Research team Julianna “Lita” Bozler, PhD, and Balint Kacsoh, PhD, formerly of Dr. Giovanni Bosco’s laboratory at the Geisel School of Medicine at Dartmouth College, released a study in July that is gaining a lot of attention in both the scientific community and the broader, mainstream media. In their article, published in eLife,they show that parental environmental exposures can influence offspring behavior for many generations using the fruit fly (Drosophila melanogaster) model system.
For the study they exposed fruit flies to one of their natural predators—a species of parasitoid wasps—who inject their eggs into fruit fly larvae, which consume the developing flies from the inside out after hatching. Upon exposure to this threat, female fruit flies are known change their egg laying habits and lay their eggs on ethanol-laden food supplies, deterring the wasp from larval infection.
Leveraging this insight, they exposed fruit flies to the wasps and collected the eggs that were laid. Half of the eggs were used to produce the next generation of flies and the other half were grown to adulthood and the egg-laying ethanol preference of the mature flies was analyzed. Shockingly, the progeny of wasp-exposed flies, never having been exposed directly to the wasps, had a preference for laying their eggs on ethanol-laden food. This preference persisted, although gradually moved towards wild-type [natural] behavior, for five generations. They discovered imprinting of a certain maternally derived locus, Neuropeptide-F (NPF), homolog of Neuropeptide-Y (NPY) in humans, as being important for this inherited behavior.
Their findings have broad implications on the long-standing question of ‘nature or nurture’ and PhD candidate in the Program in Experimental and Molecular Medicine (PEMM) program, Faith Anderson, sat down with them to learn more about their study and shed some light on this age-old debate.
Faith Anderson: Can you explain what “transgenerational inheritance of neuronally-encoded behavior” means in layman’s terms?
Julianna “Lita” Bozler: The transgenerational part just means across multiple generations and we’re changing the phenotype or behavior across not just the immediate generation but, in this example, across five generations. [As for] the neuronally-encoded behavior part… the idea here is that behavior can arise from a number of different things and our research suggested that this behavior was actually a change in the neuropil or a change in neuronal signaling, which is how we more classically like to think of behavior.
Faith: Some articles written about your work have been describing this phenomenon as inherited ‘memory.’ Do you think that’s an appropriate term?
Lita: I’m not in favor of that terminology exactly, because we specifically found that… offspring can inherit this behavior without having an effective long-term memory. It’s a bit of a misnomer, but at the same time, I can understand why people are using that as shorthand. It’s more of a molecular memory than how neurobiologists would describe memory.
Faith:What led you to study the effect of parental experiences influencing future generations?
Lita: It was round-about to be honest. At the time, we were studying the effects of stress exposures on fruit flies. In some of the sequencing data we had, we just saw a few unusual blips that got us brainstorming and we thought ‘there’s no way this would be across generations,’ but we kept coming back to it and thought ‘well that’s a really easy experiment.’ We tested it and got this positive result… so I did it again, and did it again, and finally I was completely convinced ‘there’s no way this is an accident.’ I’d like to give a lot of credit to the lab that we were in, Gio Bosco’s lab, because it was this collaborative, exciting environment where you’re allowed to have crazy ideas…and explore some of the weird biology that’s out there.
Faith: What makes this model (Drosophila) ideal for studying transgenerational inheritance?
Balint Kacsoh: One part is a short generational time. You can do transgenerational studies in mice, but it takes months for them to mature… here we can do multiple testings of various genetic backgrounds in a matter of the 10 day lifecycle of a fly. You can go through multiple genetic backgrounds and test them to actually nail down the mechanism, which is what we did in this study. The speed that we can do it in, as well as the reagents available, makes the fly the ideal system to study this.
Faith: Are these results what you were expecting?
Balint: Yes and no. I think there was definitely the surprise, first of all, that the phenomenon happened. It was also surprising that it was the [Neuropeptide-F] NPF locus itself that was getting altered, inducing those changes… we weren’t expecting the specificity of it.
Lita: There were a few times where we were like ‘wouldn’t it be really wild if these things happened but there’s no way they’re going to happen.’ That’s how the experiment started, looking at the F1 generation [first filial generation], [we thought] it’s going to be an intergenerational effect, so it’s just going to be the immediate offspring that are affected. ‘There’s no way that this is in the germline [reproductive cells], that sounds impossible!’ ‘The only place that’s ever been seen really is in a couple of plants and C. Elegans[a nematode roundworm], this is not a phenomenon seen elsewhere right?’ That was surprising to us. The other thing that was surprising to us… was when we were looking into the actual molecular passage of this transgenerational effect. I was geared up for small RNA… and then the chromosome result was very, very unexpected. This project has had some real ups and downs for us where we thought we knew what was going on…and then the data just led us in a completely different direction.
Faith: What do you think are the advantages/disadvantages of this transient effect rather than a permanent one?
Balint: Alcohol in general is not the best thing for a fly. There are developmental defects of growing up in high concentrations of ethanol, their development is slowed down and morphologic defects can happen. It makes sense that, if the predator is no longer present, you start reverting back to the wild-type state. It would not be beneficial, without a corresponding evolutionary change or resistance, to keep laying [eggs] on ethanol permanently.
Faith: Although the initial effect of this environmental stressor is seen in the mom, why do you think you see both male and female offspring capable of passing on this information?
Lita: It was a little perplexing to us. Our working theory is that there’s some sort of imprinting of the [Neuropeptide-F] NPF locus. Something needs to happen in mom, so in the female germline, to activate that imprinting… but once that’s imprinted, it’s passed onto male and female offspring. We think there’s someway of maintaining that imprinted locus, even in males, that’s being passing on to their offspring as well.
Faith: Has this phenomenon been seen in the real world before, whether in other animals or humans?
Lita: Human studies are difficult and very controversial, and I don’t want to overstate the significance of our findings and say this impacts humans health in the most direct ways. There are a number of human studies that show parental stress environments do impact at least the next generation. There was the Dutch Hunger Winter [Dutch famine of 1944-1945 near the end of World War II] and a number of other studies that show that food deprivation in one generation can have lasting metabolic changes in offspring and grandchildren.
There are some studies done in humans that link paternal post-traumatic stress disorder (PTSD) to increased anxiety in offspring, but these are human studies and we’re obviously not going to experiment that, so these are a little anecdotal. There’s some really interesting work going on in mice and other model systems where you can actually do more experimentation. All of these things are pointing generally to the idea that there is some degree of extra-environmental information that is being passed along to offspring. The field is really young so we’re still working out how much can this environmental information be fine-tuned. To my knowledge, we’re the first group to look at the effects of alcohol consumption and link the addiction pathways to transgenerational impacts, but I think more generally, other model systems have shown similar things.
Faith: What challenges did you face trying to study this phenomenon in the laboratory?
Lita: I’ll speak more to the general problems of studying behavior in lab which is that… animal behavior, in order to quantify it, needs to be reproducible. Very small changes in environment can lead to profound changes in your experimental outcomes. Keeping things so beautifully standardized across the collection of many, many generations, regardless of what system you’re working on, can be incredibly challenging.
Balint: If fly food is too dry, it can yield nutrient deprivation, which has been shown to have a trans-generational effect in flies on its own, metabolically. If the food is too wet, it’s the opposite direction. That’s something that you might think is a subtle difference, but can have big effects.
Faith: What implications do you think this work has for human health and disease? What do you think the next steps are?
Balint: I think the biggest part is that conserved region. [Neuropeptide-F] NPF [is a homolog of] [Neuropeptide Y] NPY in mammals, so it does a very similar thing; knowing that there could be imprinting on that particular region I think could be insightful for other models as well. I wouldn’t say it’s directly jumping straight to humans. We’d have to see in a more mammalian model if the NPF locus modification is conserved. I think that is something we can actually start looking at in higher order system. The general observation that the reward pathway is being altered under F1 would be a big picture idea to take onto the next step.
Lita: This might turn out to be one of those really specific responses that you only see in flies because it’s an adaptation they have to a very specific environmental stress. That being said, fruit flies have very conserved molecular mechanisms to all different orders of animals including humans… lots of things function in a very similar way. I’m hoping that somebody might continue this work and really nail down the exact mechanism of imprinting because even if it’s not the same locus that’s imprinted my guess is that if it’s possible to happen to flies, I would not be surprised if mechanistically the same thing could happen in a mammalian system. My vote would be to continue it in flies, really understand [the mechanism] in detail, and then move along to mice and other higher order systems. When we can collaborate in that way and work across model systems, we make the biggest advances.
Faith: Do you think the Neuropeptide-F (NPF) locus, and its mammalian counterpart Neuropeptide-Y (NPY), provides a rational, druggable target in diseases like addiction?
Lita: I’m going to give that a hard no. NPF/NPY governs the overall reward state of the brain. I think it’s a little too high level. I think changing overall levels in an animals’ brain might be altering some very serious cognitive functions, mood changes, the desire to eat, the desire to have social interaction, so I would be very, very cautious changing [it] directly. That’s not to say that there are not downstream effects that could be more narrowly targeted. I think that that’s something people are looking at, trying to understand the downstream circuits.
Balint: We did a pilot drug study against NPF/NPY. We have inhibitors to them and they induced ovarian apoptosis [programmed cell death]. That’s obviously a systemic drug, you’re ingesting it and it’s going everywhere, maybe if it were more localized it would have different results, but at least our foray into the drug field showed there are many other effects of altering [it].
Faith: What fields of research are you both pursuing in your post-doctoral positions? Do you have any plans of continuing this work in the future?Balint: Currently I’m in Dr. Shelley Berger’s lab pursuing work in ant epigenetics [study of heritable changes in gene expression]– so figuring out what is the epigenetic organization that dictates what caste [social order] an ant is in, giving self identify in an ant system. We have one model where an ant is able to transition from a worker to a queen and which is accompanied by activation of her ovary and a quadrupling of her lifespan. [We want to] see what changes in chromatin are occurring to give [rise to] upregulation of these protective genes. Finally, [my last project] is to figure out if we can model tumor progression in the ant. There are some human anecdotal studies… that suggest that social order modulates tumor progression, so if you’re in a more crowded region, disease state accelerates. We’re going to try to induce a tumor model in the ant and then overcrowd them or isolate them and see how tumorigenesis [tumor formation] is affected. There are some potential epigenetic changes that are transgenerational way down the line. When these workers become the queen, if it’s an aged worker, she’s not as good at transitioning to becoming a new queen, and her offspring are far fewer. There could very well be some changes in said offspring but we first need to understand six steps back before it gets there. So if one day I ever get a lab, that is on the docket to study.
Lita: Balint and I have gone to different labs at the same institute. We’re actually at the Epigenetics Institute at UPENN. We’re at least staying in the periphery of this field. I was lucky enough to get a position in a lab that is letting me charge straight into the small RNA world. I’m in the Bonasio Lab and my focus is on understanding different types of small RNAs and maybe their binding partners. I’m hoping to tie that into some behavioral, memory assays farther down the line, but right now it’s just strict RNA biochemistry and I’m loving it.
Lita and Balint graduated this past spring from the Molecular and Cellular Biology (MCB) Program in the Geisel School of Medicine at Dartmouth College. They earned their doctorates in the laboratory of Dr. Giovanni Bosco. Lita and Balint are both pursuing post-doctorate positions at the Penn Epigenetics Institute, in the laboratories of Dr. Roberto Bonasio and Dr. Shelley Berger respectively.
Visit Lita’s and Balint’s websites and follow them on Twitter @JBozler and @BalintZKacsoh to learn more about their exciting research and follow their post-doctoral journeys.