Antagonistic Roles of Dmrt1 and Foxl2 in Sex Differentiation via Estrogen Production in Tilapia as Demonstrated by TALENs

Abstract

Transcription activator-like effector nucleases (TALENs) are a powerful approach for targeted genome editing and have been proved to be effective in several organisms. In this study, we reported that TALENs can induce somatic mutations in Nile tilapia, an important species for worldwide aquaculture, with reliably high efficiency. Six pairs of TALENs were constructed to target genes related to sex differentiation, including dmrt1, foxl2, cyp19a1a, gsdf, igf3, and nrob1b, and all resulted in indel mutations with maximum efficiencies of up to 81% at the targeted loci. Effects of dmrt1 and foxl2 mutation on gonadal phenotype, sex differentiation, and related gene expression were analyzed by histology, immunohistochemistry, and real-time PCR. In Dmrt1-deficient testes, phenotypes of significant testicular regression, including deformed efferent ducts, degenerated spermatogonia or even a complete loss of germ cells, and proliferation of steroidogenic cells, were observed. In addition, disruption of Dmrt1 in XY fish resulted in increased foxl2 and cyp19a1a expression and serum estradiol-17β and 11-ketotestosterone levels. On the contrary, deficiency of Foxl2 in XX fish exhibited varying degrees of oocyte degeneration and significantly decreased aromatase gene expression and serum estradiol-17β levels. Some Foxl2-deficient fish even exhibited complete sex reversal with high expression of Dmrt1 and Cyp11b2. Furthermore, disruption of Cyp19a1a in XX fish led to partial sex reversal with Dmrt1 and Cyp11b2 expression. Taken together, our data demonstrated that TALENs are an effective tool for targeted gene editing in tilapia genome. Foxl2 and Dmrt1 play antagonistic roles in sex differentiation in Nile tilapia via regulating cyp19a1a expression and estrogen production.

Bending Genders: The Biology of Natural Sex Change in Fish

“…In males, Dmrt1 is a critical transcriptional regulator activating male-promoting genes (e.g., sox9 , sox8 ) while suppressing ovarian pathways ( foxl2 and rspo1 / wnt / β -catenin signalling) [Herpin and Schartl 2011a, b]. Dmrt1 interacts antagonistically with the female-specific transcription factor Foxl2 to influence cyp19a1a expression and control oestrogen production and gonadal fate in teleosts Li et al, 2013]. Under-or overexpression of either foxl2 or dmrt1 induces sexual cell fate reprogramming and gonadal sex reversal in mice and fish [Uhlenhaut et al, 2009;Matson et al, 2011;Li et al, 2013;Lindeman et al, 2015].…”

“…Dmrt1 interacts antagonistically with the female-specific transcription factor Foxl2 to influence cyp19a1a expression and control oestrogen production and gonadal fate in teleosts Li et al, 2013]. Under-or overexpression of either foxl2 or dmrt1 induces sexual cell fate reprogramming and gonadal sex reversal in mice and fish [Uhlenhaut et al, 2009;Matson et al, 2011;Li et al, 2013;Lindeman et al, 2015]. In gonochoristic and sex-changing fishes, cyp19a1a , foxl2, and dmrt1 expression is consistently sex-specific, depending on which gonadal phenotype is developing [Xia et al, 2007;Alam et al, 2008;Wu et al, 2008].…”

Efficient and Heritable Gene Targeting in Tilapia by CRISPR/Cas9

“…It is also an important laboratory model for understanding the developmental genetic basis of sex determination. The availability of monosex populations, together with the wholegenome sequence of Nile tilapia, has made it much easier to study the genes involved in sex determination (Soler et al 2010;M. H. Li et al 2013).…”

Large-scale tissue-specific and temporal gene expression profiles in Pengze crucian carp

“…sox9a and foxl2 were associated with the battle of sexes and the control of endogenous steroid hormone balance. Recently, cyp19a1a was found to be regulated by dmrt1 and its antagonistic gene, foxl2 (Li et al, 2013b), and foxl2 was confirmed as a cyp19a1a regulator. In the present study, the sex differentiation-related genes (Pcc-foxl2, nr5a1b, and sox9a) were significantly higher at 48 dph compared with other time points, which indicated that 48 dph was the turning point.…”