Abstract
Regenerating tissue must initiate the signaling that drives regenerative growth, and sustain that signaling long enough for regeneration to complete. How these key signals are sustained is unclear. To gain a comprehensive view of the changes in gene expression that occur during regeneration, we performed whole-genome mRNAseq of actively regenerating tissue from damaged Drosophila wing imaginal discs. We used genetic tools to ablate the wing primordium to induce regeneration, and carried out transcriptional profiling of the regeneration blastema by fluorescently labeling and sorting the blastema cells, thus identifying differentially expressed genes. Importantly, by using genetic mutants of several of these differentially expressed genes we have confirmed that they have roles in regeneration. Using this approach, we show that high expression of the gene moladietz (mol), which encodes the Duox-maturation factor NIP, is required during regeneration to produce reactive oxygen species (ROS), which in turn sustain JNK signaling during regeneration. We also show that JNK signaling upregulates mol expression, thereby activating a positive feedback signal that ensures the prolonged JNK activation required for regenerative growth. Thus, by whole-genome transcriptional profiling of regenerating tissue we have identified a positive feedback loop that regulates the extent of regenerative growth.
Highlights
The capacity to regenerate damaged or lost organs or limbs is significantly greater in some animals than others
We show that damage-induced Jun N-terminal Kinase (JNK) signaling leads to the upregulation of a gene called moladietz, which encodes a co-factor for an enzyme, NADPH dual oxidase (Duox), that generates reactive oxygen species (ROS), a key tissue-damage signal
We examined the expression of genes that regulate ROS production and removal in our transcriptional profile of the imaginal disc regeneration blastema, and found that in addition to the downregulated genes identified by the gene ontology (GO) analysis, there were ROS-regulating factors among the upregulated genes (Table 2)
Summary
The capacity to regenerate damaged or lost organs or limbs is significantly greater in some animals than others. The complementary tools available in different model organisms has enabled identification of conserved mechanisms and signaling pathways that are used in many regeneration contexts, such as WNT signaling [2,3,4,5,6,7,8], Receptor Tyrosine Kinase (RTK) signaling [9,10,11,12,13,14,15,16], Hippo signaling [17,18,19,20,21,22], and Jun N-terminal Kinase (JNK) signaling [23,24,25], as well as clear differences in regenerative mechanisms among organisms and tissues [26,27]. Model organisms that are amenable to mutagenesis, transgenics, or RNAi-mediated gene knockdown enable functional studies based on the results of transcriptional profiling. Genes regulating anterior-posterior patterning during fin regeneration were identified through transcriptional profiling of anterior and posterior portions of the blastema. Transcriptional profiling followed by functional analysis is an effective approach to identification and validation of regeneration genes
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