Organizer regions in marine colonial hydrozoans

Accepted Manuscript Title: Organizer regions in marine colonial hydrozoans Author: Tatiana Mayorova Igor Kosevich Nickolai Dulin Elizaveta Savina Yulia Kraus PII: DOI: Reference:

S0944-2006(15)00014-8 http://dx.doi.org/doi:10.1016/j.zool.2014.12.001 ZOOL 25432

To appear in: Received date: Revised date: Accepted date:

1-10-2014 20-12-2014 21-12-2014

Please cite this article as: Mayorova, T., Kosevich, I., Dulin, N., Savina, E., Kraus, Y.,Organizer regions in marine colonial hydrozoans, Zoology (2015), http://dx.doi.org/10.1016/j.zool.2014.12.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Organizer regions in marine colonial hydrozoans Tatiana Mayorovaa, Igor Kosevichb, Nickolai Dulinc, Elizaveta Savinab, Yulia Krausb,*

Koltzov Institute of Developmental Biology, Russian Academy of Sciences, ul. Vavilova 26,

ip t

a

119334 Moscow, Russia

Faculty of Biology, Lomonosov Moscow State University, Leninskie Gory 1/12, 119234

cr

b

Moscow, Russia

Cancer Research Center, The University of Chicago, 929 East 57th Street, Chicago, IL

us

c

* Corresponding author. Tel.: +7-495-9393501.

an

60637, USA

9 figures

Ac ce p

1 table

te

24 pages text

d

M

E-Mail address: [email protected], [email protected] (Y. Kraus).

1

Page 1 of 38

ABSTRACT Organizers are specific tissue regions of developing organisms that provide accuracy and robustness to the body plan formation. Hydrozoan cnidarians (both solitary and colonial) require organizer regions for maintaining the regular body patterning during continuous tissue dynamics during asexual reproduction and growth. While the hypostomal organizer of the

ip t

solitary Hydra has been studied relatively well, our knowledge of organizers in colonial hydrozoans remains fragmentary and incomplete. As colonial hydrozoans demonstrate an

cr

amazing diversity of morphological and life history traits, it is of special interest to investigate the organizers specific for particular ontogenetic stages and particular types of colonies. In the

us

present study we aimed to assess the inductive capacities of several candidate organizer regions in hydroids with different colony organization. We carried out grafting experiments on colonial hydrozoans belonging to Leptothecata and Anthoathecata. We confirmed that the

an

hypostome tip is an organizer in the colonial Anthoathecata as it is in the solitary polyp Hydra. We also found that the posterior tip of the larva is an organizer in hydroids regardless

M

of the peculiarities of their metamorphosis mode and colony structure. We show for the first time that the shoot growing tip, which can be considered a key evolutionary novelty of Leptothecata, is an organizer region. Taken together, our data demonstrate that organizers

d

function throughout the larval and polypoid stages in colonial hydroids.

te

Keywords: Cnidaria; Colonial hydrozoans; Organizer regions; Inductive capacity;

Ac ce p

Transplantation experiments

2

Page 2 of 38

1. Introduction The term ‘organizer’, which was introduced into developmental biology by vertebrate embryologists (Spemann and Mangold, 1924; Sander and Faessler, 2001), accurately describes the action of a small fragment of the dorsal blastopore lip of the gastrulating amphibian embryo: “A piece […] exerts an organizing effect on its environment in such a

ip t

way that, following its transplantation to an indifferent region of another embryo, it there causes the formation of a secondary embryo” (Spemann and Mangold, 1924). Since then,

cr

organizers are generally attributed to embryonic development. Embryonic organizers have been found and thoroughly studied in representatives of all vertebrate classes (Smith and

us

Schoenwolf, 1998) and in a number of invertebrates (Itow et al., 1991; Ransick and Davidson, 1993; Benink et al., 1997; Hudson and Lemaire, 2001; Kraus et al., 2007; Lambert, 2008; Nakamoto et al., 2011; Amiel et al., 2013). The molecular nature of many of them has been

an

deciphered (e.g., De Robertis, 2006; Sethi et al., 2009). In general, an organizer is a complex signaling center that is responsible for maintaining the molecular patterning regulating the

M

establishment of primary body axes (Garcia-Fernandez et al., 2007). Interestingly, the organizer (which remains in the active state in an adult animal) had already been discovered earlier, in 1909. In the cnidarian Hydra, transplantation of a fragment of the

d

hypostome into the body column of a host polyp provokes the formation of the secondary

te

body axis (Browne, 1909; Lenhoff, 1991; Bode, 2012). Having a unique stem cell system consisting of three self-renewing cell lineages, Hydra is capable of continuous tissue

Ac ce p

dynamics and asexual reproduction by budding (Bode, 2011). This is why adult Hydra requires its organizer region for the maintenance of normal body patterning (Bode, 2012). Hydra is a solitary freshwater hydrozoan, and its life cycle lacks the medusa and larval stages. In contrast to Hydra, almost all other hydrozoans have complex life cycles, and a majority of them forms colonies. There are two main groups of colonial hydrozoans: thecates (Leptothecata) and athecates (Anthoathecata) (Cartwright et al., 2008). The athecates form rather simple stolonial colonies in which hydranths resembling solitary polyps attach to the stolons spreading over the substrate (e.g., Clava multicornis, Fig. 1a and b). New hydranths are formed on the stolons by budding. The colonies of thecates are more complex: they consist of stolons and upright branches, or shoots. The soft tissues of thecates are completely covered by a rigid perisarc. Therefore, shoots can grow only via the shoot growing tips – specific structures resembling the shoot apices in plants. The shoot is composed of multiple similar modules called internodes; and every morphogenetic cycle of a growing tip produces 3

Page 3 of 38

one internode. In thecates, shoots can grow sympodially (e.g., Laomedea flexuosa and Gonothyraea loveni, Fig. 1c–e) or monopodially (e.g., Dynamena pumila, Fig. 1f–h) forming very complex colonies (Berking, 2006). It is likely that, similar to Hydra, colonial hydroids require organizer regions as they maintain a steady morphology during colony growth. However, information on organizers in colonial hydrozoans is very scarce.

ip t

There are two regions that have already been tested as organizers. However, it is uncertain whether these organizers are conserved among hydrozoans. The posterior tip of a hydrozoan

cr

planula larva has been shown to function as an organizer in athecates (Hydractinia echinata; Stumpf et al., 2010) and in thecates with monopodial growth (D. pumila; Kraus, 2011). In D.

us

pumila, transplantation of the posterior tip fragment into the body of a recipient planula induced the formation of a secondary body axis during metamorphosis from the larval to the polypoid stage (Kraus, 2011). It seems that the localization of an organizer in the posterior tip statement needs a broader taxonomic sampling.

an

of a planula is a characteristic feature of hydrozoans. However, a confirmation of this

M

The organizer capacity of the hypostome has been tested in the colonial athecate Cordylophora lacustris. When the tip of the hypostome was implanted into a so-called 'reconstitution mass' formed from dissociated colony tissue, this provoked the formation of a

d

hydranth (Beadle and Booth, 1938; Moore, 1952). However, differentiation of multiple

te

hydranths from a reconstitution mass is also well known for C. lacustris and C. multicornis and proceeds spontaneously (Moore, 1952; Polteva et al., 1988). This fact makes the results of

Ac ce p

grafting experiments quite ambiguous. To clarify whether the hypostome serves as an organizer region in colonial hydroids, parts of an intact colony have to be used as recipients in grafting experiments.

Analysis of the data on pattern formation in colonial hydrozoans suggests that the shoot growing tip may be considered a new and additional candidate region for an organizer. This region attracted our attention since species-specific colony patterning is provided by the morphogenetic activity of the shoot growing tips (Crowell, 1957; Wyttenbach et al., 1965; Kosevich, 1990, 1991, 2013). However, this region has never been tested in transplantation experiments, which are crucial for proving the organizer capacities of a candidate region. Thus, our study aimed to (i) provide evidence for the conservation of already known organizers among hydrozoans by increasing the taxonomic sampling; and (ii) convincingly demonstrate the organizer capacities of a new candidate organizer region. To achieve these aims, we designed a series of grafting experiments with three model species – C. multicornis 4

Page 4 of 38

(athecate), L. flexuosa and G. loveni (sympodially growing thecates) (Fig. 1a–f). We examined (1) whether the posterior tip of the planula functions as an organizer in the sympodially growing thecates similarly to the representatives of athecates and monopodially growing thecates; (2) whether the hypostome tip works as an organizer in colonial hydrozoans as it does in the solitary polyp Hydra; and (3) whether the shoot growing tip of sympodially

ip t

growing thecates is an organizer region.

cr

2. Materials and methods 2.1. Study animals

us

C. multicornis (Forsskal, 1775[RS1]) (Hydractiniidae) is an athecate colonial hydrozoan (Fig. 1a and b). G. loveni (Allman, 1859[RS2]) and L. flexuosa (Crowell, 1953[RS3]) (Campanulariidae) are sympodially growing thecates (Fig. 1c and d). As many other hydrozoans, these species

an

have reduced the medusa stage, and their gametogenesis occurs in sessile gonophores developing on male and female colonies. Embryos develop in female gonophores until the

M

planula larva stage. The planula leaves the maternal organism, attaches to an appropriate

2.2. Experimental procedures

d

substrate with its anterior pole and undergoes metamorphosis, giving rise to the young colony.

te

All experiments were performed at the Pertsov White Sea Biological Station of the Lomonosov Moscow State University (66°34'N, 33°08'E) during June and July. C.

Ac ce p

multicornis, G. loveni and L. flexuosa colonies were collected during low tide and kept in the laboratory at 18 °С under constant aeration and natural lighting conditions in natural sea water (NSW).

Planulae and colony fragments were kept in 5 ml Petri dishes at 18 °С in NSW filtered through Millipore glass filters (24 µm) (filtered sea water, FSW). To obtain a cohort of mature planulae, colonies were kept in darkness overnight. Soon after the onset of light, mature planulae left the gonophores and were collected for experiments. Planulae were stimulated to metamorphosis by treatment with CsCl (Seipp et al., 2006). We found that 30 min of treatment with a 100 mM solution of CsCl in FSW does not disturb the normal course of metamorphosis in our model species. In experiments with C. multicornis, hydranths were relaxed with 5% MgCl2 in FSW prior to the grafting operations, which permitted making a slash in the hydranth pedicel. FSW was replaced immediately after the operations. Because all hydranths recovered within a few 5

Page 5 of 38

minutes following the end of the MgCl2 treatment, it is unlikely that the relaxation affected the results of our experiments. The grafting experiments were performed using acupuncture needles and microsurgical scalpels. Unfortunately, we could not trace the fate of the transplanted grafts as none of the vital dyes we tested were retained in the tissues throughout the experiment, which lasted for

ip t

more than one day. The results of the experiments were recorded under a stereo microscope (Leica M205 A; Leica Microsystems, Wetzlar, Germany) with a built-in 10 МP digital

cr

camera.

us

2.3. Histology and electron microscopy

For light and electron microscopy, samples were fixed in 2.5% glutaraldehyde in phosphate buffer (pH 7.4; 0.83 osmol/l) (Millonig, 1964) for 1 h at 4 °C, postfixed in 1% OsO4 in the

an

same buffer (1 h, room temperature) and stored in 70% ethanol at 4 °C until further processing. The specimens were then embedded in a standard mixture of Araldite and Epon

M

(Electron Microscopy Sciences, Munich, Germany), and 1–2 µm thick semi-thin sections were stained with a mixture of toluidine blue and methylene blue (Mironov et al., 1994). Ultra-thin sections were stained with uranyl acetate and lead citrate and examined with a

d

transmission electron microscope (JEM-1011; JEOL, Akishima, Japan) with a CCD camera

Ac ce p

2.4. Statistical analysis

te

(model 782 ES500W; Gatan Inc., Tokyo, Japan).

Statistical analysis of the data was performed using Fisher's exact test (Statistica 6.0; StatSoft, Tulsa, OK, USA).

3. Results

3.1. Transplantation of the posterior tip fragment of a G. loveni planula into the body of a recipient planula

Donor and recipient planulae of G. loveni used in this experiment belonged to the same cohort. The posterior tip fragment (approximately one tenth of the larval length) was cut from a donor planula with a scalpel and inserted into a slash made in the middle part of the body of a recipient planula (Fig. 2a). Incisions without transplants were made on 20 control planulae from the same cohort. The transplantation was considered successful if a recipient planula did not reject the donor tissue during wound healing. Seven out of twenty transplantations were 6

Page 6 of 38

successful (Table 1). In four planulae, the transplanted fragments formed cone-shaped outgrowths (Fig. 3a). In another three cases, the transplanted fragments were completely incorporated into the recipient tissues (Fig. 3b). After wound healing, both experimental and control planulae were stimulated to metamorphose. All planulae settled onto the substrate (the bottom of the Petri dish) and

ip t

flattened forming an anchoring disk as planulae do during their normal development. Throughout the course of further metamorphosis, both the control planulae and the recipient

cr

planulae that had completely incorporated the graft tissues always developed a single primary shoot (Fig. 3c and d; Table 1). The four recipient planulae with a superficial location of the

us

transplanted fragments formed two primary shoots growing from the common anchoring disk (Fig. 3e and f; Table 1). These primary shoots either differed in size (Fig. 3e) or were nearly identical to each other (Fig. 3f). Since the volume of the transplanted donor tissue was smaller

an

than that of the ectopic shoot, it may be assumed that at least part of the recipient tissue was involved in the primary shoot formation.

M

Applying Fisher's exact test, we could show that the absence of secondary axes in the control group was not just a matter of chance (p < 0.05).

te

body of a recipient hydranth

d

3.2. Transplantation of the hypostome fragment from a C. multicornis donor hydranth into the The C. multicornis hydranth consists of a cone-shaped hypostome, a gastral region bearing

Ac ce p

multiple tentacles and a pedicel attached to the stolon (Fig. 1a). Experiments were performed on hydranths isolated from a colony, as isolation does not lead to hydranth regression in athecates. Donor and recipient hydranths were cut from the same colony at the base of their pedicels (Fig. 2b), placed into Petri dishes and relaxed. The hypostome tip was cut from a donor hydranth and split longitudinally into four parts with a scalpel. A slash corresponding to the size of the hypostome fragment was made in the subtentacle region of the recipient hydranth pedicel (Fig. 2b). The hypostome fragment was then pushed into this slash with an acupuncture needle.

Graft rejection occurred in 4 out of 16 recipients, but in the other 12 cases the transplantation could be considered successful (Table 1). Three days post transplantation, ectopic tentacles appeared at the grafting site in 6 out of 12 recipients (Table 1). Ectopic hypostome development (Fig. 4a and b) and secondary hydranth elongation proceeded during the next few days (Fig. 4b). 7

Page 7 of 38

Incisions without transplants were made on 16 control hydranths. These incisions never provoked the formation of ectopic hypostomes or tentacles (Fisher's exact test; p < 0.05).

3.3. Grafting of the growing tip apex into the proximal part of a recipient internode For these experiments we used G. loveni and L. flexuosa, two hydrozoan species with the

ip t

sympodial mode of growth.

The distalmost parts of shoots containing at least three internodes were isolated from the

cr

colonies and were placed into a Petri dish. The apical fragment of the growing tip was then cut from a donor shoot and was pushed into the recipient's perisarc tube through the opening

us

formed when the recipient was cut from a colony (Fig. 2c and d).

Shoots containing at least three internodes that were isolated from the colonies and kept in a Petri dish without any additional treatment served as controls (30 shoots each of G. loveni and

an

L. flexuosa). The control shoots of both species developed stolons growing from the proximal end of the proximal internode, i.e. from the cutting site (Fig. 5a, b and Table 1). These stolons

M

were completely functional, as they attached to and spread over the bottom of the Petri dish. Throughout these experiments, we observed a very high percentage of donor tissue rejections (Table 1), which can be explained at least partially by the absence of a firm contact between

d

the grafted piece and the recipient tissues during wound healing. This may be due to an

te

inappropriate orientation of the grafted piece inside the perisarc tube or to the retraction of the recipient tissue in the direction of the shoot distal end because of the continuous shoot growth

Ac ce p

(Kosevich, 2013). Only those transplantations which ended up with a fusion between the donor and the recipient tissues were considered ‘successful’. In G. loveni, 30 transplantations were performed and 10 of them were successful. Six out of those 10 proximal internodes formed stolons as in the control group, but 4 developed internode anlagen growing from the grafting site (Table 1). Eventually, these anlagen transformed into fully developed internodes looking very similar to the internodes of the recipient (Fig. 5c and d). Morphologically, the newly developed internodes were nearly identical to regular internodes, as they had a proximal annulation zone (a ring-like perisarc deformation typical of the shoots of Campanulariidae) contacting the proximal annulation zone of the recipient internode, a medial smooth zone, and a distal annulation zone bearing a hydranth (Fig. 5d). However, the proximal annulation zone of the newly developed internodes consisted of up to 13 annulations and was longer than that of the regular internodes consisting of 3 or 4 annulations (Fig. 5d). 8

Page 8 of 38

Similar results were obtained in the experiments with L. flexuosa: 30 transplantations were performed, 17 transplantations were considered successful, and 8 out of 17 recipient shoots formed internodes at the grafting site (Fig. 5e and Table 1). Control shoots of both species never developed internodes at the site of cutting (Fisher's exact test; p < 0.05).

ip t

The results obtained in this series of experiments led us to hypothesize that the shoot growing the next series of grafting experiments support this hypothesis.

cr

tip is able to act as an organizer upon transplantation to an ectopic site. The data obtained in

us

3.4. Rescue of the regressing hydranth by the implantation of an apical fragment of the shoot growing tip

an

In colonial hydrozoans, tissue regression is a normal physiological process important for colony renewal and fine tuning of the colony structure, thus allowing the colony to adapt to a changing environment (Cherry Vogt et al., 2011). The regression of hydranths and stolons has

M

been shown in many hydrozoan species (Thatcher, 1903; Crowell, 1953; Hale, 1973; Marfenin and Kosevich, 1984; Cherry Vogt et al., 2011). In L. flexuosa, several morphological stages of hydranth regression have been dissected (Huxley and de Beer, 1923;

d

Strehler, 1961). An intact feeding hydranth almost always keeps its hypostome and tentacles

te

outside the hydrotheca (Fig. 6a). The first sign of regression is a shrinking of the tentacles and the hypostome (Fig. 6b). Tentacles then start to merge with the hydranth’s gastric region (Fig.

Ac ce p

6c). Finally, the regressing hydranth transforms into a ball-like cell mass, which gradually disappears as its cells migrate into the colony. In thecates, hydranth regression can be easily provoked by its isolation from a colony. To obtain the ball-like cell masses, hydranths were cut from a colony just beneath the gastral region by a transverse cut so that they would contain no pedicel tissues and placed into a Petri dish with FSW (Figs. 2d and 6a). The isolated hydranths started regression about 12 h after having been cut, and they transformed into a ball-like cell mass during the next 6 h. In contrast to the ‘reconstitution mass’ formed from dissociated colony tissues (Beadle and Booth, 1938; Moore, 1952), the cell mass formed from a regressing hydranth died approximately 36 h post isolation. We characterized the successive stages of hydranth regression by histological and ultrastructural analyses. All changes in the morphology and tissue structure occurring in the 9

Page 9 of 38

isolated hydranths were similar to those occurring during the natural process of hydranth regression (data not shown). In a feeding hydranth, the epidermis and gastrodermis demonstrated a regular epithelial organization (Fig. 7a). The gastric cavity of the hydranth contained some food debris, but it was free of hydranth cells. Soon after the beginning of regression (12 h post isolation),

ip t

gastrodermal cells elongated their apico-basal axes and started leaving the epithelium (Fig. 7b and c). They were shed into the hydranth’s gastric cavity forming an inner cell mass (Fig. 7b).

cr

Through further regression (15 h post isolation), the hypostome vanished and the hydranth’s gastric cavity became completely filled with gastrodermal cells (Fig. 7c). During these stages

us

of regression, the epidermis and the gastrodermis preserved epithelial organization (Fig. 7b and c). During the final stage of regression (18 h post isolation), both epidermal and gastrodermal cells lost the regular arrangement typical of an epithelium, and the basal lamina

an

separating the cell layers disappeared (Fig. 7d–f). The inner cell masses consisted of tightly packed cells of gastrodermal origin (Fig. 7d–f), and many of them contained vesicles

M

resembling phagosomes (Fig. 7f).

In the rescue experiments, the apex of the shoot growing tip was implanted into the ball-like cell mass at 18 h post hydranth isolation (Fig. 6d). The volume of the implanted fragment was

d

about one fourth of the regressing hydranth.

te

Isolated hydranths kept in a Petri dish without additional treatment served as a control for the grafting experiments (Table 1). No hydranth or colony structures (stolon, growing tip etc.)

Ac ce p

ever developed from these regressing hydranths (Fisher’s exact test; p < 0.05). 12 out of 30 performed implantations were considered successful, as the recipient cell mass did not reject the growing tip of the donor and did not die. In all 12 cases, the growing tip fused with the cell mass in less than 1 h (Fig. 6e and Table 1). A structure resembling a hydranth anlage developed at the site of the implantation within about 4 h (Fig. 6f). Growth pulsations typical of a growing hydrozoan colony then started to spread over the hydranth anlage area and the cell mass area (not shown). The hydranth anlage underwent a morphogenesis similar to that observed during normal hydranth development in an intact colony (Fig. 6g–j). Cells from the cell mass appeared to be gradually incorporated into the developing hydranth while a shrinking of the cell mass volume concomitant with the growth of the hydranth anlage was observed. The hydranth anlage (but not the cell mass area) became covered with a perisarc about 16 h post implantation (Figs. 6h–j and 7g–i). Tentacle buds emerged around the hypostome about 24 h post implantation (Fig. 6i). Eventually, a 10

Page 10 of 38

completely developed hydranth formed from the cell mass within about 30 h (Figs. 6j and 7j). Histological analysis revealed that while the cell mass area did not show epithelial organization prior to apex implantation, the epithelium was immediately re-formed in the developing hydranth anlage. 12–16 h post implantation, the epidermal epithelium consisted of flattened cells, and the gastrodermal epithelium consisted of large vacuolated cells. The latter

ip t

were columnar in the hypostome and gastral regions and cuboidal in the tentacles (Fig. 7g–i). The cells in the cell mass area also re-established epithelial organization (Fig. 7g and i). The

cr

gastric cavity of the induced hydranth contained rounded non-epithelial cells until the end of gastric cavity of the regressing hydranth (Fig. 7d and k).

us

hydranth morphogenesis. However, their quantity appeared to be much lower than in the The high percentage of donor tissue rejections and the recipients’ deaths that we observed (Table 1) may be explained by the fact that the ball-like cell masses that we used in the

an

experiments were very close to the irreversible stage of tissue regression. Hence, even a slight shift in the stage of the recipients’ regression might have strongly affected the result of the

M

experiment.

Taken together, these data demonstrate that implantation of the growing tip apex into the

4. Discussion

te

hydranth.

d

otherwise dying cell mass rescues it from death and leads to the development of a new

Ac ce p

4.1. Organizer at the larval stage: the posterior tip of a planula Previously, larval organizers have been detected in the posterior tips of the planulae of the athecate Hydractinia echinata (Hydractiniidae) (Stumpf et al., 2010) and of the monopodially growing thecate Dynamena pumila (Sertulariidae) (Fig. 8) (Kraus, 2011). In the present study, we show that in G. loveni (Campanulariidae), which differs from H. echinata and D. pumila in colony structure, the posterior tip of the planula is an organizer as well (Figs. 1e, h, 2b and 4). It is likely that this feature can be regarded as conserved among hydrozoans. At this time, we cannot extend this conclusion to all cnidarians, as there are no data on the existence of larval organizers outside the hydrozoans. The presence of the organizer center in the larvae of colonial hydrozoans may be essential for the robust formation of the species-specific body plan during metamorphosis, which is accompanied by a dramatic reorganization of the body plan of the planula (e.g., Seipp et al., 2006, 2007). In colonial hydrozoans, the modes of metamorphosis are very diverse (Fig. 9). 11

Page 11 of 38

The metamorphosing planula attaches to the substrate with its aboral end (Fig. 9a). Settlement is associated with a flattening of the body, whereby the larva is gradually shortening in length and its aboral adhesive zone is enlarging (Fig. 9a and b). In colonial athecates (e.g. Hydractinia), the planula directly converts into a primary polyp (Fig. 9c). In thecates, the planula converts into a primary module of a colony instead of a primary polyp (Fig. 9d–i).

ip t

During settlement, the planula forms an anchoring disk covered with a perisarc (Fig. 9d). The growing tip, which is formed at the center of this disk (Fig. 9e), gives rise to the primary shoot

cr

of a colony, and the structure of the primary shoot depends on the mode of colony growth (cf. Fig. 9f/h and g/i) (Pyataeva, 2007; Kraus, 2011). For example, in D. pumila, the primary

us

shoot bears two hydranth anlagen, and the shoot growing tip is located between them (Fig. 9h).

Thus, in different species of colonial hydrozoans, larval organizers that are morphologically

an

very similar guide the formation of the young colonies with striking differences in colony morphology and in the level of structural complexity. Further studies on metamorphosis in

M

colonial hydrozoans are required to characterize the molecular mechanisms by which larval organizers impose their control on the morphogenetic processes. An interesting question is whether these molecular mechanisms are conserved among hydrozoans, which significantly

te

d

differ from each other in colony structure.

4.2. Organizers at the polyp stage: the hypostome

Ac ce p

The hydranths of colonial hydrozoans differ from the solitary polyps in many aspects of their life cycle. The solitary Hydra, which is in a steady state of continuous production and loss of tissues, has no defined lifetime (Martinez and Bridge, 2012). In colonial species, the lifetime of the individual hydranth is limited, and it undergoes the process of autotomy or regression (Morse, 1909; Crowell, 1953; Tardent, 1963). Moreover, thecates significantly differ from athecates in the regulation of their hydranths. Once formed, thecate hydranths do not grow, they are not able to regenerate any missing parts, they have no cell proliferation and they regress after functioning for only a week (Crowell, 1953, 1961; Kosevich, 2013). In contrast, athecate hydranths can live for more than a few months, and they are able to restore missing hypostomes or tentacles (Crowell, 1960). These facts support the hypothesis that the evolution of coloniality was accompanied by a progressive weakening of hydranth individuality (Beklemishev, 1969; Rosen, 1979; Marfenin and Kosevich, 2004). Therefore, similar body parts of thecate hydranths, athecate hydranths and solitary polyps do not necessarily possess 12

Page 12 of 38

the same capabilities, and it is not given that the hypostome of a hydranth is an a priori organizer. On the other hand, the key components of the Wnt/β-catenin signaling pathway, which has been shown to be of high importance for the Hydra hypostomal organizer (Hobmayer et al., 2000; Lengfeld et al., 2009; Bode, 2012), are also expressed in the hypostomes of the colonial

ip t

athecates H. echinata and Ectopleura larynx (Plickert et al., 2006; Müller et al., 2007; Nawrocki and Cartwright, 2013). The experiments performed on H. echinata (Duffy et al.,

cr

2010) demonstrated that Wnt signaling is indispensable for the formation of the oral structures in colonial hydroids. Indeed, inhibition of canonical Wnt-signaling by Tcf RNAi prevented

us

the formation of oral structures in both metamorphosis and regeneration of a decapitated hydranth (Duffy et al., 2010). These data suggest that the hypostomes of Hydra and of athecate hydranths might have common molecular properties.

an

In the past, grafting experiments were performed on the athecate Cordylophora lacustris (Beadle and Booth, 1938; Moore, 1952). The ‘reconstitution mass’ composed of dissociated

M

colony tissues was used as a recipient in these experiments. It was shown that the grafted hypostome became the hypostome of a newly formed hydranth, and all other parts of this hydranth’s body were formed from the cells of the reconstitution mass (Moore, 1952). Our

d

transplantation experiments on hydranths of C. multicornis confirmed that the hydranth (Figs. 2a and 4).

te

hypostome acts as an organizer in colonial athecates as it does in the solitary polyp Hydra

Ac ce p

To date, hypostomal organizers have been described in the solitary polyp Hydra (Browne, 1909; Broun et al., 2005) and in the colonial athecates C. lacustris (Beadle and Booth, 1938; Moore, 1952) and C. multicornis (present study). The question remains whether the hydranth hypostome has retained organizer capacities during the progressive evolution of coloniality. There is indirect evidence that the hydranth hypostome of thecates is an organizer as well. An experimental study performed on Eirene viridula, which forms very simple athecate-like colonies consisting of only stolons and polyps, showed that the head region of the thecate hydranth is able to affect colony patterning by producing a specific low-molecular-weight substance that is thought to inhibit the growth of new hydranth buds near a hydranth (Plickert et al., 1987). Unfortunately, the molecular signature of the hydranth hypostome has never been characterized in Leptothecata. In the future, grafting experiments on thecates of the highest level of colony integration and the lowest level of hydranth individuality should be designed and performed to clarify the organizer capacities of their hypostomes. 13

Page 13 of 38

4.3. Organizers at the polyp stage: the shoot growing tip All colonial hydrozoans can grow continuously, increasing the size and changing the shape of their colonies. Whereas all colonial hydrozoans have stolons which elongate and spread over the substrate due to the functioning of the stolon growing tips (Fig. 1b, e and h), the branched

ip t

uprights (or shoots) growing via a step-by-step addition of new internodes are a characteristic and unique feature of thecate hydrozoans (Fig. 1c and h). As the thecate colony is covered

cr

with the rigid perisarc, it can form new internodes only via the shoot growing tips.

Thus, the shoot growing tips permit the thecate colony to spread in three-dimensional space

us

and to grow in a cyclic manner characteristic of modular organisms, which has certain evolutionary and ecological advantages (Buss, 1979; Marfenin 1997; Kosevich, 2013). It appears that acquiring the shoot growing tip has given thecates the opportunity of a

an

progressive increase in the complexity of the colony structure, which became a major evolutionary trend in this group (Kosevich, 2006, 2012). Hence, the shoot growing tip can be

M

considered a key evolutionary novelty of thecate hydroids.

It is likely that the shoot growing tip plays a central role in the regulation of the speciesspecific patterning of a colony. It has been shown that the shoot growing tip can function even

d

autonomously, as a tip separated from the colony is able to complete the morphogenetic cycle

te

of internode development (Kosevich, 1990, 1991). As the main function of the organizer regions is the regulation of the body plan formation, it is reasonable to hypothesize that the

Ac ce p

shoot growing tip may possess organizer capacities. In the present study, we grafted apical fragments of L. flexuosa shoot growing tips onto the proximal end of recipient internodes and into the ball-like cell mass formed from regressing hydranths (Figs. 2c, d, 5 and 6). Unfortunately, the perisarc that covers the colony prevented any labeling of the soft tissues of the shoot growing tips and of the recipient internodes used in the grafting experiments. Attempts to label the recipient ball-like cell mass were not effective, as all vital dyes we tested were not retained in the tissues throughout the experiment (more than 24 h).

However, we have circumstantial evidence that the results of our experiments are unlikely to reflect autonomous differentiation of the graft. First, we know that there is no cell proliferation within the shoot growing tip (Hale, 1964; Wyttenbach, 1965; Kossevitch, 1999). Second, an ectopic internode growing from the proximal end of the recipient internode was always larger than the engrafted fragment in our experiments (Figs. 2c and 5d). Therefore, it 14

Page 14 of 38

is likely that the development of an ectopic internode occurred due to the induction of the recipient tissues by the graft. Similarly, the hydranth formed from the ball-like cell mass at the site of grafting was always larger than the implanted fragment (Fig. 6d), and its development was concomitant with the reduction of the cell mass (Fig. 6e–j). We propose that the development of the hydranth which is capable of reorganizing the cells from the ball-like cell mass.

ip t

anlage in this experiment represents a rescue of the regressing hydranth tissue by the graft

cr

Taking our data together, we conclude that the shoot growing tip is an organizer region. The shoot growing tip is an example of a taxonomically restricted organizer, which cannot be

us

found outside the Leptothecata. However, there is a high probability that this structure evolved on the basis of the hydranth hypostomal organizer during colony evolution. This conclusion can be drawn from the transplantation and isolation experiments. In Hydra, the

an

transplanted piece of a donor hypostomal organizer induces the formation of a secondary polyp and differentiates itself into the hypostome of this polyp (Yao, 1945; Broun and Bode,

M

2002). In our transplantation experiments, the shoot growing tip always induced the formation of the complete internode, but never the hydranth. Although the isolated shoot growing tip autonomously passes through the complete morphogenetic cycle of internode development, in

d

the end it differentiates itself into the hydranth “head” consisting of the hypostome and the

te

tentacles (Crowell, 1961; Kosevich, 2013). The molecular nature of the shoot growing tip organizer is unknown. However, given the established role of Wnt signaling in the

Ac ce p

hypostomal organizer of the colonial athecate H. echinata (Duffy et al., 2010), a similar mechanism could be involved in the functioning of the shoot growing tip organizer that evolved in the Leptothecata.

4.3. The stolon growing tip

One might wonder why the stolon growing tip was not considered as a candidate organizer region. Indeed, there are several studies that have demonstrated an inductive ability of this structure. In some hydrozoan species, the stolon growing tip is able to induce the neighboring stolon to form a lateral tip, i.e. a new branch. The stolon growing tip can act over a distance of up to 140 μm. It also attracts the induced tip, guiding its growth. Upon contact, both tips fuse, forming an anastomosis (Müller et al, 1987; Plickert, 1987; Lange and Müller, 1991). However, the inducing activity of the stolon growing tip is not conserved among hydrozoans. Stolonial anastomoses are very typical in athecates but are very rare in thecates (e.g., E. 15

Page 15 of 38

viridula) and are viewed as a species-specific (diagnostic) feature (Naumov, 1969). Our model species (G. loveni, L. flexuosa and D. pumila) did not form anastomoses either during their normal development or in the experiments when the growing tip and the host stolon were forced to come into close contact. A parallel may be drawn between the stolon growing tip and the pedal disk of the Hydra polyp. Transplantation of the pedal disk fragment to the body

ip t

column of a host Hydra leads to the formation of a secondary foot, not a secondary body column (e.g., Yao, 1945). Similarly, the stolon growing tips always induce stolons, not colony

cr

modules. Hence, we consider the stolon growing tip to be an inducer rather than an organizer.

us

4.4. Perspectives of studying organizers in colonial hydroids: continuity of the organizer region

In all cnidarian species studied in this respect, the oral–aboral polarity of the polyp could be

an

traced back to the polarity of the oocyte (Teissier, 1931; Freeman, 1980, 1981a,b; Momose and Schmid, 2006; Plickert et al., 2006; Momose and Houliston, 2007; Fritzenwanker et al.,

M

2007). The oral pole of the polyp corresponds to the animal pole of the egg where the polar bodies are given off, the female pronucleus is located and the first cleavage furrow appears. In unipolarly gastrulating species, this is also the place where the endoderm forms. In planulae

d

of all cnidarians, this pole becomes the swimming posterior end of the larva. In several

te

cnidarians, the organizer capacity of the oral region or of the prospectively oral region has been demonstrated (Browne, 1909; Beadle and Booth, 1938; Moore, 1952; Kraus et al., 2007;

Ac ce p

Stumpf et al., 2010; Kraus, 2011; present study). However, it remains unknown whether the organizing capacity is retained in a certain region of the cnidarian body throughout development. In order to address this question, the investigation of a species with documented organizers at the embryonic, larval and polyp stages is required. In Nematostella vectensis (Anthozoa), the transplantation of a small fragment of the blastopore lip induces the formation of a secondary body axis in a host embryo (Kraus et al., 2007). In hydrozoans, transplantation experiments have never been performed on embryos. However, the similarity of the molecular signatures of the Hydra hypostome, of the N. vectensis blastopore area, and of the prospective oral ends of hydrozoan embryos (Clytia hemisphaerica and H. echinata) (Lee et al., 2006; Plickert et al., 2006; Momose and Houliston, 2007; Momose et al., 2008; Bode, 2012) suggests that hydrozoan embryos may also possess organizers. The scarcity of data can be partially explained by technical difficulties. In many colonial hydroids, including our objects, gastrulation proceeds through 16

Page 16 of 38

delamination and is not restricted to any particular site in an embryo. Thus, the embryo has no morphological landmarks of the future oral pole, and direct examination of the candidate organizer region at the gastrula stage requires establishing a blastomere labeling technique suitable for embryos of the cold water species, whose development takes longer than 3 days and which, at least currently, cannot be cultured in a laboratory.

ip t

It is of particular interest to find out whether the organizer is retained throughout metamorphosis, as the fate of the planula posterior tip may depend on the structure of the

cr

young colony that is formed during metamorphosis. It is quite apparent that the posterior organizer of the larva corresponds to the hypostome of the athecate primary polyp (Fig. 9a–c).

us

It is also likely that in thecates one can trace the developmental succession of regions transforming one into the other through metamorphosis and hypothetically retaining the organizer capacity. In the sympodially growing thecates, the posterior tip of a planula gives

an

rise to the growing tip of a primary shoot, which forms the first internode and differentiates itself into the hypostome of the first hydranth (Fig. 9a, b, d, e, g and i). However, it is unlikely

M

that the larval organizer somehow contributes to the development of the first growing tip of the young sympodial colony (Fig. 9i). In the monopodially growing thecates, the developmental transition seems even more complex, as the posterior tip of a planula gives rise

d

to the growing tip of a primary shoot, which, at the end of the morphogenetic cycle, splits to

te

differentiate itself into the first growing tip of the young colony and into the hypostomes of the first pair of hydranths (Fig. 9a, b, d, e, f and h). These developmental successions reflect

Ac ce p

the increasing complexity of metamorphosis in thecates compared with athecates, and an increasing complexity of the thecate colony structure. Labeling experiments on the planulae of colonial hydroids with different morphologies of the young colony will have to be designed and performed to prove or reject our assumptions on organizer continuity. The coexistence of multiple organizer regions at the same time within the same organism is a characteristic feature of hydrozoan colonies. It is doubtful that the larval organizer or the primary organizer of the young colony contributes to the formation of all adult organizers. It is likely that colonial hydrozoans have molecular mechanisms for the de novo formation of the organizer, as a new organizer region is formed during every morphogenetic cycle providing the development of a new colony module. These mechanisms, as well as the mechanisms responsible for the coexistence and cofunctioning of multiple organizers within the same colony, have never been studied. 17

Page 17 of 38

4.5. Conclusions In the present study, we demonstrated the inductive capacities of several candidate organizer regions in hydrozoan representatives differing from each other in colony structure. We revealed that organizers function throughout the larval and polypoid stages in colonial hydroids. Investigations of the molecular mechanisms that enable the functioning, de novo

ip t

formation and maintenance of the organizer regions at different stages of the hydrozoan life

cr

cycle are required to complete the story of the organizers in colonial hydrozoans.

Acknowledgments

us

The authors thank the Electron Microscopy Laboratory of the Shared Facilities Center of Lomonosov Moscow State University (MSU) sponsored by the RF Ministry of Education and Science; the Pertsov White Sea Biological Station (WSBS, MSU); Nikolay N. Marfenin

an

(MSU) for discussing the data; Grigory Genikhovich, Stefan Jahnel (University of Vienna) for critical reading of the manuscript. We also thank three anonymous reviewers for their

M

valuable comments. Research was in part supported by a grant of the Council of the President of the Russian Federation (NSH-1801.2014.4) and by a grant of the Russian Foundation for

Ac ce p

te

d

Basic Research (RFBR) (09-04-01487).

18

Page 18 of 38

References Amiel, A.R., Henry, J.Q., Seaver, E.C., 2013. An organizing activity is required for head patterning and cell fate specification in the polychaete annelid Capitella teleta: new insights into cell–cell signaling in Lophotrochozoa. Dev. Biol. 379, 107–122. Beadle, L.C., Booth, F.A., 1938. The reorganization of tissue masses of Cordylophora

ip t

lacustris and the effect of oral cone grafts, with supplementary observations on Obelia gelatinosa. J. Exp. Biol. 15, 303–326.

cr

Beklemishev, W.N., 1969. Principles of Comparative Anatomy of Invertebrates. Vol. 1: Promorphology. Oliver and Boyd, Edinburgh.

us

Benink, H., Wray, G., Hardin, J., 1997. Archenteron precursor cells can organize secondary axial structures in the sea urchin embryo. Development 124, 3461–3470. Berking, S., 2006. Principles of branch formation and branch patterning in Hydrozoa. Int. J.

an

Dev. Biol. 50, 123–134.

Bode, H., 2011. Axis formation in hydra. Ann. Rev. Genetics 45, 105–117.

M

Bode, H.R., 2012. The head organizer in hydra. Int. J. Dev. Biol. 56, 473–478. Broun, M., Bode, H.R., 2002. Characterization of the head organizer in hydra. Development 129, 875–884.

d

Broun, M., Gee, L., Reinhardt, B., Bode, H.R., 2005. Formation of the head organizer in

te

Hydra involves the canonical Wnt pathway. Development 132, 2907–2916. Browne, E.N., 1909. The production of new hydranths in Hydra by the insertion of small

Ac ce p

grafts. J. Exp. Zool. 7, 1–23.

Buss, L.W., 1979. Bryozoan overgrowth interactions – the interdependence of competition for space and food. Nature 281, 475–477.

Cartwright, P., Evans, N.M., Dunn, C.W., Marques, A.C., Miglietta, M.P., Schuchert, P., Collins, A.G., 2008. Phylogenetics of Hydroidolina (Hydrozoa: Cnidaria). JMBA 88, 1663–1672.

Cherry Vogt, K.S., Harmata, K.L., Coulombe, H.L., Bross, L.S., Blackstone, N.W., 2011. Causes and consequences of stolon regression in a colonial hydroid. J. Exp. Biol. 214, 3197–3205. Crowell, S., 1953. The regression–replacement cycle of hydranths of Obelia and Campanularia. Physiol. Zool. 26, 319–327. Crowell, S., 1957. Differential responses of growth zones to nutritive level, age, and temperature in the colonial hydroid Campanularia. J. Exp. Zool. 134, 63–90. 19

Page 19 of 38

Crowell, S., 1961. Developmental problems in Campanularia. In: Lenhoff, H.M., Loomis, W.F. (Eds.), The Biology of Hydra and of Some Other Coelenterates. University of Miami Press, Coral Gables, pp. 297–316. De Robertis, E.M., 2006. Spemann’s organizer and self-regulation in amphibian embryos. Nat. Rev. Mol. Cell Biol. 7, 296–302.

ip t

Duffy, D.J., Plickert, G., Kuenzel, T., Tilmann W., Frank, U., 2010. Wnt signaling promotes oral but suppresses aboral structures in Hydractinia metamorphosis and regeneration.

cr

Development 137, 3057–3066.

Freeman, G., 1980. The role of cleavage in the establishment of the anterior–posterior axis of

us

the hydrozoan embryo. In: Tardent, P., Tardent, R. (Eds.), Developmental and Cellular Biology of Coelenterates. Elsevier/North-Holland, Amsterdam, pp. 97–108. Freeman, G., 1981a. The cleavage initiation site establishes the posterior pole of the

an

hydrozoan embryo. Roux's Arch. Dev. Biol. 190, 123–125.

Freeman, G., 1981b. The role of polarity in the development of the hydrozoan planula larva.

M

Roux's Arch. Dev. Biol. 190, 168–184.

Fritzenwanker, J.H., Genikhovich, G., Kraus, Y., Technau, U., 2007. Early development and 279.

d

axis specification in the sea anemone Nematostella vectensis. Dev. Biol. 310, 264–

te

Garcia-Fernandez, J., D’Aniello, S., Escriva, H., 2007. Organizing chordates with an organizer. BioEssays. 29, 619–624.

Ac ce p

Hale, L.J., 1964. Cell movements, cell division and growth in the hydroid Clytia johnstoni. J. Embryol. Exp. Morphol. 12, 517–538.

Hale, L.J., 1973. The pattern of growth of Clytia johnstoni. J. Embryol. Exp. Morphol. 29, 283–309.

Hobmayer, B., Rentzsch, F., Kuhn, K., Happel, C.M., von Laue, C.C., Snyder, P., Rothbächer, U., Holstein, T.W., 2000. WNT signalling molecules act in axis formation in the diploblastic metazoan Hydra. Nature 407, 186–189.

Hudson, C., Lemaire, P., 2001. Induction of anterior neural fates in the ascidian Ciona intestinalis. Mech. Dev. 100, 189–203. Huxley, J.S., de Beer, G.R., 1923. Studies in dedifferentiation. IV. Resorption and differential inhibition in Obelia and Campanularia. Q. J. Microsc. Sci. 67, 473–495.

20

Page 20 of 38

Itow, T., Kenmochi, S., Mochizuki, T., 1991. Induction of secondary embryos by intra- and interspecific grafts of center cells under the blastopore in horseshoe crabs. Dev. Growth Differ. 33, 251–258. Kosevich, I.A., 1990. Development of stolon’s and stem’s internodes in the hydroid genus Obelia (Campanulariidae). Vestn. Mosk. Univ. Ser. 16: Biol. No. 3, 26–32 (in

ip t

Russian).

Kosevich, I.A., 1991. Comparison of upright’s and stolon’s tips function in hydroid colony

cr

Obelia loveni (Allm.) (Hydrozoa, Campanulariidae). Vestn. Mosk. Univ. Ser. 16: Biol. No.2, 44–52 (in Russian).

us

Kosevich, I.A., 2006. Changes in the patterning of a hydroid colony. Zoology 109, 244–259. Kosevich, I.A., 2012. Morphogenetic foundations for increased evolutionary complexity in organization

of

thecate

hydroids

Leptomedusae). Biol. Bull. 39, 172–185.

shoots

(Cnidaria,

Hydroidomedusa,

an

the

Kosevich, I.A., 2013. Cell migration during growth and morphogenesis in thecate hydroids.

M

Mar. Ecol. 34 (S1), 83–95.

Kossevitch, I.A., 1999. Cell migration during growth of hydroid colony. Zh. Obshch. Biol. 60, 91–98 (in Russian).

d

Kraus, Y.A., 2011. Inductive activity of the posterior tip of planula in the marine hydroid

te

Dynamena pumila. Russ. J. Dev. Biol. 42, 92–100. Kraus, Y., Fritzenwanker, J.H., Genikhovich, G., Technau, U., 2007. The blastoporal

Ac ce p

organiser of a sea anemone. Curr. Biol. 17, 74–76.

Lambert, J.D., 2008. Mesoderm in spiralians: the organizer and the 4d cell. J. Exp. Zool. B Mol. Dev. Evol. 310, 15–23.

Lange, R.G., Müller, W.A., 1991. SIF, a novel morphogenetic inducer in Hydrozoa. Dev. Biol. 147, 121–132.

Lee, P.N., Pang, K., Matus, D.Q., Martindale, M.Q., 2006. A WNT of things to come: evolution of Wnt signaling and polarity in cnidarians. Semin. Cell Dev. Biol. 17, 157– 167.

Lengfeld, T., Watanabe, H., Simakov, O., Lindgens, D., Gee, L., Law, L., Schmidt, H.A., Özbek, S., Bode, H., Holstein, T.W., 2009. Multiple Wnts are involved in Hydra organizer formation and regeneration. Dev. Biol. 330, 186–199. Lenhoff, H.M., 1991. Ethel Browne, Hans Spemann, and the discovery of the organizer phenomenon. Biol. Bull. 181, 72–80. 21

Page 21 of 38

Marfenin, N.N., 1997. Adaptation capabilities of marine modular organisms. Hydrobiologia 355, 153–158. Marfenin, N.N., Kosevich, I.A., 1984. Biology of the hydroid Obelia loveni (Allm.): colony formation, behavior and life cycle of hydranths, reproduction. Vestn. Mosk. Univ. Ser. 16: Biol. 3, 16–24 (in Russian).

ip t

Marfenin, N.N., Kosevich, I.A., 2004. Morphogenetic evolution of hydroid colony pattern. Hydrobiologia 530/531, 319–327.

cr

Martinez, D., Bridge, D., 2012. Hydra, the everlasting embryo, confronts aging. Int. J. Dev. Biol. 56, 479–487.

us

Millonig, G., 1964. Study on the factors which influence preservation of fine structure. In: Buffa, P. (Ed.), Symposium on Electron Microscopy. Consiglio Nazionale delle Ricerche, Rome, p. 347.

an

Mironov, A.A., Komissarchik, J.J., Mironov, V.A., 1994. Methods of Electron Microscopy in Biology and Medicine. Nauka, St. Petersburg (in Russian). determinants

in

a

M

Momose, T., Houliston, E., 2007. Two oppositely localised frizzled RNAs as axis cnidarian

10.1371/journal.pbio.0050070.

embryo.

PLoS

Biol.

5:

e70,

doi:

d

Momose, T., Schmid, V., 2006. Animal pole determinants define oral–aboral axis polarity and 380.

te

endodermal cell-fate in hydrozoan jellyfish Podocoryne carnea. Dev. Biol. 292, 371–

Ac ce p

Momose, T., Derelle, R., Houliston, E., 2008. A maternally localized Wnt ligand required for axial patterning in the cnidarian Clytia hemisphaerica. Development 135, 2105–2113.

Moore, J., 1952. The induction of regeneration in the hydroid Cordylophora lacustris. J. Exp. Biol. 29, 72–93.

Morse, M., 1909. On the cause of autotomy in Tubularia. Biol. Bull., 16, 172–182. Müller, W.A., Hauch, A., Plickert, G., 1987. Morphogenetic factors in hydroids. 1. Stolon tip activation and inhibition. J. Exp. Zool. 243, 111–124.

Müller, W.A., Frank, U., Teo, R., Mokady, O., Guette, C., Plickert, G., 2007. Wnt signaling in hydroid development: ectopic heads and giant buds induced by GSK3b inhibitors. Int. J. Dev. Biol. 51, 211–220. Nakamoto, A., Nagy, L.M., Shimizu, T., 2011. Secondary embryonic axis formation by transplantation of D quadrant micromeres in an oligochaete annelid. Development 138, 283–290. 22

Page 22 of 38

Naumov, D.V., 1969. Hydroids and Hydromedusae of the USSR. Israel Program for Scientific Translations, Jerusalem. Nawrocki, A., Cartwright, P., 2013. Expression of Wnt pathway genes in polyps and medusalike structures of Ectopleura larynx (Cnidaria: Hydrozoa). Evol. Dev. 15, 373–384. Plickert, G., 1987. Low-molecular-weight factors from colonial hydroids affect pattern

ip t

formation. Roux's Arch. Dev. Biol. 196, 248–256.

Plickert, G., Heringer, A., Hiller, B., 1987. Analysis of spacing in a periodic pattern. Dev.

cr

Biol. 120, 399–411.

Plickert, G., Jacoby, V., Frank, U., Müller, W.E., 2006. Wnt signaling in hydroid

us

development: formation of the primary body axis in embryogenesis and subsequent patterning. Dev. Biol. 298, 368–378.

Polteva, D.G., Donakov, V.V., Markova, L.V., 1988. Cellular basis of morphogenesis in the

an

hydrozoan polyps Obelia loveni and Clava multicornis. In: Koltoon, V.M., Stepanyants, S.D. (Eds.), Sponges and Cnidarians. Contemporary and Prospective

M

Investigations. Zoological Institute of the Russian Academy of Sciences, Leningrad, pp. 111–116 (in Russian).

Pyataeva, S., 2007. Transformation of Anatomy and Morphology of the Marine Colonial

te

University, Moscow.

d

Hydroids (Hydrozoa) in the Course of Ontogeny. Ph.D. Thesis, Moscow State Ransick, A., Davidson, E.H., 1993. A complete second gut induced by transplanted

Ac ce p

micromeres in the sea urchin embryo. Science 259, 1134–1138.

Rosen, B.R., 1979. Modules, members and communes: a postscript introduction to social organisms. In: Larwood, G., Rosen, B.R. (Eds.), Biology and Systematics of Colonial Organisms. Academic Press, London, pp. 23–35.

Sander, K., Faessler, P., 2001. Introducing the Spemann–Mangold organizer: experiments and insights that generated a key concept in developmental biology. Int. J. Dev. Biol. 45 (S1), 1–12.

Seipp, S., Wittig, K., Stiening, B., Böttger, A., Leitz, T., 2006. Metamorphosis of Hydractinia echinata (Cnidaria) is caspase-dependent. Int. J. Dev. Biol. 50, 63–70. Seipp, S., Schmich, J., Kehrwald, T., Leitz, T., 2007. Metamorphosis of Hydractinia echinata – natural versus artificial induction and developmental plasticity. Dev. Genes Evol. 217, 385–394. 23

Page 23 of 38

Sethi, A.J., Angerer, R.C., Angerer, L.M., 2009. Gene regulatory network interactions in sea urchin

endomesoderm

induction.

PLoS

Biol.

7(2),

e1000029,

doi:

10.1371/journalpbio.1000029. Smith, J.L., Schoenwolf, G.C., 1998. Getting organized: new insights into the organizer of higher vertebrates. Curr. Top. Dev. Biol. 40, 79–111.

ip t

Spemann, H., Mangold, H., 1924. Über Induktion von Embryonalanlagen durch Implantation artfremder Organisatoren. Arch. Mikr. Anat. Entw.-Mech. 100, 599–638.

cr

Strehler, B., 1961. Aging in coelenterates. In: Lenhoff, H.M., Loomis, W.F. (Eds.), The Biology of Hydra and of Some Other Coelenterates. University of Miami Press, Coral

us

Gables, pp. 374–395.

Stumpf, M., Will, B., Wittig, K., Kasper, J., Fischer, B., Schmich, J., Seipp, S., Leitz, T., 2010. An organizing region in metamorphosing hydrozoan planula larvae –

an

stimulation of axis formation in both larval and in adult tissue. Int. J. Dev. Biol. 54, 795–802.

M

Tardent, P., 1963. Regeneration in the Hydrozoa. Biol. Rev. 3, 293–333. Thatcher, H.F., 1903. Absorption of the hydranth in hydroid polyps. Biol. Bull. 5, 295–303. Teissier, G., 1931. Étude expérimentale du developement de quelques hydraires. Ann. Sci.

d

Nat. Ser. Bot. Zool. 14, 5–60.

te

Wyttenbach, C.R., Crowell S., Suddith R.L., 1965. The cyclic elongation of stolons and uprights in the hydroid, Campanularia. Biol. Bull. 129, 429.

Ac ce p

Yao, T., 1945. Studies on the organizer problem in Pelmatohydra oligactis. I. The induction potency of the implants and the nature of the induced hydranth. J. Exp. Biol. 21, 147– 150.

24

Page 24 of 38

Figure captions Fig. 1. Representative hydrozoan species exhibiting different types of colony structure (a, c, d, f and g) and schematic drawings of their colonies (b, e, h), respectively. Clava multicornis (a and b) is an athecate hydrozoan with a simple stolonial colony. Gonothyraea loveni (c and

ip t

e) and Laomedea flexuosa (d and e) are thecate species with a sympodial mode of colony growth. Dynamena pumila (f–h) is a thecate species with a monopodial mode of colony

cr

growth. (f) Two distal modules of the shoot of a D. pumila colony. Scale bars in a, c, and d = 1 mm; in f = 250 m. Abbreviations: fh, forming hydranth; fs, forming shoot; gr, gastral

us

region of a hydranth; g, gonophores; ha, hydranth anlage; hb, hydranth bud; h, hydranth; hy, hypostome; in, internode; p, hydranth pedicel; st, stolon; stgt, stolon growing tip; s, shoot of a

an

colony; sgt, shoot growing tip; t, tentacles.

Fig. 2. Experimental design of the study. (a) Transplantation of the posterior tip of a donor

M

planula into the middle part of the body of a recipient planula. (b) Transplantation of the hypostome fragment from a donor hydranth into the subtentacle region of a recipient hydranth cut above the distal end of its pedicel. (c) Grafting of a shoot growing tip fragment into the

d

proximal part of the recipient internode. (d) Grafting of a shoot growing tip fragment into the

te

ball-like cell mass formed in the course of hydranth regression. Abbreviations: A(Ab), anterior (aboral) end of a planula; hy, hypostome; MS, metamorphosis stimulation; P(Or), tip.

Ac ce p

posterior (oral) end of a planula; RH, regression of an isolated hydranth; sgt, shoot growing

Fig. 3. Transplantation of the posterior tip of a Gonothyraea loveni donor planula into the mid-body region of the recipient planula. (a) Recipient planulae in which superficially located grafts were displaced towards the posterior pole during wound healing. White arrows point at the graft. (b) Recipient planula with a completely incorporated graft. (c and d) Outcomes of the metamorphosis typical of control planulae (c) and recipient planulae with completely incorporated grafts (d): one primary shoot grows from the anchoring disk formed in the course of planula metamorphosis. (e and f) Two primary shoots growing from the anchoring disk formed from the planula with a superficially located graft. Scale bars in a and b = 100 µm; in c–f = 200 µm. Abbreviations: A(Ab), anterior (aboral) end of a planula; ad, anchoring disk formed in the course of planula settlement and flattening; P(Or), posterior (oral) end of a 25

Page 25 of 38

planula; ps, primary shoot of a new colony growing from the anchoring disk; ps1 and ps2, recipient and ectopic primary shoots, respectively; sgt, shoot growing tip; sgt1 and sgt2, growing tips of recipient and ectopic shoots, respectively. Fig. 4. Transplantation of a hypostome fragment of the donor Clava multicornis hydranth into

ip t

the subtentacle region of the recipient hydranth. Secondary hydranths formed at the grafting sites are as follows: (a) secondary hydranth containing a hypostome and a tentacle region; (b)

cr

secondary hydranth with elongated body column. Scale bar = 1 mm. Abbreviations: gr, gastral region of a hydranth; hy1 and t1, hypostome and tentacles of the recipient hydranth; hy2 and

us

t2, hypostome and tentacles of an ectopic hydranth formed at the grafting site; p, hydranth pedicel.

an

Fig. 5. Implantation of a shoot growing tip fragment into the proximal part of the recipient internode. Shoots of Gonothyraea loveni (a) and Laomedea flexuosa (b) isolated from

M

colonies served as controls. Shoots of G. loveni (c) and L. flexuosa (e) engrafted with fragments of donor growing tips developed ectopic internodes at the grafting sites. (d) Closeup view of an ectopically induced internode of G. loveni. Inset shows the donor's shoot

d

growing tip. An apical fragment of the growing tip that was used in the grafting experiment is

te

framed. White arrows point at the cutting site (a and b) or at the cutting and implantation site (c–e). Scale bars = 2 mm (all except d); = 500 µm (d): = 50 µm (d, inset). Abbreviations: daz,

Ac ce p

distal annulation zone of the induced internode; h, hydranth; in, internode; paz, proximal annulation zone of the induced internode; paz(r), proximal annulation zone of the proximal recipient internode; rh, regressing hydranths; sgt, shoot growing tip; st, stolon; sz, smooth zone of the induced internode.

Fig. 6. Regression and restoration of Laomedea flexuosa hydranths. (a) Intact hydranth. The dotted line marks the line of the hydranth cuts in the experiments. (b and c) Successive stages of hydranth regression. (d) Ball-like cell mass formed during the last stage of regression of the isolated hydranth shown together with an apical fragment of the donor's shoot growing tip prepared for implantation. (e–j) Successive stages of hydranth formation from a ball-like cell mass engrafted with the shoot growing tip fragment: (e) 0.5 h post implantation (hpi); (f) 4 hpi; (g) 10 hpi; (h) 16 hpi; (i) 24 hpi; (j) 30 hpi. White arrowheads point at the perisarc. Scale bars = 500 µm (a); = 100 µm (b–j). Abbreviations: gr, gastral region of a hydranth; ha, 26

Page 26 of 38

hydranth anlage; hy, hypostome; cm, cell mass; sgt, shoot growing tip; t, tentacles; tb, tentacle bud. Fig. 7. Histological cross-sections and TEM micrographs of the regressing hydranths and hydranths developing from the cell masses engrafted with fragments of shoot growing tips. (a)

ip t

Cross-section through the gastral region of the intact hydranth. Black arrowhead points at the perisarc. (b) Cross-section through the gastral region of the isolated hydranth at the first stage

cr

of regression (12 h post isolation, formation of the inner cell mass). (c) Isolated hydranth at the second stage of regression (15 h post isolation, reduction of the hypostome) sectioned

us

along the oral–aboral axis. The white arrows in (b) and (c) point at the group of elongated gastrodermal cells. (d) Cross-section of the ball-like cell mass formed at the final stage of regression (18 h post isolation). (e and f) TEM of the ball-like cell mass. High magnification

an

of the superficial and inner cells (e) and the inner cells only (f). White arrowheads point at cells containing a structure resembling a phagosome. (g and j) Semi-thin sections of the

M

hydranths developing from the cell mass: 16 hpi (g) and 30 hpi (j). (h and i) TEM close-up views of the regions framed in (g). Note that only the hydranth anlage area is covered with a perisarc (black arrowheads in (h) and (i)) and that the basal lamina is re-established in the cell

d

mass area (i). (k) TEM micrograph showing cells in the gastric cavity of the forming hydranth

te

at 30 hpi. Scale bars = 100 µm (a–d, g, j); = 50 µm (e); = 5 µm (f, k). Abbreviations: bl, basal lamina; cc, cnidocytes observed in the gastric cavity of the forming hydranth; cm, cell mass

Ac ce p

area; ed, epiderm; gd, gastroderm; gc, gastric cavity; ha, hydranth anlage area; hy, hypostome; icm, inner cell mass; scm, superficial cell mass; t, tentacles. Fig. 8. Transplantation experiment performed on a planula of Dynamena pumila, a hydrozoan species with a monopodial mode of growth. (a and b) Successive developmental stages of two primary shoots growing from the anchoring disk which formed during metamorphosis of a recipient planula engrafted with the posterior tip of a donor planula. Abbreviations: ad, anchoring disk formed during planula settlement and flattening; ha, hydranth anlage area; ps1 and ps2, recipient and ectopic primary shoots, respectively; sgt, shoot growing tip. Fig. 9. Schematic drawings illustrating the metamorphosis in colonial hydrozoans: (a and b) settlement and flattening of a planula; (c) transformation of the flattened planula into a primary polyp in athecate hydrozoans; (d) development of the anchoring disc covered with a 27

Page 27 of 38

perisarc in thecate hydrozoans and (e) appearance of the shoot growing tip at the center of this disc; development of the primary shoot in thecates with (f and g) monopodial and (h and i) sympodial growth modes. Abbreviations: A(Ab), anterior (aboral) end of a planula; ad, anchoring disk formed in the course of planula settlement and flattening; h, hydranth; ha, hydranth anlage; hy, hypostome; P(Or), posterior (oral) end of a planula; sgt, newly formed

ip t

shoot growing tip of a young colony; p, perisarc; ps, primary shoot; psgt, growing tip of a

Ac ce p

te

d

M

an

us

cr

primary shoot; st, stolon.

28

Page 28 of 38

Table 1. Quantification of grafting experiments with Clava multicornis (Cm), Gonothyraea loveni (Gl), and Laomedea flexuosa (Lf).

Origin of graft

Site of grafting

Species

Number of experiments

Number of successful experiments*

Posterior tip of a planula

Middle part of the body of a planula

Gl

20

7

4 (57%)

3 (43%)

Control: incisions were made on control planulae without performing transplantations

Gl

20

-

0

20 (100%)

Hypostome fragment of a hydranth

Subtentacle region of a hydranth

Cm

16

12

6 (50%)

6 (50%)

Control: incisions were made on control hydranths without performing transplantations

Cm

-

0

16 (100%)

2

Proximal part of a shoot

3

Ac ce p

4

Ball-like cell mass formed from regressing hydranth

te

Apical fragment of the growing tip of a shoot

d

Control: shoots were isolated from colonies and kept without any additional treatment

Control: hydranths were cut from colonies just beneath the gastral region and kept without any additional treatment

16

cr

ip t

Secondary axis

No secondary axis

Gl

30

10

4 (40%)

6 (60%)

Lf

30

17

8 (47%)

9 (53%)

Lf

30

-

0

30 (100%)

Gl

30

-

0

30 (100%)

M

Apical fragment of the growing tip of a shoot

us

1

an

N

Lf

30

12

12

†

0

(100%)

Lf

50

-

0

††

50 (100%)

* The transplantation was considered successful if a recipient did not reject the donor tissue during wound healing. †

Development of a new hydranth at the site of grafting.

††

The isolated hydranths transformed into ball-like cell masses which died and disintegrated approximately 36 h post isolation. 29

Page 29 of 38

Ac ce p

te

d

M

an

us

cr

ip t

Figure1

Page 30 of 38

Ac

ce

pt

ed

M

an

us

cr

i

Figure2

Page 31 of 38

Ac

ce

pt

ed

M

an

us

cr

i

Figure3

Page 32 of 38

Ac ce p

te

d

M

an

us

cr

ip t

Figure4

Page 33 of 38

Ac ce p

te

d

M

an

us

cr

ip t

Figure5

Page 34 of 38

Ac

ce

pt

ed

M

an

us

cr

i

Figure6

Page 35 of 38

Ac

ce

pt

ed

M

an

us

cr

i

Figure7

Page 36 of 38

Ac

ce

pt

ed

M

an

us

cr

i

Figure8

Page 37 of 38

Ac ce p

te

d

M

an

us

cr

ip t

Figure9

Page 38 of 38