We present a microscopy technique that enables long-term time-lapse microscopy at single-cell resolution in moving and feeding Caenorhabditis elegans larvae. Time-lapse microscopy of C. elegans post-embryonic development is challenging, as larvae are highly motile. Moreover, immobilization generally leads to rapid developmental arrest. Instead, we confine larval movement to microchambers that contain bacteria as food, and use fast image acquisition and image analysis to follow the dynamics of cells inside individual larvae, as they move within each microchamber. This allows us to perform fluorescence microscopy of 10–20 animals in parallel with 20 min time resolution. We demonstrate the power of our approach by analysing the dynamics of cell division, cell migration and gene expression over the full ∼48 h of development from larva to adult. Our approach now makes it possible to study the behaviour of individual cells inside the body of a feeding and growing animal.

Significance An enduring mystery of development is how its timing is controlled, particularly for development after birth, where timing is highly flexible and depends on environmental conditions, such as food availability and diet. We followed timing of cell- and organism-level events in individual Caenorhabditis elegans larvae developing from hatching to adulthood, uncovering widespread variations in event timing, both between isogenic individuals in the same environment and when changing conditions and genotypes. However, in almost all cases, we found that events occurred at the same time, when time was rescaled by the duration of development measured in each individual. This observation of “temporal scaling” poses strong constraints on models to explain timing of larval development.

It is essential that correct temporal order of cellular events is maintained during animal development. During post-embryonic development, the duration of development depends on external conditions, such as food availability, diet and temperature. How timing of cellular events is impacted when the rate of development is changed is not known. We used a novel time-lapse microscopy approach to simultaneously measure the timing of oscillatory gene expression, seam cell divisions and cuticle shedding in individual animals during C. elegans larval development. We then studied how timing of these events was impacted by changes in temperature or diet, and in lin-42/Period mutants that show strongly perturbed and heterogeneous timing of larval development. We uncovered significant variability in timing between individuals under the same conditions. However, we found that changes in timing between individuals were fully explained by temporal scaling, meaning that each event occurred at the same relative time, when rescaled by the total duration of development in each individual. Upon changing conditions, we found that larval development separated into distinct epochs that differed in developmental rate. Changes in timing of individual events were fully captured by temporal scaling for events occurring within each epoch, but not for events from different epochs. Overall, our results reveal a surprisingly simple structure that governs changes in timing of development in response to environmental conditions. The unexpected observation of continued development and accurate temporal scaling in growth-arrested lin-42 mutants rules out a mechanism that explains temporal scaling by linking developmental timing to body size.

FOXO transcription factors are highly conserved effectors of insulin and insulin-like growth factor signaling, that are crucial for mounting responses to a broad range of stresses. Key signaling step is the stress-induced translocation of FOXO proteins to the nucleus, where they induce expression of stress response genes. Insulin signaling and FOXO proteins often control responses that impact the entire organism, such as growth or starvation-induced developmental arrest, but how body-wide coordination is achieved is poorly understood. Here, we leverage the small size of the nematode C. elegans, to quantify translocation dynamics of DAF-16, the sole C. elegans FOXO transcription factor, with single-cell resolution, yet in a body-wide manner. Surprisingly, when we exposed individual animals to constant levels of stress that cause larval developmental arrest, DAF-16/FOXO translocated between the nucleus and cytoplasm in stochastic pulses. Even though the occurrence of translocation pulses was random, they nevertheless exhibited striking synchronization between cells throughout the body. DAF-16/FOXO pulse dynamics were strongly linked to body-wide growth, with isolated translocation pulses causing transient reduction of growth and full growth arrest observed only when pulses were of sufficiently high frequency or duration. Finally, we observed translocation pulses of FOXO3A in mammalian cells under nutrient stress. The link between DAF-16/FOXO pulses and growth provides a rationale for their synchrony, as uniform proportions are only maintained when growth and, hence, pulse dynamics are tightly coordinated between all cells. Long-range synchronization of FOXO translocation dynamics might therefore be integral also to growth control in more complex animals.

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