16 Results
for author "Yue-wen Wang"
Figure 1Molecular characterization of zebrafish foxl2a and foxl2b. (A and B) Genomic structure and transcript schematics of zebrafish foxl2a and foxl2b. Exons and introns are shown by boxes and horizontal lines, respectively. ORFs and FHD are highlighted by black boxes and red boxes, respectively. The exon and intron size are indicated above or below as bp. (C) Phylogenetic tree of Foxl proteins in vertebrates. (D) Syntenic alignment of chromosomal regions around vertebrate foxl2 genes. Zebrafish foxl2a and foxl2b are located on chromosome 15 and 2, respectively. Chromosome segments are represented as thick lines. The conserved gene blocks are shown in matching colors and the transcription orientation are indicated by arrows. The oblique line between two genes shows their discontinuity. Gene symbols, full names, RefSeq identifications, and related references are listed in Table S2. Chr, chromosome.
Figure 2Expression characterization of zebrafish foxl2a-1, foxl2a-2, and foxl2b during ovary development and maintenance. (A) Sexually dimorphic expression between mature ovary and testis. (B) Sequential and divergent expression during ovary development and maintenance from 15 to 270 dpf. The qRT-PCR quantification of gene expression was normalized to actb1. The relative expression values were first calculated against actb1 and then against that of foxl2a-1 at 25 dpf. (C and D) Ovary section in situ localization at (C) 35 dpf and (D) 105 dpf. Bars are shown at bottom-right of the images.
Figure 3Establishment of foxl2a and foxl2b knockout mutant lines by TALEN. (A and B) The TALEN target sites of zebrafish (A) foxl2a and (B) foxl2b. The coding and untranslated exon regions are depicted as solid and open boxes, respectively. The left and right TALEN binding sites are indicated by underlining. Cleavage sites with BstN I and BsrD I in the spacer are shown by blue color, and forward and reverse primers (F primer and R primer) are indicated in the corresponding sites. (C and D) Detection of (C) foxl2a and (D) foxl2b mutants by BstN I or BsrD I digestion. The amplified fragment sizes (bp) are shown on the right. (E and F) Sequences of different indels of TALEN-induced (E) foxl2a and (F) foxl2b mutants in F0 embryos. A total of eight and six indels (number of embryos are indicated in each bracket) at targeted locus are shown for (E) foxl2a and (F) foxl2b, and the numbers at the right-hand side indicate the number of deleted base pairs. (G) Transcription-level confirmation of foxl2a and foxl2b mutants by RT-PCR. The detected primers are shown on the left, and primer P2 is specific for the deleted sequences. The actb1 was used as control. F, forward; R, reverse; M, marker.
Figure 4Disruption of foxl2a leads to similar POF in adult females. (A–D) Gross morphology, anatomical, and histological examination of adult ovaries in WT controls and foxl2a mutants (foxl2a−/−) were analyzed at (A) 105 dpf (n = 12), (B) 150 dpf (n = 6), (C) 180 dpf (n = 3), and (D) 270 dpf (n = 6). (E) GSI values in control females and foxl2a mutant females. Data are presented as mean ± SEM * P ≤ 0.05 (Duncan’s multiple range test). Bars are shown at bottom right of the images. ♀, female; ♂, male.
Figure 5Partial sex reversal toward testicular tissue in foxl2b-deficient adult females. (A–F) Gross morphology, anatomical, and histological examination of adult ovaries in controls (WT) and foxl2b mutants (foxl2b−/−) were analyzed at (A) 105 dpf (n = 13), (B) 150 dpf (n = 6), (C) 180 dpf (n = 5), and (D) 270 dpf (n = 7). Higher magnifications of the boxed areas in (C) and (D) are shown in (E) and (F). (G) GSI values in control females and foxl2b mutant females. Data are presented as mean ± SEM * P ≤ 0.05 (Duncan’s multiple range test). ♂, female; ♀, male; SD, spermatid. Bars are shown at bottom right of the images.
Figure 6Transcriptome profiling changes of gonadal development-related genes in ovaries of foxl2a−/− and foxl2b−/− mutants. (A) Relatively transcribed changes of several kinds of gonadal development-related genes between foxl2a−/− and WT ovaries or foxl2b−/− and WT ovaries at 150 dpf. (B–N) Relative expression qRT-PCR detection of the indicated genes in WT, foxl2a−/−, and foxl2b−/− ovaries from 105 to 270 dpf (n = 3). The qRT-PCR quantification of each gene expression is normalized to actb1, and the data are presented as mean ± SEM * P ≤ 0.05 (Duncan’s multiple range test).
Figure 7Transcriptome profiling changes of cyp genes in the ovaries of foxl2a−/− and foxl2b−/− mutants. (A) Relatively transcribed changes of nine cyp genes between foxl2a−/− and WT ovaries or foxl2b−/− and WT ovaries at 150 dpf. (B–J) Relative expression qRT-PCR detection of nine cyp genes in WT, foxl2a−/−, and foxl2b−/− ovaries from 105 to 270 dpf (n = 3). The qRT-PCR quantification of each gene expression is normalized to actb1, and the data are presented as mean ± SEM * P ≤ 0.05 (Duncan’s multiple range test).
Figure 8Complete sex reversal in homozygous foxl2a−/−/foxl2b−/− females. (A and B) Gross morphology, anatomical, and gonadal histology examination of (A) heterozygous double mutants (foxl2a−/−/foxl2b+/−) (n = 13) and (B) homozygous double mutants (foxl2a−/−/foxl2b−/−) (n = 3) at 105 dpf. (C) GSI in foxl2a−/−/foxl2b+/− and foxl2a−/−/foxl2b−/− mutants at 105 dpf. Data are presented as mean ± SEM * P ≤ 0.05 (Duncan’s multiple range test). (D) Gonadal development status of homozygous foxl2a−/−/foxl2b−/− individuals from young fishes to adults at 35, 40, 50, 60, 105, and 150 dpf. (E) Gonadal histology examination of heterozygous double mutants (foxl2a−/−/foxl2b+/−) and homozygous double mutants (foxl2a−/−/foxl2b−/−) at 35 dpf (n = 7), 40 dpf (n = 3), 50 dpf (n = 7), and 60 dpf (n = 9). ♀, females; ♂, males; CV, cavity; SD, spermatid. Bars are shown at bottom right of the images.
Figure 9Foxl2a and foxl2b cooperatively regulate ovary differentiation and maintenance in zebrafish. (A–V) Relative expression qRT-PCR detection of the indicated genes in ovary (O), ovotestis (O/T), and testis (T) of homozygous double mutants (foxl2a−/−/foxl2b−/−) at 60 dpf (n = 3). Gene names are indicated at the top. (W–Y) Relative expression qRT-PCR detection of (W) foxl2a-1, (X) foxl2a-2, and (Y) foxl2b in the ovaries of WT and foxl2b mutants (foxl2b−/−) or foxl2a mutants (foxl2a−/−) from 35 to 270 dpf, respectively (n = 3). The qRT-PCR quantification of each gene expression is normalized to actb1, and the data are presented as mean ± SEM * P ≤ 0.05 (Duncan’s multiple range test).