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.     

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.     

Regeneration in youngBunodactis verrucosa Pennant (actinaria,anthozoa)

Summary YoungBunodactis verrucosa Pennant at the 12 tentacle stage are employed to test the applicability of the polar coordinate model to coelenterate regeneration. The animals are cut along every radius into fragments of 3 to 9 segments. Most fragments are patent 3–4 weeks later, but small fragments have a higher mortality rate than large fragments. Some fragments do not regenerate and occasionally tentacles fuse, thereby reducing the number of segments. Small fragments tend to regenerate more tentacies than large fragments, but large fragments may regenerate great numbers of supernumerary tentacles. Twenty-two percent of the fragments restore the missing number of tentacles, while 76% of all fragments produce an even number of tentacles.Fragments restoring the correct numbers of tentacles show a marked tendency to form the correct tentacles (regulative regeneration). Fragments regenerating two less than the number of tentacles already present show a marked tendency to reproduce tentacles of the types already present (miror image formation). Other fragments produce missing segments (forward regeneration), or those already present (reverse regeneration) at lower frequencies.No fragments beginning or ending with the number 1 directive tentacle fail to regenerate entirely, while first cycle segments maximally remote from segment 1 are associated with the absence of regeneration. No fragments beginning or ending with the number 4 directive tentacle fail to undergo forward regeneration, regulate or produce a mirror image when the appropriate number of segments are regenerated. In contrast, segment 4 is associated with a low frequency of reverse regeneration, and second cycle segments cut away from immediate contact with segment 4 show an increase in the frequency of reverse regeneration. Controls through morphogenic substances rather than polar coordinates seem to explain these results. Such substances would control the number and direction of tentacle regeneration.This work was performed while the author was on sabbatical leave from the University of Pittsburgh at the Stazione Zoologica di Napoli. The author gratefully acknowledges the assistance of Mr. Ciro Gargiulo and of Ms. Gisella Princivalli. This work was supported by a travel grant from the United States Italy Cooperative Science Program of the National Science Foundation. The paper is dedicated to Dr. Alberto Monroy whose generosity made it possible     

Expression of a novel receptor tyrosine kinase gene and a paired-like homeobox gene provides evidence of differences in patterning at the oral and aboral ends of hydra

Axial patterning of the aboral end of the hydra body column was examined using expression data from two genes. One, shin guard, is a novel receptor protein-tyrosine kinase gene expressed in the ectoderm of the peduncle, the end of the body column adjacent to the basal disk. The other gene, manacle, is a paired-like homeobox gene expressed in differentiating basal disk ectoderm. During regeneration of the aboral end, expression of manacle precedes that of shin guard. This result is consistent with a requirement for induction of peduncle tissue by basal disk tissue. Our data contrast with data on regeneration of the oral end. During oral end regeneration, markers for tissue of the tentacles, which lie below the extreme oral end (the hypostome), are detected first. Later, markers for the hypostome itself appear at the regenerating tip, with tentacle markers displaced to the region below. Additional evidence that tissue can form basal disk without passing through a stage as peduncle tissue comes from LiCl-induced formation of patches of ectopic basal disk tissue. While manacle is ectopically expressed during formation of basal disk patches, shin guard is not. The genes examined also provide new information on development of the aboral end in buds. Although adult hydra are radially symmetrical, expression of both genes in the bud's aboral end is initially asymmetrical, appearing first on the side of the bud closest to the parent's basal disk. The asymmetry can be explained by differences in positional information in the body column tissue that evaginates to form a bud. As predicted by this hypothesis, grafts reversing the orientation of evaginating body column tissue also reverse the orientation of asymmetrical gene expression.     

Size and shape in the phylum Phoronida

The lophophorate phylum Phoronida consists of about 13 species, which differ in body length and width, number of longitudinal muscles, lophophore geometry and number of lophophore tentacles. In absolute terms large species have a larger body width, more tentacles, more longitudinal muscles and greater coiling of the lophophore than small species. However, size and shape analyses suggest that with increasing size: (I) the body surface area to volume ratio increases because body length increases faster than body width; (2) the relative number longitudinal muscles decreases, and (3) the relative feeding surface area of the lophophore decreases because tentacle diameter is constant while tentacle number increases at the same rate as body length and tentacle length increases more slowly than tentacle number. Coiling and spiraling of the lophophore in large species may be an attempt to compensate for this last relationship. We suggest that the habits, mode of growth and feeding mechanism of phoronids constrain size-related changes in shape.     

Formation of pattern in regenerating tissue pieces of Hydra attenuata: I. Head-body proportion regulation

The precision with which an almost uniform sheet of hydra cells develops into a complete animal was measured quantitatively. Pieces of tissue of varying dimensions were cut from the body column of an adult hydra and allowed to regenerate. The regenerated animals were assayed for number of heads (hypostomes plus tentacle rings), head attempts (body tentacles), and basal discs. To ascertain whether the head and body were reformed in normal proportions, the average number of epithelial cells in the heads and bodies was measured. Pieces of tissue, from $\text{1}\text{2}$ to $\text{1}\text{20}$ an adult in size, formed heads that were a constant fraction of the regenerate. Thus, over a 10-fold size range, a proportioning mechanism was operating to divide the tissue into head area and body area quite precisely, but appeared to reach limits at the extremes of the range. However, the regenerates were not all normal miniatures with one hypostome and one basal disc. As the width-length ratio of the cut piece was increased beyond the circumference-length ratio of the intact body column, the incidence of extra hypostomes in the “head” and body tentacles and extra basal discs in the “body” rose dramatically. A proportioning mechanism based on the Gierer-Meinhardt model for pattern formation is presented to explain the results.     

Tentacle morphogenesis in hydra

Summary Hydra oligactis exposed to 3 g/ml actinomycin D for 24 hours regenerated only the first pair of tentacles (the mid-laterals). If left uncut, actinomycin D treated animals underwent a reduction of the normal number of tentacles to two or less.Inductive activity was retained in the 2-tentacled hypostomes. However, the tentacles present exhibited reduced capacities to capture and manipulate prey.Histological studies showed that the tentacles of actinomycin D treated hydra were morphologically identical to those of the controls. The interstitial cell (I-cell) population of the treated animals, however, became depleted. Replacing the hypostome of an actinomycin D treated hydra with a normal hypostome reversed the cellular effects of actinomycin D treatment.The modifications in tentacle morphogenesis occurring after actinomycin D treatment are consistent with an impairment of hypostomal function in the animal. It is suggested that the morphological site of this malfunction may be in the nervous system.Research supported by American Cancer Society Institutional Grant No. 342-9157, USPHS Institutional Grant No. 342-9241 and by a grant from Research Corporation.Part of this work was completed while L.H. was an undergraduate research student supported by the National Science Foundation.     

Roles of Head-Activation and Head-Inhibition Potentials in Pattern Formation of Hydra: Analysis of a Multi-Headed Mutant Strain

SYNOPSIS. Lateral grafting of tissue was used to compare therelative head-activation and head-inhibition potentials of differentHydra strains. A small piece of tissue taken from one polyp,when grafted to another polyp, induces formation of a head structurewhen the relative head-activation potential of the donor tissueis sufficiently (i.e., more than some threshold value) higherthan the relative head-inhibition potential of the recipienttissue. It was found that a multi-headed mutant strain (mh-1),which produces many extra heads along its body column, has significantlyhigher head-activation and significantly lower head-inhibitionpotentials than the standard wild-type strain. This suggeststhat these potentials play important roles in hydra morphogenesis,and that an imbalance between the two potentials is responsiblefor the developmental abnormality of mh-1. The significanceof this finding is discussed in light of the "positional information"model proposed by Wolpert and his associates and the "lateralinhibition" model proposed by Gierer and Meinhardt.     

The ontogeny and maintenance of adult symmetry properties in the ctenophore, Mnemiopsis mccradyi

Ctenophores are biradially symmetrical animals. The body is composed of four identical quadrants which are organized along an oral-aboral axis. Most species have eight comb rows, two tentacles, and an apical organ (located on the aboral surface). During embryogenesis there is a fixed pattern of cleavage, a precocious specification of blastomere developmental potential, and an inability to regulate for portions of the embryo that have been removed. When blastomeres are separated at the two-cell stage each blastomere develops into a "half-animal" with four comb rows, one tentacle, and half an apical organ. In contrast, adult ctenophores regenerate readily. When an adult ctenophore is cut in half to produce "half-animals," in most cases each half regenerates the missing half. In some cases, however, bisected animals remain as "half-animals" which repair the wound site but do not replace all of the missing structures. When animals are cut in half along the tentacular or esophageal axis at different stages of embryogenesis a transition period is detected when the capacity for adult regeneration begins. This transition occurs at the time when the formation of the apical organ is complete and comb row function becomes coordinated. Embryos bisected prior to this time remain as "half-animals" even after growing to large reproductive sizes, while animals bisected after the transition period usually regenerate the missing structures within 2-3 days. When adult "half-animals" (produced by bisection either before or after the transition period) are cut into "quarter-pieces," the pieces regenerate to form either "half-animals" or whole animals. Thus, "half-animals" produced prior to the transition period--although they failed to undergo embryonic regulation--have not irreversibly lost the capacity to form whole animals if challenged to regenerate during adult stages. When aboral blastomeres destined to form the apical organ, tentacles, and comb rows are removed from early cleavage stages (prior to the transition period), the embryo does not form these structures at the appropriate time. However, the resulting deficient adults spontaneously form these structures from remaining blastomere lineages soon after hatching. These experiments suggest that as long as some quadrant-specific cells of the oral pole are present at the time of the transition period, the structures of that quadrant will be spontaneously replaced during the adult period.(ABSTRACT TRUNCATED AT 400 WORDS)     

VARIATION IN HYDRA'S TENTACLE NUMBERS AS A FUNCTION OF TEMPERATURE

Chlorohydra uiridissima whose tentacle number is altered at different temperatures, was studied to see how other developmental variables changed as a function of temperature. The results suggest that temperature is instrumental in establishing the size of bud and tentacle primordia, but the number of primordia present may play a limiting role.

Animals were cultured at 18, 23 and 28°C and shifted between the extreme temperatures. Large animals with 8 tentacles, small animals with 5 tentacles, and intermediate animals with 6 and 7 tentacles served as parents. Buds and parents were monitored daily and scored for numbers of buds and tentacles.

Temperature, not parental size, determined the size of the buds. At the lower temperature buds were produced more slowly and initiated less frequently, but occurred in greater numbers per parent and had more tentacles than at the higher temperatures. The duration of bud development also increased at lower temperature, but at the lowest temperature the duration of bud development was not correlated with tentacle numbers on buds.

Changes in the frequency of bud initiation and the duration of bud development induced by changing temperature did not parallel changes in the number of tentacles produced on buds. Animals shifted from 18°C to 28°C underwent rapid increases in the rate of bud initiation and rapid shortening in the duration of bud development, while animals shifted from 28°C to 18°C underwent equally rapid changes in the opposite directions. The number of tentacles produced on buds, however, changed slowly to that characteristic of buds acclimated to the new temperatures. The frequency of bud initiation and the duration of bud development, therefore, do not determine tentacle number.

The number of tentacles already present seems to limit possibilities for adding new tentacles. Parents with five tentacles were especially likely to undergo upward changes in their tentacle number while parents with eight tentacles were resistant to such changes.     

Inhibition of planarian regeneration by melatonin

Melatonin, which is a substance produced by the pineal body in vertebrates, inhibited regeneration in the planarian Dugesia japonica japonica Ichikawa et Kawakatsu. When decapitated planarians were maintained in a 1 mmol dm^–3 solution of melatonin, formation of the head was retarded; formation of the eyes, however, was not disturbed. Similarly in animals from which the tail was cut, regeneration of the tail was retarded if the animals were kept in melatonin solution of 1 mmol dm^–3. The effect was reversible once the melatonin was removed. Retardation of regeneration did not occur with similar application of three melatonin derivatives, serotonin hydrochloride, N-acetylserotonin, and 6-hydroxymelatonin. Melatonin endogenous to the planarian could be demonstrated by means of radio-immunoassay and was more abundant in the head region than other regions of the body. Melatonin, thus, appears to play a role in regulating regeneration in planarians and conceivably provides positional information in that process.     

The phorbol ester TPA dramatically inhibits planarian regeneration

The effect of phorbol ester TPA (12-O-tetradecanoylphorbol-13-acetate) on head regeneration in decapitated planarians (Dugesia lugubris s.1.) has been studied. TPA-treatment soon after head amputation dramatically inhibited the regenerative process. Ultrastructural analysis revealed that the migration of fixed parenchymal cells (FPCs) to the wound area was strongly activated by TPA. FPCs interacted with various types of cells inducing lysis and phagocyting cell debris. The resulting fluid was removed through diaphanous protrusions appearing at the level of the wound zone. Moreover the close association of FPCs with neoblastlike cell clusters in the parenchyma indicated their possible role in the modulation of neoblast migration.     

Differentiation pathways of ectodermal epithelial cells in hydra

The differentiation pathways of ectodermal epithelial cells in hydra were investigated. We found that under steady state conditions the ectodermal epithelial cells of the foot, the foot mucous cells, and the ectodermal epithelial cells of the tentacles, the battery cells, differentiate from gastric ectodermal ephithelial stem cells. From stem cell to the terminally differentiated state, a single cell cycle is required. The cells undergo a final round of DNA replication, double their genome to 4 n and become arrested in the G2-phase of the cell cycle. The ectodermal ephithelial cells of the hypostome, which like the tentacle cells are part of the head structure, can also arise from gastric ectodermal epithelial stem cells, but do so only during head regeneration and budding. They differentiate from stem cell to hypostomal cell in a single cell cycle, but in contrast to foot mucous and battery cells they remain capable of cell proliferation. Due to this self-renewal potential, they do not require recruitment from the gastric stem-cell pool in steady-state animals.     

Analysis of head and foot formation inHydra by means of an endogenous inhibitor

Summary In tissue regenerating the head, the ability to initiate head formation in a host increases with the time allowed for regeneration before grafting, while the foot-initiating ability decreases concomitantly. The reverse was found for tissue about to regenerate a foot. The early divergent changes thus indicated are counteracted in both head and foot regeneration by treatment with an inhibitor (Berking, 1977) in low concentrations.The inhibitor also interferes with processes which determine wether or not hypostome and tentacles are formed, and how many tentacles (if any) appear. The circumferential spacing of the tentacles was regular whether their number was normal or below normal.Secondary axes caused by implanted tissue either detach after having formed a head and a foot (i.e. behave like buds) or do not detach, having only formed a head. This alternative depends on the origin and amount of the implanted tissue and on the position of the implant within the host.The following model based on these findings is proposed: Head and foot formation start with pre-patterns which cause a continuously increasing change of the tissue's ability to initiate a head or a foot. Along the body axis this ability is determined by a graded distribution of sources. As development progresses, the high source density which accumulates in the head region causes the formation of a hypostome and tentacles; the angular spacing of tentacles is also dependent on source density. At a certain low source density foot-formation is initiated. The inhibitor counteracts the increase of source density in head-forming tissue as well as the decrease of source density in foot-forming tissue. It thus appears to be part of the mechanism which controls morphogenesis in hydra.     

Nerve cells in hydra: monoclonal antibodies identify two lineages with distinct mechanisms for their incorporation into head tissue

The relationship between populations of nerve cells defined by two monoclonal antibodies was investigated in Hydra oligactis. A population of sensory nerve cells localized in the head (hypostome and tentacles) is identified by the binding of antibody JD1. A second antibody, RC9, binds ganglion cells throughout the animal. When the nerve cell precursors, the interstitial cells, are depleted by treatment with hydroxyurea or nitrogen mustard, the JD1+ nerve cells are lost as epithelial tissue is sloughed at the extremities. In contrast, RC9+ nerve cells remain present in all regions of the animal following treatment with either drug. When such hydra are decapitated to initiate head regeneration, the new head tissue formed is again free of JD1+ sensory cells but does contain RC9+ ganglion cells. Our studies indicate that (1) nerve cells are passively displaced with the epithelial tissue in hydra, (2) JD1+ sensory cells do not arise by the conversion of body column nerve cells that are displaced into the head, whereas RC9+ head nerve cells can originate in the body column, (3) formation of new JD1+ sensory cells requires interstitial cell differentiation. We conclude from these results that the two populations defined by these antibodies are incorporated into the h ad via different developmental pathways and, therefore, constitute distinct nerve cell lineages.     

Evaluation of tentacle regeneration as a biological assay inHydra

Summary Tentacle number in non-buddingHydra attenuata, randomly selected from mass culture varies <0.5 tentacles over a 3 month period. Replicate samples of untreated regenerates (n=50–60), however, show some variability in mean tentacle number regenerated (S _x0.13–0.15). The variability is similar whether experiments are performed using randomly selected animals or animals with identical tentacle numbers. The variability is, further, not the result of profound differences in the time of tentacle initiation in individual animals.Addition of 10^–5 M glutamate or a methanol extract to the assay medium results in both an earlier appearance of tentacles and in more tentacles being regenerated during early time periods. The mean tentacle number of methanol extract-treated animals is significantly higher than the mean tentacle number of either control or glutamate-treated animals at all time periods examined.The distribution of tentacle number classes among regenerates is normal in control and glutamate-treated animals but nonparametric in methanol extract-treated animals, making statistical analysis of the data using Student'st-test in-appropriate. The usefulness of the Mann WhitneyU and Kruskal-Wallis tests is discussed, as is the appropriateness of tentacle regeneration as an assay forhydra morphogens.     

Morphogenesis of Stigmatella aurantiaca fruiting bodies.

Scanning electron micrographs of intermediate stages of fruiting body formation in the myxobacterium Stigmatella aurantiaca suggest that fruiting body formation can be divided into several stages distinguishable on the basis of the motile behavior of the cells. Aggregates formed at sites where cells glide as groups in circles or spirals. Thus, each aggregate was surrounded by a wide band of cells. Several streams of cells were pointed toward and connected to the wide band of cells at the base of the aggregate, suggesting directed cell movement toward the aggregate. The pattern of cells at the base of taller, more mature aggregates suggested that groups of cells enter the aggregate from the surrounding band of cells by changing the pitch of their movement, thus creating an ascending spiral. Stalk formation was characterized by a distinctly different pattern, which suggested that single cells emerge from the band of cells and move toward the aggregate, under it, and then vertically to create the stalk. At this stage, the aggregate appeared to be torn from the substrate as it was lifted off the surface. The cells in the completed stalks were well separated, and most had their long axes pointed in a vertical direction. A great deal of the stalk material appeared to be slime in which the cells were embedded and through which they were presumably moving in the live material. Some suggestions regarding factors that may direct the observed morphogenetic movements are discussed.     

Genetic analysis of developmental mechanisms in hydra. X. Morphogenetic potentials of a regeneration-deficient strain (reg-16)

Morphogenetic potentials of hydra tissue involved in head or foot formation were examined in a standard wild-type strain (105) and a mutant strain (reg-16) which has a very low head regenerative but a nearly normal foot regenerative capacity (T. Sugiyama and T. Fujisawa, 1977, J. Embryol. Exp. Morphol. 42, 65-77). Hydra tissue has two types of morphogenetic potentials to control head formation: the potential to form head structure (head-activation potential) and the potential to inhibit head formation (head-inhibition potential). It also has two types of morphogenetic potentials to control foot formation: foot-activation and foot-inhibition potentials. A lateral tissue grafting procedure (G. Webster and L. Wolpert, 1966, J. Embryol. Exp. Morphol. 16, 91-104), was used to examine and compare the relative levels of these potentials in the normal and the mutant strains. The potential levels were examined along the body axis of the intact animals and also in the regenerating animals after head removal. The results obtained show that the potentials involved in head formation are highly abnormal, whereas the potentials involved in foot formation are apparently normal in the mutant strain (reg-16). This suggests that the abnormal potentials are related in some way to, and may be responsible for, the reduced head regenerative capacity in the mutant strain reg-16.