Identification and functional analysis of Dmrt1 gene and the SoxE gene in the sexual development of sea cucumber, Apostichopus japonicus

Abstract

Members of the Doublesex and Mab-3-related transcription factor (Dmrt) gene family handle various vital functions in several biological processes, including sex determination/differentiation and gonad development. Dmrt1 and Sox9 (SoxE in invertebrates) exhibit a very conserved interaction function during testis formation in vertebrates. However, the dynamic expression pattern and functional roles of the Dmrt gene family and SoxE have not yet been identified in any echinoderm species. Herein, five members of the Dmrt gene family (Dmrt1, 2, 3a, 3b and 5) and the ancestor SoxE gene were identified from the genome of Apostichopus japonicus. Expression studies of Dmrt family genes and SoxE in different tissues of adult males and females revealed different expression patterns of each gene. Transcription of Dmrt2, Dmrt3a and Dmrt3b was higher expressed in the tube feet and coelomocytes instead of in gonadal tissues. The expression of Dmrt1 was found to be sustained throughout spermatogenesis. Knocking-down of Dmrt1 by means of RNA interference (RNAi) led to the downregulation of SoxE and upregulation of the ovarian regulator foxl2 in the testes. This indicates that Dmrt1 may be a positive regulator of SoxE and may play a role in the development of the testes in the sea cucumber. The expression level of SoxE was higher in the ovaries than in the testes, and knocking down of SoxE by RNAi reduced SoxE and Dmrt1 expression but conversely increased the expression of foxl2 in the testes. In summary, this study indicates that Dmrt1 and SoxE are indispensable for testicular differentiation, and SoxE might play a functional role during ovary differentiation in the sea cucumber.

Keywords: DMRT; RNAi; SoxE; gonad development; sea cucumber.

FIGURE 1

Phylogenetic analysis of the Dmrt and SOX protein family in Apostichopus japonicus (A) Dmrt.(B) SoxE. The phylogenetic tree was constructed with MEGA version 7.0 (1,000 replicates) by maximum likelihood analysis.

FIGURE 2

RT-qPCR analysis of Dmrts and SoxE Expression in adult tissues. NADH was used as internal control (A) Dmrt1. (B) Dmrt2. (C) Dmrt3a. (D) Dmrt3b. (E) Dmrt5. (F) SoxE. Data were performed from three independent experiments. Each bar represents mean ± SD. Different letters indicate significant differences between mean values (p ≤ 0.05), whilst shared letters indicate no significant difference.

FIGURE 3

Dmrt1 and SoxE expression in gonad. (A) RT-qPCR analysis of Dmrt1 expression in testis at different development stages. (B) RT-qPCR analysis of SoxE expression in testis at different development stages. (C) RT-qPCR analysis of SoxE expression in ovary at different development stages. NADH was used as internal control. Data were performed from three independent experiments. Each bar represents mean ± SD. Different letters indicate significant differences between mean values (p ≤ 0.05), whilst shared letters indicate no significant difference.

FIGURE 4

RNA interference (RNAi) of Dmrt1 in testis. RT-qPCR detected mRNA levels of Dmrt1 and other sex related genes after Dmrt1 RNAi. (A) Dmrt1. (B) SoxE. (C) foxl2. (D) piwi. NADH was used as internal control. Data were performed from three independent experiments. Each bar represents mean ± standard deviation (SD). Asterisks (*) indicate significant differences (p ≤ 0.05) between knock-down and control. dpi: days post-injection.

FIGURE 5

RNA interference (RNAi) of SoxE in testis. RT-qPCR detected mRNA levels of SoxE and other sex related genes after SoxE RNAi. (A) SoxE. (B) Dmrt1. (C) foxl2. (D) piwi. NADH was used as internal control. Data were performed from three independent experiments. Each bar represents mean ± standard deviation (SD). Asterisks (*) indicate significant differences (p ≤ 0.05) between knock-down and control. dpi: days post-injection.