Synchronizing Waves of Signaling in the Presomitic Mesoderm to Study Regulatory Strategies
Summary by Matt Monaghan: Meijer, W., Ribeiro de Andrade, V., Stelloo, S., Thomas, W., van Oostrom, M., Ilcken, E., Peters, K., Vermeulen, M., Sonnen, I., 2025. Spatiotemporal proteomics reveals dynamic antagonistic gradients shaping signallin gwaves. bioRxiv. https://www.biorxiv.org/content/10.1101/2025.09.05.674076v1
Image credit: Wikimedia Commons (Thomas Fuhrmann)
During embryo development, somitogenesis yields chunks of tissue that become muscle, skin, and vertebrae. Intersecting signaling pathways - including Wnt - guide proper development via the rhythmic clock-and-wavefront model, with a molecular ‘clock’ that oscillates in presomitic mesoderm (PSM) and a ‘wavefront’ that moves down the tissue as a result, dynamically regulating gene expression [1]. To investigate this process, past studies largely assessed mRNA expression - not protein levels - during somitogenesis. However, RNA and protein levels are not necessarily correlated, so analyzing both is important to further understand the dynamic process of somitogenesis.
To address this challenge, the Sonnen lab first performed mass spectrometry in dissected posterior, anterior, and somite regions of embryonic mouse tails [1]. They identified differential protein expression between the anterior PSM and the posterior PSM. Some identified proteins had known roles related to somitogenesis, and some had not been previously associated. Differential protein expression was identified for WNT3A, as well as the transcription factor FOXP1 and the broadly present protein linking actin cytoskeleton to plasma membrane, EZRIN.The authors concluded that gradients of these proteins may be relevant for somite formation.
Next, bulk RNA sequencing allowed the authors to compare transcriptomic and proteomic data. This showed interesting differences between RNA and protein levels based on function, with high correlations associated with morphogenesis and adhesion. This difference between RNA and protein levels shows how important it is to assess both for a more complete understanding of developmental processes.
To expand their detection capabilities in individually developing tails, the authors had to combine multiple samples. To do this, they had to address a key technical challenge: each developing tail runs on its own segmentation clock. They developed a strategy to artificially sync their clocks with micro fluidic synchronization on a chip by aligning multiple, dissected embryonic mouse tails with simultaneous application of drugs for control of signaling. By applying a Notch (gamma secretase) inhibitor, DAPT, at regular intervals, Meijer et al. could activate antagonistic signaling, locking the clocks in synchrony.
Using a neural network model to identify dynamic RNAs and proteins from -omics datasets obtained from synchronized samples, “significantly dynamic” [1] signaling mechanisms were found to be correlated to morphogenesis or stress responses, supporting their hypothesized role in somitogenesis. With this approach, they identified anti-correlated gradients of a ligand (R-SPONDIN) and receptor (leucine-rich repeat-containing G-protein-coupled receptor). They further investigated function by adding R-SPONDIN or blocking R-SPONDIN-receptor interaction. Both treatments dampened Wnt oscillations, suggesting an essential role for these gradients in somite differentiation through Wnt regulation.
This study provided a basis for how proteins throughout the cytoskeleton and membrane contribute to morphogenesis, identified spatial patterns of mRNA-protein pairs in Wnt signaling, and explored how the rhythmic Wnt signaling gradient is regulated and sharpened during somitogenesis. There is not a universal, direct correlation between protein and RNA during somitogenesis, as seen in their spatial gradient patterning work. The author’s microfluidic synchronization chip system enables further study of signaling mechanisms in somitogenesis as it relates to regenerative medicine and congenital disorders.
Meijer, W., Ribeiro de Andrade, V., Stelloo, S., Thomas, W., van Oostrom, M., Ilcken, E., Peters, K., Vermeulen, M., Sonnen, I., 2025. Spatiotemporal proteomics reveals dynamic antagonistic gradients shaping signallin gwaves. bioRxiv. https://www.biorxiv.org/content/10.1101/2025.09.05.674076v1