Patiria miniata, using single nuclear RNA sequencing (snRNA-seq) of embryonic stages. The atlas revealed 23 cell clusters covering all expected cell types from the endoderm, mesoderm, and ectoderm germ layers. In particular, four distinct neural clusters, an immune-like cluster, and distinct right and left coelom clusters were revealed as distinct cell states. A comparison with Strongylocentrotus purpuratus embryo single-cell transcriptomes was performed using 1:1 orthologs to anchor and then compare gene expression patterns. The equivalent of S. purpuratus piwil3+ Cells were not detected in P. miniata, while the Left Coelom of P. miniata has no equivalent cell cluster in S. purpuratus. These differences may reflect changes in developmental timing between these species. While considered novel morphologically, the Pigment Cells of S. purpuratus map to clusters containing Immune-like Mesenchyme and Neural cells of P. miniata, while the Skeletogenic Mesenchyme of S. purpuratus are revealed as orthologous to the Right Coelom cluster of P. miniata. These results suggest a new interpretation of the evolution of these well-studied cell types and a reflection on the definition of novel cell types.Cell types are the building blocks of metazoan biodiversity and offer a powerful perspective for inferring evolutionary phenomena. With the development of single-cell transcriptomic techniques, new definitions of cell types are emerging. This allows a conceptual reassessment of traditional definitions of novel cell types and their evolution. Research in echinoderms, particularly sea star and sea urchin embryos has contributed significantly to understanding the evolution of novel cell types, through the examination of skeletogenic mesenchyme and pigment cells, which are found in sea urchin larvae, but not sea star larvae. This paper outlines the development of a gene expression atlas for the bat sea star,  Cell types are the fundamental units of multicellular life. Understanding how novel cell types emerge is critical to understanding the relationships between cells, and hence the processes by which organisms acquire new evolutionary traits. Although fundamental to theories of morphological evolution, understanding how novel cell types evolve, or even how to define a novel cell type, is still unclear and a source of active debate . TraditiGene Regulatory Networks (GRNs) are a valuable tool for understanding the evolution of cell types within and between species, as evolutionarily related cell types are assumed to have similar regulatory relationships. Homologous cell types can therefore be identified between species by examining the expression of orthologous genes as a proxy for GRNs operating in the cells. However, the cooption of modular GRN subcircuits may frequently occur during evolution, obscuring the relationship between cells in an organism .The phylum Echinodermata, which includes species of sea stars, sea urchins, brittle stars, sea cucumbers, and feather stars, is a powerful system for studying the evolution of cell types. Their larval forms in particular have several cell types that, based on developmental, morphological, and functional criteria, are considered to be novel . One of Another novel cell type, the larval pigment cells, are unique to the sea urchins (i.e. the echinoid family). The pigment cells emerge in the second wave of mesenchymal ingression during gastrulation and embed themselves in the ectoderm of the larvae . Upon inStrongylocentrotus purpuratus, and the green sea urchin, Lytechinus variegatus, which have an especially well-resolved early developmental GRN  and cooption of the vascular endothelial growth factor receptor (vegfr)  . These r (vegfr) . vegfr ise cells .P. miniata. snRNA-seq is performed on isolated nuclei rather than whole cells (Single-cell RNA-sequencing (scRNA-seq) has revolutionized our understanding of cell-type diversity within an organism . This tele cells  and is sle cells .S. purpuratus scRNA-seq data , 15 hpf, 23 hpf, and 33 hpf], we were able to identify 15 clusters, encompassing all three germ layers and including several clusters corresponding to skeletogenic mesenchyme and pigment cells. These skeletogenic mesenchyme clusters were characterized by gene expression associated with different degrees of differentiation. Further analysis of this pilot dataset is provided in Finally, as proof of principle, we performed snRNA-seq in S. purpuratus into one atlas results in biologically meaningful clustering  (Previous work has shown that pooling scRNA-seq data from the eight-cell stage to the late gastrula of ustering . Therefo 40 hpf) . These t 40 hpf) .P. miniata genome v3.0 , are discussed in detail below and will be referenced in our subsequent comparative analysis. Four of these clusters  represent neuronal cell types, offering valuable insight into the diversity of neurons in early echinoderm embryonic development. One cluster represents a distinct immune-like population (Pmin CL 21), which is the first time this cell type has been molecularly characterized in this species. Finally, we identified two populations of coelomic cells, left and right (Pmin CL 14 and 16), indicating that these tissues are composed of transcriptomically distinct populations. A complete description of the cluster annotation justification for clusters not discussed in this paper’s body is provided in P. miniata and genes with well-documented, conserved neural function including pre-synaptic synaptotagmins, neurotransmitter receptors, and the presence of voltage-gated ion channels  are all closely associated in the UMAP projection . Their ichannels .synaptotagmin-7 (syt7), synaptotagmin-14 (syt14), synaptotagmin-1-like (syt1l), and synaptotagmin-like protein 4 (syt4l). The synaptotagmin gene family has been extensively documented to have conserved functionality in neurons across phyla  is a transcription factor expressed in all four neural clusters and expressed in cells dispersed throughout the ectoderm  is found in three of the four clusters and expressed in a manner spatially consistent with neural fate , a gene previously established as a marker of serotonergic neurons (Tryptophan 5-hydroxylase 1-like (tph1l), a gene involved in serotonin biosynthesis (SRY-box transcription factor 4 (sox4), neogenin-like (neo1l), and ELAV-like protein 1B (elavl1-b) allow us to conclude Pmin CL 17 corresponds to serotonergic neurons , the sea star ortholog of the Drosophila gene glial cells missing (Dmel_CG12245), was also identified as a statistically significant marker gene of Pmin CL 22 and showed increased expression in Pmin CL 10 and Pmin CL 18, relative to the serotonergic cluster. WMISH verified that this gene localizes to a population of cells embedded throughout the ectoderm (gcml-expressing clusters is rab3 GTPase (rab3). Double FISH , and beta-adrenergic receptor kinase 2 (adrbk2). It was also marked by the expression of thyroid hormone receptor beta (thrb) and nuclear receptor subfamily 6 group A member 1B . WMISH of these two genes confirmed localization to a subset of ectoderm, consistent with a neural population (paired box protein Pax5-like (pax5l) which was also shown via WMISH to express in cells embedded in the ectoderm ) (TUB like protein 4 (tulp4)) )  and phot(tulp4)) .homeobox protein Hox-D9-like (hoxd9a), a gene not previously examined in echinoderms. Double FISH of hoxd9a and gcml , neuronal acetylcholine receptor subunit alpha-10 (chrna10) and hormonal receptors including nr6a1b, type-1 angiotensin II receptor-associated protein-like (agtrap) and ecdysis triggering hormone receptor subtype-A (ethr-A). Several other genes related to neuroendocrine function were also present, such as endothelin-converting enzyme 2-like (ece2l)  , and pen(pendrin .serotonin N-acetyltransferase-like (aanat). Pmin CL 18 is also characterized by the expression of highly conserved muscle-associated genes such as dystrophin-related protein 2  (Scavenger receptor cysteine-rich domain superfamily protein (srcr) (mitogen-activated protein kinase-binding protein 1 (mapkbp1) (AN1-type zinc finger protein 6 (zfand6) Genes potentially related to immune function were also detected as markers including ugt2c1l) , Scavengn (srcr)  and sevemapkbp1)  and AN1-pax5l and adrbk2. Although not a statistically significant marker gene, this cluster also expresses agtrap. When we examine the normalized contribution of the three sampling time points to each cluster , transcriptional regulator ERG homolog (Pm-erg). The nuclei of this cluster also express genes involved in immune response in other echinoderms. TEK receptor tyrosine kinase (tek) was identified as a statistically significant marker. The ortholog of tek in S. purpuratus is expressed in coelomocytes and is upregulated in the apical organ during an immune challenge in adults  as a marker of this cluster. Previous work in sea urchins has shown gata3 plays a key role in blastocoelar immunocyte migration and maturation  (ras-related protein Rap-2c (rap2c) (Vitamin D3 Receptor-like (vdrl)  , ras-rel (rap2c) , and Vite (vdrl) , were alvascular endothelial growth factor receptor 1-like (vegfr1l) as a marker of Pmin CL 21. WMISH of this gene  chain-like (col4a2l), fibulin-1, collagen type IV alpha 1 chain (col4a1), mucin-16-like (muc16), teneurin-3-like (tenm3l), and signal peptide, CUB and EGF-like domain-containing protein 1 (scube1). Curiously, there are few regulatory genes shared by these populations at this time point, with only transcription factor AP4 (tfap4) and forkhead box p4 (foxp4) in common. These overlaps suggest that both coelomic pouches share similarities in tissue structure yet have distinct regulatory profiles.To examine the similarity between the marker gene sets of these two coelomic clusters, we calculated the representation factor (RF), a measure of the significance of the overlap between two sets of objects, compared to what would be expected by chance. Values greater than 1 indicate greater-than-expected overlap between two sets, while values less than 1 indicate less-than-expected overlap. These two clusters share 13 marker genes in common, with a representation factor value of 50.2 and p-val <2.921e-19, representing a high degree of similarity. Many of those shared genes are related to the extracellular matrix or cell/cell adhesion, such as paired box 6 (pax6), GATA binding protein 6 (gata6) and msh homeobox-like (msxl)  . gata6   was also identified as a marker of this right coelomic region. arid3a has been shown to be involved in the epithelial to mesenchymal transition (EMT) and primary mesenchymal formation in other echinoderms , also acts as a marker for, and transcripts localize, to the right coelomic pouch , mesenchymal blastula (18–20 hpf), and early gastrula (24 hpf) stages, are equivalent in developmental timing to the P. miniata time points used to generate the atlas here (S. purpuratus atlas using samples taken at these stages from a previously published dataset (P. miniata (see Methods).In order to compare las here . We, the dataset . Our pil dataset . ClusterS. purpuratus’ Pigment Cell and Skeletogenic Mesenchyme clusters. These cell types are novel to S. purpuratus and thus of great interest in our comparison with P. miniata. We also document the identification and annotation of the Oral Non-skeletogenic Mesenchyme and stem/germ cell like piwil3+ lineage, as they are relevant in later discussion. S. purpuratus atlas, with those also used by Below we discuss in greater depth the identification and annotation of S. purpuratus UMAP projection (Pigment Cells (Spur CL 12 and Spur CL 14) and Skeletogenic Mesenchyme (Spur CL 13) were identified as distinct annotated clusters on the ojection .ets1, aristaless-like homeobox , ALX homeobox 1-like , T-box brain transcription factor 1 (tbr), ets variant transcription factor 6 , transcriptional regulator ERG (erg), hematopoietically-expressed homeobox protein HHEX homolog (hhex), TGFB induced factor homeobox 2-like (tgif2), arid3a, and vascular endothelial growth factor receptor 1 . As expected, this cluster was also marked by genes involved in biomineralization, including 27 kDa primary mesenchyme-specific spicule protein (pm27a), spicule matrix protein SM50 (sm50), and mesenchyme-specific cell surface glycoprotein (msp130).The Skeletogenic Mesenchyme cluster (Spur CL 13) was marked by the expression of regulatory genes such as gcml, polyketide synthase 1 (pks1), dimethylaniline monooxygenase [N-oxide-forming] 2 (fmo3), and sulfotransferase (sult) . These clusters shared 107 markers including known markers of pigment cells in sea urchins, including e (sult) .(Maternae (sult)  We hypoterg, gata3, gata6, hhex, prox1, ets1, as well as genes that have been linked to immune response in echinoderms, like tek, and srcr. Other genes implicated in immune function in other systems such as two allograft inflammatory factor 1-like genes , C-type lectin domain-containing protein 141-like, and duox1 are also found in this cluster . It is also marked by the gene nanos C2HC-type zinc finger 2-like (nanos2l), which has been shown to play a role in Primordial Germ Cell (PGC) specification , removing species-specific genes and genes with 1:many orthologs from our analysis. Normalization, integration, clustering, and dimensional reduction were done according to the same pipeline used in creating our single-species atlases and are detailed in the methods section. Our analysis resulted in 15 integrated clusters  present in the integrated cluster (Int CL ID). A full table of these values is available in S. purpuratus, whose cells made up 77.95% of the cluster as opposed to P. miniata, whose nuclei made up 22.0%. The S. purpuratus components of Int CL 14 primarily originate from Spur CL 19 Undetermined  and Spur CL 22 piwil3+ Cells . From P. miniata, 11 out of 23 clusters contribute nuclei to Int CL 14, with all but one contributing 5 or fewer nuclei. Pmin CL 19 Dividing Ectoderm contributes 34 nuclei, making up 12.8% of all cells of this identity.Int CL 14 was the most species-specific integrated cluster. It had 210 cells total, making up 0.87% of the total dataset. This cluster was skewed towards P. miniata nuclei, which originated from low-level contributions from a diverse array of originally annotated clusters, in this integrated cluster indicates that there are no populations in P. miniata that closely resemble the transcriptome profile of S. purpuratus’s piwil3+ Cells and the undefined cluster. In the S. purpuratus cluster, Spur CL 19 Undetermined is dominated by the marker gene expression of cell cycle factors. Therefore, we think the expression of these factors drives clustering with both cells from Spur CL 22 piwil3+ Cells and Pmin Dividing Ectoderm in the integrated dataset. Therefore the presence of P. miniata nuclei in this cluster is likely driven by the impact of cell cycle genes, statistical noise rather than transcriptomic similarity to the piwil3+ Cells, thus making Int CL 16 the only S. purpuratus-specific transcriptomic profile present at this stage of development.We hypothesize that the low membership of piwi, nanos, and pumillo, only begin to show cell-type-specific expression in P. miniata by the late gastrula stage when the posterior endocoelom forms  do not form a novel transcriptomic cluster in the multispecies integrated clustering. These cells mainly segregate into two integrated clusters, Int CL 8 and Int CL 12 , and calumin .Amongst these markers were some shown to be involved in immune function in other animals. This gene set includes  calumin . We therP. miniata cells contributing to this cluster primarily originated from Pmin CL 10 Early Neural (48.3%), Pmin CL 18 Mixed Neural (95.9%), and Pmin CL 22 Sensory Neural (87.2%)  .S. purpuratus Pigment Cells A and B and P. miniata Non-Serotonergic Neurons had several transcription factors shared as marker genes: hoxd9a, etv1, and gcml. In S. purpuratus, gcml is a known marker of pigment cells  , identified 32 gene orthologs in common, with a representation factor of 6.4 and corresponding p-value <5.814e-18 . S. purpnt cells . Severalse (nlk) .Spur CL 14, annotated as Skeletogenic Mesenchyme, does not form a distinct transcriptomic cluster in the multi-species integration map . HoweverP. miniata cell types contributing to Int CL 7, the top contributors are Pmin CL 14 Right Coelom (83.0%) and Pmin CL 15 Foregut (30.5%) , and 3 alpha procollagen precursor (col4a1). Probable JmjC domain-containing histone demethylation protein 2 C (hnhd1c) acts as a marker in only S. purpuratus’ Skeletogenic Mesenchyme and P. miniata Foregut.When we examine  (30.5%) . A compa.819e-10 . The thrP. miniata’s Right Coelom. Of particular interest are the shared markers, arid3a, transcription factors elk1, and ets1, genes previously characterized in the skeletogenic mesenchyme GRN of S. purpuratus , 3 alpha procollagen precursor (col4a1), rap associating with DIL domain (radil), and DENN domain-containing protein 5B (dennd5b) with a representation factor of 6.3 and p-value <0.0033.Unlike the Right Coelom, there is little overlap in marker gene content between the Left Coelom and Skeletogenic Mesenchyme. These two groups share 4 marker genes in common S. purpuratus cells to that cluster, we see that there is no main contributor, rather small contributions across a wide range of S. purpuratus cell types, originating from 21 of the 23 different Spur CL clusters, with contributions ranging from 0.08% to 2.9% of a given Spur CL.We, therefore, were especially interested in comparing the cluster identity of the Left Coelom. 95.7% of Pmin CL 16 Left Coelom nuclei contribute to Int CL 10 . When weS. purpuratus at the sampled stages. Further interrogation of P. miniata’s left coelomic pouch development is needed to better understand these phenomena.There are several potential explanations for the isolation of the left coelom cells in our integrated clustering. Firstly, this could correspond to a novel cell lineage that has not been documented in sea stars. Alternatively, the transcriptomes of the homologous coelomic pouches between the species could be sufficiently divergent so that they no longer drive clustering in the integration approaches we used, possibly reflecting to the morphological uniqueness of sea star coeloms with respect to other echinoderm species . This coP. miniata. An integrated atlas was constructed using 1:1 orthologs of P. miniata and S. purpuratus to investigate and infer the changes during cell type specification that underlie the developmental origins of these novel cell types.At the heart of evolutionary developmental biology rest two questions: what is a cell type? And what is novelty? Being able to differentiate and reproducibly define cell categories is essential to understanding the evolutionary relationship between cell types and the cross-species comparisons needed to establish novelty. The identification and definition of cell types can change with new technologies as new data is brought to light. In this paper, a single-nucleus RNA-sequencing approach was used to classify cell types present during early embryonic development in the bat sea star, P. miniata, suggesting distinct synaptic vesicle trafficking in these neural types. The identification of endocrine-associated markers in two of these neural clusters  supports an emerging hypothesis of the origins of neural subtypes from secretory cells . We suggest that these genes have a conserved role in the core mesenchyme specification program present in their last common ancestor. Further work examining these cell types across species of echinoderms is needed to better define these novelties and allow for a more comprehensive characterization of their evolutionary timing.In comparison, the presumed, traditionally defined novel cell types, that is the skeletogenic mesenchyme and pigment cells in sea urchins form molecularly distinct populations in the single species atlas but lose that distinctness in our multi-species atlas where we find similarities between their transcriptomes and those of er genes , includivegfr1l in P. miniata. Previous studies have shown that an ortholog of this gene, vegfr1l  was exclusively expressed in the skeletogenic mesenchyme of brittle stars and sea urchins and was responsible for the deployment of the biomineralization gene set as was seen as a critical node of cooption , possibly another consequence of gcml expression. This suggests that these genes may play dual roles in immune and neural cells and the emergence of modern pigment cells is the result of a fusion of regulatory programs from different cell lineages. Additionally, work comparing fully differentiated neurons, immune cells and pigment cells would help elucidate any further similarities in cell function and gene expression, possibly providing insights into how novel cell types arise and develop unique phenotypes via the co-option of regulatory pathways.The pigment cell clustering with ulations . When coer phyla  leads usP. miniata and an orthology-centered approach to integrate it with the atlas of sea urchin, S. purpuratus. Single-cell transcriptomics provides a very detailed picture of gene expression and cell identity. This allows us to deeply explore concepts of cell-type identity and evolutionary novelty through the lens of gene expression and inferred GRNs operating in the cells.We have used single nuclei RNA-seq to study early embryo development in the sea star The examples uncovered in this study serve to expand our understanding of evolution of cell types among these echinoderms, and more generally highlight ways that we can think about novelty and the limitations of experimental approaches to this understanding.piwil3+ cell population in sea urchin likely points to a different developmental program and possible functional role of these cell types in these species.Our comparisons discovered examples of transcriptomically distinct cell clusters, that is piwil3+ Cells in sea urchins and Left Coelom in sea stars, with no clear orthologous cluster in the other species. These findings were unexpected and have identified previously unappreciated differences in cell types. These findings may be taken as examples of a novel cell when defined on the basis of gene expression similarities achieved by this method. Novelty in both of these cases may be relative changes in timing or heterochrony of the cell state specification. Unless snRNA-seq covers a tight time course across the entirety of development and adult tissues, and possible environmental contexts we can never truly determine novelty. However, heterochronic activation of a GRN in a new context can result in dramatic functional differences to the organism. The very early specification of a Conversely, the two cell types that we anticipated as novel based on cell morphology, function, and developmental lineage, that is skeletogenic mesenchyme and pigment cells in sea urchins had significant transcriptomic profiles in common with sea star cell clusters. This implies a shared ancestry of these cells, with an ancestral cell type possessing these regulatory profiles. In the case of skeletogenic mesenchyme and right coelom, this may include overlapping functions in EMT, cell migration, and tubulogenesis with a cooption, quite late in the developmental GRN of cassettes of biomineral genes. The most surprising find from our studies was the overlap between pigment cell cluster transcriptome and the immune and neural clusters in sea stars. This implies a more complex evolutionary scenario whereby programs from two cell types in sea stars have merged into one to produce a distinct cell morphology and function.In conclusion, our study provides new interpretations of cell type novelty and new inferences of novel cell type evolution when considering comparisons of orthologous gene expression profiles over traditional approaches.P. miniata and S. purpuratus animals were obtained by Marinus Scientific  and housed in 100-gallon tanks containing aerated artificial seawater . To induce spawning, adults were injected with 200 µM 1-methyl-adenine . The embryos were then manually dissociated using a P1000 micropipette and filtered through a 30 µM mesh filter. The sample was then centrifuged at 300 g for 5 min at 4 °C, and resuspended in artificial seawater. A single sample was then processed for bulk RNA-seq (see below).The whole-cell dissociation protocol was adapted from g) at 4 °C, and the pellet was resuspended in 1 mL HB buffer. Tip strainers (Scienceware Flowmi) were used to transfer the sample to a new tube and 9 mL ice-cold HB buffer was added to the sample and centrifuged for 5 min (3500 g) at 4 °C. The pellet was resuspended in 1 mL PBT . Samples were centrifuged for 5 min (2.4 g) in a cold room and nuclei were resuspended in 300 µL PBT. Nuclei were stained with Trypan Blue and counted in triplicate with a Countess II Automatic Cell Counter (Thermo Fisher). The resulting nuclei were resuspended to 1000 nuclei/µL.Prior to dissociation, embryos were washed twice with cold seawater by centrifuging for 5 min (22 g) at 4 °C, and resuspended in 10 mL cold HB Buffer ) and kept on ice. Washed embryos were then transferred to a 15 mL dounce, and homogenized 20 times with the loose pestle and 10 times with the tight pestle. The dounced sample was centrifuged for 5 min  sample, 5000 for the 15 hpf and 6000 for 23 hpf samples. For P. miniata, there were 3000 nuclei in the 16 hpf sample, 5000 in the 26 hpf sample, and 6000 in the 40 hpf sample. After constructing the libraries, samples were quantitated on a Tapestation using D5000 ScreenTape to confirm insert sizes of 400–500 bp. Samples were sequenced on a Novaseq 2X150 S4 lane .Single nuclei libraries were created using the Chromium Next GEM Single Cell 3’ Reagent Kit v3.1 RevD (10 X Genomics) and the Chromium X instrument (10 X Genomics) using manufacturer protocols. For An outline of the code used to create both species atlases and the integrated atlases is available in the .P. miniata genome V3.0 (GenBank assembly accession: GCA_015706575.1) and S. purpuratus genome V5.0 (GenBank assembly accession: GCA_000002235.4). The pre-mRNA indices were constructed using gene annotation files modified by an in-house script to remove intron annotations, allowing unspliced reads to align. All further analysis was conducted in R V4.0.5  to identify up to 3000 genes best suited to anchoring the datasets together using the SCT normalized values . Following integration, Principal Component Analysis was run on the composite object and significant Principle Components (PCs) were identified by plotting the standard deviation of the first 50 PCs and identifying the PCs that describe the most variability of the datasets.Using Seurat V4.0.5 , each tiP. miniata nuclei atlas used 20 PCs, our S. purpuratus nuclear atlas used 10 PCs, the S. purpuratus whole-cell atlas used 10 PCs, and our multi-species atlas used 10 PCs. Using our selected PCs, we constructed k-nearest neighbor graphs (with k=20), performed Louvian Community Assignment , and performed Uniform Manifold Approximation and Projection (UMAP) reduction in order to generate clusters. Resolution was determined for each species’ dataset individually seeking to optimize the number of clusters, number of marker genes, distribution of marker genes between clusters, and maximum modularity with 10 random starts observed with different resolution.Our To annotate cluster identity, we identified genes that differentially marked clusters using the Wilcox rank-sum test. A gene is considered a marker gene for a cluster if it is expressed in at least 5% of the cells in that cluster and has at least a 0.25 log fold-change difference in average expression between the cells in that cluster and all other cells. Adjusted p-values were calculated using Bonferroni correction and used to assess significance. We then used these marker genes to determine cluster cell-type labels by cross-referencing them with genes established in previous studies to have cell-type-specific expression.S. purpuratus genome V 5.0 and P. miniata genome V3.0 and named according to orthologs identified in the human genome. For a full description of ortholog identification see 1:1 orthologs were identified between S. purpuratus atlas at comparable time points to our P. miniata set, using the hatched blastula, mesenchymal blastula, and early gastrula stage data from a previously published dataset , 10,611 cells were selected for analysis and clustering using Seurat V4.0.5 (To facilitate this comparison we constructed an  dataset . CellRan dataset  was usedt V4.0.5 .S. purpuratus only) and rRNA transcripts. 2321 integration anchors were identified and used for dataset integration via canonical correlation analysis (CCA), using the Seurat package .To compare marker genes between different clusters, all calculated marker genes for a cluster of interest in their species-specific atlas were input into a Venn Diagram tool, housed by the Bioinformatics and Evolutionary Genomics group at the Ghent University  washed twice, then transferred into hybridization buffer  and pre-hybridized overnight at 58 °C. Following denaturation for 5 min at 65 °C, DIG-labeled probes were added to the embryos at 0.2 ng/µL. Samples were then incubated at 58 °C for 4–7 days. Embryos were washed three times with a hybridization buffer over 4–6 hr, transferred to MAB  with 0.1%Tween20 then washed four times with MAB +0.1%Tween. Samples were blocked with MAB-2% Roche Block solution for 30 min at room temperature or overnight at 4 °C. Anti-DIG AP FAB Antibody in MAB-2% Roche Block was added to the samples for a final concentration of 1:1000 and left to incubate for 2 hr at room temperature or overnight at 4 °C.Next, the samples were washed four times in MAB-0.1%Tween20 followed by three washes in AP buffer , with 15 min between washes. Samples were resuspended in color reaction solution , transferred to watch glasses, and kept in the dark at room temperature until the reaction was completed. When the color reaction concluded, embryos were washed three times in MAB-0.1%Tween20 and stored indefinitely in MAB.Embryos were evaluated using a Leica MZ 95 dissection scope. For imaging, embryos were resuspended in MAB- 50% glycerol, and photographs were taken using a Leica DMI 4000 B inverted scope and Leica Application Suite X V3.6.20104.Double FISH was performed using a method previously described in BioRender.com was used to create the schematics in Gimp was used to process microscopy images . BioRend This important study combines single cell and single nucleus transcriptomics to two echinoderm embryos to identify embryonic cell types and assess cell type evolutionary novelties. The paper provides convincing pieces of evidence in regards to the sea star embryonic cell types and their relationship to the sea urchin ones, highlighting conserved, diverged as well as novel genetic programs operating during early echinoderm development. The work will be of broad interest to developmental and evolutionary biologists. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, including Maria Ina Arnone as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Didier Stainier as the Senior Editor. The following individual involved in the review of your submission has agreed to reveal their identity: Roger Revilla-i-Domingo (Reviewer #2).Thank you for submitting your article "New hypotheses of cell type diversity and novelty from comparative single cell and nuclei transcriptomics in echinoderms" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:1) Improve validation of the P. miniata clustering by using double whole month in situ hybridization as suggested by Reviewer #1.2) Complement the integration of sea urchin and sea star datasets analysis with a method that is not limited to the 1:1 orthologs between sea urchin and sea star, using, for example, as suggested by Reviewer #2 the algorithm based on self-assembling manyfold (SAMap). This could partially address also Reviewer #3 concern about comparing a single-nuclei RNA-seq dataset with a single-cell RNA-seq dataset.3) Include a sea urchin gastrula stage in the sea urchin and sea star integrated single cell analysis or otherwise address the concerns of Reviewer #1 regarding the absence of a sea urchin gastrula stage in the analysis.4) Include clear statements and general discussion about the limitation of cell type evolutionary comparison performed as described in points 2 and 3 by Reviewer #3.Reviewer #1 (Recommendations for the authors):While the manuscript is well written, particularly in the introduction and Discussion sections, the data presented show two major weaknesses, one concerning the validation of the clustering via whole month in situ hybridization (WMISH), and the other concerning the stages of S. purpuratus chosen as homologous of the ones analyzed in P. miniata.Regarding validation by WMISH, the general point is that this reviewer would like to see more embryos/gene validation, especially for the novel marker genes identified, and, in some cases a better resolution of the mRNA localization with the help of fluorescent in situ hybridization (FISH) or double FISH. For instance, in lines 220-230, the authors make a very interesting point on novel marker genes of their neural clusters, the synaptotagmin genes, which, as shown in the dot plot reported in Figure 5a, exhibit a differential expression marking different neural cell clusters. It would be very nice to see at least a couple of FISH experiments showing these differentiating markers in situ. Double FISH for this would be perfect. Similarly, a better resolution is necessary to convince the reader about the expression patterns reported in Figure 5 E-G and those in Figure 7.Another point regarding the WMISH is that there is no information in the paper regarding the stages analyzed. I assume that the embryos in Figure 5 and 6 correspond to the mid gastrula/ 40 h P. miniata embryos that were used as the latest time point in the construction of the single cell atlas, while the ones reported in Figure 7 correspond to later gastrula stages. However, this info has to be included in the figures and/or figure legends.Regarding the stages of S. purpuratus chosen as homologous of the ones analyzed in P. miniata, as stated in the results and reported in Figure 3B the authors have used for P. miniate two blastula stages and one gastrula, 40h, which the authors correctly define as mid gastrula (line 171). However, strangely enough, they didn't include any gastrula stage for the corresponding sea urchin data sets, despite the fact the source they used for scRNA-seq data of S. purpuratus  includes two gastrula stages, one early and one late. On the contrary, the authors chose only hatched and mesenchyme blastula stages for sea urchins and justify this choice with a sentence that appears very controversial: "An extensive comparison of whole embryo RNA-seq time series data between S. purpuratus and P. miniata has shown that P. miniata develops at a faster rate and that S. purpuratus  are equivalent in developmental timing to the P.miniata time points used to generate the atlas here ." Besides the fact that P. miniata definitely develops at a slower and not faster rate compared to S. purpuratus, and that the staging reported in Gildor et al. is not the best reference to be used  also because the 40h P. miniata stage is not included in it, this statement is clearly contradictory even in respect to what stated by the authors of this paper in other parts of the manuscript. While I can agree with the fact that the so-called "mesenchyme blastula" in sea urchins can be considered as a very early gastrula (given that mesenchyme cells are ingressed in the blastocoel at this stage), no endoderm has ingressed at all at this stage. Therefore, it would be more appropriate to include in the S. purpuratus data sets a stage in which at least some part of the archenteron has ingressed. Indeed, the S. purpuratus early gastrula of the Foster et al. dataset (30h) appears as a bona fide equivalent of the 40h P. miniata gastrula stage. I believe the exclusion of a sea urchin gastrula stage in the comparative analysis introduces a strong bias and I strongly suggest redoing this analysis with the inclusion of this stage.Another general, important, although minor and easy to fix point, is the nomenclature used in the manuscript:– First of all, about gene names: throughout the manuscript, text, figures, and figure legends, a plethora of different styles are used, even within the same list (such as in the y axis of dot plots), including UPPERCASE, lowercase , capitalised words, with or without initials of species names … please choose a style and stick to it, because it is very confusing– Second, about cluster names: some choices are in my opinion confusing as they refer to a structure/ developmental stage that is not present in the data set; this is the case for example of (left and right) "coelom", which should be better referred to as "coelomic mesoderm" given that coelomic pouches appears much later compared to the stages analysed; or the case of the "immune mesenchyme", which should be better referred to as immune-like given that immune cells, to my knowledge, were never functionally identified in the P. miniata larva; I understand that for the sake of simplicity "immune mesenchyme" could be a better name, but this assumption should be clearly specified and definitely the short name "immune cluster" used in abstract and conclusions is not appropriate– Another issue concerns the use of "primary mesenchyme cells", which is quite an obsolete terminology, not very informative, and really meaningful only within the sea urchin community. Skeletogenic mesenchyme appears more appropriate for a wider audience.Two other important changes that would make the manuscript more readable are:– First, about the use of the adjusted p-value: reporting the adjusted p-value for each of the marker genes identifying each cluster appears as redundant. If a gene is a gene marker it means that it passed the adjusted p-value test and if the reader wants to know more about those figures these are all included in the tables. The result session is uselessly heavy and boring to read with all these parentheses. I strongly suggest eliminating these details in the text.– Second, in the analysis of the contribution of cells for each integrated cluster, the list of all contributions, even if they account for just one cell, again appears redundant, and the text become difficult to read  and eventually even confusing to what is the actual identity of the cluster. I would propose to decide on a certain cut-off  and list in the text only the contribution above that threshold, leaving to a supplementary figure the detailed contribution of sea urchin or sea star cluster to the integrated cluster, as done for Figure 9 and 10.Reviewer #2 (Recommendations for the authors):eLife 2021), where single-cell datasets from animals across different phyla were integrated.1. To strengthen  the claims resulting from the integration of sea urchin and sea star datasets, I recommend that the authors complement their analysis with a method that is not limited to the 1:1 orthologs between sea urchin and sea star. I would suggest, for example, the algorithm based on self-assembling manyfold (SAMap), which has been successfully employed by Musser et al. (Science 2021) to compare two sponge species  and has been described in detail by Tarashansky et al. . This would make it easier to follow the description of the integrated clusters.3. In Figure 6, the authors very nicely show that genes enriched in Pmin Cl 21 are expressed in mesenchymal populations. In particular, VEGFR1L is shown to be expressed in a population that accumulates around the base of the archenteron , similar to the localization of PMCs in S. purpuratus. According to Figure 6.D, my impression is that tek (another gene enriched in Pmin CL 21) is also expressed in the population of cells around the archenteron. Although less clear, the same might be true for ets1 . Could the authors explain whether these (and perhaps other) markers of Pmin CL 21 are expressed in the mesenchymal population around the archenteron? If they are indeed expressed in this population, can the authors comment on the significance of this?4. An interesting finding of the study by Meyer et al. is the overlap between pigment cell cluster transcriptome in sea urchins and the immune and neural clusters in sea stars. Would it be relevant to discuss this finding in the light of the hypothesis by Klimovich and Bosch  that the nervous system and the immune system have a common evolutionary origin?Reviewer #3 (Recommendations for the authors):1. Combing scRNAseq and snRNAseq data could introduce biases. Several studies have noted that seemingly subtle differences like sequencing platform or even batch can produce artificial signals within scRNAseq data, sometimes even swamping species effects . Thus, it's not clear to me that single-nucleus and single-cell RNAseq data can be combined without introducing systematic biases in the representation of transcripts present. For instance, mRNAs with short half-lives would likely be over-represented in snRNAseq compared with scRNAseq. I suspect that conclusions about overlapping clusters and the presence/absence of clusters reported in this study are generally secure. However, differences in timing and in proportions of cells in a given cluster between species may be more problematic. If the authors are aware of any empirical data that support direct comparisons between snRNAseq and scRNA seq, citing these would build confidence in the results. At a minimum, the authors should state more clearly that they are making an assumption when combining different data types in this study, and also comment on how this might affect the interpretation of results.2. Sampling only embryonic stages limits the ability to confidently identify the absence of a cell type. In an adult organ composed mostly of differentiated cells, determining the presence/absence of a cell type is fairly straightforward, assuming fairly representative cells/nuclei and reasonably high-quality data. In contrast, a gastrula-stage embryo contains very few, if indeed any, fully differentiated cells. Further, it is well established that homologous cells can differentiate at different stages of development in different species. Thus, a cell type might appear to be absent in the gastrula of one species and present in another simply because it has not yet begun to differentiate in the first species. It's a situation where the absence of evidence is not evidence of absence. This makes it impossible to reach any firm conclusions about which cell types might be unique to seastars or sea urchins. The authors are clearly aware of this limitation, e.g. lines 446-447: "thus making Int CL 16 the only S. purpuratus-specific cell type at this stage of development" (emphasis added). Here and elsewhere, the authors are good about stating this caveat. However, these statements come across as each applying to a specific case rather than a general limitation that applies to all cell types. This more general caveat should be clearly stated, as it is key to understanding what can and can't be learned from the data. This is particularly important because many non-specialists reading this paper will get lost in, or simply skip over, the numerous details presented for any given cell type and could easily miss both the individual caveats and the more general point that in no case is it possible to conclude that a particular cell type is present or absent from one species or the other.3. Data from only two species limits the ability to confidently identify the direction of evolutionary change. Even if we accept that some cells are truly present/absent, figuring out whether these cases represent novelties or losses is problematic when only examining two species: is it a gain in the species with the unique cell type or a loss in the species without? The usual approach to answering this question is getting data from an outgroup species, but in the present case, the data simply aren't available. The authors are not always clear about this limitation, for example (lines 462-463) "this cluster can be considered novel to S. purpuratus". More accurate would be to say that it is uniquely present rather than novel. It could also be the case that the ancestral echinoderm had an early specification of germ cells and that this has been lost in seastars and perhaps other echinoderms. Here again, the limitation is a general one rather than specific to this particular cell type and should be clearly stated as such. Again, this is key to understanding what can and cannot be inferred from the data.A suggestion: There may be an opportunity to make a different kind of evolutionarily inference that is more secure, namely to identify evolutionary differences in the timing of differentiation of specific cell types. These observations are interesting because they have implications for the evolution of cell fate specification mechanisms. For instance, germ cells are specified clonally and very early in sea urchins and the molecular mechanisms and cellular processes responsible are well studied. The absence of any cells with a similar transcriptional profile in seastar embryos implies no that early segregation of the germ line does not occur, and thus it is unlikely that localized maternal determinants are responsible. Given the wealth of information available about cell fate specification mechanisms in sea urchins, inferences about evolutionary differences could be made about other cell types as well. This seems to me a missed opportunity for the authors to highlight some interesting and informative aspects of their results. Essential revisions:1) Improve validation of the P. miniata clustering by using double whole month in situ hybridization as suggested by Reviewer #1.syt14 , syt7 , etv1  and got1 . These genes were identified as markers for the four neural clusters and now importantly indicate expression in patterns consistent with neural fate and a pattern that differs between the genes suggesting non overlapping of neural fates. We additionally added two color FISH for gcml + hoxd9a  and rab3 + gcml . These were important to show that hoxd9a and rab3 are coexpressed with some subsets of gcml+ cells. This supports the hypothesis suggested by the clustering of neural types in Figure 4 and Figure 5.A that gcml, rab3 and hoxd9a are are marker genes for the Sensory Neural cluster (Pm CL 22) and colocalize. There is not perfect overlap with these gene transcripts suggesting an even more complex neural cell fate than we have captured in our clustering. We also performed FISH for ets1 + tek  to support the hypothesis of Figure 4 and Figure 6.A and 6.B that ets1 and tek are coexpressed and are marker genes for immune-like mesenchyme (Pm CL 1). We show that they are exclusively overlapping.We have added single-color WMISH for the genes We have added text into the results, figure legends, and discussion to provide details about these experiments and their interpretation.2) Complement the integration of sea urchin and sea star datasets analysis with a method that is not limited to the 1:1 orthologs between sea urchin and sea star, using, for example, as suggested by Reviewer #2 the algorithm based on self-assembling manyfold (SAMap). This could partially address also Reviewer #3 concern about comparing a single-nuclei RNA-seq dataset with a single-cell RNA-seq dataset.After careful consideration we have decided to maintain our focus on 1:1 orthologs for our mapping as we believe this gives more robust results SAMap was developed for mouse/human integration (~100 Mya) and will be limited for sea star/sea urchin integration (~400 Mya). First it should be noted that our orthology mapping between these two species has been extensively performed using the principles of the Alliance of Genome Resources that at least three DIOPT tools should agree on the orthology. We have used BLAST, phylogenetic-based tools  as published in “Integration of 1: 1 orthology maps and updated datasets into Echinobase, Foley et al. Database, 2021”. We think mapping using orthologous genes provides a more reliable comparison for our integrated echinoderm datasets.S. purpuratus Piwil3+ Cells. The near absence of sea star nuclei in Int CL 14 is supported by previous gene expression studies  which show that piwil3, nanos2l and pum2 orthologs/paralogs are not expressed in these stages in P. miniata.We also show that several results from this integrated mapping corroborate observations from other data types to support that we are making meaningful findings. For example, we identify a sea urchin specific integrated cluster (Int CL 14) that corresponds to To reflect our focus on ortholog driven clustering we have changed the title to of the paperNew hypotheses of cell type diversity and novelty from orthology-driven comparative single cell and nuclei transcriptomics in echinoderms.We also comment here on the differences between single nuclei and single cell approaches. We carefully considered the approaches to isolation and thought for this and future studies that single nuclei sequencing would be less biased, robust and easier to achieve reliability; especially as researchers look to later and more complex stages and tissues. We therefore took great care to evaluate the two procedures since we do rely on comparison to scRNA sequencing. Figure 2 and Figure 2 Supplemental FIgure 1 strongly support that there are no significant differences between approaches and that they can be reliably compared. We showed bulk RNA profiles are not significantly different when obtained from whole embryos, isolated cells or isolated nuclei  and that the single nuclei atlas prepared in our studies was highly similar to the atlas previously prepared from single cells .We added the following sentence in the fourth paragraph of the results to more thoroughly clarify our position on this."These results thus strongly support that single nuclei and single-cell RNA isolation and sequencing each resulted in appropriate representative clusters thus providing confidence that either isolation method was an effective way to establish cell clusters. snRNA-seq is more likely to capture earlier transcriptional responses than scRNA-seq, but such timing differences are much smaller than the sampling times here and unlikely to affect our analyses."3) Include a sea urchin gastrula stage in the sea urchin and sea star integrated single cell analysis or otherwise address the concerns of Reviewer #1 regarding the absence of a sea urchin gastrula stage in the analysis.We have included this new time point in our analysis. In doing so we have re-clustered and re-annotated the sea urchin data and re-performed the integrated cluster analysis. This reanalysis has led to many small changes in summary statistics and specifics of clusters, and overlapping gene sets. However, the main findings of the work and conclusions of the analyses have not changed, as many of the differences in the re-analyses are of small effect.Thus, we recreated the sea urchin developmental atlas now using three time points . We now have 10,611 cells in the analysis rather than the original reported 7,370 and 23 vs 20 distinct clusters.S. purpuratus annotation in Figure 8 supplemental figure 1 and Figure 8 was updated to reflect the new cluster names.We then performed a new cluster annotation. The most significant difference with this new analysis is that we now have two rather than one pigment cell cluster, named Spur CL 12 Pigment Cells A and Spur CL 14 Pigment Cells B. Additionally small micromeres and muscle clusters were annotated. The We added additional text to more robustly explain the annotation of Spur CL 12 and 14, the two pigment cell clusters and Spur CL 13 as skeletogenic mesenchyme (previously PMCs).The text now reads:gcml, polyketide synthase 1 (pks1), dimethylaniline monooxygenase [N-oxide-forming] 2 (fmo3), and sulfotransferase (sult) . We hypothesize Spur CL 14 Pigment cells B represents a population of pigment cells in an earlier state of differentiation than Spur CL 12 Pigment Cells A, because upstream transcription factors such as etv6, gata6, and etv1 mark only Spur CL 14 Pigment Cells B. ““Two clusters were identified as belonging to the pigment cell lineage (Spur CL 12 Pigment Cells A and Spur CL 14 Pigment Cells B). These clusters shared 107 markers including known markers of pigment cells in sea urchins, including “The skeletogenic mesenchyme cluster (Spur CL 13) was marked by the expression of regulatory genes such as ets1, aristaless-like homeobox , ALX homeobox 1-like , T-box brain transcription factor 1 (tbr), ets variant transcription factor 6 , transcriptional regulator ERG (erg), hematopoietically-expressed homeobox protein HHEX homolog (hhex), TGFB induced factor homeobox 2-like (tgif2), arid3a, and vascular endothelial growth factor receptor 1 . As expected, this cluster was also marked by genes involved in biomineralization, including 27 kDa primary mesenchyme-specific spicule protein (pm27a), spicule matrix protein SM50 (sm50), and mesenchyme-specific cell surface glycoprotein (msp130).”While the annotation of the immune cell cluster did not change, we added additional text to more clearly explain the rationale for this cluster.erg, gata3, gata6, hhex, prox1, ets1, as well as genes that have been linked to immune response in echinoderms, like tek, and srcr. Other genes implicated in immune function in other systems such as two allograft inflammatory factor 1-like genes , C-type lectin domain-containing protein 141-like, and duox1 are also found in this cluster .""Another cluster of particular interest is Spur CL 17 Oral Non-skeletogenic Mesenchyme, the lineage fated to become immune coelomocyte cells. This cluster is marked by genes known to be involved in the specification of this lineage like P. miniata and S. purpuratus datasets and updated the analyses in Figures 8-12.With the new cluster analysis we then re-performed the integration between the We have used the same methodology and pipelines as previously. As requested by Reviewer 3 we have provided the code used to create and integrate the atlases in Supplementary file 7.The integrated analysis with the additional data changed some of the values and numbers of overlapping genes but no substantive differences were found in our conclusions. We identified 15 rather than 17 clusters in this new dataset.The wording has been changed in multiple places in the Results section to reflect the changed numbering of the clusters (simply a result of reclustering) and the % and numbers of overlapping genes and changes in significance. Summarized below, these differences were only slight and did not change the conclusions.Figure 9 shows that Int CL 14 is enriched with cells from Spur CL 22 piwil3+ Cells and Undetermined with very few Pmin CL 19 Dividing Ectoderm.P. miniata but now with stronger support (RF 5.2 compared to previous 5.0) although the number of genes overlapping decreased somewhat.Figure 10 shows the integrated clusters with separate contributions from Pigment A and B. Int CL 8 and 12 show contributions from both Pigment A and B (Spur). Contributions from Pmin to Int CL 8 are majority Immune-like Mesenchyme and Int CL 12 shows contributions from early, mixed and sensory neural clusters from S. purpuratus Skeletogenic Mesenchyme and P. miniata Right Coelom and Foregut. Our conclusions here therefore remain unchanged also.Figure 11 shows that Int CL 7 is primarily P. miniata Left Coelom with no predominant corresponding contributions from S. purpuratus. This is the same result also.Figure 12 shows Int CL 10 is primarily composed of 4) Include clear statements and general discussion about the limitation of cell type evolutionary comparison performed as described in points 2 and 3 by Reviewer #3.We agree that these reviewers have made important points and we have added several new sentences throughout the text and in particular into the conclusion section to address their comments.We have added into the discussion:“This suggests that these genes may play dual roles in immune and neural cells and the emergence of modern pigment cells is the result of a fusion of regulatory programs from different cell lineages. Future work comparing fully differentiated neurons, immune cells and pigment cells would help elucidate any further similarities in cell function and gene expression, possibly providing insights into how novel cell types arise and develop unique phenotypes via the co-option of regulatory pathways.”And into the conclusions“The examples uncovered in this study serve to expand our understanding of evolution of cell types among these echinoderms, and more generally highlight ways that we can think about novelty and the limitations of experimental approaches to this understanding.”piwil3+ cell population in sea urchin likely points to a different developmental program and possible functional role of these cell types in these species.”“Novelty in both of these cases may be relative changes in timing or heterochrony of the cell state specification. Unless snRNA-seq covers a tight time course across the entirety of development and adult tissues, and possible environmental contexts we can never truly determine novelty. However heterochronic activation of a GRN in a new context can result in dramatic functional differences to the organism. The very early specification of a Reviewer #1 (Recommendations for the authors):While the manuscript is well written, particularly in the introduction and Discussion sections, the data presented show two major weaknesses, one concerning the validation of the clustering via whole month in situ hybridization (WMISH), and the other concerning the stages of S. purpuratus chosen as homologous of the ones analyzed in P. miniata.Regarding validation by WMISH, the general point is that this reviewer would like to see more embryos/gene validation, especially for the novel marker genes identified, and, in some cases a better resolution of the mRNA localization with the help of fluorescent in situ hybridization (FISH) or double FISH. For instance, in lines 220-230, the authors make a very interesting point on novel marker genes of their neural clusters, the synaptotagmin genes, which, as shown in the dot plot reported in Figure 5a, exhibit a differential expression marking different neural cell clusters. It would be very nice to see at least a couple of FISH experiments showing these differentiating markers in situ. Double FISH for this would be perfect. Similarly, a better resolution is necessary to convince the reader about the expression patterns reported in Figure 5 E-G and those in Figure 7.These in situs have been added and details are provided under “essential revisions” point 1.syt14 , syt7 , etv1  and got1 . These genes were identified as markers for the four neural clusters and now importantly indicate expression in patterns consistent with neural fate and a pattern that differs between the genes suggesting non overlapping of neural fates. We additionally added two color FISH for gcml + hoxd9a  and rab3 + gcml . These were important to show that hoxd9a and rab3 are coexpressed with some subsets of gcml+ cells. This supports the hypothesis suggested by the clustering of neural types in Figure 4 and Figure 5.A that gcml, rab3 and hoxd9a are are marker genes for the Sensory Neural cluster (Pmin CL 22) with some colocalization. There is not perfect overlap with these gene transcripts suggesting an even more complex neural cell fate than we have captured in our clustering.We have added single-color WMISH of 48 hpf gastrula stage embryos for the genes ets1 + tek  to support the hypothesis of Figure 4 and Figure 6.A and 6.B that ets1 and tek are coexpressed and are marker genes for Immune-like Mesenchyme (Pm CL 1). We show that they are exclusively overlapping.We also added images from double FISH of 48 hpf gastrula stage embryos for Figure 7.D-I are WMISH of later stages of development (72 hpf). The 48 hpf gastrula stage embryos show no morphological distinction between left and right so we performed in situs at a later stage where the left/right symmetry exists to show the expression in the coeloms. WMISH served to show this asymmetrical expression.We have added text into the results, figure legends, and discussion to provide details about these experiments and their interpretation.Another point regarding the WMISH is that there is no information in the paper regarding the stages analyzed. I assume that the embryos in Figure 5 and 6 correspond to the mid gastrula/ 40 h P. miniata embryos that were used as the latest time point in the construction of the single cell atlas, while the ones reported in Figure 7 correspond to later gastrula stages. However, this info has to be included in the figures and/or figure legends.Times and stages have been added to the figure legends.Figure 5 shows 48 hpf gastrula stage embryos.Figure 6 shows 48 hpf gastrula stage embryos.Figure 7 shows 72 hpf gastrula stage embryos.Regarding the stages of S. purpuratus chosen as homologous of the ones analyzed in P. miniata, as stated in the results and reported in Figure 3B the authors have used for P. miniate two blastula stages and one gastrula, 40h, which the authors correctly define as mid gastrula (line 171). However, strangely enough, they didn't include any gastrula stage for the corresponding sea urchin data sets, despite the fact the source they used for scRNA-seq data of S. purpuratus  includes two gastrula stages, one early and one late. On the contrary, the authors chose only hatched and mesenchyme blastula stages for sea urchins and justify this choice with a sentence that appears very controversial: "An extensive comparison of whole embryo RNA-seq time series data between S. purpuratus and P. miniata has shown that P. miniata develops at a faster rate and that S. purpuratus  are equivalent in developmental timing to the P.miniata time points used to generate the atlas here ." Besides the fact that P. miniata definitely develops at a slower and not faster rate compared to S. purpuratus, and that the staging reported in Gildor et al. is not the best reference to be used  also because the 40h P. miniata stage is not included in it, this statement is clearly contradictory even in respect to what stated by the authors of this paper in other parts of the manuscript. While I can agree with the fact that the so-called "mesenchyme blastula" in sea urchins can be considered as a very early gastrula (given that mesenchyme cells are ingressed in the blastocoel at this stage), no endoderm has ingressed at all at this stage. Therefore, it would be more appropriate to include in the S. purpuratus data sets a stage in which at least some part of the archenteron has ingressed. Indeed, the S. purpuratus early gastrula of the Foster et al. dataset (30h) appears as a bona fide equivalent of the 40h P. miniata gastrula stage. I believe the exclusion of a sea urchin gastrula stage in the comparative analysis introduces a strong bias and I strongly suggest redoing this analysis with the inclusion of this stage.We have now added the sea urchin early gastrula (24 hpf) stage sample from Foster et al. to the data and these changes to the manuscript are explained in “essential revisions” point 3.We have included this new time point in our analysis. In doing so we have re-clustered and re-annotated the sea urchin data and re-performed the integrated cluster analysis. This reanalysis has led to many small changes in summary statistics and specifics of clusters, and overlapping gene sets. However, the main findings of the work and conclusions of the analyses have not changed, as many of the differences in the re-analyses are of small effect.Thus, we recreated the sea urchin developmental atlas now using three time points . We now have 10,611 cells in the analysis rather than the original reported 7,370 and 23 vs 20 distinct clusters.S. purpuratus annotation in Figure 8 Supplemental Figure 1 and Figure 8 was updated to reflect the new cluster names.We then performed a new cluster annotation. The most significant difference with this new analysis is that we now have two rather than one pigment cell cluster, named Spur CL 12 Pigment Cells A and Spur CL 14 Pigment Cells B. Additionally small micromeres and muscle clusters were annotated. The We added additional text to more robustly explain the annotation of Spur CL 12 and 14, the two pigment cell clusters and Spur CL 13 as skeletogenic mesenchyme (previously PMCs).The text now reads:etv6, gata6, and etv1 mark only Spur CL 14 Pigment Cells B. ““Two clusters were identified as belonging to the pigment cell lineage (Spur CL 12 Pigment Cells A and Spur CL 14 Pigment Cells B). These clusters shared 107 markers including known markers of pigment cells in sea urchins, including gcml, polyketide synthase 1 (pks1), dimethylaniline monooxygenase [N-oxide-forming] 2 (fmo3), and sulfotransferase (sult) . We hypothesize Spur CL 14 Pigment cells B represents a population of pigment cells in an earlier state of differentiation than Spur CL 12 Pigment Cells A, because upstream transcription factors such as “The skeletogenic mesenchyme cluster (Spur CL 13) was marked by the expression of regulatory genes such as ets1, aristaless-like homeobox , ALX homeobox 1-like , T-box brain transcription factor 1 (tbr), ets variant transcription factor 6 , transcriptional regulator ERG (erg), hematopoietically-expressed homeobox protein HHEX homolog (hhex), TGFB induced factor homeobox 2-like (tgif2), arid3a, and vascular endothelial growth factor receptor 1 . As expected, this cluster was also marked by genes involved in biomineralization, including 27 kDa primary mesenchyme-specific spicule protein (pm27 a), spicule matrix protein SM50 (sm50), and mesenchyme-specific cell surface glycoprotein (msp130).”While the annotation of the immune cell cluster did not change, we added additional text to more clearly explain the rationale for this cluster.erg, gata3, gata6, hhex, prox1, ets1, as well as genes that have been linked to immune response in echinoderms, like tek, and srcr. Other genes implicated in immune function in other systems such as two allograft inflammatory factor 1-like genes , C-type lectin domain-containing protein 141-like, and duox1 are also found in this cluster .""Another cluster of particular interest is Spur CL 17 Oral Non-skeletogenic mesenchyme, the lineage fated to become immune coelomocyte cells. This cluster is marked by genes known to be involved in the specification of this lineage like P. miniata and S. purpuratus datasets and updated the analyses in Figures 8-12.With the new cluster analysis we then re-performed the integration between the We have used the same methodology and pipelines as previously. As requested by Reviewer 3 we have provided the code used to create and integrate the atlases in Supplementary file 7.The integrated analysis with the additional data changed some of the values and numbers of overlapping genes but no substantive differences were found in our conclusions. We identified 15 rather than 17 clusters in this new dataset.The wording has been changed in multiple places in the Results section to reflect the changed numbering of the clusters (simply a result of reclustering) and the % and numbers of overlapping genes and changes in significance. Summarized below, these differences were only slight and did not change the conclusions.Figure 9 shows that Int CL 14 is enriched with cells from Spur CL 22 Piwil3+ cells and Undetermined with very few Pmin CL 19 Dividing Ectoderm.P. miniata but now with stronger support (RF 5.2 compared to previous 5.0) although the number of genes overlapping decreased somewhat.Figure 10 shows the integrated clusters with separate contributions from Pigment A and B. Int CL 8 and 12 show contributions from both Pigment A and B (Spur). Contributions from Pmin to Int CL 8 are majority Immune-like Mesenchyme and Int CL 12 shows contributions from early, mixed and sensory neural clusters from S. purpuratus Skeletogenic Mesenchyme and P. miniata Right Coelom and Foregut. Our conclusions here therefore remain unchanged also.Figure 11 shows that Int CL 7 is primarily P. miniata Left Coelom with no predominant corresponding contributions from S. purpuratus. This is the same result also.Figure 12 shows Int CL 10 is primarily composed of Another general, important, although minor and easy to fix point, is the nomenclature used in the manuscript:– First of all, about gene names: throughout the manuscript, text, figures, and figure legends, a plethora of different styles are used, even within the same list (such as in the y axis of dot plots), including UPPERCASE, lowercase , capitalised words, with or without initials of species names … please choose a style and stick to it, because it is very confusingP. miniata gene symbols for 1:1 orthologs to S. purpuratus therefore the NCBI-annotated gene symbols or locus IDs (LOCentrezid#) are provided. Supplementary Table 1 lists gene symbols and names from Echinobase, NCBI and Ensembl for all genes mentioned in this manuscript.We have followed the Echinobase Gene Nomenclature Guidelines where possible (https://www.echinobase.org/entry/static/gene/geneNomenclature.jsp). Briefly, gene symbols and names will be lowercase and italicized and follow HGNC guidelines. Echinobase currently has – Second, about cluster names: some choices are in my opinion confusing as they refer to a structure/ developmental stage that is not present in the data set; this is the case for example of (left and right) "coelom", which should be better referred to as "coelomic mesoderm" given that coelomic pouches appears much later compared to the stages analysed; or the case of the "immune mesenchyme", which should be better referred to as immune-like given that immune cells, to my knowledge, were never functionally identified in the P. miniata larva; I understand that for the sake of simplicity "immune mesenchyme" could be a better name, but this assumption should be clearly specified and definitely the short name "immune cluster" used in abstract and conclusions is not appropriateP. miniata atlas and PGCs to Piwil3+ cells in the S. purpuratus atlas.We agree that this can be misleading, but also we want to convey our best understanding of the functional types in these clusters. We have throughput therefore softened naming for example from immune to immune-like in the Added text:piwi-like RNA-mediated gene silencing 3 (piwil3). It is also marked by the gene nanos C2HC-type zinc finger 2-like (nanos2l), which has been shown to play a role in Primordial Germ Cell (PGC) specification . However, it must be noted that while this cluster could potentially be PGCs, further studies are needed to determine if this population is simply a piwil3 positive group of cells with stem cell like properties or a genuine germ cell lineage.”“Spur CL 22 is annotated as piwil3+ Cells, because they are marked by the expression of ”“Pmin CL 21 is a very spatially distinct cluster in the UMAP dimensional reduction plot  and was identified as Immune-like Mesenchyme.For the coelom clusters we are confident in the left/right symmetry based on the WMISH data of the cluster markers shown in Figure 7 of a 72 hpf late gastrula stage where the left and right coeloms are clearly distinguishable.– Another issue concerns the use of "primary mesenchyme cells", which is quite an obsolete terminology, not very informative, and really meaningful only within the sea urchin community. Skeletogenic mesenchyme appears more appropriate for a wider audience.Thank you for this comment, we have changed primary mesenchyme cells (PMCs) to skeletogenic mesenchyme throughout the manuscript.Two other important changes that would make the manuscript more readable are:– First, about the use of the adjusted p-value: reporting the adjusted p-value for each of the marker genes identifying each cluster appears as redundant. If a gene is a gene marker it means that it passed the adjusted p-value test and if the reader wants to know more about those figures these are all included in the tables. The result session is uselessly heavy and boring to read with all these parentheses. I strongly suggest eliminating these details in the text.We have removed these values throughout the text.– Second, in the analysis of the contribution of cells for each integrated cluster, the list of all contributions, even if they account for just one cell, again appears redundant, and the text become difficult to read  and eventually even confusing to what is the actual identity of the cluster. I would propose to decide on a certain cut-off  and list in the text only the contribution above that threshold, leaving to a supplementary figure the detailed contribution of sea urchin or sea star cluster to the integrated cluster, as done for Figure 9 and 10.We have removed these throughout the text.Reviewer #2 (Recommendations for the authors):1. To strengthen  the claims resulting from the integration of sea urchin and sea star datasets, I recommend that the authors complement their analysis with a method that is not limited to the 1:1 orthologs between sea urchin and sea star. I would suggest, for example, the algorithm based on self-assembling manyfold (SAMap), which has been successfully employed by Musser et al. (Science 2021) to compare two sponge species  and has been described in detail by Tarashansky et al. (eLife 2021), where single-cell datasets from animals across different phyla were integrated.We discuss this under “essential revisions” point 2 where we justify our decision to maintain the analysis on 1:1 orthologs and acknowledge this by changing the title of the paper to reflect our focus on this subset of genes.2. I am not sure that this is possible, but if it is, I would suggest visualizing the data in Supplementary Table 3 in a similar way as Figure 2B of Musser et al. (Science 2021). This would make it easier to follow the description of the integrated clusters.While we appreciate this reviewer's point after discussion among the authors, we opted for our visualization method used in Figures 9 -12 because we determined a ribbon plot linking 3 clustering methods was ultimately too complex and chaotic to effectively communicate our findings. Therefore, we opted to use bar plots to examine in depth the integrated clusters of interest while providing all the cluster composition information in a supplementary table that is more easily navigated.3. In Figure 6, the authors very nicely show that genes enriched in Pmin Cl 21 are expressed in mesenchymal populations. In particular, VEGFR1L is shown to be expressed in a population that accumulates around the base of the archenteron , similar to the localization of PMCs in S. purpuratus. According to Figure 6.D, my impression is that tek (another gene enriched in Pmin CL 21) is also expressed in the population of cells around the archenteron. Although less clear, the same might be true for ets1 . Could the authors explain whether these (and perhaps other) markers of Pmin CL 21 are expressed in the mesenchymal population around the archenteron? If they are indeed expressed in this population, can the authors comment on the significance of this?We have added more in situs and double in situs and have added text as now explained in “essential revisions” point 1.We have also added the following text:"This is an intriguing finding as it suggests that the patterning and migration cues that direct skeletogenic mesoderm cells in sea urchins are also in place in sea stars. This indicates that perhaps only the very final activation of the skeletogenesis program is present in sea urchins and that otherwise the cell lineage between these species is similar. This finding is also consistent with the findings from Cidaroid sea urchins that show that skeletogenic cells arise from a population of mesenchyme that migrates from the top of the archenteron (Erkenbrack and Davidson 2015)4. An interesting finding of the study by Meyer et al. is the overlap between pigment cell cluster transcriptome in sea urchins and the immune and neural clusters in sea stars. Would it be relevant to discuss this finding in the light of the hypothesis by Klimovich and Bosch  that the nervous system and the immune system have a common evolutionary origin?We agree that this is a very interesting finding. We have added the following text and reference into the discussion:"This supports previous work hypothesizing the close evolutionary relationship and possible functional ancestral interdependence between the immune and nervous systems ."Reviewer #3 (Recommendations for the authors):1. Combing scRNAseq and snRNAseq data could introduce biases. Several studies have noted that seemingly subtle differences like sequencing platform or even batch can produce artificial signals within scRNAseq data, sometimes even swamping species effects . Thus, it's not clear to me that single-nucleus and single-cell RNAseq data can be combined without introducing systematic biases in the representation of transcripts present. For instance, mRNAs with short half-lives would likely be over-represented in snRNAseq compared with scRNAseq. I suspect that conclusions about overlapping clusters and the presence/absence of clusters reported in this study are generally secure. However, differences in timing and in proportions of cells in a given cluster between species may be more problematic. If the authors are aware of any empirical data that support direct comparisons between snRNAseq and scRNA seq, citing these would build confidence in the results. At a minimum, the authors should state more clearly that they are making an assumption when combining different data types in this study, and also comment on how this might affect the interpretation of results.We have provided comments on this in Essential Revisions under point 2.After careful consideration we have decided to maintain our focus on 1:1 orthologs for our mapping as we believe this gives more robust results SAMap was developed for mouse/human integration (~100 Mya) and will be limited for sea star/sea urchin integration (~400 Mya). First it should be noted that our orthology mapping between these two species has been extensively performed using the principles of the Alliance of Genome Resources that at least three DIOPT tools should agree on the orthology. We have used BLAST, phylogenetic-based tools  as published in “Integration of 1: 1 orthology maps and updated datasets into Echinobase, Foley et al. Database, 2021”. We think mapping using orthologous genes provides a more reliable comparison for our integrated echinoderm datasets.S. purpuratus Piwil3+ Cells. The near absence of sea star nuclei in Int CL 14 is supported by previous gene expression studies  which show that piwil3, nanos2l and pum2 orthologs/paralogs are not expressed in these stages in P. miniata.We also show that several results from this integrated mapping corroborate observations from other data types to support that we are making meaningful findings. For example, we identify a sea urchin specific integrated cluster (Int CL 14) that corresponds to To reflect our focus on ortholog driven clustering we have changed the title to of the paper.New hypotheses of cell type diversity and novelty from orthology-driven comparative single cell and nuclei transcriptomics in echinoderms.We also comment here on the differences between single nuclei and single cell approaches. We carefully considered the approaches to isolation and thought for this and future studies that single nuclei sequencing would be less biased, robust and easier to achieve reliability; especially as researchers look to later and more complex stages and tissues. We therefore took great care to evaluate the two procedures since we do rely on comparison to scRNA sequencing. Figure 2 and Supplementary file 5 strongly support that there are no significant differences between approaches and that they can be reliably compared. We showed bulk RNA profiles are not significantly different when obtained from whole embryos, isolated cells or isolated nuclei  and that the single nuclei atlas prepared in our studies was highly similar to the atlas previously prepared from single cells .We added the following sentence in the fourth paragraph of the results to more thoroughly clarify our position on this."These results thus strongly support that single nuclei and single-cell RNA isolation and sequencing each resulted in appropriate representative clusters thus providing confidence that either isolation method was an effective way to establish cell clusters. snRNA-seq is more likely to capture earlier transcriptional responses than scRNA-seq, but such timing differences are much smaller than the sampling times here and unlikely to affect our analyses."2. Sampling only embryonic stages limits the ability to confidently identify the absence of a cell type. In an adult organ composed mostly of differentiated cells, determining the presence/absence of a cell type is fairly straightforward, assuming fairly representative cells/nuclei and reasonably high-quality data. In contrast, a gastrula-stage embryo contains very few, if indeed any, fully differentiated cells. Further, it is well established that homologous cells can differentiate at different stages of development in different species. Thus, a cell type might appear to be absent in the gastrula of one species and present in another simply because it has not yet begun to differentiate in the first species. It's a situation where the absence of evidence is not evidence of absence. This makes it impossible to reach any firm conclusions about which cell types might be unique to seastars or sea urchins. The authors are clearly aware of this limitation, e.g. lines 446-447: "thus making Int CL 16 the only S. purpuratus-specific cell type at this stage of development" (emphasis added). Here and elsewhere, the authors are good about stating this caveat. However, these statements come across as each applying to a specific case rather than a general limitation that applies to all cell types. This more general caveat should be clearly stated, as it is key to understanding what can and can't be learned from the data. This is particularly important because many non-specialists reading this paper will get lost in, or simply skip over, the numerous details presented for any given cell type and could easily miss both the individual caveats and the more general point that in no case is it possible to conclude that a particular cell type is present or absent from one species or the other.We agree with the reviewer that it can be extremely difficult to demonstrate that a cell type is entirely absent in one species and hence truly novel in the other. We think the novelty we can assess here is developmental timing and context. We have tried to be more clear about the role of timing differences in what we have found, which was also suggested by reviewer 2.Text in intro:“Their larval forms in particular have several cell types that, based on developmental, morphological, and functional criteria, are considered to be novel ."Text added to discussion:“Further work examining these cell types across species of echinoderms is needed to better define these novelties and allow for a more comprehensive characterization of their evolutionary timing.”“Additionally, work comparing fully differentiated neurons, immune cells and pigment cells would help elucidate any further similarities in cell function and gene expression, possibly providing insights into how novel cell types arise and develop unique phenotypes via the co-option of regulatory pathways.”3. Data from only two species limits the ability to confidently identify the direction of evolutionary change. Even if we accept that some cells are truly present/absent, figuring out whether these cases represent novelties or losses is problematic when only examining two species: is it a gain in the species with the unique cell type or a loss in the species without? The usual approach to answering this question is getting data from an outgroup species, but in the present case, the data simply aren't available. The authors are not always clear about this limitation, for example (lines 462-463) "this cluster can be considered novel to S. purpuratus". More accurate would be to say that it is uniquely present rather than novel. It could also be the case that the ancestral echinoderm had an early specification of germ cells and that this has been lost in seastars and perhaps other echinoderms. Here again, the limitation is a general one rather than specific to this particular cell type and should be clearly stated as such. Again, this is key to understanding what can and cannot be inferred from the data.We agree with this reviewer and have worked to be more clear and not assume direction and be more careful about what we have discussed as novel versus simply missing at this time point. We have addressed this point also in the essential revisions.We have added the following text:S. purpuratus within our sampled window.”“Therefore, this cluster can be considered novel to "Further work examining these cell types across species of echinoderms is needed to better define these novelties and allow for a more comprehensive characterization of their evolutionary timing."A suggestion: There may be an opportunity to make a different kind of evolutionarily inference that is more secure, namely to identify evolutionary differences in the timing of differentiation of specific cell types. These observations are interesting because they have implications for the evolution of cell fate specification mechanisms. For instance, germ cells are specified clonally and very early in sea urchins and the molecular mechanisms and cellular processes responsible are well studied. The absence of any cells with a similar transcriptional profile in seastar embryos implies no that early segregation of the germ line does not occur, and thus it is unlikely that localized maternal determinants are responsible. Given the wealth of information available about cell fate specification mechanisms in sea urchins, inferences about evolutionary differences could be made about other cell types as well. This seems to me a missed opportunity for the authors to highlight some interesting and informative aspects of their results.We have added some new text into the “Essential Revisions #4”. We agree that timing changes are very important in our system and should be highlighted better in our text.We have added some discussion about differences in the timing of germ line specification while also being careful to take into account the points made by Reviewer #1 about the lack of confidence in our identification in PGCs, which we have re-annotated as piwi3l+ Cells:“We hypothesize that this could be caused by the differences in the timing of PGC formation. Previous work has shown that the genes typically used to identify them, such as piwi nanos, and pumillo, only begin to show cell-type-specific expression in P. miniata by the late gastrula stage when the posterior endocoelom forms , a time point that lies outside our sampling window. Therefore, this cluster can be considered novel to S. purpuratus within our sampled window. “