Unveiling Gene Expression Dynamics during Early Embryogenesis in Cynoglossus semilaevis: A Transcriptomic Perspective

Abstract

Commencing with sperm-egg fusion, the early stages of metazoan development include the cleavage and formation of blastula and gastrula. These early embryonic events play a crucial role in ontogeny and are accompanied by a dramatic remodeling of the gene network, particularly encompassing the maternal-to-zygotic transition. Nonetheless, the gene expression dynamics governing early embryogenesis remain unclear in most metazoan lineages. We conducted transcriptomic profiling on two types of gametes (oocytes and sperms) and early embryos (ranging from the four-cell to the gastrula stage) of an economically valuable flatfish-the Chinese tongue sole Cynoglossus semilaevis (Pleuronectiformes: Cynoglossidae). Comparative transcriptome analysis revealed that large-scale zygotic genome activation (ZGA) occurs in the blastula stage, aligning with previous findings in zebrafish. Through the comparison of the most abundant transcripts identified in each sample and the functional analysis of co-expression modules, we unveiled distinct functional enrichments across different gametes/developmental stages: actin- and immune-related functions in sperms; mitosis, transcription inhibition, and mitochondrial function in oocytes and in pre-ZGA embryos (four- to 1000-cell stage); and organ development in post-ZGA embryos (blastula and gastrula). These results provide insights into the intricate transcriptional regulation of early embryonic development in Cynoglossidae fish and expand our knowledge of developmental constraints in vertebrates.

Keywords: Cynoglossidae; Cynoglossus semilaevis; early embryonic development; gametes; transcriptomics.

Figure 1

A schematic for sample collection with the number of replicates indicated (top) and the high consistency of gene expression between replicates (bottom). The degree of consistency was measured by Pearson’s correlation of gene expression between two replicates within the same sample group. The mean correlation coefficient for each sample group is presented under the box.

Figure 2

(A) Principal component analysis (PCA) based on 21,920 Cynoglossus semilaevis genes covered by at least one read. (B) Numbers of up-regulated (pink) and down-regulated (grey) differentially expressed genes (DEGs) between pairs of consecutive embryogenesis stages in C. semilaevis. Sp: sperm; Oo: oocyte; 4c: 4 cells; 32c: 32 cells; 128c: 128 cells; 1kc: 1000 cells; Bla: blastula; Gas: gastrula. (C) The five most abundant gene transcripts in different gametes and embryonic developmental stages in C. semilaevis. The potential functions of genes are indicated by color. The putative gene (gene ID: Cse_R021186, located in scaffold7208: 12,125–16,516) was structurally predicted but did not correspond to orthologs in the current NCBI-nr database; its biological function is unclear. TPM, transcripts per million.

Figure 3

(A) Hierarchical clustering of the 24 samples based on 9549 dynamically expressed genes. Three distinct clusters of samples are highlighted by different colors and named after the developmental stages, namely “Sperm” (blue), “Oocyte and pre-ZGA embryo” (red), and “Post-ZGA embryo” (yellow). (B) The correlation (Cor) between the eight sample categories and five co-expression modules. The significance (P) of Spearman’s correlation analysis is indicated by asterisks. (C) Relative expression levels of genes in different modules, represented by Z-score of transcripts per million (TPM). The title of each plot indicates the highly expressed stage(s) of gene members in the relevant module. The number of genes is shown in parentheses. Large-scale zygotic genome activation (ZGA) was inferred to occur in the blastula stage.

Figure 4

Hub genes with the top 30 intra-modular connectivity in five co-expression modules, respectively, namely Sperm (A), Maternal (B), Pre-ZGA (C), Post-ZGA (D), and Sperm–post-ZGA (E). Purple lines represent the correlation (0.95~1) between two linked genes, with line width representing value size. Genes closer to the center of the network (indicated by light yellow) have higher intra-modular connectivity.

Figure 5

The top most significant Gene Ontology (GO) biological processes which are enriched in different gene co-expression modules, respectively. GO ID of each term is indicated in parentheses. Biological processes with similar functions are clustered and represented by a single icon. FDR: false discovery rate.

Figure 6

(A) Interspecific conservation of genes in co-expression modules, compared with Danio rerio. The distribution bias of orthologs was tested by Fisher’s exact test. (B) Relative expression level of the orthologs in D. rerio during embryogenesis. The horizontal scale indicates samples taken every 40 min from fertilization. The red arrow indicates the putative ZGA of the D. rerio embryo. The D. rerio gene expression data were obtained from Levin et al. [41].