Large Scale Identification And Functional Exploration Of Alternative Splicing In Amphioxus Embryo Development

Alternative splicing confers different gene transcriptional products or gene isoforms spatiotemporally.Hence,alterantive splicing plays crucial roles in exploring diversity and dynamics of gene behaviors in biological events such as cell differentiation,cell fate determination,tissue/organ development,and so on.The analyzing from isoform level can help us understand the switch of gene functions.Amphioxus is considered as an idea animal model for investigating embryo development of organs.Study of alternative splicing during amphioxus embryo development will improves our understanding of vertebrate organ development.Imperferfect genome annotation leads a big setback to current large scale amphioxus study in precise identification and quantification of gene transcripts.To overcome this problem,we developed a computational workflow to build an integrative and high quality transcript library in basis of 16 strand-specific RNA sequencing transcriptomes determined at different Brachystoma floridae(B.floridae)embryo development stages,as well as one dataset generated by third generation sequencing with seven stages mixture RNA samples.Furthermore,we used the tool"Annocript" to compreshensively annotate the integrative transcript library.Eventually,we achived a library of 164,603 supertranscripts,189,528 distinct transcripts,and 41,157 isoforms for 16,231 genes.Using this library as reference,we precisely quantified the 16 amphioxus embryo development transcriptomes.In basis of the serial embryo development transcriptomes,we large scale detected the gene expression patterns during embryo delopment.Gene ontology(GO)enrichement analyses found the pattern-experessed genes,such as development-specific/selective genes and housekeeping genes,might play crucial roles in amphioxus embryo development.Combinding the gene expression change speed and trend,as well as KOG functional enrichment,we inferred that the maternal-zygotic transition likely started soon after fertilization and ended at 256-cell stage in amphioxus.Additionally,by application of NCA algorithm,we analyzed the activity change modes of transcription factors during amphioxus embryo development.We depicted that the transcription factors collaborated harmoniously by activation and deactivation at different development stages to ensure the embryo development at the right schedule.Finally,we portrayed the complexity of the alternative splicing landscape in amphioxus embryo development.From the perspective of alternative splicing,we described the evolutionary position of amphioxus.In particular,we found that high-incidence isoform switch occurred in the transition intervals of 256 cell-early gastrula,early gastrula-mid gastrula,and mid neurula-late nerula,indicating the progress of complex development events like organ development.Functional outcome analyses found that the genes intended to switch from protein-coding transcripts to noncoding transcripts,or long ORFs to shorter ORFs,or between different domains.In addition,we took gene KLF4 as an example to illustrate the biological significance behind the isoform switch.In summary,this study suggests potential targets for deep intevestigation of organ embryo development.It also provides data support for prompting amphioxus as ideal animal model for vertebrate organ development.