preparation-and-live-imaging-of-regenerating-hydra-spheroids

Preparation and Live Imaging of Regenerating <em>Hydra</em> Spheroids

Hydra is renowned for its exceptional regenerative capacity. Remarkably, small regenerating fragments of Hydra tissue, called Hydra spheroids, can ...

Friedrich Miescher Institute for Biomedical Research

The freshwater polyp  provides a potent model system for investigating the conditions that promote wound healing, reactivation of a developmental process and, ultimately, regeneration of an amputated body part.  polyps can also be dissociated to the single cell level and can regenerate a complete body axis from aggregates, behaving as natural organoids. In recent years, the ability to exploit  has been expanded with the advent of new live-imaging approaches, genetic manipulations that include stable transgenesis, gene silencing and genome editing, and the accumulation of high-throughput omics data. In this Primer, we provide an overview of  as a model system for studying regeneration, highlighting recent results that question the classical self-enhancement and long-range inhibition model supposed to drive  regeneration. We underscore the need for integrative explanations incorporating biochemical as well as mechanical signalling.

Model systems for regeneration: .

During C. elegans larval development, thousands of genes, accounting for >20% of the transcriptome, exhibit oscillatory expression with large amplitudes. The time of peaking varies for different genes, but expression generally peaks once per larval stage, with both the oscillation period and larval stage duration varying in concert with temperature. This and other evidence support the existence of a gene expression oscillator that functions as a developmental clock. In this article, we review what is known about the biology, architecture and possible mechanisms of this clock. We compare it to other oscillators, and highlight tools and approaches suited to its study. Finally, we point out implications of these wide-spread and dynamic changes of gene expression on any type of gene expression profiling experiment in C. elegans larvae and how such experiments need to be controlled.

Gene expression oscillations in C. elegans underlie a new developmental clock.

Mechanical input shapes cell fate decisions during development and regeneration in many systems, yet the mechanisms of this cross-talk are often unclear. In regenerating  tissue spheroids, periodic osmotically driven inflation and deflation cycles generate mechanical stimuli in the form of tissue stretching. Here, we demonstrate that tissue stretching during inflation is important for the appearance of the head organizer—a group of cells that secrete the Wnt3 ligand. Exploiting time series RNA expression profiles, we identify the up-regulation of Wnt signaling as a key readout of the mechanical input. In this system, the levels of Wnt3 expression correspond to the levels of stretching, and Wnt3 overexpression alone enables successful regeneration in the absence of mechanical stimulation. Our findings enable the incorporation of mechanical signals in the framework of  patterning and highlight the broad significance of mechanochemical feedback loops for patterning epithelial lumens.

Mechanical oscillations orchestrate axial patterning through Wnt activation in .

Cells of the freshwater cnidarian Hydra possess an exceptional regeneration ability. In small groups of these cells, organizer centers emerge spontaneously and instruct the patterning of the surrounding population into a new animal. This property makes them an excellent model system to study the general rules of self-organization. A small tissue fragment or a clump of randomly aggregated cells can form a hollow spheroid that is able to establish a body axis de novo. Interestingly, mechanical oscillations (inflation/deflation cycles of the spheroid) driven by osmosis accompany the successful establishment of axial polarity. Here we describe different approaches for generating Hydra tissue spheroids, along with imaging and image analysis techniques to investigate their mechanical behavior.

Studying Mechanical Oscillations During Whole-Body Regeneration in Hydra.

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  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  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.

The transcription factor Zic4 promotes tentacle formation and prevents epithelial transdifferentiation in .

Oscillator systems achieve synchronization when oscillators are coupled. The presomitic mesoderm is a system of cellular oscillators, where coordinated genetic activity is necessary for proper periodic generation of somites. While Notch signaling is required for the synchronization of these cells, it is unclear what information the cells exchange and how they react to this information to align their oscillatory pace with that of their neighbors. Combining mathematical modeling and experimental data, we found that interaction between murine presomitic mesoderm cells is controlled by a phase-gated and unidirectional coupling mechanism and results in deceleration of their oscillation pace upon Notch signaling. This mechanism predicts that isolated populations of well-mixed cells synchronize, revealing a stereotypical synchronization in the mouse PSM and contradicting expectations from previously applied theoretical approaches. Collectively, our theoretical and experimental findings reveal the underlying coupling mechanisms of the presomitic mesoderm cells and provide a framework to quantitatively characterize their synchronization.

Unidirectional and phase-gated signaling synchronizes murine presomitic mesoderm cells.

The spontaneous emergence of tissue patterns is often attributed to biochemical reaction-diffusion systems. In  tissue regeneration, the formation of a Wnt signaling center exemplifies this process. However, a strictly biochemical mechanism for self-organization in  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  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.

Mechanochemical patterning localizes the organizer of a luminal epithelium.

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.

Article	Year
Model systems for regeneration: . Development (Cambridge, England)\| PubMed ID: 31676551	2019
Gene expression oscillations in C. elegans underlie a new developmental clock. Current topics in developmental biology\| PubMed ID: 33992153	2021
Mechanical oscillations orchestrate axial patterning through Wnt activation in . Science advances\| PubMed ID: 34890235	2021
Studying Mechanical Oscillations During Whole-Body Regeneration in Hydra. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 35359332	2022
The transcription factor Zic4 promotes tentacle formation and prevents epithelial transdifferentiation in . Science advances\| PubMed ID: 36563144	2022
Unidirectional and phase-gated signaling synchronizes murine presomitic mesoderm cells. Developmental cell\| PubMed ID: 37098349	2023
Mechanochemical patterning localizes the organizer of a luminal epithelium. Science advances\| PubMed ID: 40561030	2025
A transcription factor toggle switch determines differentiated epidermal cell identities in Hydra. Development (Cambridge, England)\| PubMed ID: 40824680	2025

Charisios D. Tsiairis

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