Integrated Genomic and Transcriptomic Analysis Reveals a Transcription Factor Gene Set Facilitating Gonadal Differentiation in the Pacific Oyster Crassostrea gigas

Abstract

Background/objectives: The Pacific oyster Crassostrea gigas has emerged as a promising model system for sex determination studies due to its complex reproduction strategy and sex reversal. Transcription factors (TFs) play crucial roles in sex determination and gonadal differentiation. Despite previous research revealing functions of several conserved sex-determining pathway genes, such as Dmrt1, Foxl2, and SoxH, little is known about the other essential TF regulators driving C. gigas gonadal differentiation and development.

Methods: In this study, a systematic identification of TFs revealed 1167 TF genes in the C. gigas genome. Comparative transcriptome analysis of C. gigas female and male gonads demonstrated 123 differentially expressed TF genes.

Results: The majority of these sex-related TF genes were up-regulated in female or male gonads from the inactive stage to the mature stage. Moreover, this TF gene set was deeply conserved and showed similar regulation in the Kumamoto oyster Crassostrea sikamea gonads, suggesting their important regulatory roles in gonadal differentiation and development in Crassostrea oysters. Furthermore, two BTB TF gene clusters were identified in the C. gigas genome, both of which were specifically expressed in the male gonad. Gene numbers of each BTB gene cluster showed significant variations among six Crassostrea species.

Conclusions: To the best of our knowledge, this study provides the first report of the whole TF family in C. gigas. The sex-related TF gene set will be a valuable resource for further research aimed at uncovering TF gene regulatory networks in oyster sex determination and gonadal differentiation.

Keywords: Crassostrea gigas; Pacific oyster; gonadal differentiation; sex determination; transcription factor.

Figure 1

Genome-wide identification of Pacific oyster transcription factors. (A) Schematic diagram of TF identification based on C. gigas genome and annotations. (B) Summary of TF families and their gene numbers in C. gigas.

Figure 2

Distribution of transcription factor genes on ten chromosomes in C. gigas. Circos plots showed chromosome size, total gene, and TF gene density from outer to inner circles. The sliding window size was set as 1 Mb. The right table shows the average gene number per Mb sequence on ten C. gigas chromosomes.

Figure 3

Alternative splicing profiles of Pacific oyster transcription factors. (A) Summary of transcript numbers of C. gigas TF genes. (B) Alternative splicing types in C. gigas TF genes. (C) Sashimi plots of two TF genes with multiple alternative splicing sites. Numbers in the grey rectangles indicated RNA-seq reads aligned to the junction spanning the exons. ES, exon skipping; A3SS, alternative 3′ splicing site; A5SS, alternative 5′ splicing site; IR, intron retention; MXE, mutually exclusive exons; AT, alternative terminator.

Figure 4

Expression patterns of Pacific oyster transcription factors in adult tissues. (A) Pearson correlation coefficient of nine adult tissues based on total gene expression levels. (B) Number of total genes and TF genes expressed in nine tissues. (C) Diverse expression patterns of TF families in adult tissues. C. gigas TFs were classified into three groups: core, softcore, and tissue-specific. Amu: adductor muscle, Dgl: digestive gland, Mgo: male gonad, Hem: hemolymph, Fgo: female gonad, Gil: gill, Lpa: labial palp, Mou: mantle outer region, Min: mantle inner region.

Figure 5

Sex-biased expression of transcription factors in Pacific oyster gonads. (A) Upset plot of differentially expressed genes (DEGs) between female and male gonads. DEGs were generated based on two gonad transcriptome datasets from published research as described in Materials and Methods. (B) KEGG pathway enrichment of gonadal DEGs. The shared 1409 up-regulated and 894 down-regulated DEGs in the two transcriptome datasets were used for enrichment analysis. (C) Differentially expressed TF genes in female and male gonads. Five TF genes marked in blue in the bracket were up-regulated in s3F_vs_s3M group but down-regulated in F2n_vs_M2n group. Female gonads (ovary) and male gonads (testis) were used as control and experiment groups, respectively [10,23].

Figure 6

Dynamic regulation of sex-biased transcription factors during gonadal development. (A) Heatmap showing differential expression levels of sex-biased TFs during gonadal maturation. Z-score was generated using FPKM values. C. gigas gonad at the inactive stage was indicated as s0. (B) Representative TFs related to gonadal development and their DNA-binding domains. C1 to C4 corresponded to cluster1 to cluster4 in the heatmap.

Figure 7

Highly conserved regulation of sex-related transcription factors in two Crassostrea species. (A) Comparison of sex-biased TFs between Pacific oyster (C. gigas) and Kumamoto oyster (C. sikamea). BTBGC, BTB gene cluster. (B) Linear regression analysis of fold change (FC) of sex-biased TFs between C. gigas and C. sikamea. (C) Expression levels of nine C. sikamea TFs in gonads based on FPKM values. (D) RT-PCR analyses of sex-biased TFs in C. sikamea gonads. F: female gonad, M: male gonad. (E) Quantitative real-time PCR (qRT-PCR) verification of sex-biased TFs in C. sikamea gonads. Fold changes (FCs) of five TFs were determined by qRT-PCR and compared to C. sikamea and C. gigas transcriptomic results. The Log2(FC) values are shown as mean with SD (standard deviation).

Figure 8

BTB gene duplication in Crassostrea genomes. (A) Screening of testis-related TFs. A total of 37 TFs were identified to be specifically expressed in Pacific oyster male gonads. (B) Chromosomal distribution of two BTB gene clusters (BTBGCs) in C. gigas genome. XM_034465933.1 was annotated as one transcript of LOC105323166 in GCF_902806645.1, but it should be an independent BTB gene. (C) PCR examination of BTB gene duplication in Kumamoto oyster. Two primer pairs were designed based on gene models in C. gigas and were used to investigate BTB gene duplication in C. sikamea genomes (gDNA, genomic DNA). Female and male gonad cDNA samples were used as controls. Red arrowheads indicated the position of primers.