Final Report Summary - MORPHOGRAD (Biophysical study of the coupling between cell proliferation and morphogen gradients)
The Decapentaplegic (Dpp) morphogen is essential for growth control in the Drosophila wing. The host lab recently uncovered a novel temporal mechanism by which the Dpp gradient controls growth, whereby cells in the wing epithelium are subjected to a temporal increase in Dpp signalling and divide when they have perceived a fixed relative increase in signalling (of around 50 %) since the beginning of their cell cycle. As very little is known about how this process is regulated, the MORPHOGRAD project addresses several key questions:
- How do cells sense this relative Dpp increase during each cell cycle?
- What could determine the constant percentage of Dpp signalling increase that a cell needs to perceive before committing to a new round of cell division?
- What Dpp gradient conditions could lead to tumour formation?
We focused on the interaction between Dpp and the Hippo (Hpo) pathway, a recently discovered tumour suppressor pathway that is conserved in mammals. The project was approached from two main angles and its findings can be subdivided as such: 1) characterisation of a putative adaptation phenomenon between the Dpp and the Hpo pathway in a Drosophila cell line; 2) identification of the Dpp gradient parameters affected by mutations in the Hpo pathway.
1) Adaptation in biology is the phenomenon by which a system is able to sense a stimulus, to respond to it and to reset itself. The temporal growth model brought forward by the host lab is based on the ability of cells to adapt to Dpp signalling. Thus, cells need to be able to sense the increase in Dpp signalling, to respond to it by entering into mitosis once the threshold is reached but then to stop responding until the appropriate threshold is reached again. We hypothesised that the Hippo pathway might be the effector of this adaptation to Dpp. We set out to test this by monitoring the response of luminescent reporters of the Dpp and Hpo pathway after ectopic addition of Dpp in Drosophila S2 cells (Fig. 4).
We first generated various stable cell lines each expressing a different Hpo or Dpp reporter. We then optimised conditions allowing us to monitor cell responses to ectopic Dpp addition live and in real time over the course of several days. We could show that, in this system, the Hpo pathway responds to Dpp addition in a longer time scale than previously believed. Interestingly, we discovered that some Hpo reporters presented an exponential increase after Dpp addition while others were insensitive. For instance, bantam transcription reporters showed a response while a diap transcription reporter was non-responsive (Fig. 4). Thus, targets of the Hpo pathway respond differently to Dpp signals, an avenue we are currently investigating in detail.
During the course of our experiments, we uncovered an important effect of cell concentration and cell-cell contacts on the Hpo pathway activation. Contact inhibition is a central theme in the cancer field and will be pursued in the near future thanks to our sensitive and dynamic experimental set-up.
2) Drosophila larval wing cells with an impaired Hpo pathway function are known to overproliferate and lead to tumour formation in adult flies. In order to shed light on the temporal dynamics of this overgrowth phenotype, we systematically analysed several known Hpo pathway loss-of-function mutations in the wing imaginal disc (the presumptive adult wing) in order to determine which parameters of the temporal growth rule were affected. We generated several complex fly strains carrying all necessary mutations and reporters. We also developed specific ImageJ and Matlab programs in order to facilitate the statistical analysis of our samples. New reagents and reporter fly lines are also currently being generated in order to further our study of some interesting aspects that emerged during the course of those two years.
Strikingly, our first observation was that Hpo pathway-mutant wing discs were on average not bigger than control discs of the same age, contrary to what was expected in view of the literature. The resulting overgrowth of mutant discs appears to result from an elongated proliferation period, rather than from an increased proliferation rate (Figs. 1 & 2). However, mutant discs present a different anisotropy of growth from control tissues (Fig. 1). Interestingly, in all Hpo pathway mutants tested, the Dpp gradient still scales up proportionally to the growing size of the tissue, albeit with a somewhat different scaling factor for some mutants (Fig. 3).
Furthermore, we have uncovered a two-phase behaviour of the studied mutants in regard to another aspect of the Dpp temporal growth rule (Fig. 3). Indeed, in a first phase, Hpo mutant discs behave as control discs. However, after a certain time and compared to wild-type, it seems that Hpo pathway-deficient cells need to sense a smaller relative increase in Dpp signalling before being able to commit again to cell division. This, in addition to a longer proliferation period, could explain their overproliferation phenotype.
Preliminary results on the analysis of tsc2, a tumour suppressor gene independent of the Hpo pathway suggest the same trend. Consequently, such modifications of the temporal growth rule could be general principles governing tumour growth.
This project has thus enabled the elucidation of novel mechanisms involved in the regulation of proliferation in the Drosophila wing disc, thus advancing the current state-of-the-art in the growth control and tumourigenesis field. In particular, it has shed more light on the complex relationship between the Dpp and Hpo pathways, two crucial growth-controlling networks that are both implicated in human cancers. Our preliminary results that suggest that the ability to respond to a smaller temporal increase in Dpp may be a general property of cells deficient for different tumour suppressors are also particularly of interest. If this is indeed the case, this work may provide new strategies for repressing tumour growth by manipulating the ability of tumour cells to respond to Dpp family ligands. This is currently being investigated in the host lab and will soon lead to some further breakthrough in the cancer field. This work outlines the importance of understanding how normal and cancer cells compute their exposure to extracellular ligands in time and space.
- How do cells sense this relative Dpp increase during each cell cycle?
- What could determine the constant percentage of Dpp signalling increase that a cell needs to perceive before committing to a new round of cell division?
- What Dpp gradient conditions could lead to tumour formation?
We focused on the interaction between Dpp and the Hippo (Hpo) pathway, a recently discovered tumour suppressor pathway that is conserved in mammals. The project was approached from two main angles and its findings can be subdivided as such: 1) characterisation of a putative adaptation phenomenon between the Dpp and the Hpo pathway in a Drosophila cell line; 2) identification of the Dpp gradient parameters affected by mutations in the Hpo pathway.
1) Adaptation in biology is the phenomenon by which a system is able to sense a stimulus, to respond to it and to reset itself. The temporal growth model brought forward by the host lab is based on the ability of cells to adapt to Dpp signalling. Thus, cells need to be able to sense the increase in Dpp signalling, to respond to it by entering into mitosis once the threshold is reached but then to stop responding until the appropriate threshold is reached again. We hypothesised that the Hippo pathway might be the effector of this adaptation to Dpp. We set out to test this by monitoring the response of luminescent reporters of the Dpp and Hpo pathway after ectopic addition of Dpp in Drosophila S2 cells (Fig. 4).
We first generated various stable cell lines each expressing a different Hpo or Dpp reporter. We then optimised conditions allowing us to monitor cell responses to ectopic Dpp addition live and in real time over the course of several days. We could show that, in this system, the Hpo pathway responds to Dpp addition in a longer time scale than previously believed. Interestingly, we discovered that some Hpo reporters presented an exponential increase after Dpp addition while others were insensitive. For instance, bantam transcription reporters showed a response while a diap transcription reporter was non-responsive (Fig. 4). Thus, targets of the Hpo pathway respond differently to Dpp signals, an avenue we are currently investigating in detail.
During the course of our experiments, we uncovered an important effect of cell concentration and cell-cell contacts on the Hpo pathway activation. Contact inhibition is a central theme in the cancer field and will be pursued in the near future thanks to our sensitive and dynamic experimental set-up.
2) Drosophila larval wing cells with an impaired Hpo pathway function are known to overproliferate and lead to tumour formation in adult flies. In order to shed light on the temporal dynamics of this overgrowth phenotype, we systematically analysed several known Hpo pathway loss-of-function mutations in the wing imaginal disc (the presumptive adult wing) in order to determine which parameters of the temporal growth rule were affected. We generated several complex fly strains carrying all necessary mutations and reporters. We also developed specific ImageJ and Matlab programs in order to facilitate the statistical analysis of our samples. New reagents and reporter fly lines are also currently being generated in order to further our study of some interesting aspects that emerged during the course of those two years.
Strikingly, our first observation was that Hpo pathway-mutant wing discs were on average not bigger than control discs of the same age, contrary to what was expected in view of the literature. The resulting overgrowth of mutant discs appears to result from an elongated proliferation period, rather than from an increased proliferation rate (Figs. 1 & 2). However, mutant discs present a different anisotropy of growth from control tissues (Fig. 1). Interestingly, in all Hpo pathway mutants tested, the Dpp gradient still scales up proportionally to the growing size of the tissue, albeit with a somewhat different scaling factor for some mutants (Fig. 3).
Furthermore, we have uncovered a two-phase behaviour of the studied mutants in regard to another aspect of the Dpp temporal growth rule (Fig. 3). Indeed, in a first phase, Hpo mutant discs behave as control discs. However, after a certain time and compared to wild-type, it seems that Hpo pathway-deficient cells need to sense a smaller relative increase in Dpp signalling before being able to commit again to cell division. This, in addition to a longer proliferation period, could explain their overproliferation phenotype.
Preliminary results on the analysis of tsc2, a tumour suppressor gene independent of the Hpo pathway suggest the same trend. Consequently, such modifications of the temporal growth rule could be general principles governing tumour growth.
This project has thus enabled the elucidation of novel mechanisms involved in the regulation of proliferation in the Drosophila wing disc, thus advancing the current state-of-the-art in the growth control and tumourigenesis field. In particular, it has shed more light on the complex relationship between the Dpp and Hpo pathways, two crucial growth-controlling networks that are both implicated in human cancers. Our preliminary results that suggest that the ability to respond to a smaller temporal increase in Dpp may be a general property of cells deficient for different tumour suppressors are also particularly of interest. If this is indeed the case, this work may provide new strategies for repressing tumour growth by manipulating the ability of tumour cells to respond to Dpp family ligands. This is currently being investigated in the host lab and will soon lead to some further breakthrough in the cancer field. This work outlines the importance of understanding how normal and cancer cells compute their exposure to extracellular ligands in time and space.
