Transcriptome Profiling and Expression Localization of Key Sex-Related Genes in a Socially-Controlled Hermaphroditic Clownfish, Amphiprion clarkii

Abstract

Clownfish can be an excellent research model for investigating the socially-controlled sexual development of sequential hermaphrodite teleosts. However, the molecular cascades underlying the social cues that orchestrate the sexual development process remain poorly understood. Here, we performed a comparative transcriptomic analysis of gonads from females, males, and nonbreeders of Amphiprion clarkii, which constitute a complete social group, allowing us to investigate the molecular regulatory network under social control. Our analysis highlighted that the gonads of nonbreeders and males exhibited high similarities but were far from females, both in global transcriptomic profiles and histological characteristics, and identified numerous candidate genes involved in sexual development, some well-known and some novel. Significant upregulation of cyp19a1a, foxl2, nr5a1a, wnt4a, hsd3b7, and pgr in females provides strong evidence for the importance of steroidogenesis in ovarian development and maintenance, with cyp19a1a playing a central role. Amh and sox8 are two potential key factors that may regulate testicular tissue development in early and late stages, respectively, as they are expressed at higher levels in males than in females, but with slightly different expression timings. Unlike previous descriptions in other fishes, the unique expression pattern of dmrt1 in A. clarkii implied its potential function in both male and female gonads, and we speculated that it might play promoting roles in the early development of both testicular and ovarian tissues.

Anemonefishes: A model system for evolutionary genomics

“…123 Though protocols were originally established in zebrafish, in situ hybridization has also been successfully performed in anemonefish embryos 124 and different tissues. 15,74,78,80,125 Importantly, this technique revealed unique aspects of the embryogenesis of the tomato clownfish A. frenatus that suggest an evolutionary adaptability of the teleost developmental program. 124 Similarly, fluorescent in situ hybridization (FISH) has provided a detailed understanding of the visual system of various anemonefish species by visualizing and quantifying patterns in opsin gene expression.…”

“…75,76 RNA-seq and qPCR are lowcost methods that can provide high-resolution data without the need for extensive genomic resources. 76 In anemonefish, transcriptome sequencing and qPCR experiments have provided insights into gene expression changes during development 12,15,74,[77][78][79][80][81] and adaptive responses to variations in the environment. 21,[82][83][84] Numerous efforts have contributed to the paramount abundance of transcriptomic data that is currently available for different tissues from multiple species and developmental stages (reviewed in Ref.…”

“…85), which has enabled the study of specific genes involved in a variety of functions including pigmentation, 15,86 vision, 59,87 thyroid hormone regulation of metabolism, 77 and sex change. 12,74,78,80,81 Recent analysis of gene expression patterns across tissues in A. ocellaris 60 and A. clarkii 35 revealed interesting findings pertaining to the total number of genes and unique number of genes expressed per tissue. For example, ~15% of all genes (in both species) were expressed in nearly all tissues without biased expression (as determined by the tau index which quantifies tissue-specificity 88 ) and therefore considered as housekeeping genes.…”

“…Particularly, several studies have investigated developmental gene expression related to sex change. 12,74,78,80,81,[89][90][91][92][93] After a first examination of the role of the aromatase gene during sex change in A. clarkii, 74 Casas et al (2016) was the first transcriptome-wide study to provide insights into the genetic mechanisms governing social sex change and gonadal restructuring in clownfish. 12 Differential expression analyses in A. bicinctus revealed a complex genomic response of the brain associated with sex change that is subsequently transmitted to the gonads (see more in Section 4.1).…”

“…12 Differential expression analyses in A. bicinctus revealed a complex genomic response of the brain associated with sex change that is subsequently transmitted to the gonads (see more in Section 4.1). Moreover, it identified a large number of genes, some of them well-known and others novel, that facilitated further research on sex change in anemonefish 78,94 as well as in other hermaphrodite fish. 95 The clownfish is a conspicuously colored species (possessing a bright orange body with three iridescent white bars bordered with black), and understanding the molecular basis of pigmentation has also become a fundamental question of evolutionary biology.…”