Three amphioxus reference genomes reveal gene and chromosome evolution of chordates

The slow-evolving invertebrate amphioxus has an irreplaceable role in advancing our understanding into the vertebrate origin and innovations. Here we resolve the nearly complete chromosomal genomes of three amphioxus species, one of which best recapitulates the 17 chordate ancestor linkage groups. We reconstruct the fusions, retention or rearrangements between descendants of whole genome duplications (WGDs), which gave rise to the extant microchromosomes likely existed in the vertebrate ancestor. Similar to vertebrates, the amphioxus genome gradually establishes its 3D chromatin architecture at the onset of zygotic activation, and forms two topologically associated domains at the Hox gene cluster. We find that all three amphioxus species have ZW sex chromosomes with little sequence differentiation, and their putative sex-determining regions are nonhomologous to each other. Our results illuminate the unappreciated interspecific diversity and developmental dynamics of amphioxus genomes, and provide high-quality references for understanding the mechanisms of chordate functional genome evolution.

Application of CRISPR/Cas9 Nuclease in Amphioxus Genome Editing

The cephalochordate amphioxus is a promising animal model for studying the origin of vertebrates due to its key phylogenetic position among chordates. Although transcription activator-like effector nucleases (TALENs) have been adopted in amphioxus genome editing, its labor-intensive construction of TALEN proteins limits its usage in many laboratories. Here we reported an application of the CRISPR/Cas9 system, a more amenable genome editing method, in this group of animals. Our data showed that while co-injection of Cas9 mRNAs and sgRNAs into amphioxus unfertilized eggs caused no detectable mutations at targeted loci, injections of Cas9 mRNAs and sgRNAs at the two-cell stage, or of Cas9 protein and sgRNAs before fertilization, can execute efficient disruptions of targeted genes. Among the nine tested sgRNAs (targeting five genes) co-injected with Cas9 protein, seven introduced mutations with efficiency ranging from 18.4% to 90% and four caused specific phenotypes in the injected embryos. We also demonstrated that monomerization of sgRNAs via thermal treatment or modifying the sgRNA structure 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.

Application of CRISPR/Cas9 nuclease in amphioxus genome editing

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.

Integrate Heterogeneous NGS and TGS Data to Boost Genome-free Transcriptome Research

It is a long-term challenge to undertake reliable transcriptomic research under different circumstances of genome availability. Here, we newly developed a genome-free computational method to aid accurate transcriptome assembly, using the amphioxus as the example. Via integrating ten next generation sequencing (NGS) transcriptome datasets and one third-generation sequencing (TGS) dataset, we built a sequence library of non-redundant expressed transcripts for the amphioxus. The library consisted of overall 91,915 distinct transcripts, 51,549 protein-coding transcripts, and 16,923 novel extragenic transcripts. This substantially improved current amphioxus genome annotation by expanding the distinct gene number from 21,954 to 38,777. We consolidated the library significantly outperformed the genome, as well as de novo method, in transcriptome assembly from multiple aspects. For convenience, we curated the Integrative Transcript Library database of the amphioxus (http://www.bio-add.org/InTrans/). In summary, this work provides a practical solution for most organisms to alleviate the heavy dependence on good quality genome in transcriptome research. It also ensures the amphioxus transcriptome research grounding on reliable data.

Amphioxus: Beginning of Vertebrate and End of Invertebrate Type GnRH Receptor Lineage

In vertebrates, activation of the GnRH receptor is necessary to initiate the reproductive cascade. However, little is known about the characteristics of GnRH receptors before the vertebrates evolved. Recently genome sequencing was completed for amphioxus, Branchiostoma floridae. To understand the GnRH receptors (GnRHR) from this most basal chordate, which is also classified as an invertebrate, we cloned and characterized four GnRHR cDNAs encoded in the amphioxus genome. We found that incubation of GnRH1 (mammalian GnRH) and GnRH2 (chicken GnRH II) with COS7 cells heterologously expressing the amphioxus GnRHRs caused potent intracellular inositol phosphate turnover in two of the receptors. One of the two receptors displayed a clear preference for GnRH1 over GnRH2, a characteristic not previously seen outside the type I mammalian GnRHRs. Phylogenetic analysis grouped the four receptors into two paralogous pairs, with one pair grouping basally with the vertebrate GnRH receptors and the other grouping with the octopus GnRHR-like sequence and the related receptor for insect adipokinetic hormone. Pharmacological studies showed that octopus GnRH-like peptide and adipokinetic hormone induced potent inositol phosphate turnover in one of these other two amphioxus receptors. These data demonstrate the functional conservation of two distinct types of GnRH receptors at the base of chordates. We propose that one receptor type led to vertebrate GnRHRs, whereas the other type, related to the mollusk GnRHR-like receptor, was lost in the vertebrate lineage. This is the first report to suggest that distinct invertebrate and vertebrate GnRHRs are present simultaneously in a basal chordate, amphioxus.