An atlas of rabbit development as a model for single-cell comparative genomics.

Traditionally, the mouse has been the favoured vertebrate model for biomedical research, due to its experimental and genetic tractability. However, non-rodent embryological studies highlight that many aspects of early mouse development, such as its egg-cylinder gastrulation and method of implantation, diverge from other mammals, thus complicating inferences about human development. Like the human embryo, rabbits develop as a flat-bilaminar disc. Here we constructed a morphological and molecular atlas of rabbit development. We report transcriptional and chromatin accessibility profiles for over 180,000 single cells and high-resolution histology sections from embryos spanning gastrulation, implantation, amniogenesis and early organogenesis. Using a neighbourhood comparison pipeline, we compare the transcriptional landscape of rabbit and mouse at the scale of the entire organism. We characterize the gene regulatory programmes underlying trophoblast differentiation and identify signalling interactions involving the yolk sac mesothelium during haematopoiesis. We demonstrate how the combination of both rabbit and mouse atlases can be leveraged to extract new biological insights from sparse macaque and human data. The datasets and computational pipelines reported here set a framework for a broader cross-species approach to decipher early mammalian development, and are readily adaptable to deploy single-cell comparative genomics more broadly across biomedical research.

Restricted Proliferation During Neurogenesis Contributes to Regionalisation of the Amphioxus Nervous System.

The central nervous system of the cephalochordate amphioxus consists of a dorsal neural tube with an anterior brain. Two decades of gene expression analyses in developing amphioxus embryos have shown that, despite apparent morphological simplicity, the amphioxus neural tube is highly regionalised at the molecular level. However, little is known about the morphogenetic mechanisms regulating the spatiotemporal emergence of cell types at distinct sites of the neural axis and how their arrangements contribute to the overall neural architecture. In vertebrates, proliferation is key to provide appropriate cell numbers of specific types to particular areas of the nervous system as development proceeds, but in amphioxus proliferation has never been studied at this level of detail, nor in the specific context of neurogenesis. Here, we describe the dynamics of cell division during the formation of the central nervous system in amphioxus embryos, and identify specific regions of the nervous system that depend on proliferation of neuronal precursors at precise time-points for their maturation. By labelling proliferating cells in vivo at specific time points in development, and inhibiting cell division during neurulation, we demonstrate that localised proliferation in the anterior cerebral vesicle is required to establish the full cell type repertoire of the frontal eye complex and the putative hypothalamic region of the amphioxus brain, while posterior proliferating progenitors, which were found here to derive from the dorsal lip of the blastopore, contribute to elongation of the caudal floor plate. Between these proliferative domains, we find that trunk nervous system differentiation is independent from cell division, in which proliferation decreases during neurulation and resumes at the early larval stage. Taken together, our results highlight the importance of proliferation as a tightly controlled mechanism for shaping and regionalising the amphioxus neural axis during development, by addition of new cells fated to particular types, or by influencing tissue geometry.

Single-cell morphometrics reveals ancestral principles of notochord development.

Embryonic tissues are shaped by the dynamic behaviours of their constituent cells. To understand such cell behaviours and how they evolved, new approaches are needed to map out morphogenesis across different organisms. Here, we apply a quantitative approach to learn how the notochord forms during the development of amphioxus: a basally branching chordate. Using a single-cell morphometrics pipeline, we quantify the geometries of thousands of amphioxus notochord cells, and project them into a common mathematical space, termed morphospace. In morphospace, notochord cells disperse into branching trajectories of cell shape change, revealing a dynamic interplay between cell shape change and growth that collectively contributes to tissue elongation. By spatially mapping these trajectories, we identify conspicuous regional variation, both in developmental timing and trajectory topology. Finally, we show experimentally that, unlike ascidians but like vertebrates, posterior cell division is required in amphioxus to generate full notochord length, thereby suggesting this might be an ancestral chordate trait that is secondarily lost in ascidians. Altogether, our novel approach reveals that an unexpectedly complex scheme of notochord morphogenesis might have been present in the first chordates. This article has an associated 'The people behind the papers' interview.

The dorsoanterior brain of adult amphioxus shares similarities in expression profile and neuronal composition with the vertebrate telencephalon.

BACKGROUND: The evolutionary origin of the telencephalon, the most anterior part of the vertebrate brain, remains obscure. Since no obvious counterpart to the telencephalon has yet been identified in invertebrate chordates, it is difficult to trace telencephalic origins. One way to identify homologous brain parts between distantly related animal groups is to focus on the combinatorial expression of conserved regionalisation genes that specify brain regions. RESULTS: Here, we report the combined expression of conserved transcription factors known to specify the telencephalon in the vertebrates in the chordate amphioxus. Focusing on adult specimens, we detect specific co-expression of these factors in the dorsal part of the anterior brain vesicle, which we refer to as Pars anterodorsalis (PAD). As in vertebrates, expression of the transcription factors FoxG1, Emx and Lhx2/9 overlaps that of Pax4/6 dorsally and of Nkx2.1 ventrally, where we also detect expression of the Hedgehog ligand. This specific pattern of co-expression is not observed prior to metamorphosis. Similar to the vertebrate telencephalon, the amphioxus PAD is characterised by the presence of GABAergic neurons and dorsal accumulations of glutamatergic as well as dopaminergic neurons. We also observe sustained proliferation of neuronal progenitors at the ventricular zone of the amphioxus brain vesicle, as observed in the vertebrate brain. CONCLUSIONS: Our findings suggest that the PAD in the adult amphioxus brain vesicle and the vertebrate telencephalon evolved from the same brain precursor region in ancestral chordates, which would imply homology of these structures. Our comparative data also indicate that this ancestral brain already contained GABA-, glutamatergic and dopaminergic neurons, as is characteristic for the olfactory bulb of the vertebrate telencephalon. We further speculate that the telencephalon might have evolved in vertebrates via a heterochronic shift in developmental timing.

The ADAR Family in Amphioxus: RNA Editing and Conserved Orthologous Site Predictions.

RNA editing is a relatively unexplored process in which transcribed RNA is modified at specific nucleotides before translation, adding another level of regulation of gene expression. Cephalopods use it extensively to increase the regulatory complexity of their nervous systems, and mammals use it too, but less prominently. Nevertheless, little is known about the specifics of RNA editing in most of the other clades and the relevance of RNA editing from an evolutionary perspective remains unknown. Here we analyze a key element of the editing machinery, the ADAR (adenosine deaminase acting on RNA) gene family, in an animal with a key phylogenetic position at the root of chordates: the cephalochordate amphioxus. We show, that as in cephalopods, ADAR genes in amphioxus are predominantly expressed in the nervous system; we identify a number of RNA editing events in amphioxus; and we provide a newly developed method to identify RNA editing events in highly polymorphic genomes using orthology as a guide. Overall, our work lays the foundations for future comparative analysis of RNA-editing events across the metazoan tree.

Hybridization Chain Reaction for Quantitative and Multiplex Imaging of Gene Expression in Amphioxus Embryos and Adult Tissues.

In situ hybridization (ISH) methods remain the most popular approach for profiling the expression of a gene at high spatial resolution and have been broadly used to address many biological questions. One compelling application is in the field of evo-devo, where comparing gene expression patterns has offered insight into how vertebrate development has evolved. Gene expression profiling in the invertebrate chordate amphioxus (cephalochordate) has been particularly instrumental in this context: its key phylogenetic position as sister group to all other chordates makes it an ideal model system to compare with vertebrates and for reconstructing the ancestral condition of our phylum. However, while ISH methods have been developed extensively in vertebrate model systems to fluorescently detect the expression of multiple genes simultaneously at a cellular and subcellular resolution, amphioxus gene expression profiling is still based on single-gene nonfluorescent chromogenic methods, whose spatial resolution is often compromised by diffusion of the chromogenic product. This represents a serious limitation for reconciling gene expression dynamics between amphioxus and vertebrates and for molecularly identifying cell types, defined by their combinatorial code of gene expression, that may have played pivotal roles in evolutionary innovation. Herein we overcome these problems by describing a new protocol for application of the third-generation hybridization chain reaction (HCR) to the amphioxus, which permits fluorescent, multiplex, and quantitative detection of gene expression in situ, within the changing morphology of the developing embryo, and in adult tissues. A detailed protocol is herein provided for whole-mount preparations of embryos and vibratome sections of adult tissues.

Amphioxus functional genomics and the origins of vertebrate gene regulation.

Vertebrates have greatly elaborated the basic chordate body plan and evolved highly distinctive genomes that have been sculpted by two whole-genome duplications. Here we sequence the genome of the Mediterranean amphioxus (Branchiostoma lanceolatum) and characterize DNA methylation, chromatin accessibility, histone modifications and transcriptomes across multiple developmental stages and adult tissues to investigate the evolution of the regulation of the chordate genome. Comparisons with vertebrates identify an intermediate stage in the evolution of differentially methylated enhancers, and a high conservation of gene expression and its cis-regulatory logic between amphioxus and vertebrates that occurs maximally at an earlier mid-embryonic phylotypic period. We analyse regulatory evolution after whole-genome duplications, and find that-in vertebrates-over 80% of broadly expressed gene families with multiple paralogues derived from whole-genome duplications have members that restricted their ancestral expression, and underwent specialization rather than subfunctionalization. Counter-intuitively, paralogues that restricted their expression increased the complexity of their regulatory landscapes. These data pave the way for a better understanding of the regulatory principles that underlie key vertebrate innovations.

Mirnovo: genome-free prediction of microRNAs from small RNA sequencing data and single-cells using decision forests.

The discovery of microRNAs (miRNAs) remains an important problem, particularly given the growth of high-throughput sequencing, cell sorting and single cell biology. While a large number of miRNAs have already been annotated, there may well be large numbers of miRNAs that are expressed in very particular cell types and remain elusive. Sequencing allows us to quickly and accurately identify the expression of known miRNAs from small RNA-Seq data. The biogenesis of miRNAs leads to very specific characteristics observed in their sequences. In brief, miRNAs usually have a well-defined 5' end and a more flexible 3' end with the possibility of 3' tailing events, such as uridylation. Previous approaches to the prediction of novel miRNAs usually involve the analysis of structural features of miRNA precursor hairpin sequences obtained from genome sequence. We surmised that it may be possible to identify miRNAs by using these biogenesis features observed directly from sequenced reads, solely or in addition to structural analysis from genome data. To this end, we have developed mirnovo, a machine learning based algorithm, which is able to identify known and novel miRNAs in animals and plants directly from small RNA-Seq data, with or without a reference genome. This method performs comparably to existing tools, however is simpler to use with reduced run time. Its performance and accuracy has been tested on multiple datasets, including species with poorly assembled genomes, RNaseIII (Drosha and/or Dicer) deficient samples and single cells (at both embryonic and adult stage).

Gastric pouches and the mucociliary sole: setting the stage for nervous system evolution.

Prerequisite for tracing nervous system evolution is understanding of the body plan, feeding behaviour and locomotion of the first animals in which neurons evolved. Here, a comprehensive scenario is presented for the diversification of cell types in early metazoans, which enhanced feeding efficiency and led to the emergence of larger animals that were able to move. Starting from cup-shaped, gastraea-like animals with outer and inner choanoflagellate-like cells, two major innovations are discussed that set the stage for nervous system evolution. First, the invention of a mucociliary sole entailed a switch from intra- to extracellular digestion and increased the concentration of nutrients flowing into the gastric cavity. In these animals, an initial nerve net may have evolved via division of labour from mechanosensory-contractile cells in the lateral body wall, enabling coordinated movement of the growing body that involved both mucociliary creeping and changes of body shape. Second, the inner surface of the animals folded into metameric series of gastric pouches, which optimized nutrient resorption and allowed larger body sizes. The concomitant acquisition of bilateral symmetry may have allowed more directed locomotion and, with more demanding coordinative tasks, triggered the evolution of specialized nervous subsystems. Animals of this organizational state would have resembled Ediacarian fossils such as Dickinsonia and may have been close to the cnidarian-bilaterian ancestor. In the bilaterian lineage, the mucociliary sole was used mostly for creeping, or frequently lost. One possible remnant is the enigmatic Reissner's fibre in the ventral neural tube of cephalochordates and vertebrates.

Methods for generating year-round access to amphioxus in the laboratory.

Cephalochordates, commonly known as amphioxus, are key to understanding vertebrate origins. However, laboratory work suffers from limited access to adults and embryonic material. Here we report the design and experimental validation of an inland marine facility that allows establishing stable amphioxus colonies in the laboratory and obtaining embryos at any time of day and over almost the entire year, far exceeding natural conditions. This is achieved by mimicking the natural benthic environment, natural day- and moon- light, natural substrate and by providing a strictly controlled and seasonally fluctuating temperature regimen. Moreover, supplemented algae diets allow animals to refill their gonads in consecutive years. Spontaneous spawning, a major problem in previous setups, no longer occurs in our facility; instead, all breeding is induced and fertilization occurs fully in vitro. Our system makes amphioxus a standard laboratory animal model.

Molecular analysis of the amphioxus frontal eye unravels the evolutionary origin of the retina and pigment cells of the vertebrate eye.

The origin of vertebrate eyes is still enigmatic. The "frontal eye" of amphioxus, our most primitive chordate relative, has long been recognized as a candidate precursor to the vertebrate eyes. However, the amphioxus frontal eye is composed of simple ciliated cells, unlike vertebrate rods and cones, which display more elaborate, surface-extended cilia. So far, the only evidence that the frontal eye indeed might be sensitive to light has been the presence of a ciliated putative sensory cell in the close vicinity of dark pigment cells. We set out to characterize the cell types of the amphioxus frontal eye molecularly, to test their possible relatedness to the cell types of vertebrate eyes. We show that the cells of the frontal eye specifically coexpress a combination of transcription factors and opsins typical of the vertebrate eye photoreceptors and an inhibitory Gi-type alpha subunit of the G protein, indicating an off-responding phototransductory cascade. Furthermore, the pigmented cells match the retinal pigmented epithelium in melanin content and regulatory signature. Finally, we reveal axonal projections of the frontal eye that resemble the basic photosensory-motor circuit of the vertebrate forebrain. These results support homology of the amphioxus frontal eye and the vertebrate eyes and yield insights into their evolutionary origin.

CNS evolution: new insight from the mud.

Whether the highly centralised nervous systems of chordates and protostomes arose from a common ancestral precursor or independently has been a long-standing debate. Now, analysis of neural gene expression in an evolutionarily important chordate outgroup--the sand-dwelling, hemichordate acorn worms--reveals the presence of a central and peripheral nervous system, suggesting a common origin of central nervous systems.

It's a long way from amphioxus: descendants of the earliest chordate.

The origin of chordates and the consequent genesis of vertebrates were major events in natural history. The amphioxus (lancelet) is now recognised as the closest extant relative to the stem chordate and is the only living invertebrate that retains a vertebrate-like development and body plan through its lifespan, despite more than 500 million years of independent evolution from the stem vertebrate. The inspiring data coming from its recently sequenced genome confirms that amphioxus has a prototypical chordate genome with respect to gene content and structure, and even chromosomal organisation. Pushed by joint efforts of amphioxus researchers, amphioxus is now entering a new era, namely its maturation as a laboratory model, through the availability of a large amount of molecular data and the advent of experimental manipulation of the embryo. These two facts may well serve to illuminate the hidden secrets of the genetic changes that generated, among other vertebrates, ourselves.

From the American to the European amphioxus: towards experimental Evo-Devo at the origin of chordates.

Pallid anchovy fillet, friendly filtering, peacefully laying and little lancelet are some of the nicknames and adjectives the cephalochordate amphioxus has received throughout the last two centuries. Traditionally regarded as the living representative of the last ancestor of vertebrates, amphioxus has recently been promoted to the privileged position of being the most ancient chordate. The preliminary analysis of its prototypical genome is nearly completed, and its hidden secrets towards the understanding of the primitive chordate and deuterostome genomes will soon see the light. Amphioxus embryonic development and body plan have remained in evolutionary stasis since the cephalochordate lineage split from the chordate ancestor about 500 million years ago. In contrast, amphioxus research is far from being at a standstill; in Europe, thanks to the international cooperation and the Banyuls Oceanographic Station, amphioxus embryos are obtained on demand during the spawning season. We summarise here our progress towards the dream of the experimental manipulation of the amphioxus embryo, to enter the era of Experimental Evo-Devo.

The amphioxus genome illuminates vertebrate origins and cephalochordate biology.

Cephalochordates, urochordates, and vertebrates evolved from a common ancestor over 520 million years ago. To improve our understanding of chordate evolution and the origin of vertebrates, we intensively searched for particular genes, gene families, and conserved noncoding elements in the sequenced genome of the cephalochordate Branchiostoma floridae, commonly called amphioxus or lancelets. Special attention was given to homeobox genes, opsin genes, genes involved in neural crest development, nuclear receptor genes, genes encoding components of the endocrine and immune systems, and conserved cis-regulatory enhancers. The amphioxus genome contains a basic set of chordate genes involved in development and cell signaling, including a fifteenth Hox gene. This set includes many genes that were co-opted in vertebrates for new roles in neural crest development and adaptive immunity. However, where amphioxus has a single gene, vertebrates often have two, three, or four paralogs derived from two whole-genome duplication events. In addition, several transcriptional enhancers are conserved between amphioxus and vertebrates--a very wide phylogenetic distance. In contrast, urochordate genomes have lost many genes, including a diversity of homeobox families and genes involved in steroid hormone function. The amphioxus genome also exhibits derived features, including duplications of opsins and genes proposed to function in innate immunity and endocrine systems. Our results indicate that the amphioxus genome is elemental to an understanding of the biology and evolution of nonchordate deuterostomes, invertebrate chordates, and vertebrates.

A gene catalogue of the amphioxus nervous system.

The elaboration of extremely complex nervous systems is a major success of evolution. However, at the dawn of the post-genomic era, few data have helped yet to unravel how a nervous system develops and evolves to complexity. On the evolutionary road to vertebrates, amphioxus occupies a key position to tackle this exciting issue. Its "simple" nervous system basically consists of a dorsal nerve cord and a diffuse net of peripheral neurons, which contrasts greatly with the complexity of vertebrate nervous systems. Notwithstanding, increasing data on gene expression has faced up this simplicity by revealing a mounting level of cryptic complexity, with unexpected levels of neuronal diversity, organisation and regionalisation of the central and peripheral nervous systems. Furthermore, recent gene expression data also point to the high neurogenic potential of the epidermis of amphioxus, suggestive of a skin-brain track for the evolution of the vertebrate nervous system. Here I attempt to catalogue and synthesise current gene expression data in the amphioxus nervous system. From this global point of view, I suggest scenarios for the evolutionary origin of complex features in the vertebrate nervous system, with special emphasis on the evolutionary origin of placodes and neural crest, and postulate a pre-patterned migratory pathway of cells, which, in the epidermis, may represent an intermediate state towards the deployment of one of the most striking innovative features of vertebrates: the neural crest and its derivatives.

Origin and evolution of the Trk family of neurotrophic receptors.

Among the numerous tyrosine kinase receptors, those belonging to the Trk family are distinctively involved in the development of complex traits within the vertebrate nervous system. Until recently, the lack of a proper Nt/Trk system in invertebrates has lead to the belief that they were a vertebrate innovation. Recent data, however, have challenged the field, and proved that bona fide Trk receptors do exist in invertebrates. Here, we review and discuss the evolutionary history of the Trk receptor family, and draw a comprehensive scenario that situates the origin of the Nt/Trk signalling prior to the origin of vertebrates. Probably, a ProtoTrk receptor was invented by means of domain and exon shuffling from pieces of ancient genes, generating the unique combination of domains found in extant Trk receptors. It is suggestive to propose that subtle protein mutations, gene duplications, and co-options in particular territories of a primitive Nt/Trk system were instrumental to the development of a complex vertebrate nervous system.

Outlining the nascent nervous system of Branchiostoma floridae (amphioxus) by the pan-neural marker AmphiElav.

The cephalochordate amphioxus is the closest living relative of the vertebrates. The anatomy and development of the amphioxus nervous system is giving insights into the basic chordate neural organisation, prior to the increase in neural complexity that characterised the origin of vertebrates. We have analysed the expression of the pan-neural marker AmphiElav in the Florida lancelet (Branchiostoma floridae) through development and up to larval stages. AmphiElav is expressed in the developing neural tube with particular temporal and spatial dynamics that correlate with neuronal differentiation. In addition, AmphiElav is expressed in isolated epidermal cells from mid-neurula embryos. This epidermal expression probably corresponds to precursors of sensory neurons. Together with data from other neural markers, we discuss the evolutionary relevance of these neural precursors to the origin of some of the most dramatic vertebrate inventions, neural crests and placodes.

The single AmphiTrk receptor highlights increased complexity of neurotrophin signalling in vertebrates and suggests an early role in developing sensory neuroepidermal cells.

Neurotrophins (Nt) and their tyrosine kinase Trk receptors play an essential role in the development and maintenance of the complex vertebrate nervous system. Invertebrate genome sequencing projects have suggested that the Nt/Trk system is a vertebrate innovation. We describe the isolation and characterisation of the amphioxus Trk receptor, AmphiTrk. Its ancestral link to vertebrate Trk receptors is supported by phylogenetic analysis and domain characterisation. The genomic structure of AmphiTrk strongly suggests that a ProtoTrk gene emerged by means of exon-shuffling prior to the cephalochordate/vertebrate split. We also examined the physiological response of AmphiTrk to vertebrate neurotrophins, and found that despite 500 million years of divergence, AmphiTrk transduces signals mediated by NGF, BDNF, NT3 and NT4. Markedly, AmphiTrk is able to activate survival and differentiation pathways, but fails to activate the PLCgamma pathway, which is involved in synaptic plasticity in higher vertebrates. AmphiTrk is expressed during amphioxus embryogenesis in sensory neural precursors in the epidermis, which possesses single migratory cells. We propose that the duplication and divergence of the Nt/Trk system, in tandem with recruitment of the PLCgamma pathway, may have provided the genetic basis for a key aspect of vertebrate evolution: the complexity of the nervous system.