Guang Li

Decelerated genome evolution in modern vertebrates revealed by analysis of multiple lancelet genomes

Vertebrates diverged from other chordates ~500u2009Myr ago and experienced successful innovations and adaptations, but the genomic basis underlying vertebrate origins are not fully understood. Here we suggest, through comparison with multiple lancelet (amphioxus) genomes, that ancient vertebrates experienced high rates of protein evolution, genome rearrangement and domain shuffling and that these rates greatly slowed down after the divergence of jawed and jawless vertebrates. Compared with lancelets, modern vertebrates retain, at least relatively, less protein diversity, fewer nucleotide polymorphisms, domain combinations and conserved non-coding elements (CNE). Modern vertebrates also lost substantial transposable element (TE) diversity, whereas lancelets preserve high TE diversity that includes even the long-sought RAG transposon. Lancelets also exhibit rapid gene turnover, pervasive transcription, fastest exon shuffling in metazoans and substantial TE methylation not observed in other invertebrates. These new lancelet genome sequences provide new insights into the chordate ancestral state and the vertebrate evolution.

The Evolutionarily Dynamic IFN-Inducible GTPase Proteins Play Conserved Immune Functions in Vertebrates and Cephalochordates

Interferon (IFN)-inducible GTPases currently include four families of proteins: myxovirus resistant proteins (Mxs), guanylate-binding proteins (GBPs), immunity-related GTPase proteins (IRGs), and very large inducible GTPase proteins (VLIGs). They are all under conserved regulation by IFNs in humans and mice and play a critical role in preventing microbial infections. However, differences between vertebrates are poorly characterized, and their evolutionary origins have not been studied in detail. In this study, we performed comparative genomic analysis of the four families in 18 representative animals that yielded several unexpected results. Firstly, we found that Mx, GBP, and IRG protein families arose before the divergence of chordate subphyla, but VLIG emerged solely in vertebrates. Secondly, IRG, GBP, and VLIG families have experienced a high rate of gene gain and loss during the evolution, with the GBP family being lost entirely in two pufferfish and VLIG family lost in primates and carnivores. Thirdly, the regulation of these genes by IFNs is highly conserved throughout vertebrates although the VLIG protein sequences in fish have lost the first 870 amino acid residues. Finally, amphioxus IFN-inducible GTPase genes are all highly expressed in immune-related organs such as gill, liver, and intestine and are upregulated after challenge with PolyI:C and pathogens, although no IFNs or their receptors were detected in the current amphioxus genome database. These results suggest that IFN-inducible GTPase genes play conserved immune functions both in vertebrates and in cephalochordates.

Consecutive Spawnings of Chinese Amphioxus, Branchiostoma belcheri, in Captivity

Cephalochordate amphioxus is a promising model animal for studying the evolutionary and developmental mechanisms of vertebrates because its unique phylogenetic position, simple body plan and sequenced genome. However, one major drawback for using amphioxus as a model organism is the restricted supply of living embryos since they are available only during spawning season that varies from a couple of days to several months according to species. Therefore we are aiming to develop methods for obtaining viable amphioxus embryos in non-spawning season. In the current study, we found that Branchiostoma belcheri could develop their gonads and spawn consecutively in the laboratory when cultured in a low density at a high temperature (25–28°C) supplied with sufficient food and proper cleanness. Among the approximate 150 observed animals, which spawned spontaneously between November and December 2011, 10% have spawned twice, 10% three times, and 80% four times, through April 2012. The quality and quantity of the gametes reproduced in the consecutive spawning have no obvious difference with those spawned once naturally. Spawning intervals varied dramatically both among different animals (from 1 to 5 months) and between intervals of a single individual (from 27 to 74 days for one animal). In summary, we developed a method with which, for the first time, consecutive spawnings of amphioxus in captivity can be achieved. This has practical implications for the cultivation of other amphioxus species, and eventually will greatly promote the utilization of amphioxus as a model system.

Year-round reproduction and induced spawning of Chinese amphioxus, Branchiostoma belcheri, in laboratory.

Amphioxus is a best candidate for studying the evolutionary and developmental mechanisms of vertebrates, because of its vertebrate-like but much simpler morphology, embryonic development and genome structure. Producing live amphioxus embryos throughout the year is an ideal for comparative evolution and developmental studies. However, all amphioxus species have distinct breeding seasons in the wild and laboratory. We recently found that Chinese amphioxus B . belcheri could reproduce repeatedly beyond its natural breeding season when reared under proper conditions. In this study, we were able to extend further and produce embryos throughout the year from October 2011 to October 2012. We found all examined animals had spawned repeatedly during the examined period. In addition, both lancelets B . belcheri and B . japonicum could be induced to spawn by heat-shock method, although the induced spawning efficiency was not as high as that observed in the European lancelet. In general, we have succeeded in producing B . belcheri embryos almost daily throughout the year. This advancement will provide essential embryonic material for evolutionary and developmental studies, and have great implications for the cultivation and spawning induction of other amphioxus species.

An efficient microinjection method for unfertilized eggs of Asian amphioxus Branchiostoma belcheri

Amphioxus is a promising model animal for evolutionary and developmental studies. However, as an emerging model organism, amphioxus lacks most molecular techniques applied in other well-developed model animals. Microinjection is a powerful technique for gene manipulation, and thus it undoubtedly is one of useful approaches in the studies of gene function and embryonic development. Although the method has been exploited in Florida and European amphioxus, it still remains to be optimized and introduced into other amphioxus species. In order to introduce the technique into our lab, we followed and optimized the previous description and successfully performed microinjection on unfertilized eggs of Asian amphioxus Branchiostoma belcheri. We made six solutions for practice: 200xa0mM KCl, 100xa0ng/μl actin-LacZ or 100xa0ng/μl actin-RFP vectors, LacZ or RFP vectors without promoter and RFP capped mRNA. More than 99.2xa0% of eggs injected with KCl were able to be fertilized, 94.3xa0% of them could hatch normally and 55.9xa0% survived until 2-day larvae, all of which were nearly equivalent to those obtained from normally fertilized eggs. Embryos injected with two plasmid constructs also showed very high fertilizing and hatching ratios, but normally developing ratios were slightly lower than that of KCl injection. Of those injected embryos, 91.8xa0% expressed exogenous gene LacZ and 80.5xa0% exhibited foreign RFP expression, which were driven by a promoter from amphioxus β-actin gene. The data indicated a successful modified microinjection method for the unfertilized eggs of Asian amphioxus, and those modifications improved the feasibility and efficiency of microinjection on amphioxus.

Evolutionary and functional diversity of green fluorescent proteins in cephalochordates

Green fluorescent protein (GFP) has been widely used as a molecular marker in modern biological research. Before the recent report of one GFP gene in Branchiostoma floridae, GFP family members were cloned only from other two groups of species: Cnidaria and Copepoda. Here we describe the complete GFP gene repertoire of B. floridae which includes 13 functional genes and 2 pseudogenes, representing the largest GFP family found so far. Coupling with nine other GFP sequences from another two species of genus Branchiostoma and the sequences from Cnidaria and Copepoda, we made a deep-level phylogenetic analysis for GFP genes in cephalochordates and found: 1) GFP genes have experienced a divergent evolution in cephalochordates; 2) all amphioxus GFP genes form four main clades on the tree which had diverged before the radiation of the last common ancestor of all extant cephalochordates; 3) GFP genes in amphioxus shared a common ancestor with that in Copepoda rather than being derived from horizontal gene transfer, which indicates that our ancestor was derived from a fluorescent organism and lost this ability after its separation from Cephalochordata, and also makes GFP a rare gene which has a rather unusual evolutionary path. In addition, we also provided evidence indicating that GFP genes have evolved divergent functions by specializing their expression profile, and different fluorescent spectra by changing their emission peaks. These findings spark two interesting issues: what are GFP in vivo functions in cephalochordates and why they are lost in other examined deuterostomes?

Mutagenesis at Specific Genomic Loci of Amphioxus Branchiostoma belcheri Using TALEN Method

Amphioxus, also called lancelet or cephalochordate, is a promising model organism owning to its particularly evolutionary position, simple genome content and comparable body plan to that of vertebrates (Holland et al., 2004; Bertrand and Escriva, 2011). However, use of amphioxus as a model organism has been limited for many years because of lack of an efficient genomic modification method. Recently, several revolutionary gene targeting methods that could induce directed mutations, insertions and deletions at intended target sites, have been developed (Gaj et al., 2013). Among these methods, the transcription activator-like effector nuclease (TALEN) has drawn much interest because of its efficiency in generating target gene alterations, simplicity in vector design and nearly limitless in targeting range (Huang et al., 2011; Miller et al., 2011; Bedell et al., 2012; Lei et al., 2012). Up to date, this method has been shown to be effective in inducing mutations in a broad range of organisms including zebrafish, frog, rat, mouse and human (Tong et al., 2012; Gaj et al., 2013; Liu et al., 2013a), suggesting a great potential use for adopting it in amphioxus genome engineering. Here, we presented the first report of an effective TALEN-mediated genome editing method in Chinese amphioxus, Branchiostoma belcheri. We chose B. belcheri because it is the only amphioxus species which could spawn consecutively all year round (Li et al., 2012, 2013) and be raised through generations in captivity (Zhang et al., 2007). Besides, the species is one of the four amphioxus frequently used in evolutionary/developmental studies and its genome sequence (http://mosas.sysu.edu.cn/genome/gbrowser_wel.php) and embryo microinjection are available in our lab (Liu et al., 2013b).

Characterization and embryonic expression of four amphioxus Frizzled genes with important functions during early embryogenesis.

The Wnt signaling pathway plays crucial roles in the embryonic patterning of all metazoans. Recent studies on Wnt genes in amphioxus have shed important insights into the evolution of the vertebrate Wnt gene family and their functions. Nevertheless, the potential roles of Wnt family receptors encoded by Frizzled (Fz) genes in amphioxus embryonic development remain to be investigated. In the present study, we identified four amphioxus Fz genes-AmphiFz1/2/7, AmphiFz4, AmphiFz5/8, and AmphiFz9/10-and analyzed their expression patterns during amphioxus embryogenesis. We found that these four Fz genes were maternally expressed and might be involved in early animal-vegetal axis establishment. The AmphiFz1/2/7 transcripts were detected in the central dorsal neural plate, mesoderm, the Hatscheks pit, and rim of the mouth, whereas those of AmphiFz4 were detected in the mesoderm, pharyngeal endoderm, and entire gut region. AmphiFz5/8 was exclusively expressed in the anterior-most region, whereas AmphiFz9/10 was expressed in the neural plate, somites, and tail bud. The dynamic and diverse expression patterns of amphioxus Fz genes suggest that these genes are not only associated with early embryonic axis establishment but also are involved in the development of several organs in amphioxus.

Isolation and functional analysis of the promoter of the amphioxus Hsp70a gene

Amphioxus is a promising laboratorial model animal for studying the evolutionary and developmental mechanisms that appeared during the invertebrate-chordate to vertebrate transition. However, the main drawback for the use of amphioxus as a model organism is the lack of well-developed technical approaches. Conditional gene expression, as performed with thermal control, is a very useful strategy in gene function studies. To make this method possible in amphioxus studies, here we report the isolation and characterization of an amphioxus Hsp70 gene (Hsp70a) and its promoter in Chinese amphioxus (Branchiostoma belcheri). Hsp70a showed very low expression at normal temperatures but was robustly induced in animals upon heat shock. The basal cis-acting elements (CAAT and TATA), as well as four heat shock elements (HSEs), were found within the regulatory region (-1031 to -11 upstream from the start codon), but surprisingly most of the elements were located in the 5UTR region (-252 to -10). Reporter constructs, including sequences from both the transcription start site (TSS) and ATG were tested for transient expression in EPC cells and microinjected zebrafish embryos. Results suggested that the 5UTR region, which includes a TATA box at -92bp, a CAAT box at -152bp, and three HSE elements (-212 to -106), represents the core hsp promoter sequence of the B. belcheri Hsp70a gene. Therefore in this study we identified an effectively thermo-inducible promoter in amphioxus that could be used for the establishment of a conditional gene expression system in which the target gene can be regulated in a temporal- or tissue-specific way in amphioxus.

Proteomic characterization and evolutionary analyses of zona pellucida domain-containing proteins in the egg coat of the cephalochordate, Branchiostoma belcheri

BackgroundZona pellucida domain-containing proteins (ZP proteins) have been identified as the principle constituents of the egg coat (EC) of diverse metazoan taxa, including jawed vertebrates, urochordates and molluscs that span hundreds of millions of years of evolutionary divergence. Although ZP proteins generally contain the zona pellucida (ZP) structural modules to fulfill sperm recognition and EC polymerization functions during fertilization, the primary sequences of the ZP proteins from the above-mentioned animal classes are drastically different, which makes it difficult to assess the evolutionary relationships of ZP proteins. To understand the origin of vertebrate ZP proteins, we characterized the egg coat components of Branchiostoma belcheri, an invertebrate species that belongs to the chordate subphylum Cephalochordata.ResultsFive ZP proteins (BbZP1-5) were identified by mass spectrometry analyses using the egg coat extracts from both unfertilized and fertilized eggs. In addition to the C-terminal ZP module in each of the BbZPs, the majority contain a low-density lipoprotein receptor domain and a von Willebrand factor type A (vWFA) domain, but none possess an EGF-like domain that is frequently observed in the ZP proteins of urochordates. Fluorescence in situ hybridization and immuno-histochemical analyses of B. belcheri ovaries showed that the five BbZPs are synthesized predominantly in developing eggs and deposited around the extracellular space of the egg, which indicates that they are bona fide egg coat ZP proteins. BbZP1, BbZP3 and BbZP4 are significantly more abundant than BbZP2 and BbZP5 in terms of gene expression levels and the amount of mature proteins present on the egg coats. The major ZP proteins showed high polymorphism because multiple variants are present with different molecular weights. Sequence comparison and phylogenetic analysis between the ZP proteins from cephalochordates, urochordates and vertebrates showed that BbZP1-5 form a monophyletic group and share no significant sequence similarities with the ZP proteins of urochordates and the ZP3 subtype of jawed vertebrates. By contrast, small regions of homology were identifiable between the BbZP and ZP proteins of the non-jawed vertebrate, the sea lamprey Petromyzon marinus. The lamprey ZP proteins were highly similar to the ZP1 and ZP2 subtypes of the jawed vertebrates, which suggests that the ZP proteins of basal chordates most likely shared a recent common ancestor with vertebrate ZP1/2 subtypes and lamprey ZP proteins.ConclusionsThe results document the spectra of zona pellucida domain-containing proteins of the egg coat of basal chordates. Particularly, the study provides solid evidence for an invertebrate origin of vertebrate ZP proteins and indicates that there are diverse domain architectures in ZP proteins of various metazoan groups.