Patterning of the head in hydra as visualized by a monoclonal antibody: III. The dynamics of head regeneration

The dynamics of the early patterning processes leading to the regeneration of a head in tissue excised from the body column of Hydra oligactis were examined by using a monoclonal antibody, CP8. This antibody displays position-specific binding, labeling the head ectodermal epithelial cells. During regeneration of a head, antibody labeling is present well before morphological signs of the head, at a time correlated with the determination of the tissue (Javois et al., Dev. Biol., 117:607-618, '86). By quantifying antibody labeling during regeneration of three different pieces of tissue excised from the body column, it was found that the dynamics of the early patterning processes as visualized by CP8 labeling varied. The pattern of labeling observed as well as the spread of labeled tissue suggested that the amount and geometry of apical tissue in the regenerate played a critical role in the patterning processes. Contrary to the labeling pattern observed in heads which formed during bud development or which regenerated following decapitation (Javois et al., '86), not all the CP8+ tissue was confined to the head structures in these regenerates. Several alternative explanations for this surprising result are presented. The usefulness of these data in refining pattern formation models by more explicitly constraining their parameters is discussed.     

Patterning of the head in hydra as visualized by a monoclonal antibody. I. Budding and regeneration

A monoclonal antibody, CP8, has been isolated which displays a position-specific binding pattern to epithelial cells of Hydra oligactis. Antibody binding is restricted to the head of adult animals. When a new head develops during the budding process, CP8 binding is present in the area which will form the head well before morphological signs of it. Similarly, following decapitation as a new head regenerates, CP8 label appears covering a domed area at the apical end of the regenerate before tentacles evaginate delineating the head. When bud development or regeneration is complete, CP8 label is restricted to the new head. Experiments indicate the appearance of CP8 label during the formation of a head correlates closely with the patterning events which result in the determination of the tissue to form a head. The usefulness of CP8 as a diagnostic tool for exploring the dynamics of head pattern formation in hydra is discussed.     

Head activator does not qualitatively alter head morphology in regenerates ofHydra oligactis

Summary The characterization of head activator (HA) as a morphogen capable of increasing the number of tentacles regenerated by hydra was re-examined. Gastric tissue was excised from HA-treated whole animals and allowed to regenerate. At the cellular level the differentiation of head-specific ectodermal epithelial cells was monitored by quantifying monoclonal antibody, CP8, labeling. This labeling has been correlated with a rise in head activation potential and the determination of tissue to form head structures (Javois et al. 1986). At the morphological level tentacle number was monitored. HA-treated regenerates began the head patterning processes and evaginated tentacles sooner than controls but did not produce extra tentacles. The kinetics of CP8 labeling did not reveal major differences between treated and control regenerates after the initiation of head-specific epithelial cell differentiation. HA appeared to act more like a growth factor stimulating the differentiation of head-specific cell types rather than a morphogen which altered head morphology. An additional aspect of the study examined axial-specific effects of HA on the initiation and extent of head-specific epithelial cell differentiation. The cellular response of ectodermal epithelial cells to HA was dependent on their original axial location. More CP8⁺ tissue differentiated in regenerates of apical as opposed to mid-gastric origin.     

A head signal influences apical migration of interstitial cells in Hydra vulgaris

Although interstitial cells of hydra can migrate either apically or basally along the body column, there is a distinct bias toward apical cell accumulation. This apical bias could be produced by a local vectorial property of the tissue or it may be controlled by a more global property, such as a signal from the apical head region. The migration behavior of BrdU-labeled interstitial cells was examined in several types of grafts to distinguish between these two general types of migration control. Grafting BrdU-labeled midgastric region tissue into a host in either the normal or the reverse orientation had no effect on the apical bias, indicating that a local vectorial cue was probably not guiding cells apically. In grafts with heads or with feet at both ends of the body column, there was no directional bias in migration if the labeled tissue was equidistant from both ends. In the two-headed grafts, if the labeled tissue was closer to one end, there was a bias in the direction of the closer head. The results suggest that a graded signal emanating from the head creates the apical bias and may attract cells via chemotaxis. The apical bias is enhanced in decapitated animals regenerating a head, indicating that the attracting signal is present and is possibly stronger in regenerating heads. The signal for cell migration may be involved in a patterning process underlying head regeneration.     

Notch-signalling is required for head regeneration and tentacle patterning in Hydra

Local self-activation and long ranging inhibition provide a mechanism for setting up organising regions as signalling centres for the development of structures in the surrounding tissue. The adult hydra hypostome functions as head organiser. After hydra head removal it is newly formed and complete heads can be regenerated. The molecular components of this organising region involve Wnt-signalling and β-catenin. However, it is not known how correct patterning of hypostome and tentacles are achieved in the hydra head and whether other signals in addition to HyWnt3 are needed for re-establishing the new organiser after head removal. Here we show that Notch-signalling is required for re-establishing the organiser during regeneration and that this is due to its role in restricting tentacle activation. Blocking Notch-signalling leads to the formation of irregular head structures characterised by excess tentacle tissue and aberrant expression of genes that mark the tentacle boundaries. This indicates a role for Notch-signalling in defining the tentacle pattern in the hydra head. Moreover, lateral inhibition by HvNotch and its target HyHes are required for head regeneration and without this the formation of the β-catenin/Wnt dependent head organiser is impaired. Work on prebilaterian model organisms has shown that the Wnt-pathway is important for setting up signalling centres for axial patterning in early multicellular animals. Our data suggest that the integration of Wnt-signalling with Notch-Delta activity was also involved in the evolution of defined body plans in animals.     

Formation of pattern in regenerating tissue pieces of Hydra attenuata. III. The shaping of the body column

Excised pieces of hydra body tissue of varying size and shape regenerate into cylinders with a head and foot at opposite ends. The numbers of cells along the axial and circumferential dimensions were determined before, during, and after regeneration. The main process in shaping the excised tissue into a body column was found to be a rearrangement of the cells. When regenerates of different size were measured, the proportions of the body columns were found to vary, such that the smaller the animal the squatter the body column was. The presence of the head in regenerates was necessary for the formation or maintenance of the cylindrical shape, while the size of the head determined the proportions of the cylinder. The formation of a gradient of adhesivity induced by the developing head is suggested as the basis for the rearrangement of the cells into the cylindrical form.     

Ectopic pharynxes arise by regional reorganization after anterior/posterior chimera in planarians

To elucidate the mechanisms underlying pharynx regeneration in planarians, we transplanted pieces excised from various regions of the body into the prepharyngeal or postpharyngeal region, since it has been shown that such transplantation experiments can induce ectopic pharynx formation. We confirmed the ectopic formation of pharynxes by expression of the myosin heavy chain gene specific to pharynx muscles (DjMHC-A). To investigate the cellular events after grafting, we also stained such transplanted worms by in situ hybridization using neuronal cell- and mucous producing cell-type-specific marker genes which can detect formation of brain and prepharyngeal region, respectively. When the head piece was transplanted into the tail region, ectopic formation of the head, prepharyngeal and pharynx region was observed in the postpharyngeal region anterior to the graft, while these organs were formed in the reversed polarity along the anterior-posterior (A-P) axis. Furthermore, in the tail region posterior to the graft, ectopic formation of the prepharyngeal and pharynx region was observed. In the reverse combination, when a tail piece was transplanted into the prepharyngeal region, ectopic formation of prepharyngeal and pharynx region was observed in the region between the head and the graft, and an additional ectopic pharynx was also formed in reverse polarity in the region between the graft and host pharynx. These results clearly indicated that ectopic pharynxes were formed as a consequence of the regional reorganization induced by interaction between the host and graft. Furthermore, chimeric analyses demonstrated that the cells participating in ectopic pharynx formation were not exclusively derived from the host or donor cells in the worm, suggesting that the stem cells of the host and donor may change their differentiation pattern due to altered regionality. To further investigate if regional reorganization is induced after grafting, expression of a Hox gene was analyzed in the transplanted worms by whole-mount in situ hybridization. The expression of the Hox gene along the A-P axis was apparently rearranged after grafting of the head piece into the tail region. These results suggest that grafting of the head piece may rearrange the regionality of the host tail, and that stem cells in the region newly defined as pharynx-forming may start to regenerate a pharynx.     

Development of the two-part pattern during regeneration of the head in hydra

The head of a hydra is composed of two parts, a domed hypostome with a mouth at the top and a ring of tentacles below. When animals are decapitated a new head regenerates. During the process of regeneration the apical tip passes through a transient stage in which it exhibits tentacle-like characteristics before becoming a hypostome. This was determined from markers which appeared before morphogenesis took place. The first was a monoclonal antibody, TS-19, that specifically binds to the ectodermal epithelial cells of the tentacles. The second was an antiserum against the peptide Arg-Phe-amide (RFamide), which in the head of hydra is specific to the sensory cells of the hypostomal apex and the ganglion cells of the lower hypostome and tentacles. The TS-19 expression and the ganglion cells with RFamide-like immunoreactivity (RLI) arose first at the apex and spread radially. Once the tentacles began evaginating in a ring, both the TS-19 antigen and RLI+ ganglion cells gradually disappeared from the presumptive hypostome area and RLI+ sensory cells appeared at the apex. By tracking tissue movements during morphogenesis it became clear that the apical cap, in which these changes took place, did not undergo tissue turnover. The implications of this tentacle-like stage for patterning the two-part head are discussed.     

Pattern formation in hydra tissue without developmental gradients

Ring-shaped pieces of hydra tissue were excised from a specified position on a body column of 20-30 polyps and grafted together in tandem like a chain of beads. A "tandem graft" prepared in this way has the same basic tissue organization and same tube-like morphology as a normal hydra body column, but lacks the head, foot, and developmental gradients ordinarily present. Three major types of structures were formed along the length of the tandem graft: heads, buds, and feet. The relative number of these structures produced was strongly affected by the origin of the tissue used to prepare the tandem graft. Evidence was obtained which suggests that tissue originally located outside of the budding zone in intact hydra has a strong latent capacity to form a bud, and that the level of this capacity forms a gradient from the budding zone toward the hypostome. Evidence was also obtained which is consistent with the view that the head and foot forming mechanisms cross-react positively, increasing the chances for these two structures to be formed next to each other on a tandem graft.     

Characterization of the head organizer in hydra

A central process in the maintenance of axial patterning in the adult hydra is the head activation gradient, i.e. the potential to form a secondary axis, which is maximal in the head and is graded down the body column. Earlier evidence suggested that this gradient was based on a single parameter. Using transplantation experiments, we provide evidence that the hypostome, the apical part of the head, has the characteristics of an organizer in that it has the capacity to induce host tissue to form most of the second axis. By contrast, tissue of the body column has a self-organizing capacity, but not an inductive capacity. That the inductive capacity is confined to the hypostome is supported by experiments involving a hypostome-contact graft. The hypostome, but not the body column, transmits a signal(s) leading to the formation of a second axis. In addition, variations of the transplantation grafts and hypostome-contact grafts provide evidence for several characteristics of the organizer. The inductive capacity of the head and the self-organizing capacity of the body column are based on different pathways. Head inhibition, yya signal produced in the head and transmitted to the body column to prevent head formation, represses the effect of the inducing signal by interfering with formation of the hypostome/organizer. These results indicate that the organizer characteristics of the hypostome of an adult hydra are similar to those of the organizer region of vertebrate embryos. They also indicate that the Gierer-Meinhardt model provides a reasonable framework for the mechanisms that underlie the organizer and its activities. In addition, the results suggest that a region of an embryo or adult with the characteristics of an organizer arose early in metazoan evolution.     

HyBMP5-8b, a BMP5-8 orthologue, acts during axial patterning and tentacle formation in hydra

Developmental gradients play a central role in axial patterning in hydra. As part of the effort towards elucidating the molecular basis of these gradients as well as investigating the evolution of the mechanisms underlying axial patterning, genes encoding signaling molecules are under investigation. We report the isolation and characterization of HyBMP5-8b, a BMP5-8 orthologue, from hydra. Processes governing axial patterning are continuously active in adult hydra. Expression patterns of HyBMP5-8b in normal animals and during bud formation, hydra's asexual form of reproduction, were examined. These patterns, coupled with changes in patterns of expression in manipulated tissues during head regeneration, foot regeneration as well as under conditions that alter the positional value gradient indicate that the gene is active in two different processes. The gene plays a role in tentacle formation and in patterning the lower end of the body axis.     

Hym-301, a novel peptide, regulates the number of tentacles formed in hydra

Hym-301 is a peptide that was discovered as part of a project aimed at isolating novel peptides from hydra. We have isolated and characterized the gene Hym-301, which encodes this peptide. In an adult, the gene is expressed in the ectoderm of the tentacle zone and hypostome, but not in the tentacles. It is also expressed in the developing head during bud formation and head regeneration. Treatment of regenerating heads with the peptide resulted in an increase in the number of tentacles formed, while treatment with Hym-301 dsRNA resulted in a reduction of tentacles formed as the head developed during bud formation or head regeneration. The expression patterns plus these manipulations indicate the gene has a role in tentacle formation. Furthermore, treatment of epithelial animals indicates the gene directly affects the epithelial cells that form the tentacles. Raising the head activation gradient, a morphogenetic gradient that controls axial patterning in hydra, throughout the body column results in extending the range of Hym-301 expression down the body column. This indicates the range of expression of the gene appears to be controlled by this gradient. Thus, Hym-301 is involved in axial patterning in hydra, and specifically in the regulation of the number of tentacles formed.     

PI3K and ERK 1-2 regulate early stages during head regeneration in hydra

Different signaling systems coordinate and regulate the development of a multicellular organism. In hydra, the canonical Wnt pathway and the signal transduction pathways mediated by PKC and Src regulate early stages of head formation. In this paper, we present evidence for the participation of a third pathway, the PI3K-PKB pathway, involved in this process. The data presented here are consistent with the participation of ERK 1-2 as a point of convergence for the transduction pathways mediated by PKC, Src and PI3K for the regulation of the regeneration of the head in hydra. The specific developmental point regulated by them appears to be the commitment of tissue at the apical end of the regenerate to form the head organizer.     

APICAL DOMINANCE,POLARITY, AND ADVENTITIOUS GROWTH IN MARCHANTIA POLYMORPHA

Benzylaminopurine, indoleacetic acid, and the auxin inhibitors transcinnamic acid, triiodobenzoic acid, and dinitrophenol were employed to elucidate the role of apical dominance and hormones during regeneration of thalli and gemmae of Marchantia polymorpha L. The cytokinin suppressed normal gemma germination and led to the development of nodular, callus-like growths. When removed from the influence of benzylaminopurine, the site and magnitude of normal thallus outgrowths varied with the length of time that the tissue had remained on the cytokinin-containing medium. This aberrant germination was not influenced by the incorporation of indoleacetic acid into the medium. Exogenous auxin neither accelerated nor inhibited the regeneration of normal thallus growth on excised vegetative discs. Transcinnamic acid and dinitrophenol inhibited regeneration. Auxin reversed this suppression. Triiodobenzoic acid did not significantly affect regeneration. Autoradiographs demonstrated a pronounced accumulation of labelled auxin in the midribs and the acropetal regions of excised thallus discs. This evidence suggests that there is an endogenous, basipetal auxin gradient in Marchantia; that the maintenance of this gradient is vital to normal growth and regeneration of the thallus; and that high endogenous concentrations of cytokinin destroy this polarity by increasing the auxin-synthesizing capacity of the tissue.     

Microsurgery reveals regional capabilities for pattern reestablishment in somatic carrot embryos

The extent to which regions of a somatic embryo were committed to a particular developmental fate was explored by surgically removing portions of somatic embryos and observing patterns of regeneration. Through a variety of excisions that resulted in tissue slices ranging from less than 10% to nearly 90% of the original embryo mass, we observed only a few cases where such isolates completely abandoned preexisting patterns of organized growth. Instead, most subcultured portions of the embryonic axis restored all, or part of, a missing complement of the organism. At the shoot apex, a single lost cotyledon was replaced by new cotyledonary structures, although these usually occurred as multiple pairs of cotyledons. If both cotyledons were removed, secondary axes, each with its own cotyledons, typically formed at the embryo midlength. When embryos were divided into shoot and root pieces, the shoot pole usually regenerated a new root, while the original root and rapidly elongated and matured days earlier than uncut controls. Surprisingly, cotyledon regeneration from excised root sections occurred at much greater frequency when the root piece comprised only 10-25% of the embryo mass; larger portions of the root pole rarely produced recognizable shoot structures. These studies indicate that several discrete regions of the embryo are committed to specific types of patterned growth, and that continuity between certain of these regions is required for the maintenance of axial polarity.     

Formation of the head organizer in hydra involves the canonical Wnt pathway

Stabilization of beta-catenin by inhibiting the activity of glycogen synthase kinase-3beta has been shown to initiate axis formation or axial patterning processes in many bilaterians. In hydra, the head organizer is located in the hypostome, the apical portion of the head. Treatment of hydra with alsterpaullone, a specific inhibitor of glycogen synthase kinase-3beta, results in the body column acquiring characteristics of the head organizer, as measured by transplantation experiments, and by the expression of genes associated with the head organizer. Hence, the role of the canonical Wnt pathway for the initiation of axis formation was established early in metazoan evolution.     

Interactions between the Foot and the Head Patterning Systems in Hydra vulgaris

The Cnidarian, hydra, is an appealing model system for studying the basic processes underlying pattern formation. Classical studies have elucidated much basic information regarding the role of development gradients, and theoretical models have been quite successful at describing experimental results. However, most experiments and computer simulations have dealt with isolated patterning events such as the dynamics of head regeneration. More global events such as interactions among the head, bud, and foot patterning systems have not been extensively addressed. The characterization of monoclonal antibodies with position-specific labeling patterns and the recent cloning and characterization of genes expressed in position-specific manners now provide the tools for investigating global interactions between patterning systems. In particular, changes in the axial positional value gradient may be monitored in response to experimental perturbation. Rather than studying isolated patterning events, this approach allows us to study patterning over the entire animal. The studies reported here focus on interactions between the foot and the head patterning systems in Hydra vulgaris following induction of a foot in close proximity to a head, axial grafting of a foot closer to the head, or doubling the amount of basal tissue by lateral grafting of an additional peduncle-foot onto host animals. Resulting positional value changes as monitored by antigen (TS19) and gene (ks1 and CnNK-2) expression were assessed in the foot, head, and intervening tissue. The results of the experiments indicate that positional values changed rapidly, in a matter of hours, and that there were reciprocal interactions between the foot and the head patterning systems. Theoretical interpretations of the results in the form of computer simulations based on the reaction-diffusion model are presented and predict many, but not all, of the experimental observations. Since the lateral grafting experiment cannot, at present, be simulated, it is discussed in light of what has been learned from the axial grafting experiments and their simulations.     

The head organizer in Hydra

Organizers and organizing centers play critical roles in axis formation and patterning during the early stages of embryogenesis in many bilaterians. The presence and activity of an organizer was first described in adult Hydra about 100 years ago, and in the following decades organizer regions were identified in a number of bilaterian embryos. In an adult Hydra, the cells of the body column are constantly in the mitotic cycle resulting in continuous displacement of the tissue to the extremities where it is sloughed. In this context, the head organizer located in the hypostome is continuously active sending out signals to maintain the structure and morphology of the head, body column and foot of the animal. The molecular basis of the head organizer involves the canonical Wnt pathway, which acts in a self-renewing manner to maintain itself in the context of the tissue dynamics of Hydra. During bud formation, Hydra's mode of asexual reproduction, a head organizer based on the canonical Wnt pathway is set up to initiate and control the development of a new Hydra. As this pathway plays a central role in vertebrate embryonic organizers, its presence and activity in Hydra indicate that the molecular basis of the organizer arose early in metazoan evolution.     

QUANTITATIVE LAWS IN REGENERATION : III. THE QUANTITATIVE BASIS OF POLARITY IN REGENERATION.

It is well known that a long defoliated piece of stem of Bryophyllum calycinum forms shoots only at the apical or the two apical nodes, while when such a stem is cut into as many pieces as there are nodes each node produces shoots. It is shown in this paper that the dry weight of shoots produced in the apical nodes of a long piece of stem is approximately equal to the dry weight of shoots the same stem would have produced if it had been cut into as many pieces as it possesses nodes. Hence all the material which can be used for the growth of shoots goes into the most apical part of the stem and this accounts for the polar character of regeneration in this case. It seems that the mass of basal roots produced by a piece of defoliated stem also increases with the mass of the stem.     

Effect of timing of cutting on patterning and proportion regulation during regeneration of the planarian Dugesia tigrina

The time interval between cuts that are made to obtain a tissue fragment from a planarian was found to be important to the process of its regeneration. Short fragments made by two transverse cuts across the body were more likely to regenerate abnormally when the interval between the two cuts was 5 or 12 min than when it was 1.5 min. The longer intervals specifically altered the regression line in the correlation between the length:width ratio of fragments and frequency of abnormal regenerates. This effect occured regardless of which region of the body the fragment was taken from. The time interval also affected body proportioning in regenerates and to the greatest degree in fragments derived from the region located immediately behind the head. These results indicate that events occuring shortly after a cut is made in a planarian significantly affect structure patterning and proportioning of the regenerate.