The regulation of tissue proportions: analysis of progenitor behavioural dynamics in response to cell ablation in the zebrafish tailbud.

In order to form a correctly patterned embryo the behaviour of cell populations and tissues must be coordinated so that different structures form with the correct relative proportions. The fundamental body plan of the vertebrate embryo is laid down in a head to tail progression known as primary body axis elongation. This begins with the differentiation and morphogenetic events of gastrulation which initially give rise to the head and anterior trunk. During this phase of development, it has been shown that the proportions of germ-layer specification scale with the number of cells in the embryo. Cells that have not formed part of the main axis at this time remain in a structure known as the tailbud. During this later phase, the tailbud progenitors undergo morphogenesis and differentiation to form the axial tissues of the posterior trunk and tail such as neural tube and somitic mesoderm. Importantly, within the tailbud certain cells, known as neuro-mesodermal competent progenitors (NMC cells/NMPs) can differentiate into either neural or mesodermal tissue. I hypothesise that the NMC progenitor population could be maintained in the tailbud during posterior body elongation in order to facilitate the correct proportioning of neural and mesodermal tissue in response to any fluctuations in more differentiated progenitor numbers. In this thesis, I utilise two-photon laser ablation to alter the functional number of progenitor cells in the neural fated region of the zebrafish tailbud and quantify the effect this has on the behaviour of the remaining progenitors. In order to investigate this in detail I developed an open source image processing pipeline for three-dimensional nuclear segmentation, point cloud registration, and the analysis of gene expression intensities and cell tracking data. I first demonstrate that the neural tube and somitic mesoderm are proportionally reduced in elongation following ablation. Then, by combining fluorescent in situ hybridisation with live imaging I show that the progress of NMC cell differentiation is robust to ablation. However, I observe that healing of the ablation causes increased convergence of neural-fated progenitors without contribution from mesodermal-fated NMC cells. This suggests, that in this case, NMC cells are unable to regulate the proportion of neural versus mesodermal differentiation. Finally, I find that there is considerable robustness in elongation following ablation of mesodermal fated progenitors. Taken together, my analyses indicate that progenitors are not the main driver of the proportionality of tissue elongation in the tail. These results are in line with growing bodies of evidence that demonstrate that tailbud progenitors are a highly dynamic group of cells, but that zebrafish body elongation is driven primarily by more anterior multi-tissue interactions