RT-PCR and qRT-PCR analysis of candidate pluripotency genes in 10 stages of em[bryonic development of Zebrafish (ZF) and Crucian carp (CC). (A) RT-PCR analysis of 16 pluripotency-related genes in 10 stages of embryonic development of zebrafish. (B) RT-PCR analysis of eight pluripotency-related genes in 10 stages of embryonic development of crucian carp. (C) qRT-PCR analysis for candidate pluripotency genes (Nanog, Oct4, Tdgf1, Gdf3 and Klf17) through the 10 stages of ZF and CC embryos. It was clearly showed that these four genes were seemed to be mainly expressed in the pluripotent cells of undifferentiated embryos, but the expression was significantly decreased or even lost after embryonic cell differentiation. For each gene detected, the △CT value was calculated from the average CT value of the independent housekeeping gene β-actin. For each sample, At least three independent experiments were done for these results.

Expression pattern of candidate pluripotent genes in adult tissues. (A,B) RT-PCR analysis of the expression of five pluripotent genes in eight tissues of zebrafish (A) and crucian carp (B). It was clearly showed that all of these genes were no expressed in skin tissue, but mainly expressed in ovary and testis, and some genes such as Klf17 were widely expressed in the most of tissues. (C,D) qRT-PCR analysis of the expression of five pluripotent genes in eight tissues of zebrafish (C) and crucian carp (D). The results showed that the expression patterns of these genes in different tissues of zebrafish and crucian carp were similar. Oct4, Nanog and Gdf3 were mainly expressed in ovary, testis and gut, and Gdf3 was also expressed in kidney. Klf17 was expressed in all eight tissues. Tdgf1 was mainly expressed in ovary, testis and heart. (E) Analysis of mRNA levels in the ovaries and testis of zebrafish and crucian carp after fluorescence in situ hybridization with Oct4, Nanog and Gdf3 probes. It was clearly showed that these three genes were strongly expressed in early oocytes and the outermost spermatogonia of the seminal lobules. The gonads were co-stained with DAPI. At least three independent experiments were done for these results. The scale bars in ovary were equal to 200 μm, while 50 μm in testis.

Expression pattern of pluripotent candidate genes in iPS-like cells from zebrafish and crucian carp. (A) qRT-PCR analyse of pluripotent candidate genes in iPS-like cells from zebrafish (left) and crucian carp (right). The fibroblasts from zebrafish and crucian as control, respectively. It was showed that the mRNA levels of Oct4, Nanog, and Tdgf1 were higher in the iPS-like cells of zebrafish and crucian carp, while the expression level of Gdf3 was lower. (B) Immunofluorescence staining of Oct4, Nanog, and Tdgf1 iPS-like cells (passage 15) from crucian carp (Scale bars represent 20 μm). It was showed that the expression was positive in iPS-like cells, but not in fibroblasts. Data are shown as mean ± SD of values obtained from three independent experiments, and significant differences were evaluated using Student’s t test (*p < .005; **p < .001). The scale bars are equal to 20 μm.