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Stem Cell Plasticity in Mammals and Transdetermination in Drosophila: Common Themes?

^a Max Planck Institut für Immunbiologie, Freiburg, Germany;
^b Department of Zoology, University of Washington, Seattle, Washington, USA;
^c Institut für Strahlenkunde und Zellforschnung (MSZ), Universität Würzburg, Würzburg, Germany

Key Words. Mammalian stem cells • Somatic stem cells • Stem cell plasticity • Drosophila • Imaginal discs • Transdetermination

Correspondence: Albrecht M. Müller, Ph.D., Institut für Strahlenkunde und Zellforschnung (MSZ), Universität Würzburg, Versbacherstr. 5, 97078 Würzburg, Germany. Telephone: 49-931-201-3848; Fax: 49-931-201-3835/87; e mail: albrecht.mueller{at}mail.uni-wuerzburg.de

	ABSTRACT

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Stem cells have been identified in a number of mammalian tissues (e.g., bone marrow, muscle, gut, skin, and neural tissues). Until recently, it was generally believed that the differentiation potential of a mammalian somatic stem cell is restricted to one tissue only, as in the case of hematopoietic stem cells differentiating into hematopoietic cells. In this sense, somatic stem cells are limited in their differentiation potential. Several lines of evidence now challenge the idea of unilateral development. New reports show mammalian somatic stem cells can, in the course of regeneration, repopulate heterologous cell systems and therefore possess a surprisingly broad spectrum of differentiation potential. Thus, mammalian stem cells are apparently capable of fate changes between stem cell systems, although the mechanisms leading to such changes are unclear.

Mechanistic models for fate changes have been proposed in Drosophila, specifically for transdetermination of imaginal discs. Imaginal discs of the larva are the primordia of the adult exoskeleton and appendages, for example, legs, and antennae. Transplantation experiments of imaginal discs have shown that discs are determined for their disc identity. Transdetermination in Drosophila refers to cases when, after regenerative cell divisions, imaginal disc cells change from one state of determination to another, initiating a pathway of differentiation leading to structures other than those corresponding to the initial state or determination; for example, an antennal imaginal disc transdetermines to a leg imaginal disc. A fate change is thus possible in both mammalian somatic stem cells and Drosophila imaginal discs following transplantation and subsequent proliferation. Here we summarize and compare observations made in such cases of stem cell and imaginal disc differentiation.

	INTRODUCTION

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Nuclear transfer experiments have proven that the nuclei of differentiated cells, even when isolated from adult animals, are capable of totipotency when injected into enucleated oocytes. In contrast, intact somatic cells do not exhibit this capacity. In development, the totipotency of embryonic cells begins to be restricted after the blastula stage. During gastrulation, cells lose their potential to generate all cell lineages of the developing embryo. Although most cells proceed to terminal differentiation, some rare cells, known as multipotent stem cells, are maintained with a tissue-specific differentiation potential.

Stem cells are at the top of the cellular hierarchy and generate and maintain many cell systems. They are found, for example, in the developing and adult brain, liver, intestine, and hematopoietic system. Their function is to maintain the cellular homeostasis of tissues and regenerate and replace worn-out and damaged cells. Stem cell systems are composed of three different cell types: stem cells, transit cells, and differentiated cells [1]. Stem cells are defined purely functionally in that they can self-renew and generate all differentiated cells of a given stem cell system. Transit cells have limited self-renewal and multilineage differentiation potential, and differentiated cells have no self-renewal and very restricted differentiation potential.

The differentiation potential of somatic stem cells was regarded as being confined to one tissue or organ. However, recent data have shown that stem cells from one tissue can also repopulate heterologous tissues. The main goal of the present article is to review the literature published on fate changes of somatic stem cells, and compare the fate changes in somatic stem cell systems with a specific type of fate change known as transdetermination in Drosophila imaginal discs.

	SOMATIC STEM CELLS ON THE BRINK OF PLURIPOTENCY

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Recently the notion of a tissue-restricted fate of stem cells has been challenged by Bjornson et al. [2]. They isolated neural stem cells (NSCs) from fetal and adult brains, expanded them in vitro in medium containing epidermal growth factor and basic fibroblast growth factor and injected them intravenously into irradiated hosts. Analysis of NSC-derived contribution in the hematopoietic compartment 5 to 12 months post-injection revealed the presence of donor-derived blood cells in hematopoietic tissues as detected by flow cytometry and clonogenic in vitro assays. Clonally derived adult NSCs gave hematopoietic repopulation comparable to that from embryonic and adult bulk NSC clones. It is remarkable that in this case, the NSCs of neuroectodermal origin changed germ layers and contributed to the mesodermally derived hematopoietic cells. Even more surprising are results which show that NSCs from the adult mouse brain, following injection into blastocysts, contribute to the formation of chimeric embryos and give rise to all germ layers [3].

Several other recent papers further analyzing the differentiation potential of hematopoietic stem cells (HSCs) and muscle stem cells (MSCs) also support this new view that somatic stem cells can change fate. Flow cytometry (fluorescence-activated cell sorter) of Hoechst 33342-stained bone marrow cells distinguishes a side population (SP) of cells that excludes dye and is highly enriched for HSCs. The property of Hoechst dye efflux is common to HSCs of various species, suggesting this trait could be used to isolate SP cells from other tissue types as well [4]. It was reported that following transplantation into irradiated recipients, muscle SP cells or unfractionated and in vitro-cultured skeletal muscle cells contribute to the hematopoietic cell lineage [5, 6]. Gussoni and coworkers also demonstrated that muscle SP cells can, in the same animal, repopulate muscle and bone marrow, suggesting that muscle SP cells possess hematopoietic and myogenic activity [5]. In addition, bone marrow-derived SP cells were shown to contribute to muscle fibers in a Duchenne's muscular dystrophy mouse model. Furthermore, injection of unfractionated bone marrow cells reconstituted the hepatic cell lineage [7]. A transformation of "blood into brain" was shown by Eglitis and Mezey by demonstrating that cells in the bone marrow can differentiate into microglia and macroglia in the brain of adult recipient mice [8]. It is so far unclear which cell fractions in the bone marrow are the cellular origins for the hepatic oval and neural cells. However, reports demonstrating the wide differentiation potential of NSCs muscle- and bone marrow-derived SP cells strongly suggest that different somatic stem cell types can generate heterologous tissues and organs. Thus, great differentiation plasticity resides in somatic stem cells (Fig. 1).

View larger version (31K): [in this window] [in a new window]   Figure 1. Plasticity in mammalian somatic stem cells. Shown is a diagram of mammalian development starting from the zygote, via the blastula stage to a stage when germ layers and somatic stem cell systems including mature effector cells have been formed. The direction of fate changes of somatic stem cells is indicated by arrows.

	HYPOTHESES ON STEM CELL PLASTICITY

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In this context it is of importance to emphasize that a certain degree of heterogeneity is inherent to stem cell populations such that not all stem cells of a given tissue are equivalent, for example, in their cell cycle status and gene expression pattern. Indeed, heterogeneity has been reported in HSCs [9], and it is presently not known whether fate changes of somatic stem cells reflect the ability of a presently undefined subfraction of a given stem cell population.

How could fate changes of somatic stem cells be explained if it were possible to exclude heterogeneity in stem cell populations? At least four hypotheses come readily to mind. In the first, all tissues possess a common type of stem cell with the surrounding microenvironment directing differentiation into the particular progenitor and mature effector cells. This common stem cell could be a relic of early developmental stages. In a second hypothesis, stem cells revert to an ES cell-like developmental state before they generate heterologous cell systems. Alternatively, in a third hypothesis, stem cells with tissue-specific identities could be directly transformed into stem cells of other cell systems. A fourth hypothesis would predict that somatic stem cells have per se bi- or oligopotential activities such as neurohematopoietic and neuromuscular activities but are not pluripotent.

Preliminary characterizations of different types of somatic stem cells, such as HSCs, NSCs, and MSCs, revealed that there are differences as well as similarities in phenotype and functional aspects. For example, HSCs show characteristic features which distinguish them from NSCs, such as the time needed for hematopoietic reconstitution of irradiated recipients [2] and the expression of the c-kit tyrosine kinase growth factor receptor, which is expressed on HSCs but not on NSCs (Müller et al., unpublished observation). Another difference is that HSCs cannot be easily expanded in vitro whereas NSCs and MSCs can. However, analysis of muscle SP cells shows several characteristics of bone marrow-derived HSCs, such as the expression of SCA-1, the ability to efflux the Hoechst dye 33342 and the absence of hematopoietic lineage markers. The presence or absence of the c-kit receptor on muscle SP cells is controversial. A more detailed and systematic analysis of different somatic stem cell types in view of their gene expression profiles and functional properties is needed for a better understanding of the nature of stem cells and their developmental plasticity.

While observations of mammalian stem cells of one adult tissue contributing to a heterologous cell system are striking, the basis for such events is currently unclear. The phenomena themselves, however, are reminiscent of events in Drosophila collectively known as transdetermination, and concrete models for Drosophila transdetermination have been advanced.

	TRANSDETERMINATION

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Drosophila development involves three very different stages: embryonic, larval, and adult. Embryonic cells that become polyploid form the larval body. Pockets of embryonic cells remain diploid during embryogenesis and proliferate in the larval stages. Some of these diploid cells generate the imaginal discs, the anlagen of adult cuticular structures including antennae, legs, and wings. For example, there are six different leg discs: each differentiates one specific leg after metamorphosis. During metamorphosis, imaginal discs undergo eversion, synthesize pigments, and secrete cuticle for the corresponding part of the exoskeleton.

Transplantation experiments have shown that imaginal discs are determined to form specific adult structures long before metamorphosis. This disc-specific determination is mostly maintained, even after numerous extra cell divisions, and even when cells of one disc are placed next to cells from another disc [10]. However, after regenerative cell divisions, disc cells can switch their determined state, for example, leg cells switch to wing determination—this process is known as transdetermination [11]. In transdetermination experiments imaginal discs are isolated from donor larvae, mechanically fragmented and transplanted into the abdominal cavity of adult flies, and allowed to undergo regenerative growth. Subsequently these discs are reisolated and transplanted into the body cavity of host larvae where they synchronously go into metamorphosis with the host. After metamorphosis, the identity of the adult structure which differentiated from the implanted disc is identified.

All imaginal disc types can transdetermine, but the majority of regenerating disc fragments transdetermine rarely and only after many cell divisions. Therefore, in general, cell determination is properly propagated. Within each imaginal disc, specific regions are identified which, when stimulated to divide, will transdetermine in more than 60% of the cases. Such regions were termed weak points [12].

Transdetermination is a polyclonal event and not the result of either differentiation of reserve cells or somatic mutations. In addition, transdetermination does not occur at random. Figure 2 summarizes observed transdetermination events. A reciprocal transdetermination event can occur with different probabilities. For example, there is a greater chance that antenna transdetermines to wing than wing to antenna (Fig. 2). Different transdetermination events may also occur sequentially in regeneration from the same disc. For instance, transdetermination of genitalia to leg or antenna is considered a first-order transdetermination event; genitalia can also transdetermine to wing, for example, but only after going through the first-order event. In fact, even third-order transdetermination events are possible but there is a strict sequence of transdetermination steps.

View larger version (30K): [in this window] [in a new window]   Figure 2. Sequence of transdetermination events and effect of homeotic mutations and ectopic gene expression on Drosophila imaginal discs. Schema of transdetermination events from fragmentation experiments are shown in black. For each transdetermination between two structures, the length of the arrow indicates the probability of transdetermination. Dotted black lines indicate rare events. Homeotic transformation that corresponds to observed transdetermination is indicated in green letters (Antp: Antennapedia; Ubx: Ultrabithorax; pb: proboscipedia; Scr: Sex combs reduced). Red arrows and letters represent transdetermination events observed after ectopic expression of a transgene (Antp., Dll: distal-less; ey: eyeless; vg: vestigial; wg: wingless). The figure includes only those transdetermination events that are also observed in fragmentation experiments. Therefore, even though ectopic expression of ey produces eyes in many places of the animal, only wing cells transdetermine to eye after fragmentation experiments and thus are included in this figure. *Eye stands for all derivatives of the eye imaginal disc. Numbers (0-3) indicate the number of transdetermination steps necessary for genital disc cells to become thorax. The figure is adapted and modified from [12] and [18].

What happens at the molecular level to cause transdetermination? As an example, let us examine the activities of the genes wingless (wg) and decapentaplegic (dpp, known as BMP 4 in vertebrates) in the leg to wing transdetermination. In imaginal discs, the transcription factor vestigal (vg) is a molecular marker for dorsal disc identities, such as wing and haltere (Fig. 3). During development of the dorsal discs, vg expression colocalizes with wg and dpp expression. In ventral discs like the leg discs, wg and dpp do not overlap, and there is also no vg expression (Fig. 3). However, if both wg and dpp expression are experimentally sustained in the dorsal regions of intact leg discs (ventral discs), cell division is stimulated and vg transcription is activated and the leg cells transdetermine to dorsal identities such as wing and haltere in over 90% of ventral discs ([14], Fig. 3). These ventral to dorsal transdeterminations can also be induced by sustained ectopic expression of other genes in the wg and dpp signal transduction pathway. Ectopic expression of the wg signaling pathway also induces vg expression in the antenna, where transdetermination to wing is observed ([15] and Fig. 2). In addition, other first-order transdetermination events, that do not necessarily involve vg activity, occur when wg is ectopically expressed. In these cases, ectopic wg through an interaction with dpp, changes the expression of homeotic genes resulting in transdetermination ([15] and Fig. 2).

View larger version (21K): [in this window] [in a new window]   Figure 3. In mid-larval development wg and dpp expression overlap in parts of the wing disc (cross-hatched) and activate vg expression leading to wing development. When wg is ectopically expressed to overlap with dpp, vg expression is induced and leg cells transdetermine to wing.

	CONCLUSIONS

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In the experimental settings analyzing the developmental plasticity of mammalian stem cells, microenvironmental influences are central to the observed plasticity and current understanding of the molecular regulators of fate changes is elusive. In Drosophila, the molecular basis for transdetermination events has been shown to arise from the mis-expression of key regulatory genes which are either activated or repressed. The ease with which such misregulation can arise might represent developmental lineage relationships and explain the tendencies and directionality of transdetermination events in Drosophila.

Although transdetermination does not involve stem cells as the mammalian cases do, we find several parallels: A) transdetermination events and mammalian stem cell fate changes are seen under regenerative proliferation; B) progenitor cells which are able to differentiate into mature effector cells are involved, and C) fate changes do not lead to the production of intermediate cell types but produce a normal pattern of differentiated cells. In addition, in Drosophila Hox genes are primary candidates to be involved in transdetermination events (Fig. 2) and Hox genes are expressed in somatic stem cells and regulate stem cell behavior. For example, certain members of the Hox clusters are preferentially expressed in primitive hematopoietic cell types and overexpression of HOXB4 causes the selective expansion of HSCs [17].

We suggest that observations like NSC and MSC reconstitution of the hematopoietic system represent stem cell fate changes equivalent to first-order transdetermination events. Like transdetermination, we suggest that there will be an ordered sequence of possible fate changes among different types of adult stem cells and specific probabilities of fate changes towards certain cell lineages, as it might be easier for an MSC to adopt a hematopoietic rather than neural fate. Further, genes controlling pattern formation during development are involved in fate change events in mammalian somatic stem cells. Transdetermination in Drosophila may serve as a paradigm for the better understanding of mammalian stem cell biology. Trying to uncover and understand the potential of tissue-specific stem cells would obviate some of the discussion over use of totipotent embryonic cells for somatic cell therapy.

	ACKNOWLEDGMENTS

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The authors wish to thank M. C. Gibson for generating Figure 2 and M. Schubiger, J. Modolell, and A. Garcia Bellido for critical comments. GS was supported by NIH (GM58282).

	References

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