Finally, we triggered mild optoDronc activation in clones and found that their death rate 1.5-5hr later was 3.5-fold that of their WT neighbours, indicating that sublethal caspase activity can bias clone elimination in vivo.

Furthermore, we trained a time series forest machine learning model to predict the dying cell from a neighbouring pair (one dying cell and one surviving neighbour), and found that we could reach a strong predictive value using relative caspase values up to 3 hours prior to engagement in extrusion.

Among 1,130 groups of dying cells, the first dying cell had the highest cumulative caspase activity in 53% of cases, and the highest instantaneous caspase activity in 77% of cases. This suggested that relative caspase activity and past exposure to caspase can bias elimination between neighbours.

Combining optoDronc with GC3Ai we saw that cumulative caspase activity at the point of further activation is strongly predictive of cell fate at the single cell level, as the higher a cell's GC3Ai at t0 the more likely it was to die following the optoDronc pulse.

Interestingly, when we block prior caspase with RNAi and trigger optoDronc, there is still a bias towards increased death at the midline and anterior side of the tissue, indicating other unknown spatially patterned factors modulating sensitivity to caspase independently of past activation.

So could our map of caspase sensitivity be explained by the pattern of cumulative caspase activation? Indeed, the maps of deaths triggered by optoDronc in early and late pupae closely matched the underlying GC3Ai pattern, and abolishing prior caspase activity led to a more homogenous extrusion map.

To test if such priming occurs, we used optoDronc to subject one lateral domain of the tissue to two pulses of mild caspase activation separated by a number of hours (blue box), and measured the number of deaths against the control contralateral domain only subjected to the latter pulse (red box).

Compiling these maps of optoDronc-induced deaths, we saw clear hotspots of caspase sensitivity in the midline, posterior and anterior edges of the tissue. This indicated that engagement in apoptosis downstream of effector caspases can be developmentally regulated.

To test the physiological relevance of this cell-to-cell heterogeneity in caspase threshold, we used optoDronc, an optogenetic caspase, to trigger mild caspase activation everywhere in the notum and track where cells were most and least likely to die.

Cumulative caspase activity was very poorly predictive of engagement in apoptosis, while probability of death scaled quasi-linearly with instantaneous caspase activity. Nonetheless, no universal caspase threshold was found, and cells committed to die across a wide range of caspase levels.

By detecting the point at which individual tracked cells irreversibly commit to apoptotic extrusion, we searched for a shared threshold of cumulative (GC3Ai) or instantaneous (δGC3Ai) caspase activity that defines this point of commitment.

The bifurcation between survival and death was generally assumed to be driven by a threshold of caspase activity, but this was never tested quantitatively in vivo. We tracked over 15,000 cells expressing a caspase activity sensor in a developing epithelium to probe the existence of such a threshold.

The effector caspases drive cell death, but their activation can often be survived, so how do cells make this decision? Our new preprint from @levayerr.bsky.social shows that instantaneous caspase activity is important, but past activation has a key role to play!

www.biorxiv.org/content/10.1...