Cellular, Metabolic, and Developmental Dimensions of Whole-Body Regeneration in <i>Hydra</i>

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Article

Reference

Cellular, Metabolic, and Developmental Dimensions of Whole-Body Regeneration in Hydra

VOGG, Matthias, et al.

Abstract

Here we discuss the developmental and homeostatic conditions necessary for Hydra regeneration. Hydra is characterized by populations of adult stem cells paused in the G2 phase of the cell cycle, ready to respond to injury signals. The body column can be compared to a blastema-like structure, populated with multifunctional epithelial stem cells that show low sensitivity to proapoptotic signals, and high inducibility of autophagy that promotes resistance to stress and starvation. Intact Hydra polyps also exhibit a dynamic patterning along the oral-aboral axis under the control of homeostatic organizers whose activity results from regulatory loops between activators and inhibitors. As in bilaterians, injury triggers the immediate production of reactive oxygen species (ROS) signals that promote wound healing and contribute to the reactivation of developmental programs via cell death and the de novo formation of new organizing centers from somatic tissues. In aging Hydra, regeneration is rapidly lost as homeostatic conditions are no longer pro-regenerative.

VOGG, Matthias, et al . Cellular, Metabolic, and Developmental Dimensions of Whole-Body Regeneration in Hydra . Cold Spring Harbor Perspectives in Biology , 2021, no. a040725, p.

1-18

DOI : 10.1101/cshperspect.a040725 PMID : 34230037

Available at:

http://archive-ouverte.unige.ch/unige:153435

Disclaimer: layout of this document may differ from the published version.

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Cellular, Metabolic, and Developmental Dimensions of Whole-Body Regeneration in Hydra

Matthias Christian Vogg,^1,2Wanda Buzgariu,^1,2Nenad Slavko Suknovic,¹and Brigitte Galliot¹

1Department of Genetics and Evolution, Institute of Genetics and Genomics in Geneva (iGE3), Faculty of Sciences, University of Geneva, Geneva 4, Switzerland

Correspondence:[email protected]

Here we discuss the developmental and homeostatic conditions necessary forHydraregen- eration.Hydrais characterized by populations of adult stem cells paused in the G2 phase of the cell cycle, ready to respond to injury signals. The body column can be compared to a blastema-like structure, populated with multifunctional epithelial stem cells that show low sensitivity to proapoptotic signals, and high inducibility of autophagy that promotes resistance to stress and starvation. IntactHydrapolyps also exhibit a dynamic patterning along the oral–aboral axis under the control of homeostatic organizers whose activity results from regulatory loops between activators and inhibitors. As in bilaterians, injury triggers the immediate production of reactive oxygen species (ROS) signals that promote wound healing and contribute to the reactivation of developmental programs via cell death and the de novo formation of new organizing centers from somatic tissues. In agingHydra, regeneration is rapidly lost as homeostatic conditions are no longer pro-regenerative.

R

egeneration is commonly deﬁned as the reconstruction of missing 3D structures in adult organisms by the reactivation of developmental programs that are tightly controlled in space and time. In most bilaterian organisms with regenerative potential, the regenerative strategy relies on blastema formation, either from differentiated cells in the vicinity of the wound that dedifferentiate and reenter the cell cycle or alternatively transdifferentiate, or from local or distant stem cells that are activated upon injury and reenter the cell cycle (Tanaka and Reddien 2011). Blastema formation is an essential step for the regenerative program, triggering the subsequent patterning and morphogenetic

events that allow the perfect reconstruction of the amputated part. In case of the freshwater cnidarianHydra, large populations of stem cells are available along the body column of the animal, either actively proliferating or paused in the G2 phase of the cell cycle, playing an important role in the regenerative process albeit without forming a typical blastema where cells reenter the cell cycle (Buzgariu et al. 2018).

As a result of this cellular organization (i.e., the central region of the animal can be considered as a blastema-like structure), regeneration relies on the remodeling of the preexisting tissue at the wound site (Park et al. 1970; Hicklin and Wolpert 1973a).

Hydrahas a long history as a model organism in

2Co-first authors.

Editors: Kenneth D. Poss and Donald T. Fox

Advanced Online Article. Cite this article asCold Spring Harb Perspect Bioldoi: 10.1101/cshperspect.a040725

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Theory of pattern formation (Turing 1952; Wolpert 1969; Gierer and Meinhardt 1972) Low senescence aging (Brien 1953; Martínez 1998; Yoshida et al. 2006)

Reaggregation (Noda et al. 1971; Gierer and Meinhardt 1972) Nerve-free epithelial Hydra (Diehl and Burnett 1964, 1965; Campbell 1976; Sugiyama and Fujisawa 1978a) DNA labeling (Burnett et al. 1962; Campbell 1965; Plickert and Kroiher 1988) Peptide project (Takahashi et al. 1997, 2005; Fujisawa 2008) EST-RNA sequencing (Hwang et al. 2007; Hemmrich et al. 2012; Wenger and Galliot 2013; Juliano et al. 2014; Petersen et al. 2015; Wenger et al. 2016, 2019; Tomczyk et al. 2020) Proteomics (Balasubramaniam et al. 2012; Petersen et al. 2015; Lommel et al. 2018; Tomczyk et al. 2020)

Maceration of Hydra tissue (David 1973) Transient expression of reporter constructs (Bosch et al. 2002; Böttger et al. 2002; Miljkovic et al. 2002)

Genome sequencing (Chapman et al. 2010; Hamada et al. 2020) Single-cell RNA sequencing (Siebert et al. 2019)

Transplantation of organizer (Browne 1909; Rand et al. 1926; Yao 1945; Wilby and Webster 1970; MacWilliams 1983a,b; Takano and Sugiyama 1983, 1984; Shimizu 2012 Stable transgenesis (Wittlieb et al. 2006; Khalturin et al. 2007) Gene KD by RNA hairpin (Boehm et al. 2012; Franzenburg et al. 2012; Juliano et al. 2014; Klimovich et al. 2019) CRISPR/ Cas9 editing (Lommel et al. 2018)

Clonal assay for stem cells (David and Murphy 1977; David and Plotnick 1980) Gene KD by RNAi (Lohmann and Bosch 2000; Chera et al. 2006; Watanabe et al. 2014; Vogg et al. 2019b

1909 1926 1945 1952 1953 1962 1964 1965 1969 1970 1971 1972 1973 1976 1977 1978 1980 1983 1988 1993 1997 1998 1999 2000 2002 2005 2006 2007 2008 2010 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021

Gain-of-function—OE construct (Khalturin et al. 2007; Juliano et al. 2014)

Autophagy sensor (Tomczyk et al. 2020; Suknovic et al. 2021) Figure1.Timelineshowingtheemergenceofconceptsandtoolsdesignedtostudyandunderstandthemetabolic,cellular, anddevelopmentaldimensionsofHydraregeneration.(EP)Electroporation,(KD)knockdown,(OE)overexpression. (FigurebasedondatainGalliot2012.)

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theﬁeld of regeneration (Fig. 1). Here, we propose to revisit the classical knowledge aboutHydrare- generation in the light of recent observations whereHydrahomeostasis is altered and regeneration rapidly lost (Tomczyk et al. 2020; Suknovic et al. 2021) independently of alterations in the developmental mechanisms, aﬁnding that allows us to deduce the homeostatic conditions that make a robust pro-regenerative status necessary. In the second part, we will discuss the mechanisms that lead to the transformation of somatic tissues into regenerating tissues, characterized by the de novo formation of new organizer centers in an adult

organism. The third part will focus on the metabolic and cellular changes induced by amputation.

THE CELLULAR AND METABOLIC PROPERTIES THAT SUPPORT THE PRO-REGENERATIVE STATUS IN HOMEOSTATICHYDRA

A Sustained Stemness Characterizes the Central Body Column ofHydra

Hydra homeostasis is maintained by the dynamic self-renewal of its three populations of adult stem cells (ASCs) located in the central

Hypostome

Hypostome G2

Nematocytes Tentacle

Mesoglea

Hypostome G2

Gastrodermis Epidermis

Nematoblasts

Gland cells gESC apoptosis

resistant

Progenitor G2

G2

G1

G1 M

M M

Neurons

Female germ cells Male germ cells

Foot-specific G2 Neurons

Myc2, Hywi FoxO, LC3A ULK1, WIPI2 Myc2, Hywi

FoxO, LC3A ULK1, WIPI2

Myc1, FoxO, Hywi, Hyli

ISC and derivatives Apoptosis sensitive

ISC

S S

G2 G1S G2

Basal disc Budding

zone

Mucous cells G2

G1 M

S Battery cells

eESC apoptosis

resistant Tentacles

HeadBody column Gastrodem Mesoglea Epidermis

FootPeduncle

Figure 2.Anatomy and cell-type complexity of theHydramodel system. TheHydrapolyp presents an apical-to- basal polarity with three distinct and easily recognizable regions: the apex or head region, the body column, and the basal disc or foot. The head region consists of a dome-shaped structure, the hypostome, terminated by a unique opening (mouth/anus) at the tip, and a ring of tentacles at its base. The body column has a cylindrical shape terminated by the peduncle and basal disc, whose epidermal epithelial cells secrete a mucus that allows the animals to adhere to substrates.Hydratissues are organized in two myoepithelial layers, epidermis outside and gastrodermis inside, separated by an extracellular layer named mesoglea (black). The green background panels show the differentHydracell types and their distribution in the body layers. The interstitial stem cells (ISCs) give rise to nematoblasts and nematocytes (stinging cells, green), nerve cells (blue) and germ cells in the epidermis, but also to progenitors that migrate to the gastrodermis where they differentiate into gland cells ( purple), involved in food digestion, and neurons. The body column is made of epidermal and gastrodermal epithelial stem cells (eESCs, gESCs) that stop dividing when they reach the extremities. eESCs paused in G2 differentiate in tentacle- speciﬁc cells named battery cells or foot-speciﬁc cells named mucous cells in the basal disc (Dübel et al. 1987).

Some stemness regulators and autophagy markers expressed by eESCs, gESCs, and ISCs are indicated as well as the typical cycling behavior of these cells.

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part of the polyp: interstitial (ISCs), myoepithelial epidermal (eESCs), and myoepithelial gastrodermal (gESCs) stem cells (David and Plot- nick 1980; Bosch 2009; Hobmayer et al. 2012).

These three populations are not interchange- able; they distribute differently along the body column with eESCs forming the epidermis, gESC or digestive cells along the gastrodermis, and ISCs intermingled between the eESCs in the epidermal layer (Fig. 2). All epithelial cells in the body column are stem cells that actively prolif- erate, getting passively displaced toward the extremities where they stop dividing and termi- nally differentiate into head- and foot-speciﬁc epithelial cells, until they get sloughed off (Campbell 1967; Dübel and Schaller 1990; Buz- gariu et al. 2014). As typical examples, eESCs differentiate into battery cells in the tentacles, mucus gland cells in the basal disc, while gESCs are tightly packed in the hypostome where they form with the gland cells a star-shaped structure corresponding to taeniolae (Campbell and Bode 1983). In contrast, ISCs are multipotent stem cells that give rise to progenitors that differentiate along the body column into both somatic cells (i.e., gland cells, nematocytes [stinging cells], neurons) and germ cells when animals enter gametogenesis (Fig. 2). This combination of self-renewal, displacement/migration, and terminal differentiation maintainsHydraanat- omy in a unique dynamic pattern.

The recent single-cell RNA sequencing of differentiating epithelial cells and interstitial progenitors showed that the genetic modules for fate speciﬁcation vary along the body column, providing a differentiation route in ac- cordance with the position that results from cell displacement along the axis (Siebert et al.

2019). This study also suggests that the nerve cells located in the gastrodermis share a com- mon bipotent progenitor with gland cells that migrate from the epidermis into the inner layer before differentiating (see in Fig. 2). The transcriptional signature of stem cells was approached through bulk RNA sequencing (Fig. 1; Hemmrich et al. 2012; Juliano et al.

2014; Wenger et al. 2016, 2019). The transcription factor FoxO and two proto-oncogenes from the myc family, expressed in the intersti-

tial lineage for Hy-myc1and in ESCs forHy- myc2, contribute to stemness by maintaining the balance between proliferation and differentiation (Fig. 2; Hartl et al. 2010, 2014; Ambro- sone et al. 2012; Boehm et al. 2012). Also the Wnt/β-catenin signaling pathway appears to repress Hy-myc1 but not Hy-myc2, and conse- quently enhance the number of self-renewing ISCs (Hartl et al. 2019), in agreement with the Hy-myc1knockdown phenotype where proliferation of interstitial cells is promoted (Ambro- sone et al. 2012).

Other important regulators of ISCs and ESCs are the components of the PIWI-piRNA pathway, known to repress transposable elements (TEs) in both the germline and the somatic cells (Ozata et al. 2019). Two PIWI proteins named Hywi and Hyli repress TE expression in the germline and in multipotent ISCs (Fig. 2; Krish- na et al. 2013; Juliano et al. 2014; Lim et al. 2014).

In epithelial cells,Hywi directly interacts with TE RNA, and knocking downHywiincreases the TE transcript level, which drives the breakdown of epithelial cells and the subsequent disintegra- tion of the entire organism (Juliano et al. 2014;

Teefy et al. 2020). Hence, the ancestral role of the PIWI-piRNA pathway in TE repression also occurs inHydrasomatic cells,where it contributes to the maintenance of large stocks of stem cells.

G2 Pausing of Stem Cells and the Homeostatic Pro-Blastema

The epithelial and interstitial stem cell populations exhibit different self-renewal rates, 3–4 d for the ESCs, 24–30 h for the ISCs (David and Campbell 1972; Campbell and David 1974; Hol- stein and David 1990; Buzgariu et al. 2014). Both ESCs and ISCs transit brieﬂy through G1 after division, replicate their genomic DNA in about 12 h, and then pause in G2 before traversing a new mitosis. ESCs pause several days in G2, an interval that can be extended to weeks for a mi- nor fraction of quiescent ESCs or during a pro- longed starvation period (Fig. 1). In contrast, most ISCs spend 12–15 h in G2, representing about 50% of their cell-cycle length, while a

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small population of ISCs appears to remain quiescent in G2 for weeks (Govindasamy et al.

2014). As a result, the central part of the animal is deﬁned by a sustained stemness, with large populations of ASCs paused in G2. The body column can thus be compared to a blastema- like structure, populated with ASCs ready to respond to injury signals.

Three additional mechanisms linked to G2 pausing of ASCs are beneﬁcial for regenerative competence (Buzgariu et al. 2014; Sutcu and Ricchetti 2018): First, the DNA repair mechanisms that are active during the G2 phase and can ﬁx potential DNA damages (Branzei and Foiani 2008); second, the fact that G2-paused eESCs can directly undergo terminal differentiation, since it was shown that mitosis is dispen- sable (Dübel and Schaller 1990); and third, the robust resistance to cell death when cells are paused in G2 (Chera et al. 2009). This last prop- erty was also observed in human epithelial cells with stem-cell-like properties (Harper et al.

2010) and in zebraﬁsh, G2 arrest induced by the Meox homeoprotein contributes to maintain stemness in muscle stem cells (Nguyen et al.

2017). Thus, the central body column in homeostatic conditions provides a favorable framework for regeneration, since the signals induced by amputation and perceived by the stem cells paused in G2 can immediately induce either mitotic division possibly followed by a new cell cycle or terminal cell differentiation. Compared to animals where cells need to undergo dediffer- entiation or to reactivate a pool of stem cells, the presence of a homeostatic blastema-like anatomy in intactHydraexplains its ability to quickly enter the regeneration process.

Differential Sensitivity to Cell Death between ESCs and ISCs

In normal physiological conditions, cell death is important at the extremities of the animals, where it contributes to maintain homeostasis by eliminating the mature differentiated cells of the tentacles or basal disc that have a limited life span and are substituted by younger cells displaced from the body column (Campbell 1967). When this balance is perturbed by exog-

enous factors such as food limitation, injury, drugs, or heat shock, the cell death pattern is extended, occurring along the entire body (Rei- ter et al. 2012). In the body column, cells react differently to cell death stimulus and the dying cells belong predominantly to the interstitial cell lineage (nematoblasts, nematocytes, gland, and nerve cells), while ESCs appear highly resistant (Chera et al. 2009). Although several studies revealed the conservation of the apoptotic ma- chinery inHydra(Lasi et al. 2010), the molecular players that render ESCs resistant to proapoptotic signals remain largely unknown (Fig. 2).

TheHy-Bcl2-l4 protein protects eESCs against drug-induced apoptosis when constitutively overexpressed, conﬁrming the antiapoptotic function identiﬁed in bilaterians (Motamedi et al. 2019). Therefore, understanding the mechanisms that regulate cell death resistance inHy- dra could shed light on the mechanisms involved in chemotherapy resistance.

Epithelial Cells Carry the Developmental Properties inHydra

The developmental properties of epithelial cells wereﬁrst evidenced in animals that are still able to bud and regenerate after the elimination of ISCs (Fig. 1). Indeed, the depletion of ISCs by chemical treatment with nitrogen mustard (Diehl and Burnett 1965), colchicine (Marcum and Campbell 1978a), hydroxyurea (Sacks and Davis 1979), or by heat shock in the tempera- ture-sensitivesf-1mutant strain (Sugiyama and Fujisawa 1978a) does not impair patterning, tissue renewal, budding, or regeneration (Wenger et al. 2016; Buzgariu et al. 2018). Such“epithelial”

animals, lacking ISCs and the nervous system, preserve their morphogenetic properties; even if the reconstructed head is not fully functional as in the absence of neurons and nematocytes, the animals need to be force-fed to survive. Another approach relied onHydra’s ability to regenerate whole animals from reaggregated cells obtained after tissue dissociation (Gierer and Meinhardt 1972; Vogg et al. 2019a). By mixing epithelial and interstitial cells isolated from distinct strains that differ by their morphogenetic properties (i.e., budding rates or size), it was possible to produce

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chimeric animals whose morphogenetic properties correspond to that of ESCs but not ISCs (Marcum and Campbell 1978b; Sugiyama and Fujisawa 1978b; Takano and Sugiyama 1984).

In response to ISC elimination, ESCs exhibit a remarkable cellular plasticity as they rapidly modify their transcriptional program by up-regulating genes involved in neurotransmission or neurogenesis, usually expressed in neurons, gland cells, or ISCs (Wenger et al. 2016). Epi- thelial plasticity is also observed during regeneration, when shortly after amputation the epithelial cells in the vicinity of the wound change their shape, transiently lose their polarity, and convert into blastema-like cells with cytopro- tective and phagocytic functions (Chera et al.

2006, 2009). An intriguing question is whether the level of plasticity of ESCs allows transdifferentiation. So far, direct examples of epithelial transdifferentiation into any other cell type were not observed. In contrast, cases of phenotypic conversion were noticed in interstitial-derived cell types (e.g., the ganglion cells from the body column can be converted into head-speciﬁc sen- sory neurons in nerve-free regenerating animals (Koizumi et al. 1988), or the zymogen gland cells from the body column can transform into typical head mucous gland cells (Siebert et al. 2008).

Autophagy, a Key Process to Maintain Regeneration and Longevity inHydra The pioneering work of Paul Brien demonstrated the unlimited life span of severalHydraspe- cies and their capacity to maintain intact both the sexual and asexual reproduction over several years (Fig. 2, low senescence aging; Brien 1953;

Tomczyk et al. 2015). Further systematic studies on long-term survival, budding behavior, and fertility conﬁrmed the lack of senescence inHy- dra vulgaris and questioned the mechanisms that sustain this immortality (Martínez 1998;

Schaible et al. 2015; Schenkelaars et al. 2018).

The control of longevity was initially attributed to the unlimited capacity of ASCs to self-renew with FoxO responsible for the stem cell pool maintenance (Fig. 2; Boehm et al. 2012). How- ever, the work of Paul Brien (1953), further conﬁrmed by Yoshida et al. (2006) identiﬁed a

Hydra oligactisstrain, nowadays termed“cold- sensitive”(Ho_CS), which once exposed to 10°C undergoes gametogenesis within 2 wk, and shows morphological, physiological, and cellular changes assimilated to an aging phenotype as evidenced by the loss of regeneration, loss of contractility, loss of feeding behavior, and death of all animals within 3 mo (Tomczyk et al. 2015).

Comparative analyses performed on“cold-sensitive”and“cold-resistant”animals transiently expressing a tandem autophagy sensor anchored in autophagosomes through LC3A, an essential component of autophagic vacuoles, showed that autophagy is deﬁcient in epithelial cells of Ho_CS animals. This deﬁciency accounts for the loss of epithelial stem cell renewal, leading to the aging phenotype (Tomczyk et al. 2020).

This hypothesis could be tested in non-senescent H. vulgarisanimals where knocking down either WIPI2, a key component for autophagosome formation, orULK1, a kinase necessary for en- tering the autophagy process, blocks autophagy and induces aging (Tomczyk et al. 2020;

Suknovic et al. 2021). The transcriptomic analysis of these twoH. oligactisstrains also revealed a down-regulation of genes involved in stem cell maintenance in the Ho_CS strain undergoing aging (Sun et al. 2020; Tomczyk et al. 2020), supporting the link between autophagy deﬁ- ciency, and maintenance of epithelial self-renewal.

The Extracellular Matrix: A Critical Dynamic Scaffold Guiding Regeneration?

The ability to regenerate requires the presence of a functional mesoglea, an extracellular matrix (ECM) layer located all along the body between the gastrodermis and epidermis, composed of different types of ﬁbrillar collagens, laminins, proteoglycans, ﬁbronectin-like molecules, and metalloproteinases (Sarras 2012). The mesoglea is a very dynamic structure that forms a scaffold essential for ESC attachment (Sarras 2012; Berg- heim and Özbek 2019). As shown by assays performed onHydrareaggregates, the mesoglea not only acts as a physical scaffold requested for cell attachment and intercellular connection but also contributes to cell migration (Zhang and Sarras

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Homeostatic organizer A

Regenerative organizer

Derepressed organizer activity upon Sp5 silencing

Wnt Wnt3 Maintenance of an organizer

Regeneration of an organizer Foot organizer FA

Fl

Hl HA Head organizer

Sp5 Hl genes

BMP ECM Fl

Sp5 HAS-7

TSP

Fl: NBL1?

FA: BMP5-8c?

FA

Injury Cell death Wnt3

Mechanics Wnt3

?

? Regeneration

Graft Graft

Graft

Graft Induced

axis Induced

axis

Bisection

Extra head Intact

Wnt3 Reaggregation

B

C (II)

(III) (I)

(II) (I)

Figure 3.Organizers coordinate patterning in homeostatic and regeneratingHydra. (A) (I–II) Schematic representation of organizer activity in intact and regenerating animals as initially demonstrated by Ethel Browne (1909).

To trace the fate of cells, tissue from unpigmentedHydrawas transplanted into pigmented (green) animals. A secondary body axis is induced when a piece of head tissue from an intact animal (or a head-regenerating tip) is transplanted laterally into the body column of the host. Note that the induced axis is almost entirely host-derived (green arrow). (III) Silencing ofSp5de-represses organizer activity and triggers in intact and regenerating animals as well as in reaggregates the formation of ectopic axes, each axis harboring several head organizers as exempliﬁed by the emergence of ectopicWnt3-expressing clusters (blue dots indicated with red arrows). (B) The head and foot organizers produce activator ([HA] head activator, [FA] foot activator) and inhibitor ([HI] head inhibitor, [FI]

foot inhibitor) substances, whose activities are graded along the body axis. (C) (I) Organizer activity is maintained in intact animals at the apical and basal extremities by the interplay between the activator and inhibitor substances.

(II) De novo regeneration of a head organizer relies on an immediate wave of injury-induced cell death that causes the up-regulation ofWnt3expression at the regenerating tip (blue dot).Wnt3requires mechanical stimulation for a sustained expression. A cross talk between Wnt and BMP signaling pathways is likely involved in the regeneration of the foot organizer ( purple dot). The role of the extracellular matrix (ECM) in the maintenance or the activation of the organizers is largely unknown. All expression patterns are available at Hydratlas.unige.ch (Wenger et al.

2019).

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1994), cell differentiation (Yan et al. 2000a,b), and regeneration (Deutzmann et al. 2000). In vivo labeling experiments revealed that the mesoglea gets shifted toward the extremities concomitantly with epithelial cells, except in the apical part where the mesoglea remains static (Aufschnaiter et al. 2011). The mesoglea is involved in morphogenetic processes leading to head formation as pharmacological agents or blocking antibodies to ECM components inhibit regeneration (Sarras 2012; Bergheim and Özbek 2019). A recent proteomic screen performed on isolated mesoglea identiﬁed thrombospondin as a candidate repressor of Wnt signaling pathways (Lommel et al. 2018). Hence, ECM has multi- faceted roles and contributes to morphogenetic processes.

INJURY-INDUCED REGENERATION OF TWO ORGANIZER CENTERS IN BISECTED

ANIMALS

Active Patterning in Intact Animals Relies on Two Polar Homeostatic Organizers

In intact Hydra, the spatial distribution of the different cell types is actively maintained in a complex 3D pattern (e.g., for the head region, a dome structure with the mouth opening at the tip and the ring of tentacles at its basis) (Fig. 2). The process for how such spatial distributions of cells or“patterns” are generated is called patterning (Kondo and Miura 2010; Green and Sharpe 2015), a process often mediated by organizers.

Organizers are localized groups of cells that during developmental processes release inducing signals and recruit the surrounding cells to organize them into specific structures such as body parts, appendages, and organs (de Robertis 2009; Vogg et al. 2016). The concept of induction and recruit- ment was first discovered by Ethel Browne through transplantation experiments between pigmented and depigmented Hydra (Fig. 3A), and later identified in vertebrates by Spemann and Mangold (1924) who coined the name“organizer”(Browne 1909; Galliot 2012). Transplan- tation experiments also showed that the head organizer produces an activator component named head activator (HA) and an inhibitor one named

head inhibitor (HI) (Fig. 3B). HA and HI activities gradually decrease in parallel along the body axis (Rand et al. 1926; Hicklin and Wolpert 1973b; MacWilliams 1983a,b; Takano and Sugi- yama 1983; Shimizu 2012). We discuss here how the presence of active organizers in intact animals opens the ability to build in several hours de novo organizer centers in the regenerating tips.

Reestablishment of a Head Organizer in Regenerating Tips Is Measured by Transplantation

In addition to the organizer activity of the head, Browne also demonstrated that the tip of a regenerating head or a developing bud harbor a similar organizer activity (Browne 1909; Vogg et al.

2016). How body bisection leads to both wound healing and reactivation of two distinct developmental programs inHydratissues is a complex question, approached biologically over the past 110 years, and more recently completed with biochemical, genetic, and biophysical tools (Fig. 1).

MacWilliams measured the temporal and spatial regulation of each component of the head organizer activity during head regeneration and showed that the activating component is rapidly restored, within 10–12 h after midgastric bisection, while the inhibitory activity is restored more slowly, leaving sufﬁcient time for apical activation to occur. This series of transplantation experiments have demonstrated that intactHydrahas two active homeostatic organizers located at the apical and basal extremities, and that similar organizers form de novo after amputation (for re- view, see Gierer 2012; Meinhardt 2012; Vogg et al.

2016, 2019a).

Modeling the Dynamics of Organizers inHydra

How is organizer activity maintained in intact adult animals and how does it eventually orches- trate the patterning of the surrounding tissue? On the basis of data obtained fromHydratransplan- tation experiments (Webster and Wolpert 1966;

Wolpert 1969) and drawing on Turing’s reaction- diffusion theory (Turing 1952), Meinhardt and Gierer proposed a mathematical model in which

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the activator acts locally and self-catalytically, while it concomitantly activates the production of its own inhibitor, a small molecule with rapid and long-range diffusing properties (Fig. 3B,C;

Gierer and Meinhardt 1972). The distinct properties of the activator and the inhibitor together with the two regulatory loops sufﬁce to explain how the organizer activity gets maximal at the apex in case of the head organizer, and rapidly reactivates after bisection below the wound (Gierer 2012; Meinhardt 2012). The question of how subtle differences in activator and inhibitor concentrations translate into patterning signals and whether these differences are sufﬁcient to coordinate the patterning system so that morphogenesis occurs at the right place and time is still debated, and alternatives that also take into ac- count the biophysical dimension need to be considered (Veerman et al. 2021).

Characterization of the Components of the HydraHead Organizer

Over the recent decades, the challenge for the ﬁeld has been to identify the molecular components with activator and inhibitor activities.

Wnt3/β-catenin signaling has been identiﬁed as the main component of head activator, acting locally and autocatalytically (Fig. 3C; Hobmayer et al. 2000; Broun et al. 2005; Guder et al. 2006;

Lengfeld et al. 2009; Nakamura et al. 2011). RNA- seq data obtained in homeostaticHydrarevealed that the expression ofWnt3andTCFare indeed graded along the body axis (Vogg et al. 2016;

Wenger et al. 2019), while β-catenin is mainly nuclear in the head region compared to the body column and seven out of elevenWntgenes are speciﬁcally reexpressed in the regenerating head organizer,Wnt3being the earliest up-regulated Wnt gene (Hobmayer et al. 2000; Broun et al. 2005; Lengfeld et al. 2009; Wenger et al.

2019). When Wnt/β-catenin signaling is overac- tivated either by overexpressingβ-catenin or by inhibiting pharmacologically the negative regulator GSK-3β, head organizer activity is conveyed onto body column tissue (Broun et al. 2005; Gee et al. 2010). Ferenc et al. recently showed that mechanical stimulation acts as an activator of the regenerative head organizer by sustaining

Wnt3 up-regulation (Fig. 3C; Ferenc et al.

2020). This result highlights the importance of mechanical stimulus in launching head organizer activity.

The transcription factor Sp5 has been identi- ﬁed as a strong inhibitor of head formation (Vogg et al. 2019b), which fulﬁlls several expected crite- ria:Sp5shows an apical to basal graded expression and a positive regulation by Wnt3/β-catenin signaling, Sp5 silencing causes a robust multi- headed phenotype in regenerating and homeostatic animals as well as in reaggregates (Fig.

3A). As anticipated, Sp5 directly repressesWnt3 expression and acts in a negative feedback loop (Vogg et al. 2019b). However, Sp5, which likely acts cell-autonomously, does notﬁt the predic- tion that the head inhibitor is diffusible (Gierer and Meinhardt 1972; MacWilliams 1983a; Tech- nau et al. 2000). Sp5 might trigger the expression of long-range diffusible signals, or alternatively, the role of diffusible substances might be ruled out. An additional important focus is the temporal regulation ofSp5expression, which appears quite dynamic, graded in developing animals but not in adult mature ones whereSp5levels likely oscillate along the body axis.

Besides Sp5, the Dickkopf homolog DKK1/

2/4 (Guder et al. 2006), the glycoprotein thrombospondin (TSP) (Lommel et al. 2018), and the metalloproteinase HAS-7 (Ziegler et al. 2020) were proposed to act as Wnt inhibitors, potentially restricting the head organizer activity (Fig.

3C). Astacin seems to cause proteolytic degra- dation ofHydraWnt3 (Ziegler et al. 2020), suggesting that outside the head organizer region, the organizer activity is restricted by a“double lockdown” mechanism, whereby Wnt3 transcription is repressed by Sp5 and Wnt3 protein actively degraded by astacin.

Characterization of the Components of the HydraFoot Organizer

Transplantation experiments have revealed that Hydracontains two organizers, one apical in the head, and one basal in the basal disc (Hicklin and Wolpert 1973b). Much less is known about the foot organizer; the inhibition of β-catenin prevents foot regeneration (Guﬂer et al. 2018),

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several components of the BMP signaling pathway are restricted to the basal region and reexpressed during early foot regeneration, while SMAD phosphorylation is higher in basal regions indicating a higher activity of the BMP pathway basally (Wenger et al. 2019). BMP5- 8c might act as a foot activator and NBL1, a DAN-domain protein supposedly acting as a negative regulator of BMP signaling, as a foot

inhibitor (Wenger et al. 2019). Such a scenario suggests that a cross talk between Wnt/β-catenin and BMP signaling is required to set up a de novo foot organizer (Fig. 3C). The establish- ment of the foot organizer seems to be largely independent of mechanical stimuli (Ferenc et al.

2020). These results show that regenerative head and foot organizers require different sets of activators/inhibitors to launch their activity.

Patterning organizer Tip-specific

Injury induced Regenerative module Differentiation module Differentiation Proliferation

Mitotic burst migration of progenitors ROS Apoptosis

0 h 1 h 4 h 8 h

H₂O₂

MAPK ERK RSK

CREB pCREB Wnt3

O₂ H₂O₂

gESC

apoptosis

eESC

·– SOD

Wnt3

ISC + derivatives

24 h 36 h 48 h

3D morphogenesis Graded

Figure 4.Schematic representation of the metabolic and cellular events underlyingHydrahead regeneration after midgastric bisection. (Upperpanels) The patterning activity is driven by the formation of a de novo organizer that gets progressively active within theﬁrst 10–12 h of regeneration, restricted to the regenerating tip during theﬁrst 24 h. An immediate peak of reactive oxygen species (ROS) production after injury is followed by cell death, migration of interstitial progenitors to the wound, and a mitotic burst of interstitial cells. The proliferation observed after 24 h involves epithelial and interstitial cells. The differentiation of a new head with tentacle rudiments becomes visible at 40 h. (Lower panel) Scenario for the role of injury-induced ROS signals on immediate cell death in head-regenerating tips. Superoxide (O₂^.−) is symmetrically produced afterHydramid- gastric bisection predominantly in myoepithelial gastrodermal stem cells (gESCs), converted by superoxide dismutases (SODs) into H2O2that is degraded by catalase. These enzymatic activities are not equally distributed in head- and foot-regenerating tips leading to higher H₂O₂levels in head-regenerating ones. H₂O₂by activating the MAPK pathway, induces CREB phosphorylation and the subsequent up-regulation of immediate genes such asWnt3. H2O2can also contribute through paracrine signaling to trigger death of most sensitive cells (i.e., interstitial stem cells and their derivatives). Dying cells release Wnt3 and likely amplify the local production of ROS signals.

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METABOLIC CUES INDUCED BY BISECTION AND SUBSEQUENT CELLULAR RESPONSES One challenging question is the characterization of the injury-induced signals and pathways responsible for the observed asymmetric regenerative response afterHydrabisection, the upper half differentiating a basal disc and the lower half rebuilding a complete head. Below we discuss how generic or speciﬁc are the immediate metabolic and cellular responses to body bisection, and how they trigger the cellular processes that underlie regeneration.

Asymmetric Injury-Induced Cell Death Transforms Somatic Tissues into Head Organizer

After midgastric bisection, an asymmetric activation of MAPK triggers an immediate wave of cell death that activates the head regeneration program (Kaloulis et al. 2004; Chera et al.

2009, 2011). Injury-induced cell death occurs asymmetrically (i.e., massive in the apical-regenerating tips [50% of the cells die] and mini- mal in the basal-regenerating tips [<8%]). This cell death wave, which peaks after 1 to 2 h, most- ly affects the cells from the interstitial lineage, while the epithelial cells, less affected, actively contribute to the engulfment and digestion of the apoptotic bodies. This immediate wave of injury-induced apoptosis helps to locally acti- vate Wnt/β-catenin signaling in head-regenerating tips, leading to de novo head organizer formation (Figs. 3C and 4). Cell–ECM interactions play a crucial role during Hydraregeneration (Shimizu et al. 2002), and TSP, a localized component of the mesoglea, appears to directly regulate Wnt signaling (Lommel et al. 2018).

The evidence supporting this scenario are the following (Chera et al. 2009): (1) A high level of injury-induced caspase activity is necessary as exposing the animals to caspase inhibitors immediately after bisection prevents head regeneration, an effect that can be restored by adding Wnt3 protein; (2) dying cells actually play an instructive role by releasing substances includ- ing Wnt3; (3) the manual induction of apoptosis in foot-regenerating tips ectopically activates

head organizer activity, leading to the regeneration of a head in place of a foot. In response to Wnt3 release by the dying cells, an up-regulation ofWnt 3expression is detected in gESCs of apical-regenerating tips, while the surrounding interstitial cells paused in G2 enter mitosis 4–8 h after amputation and divide synchronously (Fig. 4). This mitotic burst detected by phospho- histone immunostaining or flow cytometry is followed by a peak of proliferation (S-phase cells) within the first 12 h postinjury (Chera et al. 2009; Buzgariu et al. 2018). This process, named apoptosis-induced compensatory proliferation, is not restricted to Hydra, but is also observed in various regenerative contexts (i.e., Drosophila regenerating their imaginal disc [Fox et al. 2020],Xenopustadpoles regenerating their tail [Tseng et al. 2007], or zebrafish regenerating theirfins [Vriz et al. 2014]).

The p80 ERK kinase that is asymmetrically phosphorylated immediately after bisection might play a critical role in the initiation of this cascade as it phosphorylates the CREB transcription factor, a candidate regulator ofWnt3 expression (Fig. 4; Kaloulis et al. 2004; Chera et al. 2011; Nakamura et al. 2011). By contrast, the JNK, Erk1/2, and p38 MAPK kinases appear symmetrically phosphorylated after bisection, suggesting that their activation is part of a gen- eral response associated to injury (Petersen et al.

2015; Tursch et al. 2020). An important next step will be to investigate the injury signals generated on each side of the cut.

ROS Signals as an Asymmetrical Source of Injury-Induced Signaling

ROS are universal injury-induced signals (Roj- kind et al. 2002) involved in wound healing as shown in zebrafish,Drosophila, andCaenorhab- ditis elegans(Niethammer et al. 2009; Moreira et al. 2010; Xu and Chisholm 2014). ROS signaling, which is required for attracting migratory cells of the immune system toward the wound, is also necessary to achieve regeneration in adult zebrafish regenerating theirfin andXenopustad- pole regenerating their tail (Gauron et al. 2013;

Love et al. 2013). Hydrogen peroxide (H2O2), less toxic and more stable than superoxide

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(O2.–), acts as a paracrine signal that at a high level triggers cell death, either by activating the apoptosis signal-regulated kinase (ASK/MAP3K5) (Saitoh et al. 1998; Furuhata et al. 2009) or by inactivating the MAPK phosphatase (Kamata et al. 2005; Chen et al. 2009). In injuredHydra, mitochondrial superoxide (mtO2.–) is produced immediately after injury, predominantly in the gastrodermis, at similar levels in head- and foot-regenerating tips. Pharmacological and gene-silencing approaches show that mtO2.–

plays a critical role in the wound-healing process that occurs on each side of the bisection plane (NS, unpublished). By contrast, the production of H2O2, a by-product of O2.–, is signiﬁcantly higher in head-regenerating halves, where it contributes through paracrine signaling to the high level of cell death and the activation of the head- regeneration program (Fig. 4). As in bilaterians where ROS signals trigger cell migration to the wound, ROS signals inHydraare obvious candi- dates to trigger migration of interstitial progenitors to the wound.

Cellular and Extracellular Processes that Underlie Regeneration

Concomitantly to this wave of apoptosis, the epithelial cells from the tip reorganize rapidly, the gESCs lose their apicobasal polarity and the intercellular connections as observed during regeneration from reaggregated cells, while the eESCs stretch to cover the wound (Shimizu et al. 2002). These roundish gESCs have phagocytic function and express signaling molecules necessary to establish the organizer activity. The wound closure phase lasts up to 2 h and the typical epithelial organization is resumed within 4–8 h (Chera et al. 2009). This wound-healing phase is coincident with mesoglea retraction, where ECM remodeling plays an active role with matrix metalloproteinases speciﬁc to apical or basal regeneration being up-regulated in the regenerating tips (Bergheim and Özbek 2019;

Yan et al. 2000a,b; Shimizu et al. 2002). Subse- quently, the cells from the wound start to newly synthetize the ECM components and the mesoglea layer is reestablished within 24 h postam- putation.

Since the introduction of the term “mor- phallaxis” by Morgan (1901) (i.e., a mode of regeneration that relies on tissue remodeling without cell proliferation), Hydrawas considered the archetypal model system for illustrating the concept of regeneration via remodeling of the preexisting tissue. This perception leaned on studies performed on decapitated animals, which did not identify cell division or proliferation after head removal (Park et al. 1970; Hicklin and Wolpert 1973a; Cummings and Bode 1984).

However, when animals are sectioned at the midgastric level (50% body length), sequential activation of cellular events such as cell death, cell division, and proliferation are recorded (Fig.

4). In fact, two proliferative waves were evidenced after midgastric bisection: an early one driven by the apoptotic cells as described above, and an early-late one, which consists in the ac- cumulation of both proliferating epithelial or interstitial progenitors in the presumptive head region about 30 h post-bisection (Holstein et al.

1991; Colasanti et al. 2009). This second wave is preceded by the up-regulation of 17 genes that are supposed to play a role in cell-cycle progres- sion and proliferation (Buzgariu et al. 2018).

These observations suggest that the regenerative strategies vary depending on the amputation level, fully morphallactic upon decapitation and epimorphic-like after midgastric bisection (i.e., epimorphic regeneration relies on the reactivation of cell proliferation). The abundance of ASCs in the central region and the enrichment in differentiating or differentiated cells in the proximity of the apical and basal regions actually support this view.

CONCLUDING REMARKS

As this work shows, the goal for the amputated Hydra is not to establish a blastema as most bilaterian species do, since the central region of the animal is already blastema-like in homeostatic conditions, but rather to maintain the conditions that keep animals in a pro-regenerative state. Once amputated, the challenge is to establish adequate wound-induced signaling to trigger both wound healing on each side of the wound and the asymmetric and localized trans-

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formation of epidermal and gastric layer tissues, normally dedicated to somatic functions into de novo organizing activities. We see four major themes that should be the focus of research in the coming years. Theﬁrst two concern the pro- regenerative homeostatic status that characteriz- esHydra, on the one hand identifying the molecular players that maintain stem cells in G2 with the expected properties of DNA repair, resistance to cell death, and direct differentiation, and on the other hand dissecting the link between the highly dynamic autophagy as observed in epithelial cells and their ancestral multifunctional status, a link that may have been lost with the specialization of epithelial cells during evolution. The third theme concerns the comparative analysis of molecular, biochemical, cellular, and biomechanical regulators of organizer maintenance and organizer formation in homeostatic and developmental contexts, respec- tively. The fourth theme concerns the testing of Hydraorganizer regulators in bilaterian species to see whether some of the mechanisms under- lying the reactivation of organizer activity are conserved during evolution. If so, manipulation of these activators and inhibitors in mammalian tissues could potentially release regeneration brakes.

ACKNOWLEDGMENTS

The authors acknowledge the sponsors of the Galliot laboratory, the Swiss National Science Foundation (Grants 31003_169930, 310030_

189122), the Institute of Genetics and Genomics of Geneva (iGE3) for N.S.S. fellowship, the Can- ton of Geneva, and the Claraz Donation for con- tinuous support.

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