A fundamental feature of embryonic patterning is the ability to scale and maintain stable proportions despite changes in overall size, for instance during growth. A notable example occurs during vertebrate segment formation: after experimental reduction of embryo size, segments form proportionally smaller, and consequently, a normal number of segments is formed. Despite decades of experimental and theoretical work, the underlying mechanism remains unknown. More recently, ultradian oscillations in gene activity have been linked to the temporal control of segmentation; however, their implication in scaling remains elusive. Here we show that scaling of gene oscillation dynamics underlies segment scaling. To this end, we develop a new experimental model, an ex vivo primary cell culture assay that recapitulates mouse mesoderm patterning and segment scaling, in a quasi-monolayer of presomitic mesoderm cells (hereafter termed monolayer PSM or mPSM). Combined with real-time imaging of gene activity, this enabled us to quantify the gradual shift in the oscillation phase and thus determine the resulting phase gradient across the mPSM. Crucially, we show that this phase gradient scales by maintaining a fixed amplitude across mPSM of different lengths. We identify the slope of this phase gradient as a single predictive parameter for segment size, which functions in a size- and temperature-independent manner, revealing a hitherto unrecognized mechanism for scaling. Notably, in contrast to molecular gradients, a phase gradient describes the distribution of a dynamical cellular state. Thus, our phase-gradient scaling findings reveal a new level of dynamic information-processing, and provide evidence for the concept of phase-gradient encoding during embryonic patterning and scaling.

Polarized Wnt signaling along the primary body axis is a conserved property of axial patterning in bilaterians and prebilaterians, and depends on localized sources of Wnt ligands. However, the mechanisms governing the localized Wnt expression that emerged early in evolution are poorly understood. Here we find in the cnidarian Hydra that two functionally distinct cis-regulatory elements control the head organizer-associated Hydra Wnt3 (HyWnt3). An autoregulatory element, which mediates direct inputs of Wnt/ β-catenin signaling, highly activates HyWnt3 transcription in the head region. In contrast, a repressor element is necessary and sufficient to restrict the activity of the autoregulatory element, thereby allowing the organizer-specific expression. Our results reveal that a combination of autoregulation and repression is crucial for establishing a Wnt-expressing organizing center in a basal metazoan. We suggest that this transcriptional control is an evolutionarily old strategy in the formation of Wnt signaling centers and metazoan axial patterning.regulatory DNAs | cis-regulation | enhancer | molecular evolution | axis formation

Gene expression oscillators can structure biological events temporally and spatially. Different biological functions benefit from distinct oscillator properties. Thus, finite developmental processes rely on oscillators that start and stop at specific times, a poorly understood behavior. Here, we have characterized a massive gene expression oscillator comprising > 3,700 genes in Caenorhabditis elegans larvae. We report that oscillations initiate in embryos, arrest transiently after hatching and in response to perturbation, and cease in adults. Experimental observation of the transitions between oscillatory and non-oscillatory states at high temporal resolution reveals an oscillator operating near a Saddle Node on Invariant Cycle (SNIC) bifurcation. These findings constrain the architecture and mathematical models that can represent this oscillator. They also reveal that oscillator arrests occur reproducibly in a specific phase. Since we find oscillations to be coupled to developmental processes, including molting, this characteristic of SNIC bifurcations endows the oscillator with the potential to halt larval development at defined intervals, and thereby execute a developmental checkpoint function.

SummaryIn vertebrate embryos, somites, the precursor of vertebrae, form from the presomitic mesoderm (PSM), which is composed of cells displaying signaling oscillations. Cellular oscillatory activity leads to periodic wave patterns in the PSM. Here, we address the origin of such complex wave patterns. We employed an in vitro randomization and real-time imaging strategy to probe for the ability of cells to generate order from disorder. We found that, after randomization, PSM cells self-organized into several miniature emergent PSM structures (ePSM). Our results show an ordered macroscopic spatial arrangement of ePSM with evidence of an intrinsic length scale. Furthermore, cells actively synchronize oscillations in a Notch-signaling-dependent manner, re-establishing wave-like patterns of gene activity. We demonstrate that PSM cells self-organize by tuning oscillation dynamics in response to surrounding cells, leading to collective synchronization with an average frequency. These findings reveal emergent properties within an ensemble of coupled genetic oscillators.

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Recently, three ion channel subunits of the degenerin (DEG)/ epithelial Na ؉ channel (ENaC) gene family have been cloned from the freshwater polyp Hydra magnipapillata, the Hydra Na ؉ channels (HyNaCs) 2-4. Two of them, HyNaC2 and HyNaC3, co-assemble to form an ion channel that is gated by the neuropeptides Hydra-RFamides I and II. The HyNaC2/3 channel is so far the only cloned ionotropic receptor from cnidarians and, together with the related ionotropic receptor FMRFamideactivated Na ؉ channel (FaNaC) from snails, the only known peptide-gated ionotropic receptor. The HyNaC2/3 channel has pore properties, like a low Na ؉ selectivity and a low amiloride affinity, that are different from other channels of the DEG/ENaC gene family, suggesting that a component of the native Hydra channel might still be lacking. Here, we report the cloning of a new ion channel subunit from Hydra, HyNaC5. The new subunit is closely related to HyNaC2 and -3 and co-localizes with HyNaC2 and -3 to the base of the tentacles. Coexpression in Xenopus oocytes of HyNaC5 with HyNaC2 and -3 largely increases current amplitude after peptide stimulation and affinity of the channel to Hydra-RFamides I and II. Moreover, the HyNaC2/3/5 channel has altered pore properties and amiloride affinity, more similarly to other DEG/ENaC channels. Collectively, our results suggest that the three homologous subunits HyNaC2, -3, and -5 form a peptide-gated ion channel in Hydra that could contribute to fast synaptic transmission.The DEG/ENaC 2 gene family contains ion channels that have a high selectivity for Na ϩ ions and a rather high affinity for the diuretic amiloride (apparent IC 50 ϳ 0.1-20 M). In contrast to these conserved pore properties, activating stimuli and physiological functions of DEG/ENaC channels are strikingly diverse (1); family members from Drosophila are involved in the control of locomotion (2), the liquid clearance from the trachea (3), and in the detection of pheromones (4) and salt (5); members from Caenorhabditis elegans form mechanosensitive ion channels (6); FaNaC from snails is a peptide-gated ion channel (7); and the epithelial Na ϩ channel (ENaC) and acid-sensing ion channels family members from mammals mediate Na ϩ reabsorption (8) and are proton-gated ion channels (9), respectively. The functions of many other DEG/ENaC channels are still unknown.Recently, three DEG/ENaC subunits have been cloned from the freshwater polyp Hydra (10). These subunits, HyNaC2-4, were the first DEG/ENaC channels from Cnidaria, which is an ancient phylum, where the first nervous systems are supposed to have evolved. A fourth HyNaC gene,

Hydra as a model system for regeneration

Summary Breaking bilateral symmetry is critical for the morphogenesis and placement of vertebrate organ systems. The first overt evidence of L-R asymmetry in the mouse is a rightward looping of the heart tube at early somite stages (ss) (~E8.5). Shortly thereafter, the embryo rotates 180° internalizing the gut [1, 2]. Molecular asymmetries begin earlier, with the expression of the TGFβ family member Nodal in the left lateral plate mesoderm (LPM) [3–5]. Nodal induces left LPM expression of Pitx2, a critical determinant of left sided identity, and Lefty-2, a related TGFβ family member [6–8] which antagonizes Nodal signaling [9]. Nodal signaling is confined to the left LPM through that antagonistic action of Lefty-1 secreted by cells in the left half of the prospective floor plate (PFP) [10]. The asymmetric expression of Nodal is linked to left-orientated, cilia-generated flow within a midline structure, the ventral node [11, 12]. Directional symmetry breaking is superimposed on a poorly understood Hedgehog (Hh) signaling-based mechanism that enables the establishment of L-R asymmetry. Mouse embryos lacking both Sonic hedgehog (Shh) and Indian hedgehog (Ihh), or Smoothened (Smo), an obligate membrane component in the transduction of all Hh signals, fail to activate Nodal in the LPM or Lefty-1 in the PFP, their heart remains as a linear tube and embryos die of circulatory failure shortly after E8.5 [13]. These results, together with reports of directional trafficking of Shh within membranous, nodal vesicular parcels (NVPs), have lead to a model wherein Hh signaling in the node indirectly enables the establishment of molecular asymmetry in LPM of the mammalian embryo [14]. Here, we provide evidence for a direct action of mouse Hh signaling in LPM. Further, we identify a downstream pathway that establishes a competence for Nodal activation in LPM, and as a consequence, the generation of L-R asymmetry.