genes across S. pistillata cell types. Cathepsin L Apolipoprotein A Collagen Transferrin Transferrin Peptidase_C1 (2) presents a valuable resource for the future functional coral studies, and Laminin G3 LectinC Figure 1. Life cycle of Stylophora pistillata with schematic representation of the coral anatomy and tissue Galectin Tropomyosin Minicollagen Minicollagen architecture. Ap: Actinopharynx, Me: Mesoglea, MF: Mesenterial Filaments. Per-stage single cells 2D projections (3) initiates phylogenetically informed comparative analysis of cnidarian cell types. and selected marker genes heatmaps.