Direct production of XY(DMY-) sex reversal female medaka (Oryzias latipes) by embryo microinjection of TALENs

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 XY(DMY-) 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%. XY(DMY-) mutants developed into females, laid eggs, and stably passed the Y(DMY-) chromosome to next generation. RNA-seq generated 157 million raw reads from WT male (WT_M_TE), WT female (WT_F_OV) and XY(DMY-) 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 XY(DMY-) mutant is closely related to the wnt signaling pathway. Our results provided the transcriptional profiles of XY(DMY-) mutants, revealed the mechanism between sex reversal and DMY in medaka, and suggested that XY(DMY-) medaka was a novel mutant that is useful for investigating gonadal dysgenesis in phenotypic female patients with the 46, XY karyotype.

Figure 1. The design and assembly of DMY-TALEN sites.

(A) Reconstructions of pCS2- DMY-TALENs -ELD/KKR-nanos UTR plasmids. (B) The location and sequence information of DMY-TALENs. Red uppercase letters indicate the DNA sequence of DMY-TALEN-L sites. Green uppercase letters indicate the complementary paired DNA sequence of DMY-TALEN-R sites. Lowercase letters indicate the spacer region of TALEN sites.

Figure 2. The dosage effects and efficiency evaluation of DMY-TALENs.

(A) Microinjection of TALENs mRNA into medaka. (B) Detection of mutations in TALEN targeted medaka embryos. Primers DMY F, DMY F1 and DMY R were used to amply the DMY gene. Primers DMY F and DMY R bridge both EBE regions, while primers DMY F1 and DMY R link the spacer region and the downstream EBE. DMY F and DMY R generated a 396 bp PCR fragment. DMY F1 and DMY R generated a 167 bp PCR fragment. If primer DMY F and DMY R generated a PCR fragment, while primer DMY F1 and DMY R failed to do so, this suggested that the targeted gene is disrupted by the DMY-TALEN. (C) Electrophoretic detection of mutations in TALEN-injected medaka embryos. Line 1 to 16, TALENs injected embryos. 1,3,6,11,14 show mutated embryos. (D) Sequencing detection of mutations in TALEN-induced medaka embryos. -, deleted nucleotide; lowercase letter, added nucleotide. +, insertions; Δ, deletions. (E) Evaluation of embryonic toxicity of DMY-TALENs. (F) Evaluation of embryonic toxicity of DMY-nanos3UTR-TALENs. (G) The targeting efficiency statistics of DMY- TALENs. (H) The targeting efficiency statistics of DMY-nanos-3UTR TALENs. (I) Comparative analysis of mutation rate and germline transmission rate between DMY-TALENs and DMY-nanos3UTR-TALENs.

Figure 3. Flowchart and establishment of DMY-TALENs-induced mutant lines.

(A) Flowchart of DMY-TALENs-induced mutant lines. (B) The genotypes of TALENs-induced mutations in the F0 generation. (C) The genotypes of TALENs induced F1 founders. (D) The genotypes of TALENs induced F2 mutant lines. Red lowercase letters indicate an additional nucleotide; “-” indicates a deleted nucleotide. +, insertions; Δ, deletions. F, female; M, male.

Figure 4. DMY genotyping, expression and CDS frameshift mutations of DMY mutants.

(A) Genotyping of DMY gene fragments using Li-con 4300 system. 1 to 16, randomly selected individuals in F1 generation. F, female; M, male. (B) Genomic PCR confirmed the DMY gene. M: marker DL2000; 1: WT Male; 2: WT Female; 3: Founder 2; 4: the female mutant from the F2 generation of Founder 2; 5: Founder 3; 6: the female mutant from the F2 generation of Founder 3; Ctrl: no template control. (C) Reverse transcription- PCR with DMY-specific primers. OLA Actin expression was determined for calibration. M: 250 bp Marker; 1. XY^DMY− mutant (DMYΔ11); 2. XY^DMY− mutant (DMY+16); 3. WT XY male; 4. WT XX female. (D) Numbers of germ cells in mutants and WT medaka fry at 5DAH. XX F: WT XX females; XY^DMY− F: XY^DMY− matants; XY M: WT XY males. Open circles represent the number of germ cells in individuals (N = 9). **P < 0.001. (E) The CDS frameshift mutations of TALEN-induced DMY gene. WT: CDS sequence of DMY.

Figure 5. Phenotypic identification and analysis of XY^DMY− mutants.

(A) Phenotypic diagnostics of the secondary sexual characters in XY^DMY− mutants. White dashed area shows the anal fin of medaka. *shows the dosal fin of medaka. (B) The gonad of WT and TA mutants. The red dashed area shows the gonad tissue of medaka. (C) Fluorescence in situ hybridization of the karyotypes of metaphases cell from WT male, WT female and XY mutant female. The pink signal is the male specific hybridization signal. (D) The comparative analyses of gonadosomatic index (GSI) among WT-F, WT-M and XY mutant female (TA-F). *P < 0.5; **P < 0.001. (E) The comparative analyses of oocytes maturation between WT-F and TA-F. (F) Histological analyses of gonad tissue. CN, chromatin nucleolar oocytes; PO, perinucleolar oocytes; CA, cortical alveolar oocytes; EVO, early vitellogenic oocytes; VO, vitellogenic oocytes; LVO, late vitellogenic oocytes; MO, mature oocytes.

Figure 6. Bioinformatic analyses of RNA-seq data.

(A) The number of novel transcripts in the RNA-seq data the WT female (WT_F_OV), the wild-type male (WT_M_Te) and XY^DMY− female medaka (TA_F_OV). (B) The differentially expressed transcripts between TA_F_OV and WT_F_OV/WT_M_Te. (C) Correlation of gene expression between WT_M_Te and TA_F_Ov. The up- and downregulated genes are shown in red and green, respectively. Non-differentially expressed genes are shown in blue. (D) Correlation of gene expression between WT_F_Ov and TA_F_Ov. The up- and downregulated genes are shown in red and green, respectively. Non-differentially expressed genes are shown in blue. (E) The cluster of testis-specific expressing transcripts. Cluster analyses of differentially expressed genes among WT_F_Ov, WT_M_Te and TA_F_Ov. The high- and low-expressed genes are shown form yellow to blue, corresponded to the expression level from negative 2 fold to positive 2 fold. (F) The differentially expressed transcripts among TA_F_Ov, WT_F_Ov and WT_M_Te. The up- and downregulated genes in WT_M_Te were shown in Fuchsia and yellow, respectively. The up- and downregulated genes in WT_F_Ov are shown in green and purple, respectively. TA_F_Ov ≫ WT_F_Ov means the genes were upregulated in TA_F_Ov. TA_F_Ov ≪ WT_F_Ov means the genes were downregulated in TA_F_Ov. TA_F_Ov ≫ WT_M_Te means genes were upregulated in TA_F_Ov. TA_F_Ov ≪ WT_M_Te means the genes were downregulated in TA_F_Ov. (G) SRY binding sites. (H) The SRY binding sites analyses of differentially expressed genes among TA_F_Ov, WT_F_Ov and WT_M_Te.