The overall focus of this project has been to unravel, at the single cell level, the mechanisms that allow a differentiated cell to reprogram its identity. In the process, we have set up innovative strategies to eliminate technical roadblocks: a. By adapting the DAMID technique to obtain the transcriptional dynamics of transdifferentiation and establishing a proof-of-principle both at the whole worm and unique cells levels (Gomes-Saldivar et al, 2020). b. We have engineered a cell-specific mutant for the ceh-6 gene, since loss-of-function mutations lead to early lethality (Ahier et al, 2020). c. We have set up an easy-to-implement PCR-based method to quickly genotype single nucleotide changes in different alleles (Morin et al 2020). This method is translatable to many organisms. d. We also have established an efficient workflow to isolate and purify by FACS unique cells from whole animals and set up protocols to sequence either pools of 200 C. elegans cells, or single cells (one unique cell per worm), a technical tour-de-force (in preparation).
During the entire lifetime of this project, we also have made advances in our understanding of the mechanisms at work. We identified essential and dichotomic effects of Notch activity on natural Td; in particular, Notch activity confers competence to change cellular identity in permissive cellular backgrounds, and this during a defined window of time (in preparation). In addition, we have determined that at least another natural Td event occurs in the worm, where the only other rectal cell to change its identity, named "K", does so through a cell division (K-to-DVB, Riva et al, 2021; Fig 1B). We have found that both common principles and factors exist, as well as events specific mechanisms when comparing the Y-to-PDA and K-to-DVB Tds. In the latter, the Wnt signalling pathway act in parallel of a plasticity cassette to both allow one specific K daughter to change identity and control the timing of re-differentiation (Riva et al, 2021). Thus here, both intrinsic cellular context and an extracellular signal combine to allow a cell to change its identity. Our data further suggest a model where the dynamic interplay between these factors controls the timing of re-differentiation (Fig 1C). We have further described two other natural Td in hermaphrodites, that occur though either a symmetric or asymmetric cell division (unpublished). Finally, we have found that the ability of a rectal cell to be naturally reprogrammed depends on at least two types of activities: the drivers, transcription factors which act as effectors and which activity is absolutely needed; and the licencers, nuclear factors which activity is needed to counteracts brakes to direct reprogramming (aka transdifferentiation, Becker et al, in prep). Several articles with our results have been published and several more are being written up. In addition, oral and written presentations have been made at numerous national and international meetings, both by team members or myself.
