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

Reporting period: 2018-07-01 to 2019-12-31

"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 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 is to elucidate the cellular and molecular underpinning of cellular plasticity, with a focus on transdifferentiation, 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. Our project is centered on three essential aspects of transdifferentiation (or Td), with the following objectives:
• To determine the mechanisms and cellular context that make specific cells competent to change their identity
• Data from the literature and our lab indicate that transcription factors (TFs) are critical to promote Td : What core nuclear network drives natural Td and how does it contribute to its invariant timing and efficiency?
• Postmitotic somatic identity is generally a stable feature, and is likely actively protected and maintained : What are the mechanisms responsible for this protection, and that must be turned off to allow efficient Td?
To address these questions, we will take 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. This model has been established and characterised in my 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 in the project achievement part.

The group:

Is establishing C. elegans as a unique cell plasticity model to study direct reprogramming at the single cell level in an integrated physiological context, a niche in the field. After initially describing and setting up to analyse in detail the transdifferentiation of one rectal cell, we are now characterising a number of other putative transdifferentiation events in the worm and examining their shared mechanistic aspects.

Is setting up innovative strategies to eliminate technical roadblocks:
a. boosting up the expression level of cell-specific fluorescent reporters, in order to be able to perform semi-automated screens (see Novel and/or unconventional methodologies for details on the approach);
b. adapting the DAMID technique with a spatio-temporal control, to obtain the transcriptional dynamics of transdifferentiation (see Project achievements);
c. designing 2 novel strategies, using the cas13 system or the SUNTAG system to knockdown specific genes in specific cells, and bypass complications associated with early lethality or pleiotropic effects (see Novel and/or unconventional methodologies for details).

Has set up an efficient workflow to isolate and purify by FACS single cells from whole animals.

Has set up protocols to sequence pools of 200 C. elegans cells and are developing single cells RNA Seq in the worm.

Has uncovered a Gene Regulatory Network driving the initiation of transdifferentiation, and made of conserved nuclear factors, many of which are essentials for ES cells pluripotency or iPS pluripotent reprogramming.

Has identified essential and dichotomic effects of Notch activity on natural Td; in particular, Notch activity confers competence to change cellular identity in permissive cellular background

Has identified an exciting relationship between satiety status and cellular plasticity.
We expect our results to have a two-fold impact. Firstly, our work will bring 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 single cells from whole animals represent enormous technical hurdles. We thus are developing several approaches to allow better visualisation of single cells in their live environment, perform large scale genomic approaches on given cells, and inactivate genes in specific cells in the animal, by creatively combining several new technologies. Secondly, we have obtain exciting results : on how a specific cell is endowed with the competence to change its identity, where both intrinsic cellular context and an extracellular signal combine to allow a cell to change its identity; On the existence of a Gene Regulatory Network (GRN) that promotes the initiation of transdifferentiation (Td), and that appears to be deployed dynamically in time; And on the impact of satiety status on cellular plasticity, which suggests a relay of a starvation signal from different tissues to increase the plasticity of specific cells, a signal that is then interpreted by specific cell-autonomous mechanisms. We expect to further learn : i) What specific genes both intrinsic and downstream of the extracellular Notch signal are key for the competence to change identity. ii) The detailed dynamics of the GRN and how its architecture could impact on the invariant timing of transdifferentiation, both experimentally and through modelling. iii) The core mechanisms at play during different transdifferentiation events and how, when Td involves a mitosis, cell division may impact on the process. and iv) Which organs are involved in relaying a lack-of-food signal to the Y cell, what are the pathways involved in each organ, and how the Y cell interprets this signal to decrease cell identity maintenance and allow Td to occur. We further expect this work to decipher the importance of metabolic signals and growth rate on cellular identity decisions.