Developmental Signaling in Hydra: What Does It Take to Build a “Simple” Animal?

Developmental Biology 248, 199–219 (2002) doi:10.1006/dbio.2002.0744

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Robert E. Steele1 Department of Biological Chemistry and the Developmental Biology Center, University of California, Irvine, California 92697-1700

Developmental processes in multicellular animals depend on an array of signal transduction pathways. Studies of model organisms have identiﬁed a number of such pathways and dissected them in detail. However, these model organisms are all bilaterians. Investigations of the roles of signal transduction pathways in the early-diverging metazoan Hydra have revealed that a number of the well-known developmental signaling pathways were already in place in the last common ancestor of Hydra and bilaterians. In addition to these shared pathways, it appears that developmental processes in Hydra make use of pathways involving a variety of peptides. Such pathways have not yet been identiﬁed as developmental regulators in more recently diverged animals. In this review I will summarize work to date on developmental signaling pathways in Hydra and discuss the future directions in which such work will need to proceed to realize the potential that lies in this simple animal. © 2002 Elsevier Science (USA)

“The cnidarians never stop pulling surprises.” tell us how animals were ﬁrst assembled. Over the past 15 Colin Tudge years, Hydra has moved to center stage in such studies. The Variety of Life, 2000

THE EVOLUTIONARY CONTEXT OF INTRODUCTION HYDRA DEVELOPMENTAL BIOLOGY

We know a lot about the developmental biology of a very From molecular phylogenetic studies, it is clear that the small number of animals. Concentrated efforts on several cnidarians, the ctenophores, and the sponges diverged prior model systems (especially those with tractable genetics) to the appearance of the bilaterians (Adoutte et al., 2000; have revealed in marvelous detail the circuitry which Cavalier-Smith et al., 1996; Collins, 1998; Kim et al., 1999; controls the developmental fate of individual cells and Medina et al., 2001; Wainright et al., 1993). Thus, compari- tissues. More recently the question of how organ develop- sons of the molecular features of developmental processes ment is regulated has begun to be approached. By compari- in these taxa with those in bilaterians have much to say sons of the data obtained from studies of animals in diverse about what occurred as evolution assembled the organism taxa, we can hope to determine the evolutionary routes that gave rise to modern bilaterians. Among these three taken in the formation of the extant animals. From the data taxa, the cnidarians have been the most intensively studied obtained to date, we are beginning to obtain a picture of at the molecular level. In particular, Hydra has been the how development in bilaterian animals evolved. However, most well-studied due to its long history as an experimental a considerable amount of metazoan evolution took place organism and the ease with which it can be cultured and before bilaterians appeared on the scene. It is by examining manipulated in the laboratory (Bode and Bode, 1984b; phyla which arose during this initial phase of metazoan Lenhoff, 1983). One should keep in mind, however, that evolution that one expects to ﬁnd the information that will Hydra is a highly derived cnidarian (Collins, 2000; Harrison and Westfall, 1991). It lives in fresh water, while nearly all 1 Fax: (949)824-2688. E-mail: [email protected]. other cnidarians are marine. It lacks a free-living larva,

0012-1606/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved. 199 200 Robert Steele which is present in virtually all other members of the class Hydrozoa. It lacks the medusa stage of the life cycle, which is found in many hydrozoan taxa. Thus, as is the case for many other experimental systems, efforts have been put into an organism which is tractable as a laboratory animal, not one which is most representative of its phylum. None- theless, Hydra clearly has much to tell us about evolutionary solutions to developmental problems in an animal far removed phylogenetically from those we know so well.

WHAT DOES HYDRA DEVELOPMENT INVOLVE?

The study of Hydra developmental biology is dominated by attempts to understand two phenomena. One is the basis for the establishment and maintenance of the axial pattern (Bode and Bode, 1984b). In simple terms, this can be phrased as the question “how does Hydra know to put a head at one end and a foot at the other?” The other component of the Hydra developmental equation is the question of how the various cell lineages in the adult polyp are derived from their stem cell precursors (Bode, 1996). If one could give detailed mechanistic descriptions of these two phenomena, developmental processes in the adult Hydra polyp would in essence be understood. At this point, it should be made clear that developmental biologists studying Hydra are in a peculiar position. They study developmental phenomena in the adult, although Hydra has perfectly good embryonic development (Martin et al., 1997). But because of the FIG. 1. Tissue dynamics and cell division in the adult Hydra developmental nature of the adult polyp and the variety of polyp. This ﬁgure is adapted from Campbell (1967b) and shows the ways in which it can be experimentally manipulated, vir- results of a study in which tissue movements were followed by tually all of the efforts directed at Hydra development have marking the polyp with methylene blue or grafted tentacles. The arrows indicate the starting and ending positions of the marked been directed at the adult. This puts us in the seemingly tissue, and the number of days required for transit of the marked awkward position of comparing development in the adult tissue are indicated. Blue arrows indicate the paths of marked Hydra polyp to processes occurring in embryos of other tissue which moved toward the head, red arrows indicate the paths animals. However, as discussed below, the tissue dynamics of marked tissue which moved toward the foot or onto buds, and of the Hydra polyp require that axial patterning and cell the green bracket indicates the region in which marked tissue differentiation occur continuously in the adult animal. As a showed little or no movement. The portion of the polyp colored result of this situation, it has been possible to use the adult yellow is the region in which epithelial cells are dividing (Camp- Hydra polyp to learn a great deal about developmental bell, 1967a; Holstein et al., 1991). The regions in turquoise (ten- processes in cnidarians. tacles and basal disk) are where cells are arrested in G2 of the cell The developmental dynamics of the adult Hydra polyp cycle (Du¨ bel et al., 1987). have been described in detail in a review by Bode and Bode (1984b), and they will only be summarized here (Fig. 1). The adult polyp consists of two epithelial cell layers (the ectoderm and the endoderm) which are concentrically arranged maintains its ﬁxed adult size in the face of continued to form a two-layered tube surrounding the gastric cavity. production of cells in the body column by the shunting of At the two ends of the tube are, respectively, a dome (the cells into buds, the asexual form of reproduction, and by hypostome) containing the mouth opening surrounded by a loss of cells through sloughing from the distal ends of the ring of tentacles (which together constitute the head) and a tentacles and from the foot (Campbell, 1967b, 1973). These disk of cells that allows the animal to adhere to the tissue dynamics present the Hydra polyp with one of its substratum (the foot or basal disk). All of the epithelial cells two major developmental problems. Due to the production of the body column (the part of the animal exclusive of the and loss of cells, an epithelial cell in the body column is head and foot) are mitotically active (Holstein et al., 1991). continually changing its axial position. Thus, an epithelial

The cells in the tentacles and the foot are arrested in the G2 cell that is mitotically active and located in the body phase of the cell cycle (Du¨ bel et al., 1987). The animal column will eventually ﬁnd itself crossing either into the

© 2002 Elsevier Science (USA). All rights reserved. Developmental Signaling in Hydra 201 head or the foot, where it must arrest the cell cycle and peptides have been identified by virtue of their ability to differentiate into a new type of cell, either a tentacle battery accelerate foot regeneration (Hoffmeister, 1996). cell or a foot basal disk cell. This process is even more The second approach taken to identify signaling pathway dramatically brought about when a Hydra is bisected components in Hydra has involved treating the animal through the body column. In such a case, cells located in with compounds which are known to perturb signaling the middle of the animal suddenly find themselves at the pathways in other systems. The most successful experi- ends and must adopt the appropriate developmental fates to ments in this regard involved treatment with diacylglycerol allow them to regenerate the missing structures (head and (DAG) (Mu¨ ller, 1989) or lithium chloride (Hassel et al., foot). It seems clear, then, that Hydra cells must have 1993; Hassel and Berking, 1990). As discussed below, these signaling pathways which allow them to sense their loca- compounds have dramatic effects on patterning processes tion in the animal with a high degree of accuracy and that in the Hydra polyp. such sensing must be coupled to pathways which regulate Finally, efforts to clone and characterize genes from developmental fate decisions. Hydra were begun in the late ’80s, and these efforts have The other developmental process in Hydra in which cell resulted in the identification of a number of genes for which fates must be specified is the differentiation of the various there is evidence of a role in developmental signaling products of the interstitial cell compartment (Bode, 1996; (Galliot, 2000). In the following sections, I will discuss the Campbell and David, 1974). The interstitial cell compart- results which have been gained from all three of these ment in Hydra functions much like the bone marrow of a approaches. A summary of the developmental signaling vertebrate. This compartment (Fig. 2) consists of multipo- pathways and their component molecules identified in tent stem cells which give rise to four classes of differen- Hydra is presented in Table 1. tiation products—nerves, gametes, secretory cells, and nematocytes (the “stinging” cells used for prey capture). HEAD ACTIVATOR, THE FIRST Within the gamete lineages, there are committed stem DEVELOPMENTAL SIGNALING cells, one for eggs and one for sperm (Littlefield, 1985, 1991, 1994). Homeostasis within the interstitial cell compart- MOLECULE IDENTIFIED IN HYDRA ment almost certainly requires communication between In 1973, Chica Schaller identified a protease-sensitive the cells of the compartment and the epithelial cells via activity in extracts of Hydra which accelerated regenera- signaling pathways. As is the case with the epithelial cells, tion of the head but not the foot (Schaller, 1973), which she cells within the interstitial cell compartment must also termed head activator. While HA accelerates head regenera- sense axial position. The nervous system of Hydra consists tion, as measured by counting the number of tentacles of a variety of nerve types, which express particular genes which appear 48 h following head removal (Schaller et al., (e.g., those encoding neuropeptides) dependent on their 1983), it does not affect the final number of tentacles when position within the animal (Bode, 1992). For example, there regeneration is complete (Javois and Tombe, 1991). HA are nerves which don’t express a particular neuropeptide activity was also found in extracts from another cnidarian, while in the body column but which begin expressing it the sea anemone Anthopleura elegantissima. The complete when they move into the head via the tissue displacement sequence of the sea anemone head activator was deter- process described above (Koizumi and Bode, 1986). Thus, mined, and it was found to be an 11-amino-acid peptide nerves must also have a mechanism for sensing their (Schaller and Bodenmu¨ ller, 1981). While the Hydra head position in the animal. activator peptide has never been sequenced, proteolytic Given the clear necessity that cells in the adult Hydra fragments from Hydra HA have identical amino acid com- polyp receive extracellular signaling inputs in order to positions to the corresponding fragments from sea anemone adopt and maintain the appropriate developmental fates, HA (Schaller and Bodenmu¨ ller, 1981). HA is difficult to what molecules are there that might provide such inputs? work with due to its propensity for forming inactive dimers Three approaches have been used to search for such mol- in solution (Bodenmuller et al., 1986), and efforts by several ecules. Initial efforts used biological assays to identify labs to clone the gene encoding it have been unsuccessful. activities which affected developmental processes in Hy- In addition, HA has not yet been reported to be among the dra, for example, activities which accelerated regeneration peptides sequenced by the Hydra Peptide Project (see be- of the head or the foot. Although this approach was arduous low). Using the head regeneration bioassay, Schaller and because of the large amounts of materials required to purify Gierer (1973) found that the specific activity of HA varied the active components and the relatively subtle nature of along the oral/aboral axis of the polyp, being highest in the the response, three molecules were purified to homogeneity oral quarter of the animal and lowest in the aboral quarter by using this approach. The first molecule identified was of the animal. Fractionation studies localized the biological the so-called head activator (HA) (Schaller and Bodenmu¨ l- activity to a fraction containing membraneous particles ler, 1981), a peptide which was identified by its ability to which were hypothesized to be derived from the Golgi/ increase the rate of head regeneration when applied to endoplasmic reticulum compartment (Schaller and Gierer, decapitated Hydra polyps (Schaller, 1973). In addition, two 1973). Surprisingly, fixed cells retain HA activity and den-

© 2002 Elsevier Science (USA). All rights reserved. 202 Robert Steele sity gradient centrifugation of cells from fixed, dissociated the animals were exposed to HA for 4 days. Control Hydra polyps showed that the activity was enriched in the polyps were treated identically, except that they were not fraction enriched for nerve cells (Schaller and Gierer, 1973). exposed to HA. The HA-treated polyps had formed close Attempts to examine the distribution of HA in Hydra to two buds per animal after the 4-day treatment, tissue by immunocytochemistry (Schawaller et al., 1988) whereas untreated controls had formed only one bud. have yielded results which are not easily reconciled with Interestingly, the number of epithelial cells did not the earlier data from cell fractionation studies. Monoclonal increase significantly during the 4-day period in either antibodies were raised against HA peptides coupled to the controls or the HA-treated animals. Thus, the extra keyhole limpet hemocyanin. While such antibodies stain bud formed by the HA-treated animals was not due to HA Hydra cells, the staining was primarily in interstitial cells. stimulation of cell division. Nerve cells were not stained. Interestingly, interstitial cells Finally, various studies have also indicated that HA has in the head region of buds were heavily stained. Cloning of effects on the interstitial cell compartment. During head the HA gene will allow a revisiting of these observations regeneration, about 450 new nerve cells must be formed in and should help a great deal in further dissecting the biology a period of 48 h (Bode et al., 1973). Labeling with tritiated of HA. thymidine showed that these new nerves arise from divid- If HA is required for formation and maintenance of the ing precursor cells (Schaller, 1976b). Treatment of regener- head, its apparent location in nerve cells would predict that ating polyps with HA stimulates the formation of new nerve-free Hydra should have defects in head formation. It nerves during head regeneration to an even higher degree is possible to produce nerve-free Hydra by various treat- than is seen in untreated animals (Schaller, 1976b). These ments that selectively eliminate the stem cells from the results were taken to indicate that HA acts to increase the interstitial cell compartment (Lenhoff, 1983). Depending on determination of interstitial cells to become nerve cells. the type of treatment used to produce them, such nerve-free However, additional studies have indicated that the role of animals (so-called epithelial Hydra) can be maintained HA in the nerve cell developmental pathway is likely to be indefinitely by hand feeding (Marcum, 1983), since they are more complicated than this (Hobmayer et al., 1990; unable to capture and ingest prey themselves. Surprisingly, Hoffmeister and Schaller, 1987; Holstein and David, 1986; nerve-free Hydra maintain their normal axial pattern, bud, Holstein et al., 1986). Until molecular and functional and regenerate (Marcum and Campbell, 1978). Analysis of approaches can be used to more precisely define the effects extracts of nerve-free Hydra revealed that the animals of HA on the interstitial cell lineage, it will remain unclear retained HA activity and that, in fact, the specific activity exactly how HA acts in this lineage. in such animals was eight times that seen in normal Until recently, HA stood in isolation, with no indication animals (Schaller et al., 1980). As suggested by Schaller et of the nature of the signaling pathway by which it acts. al. (1980), this result may indicate a latent capacity of the Given that HA is a neuropeptide, a G protein-coupled epithelial cells to produce HA, a capacity which is normally seven-transmembrane molecule seemed a good bet for its blocked by the nerve cells. receptor. An HA-binding protein with a Kd of about 1 nM Subsequent to identifying the HA effect on head regen- has been identified (Franke et al., 1997). Surprisingly, this eration, Schaller (1976a) demonstrated that Hydra treated binding protein is a member of the low-density lipoprotein with HA showed an increase in cell division in both the (LDL) receptor superfamily (Hampe et al., 1999); in particu- epithelial cell compartment and the interstitial cell com- lar, it is the homologue of a mammalian protein called partment, indicating that HA is a mitogen. Analysis of sorLA (Jacobsen et al., 1996). This was unexpected, particu- tentacle regeneration in HA-treated polyps has demon- larly since there is no evidence that sorLA has a signal strated that the acceleration of tentacle regeneration seen in transduction role (Jacobsen et al., 2001), although recently, such polyps was due to the generation of a larger pool of other members of the LDL receptor superfamily have been tentacle cells during regeneration via the mitogenic activity shown to have signal-transducing capacity (Herz, 2001; of HA (Hobmayer et al., 1997). Howell and Herz, 2001). Of particular interest with regard In addition to its effects on head development in the to developmental events is the finding that LRP6, a member regenerating and normal polyp, HA has been shown to of this superfamily, acts together with Frizzled as a core- stimulate budding (Hobmayer et al., 1997; Schaller, 1973), ceptor for Wnt (Pinson et al., 2000; Tamai et al., 2000; Hydra’s asexual mode of reproduction. Schaller (1973) Wehrli et al., 2000) and that LRP6 is the receptor for the treated animals with one bud for 24 h with HA and then dickkopf family of Wnt inhibitors (Mao et al., 2001; Sem- counted the number of animals which had initiated a enov et al., 2001). Since the Wnt pathway is present in second bud by the end of the 24-h treatment. Among Hydra (see below), the involvement of the HA receptor in untreated control animals, 30% had initiated a second bud this pathway is an intriguing possibility. at 24 h. For the HA-treated animals, 50% had initiated a In situ hybridization analysis with the cloned Hydra second bud at 24 h. Hobmayer et al. (1997) examined the sorLA gene demonstrated high levels of expression in the effect on budding of longer-term exposure to HA. They epithelial cells at the bases of the tentacles and a lower level collected newly dropped buds and fed them until the first of expression throughout the rest of the polyp (Hampe et al., bud began to form. At this point, feeding was stopped and 1999). Immunostaining of cells from dissociated Hydra

FIG. 2. Cell lineage compartments in Hydra. This ﬁgure shows the three cell lineage compartments in the adult Hydra polyp. The epithelial cells of the body column consist of the ectodermal and endodermal epithelial stem cells. Thus, the body column is made up entirely of stem cells which give rise to new body column epithelial cells by division and to the terminally differentiated cells of the foot and the tentacles following displacement from the body column as shown in Fig. 1. Dividing committed cells of the nerve, nematocyte, and secretory cell pathways (Bode, 1996) are not shown.

with an antibody against the sorLA protein indicated that activity has been identiﬁed in Hydra, and a cDNA encoding the protein is present in both ectodermal and endodermal a Hydra CREB protein has been isolated (Galliot et al., epithelial cells, in all of the precursor cells in the interstitial 1995). Electrophoretic mobility shift assays show that cell lineage, and in differentiated nerves (Hampe et al., CREB activity rises during the early stages (4 h) of both head 1999). This result is surprising since labeling of interstitial and foot regeneration in Hydra vulgaris and then returns to cells and nerves is not seen with in situ hybridization. In the basal level by 28 h. Hydra CREB is a substrate for addition to its transmembrane form, the Hydra sorLA protein kinase A in vitro (B. Galliot, personal communica- protein exists in a form which does not sediment at tion), suggesting that HA could modify CREB protein activ- 100,000g and which appears to lack the transmembrane and ity as a result of its effect on the cAMP level. cytoplasmic segments (Hampe et al., 1999). The function of this soluble form is unknown. Efforts to elucidate the downstream components of the THE FOOT END HA pathway have demonstrated that a 10-min exposure of intact polyps to HA results in a 2-fold increase in the cyclic In parallel with the studies in the Schaller lab on head AMP level (Galliot et al., 1995). Polyps exposed for 30 min activator, an activity which accelerated foot regeneration show cAMP levels equal to those of control animals, but not head regeneration was identiﬁed in Hydra extracts indicating that the rise in cAMP in response to HA is (Grimmelikhuijzen and Schaller, 1977). The foot activator transient. Cyclic AMP response element binding (CREB) (FA) activity was distributed in a graded manner in the adult

TABLE 1

Components identiﬁed in Hydra

Signaling Additional Developmental pathway Ligand Receptor components roles References head activator head activator sorLA CREB protein head formation, Galliot et al., 1995, budding, interstitial Hampe et al., 1999; cell development Schaller et al., 1989 Pedibin/Hym-346 pedibin/Hym-346 ? ? foot formation Grens et al., 1999; Hoffmeister, 1996 Pedin pedin ? ? foot formation Hoffmeister, 1996 HEADY HEADY ? ? head formation Lohmann and Bosch, 2000 Hym-323 Hym-323 ? ? foot formation Harafuji et al., 2001 Hym-33H Hym-33H ? ? nerve cell Takahashi et al., 1997 differentiation Hym-355 Hym-355 ? ? nerve cell Takahashi et al., 2000 differentiation Wnt Wnt Frizzled disheveled, ␤- axial patterning Hobmayer et al., 2000; catenin, GSK3, Minobe et al., 2000 Tcf/Lef TGF-␤ BMPs ? chordin, R-Smad ? B. Reinhardt and H. Bode, personal communication; B. Hobmayer, F. Rentzsch, and T. Holstein, personal communication Hedgehog Hedgehog ? Ci/Gli family ? Kaloulis, 2000 transcription factor nuclear receptor ? COUP ? ? Escriva et al., 1997 insulin ? insulin-related ? cell division, tentacle Steele et al., 1996 receptor and foot cell differentiation Shin Guard ? shin guard ? peduncle formation Bridge et al., 2000 and/or foot formation? CCK-4/Klg/Dtrk ? Lemon ? gametogenesis, budding Miller and Steele, 2000 Notch ? ? Homologues of ? P. Towb and J. Posakony, Suppressor of personal communication Hairless, Numb, and hairy/ Enhancer of Split Endothelin ? (endothelin ? ? foot development Zhang et al., 2001 converting enzyme identiﬁed) ? ? ? Src head formation Cardenas et al., 2000 ? ? ? protein kinase C head and foot Hassel, 1998; Hassel et al., development 1998; Mu¨ ller, 1989 ? ? ? Ras head development Bosch et al., 1995 Integrin/ECM extracellular integrin ? head and foot Deutzmann et al., 2000; matrix development Fowler et al., 2000; Leontovich et al., 2000; Sarras and Deutzmann, 2001; Sarras et al., 1991, 1993, 1994; Zhang et al., 1994

Note. The table lists all of the cases in which at least one component of a ligand-activated signaling pathway that is likely to play a developmental role has been identiﬁed in Hydra. In some cases, there is experimental evidence for a developmental role (e.g., head activator). In other cases, such a role is assumed based on results from other organisms.

© 2002 Elsevier Science (USA). All rights reserved. Developmental Signaling in Hydra 205 polyp, being highest at the foot end and lowest at the head findings have emerged from examination of the sequence end (Grimmelikhuijzen and Schaller, 1977). Further char- and expression of the pedibin gene. First, the predicted acterization of FA (Grimmelikhuijzen, 1979) revealed that sequence of the precursor polypeptide that yields mature it was sensitive to protease, had a low molecular weight, pedibin lacks a signal peptide. This suggests that secretion and was enriched in a nerve cell fraction isolated from of the peptide occurs via a nontraditional route. In situ dissociated Hydra by differential centrifugation. hybridization with a pedibin probe shows a high level of Hoffmeister (1989) developed methods for fractionation expression in the endoderm of the peduncle (the region of of FA-containing extracts which resulted in an enrichment the body column between the basal disk and the budding of 105-to106-fold relative to the starting extract. Using FA zone) and surprisingly in the endoderm in the proximal prepared with this approach, Hoffmeister (1989) found a portions of the tentacles. Given the role of pedibin in foot surprising range of effects. In addition to accelerating foot formation, the expression in the tentacles is unexpected. regeneration, FA caused a modest stimulation of epithelial Further studies investigating a possible role of pedibin in cell division, an increase in division of interstitial cells, and tentacle development are obviously warranted. an increase in the production of nerve cells. Studies of FA have progressed significantly recently with the demonstration that the active fraction consists of two HEADY peptides, termed pedin and pedibin (Hoffmeister, 1996). The Hydra Peptide Project (see below) has identified a peptide The Bosch laboratory at the University of Kiel has pio- termed Hym-346 which is nearly identical to pedibin, neered the use of differential display PCR to identify genes differing only in the absence of a C-terminal glutamic acid whose expression levels change in concert with develop- residue (Takahashi et al., 1997). This difference is appar- mental changes in the Hydra polyp (Bosch and Lohmann, ently due to the fact that pedibin was isolated from Hydra 1998). This approach has the potential for rapidly identify- vulgaris and Hym-346 was isolated from Hydra magnipa- ing genes involved in Hydra development without the pillata. Both pedin and pedibin/Hym-346 have similar spe- requirement for any knowledge of the products encoded by cific activities with regard to acceleration of foot regenera- these genes. Detailed studies on one gene identified using tion (Hoffmeister, 1996). Using a radioimmune assay, the this approach, a gene termed HEADY, have been published levels of pedin and pedibin/Hym-346 were determined in (Lohmann and Bosch, 2000); these studies provide a striking upper and lower halves of bisected polyps (Hoffmeister, validation of the differential display PCR approach for 1996). The level of pedibin was higher in the upper half by identifying developmental signaling molecules in Hydra. a factor of 1.5. The level of pedin was 2-fold higher in the A HEADY cDNA clone was isolated from a differential lower half than in the upper half. Treatment of Hydra display PCR screen of transcripts from aggregates of Hydra oligactis polyps with pedin stimulated the division of cells at various times after aggregation. The sequence of the interstitial cells and increased the ratio of nerve cells to clone predicts that the mature HEADY peptide is 12 amino epithelial cells (Hoffmeister, 1996). acids in length and that it arises by proteolytic cleavage and Recent studies on pedibin/Hym-346 have shown that it C-terminal amidation of a 23-amino-acid precursor. The can modify the axial patterning process. Exposure of adult HEADY gene is expressed at a very low level in intact adult polyps to pedibin or Hym-346 for 5–15 days increases the polyps, but expression is dramatically up-regulated 6 h after fraction of the body column expressing CnNK-2, a ho- the head is removed. Expression is restricted to a small meobox gene expressed in a graded fashion in the endoderm portion of the endodermal epithelium at the apex of the at the aboral end of the polyp (Grens et al., 1999). Treat- polyp. By 8 h after decapitation, HEADY expression has ment with Hym-346 also significantly increases the ability dropped significantly. No increase in the level of HEADY of segments from the upper part of the body column to RNA is associated with foot regeneration, indicating that induce a secondary foot when transplanted into host polyps induction of HEADY gene expression is not simply a (Grens et al., 1999). Aggregates of cells from dissociated response to wounding of the tissue. While the expression Hydra polyps regenerate into polyps (Gierer et al., 1972). level of the HEADY gene is very low in the adult head, the Heads form first in the aggregates, followed by foot forma- gene is expressed at elevated levels in the developing bud. tion. Aggregates were treated with Hym-346 to determine Expression is seen throughout the endoderm in early buds, whether the peptide would effect the course of foot forma- but becomes restricted to the apical endoderm as the head tion (Gierer et al., 1972). In a sample of 25 control aggre- structures begin to form. Shortly after the bud detaches gates, 31 heads and 1 foot were present after 3 days. In a from the parent, the HEADY RNA level drops to that seen sample of 27 Hym-346-treated aggregates, 4 heads and 49 in the adult polyp. feet were present after 3 days. This result provides dramatic Using an antibody against the predicted HEADY peptide support for the hypothesis that Hym-346 plays a role in foot product, the distribution of the peptide has been examined formation. (Lohmann and Bosch, 2000). The antibody gives a punctate Molecular analyses of pedibin have recently become staining pattern in the endodermal epithelium, with higher possible as a result of the cloning of a cDNA which encodes levels of staining in the body column than in the head and this peptide (Hoffmeister-Ullerich, 2001). Two surprising foot. The stained bodies were interpreted as cytoplasmic

© 2002 Elsevier Science (USA). All rights reserved. 206 Robert Steele vesicles in which the peptide is stored prior to release from Bosch and Fujisawa, 2001), its biological activity is tested the cell. Surprisingly, staining is not seen in early buds, and by using a variety of assays. These assays include testing it was not reported when staining first appears in the bud. effects on cell division, analysis of effects on gene expres- The absence of staining in the early bud, despite the sion using differential display PCR, and analysis of ef- presence of HEADY RNA, was interpreted as indicating fects on morphogenesis (e.g. budding) (Bosch and Fuji- immediate release of the peptide rather than the vesicular sawa, 2001; Takahashi et al., 1997). storage seen in the adult polyp. To date, three peptides have been reported to have RNA interference (RNAi) has been used to inhibit expres- effects on developmental processes in Hydra. Hym-323, a sion of several genes in Hydra, including HEADY (Lohm- 16-amino-acid peptide, is apparently derived from a 62- ann and Bosch, 2000; Lohmann et al., 1999; Smith et al., amino precursor by proteolytic cleavage (Harafuji et al., 2000). Double-stranded HEADY RNA was introduced into 2001). The precursor lacks a classical signal peptide polyps by electroporation, and the animals were decapitated sequence, so the route it takes out of the cell is unknown. 4 days later. Head regeneration was delayed by 2 days in the In situ hybridzation and antibody staining have been experimental animals relative to animals electroporated in used to examine the expression pattern of Hym-323 the absence of RNA, indicating that HEADY gene expres- (Harafuji et al., 2001). In situ hybridization detects Hym- sion is required for head regeneration. 323 RNA in both the ectodermal and endodermal epithe- Two other functional tests have been carried out with lial cells of the body column; no RNA is detected in the HEADY. Synthetic HEADY peptide was applied to newly tentacles or the basal disk. An anti-Hym-323 antibody detached buds for a period of 12 days. Budding of the treated stains the body column in a pattern similar to that seen animals began about 1.5 days earlier than the untreated by in situ hybridization and fails to stain the foot. controls. Animals treated with HEADY peptide for 5 days However, the antibody shows strong staining of the were used as donors for grafts to untreated animals. The tissue tentacles, where no Hym-323 RNA was detected. This from the control animals induced secondary heads in the finding is unexpected and suggests the possibility that hosts in 10% of the cases, whereas tissue from the treated Hym-323 RNA decays as cells are displaced from the animals induced secondary heads in 30% of the cases. body column into the tentacles but that the Hym-323 The data on HEADY suggest that this peptide plays an peptide does not turn over in the tentacle cells. important role in the formation of the Hydra head. How it Treatment of polyps with Hym-323 peptide followed by plays that role is still far from clear. Knowledge of the measurements of the labeling indices of cells in both the identity and distribution of the receptor for HEADY should epithelial and interstitial cell compartments showed that help a great deal in elucidating the pathway by which the peptide had no effect on cell cycle behavior in either of HEADY acts. The results from staining of polyps and buds the compartments (Harafuji et al., 2001). Treatment of with the HEADY antibody suggests that the regulation of regenerating animals with Hym-323 peptide revealed an HEADY production and release may be quite complex. The acceleration of foot regeneration, but no effect on the rate of use of a heterologous expression system in which protein head regeneration. To determine whether the target cells trafficking can be more easily investigated than in Hydra for the Hym-323 effect were in the epithelial cell compart- may be valuable in dissecting these processes. Finally, it ment or the interstitial cell compartment, polyps were will be of obvious interest to examine the expression of treated for a short term with colchicine, which kills the HEADY during embryogeneis to see whether it has a role in interstitial cell population but spares the epithelial cells the initial formation of the head. (Campbell, 1976). Hym-323 treatment accelerated foot regeneration in animals depleted of interstitial cells, indicating that the target of the peptide is in the epithelial cell THE HYDRA PEPTIDE PROJECT: WHAT compartment. IS HYDRA DOING WITH SO Additional evidence that Hym-323 is part of the foot MANY PEPTIDES? development pathway comes from grafting studies with tissue from Hym-323-treated polyps (Harafuji et al., 2001). Following up on the pioneering work of the Schaller lab Polyps treated for 3–7 days with Hym-323 were used as graft on Hydra peptides, an international consortium of re- donors. Peptide-treated tissue induced a secondary foot in searchers initiated, in 1993, a systematic and large-scale the host animal in a higher percentage of the cases than did effort to isolate peptides from Hydra and determine grafts of untreated tissue. whether they had developmental roles (Bosch and Fuji- To determine whether Hym-323 and Hym-346 act sawa, 2001; Takahashi et al., 1997). This effort, termed synergistically on foot formation, the rate of foot regen- The Hydra Peptide Project, has to date resulted in the eration was examined in polyps treated with each peptide purification of over 800 peptides from adult Hydra polyps alone and the two peptides in combination. No synergism (Bosch and Fujisawa, 2001). This is obviously a remark- was detected when the two peptides were used together able number given Hydra’s apparent simplicity. Once a (Harafuji et al., 2001). Harafuji et al. (2001) hypothesize peptide has been purified and its sequence determined that epithelial cells in the body column release Hym-323 (which has been accomplished for 40 of the peptides; peptide as they are displaced into the foot and differen-

© 2002 Elsevier Science (USA). All rights reserved. Developmental Signaling in Hydra 207 tiate into basal disk cells. In this scenario, the cells in establishing the dorsal–ventral axis during embryonic releasing the peptide would also be the targets, suggest- development (Niehrs, 1999). Thus, it was of considerable ing that Hym-323 acts in an autocrine fashion. interest to know whether evolution made this pathway Two peptides, Hym-33H and Hym-355, have been found to available early enough for it to be used by Hydra.Ina effect nerve cell development in Hydra. Hym-33H, a five- remarkable effort, Hobmayer et al. (2000) succeeded in amino-acid peptide with the sequence AALPW, was originally cloning the genes for Wnt, dishevelled, GSK3, ␤-catenin, shown to reduce specifically the labeling of nerve cells in and Tcf/Lef, nearly all the components of the Wnt signaling polyps pulse-labeled with bromodeoxyuridine (Takahashi et pathway. The Sarras lab (Minobe et al., 2000) has identified al., 1997). This finding suggested that Hym-33H inhibits the a Frizzled homologue which is the candidate Wnt receptor progression of cells in the nerve cell differentiation pathway. in Hydra; Southern blotting indicates that Hydra contains The second peptide shown to affect nerve cell differentia- only a single Frizzled gene. The expression patterns of the tion is Hym-355 (Takahashi et al., 2000). A cDNA for components of the Wnt pathway in Hydra are intriguing. Hym-355 has been cloned, and its predicted protein product The Frizzled gene is expressed strongly throughout the suggests that the Hym-355 precursor polypeptide is se- endodermal epithelium of the body column and the foot. creted by using a classical signal peptide. Proteolytic pro- Expression is much lower in the endoderm of the tentacles cessing and C-terminal amidation would give rise to the and the hypostome. Expression is also much lower in the mature nine-amino-acid peptide. In situ hybridization anal- ectodermal epithelium. The simplest interpretation of ysis shows expression of the Hym-355 gene in a subset of these data is that the endodermal epithelium is the primary the nerve cell population, with the expressing cells being target of Wnt action and that the specificity of Wnt signal- found at low density in the body column, and at higher ing is achieved by restricting distribution of the ligand or density in the tentacles and the foot (Takahashi et al., some other component of the pathway. In support of this 2000). This expression pattern has been confirmed by im- idea are the data on the expression of the Wnt gene munostaining with an anti-Hym-355 antibody. (Hobmayer et al., 2000). Wnt is expressed in a small number Bromodexoyuridine labeling studies have shown that of epithelial cells at the tip of the hypostome in both the Hym-355 does not affect the proliferation of cells in either ectoderm and the endoderm. This is the region of the Hydra the epithelial cell or the interstitial cell compartment polyp which has been shown by transplantation studies to (Takahashi et al., 2000). However, pulse labeling in the behave as an axial organizer (Broun and Bode, 2002; Yao, presence of Hym-355 peptide followed by incubation in the 1945). peptide for an additional 47 h results in a 1.5-fold increase The restoration of Wnt expression during head regenera- in the number of labeled nerve cells relative to what is seen tion also fits with Wnt being high up in the axial patterning in animals not exposed to peptide. Takahashi et al. (2000) hierarchy. Expression begins by 1 h after decaptitation suggest that this result is due to Hym-355 causing either a (Hobmayer et al., 2000). This is to be compared with the decrease in the length of S-phase or an increase in the size 30–36 h required for complete regeneration of the head in of the nerve precursor cell pool. this study. Perhaps the most rigorous test of whether the Coupling the inhibitory effect of Hym-33H (Takahashi et expression pattern of a gene is consistent with a role in axis al., 1997) with the stimulatory effect of Hym-355 provides formation in Hydra is to examine the pattern in aggregates. a potential mechanism for maintaining homeostatis in the In an aggregate, the axis has been completely disorganized nerve cell population of the adult Hydra polyp. If such a as a result of the random mixing of the cells. Wnt expres- mechanism were operating, one would predict that treat- sion is absent in newly formed aggregates but becomes ment with a combination of the two peptides should cause detectable in clusters of 10–20 cells at 24 h following nerve production to occur at the same level as seen in aggregate formation (Hobmayer et al., 2000). These clusters untreated animals. This turns out to be the case. Thus, it ultimately become located at the tips of the hypostomes of appears that the level of nerve cell production seen in a the polyps which form from the aggregates. normal adult polyp is established by a balance between a The Wnt story in Hydra is, however, not as simple at it positive feedback signal from nerves onto nerve cell precur- appears from examining the expression patterns of Wnt and sors and an inhibitory signal from epithelial cells onto these Frizzled. In a nonbudding adult polyp, Tcf/Lef expression is same cells. These findings predict that the receptors for restricted to the hypostome and the budding region of the Hym-33H and Hym-355 will be expressed on the nerve body column. The Tcf/Lef expression domain in the hypos- precursor cell population. tome is somewhat broader than the Wnt domain, and expression appears to be graded, decreasing as it moves away from the tip of the hypostome (Hobmayer et al., 2000). THE WNT PATHWAY: SIGNALING The disheveled, GSK-3, and ␤-catenin genes are expressed AT THE SUMMIT at low and uniform levels throughout the nonbudding adult polyp. During budding, expression of Tcf/Lef and ␤-catenin The Wnt signaling pathway is deployed in a large number rise in a band around the polyp at the site where the bud of developmental settings in bilaterians (Wodarz and Nusse, will emerge. Expression of ␤-catenin drops when the head of 1998). Of particular interest is the role of the Wnt pathway the bud begins to develop, and expression of Tcf/Lef be-

© 2002 Elsevier Science (USA). All rights reserved. 208 Robert Steele comes restricted to the bud head. Wnt expression starts as chordin expression begins in a small group of cells in the the bud tissue begins to evaginate from the parent’s body endodermal epithelium at the stage when evagination has column. begun. Expression is maintained throughout the bud Things get even more complicated when one examines endoderm until the head structures are formed, at which the spatial details of the Wnt expression pattern during point expression becomes restricted to the head (T. Hol- budding and regeneration (B. Hobmayer, personal commu- stein, personal communication). nication). Wnt expression is restricted to the ectodermal The data available to date do not allow one to develop a epithelium of the forming bud but later also becomes clear hypothesis about how the BMP signaling pathway expressed in the underlying endoderm as is the case in the works in Hydra. Knowledge of the expression pattern of the adult polyp. However, during head regeneration and regen- receptors for the Hydra BMP homologues should help in eration of polyps from aggregates, Wnt expression begins in this regard. The expression information on chordin and the endodermal epithelium and then later begins being R-Smad suggests that this pathway plays multiple develop- expressed in the ectodermal epithelium. The significance of mental roles in the polyp. these differences is at present unknown. The results of studies to date on the Wnt pathway in Hydra lead one to conclude that this pathway is a primary component of the axis-forming circuitry of the polyp. At the same WHAT ABOUT NOTCH? time, the details of the expression patterns of the genes encoding the pathway components indicate that further dis- Developmental processes in bilaterians make heavy use section of the pathway is not going to be simple. It has been of the Notch signaling pathway (Mumm and Kopan, 2000). possible to make soluble Wnt proteins using various cell lines Despite efforts by several labs, genes encoding Notch fam- (http://www.stanford.edu/ϳrnusse/assays/wntproteins.html), ily members have not yet been identified in cnidarians. suggesting that it may also be possible to make soluble Hydra Recently, however, genes encoding Hydra homologues of Wnt for experimental tests. The fact that Hydra consists of Drosophila Suppressor of Hairless (a transcription factor in only two epithelial layers should facilitate getting the protein the Notch pathway), hairy/Enhancer of Split (a target gene to the target cells, perhaps aided by the addition of dimethyl- in the Notch pathway), and Numb (an intracellular antag- sulfoxide to increase movement of the protein past cell junc- onist of Notch signaling) have been identified (P. Towb and tions (Fraser et al., 1987; Steele et al., 1996; Zhang and Sarras, J. Posakony, personal communication). Thus, it seems 1994). likely that the Hydra Notch pathway will be revealed in its entirely in the not too distant future.

TGF-␤ SIGNALING: IT’S THERE BUT WE DON’T YET KNOW WHAT IT DOES HEDGEHOG: NEMATODES DON’T NEED Members of the TGF-␤ superfamily play prominent de- IT, BUT HYDRA APPARENTLY DOES velopmental signaling roles in bilaterians (Massague, 1998). Genes for a type I TGF-␤ family receptor and two Smad The fourth member of the “big four” of developmental family transcription factors have been identified in the signaling pathways (the other three being the Wnt, TGB-␤, coral Acropora (Samuel et al., 2001) and a gene encoding a and Notch pathways) is the hedgehog pathway (Hammer- member of the bone morphogenetic (BMP) protein family schmidt et al., 1997). Until recently it was unclear whether has been cloned from a sea anenome (Lelong et al., 2001). the hedgehog pathway is present in Hydra. Hedgehog itself Expression data for these genes have not been reported. is absent from C. elegans, as is a gene for Smoothened, one Recently, genes for two members of the BMP family have of the components of the hedgehog receptor (Ruvkun and been identified in Hydra and are in the process of being Hobert, 1998). Genes encoding other components of the characterized (B. Reinhardt and H. Bode, personal commu- pathway, Patched (Kuwabara et al., 2000), another compo- nication). cDNA clones encoding a chordin homologue (T. nent of the hedgehog receptor, and Gli, a zinc finger Holstein, personal communication) and a R-Smad tran- transcription factor (Knight and Shimeld, 2001), are present scription factor (Hobmayer et al., 2001) have also been in C. elegans. However, the complete hedgehog pathway is isolated from Hydra. The R-Smad gene is expressed present in Drosophila, another protostome, indicating that throughout the endodermal epithelium and in the ectoder- portions of the hedgehog pathway have been secondarily mal epithelium of the body column. In addition, the lost in C. elegans. Work recently carried out in the Galliot R-Smad gene is expressed in the nematocyte differentiation lab (Kaloulis, 2000) has demonstrated that the hedgehog pathway, and its expression is up-regulated in the oocyte pathway is indeed present in Hydra. So far, genes encoding precursor cells of polyps undergoing oogenesis. Expression hedgehog itself and a member of the Gli/Ci family of of the chordin gene is restricted to the endodermal epithe- transcription factors have been isolated (Kaloulis, 2000). lium of the hypostome and the tentacles in the adult polyp The expression patterns of these genes will be of consider- (T. Holstein, personal communication). During budding, able interest.

HYDRA ON STEROIDS? dynamics of Hydra result in the continual displacement of cells toward the extremities of the polyp where they are The nuclear receptor superfamily includes an array of then lost by sloughing from the tips of the tentacles and ligand-regulated transcription factors whose members in- from the basal disk (Fig. 1). Thus, epithelial cells in the body clude steroid receptors, retinoid receptors, and a large column eventually move into the tentacles or the basal disk number of so-called orphan receptors (Owen and Zelent, where the ectodermal epithelial cells differentiate into 2000). Members of the nuclear receptor superfamily, par- nondividing cell types (Fig. 2). We have hypothesized (Steele ticularly the retinoid receptors, play crucial developmental et al., 1996) that a low level of the HTK7 receptor in the signaling roles in animals (Maden, 2000). Efforts to trace the body column results in the transduction of a signal that evolutionary history of the nuclear receptor superfamily in stimulates cells to divide and that a high level of receptor animals have so far led to the identification of a single transduces a signal that stimulates cells to differentiate. In member of this family in Hydra (Escriva et al., 1997). the case of the tentacles, the differentiation event would be Phylogenetic analysis of this Hydra receptor suggests that it conversion of body column epithelial cells into tentacle is most closely related to the COUP family within the battery cells. At the other end of the polyp, the differentia- nuclear receptor superfamily. At the moment, we know tion event would be conversion of body column epithelial nothing more than this receptor. The roles of members of cells into basal disk cells. This hypothesis is based on the COUP family in other animals have been explored and findings from studies in mammalian systems showing that found to include developmental processes in a variety of RTK levels can determine whether a cell differentiates or tissues, including the nervous system and the circulatory divides (Lillien, 1995; Marshall, 1995). system (Cooney et al., 2001). The lower level of HTK7 expression in the body column The effects of exposure of adult Hydra polyps and aggre- is proposed to be sufficient to stimulate cell division but gates of Hydra cells (which will regenerate polyps) to not to trigger cell cycle arrest and differentiation into foot various retinoids have been examined (Johnson and Chun, or tentacle cells. Our finding that treatment of Hydra with 1989). Concentrations were identified which were toxic to bovine insulin stimulates cell division of both ectodermal both the adult polyp and the cell aggregates, but no specific and endodermal epithelial cells supports this portion of the developmental effects were observed. In the absence of model (Steele et al., 1996). If our hypothesis is correct, why information regarding the presence of retinoid receptors in does exogenously added insulin affect cell division but not Hydra, it is not yet possible to determine the significance of differentiation? One would argue that the level of receptor these results. It is interesting to note, however, that treat- in the body column is such that even extra insulin supplied ment of another hydrozoan, Hydractinia, with retinoids exogenously can do no more than increase the amount of results in patterning alterations (Mu¨ ller, 1984). division since the level of receptor is too low to allow sufficient signal to cause body column cells to differentiate into basal disk or tentacle battery cells. Furthermore, in the RECEPTOR PROTEIN-TYROSINE KINASE tentacles and the foot, the receptor would act simply as a (RTK) PATHWAYS ARE THERE TOO trigger for the differentiation events but would not provide the specificity. This conclusion arises logically from the Given their importance in developmental signaling in fact that the receptor is present at high levels at both ends more recently diverged animals (Pawson and Bernstein, of the animal, yet the two extremities produce very differ- 1990; Shilo, 1992; Sternberg et al., 1995), RTKs and their ent types of cells. Thus, the specificity must be supplied by ligands are prime candidates for regulating developmental other means. Interestingly, two homeobox genes are ex- fates in Hydra. Several RTK genes have been identified in pressed in a pattern related to that of HTK7. One of the Hydra, some of which encode homologues of RTKs known genes, termed manacle, is expressed in the ring of cells at from bilaterians and others of which appear to be novel. the peduncle/basal disk boundary (Bridge et al., 2000). The One of the Hydra RTK genes, termed HTK7, encodes a other gene, termed HyAlx, is expressed in a ring at the base member of the insulin receptor family (Steele et al., 1996). of the tentacle (Smith et al., 2000). These two genes may be The highest levels of expression of the HTK7 gene are in involved in mediating the response of cells to the signaling nondividing ectodermal epithelial cells in the bases of the cascade activated by HTK7. tentacles and just above the foot basal disk (Steele et al., Two other RTK genes with possible developmental roles 1996). A lower level of expression is seen in the epithelial in Hydra are shin guard, which is expressed in a graded cells of the body column, and expression is absent from the pattern in the ectodermal epithelial cells of the pednucle basal disk and the distal parts of the tentacles. (Bridge et al., 2000), and Lemon, which is expressed in the What could be the significance of the high level of interstitial cell lineage and is up-regulated during gameto- expression of the HTK7 gene in the proximal region of the genesis and budding (Miller and Steele, 2000). Shin guard tentacles and just above the basal disk? The developmental has no homologue in other animals. Its extracellular do- dynamics of Hydra and the results of studies of receptor main contains EGF repeats and its closest relative is an- protein-tyrosine kinase signaling mechanisms in vertebrate other Hydra RTK (Reidling et al., 2000) which appears not cells suggest a possibility. As discussed earlier, the tissue to have a developmental role. Shin guard expression ap-

© 2002 Elsevier Science (USA). All rights reserved. 210 Robert Steele pears to be controlled by a signal from the foot (Bridge et al., Taken together, the data of Zhang et al. (2001) suggest 2000). Its graded expression pattern (high near the basal disk that a pathway involving an endothelin-related molecule is and low near the budding zone) is intriguing since other involved in both maintaining the state of contraction in the studies have shown that the tissue of the peduncle appar- polyp and in the formation of the foot. Identification of the ently becomes committed to foot formation as it is dis- presumed endothelin-related molecule and its receptor are placed toward the basal end of the polyp (Bode and Bode, the obvious next steps in defining how this pathway func- 1984a). Shin guard might play a role in this commitment tions in Hydra. process. The Hydra Lemon RTK (Miller and Steele, 2000) is homologous to the human CCK-4, the chicken Klg, and the SIGNALING VIA THE EXTRACELLULAR Drosophila Dtrk RTKs (Kroiher et al., 2001). These three MATRIX RTKs are unusual in that they lack kinase activity due to amino acid substitutions in the catalytic domain. This lack Although originally not thought of as a signaling entity, of activity makes their role in the cell puzzling. Lemon’s the extracellular matrix is now recognized to play an expression pattern during budding and gametogenesis sug- important role in conveying signals to cells (De Arcangelis gests the possibility that it plays a role in altering the cell and Georges-Labouesse, 2000). Such signaling is achieved cycle kinetics in the interstitial cell compartment during by interaction of the matrix molecules with receptors of the these developmental processes (Miller and Steele, 2000). integrin family. While a Hydra integrin has not yet been characterized, cDNAs for integrins from two other cnidarians (the coral Acropora and the colonial marine hydrozoan DOES HYDRA USE ENDOTHELIN AS Podocoryne) have been reported (Brower et al., 1997; Reber- A DEVELOPMENTAL SIGNALING Muller et al., 2001). The genes for several extracellular MOLECULE? matrix molecules have been cloned from Hydra and the molecules have been shown to be involved in morphogen- In efforts to identify metalloproteinases which act on the esis and cell division (Sarras and Deutzmann, 2001). Iden- extracellular matrix of Hydra, the Sarras lab has identified tification of Hydra integrins and the molecules which act a possible homologue of the vertebrate endothelin convert- downstream of them will provide the tools necessary for ing enzyme (ECE) (Zhang et al., 2001). In vertebrates, ECE investigating this important signaling pathway in greater catalyzes the final proteolytic step in the production of detail. mature endothelins (Rubanyi and Polokoff, 1994). Although their most well-studied role is the regulation of vascular tone, endothelins are apparently also involved in craniofa- A ROLE FOR SRC IN HEAD FORMATION: cial development (Clouthier et al., 1998; Kurihara et al., THE USE OF SPECIFIC INHIBITORS 1994; Yanagisawa et al., 1998). Using antibodies against human endothelin-1, Zhang et Molecules which are known to inhibit or activate signal- al. (2001) were able to detect immunoreactive material in ing pathways in other systems, such as cultured mamma- Hydra lysates. Furthermore, the immunoreactive material lian cells, are potentially powerful tools for dissecting the was enriched in the foot fraction. Upon treatment with roles of signaling pathways in Hydra. What is required is human endothelin-1 or endothelin-3, Hydra polyps con- that the target protein be present in Hydra and that it be tract to about half their normal length within 2.5 h. No well enough conserved to be affected by the molecule. This contraction was seen upon treatment with endothelin-2. In is likely to be the situation in a number of cases, but so far situ hybridization with a probe for the Hydra ECE gene we don’t know how often. Proof of principle for this showed that the gene is expressed throughout the polyp approach has been provided by a study from Cardenas et al. (Zhang et al., 2001). The highest levels of expression are (2000) using PP1, an inhibitor specific for Src family seen in the endodermal epithelial cells at the bases of the protein-tyrosine kinases (PTKs) in vertebrates (Hanke et al., tentacles and in the enodermal epithelial cells in the foot. 1996). Hydra has only a single Src family PTK, whose gene Expression of the ECE gene in the foot endoderm is a has been cloned (Bosch et al., 1989). Hydra Src is inhibited relatively early event during foot regeneration, starting at by PP1 in an in vitro kinase assay (L. Salgado, personal 3 h after removal of the foot. communication). To test the effect of PP1 on Hydra devel- To test whether ECE is required for foot formation, opment, Cardenas et al. (2000) cut off both the head and the polyps were treated with antisense phosphorothioate oligo- foot of adult Hydra polyps and followed the course of nucleotides directed against various portions of the ECE regeneration in the presence or absence of inhibitor. At a gene. The oligonucleotides were applied by localized elec- PP1 concentration of 1 ␮M, head regeneration was delayed troporation into the endodermal epithelium at the basal by 3 days relative to control animals, while foot regenera- pole following foot removal. Three of the antisense oligo- tion was only slightly delayed. The lack of an effect on foot nucleotides caused a delay in foot regeneration in a majority regeneration is not due to the absence of Src in the lower of the treated animals (Zhang et al., 2001). portion of the animal; the Src gene is expressed throughout

© 2002 Elsevier Science (USA). All rights reserved. Developmental Signaling in Hydra 211 the polyp (Miller et al., 2000). The delaying effect was not protein kinase C, is required to maintain the level of Ras2 seen if PP1 exposure began at 10 h following removal of the RNA normally found in the head. These findings raise a head, indicating that the inhibitor-sensitive step occurs number of interesting questions. If the level of Ras2 RNA is early in the regeneration process. normally the same throughout the animal, what purpose To test whether the effect of PP1 on head regeneration would the transient drop in the upper body column during was due to inhibition of cell division, Cardenas et al. (2000) head regeneration serve? What establishes the boundary examined the labeling index of cells in epithelial Hydra between the region where RNA decreases and the region treated with PP1. The polyps were decapitated, incubated where it doesn’t? Does the level of Ras2 protein drop as a for 24–48 h in the presence or absence of PP1, pulsed for 4 h result of the drop in Ras2 RNA? If so, how is the turnover of with bromodeoxyuridine, and the labeled cells were then the protein controlled? Hopefully, it will eventually be counted. No difference was seen between the treated and possible to address these questions. control animals. A final question is whether a change in the level of Ras The minimal effect of PP1 on foot regeneration in polyps protein can affect the outcome of a signaling process in from which the head and foot have been removed is perhaps which Ras is involved. That the level of Ras activity can surprising in light of data indicating that foot formation effect the outcome of a developmental process has been depends on the presence of a head (Mu¨ ller, 1990). This nicely demonstrated in Drosophila (Greenwood and Struhl, discrepancy can be explained if PP1 blocks at a point after 1997). However, it is not clear whether the level of Ras the foot “assistance” process is initiated but prior to the activity available to a pathway is necessarily directly pro- completion of head regeneration. portional to the amount of Ras protein. Expression of high By examining PP1-treated animals for expression of vari- levels of normal Ras protein under control of the hsp70 ous genes associated with head formation, it should be promoter had no effect on Drosophila development (Bishop possible to determine at what point the block to regenera- and Corces, 1988). In contrast to this result, increasing the tion occurs and therefore at what point Src is required in the gene dosage of normal Ras in C. elegans leads to a multi- regeneration process. vulval phenotype, consistent with the interpretation that increasing the level of Ras protein leads to an increase in the level of Ras activity (Sternberg and Han, 1998). Thus, ONE OF HYDRA’S RAS GENES HAS A the changes in Ras2 expression seen during head regenera- ROLE IN HEAD FORMATION tion in Hydra may very well lead to changes in the output from one or more signaling pathways. Ras is a key component in signal transduction pathways in animal cells. Its role has been particularly well worked out in pathways involving RTKs (Tan and Kim, 1999). G PROTEIN-COUPLED SEVEN- While Ras activation has not yet been functionally linked TRANSMEMBRANE RECEPTORS: WILL to any RTKs in Hydra, the expression pattern of one of the Ras genes in Hydra indicates that it plays a role in head THEY BE FOUND TO PLAY AN formation. Hydra contains at least two Ras genes (Ras1 and IMPORTANT ROLE IN HYDRA Ras2), both of which appear to be expressed througout the DEVELOPMENT? polyp (Bosch et al., 1995). The potential involvement of the Ras genes in head formation has been examined by follow- The G protein-coupled seven-transmembrane receptors ing expression during head regeneration. Ras1 and Ras2 are (GPCRs) are an ancient family predating the appearance of normally expressed at comparable levels in the head and the the first metazoans, and they are used in a variety of body. Removal of the head leads to a dramatic drop in the settings in animals. Thus, it is perhaps surprising that so far level of Ras2 RNA in the upper portion of the body column, none of this class of receptor has been identified in Hydra. while expression in the lower portion of the body column is In fact, only two members of the family have been reported unaffected (Bosch et al., 1995). The level of Ras1 RNA does in the phylum Cnidaria, one in a sea anemone (Nothacker not change in either the upper or lower part of the body and Grimmelikhuijzen, 1993) and one in Hydractinia (Gen- column of animals from which the head has been removed. Bank Accession No. AJ440242). Many of the neuropeptides By 8 h following decapitation, the Ras2 RNA level has in vertebrates act through receptors of this class, so it is returned to normal in the upper body column. hard to avoid the idea that at least some of the peptides in Treatment of adult Hydra polyps with the phorbol ester Hydra would use such receptors. This is, however, an 12-O-tetradecanoylphorbol-13-acetate (TPA) results in untested notion. The apparent signaling by head activator many of the treated polyps forming ectopic heads on the through an LDL receptor relative gives one pause in assum- body column (Mu¨ ller, 1990). Treatment of decapitated ing that GPCRs will play a major role in developmental polyps for 20 min with TPA blocked the decrease in Ras2 signaling pathways in Hydra. Efforts to identify the recep- RNA in the upper body column seen in untreated animals tors for the peptides produced by the Hydra Peptide Project (Bosch et al., 1995). These results were interpreted as and EST sequencing projects should reveal how much indicating that a signal from the head, acting through Hydra developmental signaling depends GPCR’s.

GETTING HYDRA TO MAKE EXTRA expression of such genes. Examples of results from such HEADS AND EXTRA FEET: IT’S EASIER experiments are the findings that lithium treatment shifts THAN IT MIGHT SEEM the apical expression boundaries of genes expressed in the basal part of the animal more toward the head (Bridge et al., 2000; Grens et al., 1996), in keeping with the findings that Gene cloning efforts have revealed the presence of a continuous exposure to a low concentration of lithium number of Hydra homologues of developmental signaling drives tissue toward a basal positional value (Hassel et al., molecules known from bilaterians. Ironically, however, 1993; Hassel and Berking, 1990). some of the most dramatic developmental effects associ- While DAG and lithium are valuable tools for analyzing ated with manipulation of signaling pathways in Hydra developmental processes in Hydra, we don’t yet have any have been obtained not by cloning genes and attempting to idea about what signaling pathways they perturb. The Wnt alter their expression but rather by simply incubating the pathway is one possible target due to lithium’s inhibitory animals in the presence of compounds such as diacylglyc- effects on GSK-3. The phosphoinositide cycle is likely to be erol (DAG) (Mu¨ ller, 1989, 1990) or lithium chloride (Hassel a component of more than one signaling pathway in Hydra et al., 1993; Hassel and Berking, 1990). Daily exposure to and thus it may be difficult to determine exactly what DAG leads, in about a week, to the appearance of clusters of signals are upstream of effects that DAG and/or lithium ectopic tentacles on the body column (Mu¨ ller, 1989). Sub- have on this cycle. sequently, hypostomes form in the tentacle clusters and secondary polyps arise from the treated animal. These results have been interpreted as being due to activation of protein kinase C, a reasonable hypothesis, but data that SO WHAT DO WE KNOW? prove this have not yet been obtained. Two PKC genes have been identified in Hydra (Hassel, 1998; Hassel et al., 1998). From the data currently in hand, we can begin to see the However, the proteins encoded by these genes have not yet outline of what kinds of signaling pathways it takes to build been produced in heterologous systems or purified from a Hydra. One thing that is abundantly clear is that nearly Hydra so that their response to DAG can be measured in all of the developmental pathways that are so familiar from direct biochemical tests. Hydra extracts do contain kinase studies of model bilaterians were present in the last com- activity that is stimulated by calcium plus diacylglycerol mon ancestor of these organisms and Hydra. Thus, the and phosphatidylserine (Hassel et al., 1998), as expected for basic machinery for making a metazoan was invented very PKC. early and much of the diversity we see in animal morphol- Treatment of Hydra with lithium can lead to one of two ogy must have been the result of working with this machin- results, depending on the treatment protocol that is used. ery Continuous exposure to a low lithium concentration (0.5–1 The thing that is very surprising is the abundance of mM) over several days leads to the development of ectopic peptide signaling molecules in Hydra. We are just begin- feet on the body column (Hassel and Berking, 1990). Inter- ning to understand what these molecules can do, so it is at estingly, this response to lithium is seen in some species of present difficult to place them in an evolutionary perspec- Hydra (H. vulgaris) but not in others (H. magnipapillata) tive. It will be very important to try to identify genes (Hassel and Berking, 1990). While continuous exposure to a encoding homologues of these peptides in other organisms. low concentration of lithium leads to ectopic foot forma- This will require search algorithms designed to identify tion, a short exposure (2 days) to a high concentration of short coding homologies with such motifs as proteolytic lithium, (4 mM) followed by continuous exposure to 1 mM processing signals (e.g., dibasic cleavage sites). A gene lithium, leads first to formation of ectopic heads and encoding members of the LWamide peptide family, one of tentacles in the upper body column, followed by formation the peptide families identified by the Hydra Peptide Project of ectopic feet in the lower body column (Hassel et al., (Bosch and Fujisawa, 2001; Takahashi et al., 1997), has 1993). The dramatic effects of lithium on Hydra patterning recently been identified in C. elegans (T. Fujisawa, personal are not easy to interpret mechanistically. The processes communication). The gene is expressed in the AIA inter- underlying lithium’s effects on Hydra patterning are likely neurons, which are in the nerve ring encircling the pharynx. to be complicated since lithium is potentially targeting Mutants in the C. elegans LWamide gene have been gener- more than one molecule in Hydra cells. Possible targets ated, and the resulting phenotype is in the process of being include GSK-3 and inositol monophosphatase (Phiel and characterized (T. Fujisawa, personal communication). Klein, 2001). If many of the Hydra peptides are used in other organisms Both DAG and lithium have been used extensively in (particularly those with tractable genetics), it will be very combination with analyses of gene expression to dissect interesting to see what they do there. If, on the other hand, developmental processes in Hydra. By determining how the other organisms are found to lack most of the peptides expression pattern of a developmentally important gene is present in Hydra, we will be witness to a remarkable changed in DAG- or lithium-treated polyps, one can gain experiment in evolution that will have much to say about insight into how signaling pathways may interact with the how multicellular animals operate.

WHAT CAN WE DO (OR HOPE TO DO) be brought to bear on this organism. Attempts to introduce WITH HYDRA TO DISSECT SIGNALING cloned genes into Hydra have been carried out sporadically PATHWAYS? by a number of researchers over the past 15 years. A promising initial result showed that DNA could be introduced into the cells of adult polyps by electroporation (T. On the basis of the large number of question marks in Bosch, A. Grens, H. Bode, and R. Steele, unpublished Table 1, it is obvious that we have a number of promising observations) and that low level expression of exogenous leads but that we have barely scratched the surface with genes could be obtained. However, the introduced DNA regard to understanding the signaling pathways which regu- was not retained in the polyp, being lost after a few days. late developmental processes in Hydra. How should we Recently, a GFP gene under the control of a Hydra actin proceed if we want to fully understand these pathways? A gene promoter has been shown to be expressed following review of various approaches that might be taken is given electroporation into adult polyps (C. David, personal com- below. munication). While expression is lost after a few days, as was seen in earlier studies, the use of GFP as a reporter will Genetic Screens greatly facilitate attempts to improve delivery and reten- tion of the transgene. Other possible delivery methods Efforts were initiated by Sugiyama (1983) some years ago besides electroporation include the use of pantropic retro- to apply genetic methods to understanding developmental viruses (Yee et al., 1994) and transposable elements (Rob- processes in Hydra. By crossing of animals from the same ertson, 1997). pond (to increase the chances of producing progeny which are homozygous for recessive alleles due to inbreeding), Sugiyama identified a number of interesting mutant strains. Blockage of Gene Expression Using Antisense These included, among others, a mutant in which head Oligonucleotides regeneration is inhibited (Sugiyama and Fujisawa, 1977) and mutants with altered body sizes (Takano and Sugiyama, Applications of antisense oligonucleotides as inhibitors 1985). Some of these mutants are being used in molecular of gene expression have been carried out on a variety of studies, and they can provide valuable information (Endl et developmental systems over a number of years. In some al., 1999; Technau and Bode, 1999). However, in no case do cases, the results have been convincing, while in other we know the site of the mutation and there is little hope of cases, it has been difficult to rule out the nonspecific effects obtaining this information in the foreseeable future. At that can be associated with this technology. Carefully present, no genetic screens are underway using Hydra. Such controlled experiments using phosphorthioate antisense screens face formidable roadblocks. It would be very diffi- oligonucleotides in Hydra have yielded informative results cult to obtain Hydra embryos in the numbers necessary to (Deutzmann et al., 2000; Fowler et al., 2000; Zhang et al., carry out such screens, and the hatching of the embryos 2001). Morpholino antisense oligonucleotides (Corey and occurs over a period of weeks to months following a period Abrams, 2001; Ekker, 2000; Nasevicius and Ekker, 2000) of apparent diapause. This is not to say, however, that all have not yet been successfully tested in Hydra, but their cnidarians are intractable as genetic systems. The colonial increased specificity relative to phosphorthioate oligonu- marine hydrozoan Hydractinia has been used successfully cleotides should make them useful tools for studies in in a genetic screen for the loci which control allorecogni- Hydra. tion in this genus (Mokady and Buss, 1996). The sea anemone Nematostella vectensis also shows promise as a genetic screening system since it produces large numbers of RNA Interference embryos which complete development from fertilized eggs to juvenile polyps in 1 week (Hand and Uhlinger, 1992). One of the more remarkable biological phenomena iden- Sexual maturity in Nematostella is reached in about 4 tified in recent years is RNA interference, a process in months. Thus, it seems probable that genetic screens for which double-stranded RNA produced from a cloned seg- developmental regulatory genes in a cnidarian will eventu- ment of a particular gene is capable of specifically inhibit- ally be carried out using Nematostella. Genes discovered in ing the expression of the gene from which it was derived such screens would obviously be valuable tools for further (Bosher and Labouesse, 2000). This technology has been studies in Hydra. most successfully applied in the nematode C. elegans, but the phenomenon has now been reported in a number of other animals, including Hydra. Initial results showed that The Prospects for Transgenic Hydra introduction of dsRNA from a gene expressed only in the head (the ks1 gene) into Hydra by electroporation resulted While classical genetic tools are not readily applicable to in a lowering of the level of the endogenous target mRNA Hydra, one can envision a number of informative experi- and a slowing of the process of head regeneration (Lohmann ments that could be carried out if transgenic methods could et al., 1999). Subsequently RNAi has been used successfully

© 2002 Elsevier Science (USA). All rights reserved. 214 Robert Steele with two additional Hydra genes, the HEADY peptide gene zebrafish embryos (Peterson et al., 2000). One could, for (see above) and the HyA1x homeobox gene (Smith et al., example, imagine screening with Hydra for compounds 2000). Localized electroporation of HyAlx dsRNA into early which give neomorphic phenotypes (e.g., extra heads or stage buds delayed the appearance of tentacles on the buds, feet) or which inhibit developmental processes (e.g., bud- a result in keeping with the apparent role of HyA1x in ding or regeneration). Diacylglycerol and PP1 are examples tentacle formation. of chemical compounds of the sort which one would hope While RNAi works in Hydra, the effects can be relatively to identify with a combinatorial library screen. The ability modest. Therefore, it will be useful to attempt to refine the of individual Hydra polyps to be cultured in 96-well plates technique based on biochemical parameters using extracts offers the potential for such a screen to be carried out in a as has been done in other systems (Tuschl et al., 1999). high throughput mode. The drawback of a combinatorial Secondly, epistasis experiments are needed to show that the library screen is the difficulty of identifying the target blocks achieved by RNAi are located in different positions. molecules of the active compounds. This will not be an easy undertaking given the relatively short time frames involved and the fact that in situ hybridization will be needed in most cases to follow gene expression. Nonetheless, such studies are critical if we hope to use COMBINING THE OLD AND THE NEW RNAi to dissect developmental pathways in Hydra. I will finish this review with a reminder of what enticing experimental possibilities await us when we are able to Matching Up Ligands and Receptors combine the powerful tools with which molecular biology is currently practiced with the equally powerful methods The Hydra Peptide Project and the ease of isolating the that Hydra researchers have had at their disposable in some genes for some classes of receptors (e.g., RTKs) using PCR cases since Trembley’s studies of more than 200 years ago has given us a wealth of ligands for which we as yet have no (Trembley, 1744). One can imagine a situation where graft- receptors and receptors for which we have no ligands. ing experiments will involve using pieces of tissue from a Various approaches are available for identifying both or- transgenic donor Hydra producing GFP and a nontransgenic phan receptors and orphan ligands (Cheng and Flanagan, host. It will then be possible to follow individual GFP- et al., 2001; Civelli 2001) and these should be applicable to producing cells with great precision as the cells induce a Hydra . secondary axis, are displaced through the host polyp into the foot or head, or become incorporated into a bud. Or Genomics imagine what one might see if one made a chimera in which the interstitial cell lineage produced GFP but the epithelial A great deal of molecular biological slogging to under- cells didn’t. Or let’s get really crazy and imagine an animal Hydra stand developmental signaling in could be avoided if in which a portion of the body column contains an hsp70 Hydra we had the sequence of the genome in hand. One promoter-driven transgene for Wnt but the rest of the polyp Hydra could then potentially identify the homologues of doesn’t; producing such a graft would be simple if the any developmental signaling molecules known from other transgenic animals were available. What would happen animals. In addition, it would be possible to identify mem- when such an animal is heat-shocked? Such an experiment bers of signaling molecule families which may not have probably isn’t as far away as it may seem. A variety of genes Hydra homologues in other animals (e.g. -specific RTKs and of interest have already been cloned and more are on the seven-transmembrane receptors). While there is no effort way. And the Hydra hsp70 promoter is in hand (Gellner et Hydra currently underway to sequence the genome, the al., 1992). National Science Foundation has funded a project which will result in the production of approximately 50,000 expressed sequence tags (EST’s) from Hydra. Such sequences will undoubtedly prove to be a gold mine for the Hydra ACKNOWLEDGMENTS research community as well as for other investigators interested in the evolution of metazoans. Work in my laboratory has been carried out with generous support from the National Science Foundation. I am grateful to the Screening with Combinatorial Chemical Libraries members of the Hydra research community for generously com- municating their unpublished results. This paper is dedicated to my brother David, for continually reminding me that there is a The fact that Hydra polyps are essentially naked epithelia world outside the ivory tower of academe. which live in a simple, defined culture medium would seem to make them a promising system for screening for devel- Note added in proof. A paper demonstrating expression of GFP opmental effects with combinatorial libraries of chemical transgenes has recently been published by Miljkovic et al. (Dev. compounds. This approach has been applied with success to Biol. 246, 377–390, 2002).

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