Laboratory Culture and Mutagenesis of Amphioxus (Branchiostoma floridae).

Cephalochordates (amphioxus) are invertebrate chordates closely related to vertebrates. As they are evolving very slowly, they are proving to be very appropriate for developmental genetics studies aimed at understanding how vertebrates evolved from their invertebrate ancestors. To date, techniques for gene knockdown and overexpression have been developed, but methods for continuous breeding cultures and generating germline mutants have been developed only recently. Here we describe methods for continuous laboratory breeding cultures of the cephalochordate Branchiostoma floridae and the TALEN and Tol2 methods for mutagenesis. Included are strategies for analyzing the mutants and raising successive generations to obtain homozygotes. These methods should be applicable to any warm water species of cephalochordates with a relatively short generation time of 3-4 months and a life span of 3 years or more.

Transphyletic conservation of nitric oxide synthase regulation in cephalochordates and tunicates.

Nitric oxide synthase is ubiquitously present in metazoans and is involved in a wide range of biological processes. Three distinct Nos genes have been so far identified in vertebrates exhibiting a complex expression pattern and transcriptional regulation. Nevertheless, although independent events of Nos duplication have been observed in several taxa, only few studies described the regulatory mechanisms responsible for their activation in non-vertebrate animals. To shed light on the mechanisms underlying neuronal-type Nos expression, we focused on two non-vertebrate chordates: the cephalochordate Branchiostoma lanceolatum and the tunicate Ciona robusta. Here, throughout transphyletic and transgenic approaches, we identified genomic regions in both species acting as Nos functional enhancers during development. In vivo analyses of Nos genomic fragments revealed their ability to recapitulate the endogenous expression territories. Therefore, our results suggest the existence of evolutionary conserved mechanisms responsible for neuronal-type Nos regulation in non-vertebrate chordates. In conclusion, this study paves the way for future characterization of conserved transcriptional logic underlying the expression of neuronal-type Nos genes in chordates.

The EJC component Magoh in non-vertebrate chordates.

Earliest craniates possess a newly enlarged, elaborated forebrain with new cell types and neuronal networks. A key question in vertebrate evolution is when and how this cerebral expansion took place. The exon-junction complex (EJC) plays an essential role in mRNA processing of all Eukarya. Recently, it has been proposed that the EJC represses recursive RNA splicing in Deuterostomes, with implication in human brain diseases like microcephaly and depression. However, the EJC or EJC subunit contribution to brain development in non-vertebrate Deuterostomes remained unknown. Being interested in the evolution of chordate characters, we focused on the model species, Branchiostoma lanceolatum (Cephalochordata) and Ciona robusta (Tunicata), with the aim to investigate the ancestral and the derived expression state of Magoh orthologous genes. This study identifies that Magoh is part of a conserved syntenic group exclusively in vertebrates and suggests that Magoh has experienced duplication and loss events in mammals. During early development in amphioxus and ascidian, maternal contribution and zygotic expression of Magoh genes in various types of progenitor cells and tissues are consistent with the condition observed in other Bilateria. Later in development, we also show expression of Magoh in the brain of cephalochordate and ascidian larvae. Collectively, these results provide a basis to further define what functional role(s) Magoh exerted during nervous system development and evolution.

More Than One-to-Four via 2R: Evidence of an Independent Amphioxus Expansion and Two-Gene Ancestral Vertebrate State for MyoD-Related Myogenic Regulatory Factors (MRFs).

The evolutionary transition from invertebrates to vertebrates involved extensive gene duplication, but understanding precisely how such duplications contributed to this transition requires more detailed knowledge of specific cases of genes and gene families. Myogenic differentiation (MyoD) has long been recognized as a master developmental control gene and member of the MyoD family of bHLH transcription factors (myogenic regulatory factors [MRFs]) that drive myogenesis across the bilaterians. Phylogenetic reconstructions within this gene family are complicated by multiple instances of gene duplication and loss in several lineages. Following two rounds of whole-genome duplication (2R WGD) at the origin of the vertebrates, the ancestral function of MRFs is thought to have become partitioned among the daughter genes, so that MyoD and Myf5 act early in myogenic determination, whereas Myog and Myf6 are expressed later, in differentiating myoblasts. Comparing chordate MRFs, we find an independent expansion of MRFs in the invertebrate chordate amphioxus, with evidence for a parallel instance of subfunctionalization relative to that of vertebrates. Conserved synteny between chordate MRF loci supports the 2R WGD events as a major force in shaping the evolution of vertebrate MRFs. We also resolve vertebrate MRF complements and organization, finding a new type of vertebrate MRF gene in the process, which allowed us to infer an ancestral two-gene state in the vertebrates corresponding to the early- and late-acting types of MRFs. This necessitates a revision of previous conclusions about the simple one-to-four origin of vertebrate MRFs.

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.

Evolution and development of the cartilaginous skull: From a lancelet towards a human face.

Chrondrocranium, the cartilaginous skull, is one of the major innovations that underlie evolution of the vertebrate head. Control of the induction and shaping of the cartilage is a key for the formation of the facial bones and largely defines facial shape. The appearance of cartilage in the head enabled many new functions such as protection of central nervous system and sensory structures, support of the feeding apparatus and formation of muscle attachment points ensuring faster and coordinated jaw movements. Here we review the evolution of cartilage in the cranial region and discuss shaping of the chondrocranium in different groups of vertebrates.

Canonical Wnt/ß-catenin and Notch signaling regulate animal/vegetal axial patterning in the cephalochordate amphioxus.

In bilaterians, animal/vegetal axial (A/V) patterning is a fundamental early developmental event for establishment of animal/vegetal polarity and following specification of the germ layers (ectoderm, mesoderm, endoderm), of which the evolutionary origin is enigmatic. Understanding A/V axial patterning in a basal animal from each phylum would help to reconstruct the ancestral state of germ layer specification in bilaterians and thus, the evolution of mesoderm, the third intermediate cell layer. Herein, data show that the canonical Wnt/ß-catenin (cWnt) and Notch signaling pathways control mesoderm specification from the early endomesoderm in the basal chordate amphioxus. Amphioxus belongs to the deuterostome, one of the main superphyla in Bilateria. In the present study, genes (tcf, dsh, axin, gsk3ß) encoding cWnt components were expressed in the endomesoderm during the gastrula stages. Excess cWnt signaling by BIO, a GSK3 inhibitor, expanded the expression domains of outer endomesodermal genes that include future mesodermal ones and suppressed inner endomesodermal and ectodermal genes. Interfering Notch signaling by DAPT, a γ-secretase inhibitor, resulted in decreased expression of ectodermal and endomesodermal markers. These results suggest that cWnt and Notch have important roles in mesoderm specification in amphioxus embryos. The evolution of the mesoderm is also discussed.

The conserved ancient role of chordate PIAS as a multilevel repressor of the NF-κB pathway.

In vertebrates, PIAS genes encode versatile cellular regulators, with functions extremely complex and redundant. Here we try to understand their functions from an evolutionary perspective. we evaluate the sequences, expression and molecular functions of amphioxus PIAS genes and compare them with their vertebrate counterparts. Phylogenetic analysis suggests a single PIAS gene in ancestral chordates, which has been duplicated into four families (PIAS1-4) in vertebrates by 2R-WGD but remained single in a basal chordate (amphioxus). Amphioxus PIAS encodes two variants with and without a Serine/Threonine-rich tail, which are retained in human PIAS1-3 but lost in PIAS4. We show that amphioxus PIAS binds C-terminus of NF-κB Rel and blocks the DNA binding activity. In humans, such function is retained in PIAS1, altered in PIAS4, and lost in PIAS2-3. Instead, PIAS3 has evolved new ability to inhibit Rel by binding RHD and promoting SUMOylation. We show that amphioxus PIAS also inhibits NF-κB by binding with upstream signalling adaptor TICAM-like and MyD88. Finally, we verify that human PIAS1, 3 and 4, but not 2, were capable of these newly-discovered functions. Our study offers insight into the sub- and neo-functionalization of PIAS genes and suggests a conserved ancient role for chordate PIAS in NF-κB signalling.

The Amphioxus Model System.

The amphioxus (lancelet) was first described by Pallas in 1774 and incorrectly assigned to mollusks. Since then, amphioxus attracted generations of zoologists. It took however almost one hundred years until Alexander Kowalevsky recognized that the larval stages of amphioxus had much in common with vertebrate embryos. Widely studied around 1900 as the 'elementary vertebrate', amphioxus as a model went out of fashion with the decline of comparative anatomy. Due to the scarcity of taxa at the invertebrate-to-vertebrate transition, amphioxus nevertheless remained the species with a privileged position in animal phylogeny. Its resurrection as the popular model of evolutionary developmental biology came with the advent of modern molecular biology and genomics. In the 1990s amphioxus developmental control genes were identified and characterized at a fast pace with the hope that such studies could provide novel insight into an important evolutionary transition: the origin of vertebrates. Indeed, amphioxus was found to be vertebrate-like but much simpler. Its body resembles that of the vertebrate, but it lacks most of the complexities associated with typical vertebrate organs. Its genome is only 1/6 of the human genome and it has not undergone the whole genome duplications that occurred in the vertebrate lineage. For all of these reasons, amphioxus became widely regarded as a useful proxy for the primitive ancestor of all vertebrates. A persistent problem interpreting amphioxus in the phylogenetic context is the difficulty to distinguish ancestral features, and those that are secondarily derived. There is no doubt that an integrative approach combining information from various disciplines is needed in order to help resolve such issues. Anatomy and comparative morphology has always been strong since the dawn of amphioxus research. Recent developments such as the availability of genomic sequences for three Branchiostoma species, established laboratory cultures of amphioxus that can be spawned at the investigator's will, or techniques allowing transgenesis and gene knockouts represent a major leap for studies on how the genotype generates a phenotype. These advances also enable the smooth transition of amphioxus from the model system of a distinguished past into the one with a very bright future.

The dawn of amphioxus molecular biology - a personal perspective.

The cloning and embryonic expression analysis of an amphioxus Hox gene in 1992 marked the start of molecular analysis of cephalochordate development. Other papers quickly followed, including a description of the amphioxus Hox gene cluster in 1994, fuelling a resurgence of interest in a long-forgotten animal. I describe the academic background, laboratory experiments and field work leading to the earliest publications in amphioxus molecular developmental biology and explain their scientific impact. The story of amphioxus biology in the 1990s involved collaboration, team work, opportunistic meetings, serendipity, incredulous journal editors, stingrays, airport departure lounges and popcorn.

Left-right asymmetry specification in amphioxus: review and prospects.

Extant bilaterally symmetrical animals usually show asymmetry in the arrangement of their inner organs. However, the exaggerated left-right (LR) asymmetry in amphioxus represents a true peculiarity among them. The amphioxus larva shows completely disparate fates of left and right body sides, so that organs associated with pharynx are either positioned exclusively on the left or on the right side. Moreover, segmented paraxial structures such as muscle blocks and their neuronal innervation show offset arrangement between the sides making it difficult to propose any explanation or adaptivity to larval and adult life. First LR asymmetries can be traced back to an early embryonic period when morphological asymmetries are preceded by molecular asymmetries driven by the action of the Nodal signaling pathway. This review sums up recent advances in understanding LR asymmetry specification in amphioxus and proposes upstream events that may regulate asymmetric Nodal signaling. These events include the presence of the vertebrate-like LR organizer and a cilia-driven fluid flow that may be involved in the breaking of bilateral symmetry. The upstream pathways comprising the ion flux, Delta/Notch, Wnt/ß-catenin and Wnt/PCP are hypothesized to regulate both formation of the LR organizer and expression of the downstream Nodal signaling pathway genes. These suggestions are in line with what we know from vertebrate and ambulacrarian LR axis specification and are directly testable by experimental manipulations. Thanks to the phylogenetic position of amphioxus, the proposed mechanisms may be helpful in understanding the evolution of LR axis specification across deuterostomes.

The long and winding path to understanding kidney structure in amphioxus - a review.

The history of studies on amphioxus kidney morphology is reviewed with special attention to four zoologists who made important early contributions. In 1884, Hatschek described a single anterior nephridial tubule in larval and adult amphioxus. Subsequently, in 1890, Weiss and Boveri independently found multiple branchial nephridia (morphologically similar to Hatschek's nephridium) associated with the pharyngeal gill slits. These initial discoveries set the stage for Goodrich to criticize Boveri repeatedly for the latter's contention that amphioxus nephridia develop from mesoderm and are connected to neighboring coeloms throughout the life history. In the end, Boveri was almost certainly correct about amphioxus nephridia developing from mesoderm and at least partly right about the lumen of the nephridial tubules being connected to nearby coeloms-the openings are present during larval stages but are closed off later in development. The more detailed structure of amphioxus nephridial tubules was ultimately revealed by electron microscopy. The tubule epithelium includes specialized excretory cells (cyrtopodocytes), each characterized by a basal region similar to that of a vertebrate renal podocyte and an apical region bearing a flagellar/microvillar process reminiscent of an invertebrate protonephridium. At present, in spite of considerable progress toward understanding the development and structure of amphioxus nephridia, virtually nothing is yet known about how they function, and no consensus has been reached about their phylogenetic significance.

Tracing the evolutionary origin of vertebrate skeletal tissues: insights from cephalochordate amphioxus.

Vertebrate mineralized skeletal tissues are widely considered as an evolutionary novelty. Despite the importance of these tissues to the adaptation and radiation of vertebrate animals, the evolutionary origin of vertebrate skeletal tissues remains largely unclear. Cephalochordates (Amphioxus) occupy a key phylogenetic position and can serve as a valuable model for studying the evolution of vertebrate skeletal tissues. Here we summarize recent advances in amphioxus developmental biology and comparative genomics that can help to elucidate the evolutionary origins of the vertebrate skeletal tissues and their underlying developmental gene regulatory networks (GRN). By making comparisons to the developmental studies in vertebrate models and recent discoveries in paleontology and genomics, it becomes evident that the collagen matrix-based connective tissues secreted by the somite-derived cells in amphioxus likely represent the rudimentary skeletal tissues in chordates. We propose that upon the foundation of this collagenous precursor, novel tissue mineralization genes that arose from gene duplications were incorporated into an ancestral mesodermal GRN that makes connective and supporting tissues, leading to the emergence of highly-mineralized skeletal tissues in early vertebrates.

Zoology: A New Mouth for Amphioxus.

Deuterostomes - a key subdivision of animals - are characterized by the mouth developing anteriorly as a rupture between the outer epithelium and the foregut wall. A new study of amphioxus challenges this view and proposes separate evolutionary origins of deuterostome oral openings.

Transcriptome analysis of different developmental stages of amphioxus reveals dynamic changes of distinct classes of genes during development.

Vertebrates diverged from other chordates approximately 500 million years ago and have adopted several modifications of developmental processes. Amphioxus is widely used in evolutionary developmental biology research, such as on the basic patterning mechanisms involved in the chordate body plan and the origin of vertebrates. The fast development of next-generation sequencing has advanced knowledge of the genomic organization of amphioxus; however, many aspects of gene regulation during amphioxus development have not been fully characterized. In this study, we applied high-throughput sequencing on the transcriptomes of 13 developmental stages of Chinese amphioxus to gain a comprehensive understanding of transcriptional processes occurring from the fertilized egg to the adult stage. The expression levels of 3,423 genes were significantly changed (FDR ≤ 0.01). All of these genes were included in a clustering analysis, and enrichment of biological functions associated with these clusters was determined. Significant changes were observed in several important processes, including the down-regulation of the cell cycle and the up-regulation of translation. These results should build a foundation for identifying developmentally important genes, especially those regulatory factors involved in amphioxus development, and advance understanding of the developmental dynamics in vertebrates.

A single three-dimensional chromatin compartment in amphioxus indicates a stepwise evolution of vertebrate Hox bimodal regulation.

The HoxA and HoxD gene clusters of jawed vertebrates are organized into bipartite three-dimensional chromatin structures that separate long-range regulatory inputs coming from the anterior and posterior Hox-neighboring regions. This architecture is instrumental in allowing vertebrate Hox genes to pattern disparate parts of the body, including limbs. Almost nothing is known about how these three-dimensional topologies originated. Here we perform extensive 4C-seq profiling of the Hox cluster in embryos of amphioxus, an invertebrate chordate. We find that, in contrast to the architecture in vertebrates, the amphioxus Hox cluster is organized into a single chromatin interaction domain that includes long-range contacts mostly from the anterior side, bringing distant cis-regulatory elements into contact with Hox genes. We infer that the vertebrate Hox bipartite regulatory system is an evolutionary novelty generated by combining ancient long-range regulatory contacts from DNA in the anterior Hox neighborhood with new regulatory inputs from the posterior side.

Alpha 2 macroglobulin is a maternally-derived immune factor in amphioxus embryos: New evidence for defense roles of maternal immune components in invertebrate chordate.

In fish, a series of maternal derived immune components have been identified in their eggs or embryos at very early stages, which are proposed to provide protections to themselves against pathogenic attacks from hostile environment. The phenomenon of maternal immunity has been also recorded in several invertebrate species, however, so far, very limited information about the maternal immune molecules are available. In this study, it was demonstrated maternal alpha2 macroglobulin (A2m) protein, an important innate immune factor, exists in the fertilized eggs of amphioxus Branchiostoma japonicum, an invertebrate chordate. Maternal mRNA of A2m was also detected in amphioxus embryos at very early developing stages. In addition, it was recorded that the egg lysate prepared from the newly fertilized eggs can inhibit the growth of both Gram-negative bacterium Escherichia coli and Gram-positive bacterium Staphylococcus aureus in a concentration dependent manner. The bacteriostatic activity can be reduced notably after precipitated A2m with anti-A2m antibody. Thus maternal A2m is partly attributed to the bacteriostatic activity. It was further demonstrated that recombinant A2m can bind to E. coli cells directly. All these points come to a result that A2m is a maternal immune factor existing in eggs of invertebrate chordate, which may be involved in defense their embryos against harmful microbes' attacks.

Generating amphioxus Hedgehog knockout mutants and phenotype analysis.

The amphioxus is a promising animal model for evolutionary-developmental studies due to its key position on the animal phylogenetic tree. In the present study, we reported a genetically modified amphioxus strain on the Hedgehog (Hh) gene locus using the TALEN method. The result showed that our TALEN pair injection could bring about 34% mutations in the amphioxus Hh coding region. Further analysis on the F(0) gametic DNA revealed that the mutations had entered into gametes. So, we paired one F(0) male carrying an 8 bp deletion with a wild-type (WT) female, and carefully nursed the F(1) embryos up to adulthood. We then screened F(1) individually via analyzing their genomic DNA from a tiny tail tip, and obtained eight heterozygous mutants from the F(1) offspring. Moreover, our observation on the F(2) embryos generated by mating F(1) mutants also revealed that about 25% of early larvae developed aberrantly with head and tail curving ventrally, agenesis of the mesoblastic tissue under their anterior notochord, and no mouth opening. With the larva growth, deformities (such as twist of head and tail, mouth absent, ventrally localized endostyle and gill slits) became more severe, and eventually those malformed larvae died due to no food intake. Genetic analysis showed that all these deformed embryos were homozygous mutants and the ratio of Hh hetorozygotes vs WT agreed with Mondel's law. WT amphioxus larvae are asymmetric with the mouth on the left and gill slits on the right side. However, the homozygous mutant larvae became left-right symmetric with the gill slits on the ventral side, indicating a conserved role of Hedgehog signaling in establishing the left-right embryonic axis.

Hybrids between the Florida amphioxus (Branchiostoma floridae) and the Bahamas lancelet (Asymmetron lucayanum): developmental morphology and chromosome counts.

The cephalochordate genera Branchiostoma and Asymmetron diverged during the Mesozoic Era. In spite of the long separation of the parental clades, eggs of the Florida amphioxus, B. floridae, when fertilized with sperm of the Bahamas lancelet, A. lucayanum (and vice versa), develop through embryonic and larval stages. The larvae reach the chordate phylotypic stage (i.e., the pharyngula), characterized by a dorsal nerve cord, notochord, perforate pharynx, and segmented trunk musculature. After about 2 weeks of larval development, the hybrids die, as do the A. lucayanum purebreds, although all were eating the same algal diet that sustains B. floridae purebreds through adulthood in the laboratory; it is thus unclear whether death of the hybrids results from incompatible parental genomes or an inadequate diet. The diploid chromosome count in A. lucayanum and B. floridae purebreds is, respectively, 34 and 38, whereas it is 36 in hybrids in either direction. The hybrid larvae exhibit several morphological characters intermediate between those of the parents, including the size of the preoral ciliated pit and the angles of deflection of the gill slits and anus from the ventral midline. Based on the time since the two parent clades diverged (120 or 160 million years, respectively, by nuclear and mitochondrial gene analysis), the cross between Branchiostoma and Asymmetron is the most extreme example of hybridization that has ever been unequivocally demonstrated among multicellular animals.

A comparative examination of neural circuit and brain patterning between the lamprey and amphioxus reveals the evolutionary origin of the vertebrate visual center.

Vertebrates are equipped with so-called camera eyes, which provide them with image-forming vision. Vertebrate image-forming vision evolved independently from that of other animals and is regarded as a key innovation for enhancing predatory ability and ecological success. Evolutionary changes in the neural circuits, particularly the visual center, were central for the acquisition of image-forming vision. However, the evolutionary steps, from protochordates to jaw-less primitive vertebrates and then to jawed vertebrates, remain largely unknown. To bridge this gap, we present the detailed development of retinofugal projections in the lamprey, the neuroarchitecture in amphioxus, and the brain patterning in both animals. Both the lateral eye in larval lamprey and the frontal eye in amphioxus project to a light-detecting visual center in the caudal prosencephalic region marked by Pax6, which possibly represents the ancestral state of the chordate visual system. Our results indicate that the visual system of the larval lamprey represents an evolutionarily primitive state, forming a link from protochordates to vertebrates and providing a new perspective of brain evolution based on developmental mechanisms and neural functions.