Three Copies of zbed1 Specific in Chromosome W Are Essential for Female-Biased Sexual Size Dimorphism in Cynoglossus semilaevis

Abstract

The sex chromosome, especially specific in one sex, generally determines sexual size dimorphism (SSD), a phenomenon with dimorphic sexual difference in the body size. For Cynoglossus semilaevis, a flatfish in China, although the importance of chromosome W and its specific gene zbed1 in female-biased SSD have been suggested, its family members and regulation information are still unknown. At present, three zbed1 copies gene were identified on chromosome W, with no gametologs. Phylogenetic analysis for the ZBED family revealed an existence of ZBED9 in the fish. Nine members were uncovered from C. semilaevis, clustering into three kinds, ZBED1, ZBED4 and ZBEDX, which is less than the eleven kinds of ZBED members in mammals. The predominant expression of zbed1 in the female brain and pituitary tissues was further verified by qPCR. Transcription factor c/ebpα could significantly enhance the transcriptional activity of zbed1 promoter, which is opposite to its effect on the male determinant factor-dmrt1. When zbed1 was interfered with, piwil1, esr2 and wnt7b were up-regulated, while cell-cycle-related genes, including cdk4 and ccng1, were down-regulated. Thus, zbed1 is involved in cell proliferation by regulating esr2, piwil1, cell cycle and the Wnt pathway. Further research on their interactions would be helpful to understand fish SSD.

Keywords: Cynoglossus semilaevis; sexual size dimorphism; zbed1.

Figure 1

The genomic location and sequences of Cynoglossus semilaevis zbed1 gene. (A) Location of zbed1 gene on the sex chromosome of C. semilaevis. There were three copies of zbed1 at different locations on the W chromosome and none on the Z chromosome. (B) Sequence information for C. semilaevis zbed1 cDNA and protein. The start and stop codons are shown in bold.

Figure 2

The specificity of zbed1 in the females and the confirmation of its copy number. (A) PCR experiment result for the detection of zbed1 and myh6 from genomic DNA. F1-3 and M1-3 indicated three female and male individuals, respectively. M was the maker. (B) Droplet distribution map. Zbed1, myh6 and NTC droplets were clearly distinguished under the detection channel. The blue line showed the fluorescence threshold. (C) The copy numbers of zbed1 and the single-copy gene myh6 in three female individuals.

Figure 3

Phylogenetic tree and expression heatmap of ZBED family genes. (A) Phylogenetic tree of ZBEDs from Cynoglossus semilaevis (Cs), Homo sapiens (Hs), Mus musculus (Mm), Danio rerio (Dr), Oryzias latipes (Ol), Oreochromis niloticus (On), Epinephelus lanceolatus (El), Scophthalmus maximus (Sm), Xenopus tropicalis (Xt), Bos taurus (Bt), and Pan troglodytes (Pt). The percentage of replicate trees in which associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. Eleven sub-clusters were indicated in different colors. Then, the sub-branches of Ac (ZBED1, ZBED4, ZBED6cl, ZBED6, ZBEDX, ZBED2, and ZBED3) and Buster (ZBED7, ZBED8, ZBED5, and ZBED9) transposons were clustered into two big branches. In each sub-branch of ZBED1, ZBED4, and ZBEDX, the sequences of C. semilaevis and other fishes clustered together, followed by clustering with mammalian proteins. CsZBEDs were marked in red stars. (B) Heatmap of zbed mRNA abundances in different tissues of C. semilaevis female and male individuals. FB: female brain, MB: male brain, FP: female pituitary, MP: male pituitary, FG: female gonad, MG: male gonad, FL: female liver, ML: male liver.

Figure 4

Protein interaction network among ZBED’s family members. Nodes indicate the interactive proteins; edges indicate both functional and physical protein associations; and different colors indicate the various types of interaction evidence.

Figure 5

Spatiotemporal expression patterns of zbed1 gene in C. semilaevis. The letters a–h represent significance. There are significant differences between columns with different letters in each picture. (A) Relative mRNA expression patterns of zbed1 in C. semilaevis embryos at five periods (cleavage, blastocyst, gastrula, segmentation, pharyngula and early larval). (B) Relative mRNA expression patterns of zbed1 in 11 tissues (brain, pituitary, gill, gonad, heart, intestine, kidney, liver, muscle, skin, and spleen) of female C. semilaevis. (C) Relative mRNA expression patterns of zbed1 in the brain of female C. semilaevis at six developmental stages including three-month-old (3 M), five-month-old (5 M), eight-month-old (8 M), one-year-old (1 Y), 1.5-year-old (1.5 Y), and two-year-old fish (2 Y). The data were analyzed with SPSS 25.0 (IBM Corp, Armonk, NY, USA) using one-way ANOVA and multiple comparison by Wohler and Duncan methods, and p-value < 0.05 was considered the threshold for statistical significance.

Figure 6

The knockdown effect of zbed1 on the female C. semilaevis brain cells. (A) RNAi transfection efficiency in brain cells. (B) Interference efficiency of zbed1 siRNA. (C) The expression patterns of genes in female brain cells after transfection with zbed1 siRNA. The data in (B,C) were analyzed with SPSS 25.0 (IBM Corp, Armonk, NY, USA) using t-test. The data of each downstream gene were compared with NC and p-value < 0.05 was considered the threshold for statistical significance (double asterisk, p < 0.01).

Figure 7

Structure, activity, and transcription factor analysis of C. semilaevis zbed1 promoter. (A) Sequence structure and putative transcription-factor-binding sites (i.e., pou1f1a, sox2, junB, c/ebpα, myogenin, yy1, and stat5a) on zbed1 promoter. Two primers of the promoter are shown in shadow. (B) Transcription activity of zbed1 promoter and co-transfection with transcription factors in HEK 293T cells. (C) The luciferase activity after co-transfection with the transcription factor c/ebpα and mutated zbed1 promoter. The significance is indicated by asterisks. The data in (B,C) were analyzed with SPSS 25.0 (IBM Corp, Armonk, NY, USA) using t-test. The data of each co-transfection were compared with the original promoter and p-value < 0.05 was considered the threshold for statistical significance (double asterisk, p < 0.01).