Periodic Reporting for period 1 - JNK_Life.Death (Role of dynamic JNK signalling in balancing cell removal and renewal in a stressed epithelium)
Okres sprawozdawczy: 2023-01-01 do 2024-12-31
The development of functional tissues relies on the fine balance between cell death and cell division. Little is known about how tissue size and cell numbers are maintained in stress situations when additional proliferation is needed to compensate for cell loss. The c-Jun N-terminal kinases (JNK) signalling pathway is a stress pathway that has been proposed to trigger not only cell death but also cell survival and proliferation. To decipher the regulatory events that control these diverse responses, we have set out to characterise the spatial-temporal dynamics of cell fate (proliferation, cell death, cell cycle arrest and migration) upon JNK activation. We optogenetically activate signalling in a subset of cells by expressing either a constitutively active form of the JNK Kinase hemipterous (hep). Then, we reconstruct the cell-autonomous and non-autonomous response to stress signalling. The response is very fast: we observe cell death and cell cycle arrest within two hours of Hep activation. However, the response lasts for an extended time, with a population expressing low JNK levels being observed 18 hours after the light pulse. We reconstructed the pattern of gene expression in time and space, using both biased and unbiased approaches. First, we used the Hybridization Chain Reaction to visualise the expression of genes known to be regulated by JNK signalling, such as Wg/Wnt and Dpp/BMP. Finally, we performed bulk RNA sequencing to identify the genes that could potentially account for the varied effect of JNK signalling activation. We discovered that under temporary and mild stress levels, there is no significant upregulation of morphogens/growth factors but instead of their modulators: glypicans. In the absence of glypicans, the replacement of lost cells is impaired. We hope that our results will guide further studies of more complex tissues of direct biomedical relevance.
We have developed and optimised an animal model that allows the induction of stress levels in a spatial and temporal controlled manner. Using this system, we have assessed the autonomous and non-autonomous cell response to JNK activation at different time points. We used multiple strategies including genetic manipulation, immunofluorescence, hybridization chain reaction and RNA sequencing.
The main results from this work:
1. Low JNK levels may also lead to cell death. One factor influencing this cell response is the cell cycle: G1 cells are more sensitive to stress;
2. High JNK levels do not necessarily lead to cell death after cells mount a protective response that includes G2 arrest and glypican unregulated.
3. In situations of temporary and mild stress levels, we didn’t observe significant upregulation of morphogens/growth factors but instead of their modulators: glypicans.
The main results from this work:
1. Low JNK levels may also lead to cell death. One factor influencing this cell response is the cell cycle: G1 cells are more sensitive to stress;
2. High JNK levels do not necessarily lead to cell death after cells mount a protective response that includes G2 arrest and glypican unregulated.
3. In situations of temporary and mild stress levels, we didn’t observe significant upregulation of morphogens/growth factors but instead of their modulators: glypicans.
Glypican upregulation has not yet been reported to be directly associated with JNK signalling activation. Moreover, it was quite surprising that the stressed tissue 'prioritised' upregulating the signalling modulators to the signalling molecules to compensate for cell loss and restore tissue homeostasis. Our exciting results and the novelty of our optogenetic tools are expected to be received with enthusiasm by the scientific community. The manuscript is under preparation and will be submitted within the next few months to a scientific journal of medium/high impact.