Timing is a fundamental feature of embryogenesis: the embryos of a given species develop with reproducible timing and a precise ordering of events. The underlying mechanisms that control the absolute timing of cell state changes and morphogenetic events in development are largely not understood, and crucially can be considered at a wide range of length scales, from the integration of positional cues over time in a single cell to whole-organism events such as hormone release. A key context in which cell behaviour must be coordinated with time occurs in the development of the vertebrate posterior body, which is elaborated in a strict anterior to posterior sequence. This process occurs over a period of hours to days depending upon the species, necessitating mechanisms to ensure that cells are allocated to the body axis in a controlled manner. The prevailing model for regulation of the timing of progenitor contribution to the body axis centres around a changing Hox gene expression complement in the population (‘The Hox Clock’) that can influence the timing with which cells enter the body axis (Deschamps and Duboule, 2017; Iimura and Pourquie, 2006).

In this thesis, I utilise classical experimental embryology approaches including grafting and explant culture in combination with modern techniques such as single cell transcriptomics and multiplexed in situ hybridisation to ask about the contribution of intrinsic and extrinsic influences to cell decisions during gastrulation and axis elongation. My primary experimental assay involves the heterochronic grafting of HH8 somite progenitors to a HH4 embryo. I show that HH8 somite progenitors undergo ingression through the primitive streak but are delayed relative to HH4 progenitors at the transition from ingressed to migratory mesenchyme, ultimately resulting in a more posterior axis contribution. To assess the role of Hox gene expression, I identified a set of genes with differential expression between HH4 and HH8 progenitor regions, then assayed expression of these genes in heterochronic (HH8-HH4) grafts. I found consistent support for the maintenance of the HH8 Hox profile upon grafting to the HH4 embryonic environment, suggesting that Hox regulation is population-intrinsic in this cell population. By performing grafts with variable sized pieces of donor tissue, I show that Hox profile can be uncoupled from cell behaviour in small grafts, suggesting that in addition to Hox expression, there are population size-related inputs on cell behaviour. By culturing explants on fibronectin, I show that both HH4 and HH8 MSP populations have a propensity to migrate, but HH8 populations do so with a delay and much lower tissue velocity. Finally, I present work from an additional project in which I ask about the regulation of timing upon progenitor population ablation – the surgical removal of Hensen’s node (the avian Organizer) results in the production of a morphologically normal embryo. Alexandra Neaverson and I designed and performed a single cell RNA-sequencing experiment to ask about global transcriptomic changes resulting from node ablation. We find that there are essentially no global transcriptomic changes in ablated embryos relative to control ones, and that expression of prospective neural plate marker genes is unchanged after removal of the morphological node. A possible reason for this, despite the hypothesised role for Hensen’s node in neural induction and maintenance, is that expression of many ‘node’ marker genes extends into the anterior streak, outside of the morphological node.

Together, the work described here has led to a working model for timing axial progenitor contribution to the axis where population and cell intrinsic Hox gene expression (the Hox Clock) is integrated with population size related inputs. Crucially, such a model can account for the ‘streaming’ of cells from the progenitor region (asynchronous allocation of cells to the axis) in a way that previous models could not. This is multi-scalar regulation; together with the literature review presented in Chapter 1, my work leads me to an understanding of developmental timing as a highly distributed phenomenon with aspects acting at different length scales.