Chromosome-level genome assemblies of 2 hemichordates provide new insights into deuterostome origin and chromosome evolution.

Deuterostomes are a monophyletic group of animals that includes Hemichordata, Echinodermata (together called Ambulacraria), and Chordata. The diversity of deuterostome body plans has made it challenging to reconstruct their ancestral condition and to decipher the genetic changes that drove the diversification of deuterostome lineages. Here, we generate chromosome-level genome assemblies of 2 hemichordate species, Ptychodera flava and Schizocardium californicum, and use comparative genomic approaches to infer the chromosomal architecture of the deuterostome common ancestor and delineate lineage-specific chromosomal modifications. We show that hemichordate chromosomes (1N = 23) exhibit remarkable chromosome-scale macrosynteny when compared to other deuterostomes and can be derived from 24 deuterostome ancestral linkage groups (ALGs). These deuterostome ALGs in turn match previously inferred bilaterian ALGs, consistent with a relatively short transition from the last common bilaterian ancestor to the origin of deuterostomes. Based on this deuterostome ALG complement, we deduced chromosomal rearrangement events that occurred in different lineages. For example, a fusion-with-mixing event produced an Ambulacraria-specific ALG that subsequently split into 2 chromosomes in extant hemichordates, while this homologous ALG further fused with another chromosome in sea urchins. Orthologous genes distributed in these rearranged chromosomes are enriched for functions in various developmental processes. We found that the deeply conserved Hox clusters are located in highly rearranged chromosomes and that maintenance of the clusters are likely due to lower densities of transposable elements within the clusters. We also provide evidence that the deuterostome-specific pharyngeal gene cluster was established via the combination of 3 pre-assembled microsyntenic blocks. We suggest that since chromosomal rearrangement events and formation of new gene clusters may change the regulatory controls of developmental genes, these events may have contributed to the evolution of diverse body plans among deuterostomes.

Marine Invertebrates One Cell at A Time: Insights from Single-Cell Analysis.

Over the past decade, single-cell RNA-sequencing (scRNA-seq) has made it possible to study the cellular diversity of a broad range of organisms. Technological advances in single-cell isolation and sequencing have expanded rapidly, allowing the transcriptomic profile of individual cells to be captured. As a result, there has been an explosion of cell type atlases created for many different marine invertebrate species from across the tree of life. Our focus in this review is to synthesize current literature on marine invertebrate scRNA-seq. Specifically, we provide perspectives on key insights from scRNA-seq studies, including descriptive studies of cell type composition, how cells respond in dynamic processes such as development and regeneration, and the evolution of new cell types. Despite these tremendous advances, there also lie several challenges ahead. We discuss the important considerations that are essential when making comparisons between experiments, or between datasets from different species. Finally, we address the future of single-cell analyses in marine invertebrates, including combining scRNA-seq data with other 'omics methods to get a fuller understanding of cellular complexities. The full diversity of cell types across marine invertebrates remains unknown and understanding this diversity and evolution will provide rich areas for future study.

Peripheral and central employment of acid-sensing ion channels during early bilaterian evolution.

Nervous systems are endowed with rapid chemosensation and intercellular signaling by ligand-gated ion channels (LGICs). While a complex, bilaterally symmetrical nervous system is a major innovation of bilaterian animals, the employment of specific LGICs during early bilaterian evolution is poorly understood. We therefore questioned bilaterian animals' employment of acid-sensing ion channels (ASICs), LGICs that mediate fast excitatory responses to decreases in extracellular pH in vertebrate neurons. Our phylogenetic analysis identified an earlier emergence of ASICs from the overarching DEG/ENaC (degenerin/epithelial sodium channel) superfamily than previously thought and suggests that ASICs were a bilaterian innovation. Our broad examination of ASIC gene expression and biophysical function in each major bilaterian lineage of Xenacoelomorpha, Protostomia, and Deuterostomia suggests that the earliest bilaterian ASICs were probably expressed in the periphery, before being incorporated into the brain as it emerged independently in certain deuterostomes and xenacoelomorphs. The loss of certain peripheral cells from Ecdysozoa after they separated from other protostomes likely explains their loss of ASICs, and thus the absence of ASICs from model organisms Drosophila and Caenorhabditis elegans. Thus, our use of diverse bilaterians in the investigation of LGIC expression and function offers a unique hypothesis on the employment of LGICs in early bilaterian evolution.

Most animals on Earth, from worms to chimpanzees, belong to a group known as the bilaterians. Despite their rich variety of shapes and lifestyles, all these creatures share similarities ­ in particular, a complex nervous system where neurons can quickly relay electric signals. This is made possible by a class of proteins, known as ligand-gated ion channels, which are studded through the membrane of cells. There, they help neurons efficiently communicate with each other by converting external chemical information into internal electrical signals. Yet despite their importance, how and when these proteins have evolved remains poorly understood. Marti-Solans et al. decided to explore this question by focusing on acid-sensing ion channels, a family which often forms the linchpin of bilaterian neural networks. They examined when these proteins first evolved (that is, in which putative ancestral animals) and where in the body. To do so, they combed through genetic data from all major bilaterian lineages as well as from non-biletarian groups; this included previously unexplored datasets that give insight into the type of cells in which a particular gene is active. The analyses revealed that the channels are specific to bilaterians, but that they appeared earlier than previously thought, being present in the very first members of this group. However, at this stage, the proteins were mainly located in cells at the periphery of the body rather than in those from emerging neural circuits. This suggests that the channels were co-opted by nerve cells later on, when the nervous systems became more complex. The proteins being initially located in cells at the outer edge of the body could also explain why they are absent in bilaterian creatures such as fruit flies and nematode worms; these animals all belong to a lineage where growth takes place by shedding their external layers. Acid-sensing ion channels are an important group of potential drug targets, often being implicated in pain and diseases of the nervous system. The work of Marti-Solans et al. offers an insight into the diversity of roles these proteins can play in the body, demonstrating once again how evolution can repurpose the same biophysical functions to serve a range of needs inside an organism.

Comparisons of cell proliferation and cell death from tornaria larva to juvenile worm in the hemichordate Schizocardium californicum.

BACKGROUND: There are a wide range of developmental strategies in animal phyla, but most insights into adult body plan formation come from direct-developing species. For indirect-developing species, there are distinct larval and adult body plans that are linked together by metamorphosis. Some outstanding questions in the development of indirect-developing organisms include the extent to which larval tissue undergoes cell death during the process of metamorphosis and when and where the tissue that will give rise to the adult originates. How do the processes of cell division and cell death redesign the body plans of indirect developers? In this study, we present patterns of cell proliferation and cell death during larval body plan development, metamorphosis, and adult body plan formation, in the hemichordate Schizocardium californium (Cameron and Perez in Zootaxa 3569:79-88, 2012) to answer these questions. RESULTS: We identified distinct patterns of cell proliferation between larval and adult body plan formation of S. californicum. We found that some adult tissues proliferate during the late larval phase prior to the start of overt metamorphosis. In addition, using an irradiation and transcriptomic approach, we describe a genetic signature of proliferative cells that is shared across the life history states, as well as markers that are unique to larval or juvenile states. Finally, we observed that cell death is minimal in larval stages but begins with the onset of metamorphosis. CONCLUSIONS: Cell proliferation during the development of S. californicum has distinct patterns in the formation of larval and adult body plans. However, cell death is very limited in larvae and begins during the onset of metamorphosis and into early juvenile development in specific domains. The populations of cells that proliferated and gave rise to the larvae and juveniles have a genetic signature that suggested a heterogeneous pool of proliferative progenitors, rather than a set-aside population of pluripotent cells. Taken together, we propose that the gradual morphological transformation of S. californicum is mirrored at the cellular level and may be more representative of the development strategies that characterize metamorphosis in many metazoan animals.

Patterning of cartilaginous condensations in the developing facial skeleton.

Adult endochondral bones are prefigured in the embryo as cellular condensations within fields of more loosely distributed skeletal progenitors. How these early condensations are initiated and shaped has remained enigmatic, despite the wealth of research on later stages of cartilage differentiation and endochondral ossification. Using the simple larval zebrafish facial skeleton as a model, we reevaluate the involvement of the master cartilage regulator Sox9 in shaping facial condensations and find it to be largely dispensable. We then use new lineage-tracing tools to definitively show that precartilaginous condensations originate from neighboring clusters of cells termed mesenchymal condensations. These cartilage-generating mesenchymal condensations express a cohort of transcription factors that are also expressed in odontogenic mesenchyme in mammals, including barx1, lhx6a/8a, and pax9. We hypothesized that the position of each mesenchymal condensation determines the axis of growth of its corresponding precartilaginous condensation, thus influencing its final shape. Consistent with this idea, we find that positive Fgf and inhibitory Jagged-Notch signals intersect to precisely position a mesenchymal condensation in the dorsal half of the second pharyngeal arch, with loss of pathway function leading to predictable shape changes in the resulting cartilage element. Deciphering the full array of signals that control the spatial distribution of mesenchymal condensations and regulate their maturation into precartilaginous condensations thus offers a promising approach for understanding the origins of skeletal form.

Molecular evidence for a single origin of ultrafiltration-based excretory organs.

Excretion is an essential physiological process, carried out by all living organisms, regardless of their size or complexity.1-3 Both protostomes (e.g., flies and flatworms) and deuterostomes (e.g., humans and sea urchins) possess specialized excretory organs serving that purpose. Those organs exhibit an astonishing diversity, ranging from units composed of just few distinct cells (e.g., protonephridia) to complex structures, built by millions of cells of multiple types with divergent morphology and function (e.g., vertebrate kidneys).4,5 Although some molecular similarities between the development of kidneys of vertebrates and the regeneration of the protonephridia of flatworms have been reported,6,7 the molecular underpinnings of the development of excretory organs have never been systematically studied in a comparative context.4 Here, we show that a set of transcription factors (eya, six1/2, pou3, sall, lhx1/5, and osr) and structural proteins (nephrin, kirre, and zo1) is expressed in the excretory organs of a phoronid, brachiopod, annelid, onychophoran, priapulid, and hemichordate that represent major protostome lineages and non-vertebrate deuterostomes. We demonstrate that the molecular similarity observed in the vertebrate kidney and flatworm protonephridia6,7 is also seen in the developing excretory organs of those animals. Our results show that all types of ultrafiltration-based excretory organs are patterned by a conserved set of developmental genes, an observation that supports their homology. We propose that the last common ancestor of protostomes and deuterostomes already possessed an ultrafiltration-based organ that later gave rise to the vast diversity of extant excretory organs, including both proto- and metanephridia.

Establishing typical values for hemocyte mortality in individual California mussels, Mytilus californianus.

Hemocytes are immune cells in the hemolymph of invertebrates that play multiple roles in response to stressors; hemocyte mortality can thus serve as an indicator of overall animal health. However, previous research has often analyzed hemolymph samples pooled from several individuals, which precludes tracking individual responses to stressors over time. The ability to track individuals is important, however, because large inter-individual variation in response to stressors can confound the interpretation of pooled samples. Here, we describe protocols for analysis of inter- and intra-individual variability in hemocyte mortality across repeated hemolymph samples of California mussels, Mytilus californianus, free from typical abiotic stressors. To assess individual variability in hemocyte mortality with serial sampling, we created four groups of 15 mussels each that were repeatedly sampled four times: at baseline (time zero) and three subsequent times separated by either 24, 48, 72, or 168 h. Hemocyte mortality was assessed by fluorescence-activated cell sorting (FACS) of cells stained with propidium iodide. Our study demonstrates that hemolymph can be repeatedly sampled from individual mussels without mortality; however, there is substantial inter- and intra-individual variability in hemocyte mortality through time that is partially dependent on the sampling interval. Across repeated samples, individual mussels' hemocyte mortality had, on average, a range of ~6% and a standard deviation of ~3%, which was minimized with sampling periods ≥72 h apart. Due to this intra-individual variability, obtaining ≥2 samples from a specimen will more accurately establish an individual's baseline. Pooled-sample means were similar to individual-sample means; however, pooled samples masked the individual variation in each group. Overall, these data lay the foundation for future work exploring individual mussels' temporal responses to various stressors on a cellular level.

Programmed cell removal by calreticulin in tissue homeostasis and cancer.

Macrophage-mediated programmed cell removal (PrCR) is a process essential for the clearance of unwanted (damaged, dysfunctional, aged, or harmful) cells. The detection and recognition of appropriate target cells by macrophages is a critical step for successful PrCR, but its molecular mechanisms have not been delineated. Here using the models of tissue turnover, cancer immunosurveillance, and hematopoietic stem cells, we show that unwanted cells such as aging neutrophils and living cancer cells are susceptible to "labeling" by secreted calreticulin (CRT) from macrophages, enabling their clearance through PrCR. Importantly, we identified asialoglycans on the target cells to which CRT binds to regulate PrCR, and the availability of such CRT-binding sites on cancer cells correlated with the prognosis of patients in various malignancies. Our study reveals a general mechanism of target cell recognition by macrophages, which is the key for the removal of unwanted cells by PrCR in physiological and pathophysiological processes.

Genome-wide analysis of facial skeletal regionalization in zebrafish.

Patterning of the facial skeleton involves the precise deployment of thousands of genes in distinct regions of the pharyngeal arches. Despite the significance for craniofacial development, how genetic programs drive this regionalization remains incompletely understood. Here we use combinatorial labeling of zebrafish cranial neural crest-derived cells (CNCCs) to define global gene expression along the dorsoventral axis of the developing arches. Intersection of region-specific transcriptomes with expression changes in response to signaling perturbations demonstrates complex roles for Endothelin 1 (Edn1) signaling in the intermediate joint-forming region, yet a surprisingly minor role in ventralmost regions. Analysis of co-variance across multiple sequencing experiments further reveals clusters of co-regulated genes, with in situ hybridization confirming the domain-specific expression of novel genes. We then created loss-of-function alleles for 12 genes and uncovered antagonistic functions of two new Edn1 targets, follistatin a (fsta) and emx2, in regulating cartilaginous joints in the hyoid arch. Our unbiased discovery and functional analysis of genes with regional expression in zebrafish arch CNCCs reveals complex regulation by Edn1 and points to novel candidates for craniofacial disorders.

Competition between Jagged-Notch and Endothelin1 Signaling Selectively Restricts Cartilage Formation in the Zebrafish Upper Face.

The intricate shaping of the facial skeleton is essential for function of the vertebrate jaw and middle ear. While much has been learned about the signaling pathways and transcription factors that control facial patterning, the downstream cellular mechanisms dictating skeletal shapes have remained unclear. Here we present genetic evidence in zebrafish that three major signaling pathways - Jagged-Notch, Endothelin1 (Edn1), and Bmp - regulate the pattern of facial cartilage and bone formation by controlling the timing of cartilage differentiation along the dorsoventral axis of the pharyngeal arches. A genomic analysis of purified facial skeletal precursors in mutant and overexpression embryos revealed a core set of differentiation genes that were commonly repressed by Jagged-Notch and induced by Edn1. Further analysis of the pre-cartilage condensation gene barx1, as well as in vivo imaging of cartilage differentiation, revealed that cartilage forms first in regions of high Edn1 and low Jagged-Notch activity. Consistent with a role of Jagged-Notch signaling in restricting cartilage differentiation, loss of Notch pathway components resulted in expanded barx1 expression in the dorsal arches, with mutation of barx1 rescuing some aspects of dorsal skeletal patterning in jag1b mutants. We also identified prrx1a and prrx1b as negative Edn1 and positive Bmp targets that function in parallel to Jagged-Notch signaling to restrict the formation of dorsal barx1+ pre-cartilage condensations. Simultaneous loss of jag1b and prrx1a/b better rescued lower facial defects of edn1 mutants than loss of either pathway alone, showing that combined overactivation of Jagged-Notch and Bmp/Prrx1 pathways contribute to the absence of cartilage differentiation in the edn1 mutant lower face. These findings support a model in which Notch-mediated restriction of cartilage differentiation, particularly in the second pharyngeal arch, helps to establish a distinct skeletal pattern in the upper face.