The planarian Dugesia japonica has amazing ability to regenerate a head from the anterior ends of the amputated stump with maintenance of the original anterior-posterior polarity. Although planarians present an attractive system for molecular investigation of regeneration and research has focused on clarifying the molecular mechanism of regeneration initiation in planarians at transcriptional level, but the initiation mechanism of planarian head regeneration (PHR) remains unclear at the protein level. Here, a global analysis of proteome dynamics during the early stage of PHR was performed using isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics strategy, and our data are available via ProteomeXchange with identifier PXD002100. The results showed that 162 proteins were differentially expressed at 2 h and 6 h following amputation. Furthermore, the analysis of expression patterns and functional enrichment of the differentially expressed proteins showed that proteins involved in muscle contraction, oxidation reduction and protein synthesis were up-regulated in the initiation of PHR. Moreover, ingenuity pathway analysis showed that predominant signaling pathways such as ILK, calcium, EIF2 and mTOR signaling which were associated with cell migration, cell proliferation and protein synthesis were likely to be involved in the initiation of PHR. The results for the first time demonstrated that muscle contraction and ILK signaling might played important roles in the initiation of PHR at the global protein level. The findings of this research provide a molecular basis for further unraveling the mechanism of head regeneration initiation in planarians.

Heat shock protein 70 family (HSP70s) is one of the most conserved and important group of HSPs as molecular chaperones, which plays an important role in cytoprotection, anti-apoptosis and so on. However, the molecular mechanism of HSP70s in animal regeneration remains to be delineated. In this study, we investigate the roles of HSP70s in regeneration of planarian. The four genes, Djhsp70a, Djhsp70b, Djhsp70c, and Djhsp70d of the HSP70s, are selected from the transcriptome database, because of their high expression levels in planarians. We then study the biological roles of each gene by conducting various experimental techniques, including RNAi, RT-PCR, WISH, Whole-mount immunostaining and TUNEL. The results show: (1) External stressors, such as temperature, tissue damage and ionic liquid upregulate the expression of Djhsp70s significantly. (2) The gene expression of Djhsp70s in planarians exhibits dynamic patterns. According to the result of WISH, the Djhsp70s are mainly expressed in parenchymal tissues on both sides of the body as well as blastema. It is consistent with the data of qRT-PCR. (3) After RNA interference of Djhsp70s, the worms experience cephalic regression and lysis, body curling, stagnant regeneration and death. (4) Knockdown of Djhsp70s affect the cell proliferation and apoptosis. These results suggest that Djhsp70s are not only conserved in cytoprotection, but involved in homeostasis maintenance and regeneration process by regulating coordination of cell proliferation and apoptosis in planarians.

Dugesia japonica is an excellent animal model for studying the regeneration mechanism due to its characteristics of rapid regeneration and easy breeding. PacBio sequencing was performed on the intact planarians (In) and regenerating planarians of 1 day (1d), 3 days (3d), and 5 days (5d) after amputation. The aim of this study is to deeply profile the transcriptome of D. japonica and to evaluate its regenerate changes. Using robust statistical analysis, we identified 5931, 5115, and 4669 transcripts differentially expressed between 1d and In, 3d and In, 5d and In, respectively. A total of 63 key transcripts were screened from these DETs. These key transcripts enhance the expression in different regenerate stages respectively to regulate specific processes including signal transduction, mitosis, protein synthesis, transport and degradation, apoptosis, neural development, and energy cycling. Finally, according to the biological processes involved in these potential key transcripts, we propose a hypothesis of head regeneration model about D. japonica. In addition, the weighted gene co-expression network analysis provides a new way to screen key transcripts from large amounts of data. Together, these analyses identify a number of potential key regulators controlling proliferation, differentiation, apoptosis, and signal transduction. What's more, this study provides a powerful data foundation for further research on planarians regeneration.

Planarians are the earliest free-living platyhelminthe with triploblastic and bilateral-symmetry. As an integral component of tissue homeostasis and regeneration, remodeling occurs constantly in the general planarian life history. In the present study, we isolate three planarian Dugesia japonica Atg8 genes (Djatg8-1, Djatg8-2, Djatg8-3) that show high sequence similarity with Atg8 from yeast and human. Results from whole-mount in situ hybridization indicate that Djatg8-2 and Djatg8-3 are strongly expressed in blastemas during Dugesia japonica regeneration. Using RNA interference, inhibition of Djatg8-1 gene expression has no obvious effect on planarian morphological changes. Interestingly, downregulation of Djatg8-2 gene expression in planarians results in defects in blastema regeneration and tissue regression. Furthermore, loss of Djatg8-3 expression leads to tissue degradation. Taken together, our results suggest that Djatg8-2 and Djatg8-3 play important roles in planarian remodeling during regeneration.

Heat shock protein 90 family members (HSP90s), as molecular chaperones, have conserved roles in the physiological processes of eukaryotes regulating cytoprotection, increasing host resistance and so on. However, whether HSP90s affect regeneration in animals is unclear. Planarians are emerging models for studying regeneration in vivo. Here, the roles of three hsp90 genes from planarian Dugesia japonica are investigated by WISH and RNAi. The results show that: (1) Djhsp90s expressions are induced by heat and cold shock, tissue damage and ionic liquid; (2) Djhsp90s mRNA are mainly distributed each side of the body in intact worms as well as blastemas in regenerative worms; (3) the worms show head regression, lysis, the body curling and the regeneration arrest or even failure after Djhsp90s RNAi; (4) Djhsp90s are involved in autophagy and locomotion of the body. The research results suggest that Djhsp90s are not only conserved in cytoprotection, but also involved in homeostasis maintenance and regeneration process by regulating different pathways in planarians.

FLOTILLIN-1 and FLOTILLIN-2 are membrane rafts associated proteins that have been implicated in insulin and growth factor signaling, endocytosis, cell migration, proliferation, differentiation, cytoskeleton remodeling and membrane trafficking. Furthermore, FLOTILLINs also play important roles in the progression of cancer and neurodegenerative diseases. In this study, the roles of flotillins are investigated in planarian Dugesia japonica. The results show that Djflotillin-1 and Djflotillin-2 play a key role in homeostasis maintenance and regeneration process by regulating the proliferation of the neoblast cells, they are not involved in the maintenance and regeneration of the central nervous system in planarians.

Sox genes play important roles in animal developmental processes, including embryogenesis, neural cell stemness, neurogenesis, sex determination, among others. Here, the full length sox gene in planarian Dugesia japonica, named DjsoxB, was cloned using rapid amplification of cDNA ends (RACE). Phylogenetic analysis demonstrates that DjsoxB is highly conserved evolutionarily in metazoans. Whole-mount in situ hybridization found DjsoxB mRNA to be mainly expressed in the head, intestine and mouth in both sexually mature and immature planarians. Moreover, DjsoxB transcripts were detected in the blastema after amputation and throughout the head regeneration processes. The data from real-time PCR showed that the mRNA expression levels of DjsoxB were distinctly up-regulated from 3 to 7days after amputation. These results suggest that DjsoxB gene might be active in CNS formation and functional recovery during head regeneration, maintenance of adult CNS function and the development of other tissues (e.g. intestine) in D. japonica.

The SPSB family is comprised of four highly conserved proteins, each containing a C-terminal SOCS box motif and a central SPRY domain. Presently, Spsb genes have been found in mammals and in a few invertebrates, however, the specific functions of these genes are still unknown. In this study, we identified a Spsb gene from the planarian Dugesia japonica and termed it DjSpsb. The temporal and spatial expression patterns of DjSpsb were examined in both intact and regenerative animals, and expression levels were also quantified in response to various stressors. The results show that (1) DjSpsb is highly conserved in evolutionary history in metazoans and is at closer relationship to Spsb1, Spsb2 and Spsb4; (2) DjSpsb mRNA is mainly expressed in the head and also throughout head regeneration processes, particularly, its expression up-regulated observably on day 5 after amputation; (3) DjSpsb is also expressed in the testes and yolk glands; (4) DjSpsb expression is induced by high temperature and ethanol but inhibited by high doses of ionic liquids. The date suggests that the DjSpsb gene might be active in central nervous system (CNS) formation and functional recovery during head regeneration, and it is also involved in the development of germ cells and stress responses in the planarians.