Figure 5.
PGC-specific mutation of tbx16 affects migration. (A) Genomic structure of the zebrafish tbx16 locus and generation of tbx16-gRNAs expressing stable germline. The direction of gRNA recognition sequences was indicated by arrows. E1–E9, exons; gray areas, UTRs. The low panel showed the transgenic embryos with CFP expression in the lens at 2 dpf but not at 1 dpf. (B) Procedures to obtain embryos expressing nCas9ERT2 and tbx16-gRNAs and to treat the embryos. (C) Left, morphology of WT, tbx16 whole-body knockout mutant (tbx16tsu-G115) embryos, and Tg(piwil1;nCas9ERT2-n3U;U6:tbx16-gRNAs;cryaa:CFP) embryos treated with 4-OHT at the sphere stage (PGC-KO). Right, bar chart showing genotypes of PGCs and SCs in DMSO and 4-OHT–treated groups at 24 hpf. Nclone, the number of monoclones for Sanger sequencing. (D) Ratio of PGC genotypes in PGC-KO embryos as in B. Individual PGCs were isolated from individual 4-OHT–treated Tg(piwil1;nCas9ERT2-n3U;piwil1:mCherry-CAAX-n3U;U6:tbx16-gRNAs;cryaa:CFP) embryos at 2 dpf and subjected to amplification, cloning, and sequencing of the tbx16 target region, allowing identification of each PGC’s genotype. −/−, two mutant alleles; +/−, one mutant allele; +/+, two WT alleles. No. PGCs, the number of PGCs within the corresponding embryos. Embryo: the serial number of 4-OHT–treated transgenic embryos. (E) Left, immunostaining results of Tbx16 (green) and Ddx4 (magenta) with nuclei stained by DAPI (blue) in 4-OHT– or DMSO-treated embryos from the sphere stage. Scale bar, 10 μm. Right, box plot showing the statistical results of the ratio of Tbx16 signals to DAPI in PGCs and SCs in either 4-OHT– or DMSO-treated embryos (mean ± SD). Each independent data point represents a cell. The numbers of PGCs and SCs in the 4-OHT–treated group are 20 and 31, respectively, while the numbers of PGCs and SCs in the control group are 18 and 27. P values of the t test (unpaired, two-sided) were also labeled. (F) Examples of PGC locations in 24 hpf embryos. The boxed areas in the left panel were enlarged in the right panels. In the second boxed area, PGCs were apparently mislocated in the head region. (G) Ratio of embryos with PGCs in the head in 4-OHT–treated embryos (as shown in F) and DMSO-treated control embryos. Each independent data point represents an independent experiment, totaling 15 times. P values of the t test (paired, two-sided) were also labeled. Source data are available for this figure: SourceData F5.

PGC-specific mutation of tbx16 affects migration. (A) Genomic structure of the zebrafish tbx16 locus and generation of tbx16-gRNAs expressing stable germline. The direction of gRNA recognition sequences was indicated by arrows. E1–E9, exons; gray areas, UTRs. The low panel showed the transgenic embryos with CFP expression in the lens at 2 dpf but not at 1 dpf. (B) Procedures to obtain embryos expressing nCas9ERT2 and tbx16-gRNAs and to treat the embryos. (C) Left, morphology of WT, tbx16 whole-body knockout mutant (tbx16tsu-G115) embryos, and Tg(piwil1;nCas9ERT2-n3U;U6:tbx16-gRNAs;cryaa:CFP) embryos treated with 4-OHT at the sphere stage (PGC-KO). Right, bar chart showing genotypes of PGCs and SCs in DMSO and 4-OHT–treated groups at 24 hpf. Nclone, the number of monoclones for Sanger sequencing. (D) Ratio of PGC genotypes in PGC-KO embryos as in B. Individual PGCs were isolated from individual 4-OHT–treated Tg(piwil1;nCas9ERT2-n3U;piwil1:mCherry-CAAX-n3U;U6:tbx16-gRNAs;cryaa:CFP) embryos at 2 dpf and subjected to amplification, cloning, and sequencing of the tbx16 target region, allowing identification of each PGC’s genotype. −/−, two mutant alleles; +/−, one mutant allele; +/+, two WT alleles. No. PGCs, the number of PGCs within the corresponding embryos. Embryo: the serial number of 4-OHT–treated transgenic embryos. (E) Left, immunostaining results of Tbx16 (green) and Ddx4 (magenta) with nuclei stained by DAPI (blue) in 4-OHT– or DMSO-treated embryos from the sphere stage. Scale bar, 10 μm. Right, box plot showing the statistical results of the ratio of Tbx16 signals to DAPI in PGCs and SCs in either 4-OHT– or DMSO-treated embryos (mean ± SD). Each independent data point represents a cell. The numbers of PGCs and SCs in the 4-OHT–treated group are 20 and 31, respectively, while the numbers of PGCs and SCs in the control group are 18 and 27. P values of the t test (unpaired, two-sided) were also labeled. (F) Examples of PGC locations in 24 hpf embryos. The boxed areas in the left panel were enlarged in the right panels. In the second boxed area, PGCs were apparently mislocated in the head region. (G) Ratio of embryos with PGCs in the head in 4-OHT–treated embryos (as shown in F) and DMSO-treated control embryos. Each independent data point represents an independent experiment, totaling 15 times. P values of the t test (paired, two-sided) were also labeled. Source data are available for this figure: SourceData F5.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal