Analysis of a foot regeneration deficient strain of Hydra oligactis.

A foot regeneration deficient strain of Hydra oligactis with altered size regulation was investigated. Analysis of the concentration of four morphogenetically active substances in Hydra oligactis showed that the foot regeneration deficiency was mainly due to a drastically reduced foot activator concentration. Foot activator was distributed as a very steep gradient in Hydra oligactis leading to a more severe impairment of foot regeneration the closer to the head cutting was done. The concentration of foot inhibitor was comparable, that of head activator slightly increased and that of head inhibitor reduced compared to Hydra vulgaris. Studies at the cellular level implied that the enlarged gastric region was due to an elevated level of inhibition hinting at a shift in the ratio between bound and free head inhibitor in this animal.     

Genome sizes and chromosomes in the basal metazoan Hydra

Hydras belong to one of the earliest eumetazoan animal groups, but to date very little is known about their genome sizes, gene numbers, and chromosomes. Here we provide genome size estimates and corresponding karyotypes for five Hydra species. Nuclear DNA contents were assessed by slide-based Feulgen microphotometry. Hydra oligactis possesses the largest genome of 1450 Mbp, followed by similar 1 C capacities in H. carnea (1350 Mbp), H. vulgaris (1250 Mpb) and H. circumcincta (1150 Mbp). The smallest genome of 380 Mbp was determined in H. viridissima. While the number of chromosomes is identical in all five Hydra species (2n = 30), the size of the chromosomes is strictly correlated to the size of the genome, with H. viridissima having conspicuously small chromosomes. The taxonomic and evolutionary significance of the C-value and chromosomal size variation in this ancient group of metazoans as well as its impact on genomic organization and forthcoming genome projects are discussed.     

中国绿水螅分子系统发生地位的初步探讨

目的:初步探讨中国绿水螅(Hydra sinensis)分子系统发生地位以及水螅属内部各类群系统发生关系。方法:采用酚-氯仿法提取中国绿水螅总DNA,扩增线粒体COI和16S r RNA基因片段并进行DNA序列测定,再利用Clustal及MEGA等生物信息学分析软件进行系统发生分析。结果:在本研究重建的所有系统发生树中,中国绿水螅始终与绿水螅Hydra viridissima的不同种群一起构成绿水螅单系群。同时,棕色水螅群的单系性被基于COI基因的NJ树以及基于16S r RNA基因的NJ树和ML树支持,唯独基于COI基因的ML树不支持棕色水螅群的单系发生。在基于COI基因的ML树中纤弱水螅族在系统树的基部独立为一支系,而绿水螅群和其他棕色水螅群水螅一起组成另一支系,提示纤弱水螅族水螅的系统发生地位值得进一步探讨。值得注意的是,根据本文的结果,棕色水螅群内3族的划分仍然有一定疑问。基于COI基因的NJ树和ML树支持普通水螅族、寡水螅族和纤弱水螅族各自族内的单系发生,但16S r RNA基因的NJ树和ML树中仅普通水螅族水螅聚为单系群,而寡水螅族和纤弱水螅族水螅各自并非单系发生。结论:把水螅属划分为绿水螅群及棕色水螅群有一定的合理性,但棕色水螅群内寡水螅族、普通水螅族和纤弱水螅族3族的划分还有待商榷。     

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.     

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.     

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.     

Germ cells in Hydra oligactis males. II. Evidence for a subpopulation of interstitial stem cells whose differentiation is limited to sperm production

Animals containing germline-restricted interstitial cells were obtained by treating males from a clone of Hydra oligactis with hydroxyurea (HU) to lower the interstitial population to 1 or 2 cells per animal. A 3-day HU treatment produced animals whose interstitial cells did not form somatic cells, but did produce sperm. The isolation of these cells in HU-treated animals has lead us to propose that the interstitial cell population may contain subpopulations which possess different growth dynamics and developmental potentials. Through asexual propagation, we have cloned several animals containing only sperm precursor interstitial cells and have examined the growth and differentiation behavior of these cells in offspring propagated over a 2-year period. Evidence has been obtained which demonstrates (1) the extensive self-renewal capacity of the sperm precursor interstitial cells, and (2) the restricted differentiation capacity of these interstitial stem cells. Factors which affect cells entering and traversing the spermatogenic pathway are also presented.     

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.     

Cell cycle length, cell size, and proliferation rate in hydra stem cells

We have analyzed the cell cycle parameters of interstitial cells in Hydra oligactis. Three subpopulations of cells with short, medium, and long cell cycles were identified. Short-cycle cells are stem cells; medium-cycle cells are precursors to nematocyte differentiation; long-cycle cells are precursors to gamete differentiation. We have also determined the effect of different cell densities on the population doubling time, cell cycle length, and cell size of interstitial cells. Our results indicate that decreasing the interstitial cell density from 0.35 to 0.1 interstitial cells/epithelial cell (1) shortens the population doubling time from 4 to 1.8 days, (2) increases the [3H]thymidine labeling index from 0.5 to 0.75 and shifts the nuclear DNA distribution from G2 to S phase cells, and (3) decreases the length of G2 in stem cells from 6 to 3 hr. The shortened cell cycle is correlated with a significant decrease in the size of interstitial stem cells. Coincident with the shortened cell cycle and increased growth rate there is an increase in stem cell self-renewal and a decrease in stem cell differentiation.     

Extracellular matrix (mesoglea) of Hydra vulgaris. II. Influence of collagen and proteoglycan components on head regeneration.

Hydra are characterized by having their body wall organized as an epithelial bilayer with an intervening acellular layer termed the mesoglea. As an extension of the previous study which indicated that mesoglea is a primitive basement membrane which has retained some characteristics of interstitial extracellular matrix, the present study was undertaken to analyze the role of mesoglea components during head regeneration in Hydra vulgaris. Studies were conducted that utilized drugs that affect collagen processing or secondary collagen structure (beta-aminoproprionitrile; 2,2'-dipydridyl; and cis-4-hydroxy-L-proline) and a drug that inhibits addition of glycosaminoglycan chains to proteoglycan core proteins (p-nitrophenyl-beta-D-xylopyranoside). These studies indicated that alterations in the structure of collagens or proteoglycans caused blockage of head regeneration in Hydra as monitored over a 48-hr period. Blockage of head regeneration was reversible once the drugs were removed, indicating that the drugs were not having a general toxic effect on the organism. Radiotracer studies also indicated that blockage of head regeneration was not simply due to a general depression of protein synthesis by the drugs. Various controls indicated that each drug was affecting mesoglea components under the conditions utilized in these studies. These observations indicate that preservation of normal mesoglea structure is required for Hydra head regeneration to proceed.     

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.     

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.     

Cell lineages in Hydra: isolation and characterization of an interstitial stem cell restricted to egg production in Hydra oligactis

In an attempt to isolate unipotent stem cells (progenitors to the nerve cells, nematocytes, gland cells, and gametes) from Hydra oligactis females, animals were treated with a drug (hydroxyurea, HU) that preferentially lowers or eliminates the interstitial stem cells, leaving the epithelial tissue intact. In this epithelial environment, interstitial cells remaining after treatment will proliferate and differentiate, permitting a long-term analysis of their developmental capabilities. Following treatment of females with HU, animals were isolated that contained interstitial cells that gave rise to eggs only. Two clones of animals containing these cells were propagated for several years and the growth and differentiation behavior of the interstitial cells examined in their asexually produced offspring. During this time, the cells displayed an extensive proliferative capacity (classifying them as stem cells) and remained restricted to egg differentiation. It is proposed that both the sperm- and the egg-restricted stem cells arise from a multipotent stem cell, which also gives rise to the somatic cells (see above), and that, in hydra, sex is ultimately determined by interactions between cells of the two germ cell lineages.     

Germline stem cells and sex determination in Hydra

The sex of germline stem cells (GSCs) in Hydra is determined in a cell-autonomous manner. In gonochoristic species like Hydra magnipapillata or H. oligactis, where the sexes are separate, male polyps have sperm-restricted stem cells (SpSCs), while females have egg-restricted stem cells (EgSCs). These GSCs self-renew in a polyp, and are usually transmitted to a new bud from a parental polyp during asexual reproduction. But if these GSCs are lost during subsequent budding or regeneration events, new ones are generated from multipotent stem cells (MPSCs). MPSCs are the somatic stem cells in Hydra that ordinarily differentiate into nerve cells, nematocytes (stinging cells in cnidarians), and gland cells. By means of such a backup system, sexual reproduction is guaranteed for every polyp. Interestingly, Hydra polyps occasionally undergo sex-reversal. This implies that each polyp can produce either type of GSCs, i.e. Hydra are genetically hermaphroditic. Nevertheless a polyp possesses only one type of GSCs at a time. We propose a plausible model for sex-reversal in Hydra. We also discuss so-called germline specific genes, which are expressed in both GSCs and MPSCs, and some future plans to investigate Hydra GSCs.     

强壮水螅的特征及其与寡水螅的种间差别问题

一种广泛分布于黑龙江省的大型具柄水螅被暂定为强壮水螅Hydra robusta(IT(?) 1947)。这一中国品系与IT(?)(1947)在日本报告的新种主要特征相同,包括具有精巢乳头。只是精巢乳头不稳定。在实验室条件下第一次有性生殖时每个雄体都发生精巢乳头,但第二次有性生殖时,同一群的后代可全部失去精巢乳头。这是介于强壮水螅与寡水螅Hydra oligactis Pallas(1766)之间的中间性状。从而导致作者做出结论,认为过去其他学者所主张的,以精巢乳头的有无做为鉴别特征来区分强壮水螅和寡水螅是不可靠的。因此,本文又检验了此种水螅的体细胞染色体,证明2n=30,其中第二对染色体上有明显近中位置的次缢痕,最小染色体的长度为最大染色体长度的二分之一以上。这些可做为此种水蝗的鉴别特征。对Niiyama(1944)和Datta(1970)在寡水螅上所做的染色体研究也做了比较及讨论。