Regulation of interstitial cell differentiation in Hydra attenuata. I. Homeostatic control of interstitial cell population size.

Mechanisms regulating the population size of the multipotent interstitial cell (i-cell) in Hydra attenuata were investigated. Treatment of animals with 3 cycles of a regime of 24 h in 10-2 M hydroxyurea (HU) alternated with 12 h in culture medium selectively killed 95-99% of the i-cells, but had little effect on the epithelial cells. The i-cell population recovered to the normal i-cell:epithelial cell ratio of I:I within 35 days. Continuous labelling experiments with [3H]thymidine indicate that the recovery of the i-cell population is not due to a change in the length of the cell cycle of either the epithelial cells or the interstitial cells. In control animals 60% of the i-cell population undergo division daily while 40% undergo differentiation. Quantification of the cell types of HU-treated animals indicates that a greater fraction of the i-cells were dividing and fewer differentiating into nematocytes during the first 2 weeks of the recovery after HU treatment. Therefore, the mechanism for recovery involves a shift of the 60:40 division:differentiation ratio of i-cells towards a higher fraction in division until the normal population size of the i-cells is regained. This homeostatic mechanism represents one of the influences affecting i-cell differentiation.     

Migration of multipotent interstitial stem cells in Hydra

Stem cells in Hydra represent one of the phylogenetically most ancient stem cell systems and, therefore, provide information for reconstructing the early history of stem cell control mechanisms. Hydra's interstitial stem cells are multipotent and differentiate into both somatic cell types and germ line cells. Although it is well accepted that cells of the interstitial cell lineage are migratory, the in vivo migratory potential of multipotent interstitial stem cells has never been explored. Combining in vivo tracing of genetically labeled interstitial stem cells and tissue transplantation, we show that in contrast to precursor cells, multipotent interstitial stem cells are stationary. Only when exposed to tissue depleted of the interstitial cell lineage, interstitial stem cells start to migrate and to repopulate emptied stem cell niches. We conclude that multipotent interstitial stem cells in Hydra are static and that microenvironmental cues including signals derived from the interstitial cell lineage or from niche cells can trigger a shift in collective stem cell behavior to start migration.     

Maternally localized germ plasm mRNAs and germ cell/stem cell formation in the cnidarian Clytia

The separation of the germ line from the soma is a classic concept in animal biology, and depending on species is thought to involve fate determination either by maternally localized germ plasm ("preformation" or "maternal inheritance") or by inductive signaling (classically termed "epigenesis" or "zygotic induction"). The latter mechanism is generally considered to operate in non-bilaterian organisms such as cnidarians and sponges, in which germ cell fate is determined at adult stages from multipotent stem cells. We have found in the hydrozoan cnidarian Clytia hemisphaerica that the multipotent "interstitial" cells (i-cells) in larvae and adult medusae, from which germ cells derive, express a set of conserved germ cell markers: Vasa, Nanos1, Piwi and PL10. In situ hybridization analyses unexpectedly revealed maternal mRNAs for all these genes highly concentrated in a germ plasm-like region at the egg animal pole and inherited by the i-cell lineage, strongly suggesting i-cell fate determination by inheritance of animal-localized factors. On the other hand, experimental tests showed that i-cells can form by epigenetic mechanisms in Clytia, since larvae derived from both animal and vegetal blastomeres separated during cleavage stages developed equivalent i-cell populations. Thus Clytia embryos appear to have maternal germ plasm inherited by i-cells but also the potential to form these cells by zygotic induction. Reassessment of available data indicates that maternally localized germ plasm molecular components were plausibly present in the common cnidarian/bilaterian ancestor, but that their role may not have been strictly deterministic.     

Migration and differentiation potential of stem cells in the cnidarian Hydractinia analysed in eGFP-transgenic animals and chimeras

To analyse cell migration and the differentiation potential of migratory stem cells in Hydractinia, we generated animals with an eGFP reporter gene stably expressed and transmitted via the germline. The transgene was placed under the control of two different actin promoters and the promoter of elongation factor-1α. One actin promoter (Act-II) and the EF-1α promoter enabled expression of the transgene in all cells, the other actin promoter (Act-I) in epithelial and gametogenic cells, but not in the pluripotent migratory stem cells. We produced chimeric animals consisting of histocompatible wild type and transgenic parts. When the transgene was under the control of the epithelial cell specific actin-I promoter, non-fluorescent transgenic stem cells immigrated into wild type tissue, stopped migration and differentiated into epithelial cells which then commenced eGFP-expression. Migratory stem cells are therefore pluripotent and can give rise not only to germ cells, nematocytes and nerve cells, but also to epithelial cells. While in somatic cells expression of the act-I promoter was restricted to epithelial cells it became also active in gametogenesis. The act-I gene is expressed in spermatogonia, oogonia and oocytes. In males the expression pattern showed that migratory stem cells are the precursors of both the spermatogonia and their somatic envelopes. Comparative expression studies using the promoters of the actin-II gene and the elongation factor-1α gene revealed the potential of transgenic techniques to trace the development of the nervous system.     

Stem cells and lineages of the intestine: a developmental and evolutionary perspective

The intestine consists of epithelial cells that secrete digestive enzymes and mucus (gland cells), absorb food particles (enterocytes), and produce hormones (endocrine cells). Intestinal cells are rapidly turned over and need to be replaced. In cnidarians, mitosis of differentiated intestinal cells accounts for much of the replacement; in addition, migratory, multipotent stem cells (interstitial cells) contribute to the production of intestinal cells. In other phyla, intestinal cell replacement is solely the function of stem cells entering the gut from the outside (such as in case of the neoblasts of platyhelminths) or intestinal stem cells located within the midgut epithelium (as in both vertebrates or arthropods). We will attempt in the following to review important aspects of midgut stem cells in different animal groups: where are they located, what types of lineages do they produce, and how do they develop. We will start out with a comparative survey of midgut cell types found across the animal kingdom; then briefly look at the specification of these cells during embryonic development; and finally focus on the stem cells that regenerate midgut cells during adult life. In a number of model systems, including mouse, zebrafish and Drosophila, the molecular pathways controlling intestinal stem cells proliferation and the specification of intestinal cell types are under intensive investigation. We will highlight findings of the recent literature, focusing on aspects that are shared between the different models and that point at evolutionary ancient mechanisms of intestinal cell formation.     

i-Cells and reparative morphogenesis in coelenterates

A historical survey on so called "theory of totipotent interstitial cells (i-cells)" and analysis of modern notion concerning the system of stem i-cells (SSC) in hydra and some other Coelenterata, as well as its peculiar regularity, normal and at various restorative morphogeneses are presented. Morphogenesis and space organization of polyps do not depend on the SSC, just the reverse, the SSC characteristics -- probability of selfrenovation, the TC proportion differentiating in one or other direction -- are determined by the morphogenetic process. In hydra and hydroids a possibility of reversed differentiation of the TC, with a successive change of the differentiation program is demonstrated under certain experimental conditions.     

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.     

The hydroid Hydractinia: a versatile, informative cnidarian representative

The Cnidaria represent the most ancient eumetazoan phylum. Members of this group possess typical animal cells and tissues such as sensory cells, nerve cells, muscle cells and epithelia. Due to their unique phylogenetic position, cnidarians have traditionally been used as a reference group in various comparative studies. We propose the colonial marine hydroid, Hydractinia, as a convenient, versatile platform for basic and applied research in developmental biology, reproduction, immunology, environmental studies and more. In addition to being a typical cnidarian representative, Hydractinia offers many practical and theoretical advantages: studies that are feasible in Hydra like regeneration, pattern regulation, and cell renewal from stem cells, can be supplemented by genetic analyses and classical embryology in Hydractinia. Metamorphosis of the planula larva of Hydractinia can be used as a model for cell activation and communication and the presence of a genetically controlled allorecognition system makes it a suitable model for comparative immunology. Most importantly, Hydractinia may be manipulated at most aspects of its (short) life cycle. It has already been the subject of many studies in various disciplines, some of which are discussed in this essay.     

A novel model to study the dorsolateral migration of melanoblasts

Melanocytes derived from pluripotent neural crest cells migrate initially in the dorsolateral pathway between the ectoderm and dermomyotome. To understand the role of specific proteins involved in this cell migration, we looked for a cellular model that mimics the in vivo behavior of melanoblasts, and that allows functional studies of their migration. We report here that wild-type embryonic stem (ES) cells are able to follow the ventral and dorsolateral neural crest pathways after being grafted into chicken embryos. By contrast, a mutant ES cell line deficient for beta1 integrin subunits, proteins involved in cell-extracellular interactions, had a severely impaired migratory behavior. Interestingly, ES cells deficient for Kit, the tyrosine kinase receptor for the stem cell factor (SCF), behaved similarly to wild-type ES cells. Thus, grafting mouse ES cells into chicken embryos provides a new cellular system that allows both in vitro and in vivo studies of the molecular mechanisms controlling dorsolateral migration.     

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.     

Genetic analysis of developmental mechanisms in hydra. XVIII. Mechanism for elimination of the interstitial cell lineage in the mutant strain Sf-1

The interstitial cell lineage in mutant strain sf-1 of hydra is temperature sensitive and is lost rapidly from tissue when the animal is cultured at a restrictive temperature of 23 degrees C or higher. The mechanism responsible for this cell elimination process was investigated. Sf-1 polyps were treated at a restrictive temperature of 27 degrees C for varying lengths of time, their tissues were macerated, and the resultant dissociated cells were examined for evidence of phagocytosis after Feulgen staining. It was found that large phagocytic vacuoles were present in the cytoplasm of some epithelial cells. These vacuoles contained partially degraded cells, whose nuclei had highly-condensed and intensely Feulgen-positive chromatin granules. This indicated that, as in colchicine-treated (Campbell, 1976) or starved (Bosch and David, 1984) wild-type hydra, the epithelial cells in strain sf-1 engulfed and disintegrated other cells in the phagocytic vacuoles. The incidence of phagocytosis was higher in sf-1 tissue maintained at elevated temperature than in sf-1 tissue maintained at normal temperature. However, the observed incidence was relatively low (maximally 0.14 phagocytosed cells per epithelial cell) and appeared to be too low to account for the very rapid interstitial cell loss occurring in this strain. We concluded that elimination of the interstitial cell lineage at a restrictive temperature in strain sf-1 takes place in part by phagocytosis and in part by other yet-unidentified mechanisms (cf., Marcum et al., 1980).     

Snail family members and cell survival in physiological and pathological cleft palates

Palate fusion is a complex process that involves the coordination of a series of cellular changes including cell death and epithelial to mesenchymal transition (EMT). Since members of the Snail family of zinc-finger regulators are involved in both triggering of the EMT and cell survival, we decided to study their putative role in palatal fusion. Furthermore, Snail genes are induced by transforming growth factor beta gene (TGF-beta) superfamily members, and TGF-beta(3) null mutant mice (TGF-beta(3)-/-) show a cleft palate phenotype. Here we show that in the wild-type mouse at the time of fusion, Snail is expressed in a few cells of the midline epithelial seam (MES), compatible with a role in triggering of the EMT in a small subpopulation of the MES. We also find an intriguing relationship between the expression of Snail family members and cell survival associated to the cleft palate condition. Indeed, Snail is expressed in the medial edge epithelial (MEE) cells in TGF-beta(3)-/-mouse embryo palates, where it is activated by the aberrant expression of its inducer, TGF-beta(1), in the underlying mesenchyme. In contrast to Snail-deficient wild-type pre-adhesion MEE cells, Snail-expressing TGF-beta(3) mutant MEE cells survive as they do their counterparts in the chick embryo. Interestingly, Slug is the Snail family member expressed in the chick MEE, providing another example of interchange of Snail and Slug expression between avian and mammalian embryos. We propose that in the absence of TGF-beta(3), TGF-beta(1) is upregulated in the mesenchyme, and that in both physiological (avian) and pathological (TGF-beta(3)-/-mammalian) cleft palates, it induces the expression of Snail genes promoting the survival of the MEE cells and permitting their subsequent differentiation into keratinized stratified epithelium.     

EGFR and Notch signaling respectively regulate proliferative activity and multiple cell lineage differentiation of Drosophila gastric stem cells

Quiescent, multipotent gastric stem cells (GSSCs) in the copper cell region of adult Drosophila midgut can produce all epithelial cell lineages found in the region, including acid-secreting copper cells, interstitial cells and enteroendocrine cells, but mechanisms controlling their quiescence and the ternary lineage differentiation are unknown. By using cell ablation or damage-induced regeneration assays combined with cell lineage tracing and genetic analysis, here we demonstrate that Delta (Dl)-expressing cells in the copper cell region are the authentic GSSCs that can self-renew and continuously regenerate the gastric epithelium after a sustained damage. Lineage tracing analysis reveals that the committed GSSC daughter with activated Notch will invariably differentiate into either a copper cell or an interstitial cell, but not the enteroendocrine cell lineage, and loss-of-function and gain-of-function studies revealed that Notch signaling is both necessary and sufficient for copper cell/interstitial cell differentiation. We also demonstrate that elevated epidermal growth factor receptor (EGFR) signaling, which is achieved by the activation of ligand Vein from the surrounding muscle cells and ligand Spitz from progenitor cells, mediates the regenerative proliferation of GSSCs following damage. Taken together, we demonstrate that Dl is a specific marker for Drosophila GSSCs, whose cell cycle status is dependent on the levels of EGFR signaling activity, and the Notch signaling has a central role in controlling cell lineage differentiation from GSSCs by separating copper/interstitial cell lineage from enteroendocrine cell lineage.     

Disturbing epithelial homeostasis in the metazoan Hydra leads to drastic changes in associated microbiota

Microbes have profound influence on the biology of host tissue. Imbalances in host–microbe interaction underlie many human diseases. Little, however, is known about how epithelial homeostasis affects associated microbial community structure. In Hydra , the epithelium actively shapes its microbial community indicating distinct selective pressures imposed on the epithelium. Here, using a mutant strain of Hydra magnipapillata we eliminated all derivatives of the interstitial stem cell lineage while leaving both epithelial cell lineages intact. By bacterial 16S rRNA gene analysis we observed that removing gland cells and neurones from the epithelium causes significant changes in hydra's microbial community. Absence of interstitial stem cells and nematocytes had no affect on the microbiota. When compared with controls, animals lacking neurones and gland cells showed reduced abundance of β-Proteobacteria accompanied by a significantly increased abundance of a Bacteroidetes bacterium. This previously unrecognized link between cellular tissue composition and microbiota may be applicable to understanding mechanisms controlling host–microbe interaction in other epithelial systems.     

The interstitial cells control the sexual phenotype of heterosexual chimeras of hydra

The three stem cell populations in hydra, the epithelial cells of the ectoderm and endoderm, which make up the body of the hydra, and the interstitial cells, which give rise to nerve cells, nematocytes, and gametes, were tested for their effects on determining the sexual phenotype of individuals. This was done by creating epithelial hydra, which are devoid of interstitial cells and their derivatives, of one sexual type and repopulating them with interstitial cells from individuals of the other sexual type. The resulting heterosexual chimeras were found in all cases to display the same sexual phenotype as that of the interstitial cell donor, indicating this cell type is responsible for the sex of the animal. The epithelial tissue had no influence in determining which gamete type was produced.