Quantitative study of a threshold response of ERK signalling – ERKtivation

We used in silico embryo reconstruction, quantitative imaging techniques as well as mathematical modelling to study the switch-like response of early neural induction in ascidian embryos. Ascidian embryos develop with an invariant cleavage pattern, meaning that cellular configurations are identical among embryos. At the 32-cell stage of development, the eight pairs of ectoderm cells (future epidermis and brain) are all exposed to FGF signals. However, only two specific pairs of cells respond to FGF signals to initiate neural induction and express the Otx gene. Using in silico embryo reconstruction, we quantified the areas of cell surface contact between ectoderm cells and neighbouring FGF-expressing cells. Quantitative imaging was used to measure the levels of ERK activation and Otx gene activation in ectoderm cells in control embryos and embryos in which the levels of ERK activation were experimentally modified. We also investigated the role played by the ephrin signalling pathway, which acts antagonistically to FGF signaling. Using our measurements, we constructed mathematical models to enhance our mechanistic understanding of this process.

We revealed that each ectoderm cell activates ERK to a level that mirrors its cell contact surface with FGF-expressing mesendoderm cells. This gradual interpretation of FGF inputs is followed by a threshold transcriptional response of Otx, resulting in its activation specifically in the neural precursors. At low levels of ERK, Otx is repressed by an ETS-family transcriptional repressor, ERF2. ephrin signals are critical for dampening ERK activation levels across ectoderm cells so that only neural precursors exhibit above-threshold levels, evade ERF repression, and «switch on« Otx transcription. This work helps our understanding of how noisy and graded signals can be interpreted with all or none cellular responses.

A precise understanding of the subcellular mechanisms whereby FGF- and ephrin- signals converge to control Ras activity remains to be addressed. In addition, the precise molecular and kinetic conditions leading to the threshold response of Otx transcription in response to ERK activation are not fully understood and are the topic of our new project, involving the current partners of the project.

We describe rapid and precise FGF-ERK-mediated gene activation. As the ERK signalling cascade is evolutionarily conserved and broadly implicated in development and disease, our work is likely to be broadly relevant in biology. Work from this project has been published in Developmental Cell (Williaume et al, 2021) (https://doi.org/10.1016/j.devcel.2021.09.025) and PLoS Computational Biology (Bettoni et al, 2023, In Press) (bioRxiv (https://doi.org/10.1101/2022.06.29.498205) and has been presented at numerous international conferences.

In this project, we address a long-standing problem of cell signalling: how a cell interprets a graded or variable signal to generate a threshold, or switch-like, response. The project focuses on the extracellular-signal-regulated kinase (ERK) pathway. This signalling cascade, involving Ras, Raf and MEK, is evolutionary conserved and culminates in the phosphorylation and activation of ERK. Activated ERK, in turn, phosphorylates and modulates a number of protein targets, including transcription factors, to alter their activity. The ERK signalling pathway is used repeatedly for patterning, cell fate determination and cell proliferation during embryonic development as well as homeostasis in adults. Excessive ERK signalling is associated with cancer as well as a large group of developmental syndromes in humans. Congenital malformations, including craniofacial and cardiac abnormalities, along with neurocognitive delay and an increased risk of cancer, are found in individuals with mutations in components or regulators of the ERK signalling pathway. Thus, the precise control of ERK activation levels is critical in both embryonic and adult life. Despite the profound developmental defects associated with both reduced and excessive levels of the ERK signalling, its quantitative analyses in in vivo multicellular contexts are rare and have never been conducted in relation to a threshold response. We will address how the ERK pathway exhibits a threshold response to a graded signal in the context of developing embryos. The project is a joint effort between experimental biologists and computational chemists, deploying an interdisciplinary approach including quantitative imaging, analytical chemistry, embryology, and mathematical modelling.
To address our question, we take advantage of the invariant embryogenesis of ascidians and study the switch-like response of ERK to variable levels of fibroblast growth factor (FGF) signal during neural induction. Phylogenetically, ascidians are a sister group to vertebrates and develop into a typical chordate tadpole at the larval stage. The invariant nature of ascidian embryogenesis means that cellular configurations, such as contact surfaces between neighbouring cells, are quasi-invariant among synchronised embryos. Ascidian neural induction takes place at the 32-cell stage of development. During this process, despite all sixteen ectoderm cells being neural competent and being exposed to some level of neural-inducing FGF signals, only four of them exhibit ERK activation and adopt neural fate. Our unpublished data strongly support a working hypothesis that an antagonistic regulation of Ras by opposing FGF and ephrin/Eph signals is critical for generating the threshold ERK response. In this project, we will quantitatively describe ERK activation levels, in both intact and experimentally-manipulated embryos. The quantitative measurements obtained in this analysis will be used to conduct data-driven mathematical modelling of the neural induction process. Model predictions will be experimentally challenged, enabling their further refinement. This approach should enable us to uncover the biochemical mechanism that underlies the switch-like response of ERK activation in our system. Furthermore, we will address two related issues that are also critical to our understanding of how the threshold response of ERK activation operates. In the first, we ask how ERK activation is converted to a transcriptional response, using quantitative in situ hybridisation. In the second, we will attempt to visualise the site of convergence of the FGF and ephrin signalling pathways at the subcellular level. This proposed project represents a pioneering multi-faceted and multi-disciplinary analysis of a threshold ERK response in an in vivo multicellular setting. Its successful execution would have broad-reaching implications in the fields of cell signalling, cancer biology and developmental biology.