Totipotent migratory stem cells in a hydroid

Hydroids, members of the most ancient eumetazoan phylum, the Cnidaria, harbor multipotent, migratory stem cells lodged in interstitial spaces of epithelial cells and are therefore referred to as interstitial cells or i-cells. According to traditional understanding, based on studies in Hydra, these i-cells give rise to several cell types such as stinging cells, nerve cells, and germ cells, but not to ectodermal and endodermal epithelial cells; these are considered to constitute separate cell lineages. We show here that, in Hydractinia, the developmental potential of these migratory stem cells is wider than previously anticipated. We eliminated the i-cells from subcloned wild-type animals and subsequently introduced i-cells from mutant clones and vice versa. The mutant donors and the wild-type recipients differed in their sex, growth pattern, and morphology. With time, the recipient underwent a complete conversion into the phenotype and genotype of the donor. Thus, under these experimental conditions the interstitial stem cells of Hydractinia exhibit totipotency.     

Wnt signaling controls the stem cell-like asymmetric division of the epithelial seam cells during C. elegans larval development

Metazoan stem cells repopulate tissues during adult life by dividing asymmetrically to generate another stem cell and a cell that terminally differentiates. Wnt signaling regulates the division pattern of stem cells in flies and vertebrates. While the short-lived nematode C. elegans has no adult somatic stem cells, the lateral epithelial seam cells divide in a stem cell-like manner in each larval stage, usually generating a posterior daughter that retains the seam cell fate and an anterior daughter that terminally differentiates. We show that while wild-type adult animals have 16 seam cells per side, animals with reduced function of the TCF homolog POP-1 have as many as 67 seam cells, and animals with reduced function of the β-catenins SYS-1 and WRM-1 have as few as three. Analysis of seam cell division patterns showed alterations in their stem cell-like divisions in the L2-L4 stages: reduced Wnt signaling caused both daughters to adopt non-seam fates, while activated Wnt signaling caused both daughters to adopt the seam fate. Therefore, our results indicate that Wnt signaling globally regulates the asymmetric, stem cell-like division of most or all somatic seam cells during C. elegans larval development, and that Wnt pathway regulation of stem cell-like behavior is conserved in nematodes.     

Wnt signaling in hydroid development: ectopic heads and giant buds induced by GSK-3beta inhibitors

In Hydractinia, a colonial marine hydroid representing the basal phylum Cnidaria, Wnt signaling plays a major role in the specification of the primary body axis in embryogenesis and in the establishment of the oral pole during metamorphosis. Here we report supplementing investigations on head regeneration and bud formation in post-metamorphic development. Head and bud formation were accompanied by the expression of Wnt, frizzled and Tcf. Activation of Wnt signaling by blocking GSK-3beta affected regeneration, the patterning of growing polyps and the asexual formation of new polyps in the colony. In the presence of lithium ions or paullones, gastric segments excised from adult polyps showed reversal of tissue polarity as they frequently regenerated heads at both ends. Phorbol myristate acetate, a known activator of protein kinase C increased this effect. Global activation of the Wnt pathway caused growing polyps to form ectopic tentacles and additional heads along their body column. Repeated treatment of colonies evoked the emergence of many and dramatically oversized bud fields along the circumference of the colony. These giant fields fell apart into smaller sub-fields, which gave rise to arrays of multi-headed polyps. We interpret the morphogenetic effects of blocking GSK-3beta as reflecting increase in positional value in terms of positional information and activation of Wnt target genes in molecular terms.     

Role of the cellular environment in interstitial stem cell proliferation in Hydra

Summary The role of the cellular environment on hydra stem cell proliferation and differentiation was investigated by introduction of interstitial cells into host tissue of defined cellular composition. In epithelial tissue lacking all non-epithelial cells the interstitial cell population did not grow but differentiated into nerve cells and nematocytes. In host tissue with progressively increased numbers of nerve cells growth of the interstitial cell population was positively correlated to the nerve cell density. In agreement with previous observations (Bode et al. 1976), growth of the interstitial cell population was also found to be negatively correlated to the level of interstitial cells present. The strong correlation between the growth of the interstitial cell population and the presence of interstitial cells and nerve cells implies that interstitial cell proliferation is controlled by a feedback signal from interstitial cells and their derivatives. Our results suggest that the cellular environment of interstitial cells provides cues which are instrumental in stem cell decision making. Offprint requests to: T.C.G. Bosch     

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.     

Nerve cell and nematocyte production in Hydra is deregulated by lithium ions

Summary LiCl, a well-known vegetalising agent, interferes with the commitment of stem cells to nerve cells and nematocytes in Hydra attenuata. Treatment with 20 mM LiCl inhibits commitment to nerve cells, treatment with 1 mM LiCl inhibits commitment to nematocytes. However, LiCl does not prevent stem cells committed to the nematocyte pathway from dividing and differentiating into nests of nematocytes. Following LiCl treatment, determination to nerve cells and nematocytes is triggered again. Commitment to nerve cells is strongly stimulated within the first 3 h following pulse treatment with LiCl if the animals have been fed immediately prior to treatment. In Hydra exposed to LiCl for 10 days the stem cell density is reduced by at least 90% of the initial value, and nematocytes are almost completely missing, whereas the density of nerve cells is within the normal range in animals with normal morphology. Animals which developed a transverse constriction in the middle of the body axis contain a 1.7-fold higher nerve cell density in the lower part than is observed in control animals.     

Characterization of interstitial stem cells in hydra by cloning.

A procedure has been developed for cloning interstitial stem cells from hydra. Clones are prepared by introducing small numbers of viable cells into aggregates of nitrogen mustard-inactivated host tissue. Clones derived from added stem cells are identified after 1–2 weeks of growth by staining with toluidine blue. The incidence of clones increases with increasing input of viable cells according to one-hit Poisson statistics, indicating that clones arise from single cells. After correction for cell losses in the procedure, about 1.2% of the input cells are found to form clones. This compares with estimates from in vivo experiments of about 4% stem cells in whole hydra [David, C. N., and Gierer, A. (1974). Cell cycle kinetics and development of Hydra attenuata. III. Nerve and nematocyte differentiation. J. Cell Sci.16, 359–375.]Differentiation of nematocytes and nerve cells in clones was analyzed by labeling precursors with [³H]thymidine and scoring labeled nerves and nematocytes 2 days later. Nine clones examined in this way contained both differentiated nerve cells and nematocytes, demonstrating that the interstitial stem cell is multipotent. This result suggests that the observed localization of nerve and nematocyte differentiation in whole hydra probably occurs at the level of stemcell determination. The observation that differentiated cells occur very early in clone development suggests that a stem cell's decision to proliferate or differentiate is regulated by shortrange feedback signals which are already saturated in young clones.     

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.     

Pulses of ammonia and methylamine induce down-regulation of nematocyte and nerve cell populations in Hydrozoa (Hydra; Hydractinia)

Summary Hydrozoa replace used-up nematocytes (cnidocytes) by proliferation and differentiation from interstitial stem cells (i cells). Repeated pulsed exposure ofHydra to elevated levels of unprotonated ammonia leads to successive loss of the various types of nematocytes: first of the stenoteles, then of the isorhizas and finally of the desmonemes. The loss is due to deficits in supply; the number of nematoblasts and differentiating intermediates is reduced. In the hydroidHydractinia the main process leading to numerical reduction was observed in vivo: mature nematocytes as well as precursors emigrate from their place of origin into the gastrovascular channels where they are removed by phagocytosis. This is a regular means by which these animals down-regulate an induced surplus of nematocytes. With lower effectiveness, pulses of methylamine, trimethylamine and glutamine also induce elimination of the nematocyte lineages. In the long term the population of nerve cells, which are permanently but slowly renewed from interstitial neuroblasts, decreases, too. After 2 months of daily repeated treatment the density of the Arg-Phe-amide-positive nerve cells was reduced to 50% of its normal level. Thus, ammonia induces down-regulation of all interstitial cell lineages. The temporal sequence of the ammonia-induced loss reflects the diverse rates with which the various i cell descendants normally are renewed.     

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.     

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.     

Transgenic stem cells in Hydra reveal an early evolutionary origin for key elements controlling self-renewal and differentiation

Little is known about stem cells in organisms at the beginning of evolution. To characterize the regulatory events that control stem cells in the basal metazoan Hydra, we have generated transgenics which express eGFP selectively in the interstitial stem cell lineage. Using them we visualized stem cell and precursor migration in real-time in the context of the native environment. We demonstrate that interstitial cells respond to signals from the cellular environment, and that Wnt and Notch pathways are key players in this process. Furthermore, by analyzing polyps which overexpress the Polycomb protein HyEED in their interstitial cells, we provide in vivo evidence for a role of chromatin modification in terminal differentiation. These findings for the first time uncover insights into signalling pathways involved in stem cell differentiation in the Bilaterian ancestor; they demonstrate that mechanisms controlling stem cell behaviour are based on components which are conserved throughout the animal kingdom.     

Inhibition of Wnt/β-catenin signaling by a soluble collagen-derived frizzled domain interacting with Wnt3a and the receptors frizzled 1 and 8

The Wnt/β-catenin pathway controls cell proliferation, death and differentiation. Several families of extracellular proteins can antagonize Wnt/β-catenin signaling, including the decoy receptors known as secreted frizzled related proteins (SFRPs), which have a cysteine-rich domain (CRD) structurally similar to the extracellular Wnt-binding domain of the frizzled receptors. SFRPs inhibit Wnt signaling by sequestering Wnts through the CRD or by forming inactive complexes with the frizzled receptors. Other endogenous molecules carrying frizzled CRDs inhibit Wnt signaling, such as V3Nter, which is proteolytically derived from the cell surface component collagen XVIII and contains a biologically active frizzled domain (FZC18) inhibiting in vivo cell proliferation and tumor growth in mice. We recently showed that FZC18 expressing cells deliver short-range signals to neighboring cells, decreasing their proliferation in vitro and in vivo through the Wnt/β-catenin signaling pathway. Here, using low concentrations of soluble FZC18 and Wnt3a, we show that they physically interact in a cell-free system. In addition, soluble FZC18 binds the frizzled 1 and 8 receptors' CRDs, reducing cell sensitivity to Wnt3a. Conversely, inhibition of Wnt/β-catenin signaling was partially rescued by the expression of full-length frizzled 1 and 8 receptors, but enhanced by the expression of a chimeric cell-membrane-tethered frizzled 8 CRD. Moreover, soluble, partially purified recombinant FZC18_CRD inhibited Wnt3a-induced β-catenin activation. Taken together, the data indicate that collagen XVIII-derived frizzled CRD shifts Wnt sensitivity of normal cells to a lower pitch and controls their growth.     

两种GSK-3β抑制剂对间充质干细胞神经分化的影响

目的:观察两种GSK-3β抑制剂(氯化锂和CHIR99021)对间充质干细胞向神经元样细胞分化的影响作用。方法:体外分离、扩增人胎盘间充质干细胞,分别用低浓度氯化锂和CHIR99021对细胞进行刺激,检测其对增值活力及Wnt通路中β连环蛋白调节作用的影响,并在加药条件下对细胞进行神经诱导。结果:在细胞活力相近的状况下,CHIR99021更能促进细胞分裂增殖和向神经元样细胞分化的能力,而免疫荧光结果显示,CHIR99021对Wnt通路的起效时间比氯化锂更为迟缓。结论:GSK-3β受抑制可促进间充质干细胞向神经细胞分化。在低浓度条件下,CHIR99021的抑制作用起效比氯化锂缓慢,但作用延续时间比氯化锂更长,因而对神经分化的长期促进作用也更加明显。     

Wnt signal transduction pathways

The Wnt signaling pathway is an ancient and evolutionarily conserved pathway that regulates crucial aspects of cell fate determination, cell migration, cell polarity, neural patterning and organogenesis during embryonic development. The Wnts are secreted glycoproteins and comprise a large family of nineteen proteins in humans hinting to a daunting complexity of signaling regulation, function and biological output. To date major signaling branches downstream of the Fz receptor have been identified including a canonical or Wnt/β-catenin dependent pathway and the non-canonical or β-catenin-independent pathway which can be further divided into the Planar Cell Polarity and the Wnt/Ca²⁺ pathways, and these branches are being actively dissected at the molecular and biochemical levels. In this review, we will summarize the most recent advances in our understanding of these Wnt signaling pathways and the role of these pathways in regulating key events during embryonic patterning and morphogenesis.Key words: Wnt, frizzled, dishevelled, canonical, non-canonical, β-catenin, Planar Cell Polarity     

Wnt signaling controls temporal identities of seam cells in Caenorhabditis elegans

The Wnt signaling pathway regulates multiple aspects of the development of stem cell-like epithelial seam cells in Caenorhabditis elegans, including cell fate specification and symmetric/asymmetric division. In this study, we demonstrate that lit-1, encoding the Nemo-like kinase in the Wnt/β-catenin asymmetry pathway, plays a role in specifying temporal identities of seam cells. Loss of function of lit-1 suppresses defects in retarded heterochronic mutants and enhances defects in precocious heterochronic mutants. Overexpressing lit-1 causes heterochronic defects opposite to those in lit-1(lf) mutants. LIT-1 exhibits a periodic expression pattern in seam cells within each larval stage. The kinase activity of LIT-1 is essential for its role in the heterochronic pathway. lit-1 specifies the temporal fate of seam cells likely by modulating miRNA-mediated silencing of target heterochronic genes. We further show that loss of function of other components of Wnt signaling, including mom-4, wrm-1, apr-1, and pop-1, also causes heterochronic defects in sensitized genetic backgrounds. Our study reveals a novel function of Wnt signaling in controlling the timing of seam cell development in C. elegans.     

A regulatory role of Wnt signaling pathway in the hematopoietic differentiation of murine embryonic stem cells

One of the most important issues in stem cell research is to understand the regulatory mechanisms responsible for their differentiation. An extensive understanding of mechanism underlying the process of differentiation is crucial in order to prompt stem cells to perform a particular function after differentiation. To elucidate the molecular mechanisms responsible for the hematopoietic differentiation of embryonic stem cells (ESCs), we investigated murine ES cells for the presence of hematopoietic lineage markers as well as Wnt signaling pathway during treatments with different cytokines alone or in combination with another. Here we report that Wnt/beta-catenin signaling is down-regulated in hematopoietic differentiation of murine ES cells. We also found that differentiation induced by the interleukin-3, interleukin-6, and erythropoietin combinations resulted in high expression of CD3e, CD11b, CD45R/B220, Ly-6G, and TER-119 in differentiated ES cells. A high expression of beta-catenin was observed in two undifferentiated ES cell lines. Gene and protein expression analysis revealed that the members downstream of Wnt in this signaling pathway including beta-catenin, GSK-3beta, Axin, and TCF4 were significantly down-regulated as ES cells differentiated into hematopoietic progenitors. Our results show that the Wnt/beta-catenin signaling pathway plays a role in the hematopoietic differentiation of murine ESCs and also may support beta-catenin as a crucial factor in the maintenance of ES cells in their undifferentiated state.