Periodic Reporting for period 4 - CYCLODE (Cyclical and Linear Timing Modes in Development)
Reporting period: 2022-04-01 to 2023-09-30
Organismal development requires proper temporal coordination of events such as cell differentiation, proliferation, and morphogenesis. The mechanisms that control temporal patterning remain poorly understood. In particular, we know little of the cyclical timers, or ‘clocks’, that control recurring events. Examples of repetitive developmental processes are the formation of the vertebrate spine from repetitive segments, or the molting of roundworms, which occurs regularly at the end of larval stages. Furthermore, it is unknown how clocks are started up and arrested, e.g. to ensure an appropriate number of cyclical repeats. We aim to elucidate the components, wiring, and properties of a prototypic developmental clock by studying developmental timing in the roundworm C. elegans. We build on our recent discovery that ≥20% of the worm’s transcriptome oscillates during larval development. Our aims are i) to identify components of this clock, ii) to gain insight into the system’s architecture and properties, and iii) to understand the initiation and termination of clock cycles. As developmental timing genes and rhythmic gene expression are also important for controlling stem cell fates, we foresee that the results gained will additionally reveal regulatory mechanisms of stem cells, thus advancing our fundamental understanding of animal development and future applications in regenerative medicine.
Our work relies on an ability to follow gene expression patterns and development at high temporal resolution, and ideally in parallel. Hence, we have established assays that enable us to track larval development at the single animal level but for many animals at the same time. This, together with sequencing-based analysis has enabled us to generate evidence that the gene expression oscillations in C. elegans are tightly coupled to development and indeed likely control developmental progression. We have been able to screen for factors in which oscillations are compromised and are currently characterizing genes that we think encode oscillator components. We have thus shown that GRH-1, a C. elegans Grainyhead transcription factor is required for larval viability, cuticle formation and molting. We have further shown that BLMP-1, the C. elegans Prdm1 orthologue, is required for robust oscillations and a normal tempo of development. Additionally, our analyses have provided insights into oscillator properties, constraining possible architectures as well as mathematical models and their parameters that can simulate them. Finally, we have shown that a genetic oscillator-based development clock is capable of implementing body size control, to achieve uniform animal size, without the need for explicit size check-points.
Understanding developmental clocks and oscillators requires the availability of several systems to compare communalities and idiosyncrasies, to derive general concepts and unique implementations. It is only in this way that we can obtain a more than anecdotal understanding of how such dynamic systems function, and how we can manipulate them. Moreover, a diversity of systems expands the available tool box, and thereby opportunities for unique insights. Our results have established C. elegans as a highly suitable system to study oscillations, using approaches not available in other organisms. This includes study of development and oscillatory gene expression at high temporal resolution and across many individual animals, and an ability to perform genetic screens for relevant factors. This enables mechanistic and quantitative insight into animal development at a level that has been previously impossible. We note that the factors that we have thus far identified as candidate oscillator components are well conserved in mammals including humans, where they are cell fate-specifying factors and have been linked to diverse cancers and skin defects, most notably also to rhythmic skin regeneration.