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Using a natural cellular plasticity event to decypher the cellular requirements and molecular circuitry promoting transdifferentiation at the single cell level.

Periodic Reporting for period 4 - PlastiCell (Using a natural cellular plasticity event to decypher the cellular requirements and molecular circuitry promoting transdifferentiation at the single cell level.)

Reporting period: 2020-01-01 to 2021-06-30

The last decades have seen a dramatic change in how the differentiated cellular identity is perceived. It was initially thought that once a specialised identity was acquired, this identity was a definitive and fixed property. It is now clear that differentiated cells can change their identity, whether after an experimental trigger or naturally, during development or regeneration for instance. The aim of the PlastiCell project has been to elucidate the cellular and molecular underpinnings of such natural cellular plasticity, with a focus on transdifferentiation (Td), or the conversion of one differentiated cell into a different differentiated cell type. We use for this a model of natural transdifferentiation in the worm C. elegans, because it is robust, predictable and can be followed at the single cell level in live animals, in an integrated physiological context. Our project has been centred on three essential aspects of transdifferentiation (or Td), with the following objectives: i) To determine the mechanisms and cellular context that make specific cells competent to change their identity. ii) Data from the literature and our lab indicate that transcription factors (TFs) are critical to promote Td: To determine the core nuclear network driving natural Td and how it contributes to its invariant timing and efficiency. iii) Postmitotic somatic identity is generally a stable feature, and is likely actively protected and maintained: To unravel the mechanisms responsible for this protection, and that must be turned off to allow efficient Td.
To address these questions, we have taken advantage of a natural event of direct cellular reprogramming, that occurs during the development of the worm C. elegans, whereby a rectal cell, named "Y", retracts from the rectum during mid development of the worm, and transforms into a motoneuron, named "PDA" (Fig 1A). This model has been first characterised in the laboratory, and occurs with 100% efficiency in all wild type animals. Because this event is accessible at the single cell level in live animals and is predictable, giving access to the early steps of the process, we have been able to obtain a number of mechanistic insights that represent common themes with cellular plasticity events in vertebrates, as described below.
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.
We expect our results to have a two-fold impact. Firstly, our work brings technical developments and new approaches to single cell analyses in live animals. While necessary to obtain physiologically relevant answers, addressing fundamental questions at the single cell level in a physiological environment (ie in live animals) has always been challenging, especially as tools to identify, follow over time and purify specific defined cells from whole animals represent enormous technical hurdles. Here, we have been able to extract one unique cell from each animal and examine its transcriptional content; we have done so comparing a rectal cell that later on is reprogrammed, to a rectal cell that never changes its identity. Our data highlight how similar these two cells are overall, and point to a small number of transcription factors associated with the ability to be reprogrammed. This opens the door to identifying transcriptional programmes that underline the natural transdifferentiation capability of a cell. In addition, our ability to isolate unique cells also now enables us to look at its cellular trajectory and asks whether its first differentiation into a rectal cell occurs as for other rectal cells that never change their identity. Secondly, we have obtained exciting results allowing a deeper understanding of how a specific cell is endowed with the competence to change its identity. We also have been able to explore further what nuclear events are key and define the existence of a Gene Regulatory Network (GRN) that promotes the initiation of transdifferentiation (Td), and that appears to be deployed dynamically in time; Or the chromatin mechanisms that appear to lock cell fate decisions and help maintain a cellular identity over time. Finally, the comparison of the mechanisms at play in different transdifferentiation events within one animal will be key to highlight common principles and event-specific mechanisms.