Abstract
Medaka is an ideal model for sex determination and sex reversal, such as XY phenotypically female patients in humans. Here, we assembled improved TALENs targeting the DMY gene and generated XYDMY− mutants to investigate gonadal dysgenesis in medaka. DMY-TALENs resulted in indel mutations at the targeted loci (46.8%). DMY-nanos3UTR-TALENs induced mutations were passed through the germline to F1 generation with efficiencies of up to 91.7%. XYDMY− mutants developed into females, laid eggs, and stably passed the YDMY− chromosome to next generation. RNA-seq generated 157 million raw reads from WT male (WT_M_TE), WT female (WT_F_OV) and XYDMY− female medaka (TA_F_OV) gonad libraries. Differential expression analysis identified 144 up- and 293 down-regulated genes in TA_F_OV compared with WT_F_OV, 387 up- and 338 down-regulated genes in TA_F_OV compared with WT_M_TE. According to genes annotation and functional prediction, such as Wnt1 and PRCK, it revealed that incomplete ovarian function and reduced fertility of XYDMY− mutant is closely related to the wnt signaling pathway. Our results provided the transcriptional profiles of XYDMY− mutants, revealed the mechanism between sex reversal and DMY in medaka, and suggested that XYDMY− medaka was a novel mutant that is useful for investigating gonadal dysgenesis in phenotypic female patients with the 46, XY karyotype.
Highlights
Animals, including C. elegans, Drosophila, fish, and mammals
Our results suggest that the medaka XYDMY− mutant is a novel mutant line that is useful for investigating XY to XX sex reversal and gonadal dysgenesis in phenotypically female patient with the 46, XY karyotype
The mixture containing a pair of transcription activator-like effector nucleases (TALENs) mRNAs was microinjected into one-cell stage embryos of medaka (Fig. 2A and File S1). 72 hours after microinjection, ten injected embryos were randomly pooled for extracting genomic DNA
Summary
Animals, including C. elegans, Drosophila, fish, and mammals. much is known about the master male sex-determining (SD) gene in medaka[12,14,15,16], the precise mechanisms involved in primary sex determination and sex differentiation remain undefined. It is difficult to perform specific gene targeting in medaka, because of the lack of methodologies for homologous recombination and embryonic stem cell derivation, which has impeded its use in male heterogametic (XX-XY) sex determination system studies. To better explain DMY’s regulatory functions as a transcription factor, we performed RNA-seq, a recently developed approach to transcriptome profiling based on deep-sequencing[32], and generated 157 million raw reads from WT male (WT_M_TE), WT female (WT_F_OV) and XYDMY− female medaka (TA_F_ OV) gonad libraries. These transcriptomic data will contribute to unravel the relationship and mechanism between sex reversal and the DMY gene. Our results suggest that the medaka XYDMY− mutant is a novel mutant line that is useful for investigating XY to XX sex reversal and gonadal dysgenesis in phenotypically female patient with the 46, XY karyotype
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