Characterization of Alternative Splicing (AS) Events during Chicken (Gallus gallus) Male Germ-Line Stem Cell Differentiation with Single-Cell RNA-seq.

Changhua Sun,Hongyan Sun,Guohong Chen,Kai Jin,Qisheng Zuo,Jiuzhou Song,Yani Zhang,Bichun Li

doi:10.3390/ani11051469

Abstract

Simple SummaryStudies have shown that alternative splicing (AS) has been utilized in a wide variety of life processes. However, there are very few studies on AS during germ cell development. In this study, we preliminarily investigated the variation of variable shear events during the formation of chicken germ cells through the RNA-seq data analysis of embryonic stem cells (ESCs), gonad PGCs (gPGCs), and spermatogonia stem cells (SSCs), and the critical AS mode for several crucial stage-specific genes, which were identified during germ cell development. The results of this study lay a theoretical foundation for further analysis of the regulation mechanism of key genes involved in germ cell formation.Alternative splicing (AS) is a ubiquitous, co-transcriptional, and post-transcriptional regulation mechanism during certain developmental processes, such as germ cell differentiation. A thorough understanding of germ cell differentiation will help us to open new avenues for avian reproduction, stem cell biology, and advances in medicines for human consumption. Here, based on single-cell RNA-seq, we characterized genome-wide AS events in manifold chicken male germ cells: embryonic stem cells (ESCs), gonad primordial germ cells (gPGCs), and spermatogonia stem cells (SSCs). A total of 38,494 AS events from 15,338 genes were detected in ESCs, with a total of 48,955 events from 14,783 genes and 49,900 events from 15,089 genes observed in gPGCs and SSCs, respectively. Moreover, this distribution of AS events suggests the diverse splicing feature of ESCs, gPGCs, and SSCs. Finally, several crucial stage-specific genes, such as NANOG, POU5F3, LIN28B, BMP4, STRA8, and LHX9, were identified in AS events that were transmitted in ESCs, gPGCs, and SSCs. The gene expression results of the RNA-seq data were validated by qRT-PCR. In summary, we provided a comprehensive atlas of the genome-wide scale of the AS event landscape in male chicken germ-line cells and presented its distribution for the first time. This research may someday improve treatment options for men suffering from male infertility.

Highlights

Alternative splicing (AS) is an important, co-transcriptional, and post-transcriptional regulatory mechanism that results in multiple mRNA and protein isoforms from a single gene
embryonic stem cells (ESCs) was separated from newly laid fertilized eggs and digested with trypsin-EDTA (0.25%) for 3 min. gonad primordial germ cells (gPGCs) were isolated from eggs which had been incubated for 4.5 days
To study AS events in male chicken germ cell differentiation, we performed genomewide RNA-seq for ESCs, gPGCs, and spermatogonia stem cells (SSCs) isolated from chicken embryonic blastoderms (E0, HH stage X), gonads (E4.5, HH stage 28–30), and testes (E18.5, HH stage 44), respectively (Figure 1a)

Summary

Introduction

Alternative splicing (AS) is an important, co-transcriptional, and post-transcriptional regulatory mechanism that results in multiple mRNA and protein isoforms from a single gene. Protein isoforms show different or mutually antagonistically functional and structural characteristics, which play critical roles in cellular processes, such as cell signaling and differentiation [1,2,3]. AS is the fundamental cause of significantly fewer genes than protein species in eukaryotes. AS events are involved in many physiological processes, as well as cell differentiation, apoptosis, and development. Abnormal AS events will cause deviant processes by changing gene expression products [8,9,10,11]. Studying AS in biological processes is vitally important and essential

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