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.     

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.     

Growth regulation of the interstitial cell population in hydra : I. Evidence for global control by nerve cells in the head

The interstitial cells of hydra form a multipotent stem cell system, producing terminally differentiated nerve cells and nematocytes during asexual growth. Under well-fed conditions the interstitial cell population doubles in size every 4 days. We have investigated the possible role of nerve cells in regulating this behavior. Nerve cells are normally found in highest concentrations in the head region of hydra, while interstitial cells are primarily located in the body column. Our experimental approach was to construct, by grafting, animals in which the density of nerve cells varied in (1) the head region, or (2) the body column. The growth of the interstitial cell population was then measured in these hydra. The results indicate that differences in head nerve cell density are closely correlated with how fast the interstitial cell population increases in size. Variations in the level of either nerve cells or interstitial cells in the body column showed no such correlation. These findings suggest the existence of a signaling mechanism in the head region. This signal, which is a function of the density of head nerve cells, emanates from the head tissue and exerts global control on the growth of the interstitial cell population in the body column.     

Nerve commitment during head regeneration in hydra.

Most important event in head regeneration in hydra is a wave of conversion of many interstitial cells into nerve cells. Experimental evidence lends support to the idea that the commitment of interstitial cells into nerve cells is the first morphogenetic prerequisite for emergence of head structures, when the number of nerve cells increases. This increase in nerve cells is delayed when regeneration occurs at a site lower in the body column.     

Stimulation of head-specific nerve cell formation in Hydra by pulses of diacylglycerol

In Hydra magnipapillata, repeated pulses of diacylglycerol (DG) induce a lengthening of the body column and the formation of ectopic head structures (Müller, 1989). In the present study, seven pulses given on 7 successive days led, in the gastric region, to a 1.87-fold increase in the number of epithelial cells from 16,200 to 30,400; a 4.6-fold increase in the total number of nerve cells from 2900 to 13,400; and an 18-fold increase of RF-amide immunopositive nerve cells from 100 to 1800. This subset of neurons, which is normally distributed in the form of a density gradient having its high point around the mouth and ending below the tentacle whorl in the upper gastric column, displays an altered pattern in DG-treated animals. While the density peak in the original head persisted, a second peak developed in the lower gastric region at the site of imminent ectopic tentacle formation, reflecting the local increase in positional value. Thus, the temporal sequence in which DG-induced ectopic apical body structures arise is a function of the rise in positional value and reflects the normal spatial sequence of these structures along the body column: first RF-amide neurons appear, then tentacles, and finally hypostomes.     

Interstitial cell migration in Hydra attenuata. I. Quantitative description of cell movements

The interstitial cell system of hydra contains multipotent stem cells which can form at least two classes of differentiated cell types, nerves and nematocytes. The amount of nerve and nematocyte production varies in an axially dependent pattern along the body column. Some interstitial cells can migrate, which makes it conceivable that this observed pattern of differentiation is not the result of regionally specified stem cell commitment, but rather arises by the selective movement of predetermined cells to the correct site prior to expression. To assess this latter possibility quantitative information on the dynamics of interstitial cell migration was obtained. Epithelial hydra were grafted to normal animals in order to measure (1) the number of cells migrating per day, (2) the location of these cells within the host tissue, and (3) the axial directionality of this movement. Tissue properties such as axial position and the density of cells within the interstitial spaces of the host were also tested for their possible influence on migration. Results indicate that there is a considerable traffic of migrating interstitial cells and this movement has many of the characteristics necessary to generate the position-dependent pattern of nerve differentiation.     

Neuron differentiation in hydra involves dividing intermediates

The neuron differentiation pathway in hydra is usually assumed to be the following. A multipotent stem cell among the large interstitial cells becomes committed to neuron differentiation and divides. The two daughter cells, which are postmitotic small interstitial cells, subsequently differentiate into neurons. Herein the neuron pathway of the lower peduncle of Hydra oligactis was examined in some detail. In this region a substantial amount of neuron differentiation takes place, but very few large interstitial cells are present. It was found that small interstitial cells, which are capable of dividing, differentiate into neurons. The minimum time required to traverse the pathway from S phase of the last proliferating intermediate to a neuron is 18 hr. Thus, the neuron differentiation pathway in the lower peduncle involves dividing intermediates and is therefore more complex than usually assumed. Evidence for dividing small interstitial cells in the head, where the highest rate of neuron differentiation occurs, suggests that this more complex pathway may be common to all regions of the animal. A consequence of this finding is that the body of evidence concerning the commitment of multipotent stem cells to neurons and the control of this commitment requires reinterpretation.     

FMRfamide-like immunoreactivity in the ventral nerve cord of the larval eastern spruce budworm,Choristoneura fumiferana (clemens) (Lepidoptera : Tortricidae)

Distribution of FMRFamide-like immunoreactivity was examined in the larval ventral nerve cord of the eastern spruce budworm, Choristoneura fumiferana (Lepidoptera : Tortricidae). Indirect immunofluorescent methods revealed the existence of 3 groups of FLI neurons in each ganglion. The neurons are distributed in a bilaterally symmetrical fashion at the anterodorsal, midlateral and posteroventral regions of the ganglia. There are 4 FMRFamide-like immunoreactive fiber tracts on the dorsal surface of the ganglia to which the anterodorsal FLI neurons project ipsilaterally, while the midlateral pair projects both ipsi-, and contralaterally. The last abdominal ganglion (AG8) has 4 additional pairs of FLI neurons; and axons from some of these extend into the median abdominal nerve, which suggests some role for this neuropeptide in the control of posterior structures of the larva.     

A novel neuropeptide (FRamide) family identified by a peptidomic approach in Hydra magnipapillata

In the course of systematic identification of peptide signaling molecules combined with the expressed sequence tag database from Hydra, we have identified a novel neuropeptide family that consists of two members with FRamide at the C-terminus; FRamide-1 (IPTGTLIFRamide) and FRamide-2 (APGSLLFRamide). The precursor sequence deduced from cDNA contained a single copy each of FRamide-1 and FRamide-2 precursor sequences. Expression analysis by whole-mount in situ hybridization showed that the gene was expressed in a subpopulation of neurons that were distributed throughout the body from tentacles to basal disk. Double in situ hybridization analysis showed that the expressing cell population was further subdivided into one population consisting of neurons expressing both the FRamide and Hym176 (neuropeptide) genes and the other consisting of neurons expressing only the FRamide gene. FRamide-1 evoked elongation of the body column of 'epithelial' Hydra that was composed of epithelial cells and gland cells but lacked all the cells in the interstitial stem cell lineage, including neurons. In contrast, FRamide-2 evoked body column contraction. These results suggest that both of the neuropeptides directly act on epithelial cells as neurotransmitters and regulate body movement in an axial direction.     

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.     

Putative intermediates in the nerve cell differentiation pathway in hydra have properties of multipotent stem cells

We have investigated the properties of nerve cell precursors in hydra by analyzing the differentiation and proliferation capacity of interstitial cells in the peduncle of Hydra oligactis, which is a region of active nerve cell differentiation. Our results indicate that about 50% of the interstitial cells in the peduncle can grow rapidly and also give rise to nematocyte precursors when transplanted into a gastric environment. If these cells were committed nerve cell precursors, one would not expect them to differentiate into nematocytes nor to proliferate apparently without limit. Therefore we conclude that cycling interstitial cells in peduncles are not intermediates in the nerve cell differentiation pathway but are stem cells. The remaining interstitial cells in the peduncle are in G1 and have the properties of committed nerve cell precursors (Holstein and David, 1986). Thus, the interstitial cell population in the peduncle contains both stem cells and noncycling nerve precursors. The presence of stem cells in this region makes it likely that these cells are the immediate targets of signals which give rise to nerve cells.     

Axial Patterning in Hydra

Morphogen gradients play an important role in pattern formation during early stages of embryonic development in many bilaterians. In an adult hydra, axial patterning processes are constantly active because of the tissue dynamics in the adult. These processes include an organizer region in the head, which continuously produces and transmits two signals that are distributed in gradients down the body column. One signal sets up and maintains the head activation gradient, which is a morphogenetic gradient. This gradient confers the capacity of head formation on tissue of the body column, which takes place during bud formation, hydra''s mode of asexual reproduction, as well as during head regeneration following bisection of the animal anywhere along the body column. The other signal sets up the head inhibition gradient, which prevents head formation, thereby restricting bud formation to the lower part of the body column in an adult hydra. Little is known about the molecular basis of the two gradients. In contrast, the canonical Wnt pathway plays a central role in setting up and maintaining the head organizer.Morphogen gradients play a critical role in the early stages of embryogenesis in a number of metazoans in that they initiate and are involved in axial patterning processes. Such a gradient also plays a role in axial patterning in hydra, a primitive metazoan. However, unlike in most metazoans, this gradient is continuously active in an adult hydra as part of the tissue dynamics of the adult animal.The structure of a hydra is fairly simple (Fig. ​(Fig.1).1). It consists of a single axis with radial symmetry, which contains a head, body column, and foot along the axis. The head consist of two parts: the hypostome in the apex, and the tentacle zone from which the tentacles emerge in the basal part of the head. The body column has three parts: the gastric region and peduncle in the apical, and basal parts with a budding zone between the gastric region and peduncle. Buds, hydra''s mode of asexual reproduction, emerge from the budding zone between the gastric region and peduncle.Open in a separate windowFigure 1.Longitudinal cross section of an adult hydra. The multiple regions are labeled. The two protrusions from the body column are early and late stages of bud development. The arrows indicate the direction of tissue displacement. (Reprinted from Bode 2001.)Three cell lineages are involved. The axis consists of a cylindrical shell that is made up of two concentric epithelial layers, the ectoderm and endoderm, which are separated by a basement membrane. Interspersed among the epithelial cells of both layers are the cells of the third lineage, the interstitial cell lineage. It consists of interstitial cells, which are multipotent stem cells (David and Murphy 1977), located primarily in the ectoderm throughout the body column. They give rise to neurons, secretory cells, and nematocytes, which are the stinging cells that are typical of cnidarians, as well as gametes when a hydra undergoes sexual reproduction (David and Murphy 1977).In an adult hydra, the epithelial cells of both layers are constantly in the mitotic cycle (David and Campbell 1972; Campbell and David 1974). The expanding tissue in the upper part of the body column is continuously displaced apically into the head (Fig. 1). Once there, it is displaced onto and along the tentacles or into the hypostome, and eventually sloughed when reaching the extremities (Campbell 1967; Otto and Campbell 1977). Tissue in the remainder of the body column is displaced basally either onto developing buds, or further down onto the foot, where it is sloughed at the bottom of the foot. Thus, the tissues of an adult hydra are continuously in a steady state of production and loss. As a hydra has no defined lifetime (Martinez 1998), this activity goes on indefinitely.     

Genetic Analysis of Developmental Mechanisms in Hydra. XV. Nematocyte Differentiation in Strains with Altered Developmental Gradients*

Nematocyte differentiation from the interstitial stem cells in hydra occurs non-uniformly along the body column. The relative ratios of the 4 nematocyte types produced vary gradually from head to foot along the body axis (Bode and Smith, 1977). To find out whether this regional variation in nematocyte differentiation along the body column is related to the gradients of the head-activation and head-inhibition potentials, nematocyte differentiation patterns were examined in strains which have significantly different developmental gradients along their body columns. Five strains of hydra, including a wild-type, two mutant strains and two chimeric (mutnt/wild-type) strains, were investigated. It was found that the regional variations in the nematocyte differentiation were similar in all the strains examined, and that no significant differences of the variation existed that could be attributed to the differences of the developmental gradients in these strains. This suggests that nematocyte differentiation is strongly affected by the axial position along the body column, but that the gradients of the morphogenetic potentials involved in head formation are not involved in this effect. Instead, some other parameter(s) of axial position not directly associated with these gradients must be responsible for the positional effect on nematocyte differentiation.     

The timing of initial neuropeptide expression by an identified insect neuron does not depend on interactions with its normal peripheral target.

To study the developmental regulation of a neuropeptide phenotype, we have analyzed the biochemical and morphological differentiation of two identifiable neurons in embryos of the moth, Manduca sexta. The central cell, CF, and the peripheral cell, L1, are both neuroendocrine neurons that express neuropeptides related to the molluscan tetrapeptide FMRFamide. Both neurons project axons to the transverse nerve in each thoracic segment. Within the CF and L1 cells, neuropeptide-like immunoreactivity was localized to secretory granules that had cell-specific morphologies and sizes. The onset of neuropeptide expression in the two cell types displayed a similar pattern: immunoreactivity was first detected in distal processes and soon after within cell bodies. However, the onsets occurred at different times: for the CF cell, neuropeptides were first seen at 60%-63% of embryonic development, after the neuron had extended a long axon into the periphery, while L1 neuropeptide expression began at approximately 42%, as it first extended its growth cone. These times were related in that they corresponded to the arrival times of the respective growth cones at a similar position in the developing peripheral nerve. Within this region of the nerve, the growth cones of both cell types-exhibited a transient and cell-specific interaction with an identified mesodermal cell, called the Syncytium. Like the L1 and B neurons (Carr and Taghert, 1988b), the CF growth cones typically grew past this cell, yet remained attached to it by lamellipodial and filopodial processes of the axon. Ultrastructurally, the interaction involved filopodial adhesion to and insertion within the Syncytial cell. Two other nonneuroendocrine cell types grew axons past this same region, but showed no such tendencies. To test the hypothesis that the morphological and biochemical differentiation of these cells was somehow linked, central ganglia were isolated (as individuals or connected as ganglionic chains) in tissue culture, prior to the time when CF growth cones entered the periphery and prior to the development of CF neuropeptide expression. In the majority of cases, CF neurons nevertheless displayed their neuropeptide phenotype at a normal and cell-specific stage. We conclude that the initiation of neuropeptide expression is highly correlated with schedules of morphological differentiation in these neurons, but that, in the case of the CF neuron, it is not regulated by interactions of the growth cone with peripheral structures.     

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。     

Neuronal cell death during metamorphosis of Hydractina echinata (Cnidaria, Hydrozoa)

In planula larvae of the invertebrate Hydractinia echinata (Cnidaria, Hydrozoa), peptides of the GLWamide and the RFamide families are expressed in distinct subpopulations of neurons, distributed in a typical spatial pattern through the larval body. However, in the adult polyp GLWamide or RFamide-expressing cells are located at body parts that do not correspond to the prior larval regions. Since we had shown previously that during metamorphosis a large number of cells are removed by programmed cell death (PCD), we aimed to analyze whether cells of the neuropeptide-expressing larval nerve net are among those sacrificed. By immunohistochemical staining and in situ hybridization, we labeled GLWamide- and RFamide-expressing cells. Double staining of neuropeptides and degraded DNA (TUNEL analysis) identified some neurosensory cells as being apoptotic. Derangement of the cytoplasm and rapid destruction of neuropeptide precursor RNA indicated complete death of these particular sensory cells in the course of metamorphosis. Additionally, a small group of RFamide-positive sensory cells in the developing mouth region of the primary polyp could be shown to emerge by proliferation. Our results support the idea that during metamorphosis, specific parts of the larval neuronal network are subject to neurodegeneration and therefore not used for construction of the adult nerve net. Most neuronal cells of the primary polyp arise by de novo differentiation of stem cells commited to neural differentiation in embryogenesis. At least some nerve cells derive from proliferation of progenitor cells. Clarification of how the nerve net of these basal eumetazoans degenerates may add information to the understanding of neurodegeneration by apoptosis as a whole in the animal kingdom.     

Role of interstitial cell migration in generating position-dependent patterns of nerve cell differentiation in Hydra

The role of interstitial cell migration in the formation of newly differentiated nerve cells was examined during head regeneration in Hydra magnipapillata. When distal tissue was removed from the body of a wild-type strain (105), nerve cell differentiation occurred at a rapid rate during the first 48 hr of regeneration, slowing after this point. Rapid nerve cell differentiation was due primarily to migration of interstitial cells, some of which appeared to be nerve cell precursors, into the regenerating head. The migration decreased considerably after the first 48 hr of regeneration. In reg-16, a mutant strain deficient in head regeneration, no migration of interstitial cells and hence no new nerve cell differentiation were observed in the regenerating tip. However, the interstitial cells of reg-16 were observed to migrate into regenerating tissue of strain 105. These observations suggest that the migration of nerve cell precursors plays an important role when the new nerve net is being established during head regeneration.     

Nerve commitment in Hydra: I. Role of morphogenetic signals

The kinetics of nerve commitment during head regeneration in Hydra were investigated using a newly developed assay for committed cells. Committed nerve precursors were assayed by their ability to continue nerve differentiation following explanation of small pieces of tissue. Committed nerve precursors appear at the site of regeneration within 6 hr after cutting and increase rapidly. The increase is localized to the site of regeneration and does not occur at proximal sites in the body column of the regenerate. The increase is delayed about 8–12 hr when regeneration occurs at sites lower in the body column. The results show, furthermore, that redistribution of committed precursors does not play a major role in the pattern of nerve differentiation during regeneration. Since the increase in committed nerves coincides with the increase in morphogenetic potential of the regenerating tissue, the results strengthen the idea that morphogenetic signals are involved directly in the control of nerve commitment in Hydra.     

The timing of initial neuropeptide expression by an identified insect neuron does not depend on interactions with its normal peripheral target

To study the developmental regulation of a neuropeptide phenotype, we have analyzed the biochemical and morphological differentiation of two identifiable neurons in embryos of the moth, Manduca sexta. The central cell, CF, and the peripheral cell, L1, are both neuroendocrine neurons that express neuropeptides related to the molluscan tetrapeptide FMRFamide. Both neurons project axons to the transvers nerve in each thoracic segment. Within the CF and L1 cells, neuropeptide-like immunoreactivity was localized to secretory granules that had cell specific morphologies and sizes. The onset of neuropeptide expression in the two cell types displayed a similar pattern: immunoreactivity was first detected in distal processes and soon after within cells bodies. However, the onsets occurred at different times: for the CF cell, neuropeptides were first seen at 60%-63% of embryonic development, after the neuron had extended a long axon into the periphery, while L1 neuropeptide expression began at ～42%, as it first extended its growth cone. These times were related in that they corresponded to the arrival times of the respective growth cones at a similar position in the developing peripheral nerve. Withinthis region of the nerve, the growth cones of both cell typesexhibited a transient and cell-specific interaction with an identified mesodermal cell, called the Syncytium. Like the L1 and B neurons (Carr and Taghert, 1988b), the CF growth cones typically grew past this cell, yet remained attached to it by lamellipodial and filopodial processes of the axon. Ultrastructurally, the interaction involved filopodial adhesion to and insertion within the Syncytial cell. Two other nonneuroendocrine cell types grew axons past this same region, but showed no such tendencies. To test the hypothesis that the morphological and biochemical differentiation of these cells was somehow linked, central ganglia were isolated (as individuals or connected as ganglionic chains) in tissue culture, prior to the time when CF growth cones entered the periphery and prior to the development of CF neuropeptide expression. In the majority of cases, CF neurons nevertheless displayed their neuropeptide phenotype at a normal and cell-specific stage. We conclude that the initiation of neuropeptide expression is highly correlated with schedules of morphological differentiation in these neurons, but that, in the case of the CF neuron, it is not regulated by interactions of the growth cone with peripheral structures.