Branchiostoma Floridae Research Articles

Epoxyalcohol Synthase Branch of Lipoxygenase Cascade.

Oxylipins are one of the most important classes of bioregulators, biosynthesized through the oxidative metabolism of unsaturated fatty acids in various aerobic organisms. Oxylipins are bioregulators that maintain homeostasis at the cellular and organismal levels. The most important oxylipins are mammalian eicosanoids and plant octadecanoids. In plants, the main source of oxylipins is the lipoxygenase cascade, the key enzymes of which are nonclassical cytochromes P450 of the CYP74 family, namely allene oxide synthases (AOSs), hydroperoxide lyases (HPLs), and divinyl ether synthases (DESs). The most well-studied plant oxylipins are jasmonates (AOS products) and traumatin and green leaf volatiles (HPL products), whereas other oxylipins remain outside of the focus of researchers' attention. Among them, there is a large group of epoxy hydroxy fatty acids (epoxyalcohols), whose biosynthesis has remained unclear for a long time. In 2008, the first epoxyalcohol synthase of lancelet Branchiostoma floridae, BfEAS (CYP440A1), was discovered. The present review collects data on EASs discovered after BfEAS and enzymes exhibiting EAS activity along with other catalytic activities. This review also presents the results of a study on the evolutionary processes possibly occurring within the P450 superfamily as a whole.

Serial block-face scanning electron microscopy of the tail tip of post-metamorphic amphioxus finds novel myomeres with odd shapes and unusually prominent sclerocoels.

Serial block-face scanning electron microscopyof the tail tip of post-metamorphic amphioxus (Branchiostoma floridae) revealed some terminal myomeres never been seen before with other techniques. The morphology of these myomeres differed markedly from the chevron shapes of their more anterior counterparts. Histologically, these odd-shaped myomeres ranged from empty vesicles bordered by undifferentiated cells to ventral sacs composed of well-developed myotome, dermatome, and sclerotome. Strikingly, several of these ventral sacs gave rise to a nipple-like dorsal projection composed either entirely of sclerotome or a mixture of sclerotome and myotome. Considered as a whole, from posterior to anterior, these odd-shaped posterior myomeres suggested that their more substantial ventral part may represent the ventral limb of a chevron, while the delicate projection represents a nascent dorsal limb. This scenario contrasts with formation of chevron-shaped myomeres along most of the antero-posterior axis. Although typical chevron formation in amphioxus is surprisingly poorly studied, it seems to be attained by a dorso-ventral extension of the myomere accompanied by the assumption of a V-shape; this is similar to what happens (at least superficially) in developing fishes. Another unusual feature of the odd-shaped posterior myomeres of amphioxus is their especially distended sclerocoels. One possible function for these might be to protect the posterior end of the central nervous system from trauma when the animals burrow into the substratum.

Comparison of Evolutionary Relationships between Branchiostoma floridae, Ciona intestinalis, and Homo sapiens Globins Provide Evidence of Gene Co-Option and Convergent Evolution.

Globins have been studied as model proteins to elucidate the principles of protein evolution. This was achieved by understanding the relationship between amino acid sequence, three-dimensional structure, physicochemical properties, and physiological function. Previous molecular phylogenies of chordate globin genes revealed the monophyletic evolution of urochordate globins and suggested convergent evolution. However, to provide evidence of convergent evolution, it is necessary to determine the physicochemical and functional similarities between vertebrates and urochordate globins. In this study, we determined the expression patterns of Ciona globin genes using real-time RT-PCR. Two genes (Gb-1 and Gb-2) were predominantly expressed in the branchial sac, heart, and hemocytes and were induced under hypoxia. Combined with the sequence analysis, our findings suggest that Gb-1/-2 correspond to vertebrate hemoglobin-α/-β. However, we did not find a robust similarity between Gb-3, Gb-4, and vertebrate globins. These results suggested that, even though Ciona globins obtained their unique functions differently from vertebrate globins, the two of them shared some physicochemical features and physiological functions. Our findings offer a good example for understanding the molecular mechanisms underlying gene co-option and convergence, which could lead to evolutionary innovations.

SAT026 Melanocortin Receptors In The Earliest Chordates?

Abstract Disclosure: R. Ji: None. S. Jiang: None. G. Kleinau: None. P. Scheerer: None. Y. Tao: None. Melanocortin receptors (MCRs) genes have been identified to be functional in the genome of early chordates, e.g. the cyclostomata, around 360 million years ago. Whether it appears in the most ancient chordates, such as cephalochordate and urochordata (500 million years ago), however, remained unknown until now. Herein, therefore, we studied experimentally four putative, sequence based predicted melanocortin-4 receptor (mc4r) like receptors and one putative MC1R-like receptor from urochordata and cephalochordate (from NCBI database), including Styela clava (sc), Ciona intestinalis (ci), Branchiostoma floridae (fl), and Branchiostoma belcheri (bb). No genes coding the endogenous MCR ligands were observed in these species. We further expressed these putative receptor plasmids in HEK293T cells and stimulated with α-melanocyte stimulating hormone (α-MSH) and synthetic MCR ligands to verify whether these receptors are functional MCRs. Our results showed that four receptors (so far named flMC4R, bbMC4R-1, bbMC4R-2, and ciMC1R) had high cell surface expression in HEK293T cells, whereas scMC4R had low cell surface expression. Four receptors (except scMC4R) showed high basal cAMP signaling, suggesting that these receptors are likely coupled to the stimulatory Gs protein. However, no specific binding to the superpotent α-MSH analog, NDP-MSH, were observed at these five receptors, and in accordance they had no α-MSH-induced cAMP signaling coupled to Gs stimulation. Additionally, all five receptors had low basal and no α-MSH-stimulated ERK1/2 signaling. Four human MC4R inverse agonists, including Agouti-related protein and three small molecular compounds (Ipsen5i, MCL0020, and ML00253764), had no effect on cAMP and ERK1/2 signaling of the four predicted MC4R-like receptors. In summary, our results suggested that the four predicted MCR-like receptors (except scMC4R) in ancient chordates are indeed functional receptors, with high constitutive activity, but they are all not MCR-like receptors based on our experimental data with common endogenous and synthetic MCR ligands. We hypothesize that these receptors might be MCR-like receptors, in that receptors might appear earlier than appearance of endogenous ligands; or these receptors might be other ancient G protein-coupled receptors (GPCRs) with other unidentified ligands. However, further studies are needed to identify the ancient GPCR system, which may include studies on the appearance of ligands or precursors. Presentation: Saturday, June 17, 2023

Discovery of Paralogous GnRH and Corazonin Signaling Systems in an Invertebrate Chordate.

Gonadotropin-releasing hormone (GnRH) is a key regulator of reproductive function in vertebrates. GnRH is related to the corazonin (CRZ) neuropeptide which influences metabolism and stress responses in insects. Recent evidence suggests that GnRH and CRZ are paralogous and arose by a gene duplication in a common ancestor of bilaterians. Here, we report the identification and complete characterization of the GnRH and CRZ signaling systems in the amphioxus Branchiostoma floridae. We have identified a novel GnRH peptide (YSYSYGFAP-NH2) that specifically activates two GnRH receptors and a CRZ peptide (FTYTHTW-NH2) that activates three CRZ receptors in B. floridae. The latter appear to be promiscuous, as two CRZ receptors can also be activated by GnRH in the physiological range. Hence, there is a potential for cross-talk between these closely related signaling systems. Discovery of both the GnRH and CRZ signaling systems in one of the closest living relatives of vertebrates provides a framework to discover their roles at the transition from invertebrates to vertebrates.

The long protostomic-type cytoplasmic intermediate filament (cIF) protein in Branchiostoma supports the phylogenetic transition between the protostomic- and the chordate-type cIFs.

We identified 23 and 20 cytoplasmic IF (cIF) genes in the two Branchiostoma belcheri and Branchiostoma lanceolatum cephalochordates, respectively. Combining these results with earlier data on the related Branchiostoma floridae, the following conclusions can be drawn. First, the Branchiostoma N4 protein with a long lamin-like coil 1B segment is the only protostomic-type cIF found so far in any analysed chordate or vertebrate organism. Second, Branchiostoma is the only organism known so far containing both the long protostomic- and the short chordate-prototypes of cIFs. This finding provides so far missing molecular evidence for the phylogenetic transition between the protostomic- and the chordate-type IF sequences at the base of the cephalochordates and vertebrates. Third, this finding also brings some support for another hypothesis, that the long protostomic-type cIF is subjected to evolutionary constraints in order to preclude inappropriate interactions with lamin and that the latter complexes might be prevented by a several heptad-long rod deletion, which released the selective constraints on it and promoted, at least in part, its expansion in nematodes, cephalochordates, and in vertebrates. Finally, here-presented data confirmed our previous results that cephalochordates do not have any vertebrate type III or type IV IF homolog.

Identification of a novel amphioxus leucine-rich repeat receptor involved in phagocytosis reveals a role for Slit2-N-type LRR in bacterial elimination

The basal chordate amphioxus is a model for tracing the origin and evolution of vertebrate immunity. To explore the evolution of immunoreceptor signaling pathways, we searched the associated receptors of the amphioxus Branchiostoma belcheri (Bb) homolog of immunoreceptor signaling adaptor protein Grb2. Mass-spectrum analysis of BbGrb2 immunoprecipitates from B.belcheri intestine lysates revealed a folate receptor (FR) domain- and leucine-rich repeat (LRR)-containing protein (FrLRR). Sequence and structural analysis showed that FrLRR is a membrane protein with a predicted curved solenoid structure. The N-terminal Fr domain contains very few folate-binding sites; the following LRR region is a Slit2-type LRR, and a GPI-anchored site was predicted at the C-terminus. RT-PCR analysis showed FrLRR is a transcription-mediated fusion gene of BbFR-like and BbSlit2-N-like genes. Genomic DNA structure analysis implied the B.belcheri FrLRR gene locus and the corresponding locus in Branchiostoma floridae might be generated by exon shuffling of a Slit2-N-like gene into an FR gene. RT-qPCR, immunostaining, and immunoblot results showed that FrLRR was primarily distributed in B.belcheri intestinal tissue. We further demonstrated that FrLRR localized to the cell membrane and lysosomes. Functionally, FrLRR mediated and promoted bacteria-binding and phagocytosis, and FrLRR antibody blocking or Grb2 knockdown inhibited FrLRR-mediated phagocytosis. Interestingly, we found that human Slit2-N (hSlit2-N) also mediated direct bacteria-binding and phagocytosis which was inhibited by Slit2-N antibody blocking or Grb2 knockdown. Together, these results indicate FrLRR and hSlit2-N may function as phagocytotic-receptors to promote phagocytosis through Grb2, implying the Slit2-N-type-LRR-containing proteins play a role in bacterial binding and elimination.

Ion regulation at gills precedes gas exchange and the origin of vertebrates.

Gas exchange and ion regulation at gills have key roles in the evolution of vertebrates1-4. Gills are hypothesized to have first acquired these important homeostatic functions from the skin in stem vertebrates, facilitating the evolution of larger, more-active modes of life2,3,5. However, this hypothesis lacks functional support in relevant taxa. Here we characterize the function of gills and skin in a vertebrate (lamprey ammocoete; Entosphenus tridentatus), a cephalochordate (amphioxus; Branchiostoma floridae) and a hemichordate (acorn worm; Saccoglossus kowalevskii) with the presumed burrowing, filter-feeding traits of vertebrate ancestors6-9. We provide functional support for a vertebrate origin of gas exchange at the gills with increasing body size and activity, as direct measurements in vivo reveal that gills are the dominant site of gas exchange only in ammocoetes, and only with increasing body size or challenges to oxygen supply and demand. Conversely, gills of all three taxa are implicated in ion regulation. Ammocoete gills are responsible for all ion flux at all body sizes, whereas molecular markers for ion regulation are higher in the gills than in the skin of amphioxus and acorn worms. This suggests that ion regulation at gills has an earlier origin than gas exchangethat is unrelated to vertebrate size and activity-perhaps at the very inception of pharyngeal pores in stem deuterostomes.

A novel violet fluorescent protein contains a unique oxidized tyrosine as the simplest chromophore ever reported in fluorescent proteins.

We describe an engineered violet fluorescent protein from the lancelet Branchiostoma floridae (bfVFP). This is the first example of a GFP-like fluorescent protein with a stable fluorescent chromophore lacking an imidazolinone ring; instead, it consists of oxidized tyrosine 68 flanked by glycine 67 and alanine 69. bfVFP contains the simplest chromophore reported in fluorescent proteins and was generated from the yellow protein lanFP10A2 by two synergetic mutations, S148H and C166I. The chromophore structure was confirmed crystallographically and by high-resolution mass spectrometry. The photophysical characteristics of bfVFP (323/430 nm, quantum yield 0.33, and Ec 14,300 M-1 cm-1 ) make it potentially useful for multicolor experiments to expand the excitation range of available FP biomarkers and Förster resonance energy transfer with blue and cyan fluorescent protein acceptors.

An Updated Staging System for Cephalochordate Development: One Table Suits Them All.

Chordates are divided into three subphyla: Vertebrata, Tunicata, and Cephalochordata. Phylogenetically, the Cephalochordata, more commonly known as lancelets or amphioxus, constitute the sister group of Vertebrata and Tunicata. Lancelets are small, benthic, marine filter feeders, and their roughly three dozen described species are divided into three genera: Branchiostoma, Epigonichthys, and Asymmetron. Due to their phylogenetic position and their stereotypical chordate morphology and genome architecture, lancelets are key models for understanding the evolutionary history of chordates. Lancelets have thus been studied by generations of scientists, with the first descriptions of adult anatomy and developmental morphology dating back to the 19th century. Today, several different lancelet species are used as laboratory models, predominantly for developmental, molecular and genomic studies. Surprisingly, however, a universal staging system and an unambiguous nomenclature for developing lancelets have not yet been adopted by the scientific community. In this work, we characterized the development of the European lancelet (Branchiostoma lanceolatum) using confocal microscopy and compiled a streamlined developmental staging system, from fertilization through larval life, including an unambiguous stage nomenclature. By tracing growth curves of the European lancelet reared at different temperatures, we were able to show that our staging system permitted an easy conversion of any developmental time into a specific stage name. Furthermore, comparisons of embryos and larvae from the European lancelet (B. lanceolatum), the Florida lancelet (Branchiostoma floridae), two Asian lancelets (Branchiostoma belcheri and Branchiostoma japonicum), and the Bahamas lancelet (Asymmetron lucayanum) demonstrated that our staging system could readily be applied to other lancelet species. Although the detailed staging description was carried out on developing B. lanceolatum, the comparisons with other lancelet species thus strongly suggested that both staging and nomenclature are applicable to all extant lancelets. We conclude that this description of embryonic and larval development will be of great use for the scientific community and that it should be adopted as the new standard for defining and naming developing lancelets. More generally, we anticipate that this work will facilitate future studies comparing representatives from different chordate lineages.

Filter feeding, deviations from bilateral symmetry, developmental noise, and heterochrony of hemichordate and cephalochordate gills.

We measured gill slit fluctuating asymmetry (FA), a measure of developmental noise, in adults of three invertebrate deuterostomes with different feeding modes: the cephalochordate Branchiostoma floridae (an obligate filter feeder), the enteropneusts Protoglossus graveolens (a facultative filter feeder/deposit feeder) and Saccoglossus bromophenolosus (a deposit feeder). FA was substantially and significantly low in B. floridae and P. graveolens and high in S. bromophenolosus. Our results suggest that the gills of species that have experienced a relaxation of the filter feeding trait exhibit elevated FA. We found that the timing of development of the secondary collagenous gill bars, compared to the primary gill bars, was highly variable in P. graveolens but not the other two species, demonstrating an independence of gill FA from gill bar heterochrony. We also discovered the occasional ectopic expression of a second set of paired gills posterior to the first set of gills in the enteropneusts and that these were more common in S. bromophenolosus. Moreover, our finding that gill slits in enteropneusts exhibit bilateral symmetry suggests that the left‐sidedness of larval cephalochordate gills, and the directional asymmetry of Cambrian stylophoran echinoderm fossil gills, evolved independently from a bilaterally symmetrical ancestor.

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 3-4months and a life span of 3years or more.

Comparative Neurobiology of Biogenic Amines in Animal Models in Deuterostomes

In this study, we review the current knowledge on the presence of biogenic amines and their potential role as neurotransmitters in the nervous system of three groups of invertebrate deuterostomes: tunicates, cephalochordates and echinoderms. In addition to a general overview of biogenic amines in each subphylum we point most of our attention on a few species, as the sea squirts Ciona intestinalis, Ciona robusta, Phallusia mammillata and Ciona savignyi (tunicates), the lancelet amphioxus Branchiostoma lanceolatum and Branchiostoma floridae (cephalochordates), and the sea urchin Strongylocentrotus purpuratus (echinoderms). The choice of these species lies in the fact that they are currently the most studied invertebrate deuterostomes in the research field of Evolutionary Developmental biology (EvoDevo). Providing a comparative picture of the expression and role of neurotransmitters in deuterostomes will contribute to understand the evolution of these neural signalling systems. Such an approach represents a new frontier of comparative neuroanatomy and neurobiology, and a prerequisite to uncover the homology of neuronal structures and circuits in deuterostomes with such a different body plan organization and complexity.

Mutation of amphioxus Pdx and Cdx demonstrates conserved roles for ParaHox genes in gut, anus and tail patterning

BackgroundThe homeobox genes Pdx and Cdx are widespread across the animal kingdom and part of the small ParaHox gene cluster. Gene expression patterns suggest ancient roles for Pdx and Cdx in patterning the through-gut of bilaterian animals although functional data are available for few lineages. To examine evolutionary conservation of Pdx and Cdx gene functions, we focus on amphioxus, small marine animals that occupy a pivotal position in chordate evolution and in which ParaHox gene clustering was first reported.ResultsUsing transcription activator-like effector nucleases (TALENs), we engineer frameshift mutations in the Pdx and Cdx genes of the amphioxus Branchiostoma floridae and establish mutant lines. Homozygous Pdx mutants have a defect in amphioxus endoderm, manifest as loss of a midgut region expressing endogenous GFP. The anus fails to open in homozygous Cdx mutants, which also have defects in posterior body extension and epidermal tail fin development. Treatment with an inverse agonist of retinoic acid (RA) signalling partially rescues the axial and tail fin phenotypes indicating they are caused by increased RA signalling. Gene expression analyses and luciferase assays suggest that posterior RA levels are kept low in wild type animals by a likely direct transcriptional regulation of a Cyp26 gene by Cdx. Transcriptome analysis reveals extensive gene expression changes in mutants, with a disproportionate effect of Pdx and Cdx on gut-enriched genes and a colinear-like effect of Cdx on Hox genes.ConclusionsThese data reveal that amphioxus Pdx and Cdx have roles in specifying middle and posterior cell fates in the endoderm of the gut, roles that likely date to the origin of Bilateria. This conclusion is consistent with these two ParaHox genes playing a role in the origin of the bilaterian through-gut with a distinct anus, morphological innovations that contributed to ecological change in the Cambrian. In addition, we find that amphioxus Cdx promotes body axis extension through a molecular mechanism conserved with vertebrates. The axial extension role for Cdx dates back at least to the origin of Chordata and may have facilitated the evolution of the post-anal tail and active locomotion in chordates.

Tracing the Origins of the Pituitary Adenylate-Cyclase Activating Polypeptide (PACAP).

Pituitary adenylate cyclase activating polypeptide (PACAP) is a well-conserved neuropeptide characteristic of vertebrates. This pluripotent hypothalamic neuropeptide regulates neurotransmitter release, intestinal motility, metabolism, cell division/differentiation, and immunity. In vertebrates, PACAP has a specific receptor (PAC1) but it can also activate the Vasoactive Intestinal Peptide receptors (VPAC1 and VPAC2). The evolution of the vertebrate PACAP ligand – receptor pair has been well-described. In contrast, the situation in invertebrates is much less clear. The PACAP ligand – receptor pair in invertebrates has mainly been studied using heterologous antibodies raised against mammalian peptides. A few partial PACAP cDNA clones sharing >87% aa identity with vertebrate PACAP have been isolated from a cnidarian, several protostomes and tunicates but no gene has been reported. Moreover, current evolutionary models of the peptide and receptors using molecular data from phylogenetically distinct invertebrate species (mostly nematodes and arthropods) suggests the PACAP ligand and receptors are exclusive to vertebrate genomes. A basal deuterostome, the cephalochordate amphioxus (Branchiostoma floridae), is the only invertebrate in which elements of a PACAP-like system exists but the peptides and receptor share relatively low sequence conservation with the vertebrate homolog system and are a hybrid with the vertebrate glucagon system. In this study, the evolution of the PACAP system is revisited taking advantage of the burgeoning sequence data (genome and transcriptomes) available for invertebrates to uncover clues about when it first appeared. The results suggest that elements of the PACAP system are absent from protozoans, non-bilaterians, and protostomes and they only emerged after the protostome-deuterostome divergence. PACAP and its receptors appeared in vertebrate genomes and they probably shared a common ancestral origin with the cephalochordate PACAP/GCG-like system which after the genome tetraploidization events that preceded the vertebrate radiation generated the PACAP ligand and receptor pair and also the other members of the Secretin family peptides and their receptors.

Agglutination of plasma, hemocyanin, and separated hemocyanin from the hemolymph of the freshwater prawn Macrobrachium rosenbergii (De Man, 1879) (Decapoda: Caridea: Palaemonidae)

AbstractHemocyanin, the predominant protein in the hemolymph of crustaceans, was isolated from the plasma of the giant freshwater prawn Macrobrachium rosenbergii (De Man, 1879) and investigated, with its separated proteins MrHc1, 2, 3, and 4 (60, 114, 50, and 325 kDa, respectively) for hemagglutination (HA) activity. Hemocyanin against erythrocytes from human A, B, and O blood groups, mouse, rat sheep, water buffalo, and rabbit showed HA titer values higher than plasma. Of the four proteins isolated from hemocyanin, MrHc1 alone appeared to agglutinate the tested erythrocytes. Evaluated specific hemagglutination activity was higher for hemocyanin when compared to plasma. Sugar binding ability with N-acetylated sugars observed for plasma was restricted to N-acetyl neuraminic acid for hemocyanin and MrHc1. Inhibition of hemagglutination (HAI) by bovine submaxillary mucin (BSM) further defined the agglutination ability of hemocyanin and MrHc1. The hemocyanin protein MrHc1 on matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI–TOF/MS) and search in MASCOT database showed homology to hypothetical protein of Branchiostoma floridae Hubbs, 1922 (Cephalochordata). The results clearly indicated that the structure of the hemocyanin had evolved binding sites for sugar or sugar-linkages independently lacking homology with other hemocyanin or lectin-like proteins. The immunogenicity of this hemocyanin probably has biomedical applications as carrier adjuvants, which needs further investigation.

Evolutionary Analysis of the Zinc Finger and Homeoboxes Family of Proteins Identifies Multiple Conserved Domains and a Common Early Chordate Ancestor.

The Zinc Fingers and Homeoboxes (Zhx) proteins, Zhx1, Zhx2, and Zhx3, comprise a small family of proteins containing two amino-terminal C2–H2 zinc fingers and four or five carboxy-terminal homeodomains. These multiple homeodomains make Zhx proteins unusual because the majority of homeodomain-containing proteins contain a single homeodomain. Studies in cultured cells and mice suggest that Zhx proteins can function as positive or negative transcriptional regulators. Zhx2 regulates numerous hepatic genes, and all three Zhx proteins have been implicated in different cancers. Because Zhx proteins contain multiple predicted homeodomains, are associated with interesting physiological traits, and seem to be only present in the vertebrate lineage, we investigated the evolutionary history of this small family by comparing Zhx homologs from a wide range of chordates. This analysis indicates that the zinc finger motifs and homeodomains are highly similar among all Zhx proteins and also identifies additional Zhx-specific conserved regions, including a 13 amino acid amino-terminal motif that is nearly identical among all gnathostome Zhx proteins. We found single Zhx proteins in the sea lamprey (Petromyzon marinus) and in the nonvertebrate chordates sea squirt (Ciona intestinalis) and lancelet (Branchiostoma floridae); these Zhx proteins are most similar to gnathostome Zhx3. Based on our analyses, we propose that a duplication of the primordial Zhx gene gave rise to Zhx3 and the precursor to Zhx1 and Zhx2. A subsequent tandem duplication of this precursor generated Zhx1 and Zhx2 found in gnathostomes.

A ZZ/ZW Sex Chromosome System in Cephalochordate Amphioxus.

Sex determination is remarkably variable among animals with examples of environmental sex determination, male heterogametic (XX/XY) and female heterogametic (ZZ/ZW) chromosomal sex determination, and other genetic mechanisms. The cephalochordate amphioxus occupies a key phylogenetic position as a basal chordate and outgroup to vertebrates, but its sex determination mechanism is unknown. During the course of generating Nodal mutants with transcription activator-like effector nucleases (TALENs) in amphioxus Branchiostoma floridae, serendipitously, we generated three mutant strains that reveal the sex determination mechanism of this animal. In one mutant strain, all heterozygous mutant offspring over three generations were female and all wild-type descendants were male. This pattern suggests the Nodal allele targeted is on a female-specific W chromosome. A second mutant showed the same W-linked inheritance pattern, with a female heterozygote passing the mutation only to daughters. In a third mutant strain, both male and female offspring could be heterozygous, but a female heterozygote passed the mutation only to sons. This pattern is consistent with the targeted allele being on a Z chromosome. We found an indel polymorphism linked to a Nodal allele present in most females, but no males in our cultured population. Together, these results indicate that Nodal is sex chromosome-linked in B. floridae, and that B. floridae has a ZZ/ZW sex chromosome system.

LanceletDB: an integrated genome database for lancelet, comparing domain types and combination in orthologues among lancelet and other species.

Lancelet (amphioxus) represents the most basally divergent extant chordate (cephalochordates) that diverged from the other two chordate lineages (urochordates and vertebrates) more than half a billion years ago. As it occupies a key position in evolution, it is considered as one of the best proxies for understanding the chordate ancestral state. Thus, the construction of a database with multiple lancelet genomes and gene annotation data, including protein domains, is urgently needed to investigate the loss and gain of domains in orthologues among species, especially ancient domain types (non-vertebrate-specific domains) and novel domain combination, which is helpful for providing new insight into the chordate ancestral state and vertebrate evolution. Here, we present an integrated genome database for lancelet, LanceletDB, which provides reference haploid genome sequence and annotation data for lancelet (Branchiostoma belcheri), including gene models and annotation, protein domain types, gene expression pattern in embryogenesis, different expression sequence tag sets and alternative polyadenylation (APA) sites profiled by the sequencing APA sites method. Especially, LanceletDB allows comparison of domain types and combination in orthologues among type species so as to decode the ancient domain types and novel domain combination during evolution. We also integrated the released diploid lancelet genome annotation data (Branchiostoma floridae) to expand LanceletDB and extend its usefulness. These data are available through the search and analysis page, basic local alignment search tool page and genome browser to provide an integrated display.

Functional conserved non-coding elements among tunicates and chordates

Non-coding regions with dozens to several hundred base pairs of extreme conservation have been found in all metazoan genomes. The distribution of these conserved non-coding elements (CNE) within and across genomes has suggested that many of them may have roles as transcriptional regulatory elements. A combination of bioinformatics and experimental approaches can be used to identify CNEs with regulatory activity in phylogenetically distant species. Nevertheless, the high divergent rate of genomic sequences of several organisms, such as tunicates, complicates the characterization of these conserved elements and very few examples really may prove their functional activity.We used a comparative approach to facilitate the identification of CNEs among distantly related or highly divergent species and experimentally demonstrated the functional significance of these novel CNEs.We first experimentally tested, in C. robusta and D. rerio transgenic embryos, the regulatory activity of conserved elements associated to genes involved in developmental control among different chordates (Homo sapiens and Danio rerio for vertebrates, Ciona robusta and Ciona savignyi for tunicates and Branchiostoma floridae for cephalochordates). Once demonstrated the cross-species functional conservation of these CNEs, the same gene loci were used as references to locate homologous regions and possible CNEs in available tunicate genomes.Comparison of tunicate-specific and chordate-specific CNEs revealed absence of conservation of the regulatory elements in spite of conservation of regulatory patterns, likely due to evolutionary specification of the respective developmental networks. This result highlights the importance of an integrative in-silico/in-vivo approach to CNEs investigation, encompassing both bioinformatics, essential for putative CNEs identification, and laboratory experiments, pivotal for the understanding of CNEs functionality.