Developmental reprogramming following prenatal acoustic signals

In both humans and birds, parents may pass along information to help their offspring adapt to conditions in the world; some signals are transmitted even before birth, causing changes in development, physiology and behaviour that can last the entire lifespan. Recently, project partners Mariette & Buchanan (Science, 2016) discovered a highly novel example of such a process in the zebra finch (a songbird), a well-established experimental model organism for neuroscience, behaviour and genomics. Working with wild-derived birds in Australia, they found that parents pass along "weather reports"- signals that alter the developmental trajectory of their offspring so they will respond adaptively to temperature conditions upon hatching. Amazingly, these reports are delivered via vocal signals transmitted while the offspring are still in the egg. Altered developmental programming can be detected within a day after hatching as a change in the temperature dependence of growth rate and begging behaviour. Mariette & Buchanan showed that exposure to these parental signals in ovo has lifelong consequences on adaptive fitness and behaviour, leading to enhanced reproductive success of the offspring after they mature when conditions are hot.

How can relatively brief exposures to an acoustic signal lead to such profound and lasting changes in development, physiology and fitness? This very basic question has broad implications, ranging from how environmental information gets transferred across generations, to how developmental pathways may be shaped and reprogrammed, and how organisms might adapt to climate change. To answer this, we will apply the extensive expertise in zebra finch genomics and neuroscience developed by the PI and his colleagues over the last three decades.

First, we will determine where and how the embryo initially "hears" the incubation call. We will use techniques well established in our prior work for measuring and localizing dynamic gene activity in the zebra finch brain, to map sites in the embryo that first respond to the sound of incubation calls. Accomplishing this will establish the mechanism of primary reception for incubation call signals, and will provide a fresh look at the earliest stages of auditory development in this important model for auditory communication and learning.

Second, we will test the hypothesis that incubation call exposure specifically leads to altered physiological responses to high temperatures in nestlings. This hypothesis defines a physiological output and provides a functional explanation for the evolution and maintenance of parental call-dependent reprogramming. Working with project partners in Australia (who are providing their resources, expertise and training at no cost to this BBSRC project), we will collect physiological indicators of heat tolerance (body temperature, metabolic rate, endocrine measures) in nestlings that had been exposed to incubation calls before hatching followed by chronic or acute temperature elevations in the nest. This aim also links to emerging evidence that developmental reprogramming of heat tolerance may occur in poultry.

Third, to identify the regulatory mechanisms (and specific genes) that underlie the persistence of reprogramming, we will test for sustained epigenetic responses (changes in gene expression and DNA methylation) following embryonic incubation call exposure. Using a combination of high-throughput screening and targeted gene-specific methods, we will look for effects both in the whole brain and specifically in the hypothalamus, and in both embryos and older animals.

Together these data will address for the first time the mechanisms underlying prenatal communication and their fundamental importance for developmental trajectories and individual fitness. In doing so our project will address the mechanisms at the heart of genotype x environment interactions, so prevalent throughout biology.

Zebra finches in Australia emit "incubation calls" to their eggs that reprogram how the embryos develop and behave after hatching (Mariette & Buchanan, Science 2016). How can brief exposures in the egg to a particular sound lead to a lasting adaptive change in development and behaviour? To answer this, we will pursue three aims.

Aim 1 will test the hypothesis that incubation calls elicit an acute neural response in the embryo, and localize this response and determine its stimulus specificity. For this we will use in situ hybridization (ISH) and quantitative PCR (qPCR) for immediate early genes (IEGs), and complement this with an unbiased genome-wide analysis of RNA (RNAseq) to allow for the possibility of novel response patterns in this developmental context.

Aim 2 will test the hypothesis that incubation call exposure leads to improved resistance to high temperatures, and probe the underlying neural mechanism. We will experimentally manipulate exposures to incubation calls in eggs, before they hatch, then place the hatchlings in nests at different temperatures, measuring physiological responses including body temperature, metabolism, corticosterone levels and (in Aim 3) BDNF expression in the hypothalamus.

Aim 3 will probe the mechanism for the lifelong persistence of reprogramming effects; we focus on the hypothesis that the reprogrammed state is associated with stable, epigenetic changes in gene expression and DNA methylation in the brain. We will test by performing genome-wide analyses of both gene expression (RNAseq) and DNA methylation (RRBS) at onset of reprogramming in the embryo brain, and again during nestling development. Our approach will include both whole-brain assays and focal studies of the hypothalamus, following a precedent in poultry for epigenetic effects on the BDNF gene in response to perinatal heat exposure. Any treatment-dependent effects will be followed into adults to determine whether such effects persist across the lifespan

This project is an example of "frontier bioscience," which is a major BBSRC focus (http://www.bbsrc.ac.uk/research/frontier-bioscience/). Using sophisticated tools in genomics and physiology, we are asking fundamental questions about how information from the external environment, channeled through parental signals, can lead to a lasting adaptive change in growth and behaviour. The research is relevant to the BBSRC Research Priority "Healthy ageing across the lifecourse" and especially to the stated interest on "The role of epigenetic effects in development and ageing across the lifecourse." Within this priority, it also links to "development of appropriate model organisms and systems"; to "the identification of 'critical periods' during the lifespan which may be particularly susceptible to biological influences/exposures"; and to "the development and validation of appropriate outcome measures, such as biomarkers" -as we are developing a novel animal model of prenatal sensitivity to environmental signals, and will identify molecular/epigenetic indicators of the responses to those signals which might have future applications in monitoring healthy adaptations in other contexts and organisms.

The work resonates with the "Systems approaches to the biosciences" priority, as we are working to integrate activities across the nervous system, across the genome and across development, to understand functional transitions in developmental trajectories.

The work also addresses the "International Partnerships" priority as the work will constitute UK participation in joint research activity with partners outside the UK. It also potentially contributes to strategies to realise Millennium Development Goals, specifically in the area of climate change, by identifying and dissecting a novel biological mechanism that supports rapid adaptation to environmental warming.

Finally, the work has potential for economic impact in the "Agriculture and Food Security" strategic priority, in the context of commercial poultry production. Thermal acclimation is a significant concern for animal producers, especially with the economic incentive and pressure from the public to limit animal loss during heat waves and air conditioning infrastructure failure (not to mention concerns about long-term climate change). In the USA, heat stress has been estimated to result in an annual economic loss to poultry production equivalent to more than £100 million (Lara & Rostagno, 2013). Efforts have been underway for many years to induce heat resistance in chicken by acclimating them to heat during embryogenesis - however, low hatching success and low body mass are also well known consequences of excessive heat exposure during embryogenesis (Loyau et al., 2015). Anne Collin and colleagues have shown shifts in gene expression in liver and muscle in chickens that have been acclimated to heat, suggesting a role for epigenetic processes (Loyau et al., 2016). The zebra finch precedent raises the provocative possibly that specialized acoustic signals might be used to shape developmental trajectories in other birds (current poultry practices do not involve any control over acoustic environment). But even if acoustic signals prove irrelevant to poultry development, the work here could benefit efforts to improve poultry by defining central epigenetic pathways for adaptation to thermal stress.

Lara, L., & Rostagno, M., 2013. Impact of heat stress on poultrypProduction. Animals 3, 356-369.
Loyau, T., et al., 2015. Cyclic variations in incubation conditions induce adaptive responses to later heat exposure in chickens: a review. Animal 9, 76-85.
Loyau, T., et al., 2016. Thermal manipulation of the chicken embryo triggers differential gene expression in response to a later heat challenge. BMC Genomics 17, 329.