Patterns and Crucial Regulation of Alternative Splicing During Early Development in Zebrafish

The flow of genomic information, from DNA to RNA to protein, is a highly complex process in organisms. One of the important mechanisms of this complexity is alternative splicing (AS), which enables organisms to produce an assortment of transcripts and proteins by using relatively few protein-coding genes.1 AS can generate mRNAs that differ in their untranslated regions (UTRs) or coding sequence through a series of mechanisms,2 including skipped exons (SE, the exon either excluded or included from the mRNA), alternative 5′ and 3′ splice site events (A5′SS and A3′SS, extended or shortened of exon boundary on either the 5′- or 3′-end), mutually exclusive exons (MXE, one exon is skipped and the other is retained or vice versa), and retained intron (RI, intron included from the mRNA). The AS mechanism is vital in the biological process of the organism as a major source of molecular and functional diversity. Analysis of AS revealed that approximately 90–95% of human multi-exon genes underwent some levels of AS.3 AS is particularly important during development when differentiated tissues manufacture proteins with different functions from the same pre-mRNA, such as the brain,4 muscle,5 heart,6 liver,7 and others.8 Thus, AS as a regulator of development and tissue identity plays critical physiological functions in different developmental processes in organisms.8

The zebrafish (Danio rerio) possesses high morphological and genomic conservation with humans. It is a unique vertebrate model system and has been widely used in evolutionary developmental biology studies.9 During the development, embryogenesis through a prolonged and sophisticated process that involves dramatic morphological and functional changes in the embryo. A sequential series of dynamic processes included precise spatial and temporal regulation of gene expression. Knowledge of the genomic organization of zebrafish has been advanced due to the fast development of next-generation sequencing.10 However, many aspects of post-transcriptional regulation such as AS during zebrafish development have not been fully characterized. A few previous baselines AS studies on zebrafish focused on certain aspects such as organ development,11 sexual development and determination,12 and environmental stress response.13 The maternal-to-zygotic transition (MZT) represents a critical milestone in early zebrafish embryogenesis, and the activation occurs as late as cell divisions 6–8 cell stage in zebrafish, which significantly differs from the mammals (4–16 cell stages).14 During this period, maternally provided proteins and RNAs were degraded and replaced by products of the zygotic genome. However, the AS pattern changes during this period are not well reported in previous studies. These earlier studies were limited largely to individual experiments focusing on known candidate genes, and it remains unclear when and how AS occurs during zebrafish embryonic development.

AS is pervasive in fish genomes, yet different development stage comparison was largely restricted to fish. The functionality of most AS events remains unclear. Although the transcriptome of various developmental stages of zebrafish has been well characterized,15 the regulation of AS and AS genes involved in development remains elusive. To address such critical question, in the present study, we generated developmental AS events of 18 different development stages based on extensive and publicly available RNA-seq datasets.15 This gives detailed coverage of all the critical developmental processes taking place during this period. Specifically, developmental AS events and gene expression profiles during embryonic development indicated when and how cell fate decisions were made. Thus, a more comprehensive analysis of the AS control in zebrafish development is helpful to improve our understanding of the possible interface between embryonic development and AS regulatory mechanism. The main focus of this study was to investigate the global changes on developmental patterns of AS across different developmental stages of zebrafish.

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