Unique gene expression profile of the proliferating Xenopus tadpole tail blastema cells deciphered by RNA-sequencing analysis

Abstract

Organ regenerative ability depends on the animal species and the developmental stage. The molecular bases for variable organ regenerative ability, however, remain unknown. Previous studies have identified genes preferentially expressed in the blastema tissues in various animals, but transcriptome analysis of the isolated proliferating blastema cells has not yet been reported. In the present study, we used RNA-sequencing analysis to analyze the gene expression profile of isolated proliferating blastema cells of regenerating Xenopus laevis tadpole tails. We used flow cytometry to isolate proliferating cells, and non-proliferating blastema cells, from regenerating tadpole tails as well as proliferating tail bud cells from tail bud embryos, the latter two of which were used as control cells, based on their DNA content. Among the 28 candidate genes identified by RNA-sequencing analysis, quantitative reverse transcription-polymerase chain reaction identified 10 genes whose expression was enriched in regenerating tadpole tails compared with non-regenerating tadpole tails or tails from the tail bud embryos. Among them, whole mount in situ hybridization revealed that chromosome segregation 1-like and interleukin 11 were expressed in the broad area of the tail blastema, while brevican, lysyl oxidase, and keratin 18 were mainly expressed in the notochord bud in regenerating tails. We further combined whole mount in situ hybridization with immunohistochemistry for the incorporated 5-bromo-2-deoxyuridine to confirm that keratin 18 and interleukin 11 were expressed in the proliferating tail blastema cells. Based on the proposed functions of their homologs in other animal species, these genes might have roles in the extracellular matrix formation in the notochord bud (brevican and lysyl oxidase), cell proliferation (chromosome segregation 1-like and keratin 18), and in the maintenance of the differentiation ability of proliferating blastema cells (interleukin 11) in regenerating tadpole tails.

Fig 1. BrdU-immunohistochemistry of the proliferating cells in amputated tail stumps and tail buds.

Sagittal sections from (A-D) tails stumps at 1, 2, 3, and 4 dpa of St. 49-53 tadpoles or (G) a tail bud of St. 35-39 tadpole were subjected to immunohistochemistry using anti-BrdU antibody (red), and the nuclei were counterstained with Hoechst 33342 (blue). BrdU-positive cells were enriched in the tail blastema at 3 dpa, and in the tail bud. (E) Schematic drawing of a 3-dpa tail stump. (F) A magnified view of the region delineated by the square in panel (E). Anterior is to the left, and dorsal is up. Scale bars indicate 100 μm. df, dorsal fin; s, spinal cord; n, notochord; b, blastema; vf, ventral fin; sa, spinal cord ampulla; nb, notochord bud; w, wound epithelium.

Fig 2. Isolation of proliferating and non-proliferating tail blastema cells and proliferating tail bud cells using flow cytometry.

(A) A schematic drawing of the cell preparation procedure. Xenopus images were obtained from [18] and modified. (B,C) Cell cycle analysis of cells in (B) tail blastemas and (C) tail buds using flow cytometry. Horizontal axes represent relative fluorescent intensity of Hoechst blue, which reflects DNA contents, and vertical axes represent relative cell counts. The cells were classified into G₀/G₁ phases (major peaks), and S/G₂/M phases (whose DNA contents were higher than those of cells in the major peak and lower than twice of those of cells in the major peak) based on the DNA content. Cell fractions colored in red (‘2X-tail blastema’ cell fraction), blue (‘4X-tail blastema’ cell fraction), and green (‘4X-tail bud’ cell fraction), respectively, were collected and subjected to RNA-sequence analysis. (D) Comparison of expression levels of cell cycle markers. Relative expression levels were estimated from RNA-sequencing data, taking the values in the ‘2X-tail blastema’ cell fraction as 1. Expression levels of ef1α, a housekeeping gene, were almost the same in all samples.

Fig 3. RNA-sequencing analysis of genes preferentially expressed in proliferating or non-proliferating tail blastema cells as well as proliferating tail bud cells.

Genes preferentially expressed in each cell fraction were identified by RNA-sequencing analysis. (A) Genes that are up-regulated for more than 2-folds in R4 than in R2 or E4. (B) Genes down-regulated for more than 2-folds in R4 than in R2 or E4. (C,D) Genes whose FPKMs in R4 were larger than 1 (C) or 10 (D). (E) Flow chart for identification of genes expressed preferentially in the proliferating tail blastema cells.

Fig 4. Identification of genes expressed preferentially in the tail blastema by qRT-PCR.

The relative expression levels of the 10 genes which show the blastema-selective expression by qRT-PCR using RNAs extracted from normal tails (orange), regenerating tails (blue), and tail buds (green). The vertical axis represents relative expression levels calculated by taking the value of blastemas as 1, following normalization using those of ef1α. (mean ± SE, n = 4) *P < 0.05, Dunnett’s test. Abbreviations: cse1l, chromosome segregation 1-like; l1td1, LINE-1 type transposase domain-containing protein 1; oax, oocyte activation in Xenopus.

Fig 5. Expression analysis of candidate genes in regenerating tadpole tails by whole mount in situ hybridization.

(A-E) Whole mount in situ hybridization using 3-dpa regenerating tails of St. 49-53 tadpoles for (A) interleukin 11, (B) keratin 18, (C) brevican, (D) cse1l, and (E) lysyl oxidase (blue/purple). (F-M) Sagittal sections of tail blastema from 3-dpa regenerating tails of St. 49-53 tadpoles double stained by whole mount in situ hybridization for (F-I) interleukin 11 and (J-M) keratin 18 (magenta) and BrdU-immunohistochemistry (green). (G) and (K) show magenta channels (mRNA), (H) and (L) show green channels (BrdU), (I) and (M) show blue channels (nuclei stained with Hoechst 33342), and (F) and (J) show merged images. Note that mRNA signals detected in the cytoplasm do not exactly merge with BrdU-signals detected in the nuclei. White broken lines indicate cell populations that highly expressed the genes. Anterior is to the left, and dorsal is up. Scale bars indicate 500 μm in (A-E) and 100 μm in (F-M).