Clarity in the cold

At 18ºC, embryos without Klar fail to develop normally. Far right: normal embryo (with Klar) at 18ºC. IMAGE: EMBL/I. GASPAR.
At 18ºC, embryos without Klar fail to develop normally. Far right: normal embryo (with Klar) at 18ºC. IMAGE: EMBL/I. GASPAR.

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How fruit flies thrive in colder climes: the value of precisely controlled experiments and detailed analysis, revealed in a study by EMBL Heidelberg scientists.

Like all insects, the fruit fly Drosophila melanogaster is a ‘cold-blooded’ animal. The term is a bit of a misnomer; it doesn’t necessarily mean that those animals are always cold, but rather that they cannot control their body temperature. If a fruit fly is sitting in a lab at 25ºC – which seems to be their preferred temperature – essentially all the cells in its body will be at 25ºC. If room temperature drops to 18ºC, so does the temperature inside the fly’s cells. The cells that make up its compound eyes will be at 18ºC, its stomach cells will be at 18ºC, and if it’s a female, the egg cells in its ovaries will be developing at 18ºC. This inability to keep a constant body temperature poses problems, as many of the enzymes that carry out vital processes within those cells are sensitive to temperature fluctuations.

Anne Ephrussi’s lab at EMBL Heidelberg and colleagues at Rochester University in the USA have now discovered one way in which the fruit fly manages to lay viable eggs not only in its preferred conditions, but also at lower temperatures.

The group’s main focus is on how some RNA molecules get placed in particular positions in cells, as this can be essential for cell function and embryo development. One model used for these studies is a molecule called oskar, which acts as a determinant in the egg cell, defining the region that will become the fly embryo’s posterior. oskar is carried to the right place in the cell in the form of an RNA template, which is then translated into protein on the spot, where it is needed. In 2011, Imre Gaspar, a postdoc in the Ephrussi lab, got to talking to Michael Welte from Rochester University, when the latter came to Heidelberg to give a talk. Welte told Gaspar his lab had found that a protein called Klar accumulates in the same place as Oskar, at the same time. When he added that they had some indications that without Klar, Oskar didn’t behave ‘normally’, Gaspar’s curiosity was spiked. Welte went back to the US, and what would become a four-year collaboration got under way, with Gaspar aiming to find out exactly what the connection was.“It required a lot of detailed analysis to figure out exactly what was going on, because our initial ideas were completely wrong,” says Gaspar.

It required a lot of detailed analysis to figure out exactly what was going on, because our initial ideas were completely wrong

Klar was first discovered three decades ago by EMBL alumni Christiane Nüsslein-Volhard and Eric Wieschaus, as part of work that eventually earned them a Nobel prize. They named it based on the German word for ‘clear-sightedness’, since embryos without Klar are transparent. This transparency arises from an inability to transport lipid droplets properly, and the scientists reasoned that, in egg cells that lack Klar, oskar too might fail to get to where it was needed. The scientists’ initial data seemed to confirm this assumption, but when Gaspar delved deeper he found that the effects were more subtle – and complex.

“Somehow, in the absence of Klar, at 25 degrees-ish, all these processes are functioning at rates that are compatible with each other – things are working in sync,” says Ephrussi. “But if one then goes to 18 degrees, things go wrong.” Despite originating from tropical climes, Drosophila melanogaster can thrive at 18ºC. However, at this temperature, in flies engineered to have no Klar, oskar RNA is carried to the egg cell’s posterior pole, but not all that RNA stays there. The surplus oskar RNA is taken elsewhere, but is already primed for translating into protein, so the protein ends up accumulating in other parts of the egg cell – at the risk of rendering it unviable or causing major developmental defects.

This incoordination comes about, the scientists surmise, because the drop in temperature has a more dramatic effect on the anchoring machinery than on the transport network. “If one process slows down more than the other, you may try to live with it – but you are probably going to fail,” Gaspar explains. “The alternative is you have to find a dedicated regulator that brings down the other process; and that’s what Klar does.” In short, when a fruit fly egg cell is subjected to low temperatures, Klar cranks oskar transport down to almost nothing, to keep delivery of oskar at a rate that the anchoring machinery – which is considerably slowed by the temperature drop – is capable of handling.

If one process slows down more than the other, you may try to live with it – but you are probably going to fail

Klar also coordinates the transport of other cargoes within cells, so it may very well be that this protein helps keep these processes in sync, too – and not just in Drosophila melanogaster. For instance, a separate study published just weeks before Gaspar’s showed that the embryos of several Drosophila species can develop at a wide range of temperatures. And, since many other events that are crucial to development also involve several steps driven by different molecular machines, it seems likely that there are other enzymes besides Klar whose role is to keep such processes in synch. Such molecules are likely key to enabling ‘cold blooded’ animals to deal with the vagaries of changing environments, and to Gaspar they raise a tantalising question: how do they sense temperature shifts?

Although understanding what this particular mini-thermometer does when it feels the cold took years of work, elaborate experiments and complex analysis, ultimately the findings have an element of simplicity, the scientists say. “Fruit-flies survive even without Klar, so the ancestors of modern flies could have done without it. But when Klar evolved in some of those flies, they became able to regulate this process, and lay more embryos that eventually hatched. So they will have had more progeny, and that’s how they will quickly have overgrown those that could not. So in terms of evolution, this process can be understood very simply,” Gaspar humbly concludes.

At 18ºC, with Klar (top), oskar transport is slowed down to almost nothing (yellow lines track movement of oskar), while without Klar (bottom) there is considerably more movement

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