Plasticity in the nervous system of adult hydra. II. Conversion of ganglion cells of the body column into epidermal sensory cells of the hypostome

Due to the tissue dynamics of hydra, every neuron is constantly changing its location within the animal. At the same time specific subsets of neurons defined by morphological or immunological criteria maintain their particular spatial distributions, suggesting that neurons switch their phenotype as they change their location. A position-dependent switch in neuropeptide expression has been demonstrated. The possibility that ganglion cells of the body column are converted into epidermal sensory cells of the head was examined using a monoclonal antibody, TS33, whose binding is restricted to a subset of epidermal sensory cells of the hypostome, the apical end of the head. When animals devoid of interstitial cells, which are the nerve cell precursors, were decapitated and allowed to regenerate, they formed TS33+ epidermal sensory cells. As this latter cell type is not found in the body column, and the interstitial cell-free animals contained only epithelial cells and ganglion cells in the part of the ectoderm that formed the head during regeneration, the TS33+ epidermal sensory cells most likely arose from the TS33- ganglion cells. The observation of epidermal sensory cells labeled with both TS33 and TS26, a monoclonal antibody that binds to ganglion cells, in regenerating and normal heads provides further support. The double-labeled cells are probably in transition from a ganglion cell to an epidermal sensory cell. These results provide a second example of position-dependent changes in neuron phenotype, and suggest that the differentiated state of a neuron in hydra is only metastable with regard to phenotype.     

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.     

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.     

The influence of tissue polarity on nematocyte migration in Hydra attenuata

Influences underlying the direction of nematocyte migration in hydra were studied. Nematocytes arise by interstitial cell differentiation in the body column, and then up to 80% migrate into the ectodermal epithelial cells of the tentacles. The migration of these cells, which is clearly apically directed, may be due either to a chemotactic attraction into the hypostome and tentacles, or to a property inherent in the tissue of the body column, such as the regeneration polarity. To distinguish between these two possibilities, the rates of accumulation of ³H-proline-labeled desmoneme and stenotele nematocytes in unlabeled heads (hypostome and tentacles) grafted either basally or apically to the labeled body column were compared. Basally grafted heads, if left in place for an appropriate length of time, reversed the regeneration polarity of the tissue. In all experiments the direction of desmoneme migration was correlated with the direction (apical or basal) of the regeneration polarity of the tissue. Further, the kinetics of polarity reversal were modified by varying the grafting procedure or the environmental conditions. In every case the kinetics of reversal of desmoneme migration also paralleled the kinetics of reversal of tissue polarity. The results suggest that the direction of desmoneme migration is influenced by the regeneration polarity of the tissue. Stenotele migration was largely unaffected by tissue polarity, but behaved as though chemotactically attracted to the head.     

Proportioning a Hydra

SYNOPSIS. Pieces of hydra tissue of various sizes and shapeswere cut from the body columns of adult hydra and allowed toregenerate. The proportions of the resulting animals were determinedfirst by counting cells in the head and body, and secondly bymeasuring the structures directly using an ocular micrometer. Head-body proportions were found to be constant over a tenfoldsize range. Very small regenerates had a larger head fractionand large budding regenerates had a smaller head fraction. Extrastructures developed in certain shape pieces, but total head-bodytissue remained proportional. More detailed measurement of thehead showed that the hypostome regulated only slightly withtotal size change, while the tentacle tissue varied considerablyto maintain the head-body ratio. This suggested that the patterningof the hypostome and the tentacles might involve separate processes,with the latter being responsible for proportion regulation.While the body mass appeared to be determined by the proportioningmechanism, its circumference was related to the circumferenceof the hypostome, suggesting a causal relationship between thetwo organizers and the column shaping. The basal disc remainedproportional to the body except in the smallest pieces. A Gierer-Meinhardtpattern formation scheme could account for the results found.     

Antisera to the sequence Arg-Phe-amide visualize neuronal centralization in hydroid polyps

Summary Antisera to the sequence Arg-Phe-amide (RF-amide) have a high affinity to the nervous system of fixed hydroid polyps. Whole-mount incubations of several Hydra species with RFamide antisera visualize the three-dimensional structure of an ectodermal nervous system in the hypostome, tentacles, gastric region and peduncle. In the hypostome of Hydra attenuata a ganglion-like structure occurs, consisting of numerous sensory cells located in a region around the mouth opening and a dense plexus of processes which project mostly radially towards the bases of the tentacles. In Hydra oligactis an ectodermal nerve ring was observed lying at the border of hypostome and tentacle bases. This nerve ring consists of a few large ganglion cells with thick processes forming a circle around the hypostome. This is the first direct demonstration of a nerve ring in a hydroid polyp.Incubation of Hydractinia echinata gastrozooids with RFamide antisera visualizes an extremly dense plexus of neuronal processes in body and head regions. A ring of sensory cells around the mouth opening is the first group of neurons to show RFamide immunoreactivity during the development of a primary polyp. In gonozooids the oocytes and spermatophores are covered with strongly immunoreactive neurons.All examples of whole-mount incubations with RF-amide antisera clearly show that hydroid polyps have by no means a diffuse nerve net, as is often believed, and that neuronal centralization and plexus formation are common in these animals. The examples also show that treatment of intact fixed animals with RFamide antisera is a useful technique to study the anatomy or development of a principal portion of the hydroid nervous system.     

Expression and developmental regulation of the Hydra-RFamide and Hydra-LWamide preprohormone genes in Hydra: evidence for transient phases of head formation

Hydra magnipapillata has three distinct genes coding for preprohormones A, B, and C, each yielding a characteristic set of Hydra-RFamide (Arg-Phe-NH2) neuropeptides, and a fourth gene coding for a preprohormone that yields various Hydra-LWamide (Leu-Trp-NH2) neuropeptides. Using a whole-mount double-labeling in situ hybridization technique, we found that each of the four genes is specifically expressed in a different subset of neurons in the ectoderm of adult Hydra. The preprohormone A gene is expressed in neurons of the tentacles, hypostome (a region between tentacles and mouth opening), upper gastric region, and peduncle (an area just above the foot). The preprohormone B gene is exclusively expressed in neurons of the hypostome, whereas the preprohormone C gene is exclusively expressed in neurons of the tentacles. The Hydra-LWamide preprohormone gene is expressed in neurons located in all parts of Hydra with maxima in tentacles, hypostome, and basal disk (foot). Studies on animals regenerating a head showed that the prepro-Hydra-LWamide gene is expressed first, followed by the preprohormone A and subsequently the preprohormone C and the preprohormone B genes. This sequence of events could be explained by a model based on positional values in a morphogen gradient. Our head-regeneration experiments also give support for transient phases of head formation: first tentacle-specific preprohormone C neurons (frequently associated with a small tentacle bud) appear at the center of the regenerating tip, which they are then replaced by hypostome-specific preprohormone B neurons. Thus, the regenerating tip first attains a tentacle-like appearance and only later this tip develops into a hypostome. In a developing bud of Hydra, tentacle-specific preprohormone C neurons and hypostome-specific preprohormone B neurons appear about simultaneously in their correct positions, but during a later phase of head development, additional tentacle-specific preprohormone C neurons appear as a ring at the center of the hypostome and then disappear again. Nerve-free Hydra consisting of only epithelial cells do not express the preprohormone A, B, or C or the LWamide preprohormone genes. These animals, however, have a normal phenotype, showing that the preprohormone A, B, and C and the LWamide genes are not essential for the basic pattern formation of Hydra.     

Cngsc, a homologue of goosecoid, participates in the patterning of the head, and is expressed in the organizer region of Hydra.

We have isolated Cngsc, a hydra homologue of goosecoid gene. The homeodomain of Cngsc is identical to the vertebrate (65-72%) and Drosophila (70%) orthologues. When injected into the ventral side of an early Xenopus embryo, Cngsc induces a partial secondary axis. During head formation, Cngsc expression appears prior to, and directly above, the zone where the tentacles will emerge, but is not observed nearby when the single apical tentacle is formed. This observation indicates that the expression of the gene is not necessary for the formation of a tentacle per se. Rather, it may be involved in defining the border between the hypostome and the tentacle zone. When Cngsc(+) tip of an early bud is grafted into the body column, it induces a secondary axis, while the adjacent Cngsc(-) region has much weaker inductive capacities. Thus, Cngsc is expressed in a tissue that acts as an organizer. Cngsc is also expressed in the sensory neurons of the tip of the hypostome and in the epithelial endodermal cells of the upper part of the body column. The plausible roles of Cngsc in organizer function, head formation and anterior neuron differentiation are similar to roles goosecoid plays in vertebrates and Drosophila. It suggests widespread evolutionary conservation of the function of the gene.     

水螅异常极性体轴的形成及矫正进程的观察

目的:如何建立和维持体轴是一个基本的发育生物学问题,而淡水水螅是适合进行形态发生和个体发育调控机制研究的重要模式生物。本文观察了大乳头水螅异常极性体轴的形成及矫正进程,初步探讨水螅极性体轴的维持和调控机制。方法:先切取水螅的整个头部,再获得带二根触手的口区组织。通过ABTS细胞化学染色法检测水螅基盘分子标志物过氧化物酶的表达,判别水螅基盘组织(水螅足区的末端)是否形成。结果:从40块口区组织再生得到的水螅个体中有1例极性体轴发育异常的个体,其身体两端均发育成头区,且两端的头区均具有捕食能力。随后水螅其中一端头区的触手逐渐萎缩、退化,最终该端头区转化成具有吸附能力的基盘组织。结论:水螅组织的再生涉及极性体轴的重建,而一些特殊因素可能造成临时性的水螅极性体轴调控紊乱。本研究表明水螅具备自我矫正异常极性体轴的能力。另外,本研究结果显示水螅触手可以萎缩直至退化,该现象涉及的细胞学过程可能是非常复杂的,有可能涉及到触手细胞的凋亡转化过程,也可能是触手的高度分化细胞仍然具备去分化能力、去分化后再转移到身体其他地方,其具体机制值得进一步探究。     

A three dimensional serial reconstruction of neuronal distributions in the hypostome of a Hydra

The numbers, types, and distributions of neurons in a hypostome of Hydra littoralis were determined from electron micrographs of serial (0.25 μm thick) sections. In 1,080 serial sections examined we found 75 sensory cells and 949 centrally located ganglion cells. More than 96% of the 1,024 neurons identified had a single cilium. Sensory cells were most numerous near the apex of the hypostome. Proceeding away from the apex, they steadily decreased in numbers; at 120 μm they were no longer observed. Ganglion cells were bimodally distributed; some were associated with sensory cells at the apex, but most were found at the sites of tentacle origin. We observed, throughout the hypostome, a total of 64 neuronal clusters (three or more contiguous neurons), with an average of five and a maximum of 11 neurons in a cluster. Clusters were distributed similarly to ganglion cells: an initial concentration of clusters near the apex; the majority at the hypostometentacle junctions. Each neuron identified was traced through succeeding sections in which it was observed. We used a three coordinate system to create a three-dimensional reconstruction of the neuronal locations in the hypostome. Although the functional significance of the neuronal distributions we observed is unknown, we suggest that neurons at the apex of the hypostome transduce sensory information involved in feeding behavior. The neuronal concentrations at sites of tentacle origin may be responsible for initiating Contraction Burst Pulses associated with rhythmic behavioral patterns of Hydra or coordinating tentacle movements involved in prey capture, ingestion or locomotion.     

A subset of cells in the nerve net of Hydra oligactis defined by a monoclonal antibody: its arrangement and development

A monoclonal antibody, termed JD1, was generated that bound to a subset of the nerve cells in the hypostome and tentacles of Hydra oligactis. Using a whole-mount technique the spatial pattern of the subset of nerve cells and their processes could be clearly visualized using indirect immunofluorescence. The subset largely corresponds to the epidermal sensory cells. Using the same technique the development of the pattern during head regeneration and budding was examined. The appearance of the nerve cells coincides with the formation of both the tentacles and hypostome. When head regeneration does not occur, JD1+ cells do not appear suggesting the differentiation of JD1+ cells is an integral event in head formation dependent on antecedent patterning processes.     

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.     

Regeneration of proximal and distal part of hydra body cut in the middle of gastral cavity and treated with dactinomycine

Hydras were cut in the middle of the gastral part of the body. The part with the hypostome is marked as H, and the one with the foot as P. Both parts were treated with actinomycine D in 0,5 mg : 200 ml water solution. H-parts are much more sensitive to the effect of actinomycine than P-parts, and P lives considerably longer. It is supposed that such reaction are the result of specificity of H and P cell composition, and of the growth direction which is characteristic of hydra in general. H-part has a proportionally greater number of differentiated cells and this relatively smaller number of non-differentiated cells is spent in it quicker than in P-part in which they are more numerous. The growth direction has a decisive influence on further life of H- respectively P-part. Namely, H- in growth direction does not have any damaged body regions (hypostome and tentacles are intact) and it lacks the amputated P-part i.e. gastral region with foot: the region which is on the opposite side of growth direction of hydra. H-part has all the characteristic cells of this body region, so after amputation mostly it does not change. Unfavourable effect of citostatic manifests sooner and H-part desintegrates quicker. On the contrary, P-part lacks the hypostome with tentacles, and these are the body parts in the growth direction. Zimogen cells can dedifferentiate and differentiate. The hypostome and tentacles regenerate as far as is allowed by actinomycine.     

Formation of pattern in regenerating tissue pieces of Hydra attenuata: II. Degree of proportion regulation is less in the hypostome and tentacle zone than in the tentacles and basal disc

The relative sizes of the various structures in Hydra attenuata were compared over a broad range of animal sizes to determine in detail the ability to regulate proportions during regeneration. The three components of the head, namely hypostome, tentacles, and tentacle zone from which the tentacles emerge, the body column, and the basal disc were all measured separately. Ectodermal cell number was used as the measure of size. The results showed that the basal disc proportioned exactly over a 40-fold size range, and the tentacle tissue proportioned exactly over a 20-fold size range. In contrast, the hypostome and tentacle zone proportioned allometrically. With decreasing size, the hypostome and tentacle zone became an increasing fraction of the animal at the expense of body tissue, and in the very smallest regenerates at the expense of tentacle tissue. In their current form, the reaction-diffusion models proposed for pattern regulation in hydra are not consistent with the data.     

Oriented migration of interstitial cells and nematocytes inHydra attenuata

Summary The migratory properties of hydra cells within the tissue were studied. The extent and direction of cell migration were examined in budding, non-budding, and regenerating animals. Nematocytes and a small number of single big interstitial cells (the multipotent interstitial cells) actively migrate preferentially in an apical direction. Basal migration of these cells occurs only when a bud is present and, in which case, the cells migrate into the developing bud. The regeneration of the hypostome and tentacles does not affect cell migration in either direction, except for apical migration of stenotele nematocytes, which was markedly reduced.This research was supported by National Science Foundation Grant (GB 29284), National Institute of Health Grant (HD 08086-01), and N.I.H. Public Health Service Training Grant (HD 00347).     

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.     

Effect of inhibitors of synthesis of dopamine and its antagonists on Hydra attenuata regeneration

Obvious inhibition of the hydra regeneration with no subsequent morphological abnormalities, was shown to occur when using alpha-methylthyrosine and 3 Jthyrosine. alpha-Methyldopa induced a slight inhibition but a considerable morphological change: ectopic tentacles, projections, bipolar forms in the gastric fragment. The apical and basal fragments did not suffer. The role of neurotransmitters in the hydra morphogenesis is discussed.     

OBSERVATIONS ON SUPERNUMERARY HEAD FORMATION INDUCED BY LITHIUM CHLORIDE TREATMENT IN THE REGENERATING HYDRA, PELMATOHYDRA ROBUSTA

Lithium chloride treatment of hydras cut just proximal to the tentacle circle and just distal to the budding region induces a supernumerary head at the proximal cut surface. Such a supernumerary head does not appear in the normal course of regeneration. The bipolar hydra thus formed persists for several weeks and later separates to form two normal individuals. The supernumerary head is not formed at the proximal cut surface when the hydra is transected just distal to the budding zone and the distal portion is allowed to regenerate in the Li-containing medium. LiCl has a slight inhibitory effect on the regeneration of hypostomes or tentacles when the animal is cut at the base of the hypostome.     

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.     

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.