The genomic and cellular basis of biosynthetic innovation in rove beetles.

How evolution at the cellular level potentiates macroevolutionary change is central to understanding biological diversification. The >66,000 rove beetle species (Staphylinidae) form the largest metazoan family. Combining genomic and cell type transcriptomic insights spanning the largest clade, Aleocharinae, we retrace evolution of two cell types comprising a defensive gland-a putative catalyst behind staphylinid megadiversity. We identify molecular evolutionary steps leading to benzoquinone production by one cell type via a mechanism convergent with plant toxin release systems, and synthesis by the second cell type of a solvent that weaponizes the total secretion. This cooperative system has been conserved since the Early Cretaceous as Aleocharinae radiated into tens of thousands of lineages. Reprogramming each cell type yielded biochemical novelties enabling ecological specialization-most dramatically in symbionts that infiltrate social insect colonies via host-manipulating secretions. Our findings uncover cell type evolutionary processes underlying the origin and evolvability of a beetle chemical innovation.

Analysis and Visualization of Single-Cell Sequencing Data with Scanpy and MetaCell: A Tutorial.

The emergence and development of single-cell RNA sequencing (scRNA-seq) techniques enable researchers to perform large-scale analysis of the transcriptomic profiling at cell-specific resolution. Unsupervised clustering of scRNA-seq data is central for most studies, which is essential to identify novel cell types and their gene expression logics. Although an increasing number of algorithms and tools are available for scRNA-seq analysis, a practical guide for users to navigate the landscape remains underrepresented. This chapter presents an overview of the scRNA-seq data analysis pipeline, quality control, batch effect correction, data standardization, cell clustering and visualization, cluster correlation analysis, and marker gene identification. Taking the two broadly used analysis packages, i.e., Scanpy and MetaCell, as examples, we provide a hands-on guideline and comparison regarding the best practices for the above essential analysis steps and data visualization. Additionally, we compare both packages and algorithms using a scRNA-seq dataset of the ctenophore Mnemiopsis leidyi, which is representative of one of the earliest animal lineages, critical to understanding the origin and evolution of animal novelties. This pipeline can also be helpful for analyses of other taxa, especially prebilaterian animals, where these tools are under development (e.g., placozoan and Porifera).

Brief History of Ctenophora.

Ctenophores are the descendants of the earliest surviving lineage of ancestral metazoans, predating the branch leading to sponges (Ctenophore-first phylogeny). Emerging genomic, ultrastructural, cellular, and systemic data indicate that virtually every aspect of ctenophore biology as well as ctenophore development are remarkably different from what is described in representatives of other 32 animal phyla. The outcome of this reconstruction is that most system-level components associated with the ctenophore organization result from convergent evolution. In other words, the ctenophore lineage independently evolved as high animal complexities with the astonishing diversity of cell types and structures as bilaterians and cnidarians. Specifically, neurons, synapses, muscles, mesoderm, through gut, sensory, and integrative systems evolved independently in Ctenophora. Rapid parallel evolution of complex traits is associated with a broad spectrum of unique ctenophore-specific molecular innovations, including alternative toolkits for making an animal. However, the systematic studies of ctenophores are in their infancy, and deciphering their remarkable morphological and functional diversity is one of the hot topics in biological research, with many anticipated surprises.

Molecular profiling of sponge deflation reveals an ancient relaxant-inflammatory response.

A hallmark of animals is the coordination of whole-body movement. Neurons and muscles are central to this, yet coordinated movements also exist in sponges that lack these cell types. Sponges are sessile animals with a complex canal system for filter-feeding. They undergo whole-body movements resembling "contractions" that lead to canal closure and water expulsion. Here, we combine live 3D optical coherence microscopy, pharmacology, and functional proteomics to elucidate the sequence and detail of shape changes, the tissues and molecular physiology involved, and the control of these movements. Morphometric analysis and targeted perturbation suggest that the movement is driven by the relaxation of actomyosin stress fibers in epithelial canal cells, which leads to whole-body deflation via collapse of the incurrent and expansion of the excurrent canal system. Thermal proteome profiling and quantitative phosphoproteomics confirm the control of cellular relaxation by an Akt/NO/PKG/PKA pathway. Agitation-induced deflation leads to differential phosphorylation of proteins forming epithelial cell junctions, implying their mechanosensitive role. Unexpectedly, untargeted metabolomics detect a concomitant decrease in antioxidant molecules during deflation, reflecting an increase in reactive oxygen species. Together with the secretion of proteinases, cytokines, and granulin, this indicates an inflammation-like state of the deflating sponge reminiscent of vascular endothelial cells experiencing oscillatory shear stress. These results suggest the conservation of an ancient relaxant-inflammatory response of perturbed fluid-carrying systems in animals and offer a possible mechanism for whole-body coordination through diffusible paracrine signals and mechanotransduction.

Molecular profiling of sponge deflation reveals an ancient relaxant-inflammatory response.

A hallmark of animals is the coordination of whole-body movement. Neurons and muscles are central to this, yet coordinated movements also exist in sponges that lack these cell types. Sponges are sessile animals with a complex canal system for filter-feeding. They undergo whole-body movements resembling "contractions" that lead to canal closure and water expulsion. Here, we combine 3D optical coherence microscopy, pharmacology, and functional proteomics to elucidate anatomy, molecular physiology, and control of these movements. We find them driven by the relaxation of actomyosin stress fibers in epithelial canal cells, which leads to whole-body deflation via collapse of the incurrent and expansion of the excurrent system, controlled by an Akt/NO/PKG/A pathway. A concomitant increase in reactive oxygen species and secretion of proteinases and cytokines indicate an inflammation-like state reminiscent of vascular endothelial cells experiencing oscillatory shear stress. This suggests an ancient relaxant-inflammatory response of perturbed fluid-carrying systems in animals.

Evolution of homology: From archetype towards a holistic concept of cell type.

The concept of homology lies in the heart of comparative biological science. The distinction between homology as structure and analogy as function has shaped the evolutionary paradigm for a century and formed the axis of comparative anatomy and embryology, which accept the identity of structure as a ground measure of relatedness. The advent of single-cell genomics overturned the classical view of cell homology by establishing a backbone regulatory identity of cell types, the basic biological units bridging the molecular and phenotypic dimensions, to reveal that the cell is the most flexible unit of living matter and that many approaches of classical biology need to be revised to understand evolution and diversity at the cellular level. The emerging theory of cell types explicitly decouples cell identity from phenotype, essentially allowing for the divergence of evolutionarily related morphotypes beyond recognition, as well as it decouples ontogenetic cell lineage from cell-type phylogeny, whereby explicating that cell types can share common descent regardless of their structure, function or developmental origin. The article succinctly summarizes current progress and opinion in this field and formulates a more generalistic view of biological cell types as avatars, transient or terminal cell states deployed in a continuum of states by the developmental programme of one and the same omnipotent cell, capable of changing or combining identities with distinct evolutionary histories or inventing ad hoc identities that never existed in evolution or development. It highlights how the new logic grounded in the regulatory nature of cell identity transforms the concepts of cell homology and phenotypic stability, suggesting that cellular evolution is inherently and massively network-like, with one-to-one homologies being rather uncommon and restricted to shallower levels of the animal tree of life.

Evolutionary assembly of cooperating cell types in an animal chemical defense system.

How the functions of multicellular organs emerge from the underlying evolution of cell types is poorly understood. We deconstructed evolution of an organ novelty: a rove beetle gland that secretes a defensive cocktail. We show how gland function arose via assembly of two cell types that manufacture distinct compounds. One cell type, comprising a chemical reservoir within the abdomen, produces alkane and ester compounds. We demonstrate that this cell type is a hybrid of cuticle cells and ancient pheromone and adipocyte-like cells, executing its function via a mosaic of enzymes from each parental cell type. The second cell type synthesizes benzoquinones using a chimera of conserved cellular energy and cuticle formation pathways. We show that evolution of each cell type was shaped by coevolution between the two cell types, yielding a potent secretion that confers adaptive value. Our findings illustrate how cooperation between cell types arises, generating new, organ-level behaviors.

Ciliary photoreceptors in sea urchin larvae indicate pan-deuterostome cell type conservation.

BACKGROUND: The evolutionary history of cell types provides insights into how morphological and functional complexity arose during animal evolution. Photoreceptor cell types are particularly broadly distributed throughout Bilateria; however, their evolutionary relationship is so far unresolved. Previous studies indicate that ciliary photoreceptors are homologous at least within chordates, and here, we present evidence that a related form of this cell type is also present in echinoderm larvae. RESULTS: Larvae of the purple sea urchin Strongylocentrotus purpuratus have photoreceptors that are positioned bilaterally in the oral/anterior apical neurogenic ectoderm. Here, we show that these photoreceptors express the transcription factor Rx, which is commonly expressed in ciliary photoreceptors, together with an atypical opsin of the GO family, opsin3.2, which localizes in particular to the cilia on the cell surface of photoreceptors. We show that these ciliary photoreceptors express the neuronal marker synaptotagmin and are located in proximity to pigment cells. Furthermore, we systematically identified additional transcription factors expressed in these larval photoreceptors and found that a majority are orthologous to transcription factors expressed in vertebrate ciliary photoreceptors, including Otx, Six3, Tbx2/3, and Rx. Based on the developmental expression of rx, these photoreceptors derive from the anterior apical neurogenic ectoderm. However, genes typically involved in eye development in bilateria, including pax6, six1/2, eya, and dac, are not expressed in sea urchin larval photoreceptors but are instead co-expressed in the hydropore canal. CONCLUSIONS: Based on transcription factor expression, location, and developmental origin, we conclude that the sea urchin larval photoreceptors constitute a cell type that is likely homologous to the ciliary photoreceptors present in chordates.

Mapping single-cell atlases throughout Metazoa unravels cell type evolution.

Comparing single-cell transcriptomic atlases from diverse organisms can elucidate the origins of cellular diversity and assist the annotation of new cell atlases. Yet, comparison between distant relatives is hindered by complex gene histories and diversifications in expression programs. Previously, we introduced the self-assembling manifold (SAM) algorithm to robustly reconstruct manifolds from single-cell data (Tarashansky et al., 2019). Here, we build on SAM to map cell atlas manifolds across species. This new method, SAMap, identifies homologous cell types with shared expression programs across distant species within phyla, even in complex examples where homologous tissues emerge from distinct germ layers. SAMap also finds many genes with more similar expression to their paralogs than their orthologs, suggesting paralog substitution may be more common in evolution than previously appreciated. Lastly, comparing species across animal phyla, spanning sponge to mouse, reveals ancient contractile and stem cell families, which may have arisen early in animal evolution.

Evolution of Multicellular Complexity in The Dictyostelid Social Amoebas.

Multicellularity evolved repeatedly in the history of life, but how it unfolded varies greatly between different lineages. Dictyostelid social amoebas offer a good system to study the evolution of multicellular complexity, with a well-resolved phylogeny and molecular genetic tools being available. We compare the life cycles of the Dictyostelids with closely related amoebozoans to show that complex life cycles were already present in the unicellular common ancestor of Dictyostelids. We propose frost resistance as an early driver of multicellular evolution in Dictyostelids and show that the cell signalling pathways for differentiating spore and stalk cells evolved from that for encystation. The stalk cell differentiation program was further modified, possibly through gene duplication, to evolve a new cell type, cup cells, in Group 4 Dictyostelids. Studies in various multicellular organisms, including Dictyostelids, volvocine algae, and metazoans, suggest as a common principle in the evolution of multicellular complexity that unicellular regulatory programs for adapting to environmental change serve as "proto-cell types" for subsequent evolution of multicellular organisms. Later, new cell types could further evolve by duplicating and diversifying the "proto-cell type" gene regulatory networks.

Tracing cell-type evolution by cross-species comparison of cell atlases.

Cell types are the basic building units of multicellular life, with extensive diversities. The evolution of cell types is a crucial layer of comparative cell biology but is thus far not comprehensively studied. We define a compendium of cell atlases using single-cell RNA-seq (scRNA-seq) data from seven animal species and construct a cross-species cell-type evolutionary hierarchy. We present a roadmap for the origin and diversity of major cell categories and find that muscle and neuron cells are conserved cell types. Furthermore, we identify a cross-species transcription factor (TF) repertoire that specifies major cell categories. Overall, our study reveals conservation and divergence of cell types during animal evolution, which will further expand the landscape of comparative genomics.

The origin of animals: an ancestral reconstruction of the unicellular-to-multicellular transition.

How animals evolved from a single-celled ancestor, transitioning from a unicellular lifestyle to a coordinated multicellular entity, remains a fascinating question. Key events in this transition involved the emergence of processes related to cell adhesion, cell-cell communication and gene regulation. To understand how these capacities evolved, we need to reconstruct the features of both the last common multicellular ancestor of animals and the last unicellular ancestor of animals. In this review, we summarize recent advances in the characterization of these ancestors, inferred by comparative genomic analyses between the earliest branching animals and those radiating later, and between animals and their closest unicellular relatives. We also provide an updated hypothesis regarding the transition to animal multicellularity, which was likely gradual and involved the use of gene regulatory mechanisms in the emergence of early developmental and morphogenetic plans. Finally, we discuss some new avenues of research that will complement these studies in the coming years.

A flagellate-to-amoeboid switch in the closest living relatives of animals.

Amoeboid cell types are fundamental to animal biology and broadly distributed across animal diversity, but their evolutionary origin is unclear. The closest living relatives of animals, the choanoflagellates, display a polarized cell architecture (with an apical flagellum encircled by microvilli) that resembles that of epithelial cells and suggests homology, but this architecture differs strikingly from the deformable phenotype of animal amoeboid cells, which instead evoke more distantly related eukaryotes, such as diverse amoebae. Here, we show that choanoflagellates subjected to confinement become amoeboid by retracting their flagella and activating myosin-based motility. This switch allows escape from confinement and is conserved across choanoflagellate diversity. The conservation of the amoeboid cell phenotype across animals and choanoflagellates, together with the conserved role of myosin, is consistent with homology of amoeboid motility in both lineages. We hypothesize that the differentiation between animal epithelial and crawling cells might have evolved from a stress-induced switch between flagellate and amoeboid forms in their single-celled ancestors.

NvPOU4/Brain3 Functions as a Terminal Selector Gene in the Nervous System of the Cnidarian Nematostella vectensis.

Terminal selectors are transcription factors that control the morphological, physiological, and molecular features that characterize distinct cell types. Here, we show that, in the sea anemone Nematostella vectensis, NvPOU4 is expressed in post-mitotic cells that give rise to a diverse set of neural cell types, including cnidocytes and NvElav1-expressing neurons. Morphological analyses of NvPOU4 mutants crossed to transgenic reporter lines show that the loss of NvPOU4 does not affect the initial specification of neural cells. Transcriptomes derived from the mutants and from different neural cell populations reveal that NvPOU4 is required for the execution of the terminal differentiation program of these neural cells. These findings suggest that POU4 genes have ancient functions as terminal selectors for morphologically and functionally disparate types of neurons and they provide experimental support for the relevance of terminal selectors for understanding the evolution of cell types.

Comparison of differentiation gene batteries for migratory mechanosensory neurons across bilaterians.

In embryos of distantly related bilaterian phyla, their lateral neural borders give rise to the peripheral nervous system elements, including various mechanosensory cells derived from migratory precursors, such as hair cells and dorsal root ganglion (DRG) neurons in vertebrates, bipolar tail neuron (BTN) in Ciona, chordotonal organ in Drosophila, and AVM/PVM in Caenorhabditis elegans. Developmental genetics studies had revealed a couple of transcription factors (TFs) regulating differentiation of mechanosensory cells shared by vertebrates and arthropods. However, unbiased systematic profiling of regulators is needed to demonstrate conservation of differentiation gene batteries for mechanosensory cells across bilaterians. At first, we observed that in both C. elegans Q neuroblasts and Drosophila lateral neuroectoderm, conserved NPB specifier Msx/vab-15 regulates Atoh1/lin-32, supporting the homology of mechanosensory neuron development in lateral neural border lineage of Ecdysozia. So we used C. elegans as a protostomia model. Single-cell resolution expression profiling of TFs and genetic analysis revealed a differentiation gene battery (Atonh1/lin-32, Drg11/alr-1, Gfi1/pag-3, Lhx5/mec-3, and Pou4/unc-86) for AVM/PVM mechanosensory neurons. The worm-gene battery significantly overlaps with both that of placode-derived Atonh1/lin-32-dependent hair cells and that of NPB-derived Neurogenin-dependent DRG neurons in vertebrates, supporting the homology of molecular mechanisms underlying the differentiation of neural border-derived mechanosensory cells between protostome and deuterostome. At last, Ciona BTN, the homolog of vertebrate DRG, also expresses Atonh1/lin-32, further supporting the homology notion and indicating a common origin of hair cells and DRG in vertebrate lineage.

Molecular evolution of gland cell types and chemical interactions in animals.

Across the Metazoa, the emergence of new ecological interactions has been enabled by the repeated evolution of exocrine glands. Specialized glands have arisen recurrently and with great frequency, even in single genera or species, transforming how animals interact with their environment through trophic resource exploitation, pheromonal communication, chemical defense and parental care. The widespread convergent evolution of animal glands implies that exocrine secretory cells are a hotspot of metazoan cell type innovation. Each evolutionary origin of a novel gland involves a process of 'gland cell type assembly': the stitching together of unique biosynthesis pathways; coordinated changes in secretory systems to enable efficient chemical release; and transcriptional deployment of these machineries into cells constituting the gland. This molecular evolutionary process influences what types of compound a given species is capable of secreting, and, consequently, the kinds of ecological interactions that species can display. Here, we discuss what is known about the evolutionary assembly of gland cell types and propose a framework for how it may happen. We posit the existence of 'terminal selector' transcription factors that program gland function via regulatory recruitment of biosynthetic enzymes and secretory proteins. We suggest ancestral enzymes are initially co-opted into the novel gland, fostering pleiotropic conflict that drives enzyme duplication. This process has yielded the observed pattern of modular, gland-specific biosynthesis pathways optimized for manufacturing specific secretions. We anticipate that single-cell technologies and gene editing methods applicable in diverse species will transform the study of animal chemical interactions, revealing how gland cell types are assembled and functionally configured at a molecular level.

Chromophore-Independent Roles of Opsin Apoproteins in Drosophila Mechanoreceptors.

Rhodopsins, the major light-detecting molecules of animal visual systems [1], consist of opsin apoproteins that covalently bind a retinal chromophore with a conserved lysine residue [1, 2]. In addition to capturing photons, this chromophore contributes to rhodopsin maturation [3, 4], trafficking [3, 4], and stabilization [5], and defects in chromophore synthesis and recycling can cause dysfunction of the retina and dystrophy [6-9]. Indications that opsin apoproteins alone might have biological roles have come from archaebacteria and platyhelminths, which present opsin-like proteins that lack the chromophore binding site and are deemed to function independently of light [10, 11]. Light-independent sensory roles have been documented for Drosophila opsins [12-15], yet also these unconventional opsin functions are thought to require chromophore binding [12, 13, 15]. Unconjugated opsin apoproteins act as phospholipid scramblases in mammalian photoreceptor disks [16], yet chromophore-independent roles of opsin apoproteins outside of eyes have, to the best of our knowledge, hitherto not been described. Drosophila chordotonal mechanoreceptors require opsins [13, 15], and we find that their function remains uncompromised by nutrient carotenoid depletion. Disrupting carotenoid uptake and cleavage also left the mechanoreceptors unaffected, and manipulating the chromophore attachment site of the fly's major visual opsin Rh1 impaired photoreceptor, but not mechanoreceptor, function. Notwithstanding this chromophore independence, some proteins that process and recycle the chromophore in the retina are also required in mechanoreceptors, including visual cycle components that recycle the chromophore upon its photoisomerization. Our results thus establish biological function for unconjugated opsin apoproteins outside of eyes and, in addition, document chromophore-independent roles for chromophore pathway components.

Cnidarian Cell Type Diversity and Regulation Revealed by Whole-Organism Single-Cell RNA-Seq.

The emergence and diversification of cell types is a leading factor in animal evolution. So far, systematic characterization of the gene regulatory programs associated with cell type specificity was limited to few cell types and few species. Here, we perform whole-organism single-cell transcriptomics to map adult and larval cell types in the cnidarian Nematostella vectensis, a non-bilaterian animal with complex tissue-level body-plan organization. We uncover eight broad cell classes in Nematostella, including neurons, cnidocytes, and digestive cells. Each class comprises different subtypes defined by the expression of multiple specific markers. In particular, we characterize a surprisingly diverse repertoire of neurons, which comparative analysis suggests are the result of lineage-specific diversification. By integrating transcription factor expression, chromatin profiling, and sequence motif analysis, we identify the regulatory codes that underlie Nematostella cell-specific expression. Our study reveals cnidarian cell type complexity and provides insights into the evolution of animal cell-specific genomic regulation.

Phenotypic Convergence: Distinct Transcription Factors Regulate Common Terminal Features.

Transcription factors regulate the molecular, morphological, and physiological characteristics of neurons and generate their impressive cell-type diversity. To gain insight into the general principles that govern how transcription factors regulate cell-type diversity, we used large-scale single-cell RNA sequencing to characterize the extensive cellular diversity in the Drosophila optic lobes. We sequenced 55,000 single cells and assigned them to 52 clusters. We validated and annotated many clusters using RNA sequencing of FACS-sorted single-cell types and cluster-specific genes. To identify transcription factors responsible for inducing specific terminal differentiation features, we generated a "random forest" model, and we showed that the transcription factors Apterous and Traffic-jam are required in many but not all cholinergic and glutamatergic neurons, respectively. In fact, the same terminal characters often can be regulated by different transcription factors in different cell types, arguing for extensive phenotypic convergence. Our data provide a deep understanding of the developmental and functional specification of a complex brain structure.

Pervasive Correlated Evolution in Gene Expression Shapes Cell and Tissue Type Transcriptomes.

The evolution and diversification of cell types is a key means by which animal complexity evolves. Recently, hierarchical clustering and phylogenetic methods have been applied to RNA-seq data to infer cell type evolutionary history and homology. A major challenge for interpreting this data is that cell type transcriptomes may not evolve independently due to correlated changes in gene expression. This nonindependence can arise for several reasons, such as common regulatory sequences for genes expressed in multiple tissues, that is, pleiotropic effects of mutations. We develop a model to estimate the level of correlated transcriptome evolution (LCE) and apply it to different data sets. The results reveal pervasive correlated transcriptome evolution among different cell and tissue types. In general, tissues related by morphology or developmental lineage exhibit higher LCE than more distantly related tissues. Analyzing new data collected from bird skin appendages suggests that LCE decreases with the phylogenetic age of tissues compared, with recently evolved tissues exhibiting the highest LCE. Furthermore, we show correlated evolution can alter patterns of hierarchical clustering, causing different tissue types from the same species to cluster together. To identify genes that most strongly contribute to the correlated evolution signal, we performed a gene-wise estimation of LCE on a data set with ten species. Removing genes with high LCE allows for accurate reconstruction of evolutionary relationships among tissue types. Our study provides a statistical method to measure and account for correlated gene expression evolution when interpreting comparative transcriptome data.