Transcriptomic profile of early zebrafish PGCs by single cell sequencing.

Xiaoyuan Zhang,Shifeng Li,Yiping Li,Ronghong Li,Yunbin Zhang,Xintian Li,Sheng-Ping Lucinda Hwang

doi:10.1371/journal.pone.0220364

Abstract

Single cell RNA-seq is a powerful and sensitive way to capture the genome-wide gene expression. Here, single cell RNA-seq was utilized to study the transcriptomic profile of early zebrafish PGCs (primordial germ cells) at three different developmental stages. The three stages were 6, 11 and 24 hpf (hours post fertilization). For each developmental stage, three zebrafish PGCs from one embryo were collected, and 9 samples in total were used in this experiment. Single cell RNA-seq results showed that 5099–7376 genes were detected among the 9 samples, and the number of expressed genes decreased as development progressed. Based on the gene expression pattern, samples from 6 and 11 hpf clustered closely, while samples from 24 hpf were more dispersed. By WGCNA (weighted gene co-expression network analysis), the two biggest modules that had inverse gene expression patterns were found to be related to PGC formation or migration. Functional enrichment analysis for these two modules showed that PGCs mainly conducted migration and cell division in early development (6/11 hpf) and translation activity became active in late development (24 hpf). Differentially expressed gene analyses showed that more genes were downregulated than upregulated between two adjacent stages, and genes related to PGC formation or migration reported by previous studies decreased significantly from 11 to 24 hpf. Our results provide base knowledge about zebrafish PGC development at the single cell level and can be further studied by other researchers interested in biological development.

Highlights

As early as 1930s, the zebrafish (Danio rerio) has been used for embryological research, and in 1980s, zebrafish was used as a genetically tractable organism [1, 2]
Based on the gene expression pattern and functional enrichment analysis in the turquoise and blue modules, we found that the main task for PGCs were cell division and migration from 6 to 11 hpf, while translation become the main task from 24 hpf and later
Zebrafish PGCs were separated and sequenced to explore their dynamic transcriptomes during early development. 5099–7376 genes were expressed from 6 to 24 hpf, and the number of genes expressed decreased in the course of development

Summary

Introduction

As early as 1930s, the zebrafish (Danio rerio) has been used for embryological research, and in 1980s, zebrafish was used as a genetically tractable organism [1, 2]. Zebrafish PGC development and migration the reference genomes of human and zebrafish shows that about 70% of human genes have orthologs in zebrafish [1], so zebrafish has been widely used to study human genetic diseases, such as pediatric disease, cancer, immunological disease, inflammatory bowel disease and so on [2,3,4,5]. Thanks to transparent embryos, small size, external fertilization and obtainable fertilized eggs, zebrafish becomes an excellent model organism for studying vertebrate development [2, 3, 6, 7]. In 2005, Blaser et al successfully constructed a transgenic zebrafish whose PGCs could fluoresce green, and this technique made the separation of PGCs from embryos easy and was soon adopted by lots of researchers [18]

Methods

Results

Conclusion