Living systems exhibit an unmatched complexity, due to countless, entangled interactions across scales. Here, we aim to understand a complex system, that is, segmentation timing in mouse embryos, without a reference to these detailed interactions. To this end, we develop a coarse-grained approach, in which theory guides the experimental identification of the segmentation clock entrainment responses. We demonstrate period- and phase-locking of the segmentation clock across a wide range of entrainment parameters, including higher-order coupling. These quantifications allow to derive the phase response curve (PRC) and Arnold tongues of the segmentation clock, revealing its essential dynamical properties. Our results indicate that the somite segmentation clock has characteristics reminiscent of a highly non-linear oscillator close to an infinite period bifurcation and suggests the presence of long-term feedbacks. Combined, this coarse-grained theoretical-experimental approach reveals how we can derive simple, essential features of a highly complex dynamical system, providing precise experimental control over the pace and rhythm of the somite segmentation clock.

The molecular mechanisms that maintain cellular identities and prevent dedifferentiation or transdifferentiation remain mysterious. However, both processes are transiently used during animal regeneration. Therefore, organisms that regenerate their organs, appendages, or even their whole body offer a fruitful paradigm to investigate the regulation of cell fate stability. Here, we used Hydra as a model system and show that Zic4, whose expression is controlled by Wnt3/β-catenin signaling and the Sp5 transcription factor, plays a key role in tentacle formation and tentacle maintenance. Reducing Zic4 expression suffices to induce transdifferentiation of tentacle epithelial cells into foot epithelial cells. This switch requires the reentry of tentacle battery cells into the cell cycle without cell division and is accompanied by degeneration of nematocytes embedded in these cells. These results indicate that maintenance of cell fate by a Wnt-controlled mechanism is a key process both during homeostasis and during regeneration.

Studies on the impact of metabolism on cell fate decisions are seeing a renaissance. However, a key challenge remains to distinguish signaling functions of metabolism from its canonical bioenergetic and biosynthetic roles, which underlie cellular homeostasis. Here, we tackled this challenge using mouse embryonic axis segmentation as an experimental model. First, we found that energetically subminimal amounts of glucose can support ongoing segmentation clock activity, providing evidence that glycolysis exerts a signaling function. Using a dynamical systems approach based on entrainment, we identified fructose 1,6-bisphosphate (FBP) as the potential signaling metabolite. Functionally, we demonstrated that glycolytic flux/FBP control the segmentation clock period and Wnt signaling in an anticorrelated manner. Critically, we showed that the slow segmentation clock phenotype caused by elevated glycolysis is mediated by Wnt signaling rather than cellular bioenergetic and biosynthetic state. Combined, our results demonstrate a modular organization of metabolic functions, revealing a signaling module of glycolysis that can be decoupled from its canonical metabolic functions.

The question of how metabolism impacts development is seeing a renaissance [1, 2]. How metabolism exerts instructive signaling functions is one of the central issues that need to be resolved. We tackled this question in the context of mouse embryonic axis segmentation. Previous studies have shown that changes in central carbon metabolism impact Wnt signaling [3–6] and the period of the segmentation clock [7], which controls the timing of axis segmentation. Here, we reveal that glycolysis tunes the segmentation clock period in an anti-correlated manner: higher glycolytic flux slows down the clock, and vice versa. Transcriptome and gene regulatory network analyses identified Wnt signaling and specifically the transcription factor Tcf7l2, previously associated with increased risk for diabetes [8, 9], as potential mechanisms underlying flux-dependent control of the clock period. Critically, we show that deletion of the Wnt antagonist Dkk1 rescued the slow segmentation clock phenotype caused by increased glycolysis, demonstrating that glycolysis instructs Wnt signaling to control the clock period. In addition, we demonstrate metabolic entrainment of the segmentation clock: periodic changes in the levels of glucose or glycolytic sentinel metabolite fructose 1,6-bisphosphate (FBP) synchronize signaling oscillations. Notably, periodic FBP pulses first entrained Wnt signaling oscillations and subsequently Notch signaling oscillations. We hence conclude that metabolic entrainment has an immediate, specific effect on Wnt signaling. Combined, our work identifies a glycolysis-FBP-Wnt signaling axis that tunes developmental timing, highlighting the instructive signaling role of metabolism in embryonic development.

Hydra head formation depends on an organizing center in which Wnt/β-catenin signaling, that plays an inductive role, positively regulates Sp5 and Zic4, with Sp5 limiting Wnt3/β-catenin expression and Zic4 triggering tentacle formation. Using transgenic lines in which the HySp5 promoter drives eGFP expression in either the epidermis or gastrodermis, we show that Sp5 promoter activity is differentially regulated in each epithelial layer. In intact animals, epidermal HySp5:GFP activity is strong apically and weak along the body column, while in the gastrodermis, it is maximal in the tentacle ring region and maintained at a high level along the upper body column. During apical regeneration, HySp5:GFP is activated early in the gastrodermis and later in the epidermis. Alsterpaullone treatment induces a shift in apical HySp5:GFP expression towards the body column where it forms transient circular figures in the epidermis. Upon β-catenin(RNAi), HySp5:GFP activity is down-regulated in the epidermis while bud-like structures expressing HySp5:GFP in the gastrodermis develop. Sp5(RNAi) reveals a negative Sp5 autoregulation in the epidermis, but not in the gastrodermis. These differential regulations in the epidermis and gastrodermis highlight the distinct architectures of the Wnt/β-catenin/TCF/Sp5/Zic4 network in the hypostome, tentacle base and body column of intact animals, as well as in the buds and apical and basal regenerating tips.