RESEARCH

RESEARCH

Current projects

Current projects_ERK.pdf

ERK signalling in the Naïve to Formative transition

MAPK/ERK signalling activity is crucial in many cell state transitions, including exit from Naïve pluripotency. I am exploring two main questions:

  1. The role of ERK negative feedback regulators in transition.

  2. The molecular mechanism behind ERK-mediated cell state transitions.


What we learnt:

  • The levels of ERK determine the rate of cell state transitions (Figure 1A).

  • At the population level, ERK is dynamic (Figure 1B).

  • Interfering with negative feedback systems (such as RSK) results in faster and more synchronised differentiation (Figure 1C-D).

ERK signalling regulates both the exit from Naive pluripotency AND the entry into Formative pluripotency, but these two events are separable. Combining pharmacological and genetic approaches, we can uncouple cell state exit and entry, and leave cells stranded in between cell states (unpublished).

Figure 1 (A) The rate of differentiation depends on the levels of pERK. Higher levels of pERK result in faster differentiation: at 30hrs, there is a lower percentage of undifferentiated (Rex1::GFPd2 positive) cells. (unpublished) (B) ERK activity determined by the pERK/ERK ratio and target gene expression (Dusp6 and Etv4) is dynamic during exit from the ESC state. (C) Schematic diagram of the signalling environment in either wild type (WT) or RSK mutant or inhibited cells. (D) RSK inhibition results in higher average levels of pERK, as well as faster and more synchronous downregulation of the Rex1::GFPd2 (undifferentiated) reporter. B-D adapted from Nett*, Mulas* et al. 2018.

Entropy, asynchrony and technical noise in cell state transitions

We are working on the hypothesised that transition points, associated with the dissolution of regulatory networks as cells change state, resulting in maximisation of relative entropy of the transcriptional landscape (Figure 2). To isolate true biological variability, we need to remove two confounding variables:

  1. Technical noise, caused by batch effect, PCR amplification bias, etc.

  2. Temporal heterogeneity, arising from non-synchronised differentiation.

To test our hypothesis, we are using a system in which we determine cell state independently of transcriptional profile. Therefore, we can synchronise cells according to developmental time and determine the precise transition points. Working alongside James Baye (Chalut lab) and Bernat Corominas-Murtra and Edouard Hannezo (IST, Austria), we are developing theoretical frameworks to remove technical and temporal noise to isolate true biological variability.

Entropy synchrony noise.pdf
Figure 2 – Transitions points (red box) might be characterised by high variance and high entropy. To dissect this biological variability, it is important first to remove technical noise and temporal heterogeneity.

Past research

Table of Content copy.pdf
Figure 3. Summary of the key aspects of the paper.

Microfluidics for time-resolved biology

We design a new cell encapsulating system and microfluidic platform to track and retrieve dynamic events (Figure 3). We screen for hydrogel conditions that support ES cell differentiation in 3D beads. Then, we design a microfluidic culture and imaging platform, where each bead/cell can be cultured in isolation and tracked over time. Finally, we show that we can track cells over time as they undergo differentiation, and we can retrieve particular events of interest for downstream functional or molecular studies without disrupting the rest. More info here.

Oct4 post-implantation copy.pdf
Figure 4. (A) At E6.75, Nanog expression marks the posterior domain, where the primitive streak arises, while Sox2 is expressed more anteriorly. In conditional Oct4 mutant embryos, an ectopic posterior-like domain appears anteriorly. This new domain also expressed T and Mixl1. (B) Transverse sections at E7.0. Mutant embryos fail to downregulate E-cadherin when cells exit the primitive streak.

Role of Oct4 in the post-implantation embryo

Oct4 is a key transcription factor expressed during all pluripotent stages of embryonic development. We used a conditional knock-out strategy to delete Oct4 in the early post-implantation epiblast. We show that Oct4 orchestrates multiple fates and enables expansion, correct patterning and lineage choice in the post-implantation epiblast. In particular, the mutant embryos show:

  • Disrupted gene expression domains of lineage markers: definitive endoderm expands at the expense of mesoderm; the anterior-posterior axis is positioned more distally, and an ectopic posterior-like domain appears anteriorly (Figure 4A).

  • The primitive streak forms in the presumptive proximal-posterior region but the epithelial-to-mesenchymal transition is impeded by an increase of E-cadherin (Figure 4B), leading to complete tissue disorganisation and failure to generate germ layers.

More info here.

Nodal paper copy.pdf

Acquisition of multilineage competence upon exit from naive pluripotency, and dependence of endogenous Nodal signalling

We investigated the early events that lead to exit from the naïve state and lineage choice. We show that the first cells to irreversibly exit the ES cell state in the absence of exogenous cytokines are pluripotent and have acquired the capacity to respond to lineage inducing signals (Figure 5A). Autocrine Nodal delays neural specification and ensures multi-lineage capability (Figure 5B). Read more about it here.

Figure 5. (A) The first cells to exit the naïve pluripotent cell state have acquired the capacity to differentiate towards all germ layers, including the germline, as shown by Blimp1 and Oct4 staining. Naïve ES cells cannot respond directly to these signals. (B) Endogenous Nodal signalling preserves pluripotency upon exit from the naive ES cell state. Inhibition of Nodal results in accelerated differentiation (cells downregulate Oct4 and upregulate Sox1 faster).