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.

Three amphioxus reference genomes reveal gene and chromosome evolution of chordates.

The slow-evolving invertebrate amphioxus has an irreplaceable role in advancing our understanding of the vertebrate origin and innovations. Here we resolve the nearly complete chromosomal genomes of three amphioxus species, one of which best recapitulates the 17 chordate ancestor linkage groups. We reconstruct the fusions, retention, or rearrangements between descendants of whole-genome duplications, which gave rise to the extant microchromosomes likely existed in the vertebrate ancestor. Similar to vertebrates, the amphioxus genome gradually establishes its three-dimensional chromatin architecture at the onset of zygotic activation and forms two topologically associated domains at the Hox gene cluster. We find that all three amphioxus species have ZW sex chromosomes with little sequence differentiation, and their putative sex-determining regions are nonhomologous to each other. Our results illuminate the unappreciated interspecific diversity and developmental dynamics of amphioxus genomes and provide high-quality references for understanding the mechanisms of chordate functional genome evolution.

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.

Evolution of genetic networks underlying the emergence of thymopoiesis in vertebrates.

About 500 million years ago, a new type of adaptive immune defense emerged in basal jawed vertebrates, accompanied by morphological innovations, including the thymus. Did these evolutionary novelties arise de novo or from elaboration of ancient genetic networks? We reconstructed the genetic changes underlying thymopoiesis by comparative genome and expression analyses in chordates and basal vertebrates. The derived models of genetic networks were experimentally verified in bony fishes. Ancestral networks defining circumscribed regions of the pharyngeal epithelium of jawless vertebrates expanded in cartilaginous fishes to incorporate novel genes, notably those encoding chemokines. Correspondingly, novel networks evolved in lymphocytes of jawed vertebrates to control the expression of additional chemokine receptors. These complementary changes enabled unprecedented Delta/Notch signaling between pharyngeal epithelium and lymphoid cells that was exploited for specification to the T cell lineage. Our results provide a framework elucidating the evolution of key features of the adaptive immune system in jawed vertebrates.

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.

Molecular asymmetry in the cephalochordate embryo revealed by single-blastomere transcriptome profiling.

Studies in various animals have shown that asymmetrically localized maternal transcripts play important roles in axial patterning and cell fate specification in early embryos. However, comprehensive analyses of the maternal transcriptomes with spatial information are scarce and limited to a handful of model organisms. In cephalochordates (amphioxus), an early branching chordate group, maternal transcripts of germline determinants form a compact granule that is inherited by a single blastomere during cleavage stages. Further blastomere separation experiments suggest that other transcripts associated with the granule are likely responsible for organizing the posterior structure in amphioxus; however, the identities of these determinants remain unknown. In this study, we used high-throughput RNA sequencing of separated blastomeres to examine asymmetrically localized transcripts in two-cell and eight-cell stage embryos of the amphioxus Branchiostoma floridae. We identified 111 and 391 differentially enriched transcripts at the 2-cell stage and the 8-cell stage, respectively, and used in situ hybridization to validate the spatial distribution patterns for a subset of these transcripts. The identified transcripts could be categorized into two major groups: (1) vegetal tier/germ granule-enriched and (2) animal tier/anterior-enriched transcripts. Using zebrafish as a surrogate model system, we showed that overexpression of one animal tier/anterior-localized amphioxus transcript, zfp665, causes a dorsalization/anteriorization phenotype in zebrafish embryos by downregulating the expression of the ventral gene, eve1, suggesting a potential function of zfp665 in early axial patterning. Our results provide a global transcriptomic blueprint for early-stage amphioxus embryos. This dataset represents a rich platform to guide future characterization of molecular players in early amphioxus development and to elucidate conservation and divergence of developmental programs during chordate evolution.

Co-option of the PRDM14-CBFA2T complex from motor neurons to pluripotent cells during vertebrate evolution.

Gene regulatory networks underlying cellular pluripotency are controlled by a core circuitry of transcription factors in mammals, including POU5F1. However, the evolutionary origin and transformation of pluripotency-related transcriptional networks have not been elucidated in deuterostomes. PR domain-containing protein 14 (PRDM14) is specifically expressed in pluripotent cells and germ cells, and is required for establishing embryonic stem cells (ESCs) and primordial germ cells in mice. Here, we compared the functions and expression patterns of PRDM14 orthologues within deuterostomes. Amphioxus PRDM14 and zebrafish PRDM14, but not sea urchin PRDM14, compensated for mouse PRDM14 function in maintaining mouse ESC pluripotency. Interestingly, sea urchin PRDM14 together with sea urchin CBFA2T, an essential partner of PRDM14 in mouse ESCs, complemented the self-renewal defect in mouse Prdm14 KO ESCs. Contrary to the Prdm14 expression pattern in mouse embryos, Prdm14 was expressed in motor neurons of amphioxus embryos, as observed in zebrafish embryos. Thus, Prdm14 expression in motor neurons was conserved in non-tetrapod deuterostomes and the co-option of the PRDM14-CBFA2T complex from motor neurons into pluripotent cells may have maintained the transcriptional network for pluripotency during vertebrate evolution.This article has an associated 'The people behind the papers' interview.

BMP controls dorsoventral and neural patterning in indirect-developing hemichordates providing insight into a possible origin of chordates.

A defining feature of chordates is the unique presence of a dorsal hollow neural tube that forms by internalization of the ectodermal neural plate specified via inhibition of BMP signaling during gastrulation. While BMP controls dorsoventral (DV) patterning across diverse bilaterians, the BMP-active side is ventral in chordates and dorsal in many other bilaterians. How this phylum-specific DV inversion occurs and whether it is coupled to the emergence of the dorsal neural plate are unknown. Here we explore these questions by investigating an indirect-developing enteropneust from the hemichordate phylum, which together with echinoderms form a sister group of the chordates. We found that in the hemichordate larva, BMP signaling is required for DV patterning and is sufficient to repress neurogenesis. We also found that transient overactivation of BMP signaling during gastrulation concomitantly blocked mouth formation and centralized the nervous system to the ventral ectoderm in both hemichordate and sea urchin larvae. Moreover, this mouthless, neurogenic ventral ectoderm displayed a medial-to-lateral organization similar to that of the chordate neural plate. Thus, indirect-developing deuterostomes use BMP signaling in DV and neural patterning, and an elevated BMP level during gastrulation drives pronounced morphological changes reminiscent of a DV inversion. These findings provide a mechanistic basis to support the hypothesis that an inverse chordate body plan emerged from an indirect-developing ancestor by tinkering with BMP signaling.

Evidence for BMP-mediated specification of primordial germ cells in an indirect-developing hemichordate.

Primordial germ cells (PGCs) are specified during development by either one of two major mechanisms, the preformation mode or the inductive mode. Because the inductive mode is widely employed by many bilaterians and early branching metazoan lineages, it has been postulated as an ancestral mechanism. However, among the deuterostome species that have been studied, invertebrate chordates use the preformation mode, while many vertebrate and echinoderm species are known to utilize an inductive mechanism, thus leaving the evolutionary history of PGC specification in the deuterostome lineage unclear. Hemichordates are the sister phylum of echinoderms, and together they form a clade called Ambulacraria that represents the closest group to the chordates. Thus, research in hemichordates is highly informative for resolving this issue. In this study, we investigate the developmental process of PGCs in an indirect-developing hemichordate, Ptychodera flava. We show that maternal transcripts of the conserved germline markers vasa, nanos, and piwi1 are ubiquitously distributed in early P. flava embryos, and these genes are coexpressed specifically in the dorsal hindgut starting from the gastrula stage. Immunostaining revealed that Vasa protein is concentrated toward the vegetal pole in early P. flava embryos, and it is restricted to cells in the dorsal hindgut of gastrulae and newly hatched larvae. The Vasa-positive cells later contribute to the developing trunk coeloms of the larvae and eventually reside in the adult gonads. We further show that bone morphogenetic protein (BMP) signaling is required to activate expression of the germline determinants in the gastrula hindgut, suggesting that PGC specification is induced by BMP signaling in P. flava. Our data support the hypothesis that the inductive mode is a conserved mechanism in Ambulacraria, which might even trace back to the common ancestor of Deuterostomes.

Evolution of the new vertebrate head by co-option of an ancient chordate skeletal tissue.

A defining feature of vertebrates (craniates) is a pronounced head that is supported and protected by a robust cellular endoskeleton. In the first vertebrates, this skeleton probably consisted of collagenous cellular cartilage, which forms the embryonic skeleton of all vertebrates and the adult skeleton of modern jawless and cartilaginous fish. In the head, most cellular cartilage is derived from a migratory cell population called the neural crest, which arises from the edges of the central nervous system. Because collagenous cellular cartilage and neural crest cells have not been described in invertebrates, the appearance of cellular cartilage derived from neural crest cells is considered a turning point in vertebrate evolution. Here we show that a tissue with many of the defining features of vertebrate cellular cartilage transiently forms in the larvae of the invertebrate chordate Branchiostoma floridae (Florida amphioxus). We also present evidence that during evolution, a key regulator of vertebrate cartilage development, SoxE, gained new cis-regulatory sequences that subsequently directed its novel expression in neural crest cells. Together, these results suggest that the origin of the vertebrate head skeleton did not depend on the evolution of a new skeletal tissue, as is commonly thought, but on the spread of this tissue throughout the head. We further propose that the evolution of cis-regulatory elements near an ancient regulator of cartilage differentiation was a major factor in the evolution of the vertebrate head skeleton.

Retinoic acid signaling and neurogenic niche regulation in the developing peripheral nervous system of the cephalochordate amphioxus.

The retinoic acid (RA) signaling pathway regulates axial patterning and neurogenesis in the developing central nervous system (CNS) of chordates, but little is known about its roles during peripheral nervous system (PNS) formation and about how these roles might have evolved. This study assesses the requirement of RA signaling for establishing a functional PNS in the cephalochordate amphioxus, the best available stand-in for the ancestral chordate condition. Pharmacological manipulation of RA signaling levels during embryogenesis reduces the ability of amphioxus larvae to respond to sensory stimulation and alters the number and distribution of ectodermal sensory neurons (ESNs) in a stage- and context-dependent manner. Using gene expression assays combined with immunohistochemistry, we show that this is because RA signaling specifically acts on a small population of soxb1c-expressing ESN progenitors, which form a neurogenic niche in the trunk ectoderm, to modulate ESN production during elongation of the larval body. Our findings reveal an important role for RA signaling in regulating neurogenic niche activity in the larval amphioxus PNS. Although only few studies have addressed this issue so far, comparable RA signaling functions have been reported for neurogenic niches in the CNS and in certain neurogenic placode derivatives of vertebrates. Accordingly, the here-described mechanism is likely a conserved feature of chordate embryonic and adult neural development.

Reiterative use of FGF signaling in mesoderm development during embryogenesis and metamorphosis in the hemichordate Ptychodera flava.

BACKGROUND: Mesoderm is generally considered to be a germ layer that is unique to Bilateria, and it develops into diverse tissues, including muscle, and in the case of vertebrates, the skeleton and notochord. Studies on various deuterostome animals have demonstrated that fibroblast growth factor (FGF) signaling is required for the formation of many mesodermal structures, such as vertebrate somites, from which muscles are differentiated, and muscles in sea urchin embryos, suggesting an ancient role of FGF signaling in muscle development. However, the formation of trunk muscles in invertebrate chordates is FGF-independent, leading to ambiguity about this ancient role in deuterostomes. To further understand the role of FGF signaling during deuterostome evolution, we investigated the development of mesodermal structures during embryogenesis and metamorphosis in Ptychodera flava, an indirect-developing hemichordate that has larval morphology similar to echinoderms and adult body features that are similar to chordates. RESULTS: Here we show that genes encoding FGF ligands, FGF receptors and transcription factors that are known to be involved in mesoderm formation and myogenesis are expressed dynamically during embryogenesis and metamorphosis. FGF signaling at the early gastrula stage is required for the specification of the mesodermal cell fate in P. flava. The mesoderm cells are then differentiated stepwise into the hydroporic canal, the pharyngeal muscle and the muscle string; formation of the last two muscular structures are controlled by FGF signaling. Moreover, augmentation of FGF signaling during metamorphosis accelerated the process, facilitating the transformation from cilia-driven swimming larvae into muscle-driven worm-like juveniles. CONCLUSIONS: Our data show that FGF signaling is required for mesoderm induction and myogenesis in the P. flava embryo, and it is reiteratively used for the morphological transition during metamorphosis. The dependence of muscle development on FGF signaling in both planktonic larvae and sand-burrowing worms supports its ancestral role in deuterostomes.

Regulatory circuit rewiring and functional divergence of the duplicate admp genes in dorsoventral axial patterning.

The spatially opposed expression of Antidorsalizing morphogenetic protein (Admp) and BMP signals controls dorsoventral (DV) polarity across Bilateria and hence represents an ancient regulatory circuit. Here, we show that in addition to the conserved admp1 that constitutes the ancient circuit, a second admp gene (admp2) is present in Ambulacraria (Echinodermata+Hemichordata) and two marine worms belonging to Xenoturbellida and Acoelomorpha. The phylogenetic distribution implies that the two admp genes were duplicated in the Bilaterian common ancestor and admp2 was subsequently lost in chordates and protostomes. We show that the ambulacrarian admp1 and admp2 are under opposite transcriptional control by BMP signals and knockdown of Admps in sea urchins impaired their DV polarity. Over-expression of either Admps reinforced BMP signaling but resulted in different phenotypes in the sea urchin embryo. Our study provides an excellent example of signaling circuit rewiring and protein functional changes after gene duplications.

Reproductive periodicity, spawning induction, and larval metamorphosis of the hemichordate acorn worm Ptychodera flava.

The indirect-developing enteropneust acorn worm Ptychodera flava has been used as a hemichordate model system for studying the developmental evolution of deuterostome body plans and the origins of chordate characteristics. However, research progress has been hindered by the limited accessibility of its embryonic materials and metamorphosing larvae. In this study, we identified an abundant population of P. flava in Penghu, Taiwan, and examined the feasibility of using this animal for developmental studies. Through histological examination, we established that the reproductive season of this population is between September and December, with a peak breeding period in October and November. In addition, we have developed new procedures that can induce P. flava spawning at any time of the day during the breeding season, with a higher successful rate than that achieved using a previously published method. Moreover, the culturing system we developed enables rearing of P. flava larvae through various planktonic stages and eventual metamorphosis into benthic juveniles, all under laboratory conditions. We anticipate that the animal resources and new technical procedures reported here will further facilitate the use of P. flava as a model organism for evolutionary and developmental biology research.

A novel N-terminal motif is responsible for the evolution of neural crest-specific gene-regulatory activity in vertebrate FoxD3.

The neural crest is unique to vertebrates and has allowed the evolution of their complicated craniofacial structures. During vertebrate evolution, the acquisition of the neural crest must have been accompanied by the emergence of a new gene regulatory network (GRN). Here, to investigate the role of protein evolution in the emergence of the neural crest GRN, we examined the neural crest cell (NCC) differentiation-inducing activity of chordate FoxD genes. Amphioxus and vertebrate (Xenopus) FoxD proteins both exhibited transcriptional repressor activity in Gal4 transactivation assays and bound to similar DNA sequences in vitro. However, whereas vertebrate FoxD3 genes induced the differentiation of ectopic NCCs when overexpressed in chick neural tube, neither amphioxus FoxD nor any other vertebrate FoxD paralogs exhibited this activity. Experiments using chimeric proteins showed that the N-terminal portion of the vertebrate FoxD3 protein is critical to its NCC differentiation-inducing activity. Furthermore, replacement of the N-terminus of amphioxus FoxD with a 39-amino-acid segment from zebrafish FoxD3 conferred neural crest-inducing activity on amphioxus FoxD or zebrafish FoxD1. Therefore, fixation of this N-terminal amino acid sequence may have been crucial in the evolutionary recruitment of FoxD3 to the vertebrate neural crest GRN.

BMP and Delta/Notch signaling control the development of amphioxus epidermal sensory neurons: insights into the evolution of the peripheral sensory system.

The evolution of the nervous system has been a topic of great interest. To gain more insight into the evolution of the peripheral sensory system, we used the cephalochordate amphioxus. Amphioxus is a basal chordate that has a dorsal central nervous system (CNS) and a peripheral nervous system (PNS) comprising several types of epidermal sensory neurons (ESNs). Here, we show that a proneural basic helix-loop-helix gene (Ash) is co-expressed with the Delta ligand in ESN progenitor cells. Using pharmacological treatments, we demonstrate that Delta/Notch signaling is likely to be involved in the specification of amphioxus ESNs from their neighboring epidermal cells. We also show that BMP signaling functions upstream of Delta/Notch signaling to induce a ventral neurogenic domain. This patterning mechanism is highly similar to that of the peripheral sensory neurons in the protostome and vertebrate model animals, suggesting that they might share the same ancestry. Interestingly, when BMP signaling is globally elevated in amphioxus embryos, the distribution of ESNs expands to the entire epidermal ectoderm. These results suggest that by manipulating BMP signaling levels, a conserved neurogenesis circuit can be initiated at various locations in the epidermal ectoderm to generate peripheral sensory neurons in amphioxus embryos. We hypothesize that during chordate evolution, PNS progenitors might have been polarized to different positions in various chordate lineages owing to differential regulation of BMP signaling in the ectoderm.

On a possible evolutionary link of the stomochord of hemichordates to pharyngeal organs of chordates.

As a group closely related to chordates, hemichordate acorn worms are in a key phylogenic position for addressing hypotheses of chordate origins. The stomochord of acorn worms is an anterior outgrowth of the pharynx endoderm into the proboscis. In 1886 Bateson proposed homology of this organ to the chordate notochord, crowning this animal group "hemichordates." Although this proposal has been debated for over a century, the question still remains unresolved. Here we review recent progress related to this question. First, the developmental mode of the stomochord completely differs from that of the notochord. Second, comparison of expression profiles of genes including Brachyury, a key regulator of notochord formation in chordates, does not support the stomochord/notochord homology. Third, FoxE that is expressed in the stomochord-forming region in acorn worm juveniles is expressed in the club-shaped gland and in the endostyle of amphioxus, in the endostyle of ascidians, and in the thyroid gland of vertebrates. Based on these findings, together with the anterior endodermal location of the stomochord, we propose that the stomochord has evolutionary relatedness to chordate organs deriving from the anterior pharynx rather than to the notochord.

The amphioxus genome and the evolution of the chordate karyotype.

Lancelets ('amphioxus') are the modern survivors of an ancient chordate lineage, with a fossil record dating back to the Cambrian period. Here we describe the structure and gene content of the highly polymorphic approximately 520-megabase genome of the Florida lancelet Branchiostoma floridae, and analyse it in the context of chordate evolution. Whole-genome comparisons illuminate the murky relationships among the three chordate groups (tunicates, lancelets and vertebrates), and allow not only reconstruction of the gene complement of the last common chordate ancestor but also partial reconstruction of its genomic organization, as well as a description of two genome-wide duplications and subsequent reorganizations in the vertebrate lineage. These genome-scale events shaped the vertebrate genome and provided additional genetic variation for exploitation during vertebrate evolution.

Tracing the evolutionary origin of chordate somites in the hemichordate Ptychodera flava.

Metameric somites are a novel character of chordates with unclear evolutionary origins. In the early branching chordate amphioxus, anterior somites are derived from the paraxial mesodermal cells that bud off the archenteron (i.e., enterocoely) at the end of gastrulation. Development of the anterior somites requires FGF signaling, and distinct somite compartments express orthologs of vertebrate non-axial mesodermal markers. Thus, it has been proposed that the amphioxus anterior somites are homologous to the vertebrate head mesoderm, paraxial mesoderm and lateral plate mesoderm. To trace the evolutionary origin of somites, it is essential to study the chordates' closest sister group, Ambulacraria, which includes hemichordates and echinoderms. The anterior coeloms of hemichordate and sea urchin embryos (respectively called protocoel and coelomic pouches) are also formed by enterocoely and require FGF signals for specification and/or differentiation. In this study, we applied RNA-seq to comprehensively screen for regulatory genes associated with the mesoderm-derived protocoel of the hemichordate Ptychodera flava. We also used a candidate gene approach to identify P. flava orthologs of chordate somite markers. In situ hybridization results showed that many of these candidate genes are expressed in distinct or overlapping regions of the protocoel, which indicates that molecular compartments exist in the hemichordate anterior coelom. Given that the hemichordate protocoel and amphioxus anterior somites share a similar ontogenic process (enterocoely), induction signal (FGF), and characteristic expression of orthologous genes, we propose that these two anterior coeloms are indeed homologous. In the lineage leading to the emergence of chordates, somites likely evolved from enterocoelic, FGF-dependent, and molecularly compartmentalized anterior coeloms of the deuterostome last common ancestor.

Asymmetric segregation of maternal mRNAs and germline-related determinants in Cephalochordate embryos: implications for the evolution of early patterning events in chordates.

How animal embryos determine their early cell fates is an important question in developmental biology. In various model animals asymmetrically localized maternal transcripts play important roles in axial patterning and cell fate specification. Cephalochordates (amphioxus), which have three living genera (Asymmetron, Epigonichthys, Branchiostoma), are an early branching chordate lineage and thus occupy a key phylogenetic position for understanding the evolution of chordate developmental mechanisms. It has been shown that in the zygote of Brachiostoma amphioxus, which possess bilateral gonads flanking both sides of their trunk region, maternal transcripts of germline determinants form a compact granule. During early embryogenesis this granule is inherited by a single blastomere that subsequently gives rise to a cluster of cells displaying typical characteristics of primordial germ cells (PGC). These PGCs then come to lie in the tailbud region and proliferate during posterior elongation of the larva to join in the gonad anlagen at the ventral tip of the developing myomeres in amphioxus larvae. However, in Asymmetron and Epigonichthys amphioxus, whose gonads are present only on the right side of their body, nothing is known about their PGC development or the cellular/morphogenetic processes resulting in the asymmetric distribution of gonads. Using conserved germline determinants as markers, we show that similarly to Brachiostoma amphioxus, Asymmetron also employ a preformation mechanism to specify their PGCs, suggesting that this mechanism represents an ancient trait dating back to the common ancestor of Cephalochordates. Surprisingly, we found that Asymmetron PGCs are initially deposited on both sides of the body during early larval development; however, the left side PGCs cease to exist in young juveniles, suggesting that PGCs are eliminated from the left body side during larval development or following metamorphosis. This is reminiscent of the PGC development in the sea urchin embryo, and we discuss the implications of this observation for the evolution of developmental mechanisms.