Awakenings: Stem Cell Style

Jack Craig

In the semitropical South-East of the United States there’s a plant called the miracle fern (Pleopeltis polypodioides). In adverse conditions, it’s capable of curling up, drying out and remaining dormant for years on end, only to spring back to life when the weather improves. It can do this any number of times, entering and exiting “hibernation” at will.

In the fruit fly Drosophila melanogaster, neural stem cells function very much the same way. As stem cells, they’re capable transforming into a number of different cell types as they mature, and during the embryonic phase, when the fly is still an egg, they undergo a phase of rapid growth. At this point in the fly’s development, before it even hatches, large parts of its central nervous system are developing for the first time, readying the animal for its time as a larva. Once this early development is done, however, the stem cells go dormant just like the miracle fern. 24hourslater,once the egg has hatched, they reactivate, and begin proliferation anew. In a 2010 research article, James Chell and Andrea Brand of the University of Cambridge were curious to discover exactly how this reactivation occurs.

It was known going into the project that an organ called the “fat body” exudes a chemical signal requisite for stem cell reawakening only when the larval fly receives a diet rich in nutritious amino acids, the building blocks of proteins. This makes sense, as the sole purpose of the larval stage is to build up body mass and store energy for the eventual transformation into an adult fly. Interestingly, the wingless, worm-like larvae actually contain all their adult appendages stored in their bodies as “imaginal discs,” which are like Ikea flat-packs. When the larvae are sufficiently well fed, they enter a cocoon and the imaginal discs expand like an accordion into new legs and wings. The fly then emerges from its pupa like a small, hairy butterfly.

But where does the fat-body messenger go? On which cells does the signal act in order for it to take effect? Chell and Brand managed to identify the specific type of cells, called glia, on which this signal has its effect. These particular glia form a network around the neural stem cells, and, when they receive a signal from the fat body, they in turn produce a signal of their own in the form of insulin-like peptides (basically just signaling proteins) called dILPs. The dILPs, then are crucial for reactivation of the dormant stem cells. The system works like a series of smoke signals: each cell type perceives one signal, interprets it, then sends a signal of its own. In very simple terms, the fat body perceives a rich diet and signals the glial cells, which then (through possible intermediates) signal the stem cells to reactivate.

To prove that a process or compound is the cause of a certain behavior or biological process, three criteria must be met: presence, necessity and sufficiency. First, the presumed signal or other substance of interest must actually be in the affected region at the right time (presence). Second, if it’s removed, the process it is supposed to be causing must stop (necessity). Finally, introduction of the signal can restore the process to normal or trigger it in an unexpected place (sufficienty). Chell and Brand covered each of these bases, demonstrating that the dILPs released by their glial cells as well as a related pathway responsible for the “PI3K” signal are, in fact, crucial parts of the stem cell reactivation puzzle.

First, a mutant fly line was created in which cells producing dILPs would be fluorescently tagged under the microscope for easy viewing. These flies were then inspected in the larval stage, just as their stem cells were beginning to reawaken. Sure enough, the glial cells lit up brightly, indicating the presence of dILP signals. Flies similarly tagged for PI3K lit up as well, suggesting that the PI3K pathway is active in awakening stem cells. The requirement for presence, then was met.

Next, to prove that dILPs are necessary for stem cell proliferation, a “null-mutant” was created, in which four separate dILPs were rendered inactive. These mutants were developmentally retarded, already an indication of the necessity of dILPs, but when they were examined later in development, their stem cells had still failed to grow to full size. This clearly implies some degree of necessity. To test the same criterion for the PI3K pathway, yet another mutant was created that over-expressed a signal inhibiting PI3K, essentially sabotaging its own pathway. Once again, the neural stem cells failed to develop normally, indicating that the pI3K pathway, appeared to respond to the dILPs and was necessary for the “awakening” of neural stem cells.

Finally, to test sufficiency, flies were fed on a diet of pure sucrose, a sugar completely free of the amino acids usually required for stem cell activation. These flies were then forced to produce dILPs via artificial means, even though they would typically not be expressed due to poor diet. Sure enough, the stem cells actually did reactivate at the appropriate time even without the natural cue of a healthy diet, suggesting that the dILPs alone were sufficient for reactivation. Similarly, a key component in the PI3K pathway was artificially up-regulated in flies fed no amino acids, and they too were able to carry out nearly normal development complete with stem cell reactivation.

Every rigorous scientific study is only as good as its controls. The authors needed to raise a number of flies on a sucrose-only diet, controlling for the effects of a sugar only diet that was used to test sufficiency of dILPs and PI3K signaling. Only because the stem cells in these controls failed to reactivate could Chell and Brand conclude that they’d isolated the molecular basis for neural stem cell awakening.

Another point to address was the original assumption that dILPs were activated in response to diet. To demonstrate the solid basis for their assumptions, the intrepid authors compared fly larvae raised both on protein-rich and sucrose-only diets using dILP fluorescent live imaging techniques as described above. Sure enough, the amino acid-deprived flies just didn’t light up like their natural counterparts due to a severe lack of dILPs, further cementing the study’s assumptions as fact (or as close to it as science is ever willing to go!)
Thus, one component of an obtuse developmental quirk of the fruit fly was partially elucidated. This is great for the fly, and suggests that diet is a powerful influence on the ability to renew and reawaken stem cells.

What does this mean for us humans, though? Is it just a fly study for fly biologists? The answer is stem cells. Stem cells, though often a poorly-defined family of cells, are immensely interesting to scientist for their ability to develop into any number of different cell types (which, for the record, most cells can’t do). And they’re incredibly important for studying diseases and developing treatments. Although stem cells may not be the panacea some have claimed, there exists the very real possibility that stem cells could be used to re-grow damaged human tissue. Where transplants and reconstructions fail, your own cells could be used to grow all new body parts. This study brings us one step closer to learning how to switch stem cells on and off at will. The focus of this study isn’t just some generic stem cell but neural stem cells, which  could be used for the recovery of a damaged or degenerating central nervous system. So while the present study is, superficially, about fruit fly development, there’s the very real implication that, in the foreseeable future, we could be applying fly studies to human subjects. What we learn now is just the background information for the real work that will be done in the coming years.

Click here to access the research article on which this piece was based. You can also visit the Brand Lab website to learn more about their on-going work.

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