Analyses of the Dmrt family in a decapod crab, Eriocheir sinensis uncover new facets on the evolution of DM domain genes

Abstract

DM domain genes are a group of transcription factors that are integral to sexual development and its evolution in metazoans. Their functions and regulatory mechanisms are not well understood in Malacostraca (crabs and crayfish) while these sex regulators have been widely identified in the past decade. In this study, the Dmrt family was investigated in the decapod crab, Eriocheir sinensis. We find that most members of the EsDmrt family begin to enrich around the juvenile 1 stage. In reproductive organs, EsDsx1, EsDsx2, EsiDMY and EsiDmrt1a highly express in the male-specific androgenic gland (AG), while EsDmrt-like, EsDsx-like, EsDmrt11E, and EsiDmrt1b show relatively high expression in testis. Also, we find the highly aberrant expression of EsiDMY and EsiDmrt1a in the chimeric AG, strongly indicating their function in AG development. Moreover, RNA interference of EsDsx1, EsiDMY, and EsiDmrt1a results in a significant decrease in transcription of the Insulin-like androgenic hormone (IAG), respectively. Our findings suggest that Dmrt genes in E. sinensis primarily function in male sexual differentiation, especially in AG development. Besides, this study identifies two unique groups of Dmrt genes in Malacostraca: Dsx and iDmrt1. In Malacostraca Dsx, we uncover a cryptic mutation in the eight zinc motif-specific residues, which were firmly believed to be invariant across the Dmrt family. This mutation sets the Malacostraca Dsx apart from all the other Dmrt genes and implies a different way of transcriptional regulation. Genes from the iDmrt1 group show phylogenetical limitation to the malacostracan species and underwent positive selection, suggesting their highly specialized gene function to this class. Based on these findings, we propose that Dsx and iDmrt1 in Malacostraca have developed unique transcriptional regulation mechanisms to facilitate AG development. We hope that this study would contribute to our understandings of sexual development in Malacostraca and provide new insights into the evolutionary history of the Dmrt family.

Keywords: Dmrt; Eriocheir sinensis; evolution; malacostraca; sexual development.

FIGURE 1

Characterization of Dmrt family genes in E. sinensis. (A) Structure features of nine EsDmrt genes. All elements are shown to scale except for introns. The alternative splicing events of EsDsx1 and EsiDMY are displayed above the genes with dotted lines. (B) Alignment of deduced amino acid sequences of DM domains (N-terminal domain of EsiDmrt1a, EsiDmrt1b and EsiDMY are used in this alignment). Identical amino acids are highlighted in black and similar ones in grey. Eight conserved motif-specific residues are indicated with asterisks and the point mutation in EsDsx1 and EsDsx2 is indicated with a top solid triangle. (C) Genome loci of nine EsDmrt genes. Except for the EsDsx-like, all genes have their clear chromosome locations.

FIGURE 2

Molecular phylogeny of Dmrt family genes in E. sinensis. Phylogenetic analysis was based on amino acid sequences of DM domain and performed by IQ-TREE following multiple sequence alignment using MAFFT. The maximum-likelihood method was applied. The 91 taxonomic units conducted for this analysis are listed in Supplementary Table S4. Numerical value on each node indicates bootstrap value that is >70%. EsDmrt genes are indicated with brown dots. Malacostraca Dsx and iDmrt1 groups are colored in light steel blue and light coral.

FIGURE 3

Expression level of the Dmrt family genes in three different transcriptomic libraries of E. sinensis. (A) Expression pattern of EsDmrt genes in embryonic, larval and juvenile stages. The heatmap was shown as normalized TPM value. (B) Expression pattern of EsDmrt genes (including splicing isoforms) in reproductive organs (AG, testis and ovary). The bar plot was shown as TPM value. Vertical bars represented mean ± SD, n = 3. Results were analyzed by one-way ANOVA and different letters (a-d) above the bars presented significant differences between groups (p < 0.05). AG_SY, androgenic gland at synthesis stage; AG_SE, androgenic gland at secretion stage. (C) Expression pattern of the EsDmrt genes in normal and chimeric E. sinensis. The bar plot was shown as TPM value. Vertical bars represented mean ± SD, n = 3. Tissues from left to right: AG-androgenic gland (chimeric, normal male); ovary (chimeric, normal female); eyestalk (chimeric, normal female and male). Tissue and organ from chimeric, normal female and male were colored with purple, red and blue. Vertical bars represented mean ± SD, n = 3. Results were analyzed by independent T-test comparing chimeric and normal tissue and organ (*p < 0.05, **p < 0.01).

FIGURE 4

Function of Dmrt genes for IAG expression in E. sinensis. Transcript abundance was measured through RT-qPCR. Box plot is show as log-scale relative values of expression levels to the reference gene, Esβ-actin. Each dot represents the expression level of each individual. Independent T-test was performed to ascertain the difference between siRNA and siControl groups, with significance levels indicated (*p < 0.05, **p < 0.01) Statistical results are listed in the Supplementary Table S6. Expression levels of EsDmrt genes (A) and EsIAG gene (B) in RNAi and control groups are shown separately.

FIGURE 5

Consensus analysis of DM domains of the Dmrt gene family. Less conserved points are discarded. The cryptic mutation in Malacostraca Dsx is annotated below. Amino acids are colored according to their chemical properties: red (D, E) for acidic, green (C, G, S, T, Y) for polar, blue (H, K, R) for basic, black (A, F, I, L, M, V, W) for hydrophobic, and purple (Q, N) for neutral. n: number of unique sequences. m: number of different species.

FIGURE 6

Ancestral sequence reconstruction of the DM domain from Dsx in Pancrustacea. (A) Alignment of the ancestral amino acid sequences of the DM domain from different common ancestor. Positions assumed to be positively selected are colored in red. Conserved residues are indicated with asterisks and mutation is indicated with a triangle. Es_Dsx1, Dsx1 of E. sinensis, is shown as a reference. (B) Predicted protein structures of Dsx in different common ancestors. The phylogenetic relationship is based on topology from Rota-Stabelli et al. (2013). 3D images in the midst indicate modelled proteins of DM domains with the motif-specific residues highlighted in red (cysteine) and yellow (histidine). Chelation of zinc atoms is visualized as a result of ZincExplore.

FIGURE 7

Diagram of the evolutionary history of Dsx and iDmrt1 proposed in this study. The upper topological structure and annotations describe possible statuses of Dmrt in different common ancestors. The lower part compares the transcriptional regulation of Dsx in Malacostraca, Hexapoda and Branchiopoda. Previous research has suggested that heterodimer formation of Dmrt proteins may contribute to transcriptional regulation (Murphy et al., 2007). Mutations in the DM domain and the lack of an oligomerization domain (Zheng et al., 2020) suggest that Malacostraca Dsx may not directly bind to, but rather heterodimerize on DNA with another Dmrt protein, which, according to our evolutionary analysis, could be iDmrt1. Figure created using BioRender.