In task 1, we evaluated the requirement of the APC/C for the formation of the photo detecting cells, the photoreceptors. We found that when the function of the APC/C is compromised, the cells that normally become photoreceptors fail to acquire their fate and unspecialised masses of cells are formed in the middle of the adult fly eyes. Further analysis revealed that this defect is caused by an abnormal activation of the Wingless/Wnt (Wg/Wnt) signalling pathway. An intrinsic mechanism that through the secretion of a specific molecule called Wingless by the neighbouring cells, instruct the receiving cells to keep their proliferative state.
In task 2, we aimed to identify the targets of the APC/C that when APC/C function is compromised, accumulate to cause the activation of the Wg signalling pathway. To this end, we employed a mixture of classical genetic approaches with biochemical approaches. Through these approaches, we successfully identified a cohort of proteins that present the APC/C degrons and with the potential to affect the transition from the unspecialised dividing cells to non-dividing specialised cells. Next, to identify the direct targets of the APC/C, we downregulated these candidates individually and tested whether their reduction can prevent the abnormal Wg activation caused by the inactivation of the APC/C. Through this analysis, we showed that the mitotic kinase Nek2 is directly targeted for degradation by the APC/C upon its activation with the co-activator Fzr/Cdh1.
Finally, in Task 3, we found that Nek2 is the protein that mediates the control of the Wg signalling pathway by the APC/C. By observing the behaviour of fluorescently labelled Nek2 in the eye tissue, we found that Nek2 is specifically degraded by the APC/C in the transitional zone between unspecialised cells and differentiated cells, called the morphogenetic furrow, where cells temporally stop dividing and stay in the G1 cell cycle phase. It is known that the Wg signalling pathway functions to keep the cells dividing and preventing cells to become specialised into photoreceptors. We found that when APC/C function is compromised in the transitional zone, it accumulates Nek2. This Nek2 accumulation, in turn, abnormally activates Wg signalling, which prevents cells to become photoreceptors. Altogether, we uncovered a novel function of the APC/C, by which APC/C keeps in check Wg signalling to initiate the specialisation of photoreceptors whilst stopping these cells dividing further.
In addition, we also found the evidence that this novel function of the APC/C is regulated by the Dpp signalling pathway, another signalling pathway known to promote specialisation process and antagonise Wg signalling in the transitional zone. We showed that for differentiation to proceed, Dpp signalling induces the stabilisation of a component of the APC/C. Moreover, if APC/C function is inhibited in the cells responding to Dpp, it activates Wg signalling. Thus, our results suggest that Dpp signalling uses the APC/C to inhibit Wg signalling.
