Final Report Summary - NOTCH AND CELL ARCH (Understanding Notch function in cell architecture regulation)
Notch signalling is a local cell communication mechanism highly conserved throughout the animal kingdom. It is implicated in a variety of developmental and physiological processes and aberrant Notch activity is linked to many different diseases. To date, the main focus of Notch activation has been on its roles in regulating cell fates. However, it is becoming evident that it can exert effects on cell behaviour independent of changes in cell fate. The goal of this project was to investigate the hypothesis that Notch signalling impacts on cell architecture, through direct regulation of target genes involved in cytoskeletal regulation.
1. Characterisation of new Notch cytoarchitecture targets
Utilising data from genome-wide analysis of Notch regulated genes, I compiled a list of genes that encoded key proteins involved in cell morphology. These were categorised according to whether they showed changes in expression and whether they were located in proximity to genomic region occupied by the transcription factor Su(H). Su(H) is the transcription factor mediating Notch response and the presence of a Su(H) binding region in the vicinity of a gene suggests that this gene can a direct Notch target. Using GO term analyses I identified approx. 30 potential Notch targets with function related to cell behaviour regulation that fulfilled the criteria of being up-regulated and being in proximity to Su(H) binding in at least one cell type.
2. Functional test of target gene function
Three different assays were chosen to investigate the relevance of Notch targets in cytoskeletal regulation:
(i) border cell migration during oogenesis, where the functions could be analysed in post mitotic migratory cells;
(ii) adult myogenesis, where Notch signalling is activated in migrating and fusing myoblasts;
(iii) dorsal ventral boundary of the wing disc where Notch acts by inducing the formation of an actomyosin 'fence' to keep the two compartments separated.
The strategy was to test the consequences of knocking down Notch targets on each of these specific processes by directing expression of RNAi to specific sets of cells. The border cell migration assay had many advantages, because of the ability to access function in the absence of mitosis, but it proved the least robust and reproducible. We therefore focussed on the other two assays. Circa 70 different UAS-RNAi lines (2-3 per genes) were obtained and crossed with the appropriate driver to direct expression in the Adult muscle progenitors (AMPs). We identified nine genes whose depletion induced phenotypes suggesting defects in muscle formation. These were therefore candidates to mediate effects downstream of Notch in these cells. Furthermore, the regulatory regions associated with three of these genes also exhibited binding by Twist, which was shown in the lab to act together with Notch in regulating targets in the AMPs. These three genes (Reck, Rhea/Talin and Trio) were therefore the top candidates for Notch regulation in myoblasts and their functions were subsequently explored in more depth.
The function of the same set of 30 putative target genes was also analysed in regulating the boundary between the dorsal and ventral compartments in the wing disc. From this analysis, two genes (Moesin and Chickadee) were found to be required for the proper organisation of the boundary. Their precise function and whether they are Notch targets in this context remain to be fully established.
3) Understand to what extent Notch can directly control the expression of cytoskeletal and cell behaviour regulators
Functional assays indicated the relevance of putative targets to specific processes. To demonstrate that the identified genes can be directly regulated by Notch, I have analysed the regulation by Notch of our 3 top candidates. The most robust results were obtained with Reck where I found that the combined activities of Notch and Twist were able to induce ectopic expression of Reck. In the same assays Rhea/Talin and Trio expression was at best only subtly upregulated by the activation of Notch suggesting that other factors might be required.
To analyse further the response of these genes to Notch activation, I have tested whether the genomic regions occupied by Su(H) and Twist can function as enhancers. These regions were cloned upstream of a reporter gene (such as GFP) and the response of this reporter to Notch activation in vivo assayed. This necessitates the generation of transgenic lines expressing the reporters, which have successfully been generated. I am currently analysing the expression and regulation by Notch of these sequences.
1. Characterisation of new Notch cytoarchitecture targets
Utilising data from genome-wide analysis of Notch regulated genes, I compiled a list of genes that encoded key proteins involved in cell morphology. These were categorised according to whether they showed changes in expression and whether they were located in proximity to genomic region occupied by the transcription factor Su(H). Su(H) is the transcription factor mediating Notch response and the presence of a Su(H) binding region in the vicinity of a gene suggests that this gene can a direct Notch target. Using GO term analyses I identified approx. 30 potential Notch targets with function related to cell behaviour regulation that fulfilled the criteria of being up-regulated and being in proximity to Su(H) binding in at least one cell type.
2. Functional test of target gene function
Three different assays were chosen to investigate the relevance of Notch targets in cytoskeletal regulation:
(i) border cell migration during oogenesis, where the functions could be analysed in post mitotic migratory cells;
(ii) adult myogenesis, where Notch signalling is activated in migrating and fusing myoblasts;
(iii) dorsal ventral boundary of the wing disc where Notch acts by inducing the formation of an actomyosin 'fence' to keep the two compartments separated.
The strategy was to test the consequences of knocking down Notch targets on each of these specific processes by directing expression of RNAi to specific sets of cells. The border cell migration assay had many advantages, because of the ability to access function in the absence of mitosis, but it proved the least robust and reproducible. We therefore focussed on the other two assays. Circa 70 different UAS-RNAi lines (2-3 per genes) were obtained and crossed with the appropriate driver to direct expression in the Adult muscle progenitors (AMPs). We identified nine genes whose depletion induced phenotypes suggesting defects in muscle formation. These were therefore candidates to mediate effects downstream of Notch in these cells. Furthermore, the regulatory regions associated with three of these genes also exhibited binding by Twist, which was shown in the lab to act together with Notch in regulating targets in the AMPs. These three genes (Reck, Rhea/Talin and Trio) were therefore the top candidates for Notch regulation in myoblasts and their functions were subsequently explored in more depth.
The function of the same set of 30 putative target genes was also analysed in regulating the boundary between the dorsal and ventral compartments in the wing disc. From this analysis, two genes (Moesin and Chickadee) were found to be required for the proper organisation of the boundary. Their precise function and whether they are Notch targets in this context remain to be fully established.
3) Understand to what extent Notch can directly control the expression of cytoskeletal and cell behaviour regulators
Functional assays indicated the relevance of putative targets to specific processes. To demonstrate that the identified genes can be directly regulated by Notch, I have analysed the regulation by Notch of our 3 top candidates. The most robust results were obtained with Reck where I found that the combined activities of Notch and Twist were able to induce ectopic expression of Reck. In the same assays Rhea/Talin and Trio expression was at best only subtly upregulated by the activation of Notch suggesting that other factors might be required.
To analyse further the response of these genes to Notch activation, I have tested whether the genomic regions occupied by Su(H) and Twist can function as enhancers. These regions were cloned upstream of a reporter gene (such as GFP) and the response of this reporter to Notch activation in vivo assayed. This necessitates the generation of transgenic lines expressing the reporters, which have successfully been generated. I am currently analysing the expression and regulation by Notch of these sequences.