The mechanical characteristics and differentiation potential of the axial progenitor region in vertebrate embryos

Abstract

Vertebrate embryos grow and pattern their body axis in an anterior-to-posterior direction, relying on a pool of multipotent cells at the posterior end. This region is known as the progenitor zone (PZ) and contains neural tube (NT) and presomitic mesoderm (PSM) progenitors, as well as a bi-potent population of cells that contributes to both: the Neuro-Mesodermal Competent cells (NMCs). The NMC region is defined by the co-expression of the transcription factors Sox2 and T/Brachyury; it is highly dynamic, expanding and later depleting as axis elongation progresses. The dynamics of WNT and FGF signalling pathways are believed to regulate NMC fate specification. In addition, the progenitor region shows position-dependent plasticity, i.e. the location of cells within the region influences the fate choice the cell makes. Initially attributed to differences in the signalling environment, the dynamic nature of the PZ led me to hypothesise a differential stiffness gradient within the PZ that contributes to positional information. To test this, I investigated the pattern of stiffness in the PZ and evaluated how disrupting it influences the differentiation potential of NMCs. Firstly, I measured the PZ and surrounding tissues using atomic force microscopy (AFM). I showed that the notochord, the NMCs, and the PSM are distinct mechanically; with the notochord being the stiffest and the PSM the softest. In addition, I observe an anterior-to-posterior reducing stiffness gradient within the NMC region. These findings are recapitulated with our lab’s custom-built Tissue Force Microscope (TiFM), a complementary method of mechanical measurements that probes the NMC region directly. To identify the underlying effectors of these stiffness differences, I looked at the PZ’s extracellular cellular matrix (ECM) composition. I found the region devoid of ECM fibres but with fibronectin, laminin and fibrillin puncta. A general inhibitor of matrix metalloproteinases was used on the embryos to prevent ECM degradation and thus increase stiffness. Surprisingly, AFM measurements show a decrease in measured stiffness in the NMC region and a loss of the previously described stiffness gradient. The addition of the inhibitor impairs proper axis elongation, and gene expression analysis indicates high SOX2 levels at more posterior regions of the PZ, suggesting a differentiation bias towards neural fates. To further test the role of environmental stiffness on NMC fate, I created NMCs in vitro from mouse embryonic stem cells and cultured them on substrates of different stiffness. My findings showed a decrease in co-expression of SOX2 and BRA in stiffer substrates. Together, my work suggests that the mechanical environment in the PZ is a key contributor to body axis patterning. Disruption of the mechanical environment leads to defects in elongation and changes in the differentiation potential of PZ cells.