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.

Gain of gene regulatory network interconnectivity at the origin of vertebrates.

SignificanceIn this manuscript, we address an essential question in developmental and evolutionary biology: How have changes in gene regulatory networks contributed to the invertebrate-to-vertebrate transition? To address this issue, we perturbed four signaling pathways critical for body plan formation in the cephalochordate amphioxus and in zebrafish and compared the effects of such perturbations on gene expression and gene regulation in both species. Our data reveal that many developmental genes have gained response to these signaling pathways in the vertebrate lineage. Moreover, we show that the interconnectivity between these pathways is much higher in zebrafish than in amphioxus. We conclude that this increased signaling pathway complexity likely contributed to vertebrate morphological novelties during evolution.

Cell cycle transcriptomics of Capsaspora provides insights into the evolution of cyclin-CDK machinery.

Progression through the cell cycle in eukaryotes is regulated on multiple levels. The main driver of the cell cycle progression is the periodic activity of cyclin-dependent kinase (CDK) complexes. In parallel, transcription during the cell cycle is regulated by a transcriptional program that ensures the just-in-time gene expression. Many core cell cycle regulators are widely conserved in eukaryotes, among them cyclins and CDKs; however, periodic transcriptional programs are divergent between distantly related species. In addition, many otherwise conserved cell cycle regulators have been lost and independently evolved in yeast, a widely used model organism for cell cycle research. For a better understanding of the evolution of the cell cycle regulation in opisthokonts, we investigated the transcriptional program during the cell cycle of the filasterean Capsaspora owczarzaki, a unicellular species closely related to animals. We developed a protocol for cell cycle synchronization in Capsaspora cultures and assessed gene expression over time across the entire cell cycle. We identified a set of 801 periodic genes that grouped into five clusters of expression over time. Comparison with datasets from other eukaryotes revealed that the periodic transcriptional program of Capsaspora is most similar to that of animal cells. We found that orthologues of cyclin A, B and E are expressed at the same cell cycle stages as in human cells and in the same temporal order. However, in contrast to human cells where these cyclins interact with multiple CDKs, Capsaspora cyclins likely interact with a single ancestral CDK1-3. Thus, the Capsaspora cyclin-CDK system could represent an intermediate state in the evolution of animal-like cyclin-CDK regulation. Overall, our results demonstrate that Capsaspora could be a useful unicellular model system for animal cell cycle regulation.

Transfection of Capsaspora owczarzaki, a close unicellular relative of animals.

How animals emerged from their unicellular ancestor remains a major evolutionary question. New genome data from the closest unicellular relatives of animals have provided important insights into the evolution of animal multicellularity. We know that the unicellular ancestor of animals had an unexpectedly complex genetic repertoire, including many genes that are key to animal development and multicellularity. Thus, assessing the function of these genes among unicellular relatives of animals is key to understanding how they were co-opted at the onset of the Metazoa. However, such analyses have been hampered by the lack of genetic tools. Progress has been made in choanoflagellates and teretosporeans, two of the three lineages closely related to animals, whereas no tools are yet available for functional analysis in the third lineage: the filastereans. Importantly, filastereans have a striking repertoire of genes involved in transcriptional regulation and other developmental processes. Here, we describe a reliable transfection method for the filasterean Capsaspora owczarzaki We also provide a set of constructs for visualising subcellular structures in live cells. These tools convert Capsaspora into a unique experimentally tractable organism to use to investigate the origin and evolution of animal multicellularity.

Hemichordate cis-regulatory genomics and the gene expression dynamics of deuterostomes.

Deuterostomes are one major group of bilaterians composed by hemichordates and echinoderms (collectively called Ambulacraria) and chordates. Comparative studies between these groups can provide valuable insights into the nature of the last common ancestor of deuterostomes and that of bilaterians. Indirect development of hemichordates, with larval phases similar to echinoderms and an adult body plan with an anteroposterior polarity like chordates and other bilaterians, makes them a suitable model for studying the molecular basis of development among deuterostomes. However, a comprehensive, quantitative catalogue of gene expression and chromatin dynamics in hemichordates is still lacking. In this study, we analysed the transcriptomes and chromatin accessibility of multiple developmental stages of the indirect-developing hemichordate Ptychodera flava. We observed that P. flava development is underpinned by a biphasic transcriptional program probably controlled by distinct genetic networks. Comparisons with other bilaterian species revealed similar transcriptional and regulatory dynamics during hemichordate gastrulation, cephalochordate neurulation and elongation stages of annelids. By means of regulatory networks analysis and functional validations by transgenesis experiments in echinoderms, we propose that gastrulation is the stage of highest molecular resemblance in deuterostomes and that much of the molecular basis of deuterostome development was probably present in the bilaterian last common ancestor.

Annelid adult cell type diversity and their pluripotent cellular origins.

Many annelids can regenerate missing body parts or reproduce asexually, generating all cell types in adult stages. However, the putative adult stem cell populations involved in these processes, and the diversity of cell types generated by them, are still unknown. To address this, we recover 75,218 single cell transcriptomes of the highly regenerative and asexually-reproducing annelid Pristina leidyi. Our results uncover a rich cell type diversity including annelid specific types as well as novel types. Moreover, we characterise transcription factors and gene networks that are expressed specifically in these populations. Finally, we uncover a broadly abundant cluster of putative stem cells with a pluripotent signature. This population expresses well-known stem cell markers such as vasa, piwi and nanos homologues, but also shows heterogeneous expression of differentiated cell markers and their transcription factors. We find conserved expression of pluripotency regulators, including multiple chromatin remodelling and epigenetic factors, in piwi+ cells. Finally, lineage reconstruction analyses reveal computational differentiation trajectories from piwi+ cells to diverse adult types. Our data reveal the cell type diversity of adult annelids by single cell transcriptomics and suggest that a piwi+ cell population with a pluripotent stem cell signature is associated with adult cell type differentiation.

Annelid adult cell type diversity and their pluripotent cellular origins.

Annelids are a broadly distributed, highly diverse, economically and environmentally important group of animals. Most species can regenerate missing body parts, and many are able to reproduce asexually. Therefore, many annelids can generate all adult cell types in adult stages. However, the putative adult stem cell populations involved in these processes, as well as the diversity of adult cell types generated by them, are still unknown. Here, we recover 75,218 single cell transcriptomes of Pristina leidyi, a highly regenerative and asexually-reproducing freshwater annelid. We characterise all major annelid adult cell types, and validate many of our observations by HCR in situ hybridisation. Our results uncover complex patterns of regionally expressed genes in the annelid gut, as well as neuronal, muscle and epidermal specific genes. We also characterise annelid-specific cell types such as the chaetal sacs and globin+ cells, and novel cell types of enigmatic affinity, including a vigilin+ cell type, a lumbrokinase+ cell type, and a diverse set of metabolic cells. Moreover, we characterise transcription factors and gene networks that are expressed specifically in these populations. Finally, we uncover a broadly abundant cluster of putative stem cells with a pluripotent signature. This population expresses well-known stem cell markers such as vasa, piwi and nanos homologues, but also shows heterogeneous expression of differentiated cell markers and their transcription factors. In these piwi+ cells, we also find conserved expression of pluripotency regulators, including multiple chromatin remodelling and epigenetic factors. Finally, lineage reconstruction analyses reveal the existence of differentiation trajectories from piwi+ cells to diverse adult types. Our data reveal the cell type diversity of adult annelids for the first time and serve as a resource for studying annelid cell types and their evolution. On the other hand, our characterisation of a piwi+ cell population with a pluripotent stem cell signature will serve as a platform for the study of annelid stem cells and their role in regeneration.

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.