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The cephalochordate amphioxus is a promising animal model for studying the origin of vertebrate due to its key phylogenetic position among chordates. Although transcription activator-like effector nucleases (TALENs) has been adopted in amphioxus genome editing, its labor intensive construction of the TALEN proteins limits its usage in many laboratories. We here report an application of the CRISPR/Cas9 system, a more amenable genome editing method, in this group of animals. Our data show that while co-injection of Cas9 mRNAs and sgRNAs into amphioxus unfertilized eggs causes no detectable mutations at targeted loci, injections of Cas9 mRNAs and sgRNAs at two cell stage, or of Cas9 protein and sgRNAs before fertilization can executes efficient disruptions of targeted genes. Among the nine tested sgRNAs (targeting five genes) co-injected with Cas9 protein, seven introduced mutations at efficiency ranging from 18.4% to 90% and four caused specific phenotypes in the injected embryos. We also demonstrate that monomerization of sgRNAs via thermal treatment or integration of sgRNAs into a longer backbone could increase mutation efficacies. Our study will not only promote application of genome editing method in amphioxus research, but also provide valuable experiences for other organisms in which the CRISPR/Cas9 system has not been successfully applied.

Cephalochordate amphioxus contain two Brachyury genes (AmphiBra1 and AmphiBra2). Using probes from the highly conserved coding regions, a summation of their expression profiles in amphioxus embryos have been investigated by several previous studies. However, their respective expression patterns have not been determined up to date. We here address this issue using both qRT-PCR and in situ hybridization methods (with probes from the divergent untranslated regions). qRT-PCR detected a very low maternal expression for AmphiBra2, but not for AmphiBra1. Zygotic expression of both genes are activated around early gastrula stage and change in a similar pattern at subsequent stages. However, compared to AmphiBra1, the expression level of AmphiBra2 is much higher in all examined stages of embryos; in some extreme cases an over fifty-times difference is observed. In situ hybridization and embryonic sections reveal that while AmphiBra2 is highly expressed in the blastopore, the tail bud and the notochord, AmphiBra1 is weakly transcribed only in the notochord. Our results show that the two Brachyury genes, resulted from a lineage-specific duplication in amphioxus, have evolved different embryonic expression profiles.

The systematic position of the amphioxus species with dextral gonads distributed in the southeast Pacific and Indian Oceans has remained to be clarified due to the adoption of different names by different researchers. Mitochondrial (mt) DNA is generally considered to be a powerful molecular marker in taxonomic studies. For a reliable systematics of the amphioxi collected from the South China Sea, we sequenced the complete mtDNA from a single specimen and compared it with those of the other eight amphioxus species. The present mtDNA genome contains 13 protein-coding genes, 22 tRNA genes, and two rRNA genes, with the same gene order as those in Branchiostoma and Epigonichthys, which, however, is different from that in Asymmetron. Based on our morphological data (including measurements of some characters) and the features of the mt genome, together with the distribution records of the dextral-gonad amphioxi, we conclude that the present mtDNA sequence most likely represents that of E. cultellus.

In addition to protein coding sequence, the organism genome contains a significant amount of regulatory DNA. Comparative genomics reveals that the organism genomes of vertebrates, tunicate, cephalochordate, flies, and nematodes contain cis-regulatory elements with highly conserved non-coding elements (CNEs). CNEs that cluster around trans-dev genes are part of core gene regulatory networks (GRNs), and usually, they can act as transcriptional enhancers. In this review, we described the identification of CNEs and summarized their key properties across the metazoans, and then discussed the evolution of CNEs after large-scale genome duplication events and the role of CNEs in the evolution of ani-mal body plan.

Endogenous green fluorescent proteins (GFPs) have recently been found in amphioxus, where different characteristics of light production in different development stages and between different individuals have manifested. We identified the GFP genes in an amphioxus species Branchiostoma belcheri and monitored real time fluorescence signals of GFPs during different developmental stages to provide an insight on the GFP function in amphioxus. We found there are at least 12 endogenous GFP genes in amphioxus genome, and fluorescent expression changes in body position during different developmental stages. Additionally, GFP expression after metamorphosis development differed significantly among different amphioxus individuals, suggesting that there are multiple GFP homologous genes responsible for fluorescent expression. Expression levels of these genes varied significantly during different developmental stages, indicating that different GFP genes may have their unique functions in the development of amphioxus during specific phases of growth.

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 5'UTR 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 5'UTR 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.

National Natural Science Foundation of China [30830023]; National High Technology Research and Development Program of China (863 Program) [2008AA092602]; Ministry of Education, China [20070384041]

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.

Identification of conserved genomic regions within and between different genomes is crucial when studying genome evolution. Here, we described regions of strong synteny conservation between vertebrate deuterostomes (tetrapods and teleosts) and invertebrate deuterostomes (amphioxus and sea urchin). The shared gene contents across phylogenetically distant species demonstrate that the conservation of the regions stemmed from an ancestral segment instead of a series of independent convergent events. Comparison of the syntenic regions allows us to postulate the primitive gene organization in the last common ancestor of deuterostomes and the evolutionary events that occurred to the 3 distinct lineages of sea urchin, amphioxus, and vertebrates after their separation. In addition, alignment of the syntenic regions led to the identification of 8 noncoding evolutionarily conserved regions shared between amphioxus and vertebrates. To our knowledge, this is the first report of conserved noncoding sequences shared by vertebrates and nonvertebrates. These noncoding sequences have high possibility of being elements that regulate neighboring genes. They are likely to be a factor in the maintenance of conserved synteny over long phylogenetic distance in different deuterostome lineages.

Amphioxus is a promising new animal model for developmental biology. To develop molecular tools for this model, we characterized the promoter region of a cytoplasmic β-actin gene (Bb-actin-6-2) from the Chinese amphioxus Branchiostoma belcheri. In situ hybridization and real time-quantitative PCR analyses showed that this gene is expressed in many tissues throughout embryonic development. Cloning of cDNA revealed two isoforms with distinct transcription start sites. Isoform #1 exhibits a similar exon/intron and regulatory element organization to that of vertebrate β-actin, whereas isoform #2 lacks the first exon of isoform #1 and recruits its first intron as a promoter. The activities of upstream promoter regions in the two isoforms were examined using the lacZ reporter system in amphioxus embryos. The proximal promoter of isoform #1 drove reporter gene expression broadly in 58.6 % of injected embryos. That of isoform #2 exhibited much higher activity (91.5 %) than that of isoform #1 or the human EF-1-α gene (38.2 %). We determined the minimal promoter regions of the two isoforms via functional analysis. These two regions, alone or inserted a random DNA fragment upstream, had no detectable activity, but when an upstream enhancer was inserted, the promoters directed reporter gene expression in 61.0 and 93.8 %, respectively, of injected embryos in a tissue-specific manner. Our study not only provides insight into the regulatory mechanism underlying amphioxus Bb-actin-6-2 gene expression, but also identifies two sets of efficient proximal and minimal promoters. These promoters could be used to construct gene expression vectors for transgenic studies using amphioxus as a model.

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: 200 mM KCl, 100 ng/μl actin-LacZ or 100 ng/μl actin-RFP vectors, LacZ or RFP vectors without promoter and RFP capped mRNA. More than 99.2 % of eggs injected with KCl were able to be fertilized, 94.3 % of them could hatch normally and 55.9 % 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, 91.8 % expressed exogenous gene LacZ and 80.5 % 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.

Amphioxus is a good model organism for understanding the origin and developmental mechanism of vertebrates owing to its important evolutionary position. During the developmental process of amphioxus embryo, the neurula is a crucial stage because of neural tube and notochord formation as well as somite emergence at this stage. In order to isolate genes up-regulated at the neurula stage, we constructed an 11-hour neurula subtracted cDNA library of amphioxus Branchiostoma belcheri and sequenced 204 ESTs representing 82 contigs. Comparative analysis revealed that 55% of those contigs were homologous to various known genes while 45% of them had no significant similarity to any known genes. Those observations imply that the un-identified ESTs might contain some new genes which are involved in the development of amphioxus neurula. Real-time quantitative PCR (RTqPCR) indicated that the expression levels of 14 genes are up-regulated after gastrulation among 20 assayed genes. Of those up-regulated genes, we further cloned and sequenced the full-length of fatty acid binding protein gene (AmphiFABP). The deduced protein sequence was similar to that of vertebrate brain FABP and heart FABP, and in situ hybridization displayed that AmphiFABP, similar to their vertebrate cognates, was expressed not only in nervous system but also in embryonic somite and gut, hinting a multifunctional property of AmphiFABP in amphioxus.

Previous studies showed that the vertebrate ABCA subfamily, one subgroup of the ATP-binding-cassette superfamily, has evolved rapidly in terms of gene duplication and loss. To further uncover the evolutionary history of the ABCA subfamily, we characterized ABCA members of two amphioxus species (Branchiostoma floridae and B. belcheri), the closest living invertebrate relative to vertebrates. Phylogenetic analysis indicated that these two species have the same set of ABCA genes (both containing six members). Five of these genes have clear orthologs in vertebrate, including one cephalochordate-specific duplication and one vertebrate-specific duplication. In addition, it is found that human orthologs of amphioxus ABCA1/4/7 and its neighboring genes mainly localize on chromosome 1, 9, 19 and 5. Considering that most of analyzed amphioxus genes have clear orthologs in zebrafish, we conclude these four human paralogous regions might derive from a common ancestral region by genome duplication occurred prior to teleost/tetrapod split. Therefore, the present results provide new evidence for 2R hypothesis.

Amphioxus is a crucial organism for the study of vertebrate evolution. Although a genomic BAC library of Branchiostoma floridae has been constructed, we report here another BAC library construction of its distant relative species Branchiostoma belcheri. The amphioxus BAC library established in present study consists of 45,312 clones arrayed in one hundred and eighteen 384-well plates. The average insert fragment size was 120 kb estimated by Pulsed Field Gel Electrophoresis (PFGE) analysis of 318 randomly selected clones. The representation of the library is about 12 equivalent to the genome, allowing a 99.9995% probability of recovering any specific sequence of interest. We further screened the library with 4 single copied Amphi-Pax genes and identified total of 26 positive clones with average of 6.5 clones for each gene. The result indicates this library is well suited for many applications and should also serve as a useful complemental resource for the scientific community.

Until now all amphioxus living in Xiamen waters have been regarded as Branchiostoma belcheri without any suspicion. However, a study based on Cyt b gene sequence comparisons of Branchiostoma belcheri (Oucuo in Xiamen waters) and Branchiostoma belcheri tsingtauense (Japanese waters) showed unexpected high divergence suggesting that the taxonomic status of Xiamen amphioxus should be reevaluated. In order to clarify this issue, we collected the animals from two sampling sites (Oucuo and Huangcuo in Xiamen waters), and compared their morphologies (meristic and non-meristic) as well as the complete sequences of their 12S rRNA genes. The samples could be distinguished by six of the non-meristic traits and five of the meristic traits. Moreover, the genetic distance based on 12S rRNA gene between Oucuo and Huangcuo amphioxus is 21.13%, but that between Huangcuo and Japanese amphioxus is only 0.56%. Our results suggest that original subspecies B. belcheri tsingtauense should be elevated to species level, becoming B. tsingtauense. Therefore, two species of genus Branchiostoma are living in Xiamen waters. One is the original species B. belcheri (Oucuo) and the other is B. tsingtauense (Huangcuo).

The mitochondrial DNA of the rice frog, Fejervarya limnocharis (Amphibia, Anura), was obtained using long-and-accurate polymerase chain reaction (LA-PCR) combining with subcloning method. The complete nucleotide sequence (17,717 bp) of mitochondrial genome was determined subsequently. This mitochondrial genome is characterized by four distinctive features: the translocation of ND5 gene, a cluster of rearranged tRNA genes (tRNA Thr , tRNA Pro , tRNA Leu (CUN) ), a tandem duplication of tRNA Met gene, and eight large 89-bp tandem repeats in the control region, as well as three short noncoding regions containing two repeated motifs existing in the gene cluster of ND5/tRNA Thr /tRNA Pro /tRNA Leu /tRNA Phe . The tandem duplication of gene regions followed by deletions of supernumerary genes can be invoked to explain the shuffling of tRNA Met and a cluster of tRNA and ND5 genes, as observed in this study. Both ND5 gene translocation and tandem duplication of tRNA Met were first observed in the vertebrate mitochondrial genomes.