Charisios D. Tsiairis

The Wnt signaling pathway is a conserved regulator of tissue patterning and regeneration, yet how cells interpret dynamic Wnt inputs to generate robust developmental outcomes remains poorly understood. Here, we establish the first optogenetically activatable Hydra line, enabling precise temporal control of canonical Wnt signaling in vivo . Optogenetic stimulation induced dose-dependent patterning phenotypes whose rate of progression scaled with stimulation intensity. Transcriptomic analysis revealed that distinct combinations of signal intensity and duration converged onto shared transcriptional trajectories and could be described by an effective exposure metric that represents the cumulative signaling input. Functionally, Wnt activation rescued head regeneration under conditions that normally prevent organizer formation, and equivalent regenerative outcomes could be achieved through either strong, short-lived stimulation or weaker, prolonged activation. Together, our results indicate that Hydra tissues decode Wnt signaling through temporal integration of cumulative pathway activity, progressively accumulating transcriptional responses until patterning thresholds are reached. These findings establish a quantitative framework for understanding how dynamic morphogen signaling is translated into stable developmental decisions during regeneration.

Regeneration can proceed through epimorphosis, involving regrowth of lost structures at the amputation site in a proximal-to-distal sequence, or through morphallaxis, which is characterized by extensive tissue remodeling with minimal cell proliferation. While the mechanisms underlying epimorphic regeneration are well studied, the spatiotemporal dynamics of morphallaxis remain less understood. Here we show, using time-course single-cell RNA sequencing (scRNA-seq) of regenerating Hydra tissue, that morphallaxis involves a temporal sequence in the re-emergence of terminal and subterminal cell identities, with transcriptional signatures associated with proximal structures appearing earlier than those of distal structures. We further find that Wnt signaling remains dynamically active at the amputation site throughout regeneration, with its composition changing over time, consistent with a transient regulatory state. Together, these findings provide a high-resolution view of dynamic cell state transitions and signaling activity during morphallactic regeneration and offer a framework for exploring how organizer function and axial patterning are re-established in Hydra.

The presomitic mesoderm is a transient embryonic tissue patterned along the anterior-posterior axis of vertebrates that periodically generates somites at its anterior end. Somitogenesis is driven by waves of gene expression that propagate from the posterior to the anterior end of the presomitic mesoderm under the control of the segmentation clock, a molecular oscillator. Using a mouse primary culture system, we performed a small-scale chemical screen targeting pathways implicated in cellular mechanics and we identified Rac1-dependent signaling as a modulator of segmentation clock dynamics. Pharmacological perturbation of Rac1 signaling reproducibly slowed oscillatory dynamics without disrupting intercellular synchrony. These changes were reversible and associated with increased somite length and reduced somite number. In addition, inhibition of Rac1 signaling was associated with changes in the expression of selected Wnt and Notch target genes in tail explants. These findings identify Rac1-dependent signaling as a candidate modulator of segmentation clock dynamics and somite morphogenesis, linking mechanical and molecular aspects of segmentation dynamics.

Hydra is renowned for its exceptional regenerative capacity. Remarkably, small regenerating fragments of Hydra tissue, called Hydra spheroids, can establish an organizer de novo. Once established, this organizer directs the surrounding tissue to differentiate into the appropriate oral structures. Within days, the spheroid can develop into a small yet fully patterned polyp. The relatively short timescale of this process enables continuous time-lapse imaging of the entire process, offering a unique opportunity to study the spatiotemporal dynamics of regeneration. Here, we present a detailed workflow and show representative results for generating time-lapse datasets of regenerating Hydra spheroids. The protocol includes a detailed procedure for spheroid generation and guidelines for selecting optimal samples for imaging. We discuss mounting strategies suitable for the long-term imaging of live samples and demonstrate imaging approaches using both spinning disk confocal and light-sheet microscopes. The principles described here are broadly applicable to other model systems in which dynamic morphogenetic processes can be studied using long-term imaging.

Hydra is renowned for its exceptional regenerative capacity. Remarkably, small regenerating fragments of Hydra tissue, called Hydra spheroids, can establish an organizer de novo. Once established, this organizer directs the surrounding tissue to differentiate into the appropriate oral structures. Within days, the spheroid can develop into a small yet fully patterned polyp. The relatively short timescale of this process enables continuous time-lapse imaging of the entire process, offering a unique opportunity to study the spatiotemporal dynamics of regeneration. Here, we present a detailed workflow and show representative results for generating time-lapse datasets of regenerating Hydra spheroids. The protocol includes a detailed procedure for spheroid generation and guidelines for selecting optimal samples for imaging. We discuss mounting strategies suitable for the long-term imaging of live samples and demonstrate imaging approaches using both spinning disk confocal and light-sheet microscopes. The principles described here are broadly applicable to other model systems in which dynamic morphogenetic processes can be studied using long-term imaging.

The presomitic mesoderm (PSM) is a transient embryonic tissue patterned along the anterior-posterior (A/P) axis of vertebrates that periodically generates somites at its anterior end. This process, termed somitogenesis, is driven by waves of gene expression that travel from the posterior to the anterior end of the PSM under the contol of the segmentation clock, a molecular oscillator. Using a primary culture system, we performed a small-scale chemical screen targeting cellular mechanical properties and we identified Rac1, a small GTPase, as a key regulator. Inhibition of Rac1 markedly altered the periodicity of gene expression waves and disrupted somite spatial organization. Further analyses showed that Rac1 inhibition modified gene expression patterns, notably affecting markers of the Wnt and Notch pathways which are central regulators of segmentation. These findings reveal a previously unrecognized role for Rac1-mediated signaling in coordinating the mechanical and molecular aspects of segmentation dynamics.

In Hydra, a simple cnidarian model, epithelio-muscular cells shape and maintain body architecture through continuous renewal. Undifferentiated cells from the mid-body region migrate passively toward the extremities, replacing shed cells and acquiring region-specific identities. This ongoing turnover, together with the Hydra stable axial organization, provides a powerful model to study how cell type specification is integrated with body patterning. Yet, the molecular mechanisms that govern epithelial identity remain largely unknown. Here, we identify a double-negative feedback loop between the transcription factors Zic4 and Gata3 that functions as a toggle switch to control terminal epidermal cell fates. Zic4, activated by Wnt signaling from the head organizer, induces battery cell specification in tentacles. In contrast, Gata3 promotes basal disk cell identity at the aboral end. These factors are mutually repressive: silencing one leads to expansion of the other's domain and ectopic cell fate induction. Notably, simultaneous knockdown of both restores normal patterning, suggesting that identity is dictated by their relative balance rather than absolute levels. Our study reveals how opposing transcriptional signals coordinate epithelial identity with axial patterning at Hydra poles.

The spontaneous emergence of tissue patterns is often attributed to biochemical reaction-diffusion systems. In Hydra tissue regeneration, the formation of a Wnt signaling center exemplifies this process. However, a strictly biochemical mechanism for self-organization in Hydra remains elusive. In this study, we investigated mechanical stimuli and identified a positive feedback loop between Wnt signaling and tissue stretching. We developed a mathematical model of mechanochemical pattern formation in a closed elastic shell, representing regenerating Hydra epithelial spheroids. Our model explains how mechanical forces drive axis formation and predict the organizer’s location under various perturbations. Validation by partially confining regenerating tissues showed that the organizer forms in regions with the greatest stretching potential. This work highlights a versatile mechanochemical mechanism for luminal epithelium patterning, which is relevant across various biological systems.

In Hydra , a simple cnidarian model, epithelio-muscular cells play a crucial role in shaping and maintaining the body architecture. These cells are continuously renewed as undifferentiated cells from the body’s mid-region get displaced toward the extremities, replacing shed, differentiated cells and adopting specific identities. This ongoing differentiation, coupled with the maintenance of distinct anatomical regions, provides an ideal system to explore the relationship between cell type specification and axial patterning. However, the molecular mechanisms governing epithelial cell identity in Hydra remain largely unknown. In this study, we describe a double-negative feedback loop between the transcription factors Zic4 and Gata3 that functions as a toggle switch to control epidermal cell fate. Zic4 is activated by Wnt signaling from the mouth organizer and triggers battery cell specification in tentacles. In contrast, Gata3 promotes basal disk cell identity at the aboral end. Functional analyses demonstrate that Zic4 and Gata3 are mutually antagonistic; suppression of one leads to the dominance of the other, and vice versa , resulting in ectopic cell specification. Notably, simultaneous knockdown of both factors rescues the phenotype, indicating that it is the balance between these transcription factors, rather than their absolute levels, that dictates cell identity. This study highlights the mechanisms by which distinct cellular identities are established at Hydra body termini and reveals how cell fate decisions are coordinated with axial patterning.

The spontaneous emergence of tissue patterns is often attributed to biochemical reaction-diffusion systems. In Hydra tissue regeneration, the formation of a Wnt signaling center serves as a well-known example of such a process. However, despite extensive research, a strictly biochemical mechanism for self-organization in Hydra remains elusive. In this study, we investigated mechanical stimuli and identified a positive feedback loop between Wnt signaling and tissue stretching. We developed a mathematical model of mechanochemical pattern formation in a closed elastic shell, representing regenerating Hydra epithelial spheroids. Our model explains how mechanical forces drive axis formation and predicts the organizer’s location under various perturbations, providing a more comprehensive understanding of the spatial dynamics involved. Validation by partially confining regenerating tissues showed that the organizer indeed forms in regions with the greatest stretching potential. This work highlights a novel mechanochemical mechanism for luminal epithelium patterning, suggesting that mechanical forces, in addition to biochemical signals, play a crucial role in tissue regeneration and axis specification. Our findings offer broader implications for the role of mechanical forces in tissue organization in various biological systems, opening new avenues for investigating mechanochemical feedback in development and regeneration.