During gastrulation of chicken embryo the three major germ layers, ectoderm, mesoderm and endoderm, are formed and organised into the correct topological order. A key event of the gastrulation is formation of the primitive streak that is characterised by large scale vortical flows that involve an order of >200,000 cells. During this self-organised process individual epithelial cells are migrating in a highly coordinated manner. The cell behaviours driving the flows and migration are unknown due to the lack of high resolution time lapse sequences showing each individual cell and its behaviour in detail. Several hypotheses including cell-cell intercalation, chemotaxis and oriented cell divisions, have been put forward to explain how individual cells induce the highly coordinated formation of the primitive streak. Furthermore, another open question is which of the observed motions of cells are active and which occur passively as a result from the active motions.To answer these questions we use light sheet microscopy to acquire large volumetric time-lapse image sequences covering more than half of the chick embryo. We developed an algorithm to automatically extract the apical cell surface from the acquired volumetric image sequences. Manual tracking and segmentation of all the individual cells from the embryo wide image sequences is unfeasible. To solve this problem, we developed algorithms to segment cell membranes and to automatically track each individual cell during primitive streak formation. This provided highly detailed data of position, size, cross sectional area and neighbours of each cell throughout the time sequence. The quality of tracking achieved by the automatic tracking algorithm used was validated by comparing the automatically obtained tracks with tracks from the semi-automatically annotated domains. Large scale high resolution imaging and automatic tracking has not been performed previously in chicken epiblast and this provided a good basis for quantitative analysis of tissue and cell behaviours.Analysis of the cell tracking data reveals a set of mesendoderm cells performing cell intercalation, which drives elongation of the primitive streak. The intercalation process also pulls cells from the lateral positions to the midline of the embryo and causes tissue to buckle in the anterior end of the forming primitive streak. Thus intercalation appears to drive the observed vortical tissue flows. Simultaneously with the intercalation, a set of mesendoderm cells undergoes apical contraction culminating in ingression in the site of primitive streak. This weaker tissue deformation process partially counteracts the elongation of the primitive streak but facilitates pulling of cells towards the midline. Additionally, we found that at the site of the primitive streak both apical cell shapes and orientation of cell divisions are polarised perpendicular to the streak. This implies that that oriented cell divisions are unlikely to drive the primitive streak formation.
|Date of Award||2015|
|Supervisor||Kees Weijer (Supervisor)|