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

Hiroshi Tsujioka,Yuki Katou,Takekazu Kunieda,Katsuhiko Shirahige,Takeo Kubo,Robert E Steele

doi:10.1371/journal.pone.0111655

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.

Highlights

Organ regenerative ability varies depending on the animal species and developmental stage [1]
To isolate proliferating tadpole tail blastema cells, we first used BrdU immunohistochemistry to determine the period after tail amputation when the proliferating cells were most enriched in the tail blastema
We isolated the cell fraction that corresponds to the G0/G1 phases from the tail blastema sample as a control for the non-proliferating tail blastema cells, as well as the cell fraction that corresponds to the S/G2/M phases

Summary

Introduction

Organ regenerative ability varies depending on the animal species and developmental stage [1]. Recent studies have performed transcriptome analyses of the regenerating tissues of amputated organ stumps, using frog tails or limbs or axolotl limbs [8,9,10,11,12,13,14]. These studies have identified many genes involved in regeneration, such as secreted frizzled-related protein 2 [8], mesendoderm nuclear factor [9], neuronal nitric oxide synthase [10], gremlin [11], prostate stem cell antigen [13], and patched-2 [14]. Further characterization of the early processes involved in regenerating organ/tissues will provide important insight into the variable regenerative ability

Objectives

Methods

Results

Conclusion