Holstein_et_al-2003-Developmental_Dynamics.pdf

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REVIEWS A PEER REVIEWED FORUM

Cnidarians: An Evolutionarily Conserved ModelSystem for Regeneration?T.W. Holstein,1* E. Hobmayer,1,2 and U. Technau1

Cnidarians are among the simplest metazoan animals and are well known for their remarkable regeneration capacity.

They can regenerate any amputated head or foot, and when dissociated into single cells, even intact animals will

regenerate from reaggregates. This extensive regeneration capacity is mediated by epithelial stem cells, and it is

based on the restoration of a signaling center, i.e., an organizer. Organizers secrete growth factors that act as

long-range regulators in axis formation and cell differentiation. In Hydra, Wnt and TGF-beta/Bmp signaling pathways

are transcriptionally up-regulated early during head regeneration and also define the Hydra head organizer created

by de novo pattern formation in aggregates. The signaling molecules identified in Cnidarian regeneration also act in

early embryogenesis of higher animals. We suppose that they represent a core network of molecular interactions,

which could explain at least some of the mechanisms underlying regeneration in vertebrates. Developmental

Dynamics 226:257–267, 2003. © 2003 Wiley-Liss, Inc.

Key words: Cnidaria; Hydra; regeneration; organizer; self organisation; wnt/wg signaling

Received 17 September 2002; Accepted 31 October 2002

INTRODUCTION

The freshwater polyp Hydra , a mem-ber of the ancient phylum Cnidaria,

is famous for its regenerative capac-ity. Just like the multiheaded monsterin Greek mythology that grew two

new heads for every one cut off, acnidarian polyp can regenerate anew head after decapitation. Cni-darians are among the simplest liv-ing metazoans and evolved approx-imately 700 million years ago (Bridgeet al., 1995; Conway Morris, 2000;Nielsen, 2001; Petersen and Eernisse,2001). They consist of two body lay-ers, an outer ectoderm and an innerendoderm, separated by an extra-

cellular matrix (mesoglea), and theyrepresent the first animals with a de-fined body axis and a nervous sys-tem.

The regenerative capacity of cni-

darians is remarkable. Hydra polyps

can be dissociated into single cells

that can regenerate as reaggre-

gates into an intact animal within afew days. Cnidarian regeneration

occurs by morphallaxis, i.e., a pro-

cess of repatterning of the existing

tissue without the necessity of cell

proliferation. This appears to be fun-

damentally different from regenera-

tion in vertebrates, where wound

closure is followed by blastema for-

mation during which cells beneath

the wound epidermis dedifferenti-

ate, start to divide, and transdiffer-

entiate (Lo et al., 1993; Brockes,

1997). However, recent data indi-cate that regeneration of cnidarian

tissue shares more similarities to ver-

tebrate (urodele) regeneration than

previously thought. In this review, we

focus on the molecular regulation of

Hydra head regeneration in com-

parison to vertebrate systems. A

comprehensive treatment of classictransplantation experiments, theo-

retical models, and molecular data

in Hydra is found in the review of H.

Bode (this issue).

At present, it is unclear to what

extent “adult” stem cells are in-

volved in the regeneration process.

Such stem cells have been found

even in some mammalian tissues,

and they have a capacity for devel-

oping into a limited number of differ-

ent cell types (for review, see Sto-

cum, 2001). Of interest, stem cells incnidarians also mediate the mor-

phogenetic plasticity of the tissue.

There are two epithelial stem cell

1Department of Biology, Darmstadt University of Technology, Darmstadt, Germany2Zoological Institute, University of Innsbruck, Innsbruck, AustriaGrant sponsor: Deutsche Forschungsgemeinschaft.*Correspondence to: Thomas W. Holstein, Molekulare Zellbiologie, Technische Universität, Schnittspahnstr. 10, 64287 Darmstadt,Germany. E-mail: [email protected]

DOI 10.1002/dvdy.10227

DEVELOPMENTAL DYNAMICS 226:257–267, 2003

© 2003 Wiley-Liss, Inc.


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populations, an ectodermal and an

endodermal one, which continu-

ously differentiate into head- and

foot-specific tissue, which is

sloughed off at both ends of the

body axis (Campbell, 1967a,b;

David and Campbell, 1972). A thirdstem cell system that is present at

least in Hydra and some other Hy-

drozoans gives rise mainly to nerve

cells, nematocytes, and gland cells,

but it is not required for regeneration

(David and Gierer, 1974).

In all regenerating tissue, a major

question is what instructs the cells in-

volved in regenerative processes

and which gene products are re-

sponsible for the induction of regen-

eration. The application of molecu-

lar and genetic techniques has

shown that several crucial genes ofearly embryogenesis is evolutionarily

conserved between vertebrates

and insects. Although little is known

to date about cnidarian embryo-

genesis on the molecular level, new

molecular data indicate that some

of the homologous genes involved

in bilaterian embryogenesis act dur-

ing cnidarian regeneration. There-

fore, the intriguing possibility exists

that a common set of genes might

control at least the early steps of the

regeneration process in cnidarians

and bilaterians. We presume thatthe high or even unlimited regener-

ative capacity characteristic for

cnidarians reflects the properties of

an ancient patterning system that

can generate complete structures

(whole organisms), starting from a

broad range of initial conditions. It is

plausible that molecular patterning

systems capable of extremely robust

and flexible self-organisation might

have been selected during early

metazoan evolution and became

conserved in higher animals. This re-view, therefore, mainly emphasizes

the cellular and molecular dynamics

of this self-organisation system dur-

ing regeneration, and it represents

essentially one lab’s view of the

problem. We presume that the sig-

naling molecules identified in cni-

darian regeneration represent a

core network of molecular interac-

tions that could be responsible for at

least some of the mechanisms un-

derlying regeneration in vertebrates,

e.g., limb regeneration (Brockes,1997; Gardiner et al., 1999).

CNIDARIAN’S REGENERATIVE

CAPACITY IS BASED ON A

HIGH MORPHOGENETIC

PLASTICITY OF THE TISSUE

Epithelial Stem Cells

Most cnidarian polyps and evensome medusae propagate asexu-ally, so that they are in a steadystate of constant growth and tissueturnover. In Hydra polyps, it hasbeen shown that both layers of thebody wall, the ectoderm and theendoderm, are comprised by divid-ing epithelial stem cells in whichnewborn cells are passively dis-

placed upward to form the stingingtentacles, downward to form thefoot, or bud off at the sides to makereplica animals (Campbell, 1967a,b;for review see Bode and Bode,1984). An important consequence isthat the passively displaced cells

have to assess their relative positionin the organism continuously.Hence, patterning systems neces-sary to provide this information arecontinuously active in Hydra polyps.By contrast, in mammals, most ofthese morphogenetic signals are

mainly active only during embroy-genesis.

During regeneration, these mor-phogenetic signals can be acti-vated or enhanced at the site ofwounding. Figure 1 summarizes themajor events during the regenera-tion process in Hydra and other Cni-darians. When Hydra is cut in half,

the upper half containing the headwill regenerate a new foot, and thelower half containing the foot will re-generate a new head. Regenera-tion is a rapid process. After wound

closure, which takes approximately1–3 hr, the tentacles of a new headdifferentiate within 36 hr and a re-generating foot becomes stickyagain within 30 hr (Hoffmeister andSchaller, 1985). Far less is knownabout foot regeneration, yet themechanisms of head and foot re-generation are probably similar, al-though there is some evidence thatthe head system has some support-ive function for the foot system (Mül-

ler, 1990, 1995, 1996; Lee and Javois,

1993; Forman and Javois, 1999;Javois and Frazier-Edwards, 1991;

Schiliro et al., 1999). However, themolecular basis for this phenome-non is completely unclear. If a Hydra is cut into several pieces, the middleportions will regenerate both headsand feet at their appropriate ends,maintaining the initial polarity (Mar-cum et al., 1977). By comparison, anisolated foot or head alone cannotregenerate an intact animal, only ifa head is transplanted on a foot, themissing body region will be interca-lated (Holstein and David, 1990).

Morphallaxis or Epimorphosis?

Classic experiments using Hydra pol-yps that were either x-ray irradiated

(Hicklin and Wolpert, 1973; Nodaand Egami, 1975) or treated with theS-phase blocking agent hydroxyu-rea before regeneration haveshown that cell division is not re-quired for the formation of a new

head (Cummings and Bode, 1984).This finding has led to the conclusionthat regeneration in Hydra is mor-phallactic (Gilbert, 2000; Wolpert,2002). However, the cellular dynam-ics appear to be more complicated.Figure 2A shows that, 12 hr afterhead removal, the regenerating tip

is completely free of S-phase cells(Park et al., 1970; Holstein et al., 1991;Fig. 2B), but by 30 hr, the pattern hascompletely changed, and the re-generating tip is more strongly la-beled than the gastric tissue (Fig.2C). This finding demonstrates dra-matic effects on the cell cycle andproliferation at the regenerating site.

Whether the resumption of mitosis isdue to a decay of the inhibitory ef-fect or a release of a stimulatory sig-nal is not clear. Molecules of the ex-tracellular matrix (ECM) also appear

to contribute to the changes in theproliferation patterns during Hydra head regeneration (Sarras et al.,1991, 1993; Yan et al., 1995, 2000;Shimizu et al., 2002). Accordingly, aloss of the ECM could be related tothe reduced mitotic activity and arestoration of the ECM to the deliv-ery of mitogenic factors. Of interest,newborn buds also exhibit a dra-matic increase in cell proliferation,and the head region is character-

ised by a continuous high level of

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proliferating cells (Holstein et al.,1991). This finding indicates that theloss and restoration of ECM upon

wounding is not the only prerequisitefor the effect on the cell proliferationduring regeneration. It is worth not-ing that a similar correlation be-tween regeneration and prolifera-tion was also found in othercnidarians, e.g., the Cubopolyp

Carybdea marsupialis (Holstein andStangl, manuscript in preparation).

Factors that stimulate mitosis ofepithelial cells have been de-

scribed. Schaller and coworkers(Schaller, 1976; Schaller et al., 1977,

1990) have shown that, if Hydra istreated with low concentrations of aneuropeptide, the head activator,

mitosis is stimulated in epithelial cells.A local release of this factor couldlead to an increased level of mitosisduring regeneration. Hobmayer etal. (1997) demonstrated that headactivator treatment stimulates epi-thelial cell division and induces theformation of more tentacle-specificepithelial cells during regeneration.Consistent with these findings, wealso found that inhibition of cell

proliferation by aphidicolin or hy-droxyurea treatment leads to an in-

Fig. 1. Major processes during cnidarian regeneration on the cellular and molecularlevel. The minimal steps necessary for cnidarian regeneration (solid arrows) involve (1)wound closure of the epithelia followed by (2) the establishment of an organizer and theinstruction of epithelial stem cells, and (3) subsequent morphogenesis. There are alsononobligatory steps in cnidarian regeneration (dotted arrows), e.g., dedifferentiation interminally differentiated tissue of medusae (Schmid, 1992) and cell proliferation in Hydra polyps (Holstein et al., 1991), which could be related to the blastema formation invertebrates.

Fig. 2. Changes in the pattern of epithelialcell cycling during head regeneration. A:Interstitial cell-free polyps were cut at 60–70% body length, pulse-labeled with bro-modeoxyuridine (BrdU) at the times indi-cated, and processed for BrdU visualizationby indirect immunofluorescence (cameralucida drawings). B,C: Photomicrographs il-lustrating the pattern of epithelial cell prolif-eration during head regeneration at 12 hr(b) and at 36 hr (c). Academic Press (fromHolstein et al., 1991).

Fig. 3. Local up-regulation of Hy -Cat ,HyTcf, and HyWnt expression in head re-generating tips. Whole-mount in situ hybrid-izations at 1 and 48 hr after head removal.The Wnt signaling cascade is up-regulatedduring head regeneration. HyWnt is ex-pressed in a group of 15–50 cells definingthe head organizer region at the tip of thehypostome. MacMillan Press (from Hob-mayer et al., 2000).

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complete regeneration of headstructures after 36 hr (Hobmayer andHolstein, unpublished observations).Therefore, we presume that morpho-genetic signals and growth factorsthat are released during head re-generation in cnidarians can also in-duce cell proliferation, which is re-quired for a complete regenerationof the full-sized structure (Fig. 1).

Dedifferentiation as a

Necessary Step in

Regeneration?

Although most polyps grow con-stantly, medusae normally exhibitonly a limited capacity to grow,which is related to the differentiationof a sexually mature animal (there is

an exception from this rule, becausein some hydrozoan life cycles, me-dusae can propagate asexually,e.g., Rathkea and Sarsia ; for review,see Tardent, 1978). Nevertheless,even terminally differentiated me-dusa tissue can regenerate (Fig. 1).Pioneering studies of the jellyfish

Podocoryne carnea showed thatthe muscle tissue of adult medusaetransdifferentiates into several differ-ent cell types during regeneration

(for review, see Schmid, 1992;Schmid and Reber-Müller, 1995).

When striated muscle cells were ex-planted from the subumbrella andcultured in the presence of ECM de-grading enzymes, diacylglycerol, orthe phorbol ester 12-O-tetradeca-noylphorbol-13-acetate, they dedif-ferentiated and started to prolifer-ate 24–48 hr later (Schmid et al.,1998). Such cells formed flagellaefirst, which can be interpreted as asign for the naive state of this tissue,and later they differentiated intosmooth muscle cells, sensory cells, ornematocytes (Alder and Schmid,

1987). During transdifferentiationseveral mesodermal genes, whichare specific for striated muscle differ-entiation, were turned off (Müller etal., 1999; Yanze et al., 1999; Spring etal., 2000). This finding is in accordwith the morphologic data.

Cnidarian vs. Vertebrate

(Urodele) Regeneration

A comparison of the basic featuresof cnidarian regeneration with the

regeneration process in vertebratesindicates that both systems sharesome common principles, despitethe prevailing view that one is mor-phallactic (Cnidaria) and one is epi-

morphic (vertebrates). The remark-able regeneration capacity ofurodele amphibians involves the lo-cal dedifferentiation of stump tissue

to form a blastema and new growthwithin the blastema to form distalstructures (for review, see Gardinerand Bryant, 1996; Brockes, 1997;

Gardiner et al., 1999; Wolpert, 2002).Yet, recent elegant molecular andgenetic data suggests that limb budformation and regeneration in verte-brates involves a prepatterning of

the whole limb at an early stage in asmall morphogenetic field (pre-

specification model), rather than adistal transformation (progress zone

model) of the growing blastema(Sun et al., 2002; Dudley et al., 2002;Duboule, 2002). This finding suggeststhat, also in vertebrate limb forma-

tion and regeneration, patterning ofa morphogenetic field occurs onlyat small scale and growth is neededonly to add in cells to produce astructure of larger size. The reason forthis may lie in the fact that morpho-

gens can act only over a distance ofseveral cell diameters and a maxi-

mum of 300 m.In cnidaria, neither local dediffer-

entiation nor blastema formationare obligatory steps in the regener-ation process, which is probably due

to the high morphogenetic plasticityof the tissue. Epithelial stem cells arealways competent for morphoge-netic signals released at the site ofwounding, and they can differenti-ate even when cell cycling isblocked. Hence, it appears that, in

Hydra , cell division is not completelyindispensable for the regeneration

of the complete structure. On theother hand, in vertebrates, growth ofthe blastema might only be neces-sary to enlarge an already pat-terned field. Thus, Hydra and verte-

brate regeneration might sharemore features than commonlythought. The crucial and obligatorystep during regeneration in both sys-tems is a prepatterning of the regen-erating tissue. Regeneration always

gives rise to structures with positionalvalues proximal to the site of regen-

eration. In cnidarians there is sub-stantial evidence that an apical sig-naling center, the head organizer, isthe driving force for this process. In afirst step, this organizer has to be re-established at the regenerating tip.Then, signals emanating from this or-ganizing center pattern and re-specify the tissue proximal to thewounding site. This emphasizes theimportance of the reestablishmentof an organizer during the initialsteps in regeneration. In the follow-ing, we will discuss the molecularfeatures of the Hydra head orga-nizer and how it is reestablished dur-

ing regeneration, particularly in re-aggregates.

HEAD REGENERATION IN HYDRA

IS DRIVEN BY THE RESTORATIONOF AN APICAL SIGNALING

CENTER, THE HEAD ORGANIZER

The capacity to regenerate a head ishigher at the apical end than at the

basal end of Hydra’s body axis (Web-ster, 1966a,b; Wilby and Webster,1970a,b; Wolpert et al., 1971, 1972;MacWilliams, 1983b; Technau andHolstein, 1995). Transplantation exper-iments have shown that the peak ofthis activity is localized in the hypos-tome. A small piece of tissue from the

hypostome induces a secondarybody axis when grafted laterally toanother polyp (Browne, 1909; Mutz,1930; Yao, 1945; Broun and Bode,2002). Hence, in terms of organizer ac-tivity, Hydra’s hypostomal tissue isequivalent to the dorsal lip of the frogembryo, the Spemann-Mangold or-ganizer, which also induces a second-

ary body axis when grafted to theventral side of the embryo (Spemannand Mangold, 1924).

Until recently, it was rather unclearwhen and how the organizer and its

molecular composition arose duringanimal evolution (Harland and Ger-hart, 1997; Knoll and Caroll, 1999).However, the discovery that thesame set of genes is active in theorganizer of all vertebrates sug-gested that basic features of signal-ing centers acting as an organizermight have arisen earlier in meta-zoan evolution. Potential signalingmolecules that could act as diffus-ible morphogens similar to those in

vertebrates have been identified re-

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cently in Hydra (Hobmayer et al.,2000). It is likely that the gradient of

inductive capacity is mediated by agradient of these signaling mole-cules released from the Hydra headorganizer.

A Wnt ligand (HyWnt ) and the cyto-plasmic mediators Dishevelled (HyDsh ),GSK3 (HyGSK3 ), and -Catenin (Hy -Cat ) were cloned from Hydra (Hob-mayer et al., 2000). In a two-hybridscreen with Hydra -Catenin as bait,the transcriptional coactivator Tcf(HyTcf ) was also identified (Hob-mayer et al., 2000). A Hydra memberof the family of Frizzled receptors

was identified by Minobe et al.(2000). Hence, the core Wnt path-way is present in Hydra . In situ hybrid-ization revealed that Wnt signalingacts in axial patterning and duringhead regeneration in Hydra . HyWnt is expressed in a small number of ec-todermal and endodermal epithelialcells in the apical tip of the hypos-tome, which represents the Hydra head organizer. HyTcf expression isalso restricted to the hypostome ofthe polyp, but the HyTcf expressiondomain is broader than the HyWnt

spot encompassing the entire hy-postome and, thereby, possibly de-marcating the range of action ofthe HyWnt ligand (Hobmayer et al.,2000). During head regeneration,

HyWnt , HyTcf , and Hy -cat areamong the earliest genes to be up-regulated, within 30– 60 min afterwound healing (Fig. 3). In the bud-ding zone, where the new body axisof the daughter polyp is initiated

(Otto and Campbell, 1977), activa-tion of the HyWnt pathway also starts

with an up-regulation of Hy -Cat and HyTcf and is followed by HyWnt expression in a spot of 10–15 cells

(Hobmayer et al., 2000). These dataindicate a pivotal role for the mem-bers of the Wnt-pathway in settingup the Hydra head organizer.

There is also evidence for a sec-ond major signaling system in cni-

darians, i.e., the TGF/Bmp signalingpathway and its antagonist Chordin (Samuel et al., 2001; Lelong et al.,2001; Hobmayer et al., 2001; Hay-ward et al., 2002), which are in-volved in early embryonic axis for-mation of vertebrates. A Bmp ligand(Reinhardt and Bode, personal com-

munication), a highly conserved re-ceptor-regulated Smad1 homo-logue (Hobmayer et al., 2001a), andthe Bmp antagonist Chordin (Rent-zsch, Hobmayer, and Holstein, un-published observations) have been

found in Hydra . The expression pat-terns of HySmad1 and Chordin dur-ing regeneration are consistent withthe hypothesis that Bmp signaling issuppressed by Chordin, which wouldindicate a conservation of the mo-lecular interactions of dorsoventralpatterning from Hydra to verte-brates (for review, see DeRobertisand Bouwmeester, 2001; Shilo, 2001).

These data demonstrate that at

least two major signaling systemsthat are responsible for the function

Fig. 4. Regeneration of intact Hydra pol-yps from reaggregates of dissociated sin-gle cells. Formation of the tissue bilayer byectoderm- and endoderm-specific cell sort-ing mechanisms is finished within 24 hr. De-velopment of polyp structures occurs within96 hr.

Fig. 5. Head induction by activated cellclusters. A: A two-headed aggregate (96hr) with a head containing a green-labeled60-m cluster of aggregated cells from dis-sociated 12-hr regenerating tip tissue. B: Ef-ficiency of cell clusters to induce head for-mation. Head formation frequency ofsingle cells (30 m) and different cell clustersizes were scored 80 hr after aggregation incarrier tissue derived from whole polyps(filled circles, hatched line) and polypslacking the upper fifth (filled triangles, solidline; P 0.001; n 28–53; means [SEM,three experiments]). Control clusters de-rived from the corresponding carrier tissueare indicated by open circles and opentriangles. C: Effect of head inhibition in ag-gregates. Aggregates that contained com-peting 120-m and 60-m cell clusters in asingle aggregate. A 120-m cell cluster in-hibited the formation of a head, the 60-mcell cluster; the correlation of head forma-tion frequency of 60 m cell clusters withtheir distance from the nearest head isshown. A–C from Proceedings of the Na-tional Academy of Sciences USA 2000;97:12127–12131. Scale bar 200 m in A.

Fig. 6. Expression dynamics of HyTcf, Hy- Wnt , and HyBra1 during aggregate devel-opment. In situ hybridization reveals pat-terning events during head organizerformation. HyWnt and HyBra1 appear simul-taneously in small spots (24 hr). which en-

large during later stages (96 hr), and pre-cede formation of morphologic headstructures by approximately 2–3 days. Allspots eventually develop into heads. Pro-ceedings of the National Academy of Sci-ences USA 2000;97:12127–12131 and Nature2000;407:186–189.



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of the vertebrate organizer (DeRob-ertis and Sasai, 1996; DeRobertis andBouwmeester, 2001) are alreadypresent in Hydra . This finding sug-gests that the core Wnt signalingpathway as well as the TGF/Bmpsignaling pathway and its antago-nist Chordin were present in thecommon ancestor of diploblasticcnidarians and the triploblastic Bila-teria and, hence, most likely were abasic feature of early multicellularanimals. Notably, a TGF-beta recep-tor was found in sponges (Suga etal., 1999), although its expression isunclear to date.

It should be also pointed out thattranscription factors that play a rolein the vertebrate organizer havebeen isolated from Hydra , such as

the HNF3 homolog budhead (Mar-tinez et al., 1997), the homeoboxgene goosecoid (Broun et al., 1999),and the T-box gene Brachyury (Technau and Bode, 1999). Thesegenes are all expressed in the orga-nizer region in Hydra (for review, seeGalliot, 2000) and may have a func-tion in regulatory feedback loopstogether with the Wnt and TGF sig-naling cascades during head regen-eration.

REGENERATION OF THE HEAD

ORGANIZER FROMREAGGREGATED SINGLE CELLS

Hydra can be completely dissoci-ated into single cells and will regen-erate intact animals within 3 to 4days (Fig. 4) (Noda, 1971; Gierer etal., 1972). After dissociation into a

single cell suspension and subse-quent reaggregation, all existinggradients of the polyp and any po-sitional information are destroyedand have to be reestablished(Gierer et al., 1972; Sato et al., 1992;

Technau and Holstein, 1992). This ex-perimental system is unique in that itis possible to analyze regenerationfrom the very beginning and underconditions of de novo pattern for-mation on the cellular and molecu-lar level.

Reaggregation proceeds througha well-defined sequence of mor-phogenetic processes: initial cell ad-hesion, ecto–endo cell sorting, for-

mation of the epithelial bilayer,differentiation of a head and foot,

and finally separation into intact

polyps. To establish the epithelial bi-layer configuration, three interactiontypes are necessary: ecto–ecto,endo–endo, and ecto–endo cell in-

teractions. The formation of homo-typic ectodermal and homotypicendodermal aggregates was firstobserved during rotary culture of dis-

sociated cell suspensions (Technauand Holstein, 1992) and confirmedby laser-cell trapping experiments,where the adhesive forces between

individual cells were directly deter-mined (Sato-Maeda et al., 1994).Pairs of endodermal cells exhibitedstronger adhesive forces than pairsof ectodermal epithelial cells, and

there was no initial heterotypic inter-action between individual ectoder-

mal and endodermal cells. Hob-mayer et al. (2001b) used rotary

culture of dissociated cell suspen-sions and found that aggregation ofepithelial cells proceeded in twosteps: first homotypic (ecto–ectoand endo– endo) interactions cre-

ated small cell clusters, then hetero-typic interactions between ectoder-mal and endodermal cell clustersled to the formation of larger aggre-gates. This switch from homotypic to

heterotypic interaction occurred ata critical aggregate size of 10–20

epithelial cells and indicates thatadhesive forces between ectoder-mal and endodermal cells becamesignificantly stronger than adhesiveforces between either ectodermal

or endodermal cells (Hobmayer etal., 2001b). At present it is unclearwhether this change in the cell–cellaffinities in Hydra reaggregates canbe explained by a depletion of alimited pool of cell adhesion mole-cules, a redistribution and clustering

of preexisting heterotypic adhesionmolecules (Grawe et al., 1996), or

the new expression of heterotypicadhesion molecules due to an acti-vation of intracellular signaling cas-cades in homotypic aggregates(Fagotto and Gumbiner, 1996).

The formation of ecto–endoder-mal cell clusters finally leads to theformation of ectodermal andendodermal tissue layers. During thisepithelial sheet formation, the ecto-dermal tissue layer begins an epi-boly-like movement to spread over

the endoderm (Kishimoto et al.,

1996). In parallel, the endodermal

layer organizes beneath an intactectodermal layer (Murate et al.,1997), suggesting that the formationof both epithelial layers is driven by

the ectoderm. This dramatic processof cell sorting and restoration of cellpolarity are completed within thefirst 12 hr of the reaggregation pro-

cess (Gierer et al., 1972; Technauand Holstein, 1992). Once the ecto-dermal and endodermal layers areestablished, no further rearrange-ment occurs. With respect to their

original axial position in Hydra , nocell sorting has been observed (Satoet al., 1992; Technau and Holstein,1992). This finding indicates that, af-

ter dissociation into single cells, thereis no predisposition of erstwhile head

cells to sort out into head tissue andthat the formation of new activation

centers and head organizers occursby true de novo pattern formation(Gierer et al., 1972; Technau andHolstein, 1992).

In further experiments, it was shown

that a community effect regulates theformation of activation centers in Hy- dra (Technau et al., 2000). Labeledcell clusters were produced from re-generating stumps that have a high

competence for head induction(MacWilliams, 1983b). The regenerat-

ing tissue was dissociated into singlecells, aggregated in rotary culture,and the resulting cell clusters werefractionated by size (Technau et al.,2000). Small labeled cell clusters con-

sisting of 10–15 cells (60 m in diame-ter) were added to an unlabeled cellsuspension, and approximately half ofthem were found to be present in adeveloping head after reaggrega-tion. The labeled cells were confinedto the hypostome while the tentacles

were formed by the host tissue (Fig.5A,B). This finding shows that a cluster

of only 10 to 15 cells is necessary andsufficient to instruct and recruit sur-rounding host tissue and initiate theformation of a new head, which is thedefinition of an organizer sensu strictu .

Single cells or very small clusters (30m in diameter) consisting of a fewepithelial cells have virtually no ele-vated capacity of induction. Thesedata demonstrate that a communityeffect (Gurdon et al., 1993) between

these cells is essential to create a sta-ble signaling center. Further experi-

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ments indicated an activation rangeof approximately 45 m (two to threeepithelial cell diameters; Technau etal., 2000), which is in the estimateddiffusion range of known morpho-gens, i.e., wnt/wingless in Drosophila (Gurdon and Bourillot, 2001; see be-low).

AUTOCATALYTIC SHORT-RANGE

HEAD ACTIVATION DURING

REGENERATION BY THE WNT-

PATHWAY?

That small clusters of cells can in-duce surrounding tissue to differenti-

ate into head tissue suggests thatdiffusible morphogens like Wnt mightplay an instructive role in the activa-tion process. The expression patternof HyWnt was examined in early re-aggregates (Hobmayer et al., 2000;

Technau et al., 2000) and found tooccur in small spots comprising onlya few epithelial cells (Fig. 6) by 24 hr.At this time, cells have completelysorted out into ectodermal andendodermal layers (Gierer et al.,1972; Technau and Holstein, 1992),indicating that HyWnt activation re-quires intact epithelial tissue. By96 hr, the HyWnt expression domainshave enlarged to their final size in

future hypostomes (Fig. 6). The size ofearly HyWnt spots is 50–60 m,

which corresponds to the minimalcluster size that can act as an orga-nizer (see Fig. 5B).

Reaction-diffusion models of pat-tern formation predict an autocata-

lytic feedback loop during the acti-vation process. Preliminary datasuggest a possible feedback controlin the HyWnt pathway (Fig. 7). Hy - cat and HyTcf are expressed uni-formly throughout aggregates andlater become restricted to domainswhere new heads are being formed

(Fig. 6). A uniform, but high level of

HyTcf and Hy -Cat might provide acompetence to cells to produce Hy- Wnt . Activation of HyWnt might be astochastic process which is initiated

in single cells, but only maintained if,by chance, neighboring cells also

express HyWnt . Alternatively, HyWnt might activate and stabilize its own

expression directly by means of itstranscriptional mediators Hy -Cat and HyTcf and later become re-stricted to domains where new

heads are being formed. Notably,the expression of HyWnt always pre-ceded the apparent restriction ofdomains in the initially symmetricalenvironment of an aggregate, andall HyWnt domains finally form a

head (Technau et al., 2000). Bothscenarios are consistent with the

idea of an autocatalytic feedbackloop and that HyWnt is a direct tar-get gene of an active Hy -Cat/ HyTcf complex. This finding is in linewith findings from Drosophila , where

autocatalytic self-activation of Wgand a functional Tcf-binding site inthe Wg promoter have been dem-onstrated (van de Wetering et al.,1997; Lessing and Nusse, 1998).

There is additional evidence that

HyWnt might be coupled also by a

positive feedback with another earlyhead gene, HyBra1 (Fig. 7), a Hydra

homologue of the T-box geneBrachyury (Technau and Bode,1999). In aggregates, size and timeof appearance of small HyBra1-positive spots are equivalent to the

HyWnt expression dynamics. Inter-estingly, HyBra1 also shows synex-pression with HyWnt during buddingand head regeneration as well as inadult polyps, although the HyBra1-positive domain in the steady state

hypostome is broader than the Hy- Wnt -positive domain (Technau and

Bode, 1999). A putative Tcf-bindingsite has been identified recently inthe HyBra1 promoter (Technau, un-published data), which supports theidea that Brachyury and Wnt aremembers of a synexpression groupin Hydra . In mouse embryos andmouse cell lines, Brachyury is a directtarget gene of Wnt3a signaling (Liuet al., 1999; Galceran et al., 2001),and Brachyury itself activates tran-scription of Wnt11 in Xenopus (Tadaand Smith, 2000). Direct experimen-tal proof for such a feedback loop in

Hydra by testing the effect of exog-enous HyWnt on HyBra1 expression

or by loss-of-function experimentswith the HyBra1 gene would be ofparticular importance.

SIZE CONTROL DURINGREGENERATION BY LONG-

RANGE INHIBITION

Patterning processes have to be re-stricted to the regenerating tissue.

On the theoretical level, reaction–diffusion mechanisms (Turing, 1952)predict that an inhibitor is producedby the activation center and trans-mitted to the surrounding tissue toprevent the initiation of another ac-tivation center (Gierer and Mein-hardt, 1972; Meinhardt, 1982, 1993).

Transplantation experiments usingintact Hydra have provided strongevidence for such an inhibitory gra-dient extending from head into bodycolumn (MacWilliams, 1983a,b). Therange of inhibition in regeneratingaggregates was determined by in-troducing cell clusters of differentsize into a host aggregate where

larger cell clusters (120 m) exertedan inhibitory influence on the smallerclusters (60 m). It was found thatapproximately 50% of the small clus-ters were not involved in head for-

mation at a distance of 600 m fromthe large clusters, whereas essen-tially all of them were in heads at1,000 m from the large clusters, in-dicating an effective range of inhi-bition of approximately 800–900 m(Technau et al., 2000). By compari-son, the range of activation was ap-proximately 45 m, hence, 20shorter, which fits with theoretical

predictions (Gierer and Meinhardt,1972; MacWilliams, 1982).

The molecular nature of the inhibit-

Fig. 7. Model of putative positive feed-back in Hydra Wnt signaling. Preliminaryevidence and comparison with highermetazoans support the view that autocata-lytic self-activation of the Wnt-pathway and

a feedback between HyWnt and the tran-scription factor HyBra1 are involved in es-tablishment and maintenance of the HyWntsignaling cascade (multiple arrows).



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ing gradient is currently unclear. Usingantibodies to the gap junction pro-teins, Fraser et al. (1987) could perturbthe head inhibition gradient in graft-ing operations, suggesting that the in-hibition gradient is mediated by cell–cell communication by means of gap

junctions. However, the inhibition gra-dient might also involve long-rangemorphogens regulating the propertiesof epithelial cells. In the Drosophila wing disc and the amphibian blastulaanimal cap (Day and Lawrence,2000; Lawrence, 2001), members ofthe TGF/Bmp family act as long-range morphogens up to 300 m

and concentration-dependent ef-fects have been confirmed (Gurdonand Bourillot, 2001). Changes in pro-duction of Dpp, the Drosophila Bmp

homolog, can substantially redesignthe Drosophila wing, indicating along-range action. However, recentstudies using GFP-Dpp constructs sug-gest a more complex mode of gradi-ent formation, including endocytotictrafficking and degradation (Entchevet al., 2000). The antagonistic factor toDpp/BMP2-4 is Sog/Chordin, whichforms an opposing gradient. In Xeno- pus embryos the Chordin gradientcan have a range of at least 450 m

when overexpressed, although its invivo range, which is restricted by the

metalloprotease Xolloid, appears tobe smaller (Blitz et al., 2000). Recently,it has been shown directly in Drosoph- ila that Sog forms a protein gradient indorsal cells of the embryo (Srinivasanet al., 2002). On the dorsal side, Tolloid(Tld) degradation and a dynamin-de-pendent retrieval of Sog act as a dor-sal sink for active Sog (Srinivasan et al.,2002). This long-range activity of Sog/Chordin and the related degradationby Tolloid/Xolloid could be an impor-tant component of the long-range in-hibition phenomena and size control

of Hydra during regeneration.

PERSPECTIVE

Earlier work has suggested that regen-eration by morphallaxis (as found in

Hydra ) and epimorphosis (as found invertebrates) are fundamentally differ-ent. New experimental results from Hy- dra and vertebrates reveal, however,

that regeneration in these evolution-arily extremely distant phyla sharesome similarities (see Fig. 1).

In an initial phase of regeneration,after wound closure, epithelial stemcells can respond to changes ofpatterning signals at the woundingsite. If the cells are differentiated, as

is the case in medusae of Podoc-

oryne (Schmid, 1992), they first haveto dedifferentiate to adopt a newfate. However, this dedifferentiation

appears not to be an obligatorystep, as there is no evidence for it in

Hydra . Because all epithelial stemcells along the body column of Hy- dra are competent to respond tothe regeneration signal, it is an im-portant and unsolved questionwhether this locally restricted re-sponse is due to a positive stimula-

tory signal or to a release of an in-hibitory signal at the regenerating

site.In the next phase of regeneration,

i.e., the formation of an organizerand the establishment of a prepat-tern, substantial progress has beenmade on the molecular level. It isstriking to note that a set of highly

conserved genes, i.e., the Wnt andTGF-beta pathways as well as mem-bers of the T-box gene family, areinvolved in cnidarian regeneration.The data reviewed here indicate

that Hydra , a representative of oneof the oldest metazoan phyla, uses

these genes in a signaling center forregulating the establishment and re-generation of its major body axis.These genes also have a crucial rolein the patterning of higher animals.

This finding indicates the antiquity ofthis patterning system and points to-ward an origin of signaling centers inthe earliest multicellular animals.Eventually, patterning signals haveto be translated into morphogenesisand differentiation of cells. (Non-ca-

nonical) Wnt-signaling and the T-boxtranscription factor Brachyury are

good candidates for mediating pat-terning to morphogenesis. In chor-dates, Brachyury is a target gene ofWnt, TGF-, and FGF signaling and atranscriptional activator of many

genes involved in convergence andextension, cell adhesion, and cy-toskeleton (Tada et al., 1998; Taka-hashi et al., 1999; Tada and Smith,2000).

At present, we are far away from a

comprehensive view of the geneticnetwork controlling regeneration

and the reestablishment of a bodyaxis in cnidarians. Genomic ap-proaches, screens to identify the ex-

tracellular antagonists of signalingmolecules, and promotor analyses

of the involved genes will help tounderstand this genetic network. Thisprogress will lead to the identifica-

tion of cell-type–specific down-stream genes, and to a better un-

derstanding of the question to whatextent cell proliferation is involved inthe cnidaria regeneration.

Another open question, not ad-dressed in this review, is how peptidesignaling molecules are related to

the known signal transduction path-ways. These peptides also affect cni-

darian regeneration (Schaller, 1973;Endl et al., 1999; Hampe et al., 1999;

see the review of Fujisawa in this is-sue) and may represent a phyloge-netically ancient feature of cnidar-

ians. However, it is totally unclear atpresent to what extent these cnidar-ian peptides have homologues in

vertebrates. A gene encodingmembers of the LWamide peptide

family, one of the peptide familiesidentified by the Hydra PeptideProject (Takahashi et al., 1997; Bosch

and Fujisawa, 2001), has been iden-tified recently in Caenorhabditis el- egans (see review of Fujiswa in this

issue). Thus, signaling peptides easilycould have been overlooked with

algorithms that are normally used insequencing projects.

ACKNOWLEDGMENTWe thank C.N. David (Munich) for his

critical comments on the manu-script.

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