AbstractDuring gastrulation in the chicken embryo, the mesoderm and endoderm progenitors migrate in a collective manner. They ingress through the primitive streak and follow routes that reflect their developmental fate. Despite its importance for the formation of the body plan, the mechanisms that define this process remain elusive. Previously, the introduction of external sources of growth factors into the embryo impaired the mesendoderm trajectories. However, the cellular responses were assessed in vivo and due to the complexity of the environment, they could not be fully interpreted. This is because the introduced ligands might have triggered the secretion of secondary factors to which the cells responded. In the present study, we aimed to directly expose the mesendoderm progenitors to gradients of potential chemoattractants and assess their migratory behaviour in real-time. We established an in vitro assay that enables the isolation of the cells from the embryo with minimal damage and their preservation in serum-free conditions. We then used the Dunn chemotaxis chamber to expose the extracted cells to linear gradients of chemotactic factors proposed by the previous in vivo studies. This showed that unlike their effects in the embryo, Fgf 4 and 8, mostly enhanced motility of the isolated cells with weak effects on their directionality. Vegf acted as a chemoattractant for the cells of the posterior area, while Pdgf strongly inhibited basal motility.
Notably, the extracted cells performed accurate chemotaxis toward fetal bovine serum (FBS). Unlike typical cases of mesenchymal chemotaxis, the response was not mediated by tyrosine kinase (RTK) signalling. Inhibition of major classes of RTKs expressed in chicken gastrulation failed to abolish chemotaxis, however the serum response was sensitive to pertussis toxin (PTX), a potent inhibitor of Gai/o signalling. Uniform FBS concentration triggered the outward dispersal of mesendoderm, but only in the absence of PTX. This suggested that GPCR mediate the ability of the mesendoderm cells to deplete the active component and form self-generated gradients. The present study sheds light onto the mechanisms of mesendoderm migration and for the first time provides direct evidence of chemotaxis. The identification of the chemoattractant in serum would help to validate the role of guidance cues in vivo and elucidate a fundamental developmental process.
|Date of Award||2016|
|Supervisor||Kees Weijer (Supervisor)|
- Chicken embryo