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Elucidating novel post-transcriptional regulatory mechanisms in neural development

Periodic Reporting for period 1 - Brain_stability (Elucidating novel post-transcriptional regulatory mechanisms in neural development)

Reporting period: 2017-07-01 to 2019-06-30

The development of the brain is one of the most complex examples of tissue building in the animal world. During brain development, a small population of neural stem cells gives rise to a plethora of specialized cell types, each linked to its neighbours by an intricate web of connections. It is not understood how these cell types are produced at the appropriate time in development and how each progenitor cell chooses its fate at the precise place and time. Most studies on this topic have focused on identifying specific master regulators, known as transcription factors, that are required to specify particular cell lineages. However, in many cases it is not known what regulates the transcription factors themselves. How are they activated in the correct cell types, and how is their activity repressed in other cell types? We hypothesized that different levels of gene regulation, some of them occurring post-transcriptionally and outside the direct purview of transcription factors, could provide the more fine-grained control of development that is needed in this context.

The main objective of this project was to determine whether post-transcriptional regulation, at the level of RNA stability, contributes to developmental decisions during brain development. This objective was split into several sub-objectives. Firstly, we aimed to measure RNA stability during brain development using the fruit fly larval brain as a model. Secondly, we sought to identify potential regulators of RNA stability using this dataset. Finally, we aimed to discover the functional relevance of RNA stability regulation for brain development using the power of fly genetics and the sophisticated assays we have at our disposal, including live explant brain culture.

The brain is the most complicated human organ, but it is also the most quintessentially human. As a species, our drive to learn more about the brain stems from deep existential questions and not scientific fervour alone. The more we learn about the brain, the closer we are to understanding our inner thoughts and how we interact with each other. These findings have potential to influence every sphere of society, including education, law, and politics. Understanding the development, or initial wiring of the system, is the foundation of brain science and greatly complements the work of neuroscientists who focus on the functioning of the adult brain. Even apart from its value in the broader context of advancing neuroscience, studying brain development has key societal implications. The proliferation of neural cells is precisely regulated during brain development and misregulation of these molecular pathways can lead to childhood diseases in humans, such as brain tumours or developmental delay. We have identified and are characterizing molecular pathways that we believe to be important for limiting the proliferation potential of neural stem cells and other pathways that we believe to be important for specifying the birth of a particular cell type. In the future, the results of our studies may help pharmaceutical companies select drug targets for the treatment of these disorders.
We have discovered that RNA stability varies widely in the developing brain and likely acts a regulatory strategy to limit gene expression to specific cell lineages, allowing creation of a complex tissue. Intriguingly, mRNAs encoding master regulators called transcription factors are degraded more rapidly than the average mRNA. mRNAs encoding transcription factors that are important for specifying either stem cell fate or the early steps of differentiation are especially unstable, even when compared to other transcription factor mRNAs. We are completing functional studies to determine which of these mRNAs cause uncontrolled cell proliferation or inappropriate differentiation when their degradation is prevented by artificial means. These experiments mark a paradigm shift in developmental neurobiology, which is largely focused on defining DNA-based regulatory strategies, rather than RNA-driven ones.

We are preparing two publications to disseminate our results: one focused on technical improvements we made to existing RNA metabolic labelling and sequencing workflows and one focused on the decay of mRNAs encoding transcription factors and its role in cell fate decisions during brain development.
During this project, we pushed beyond previous technological limitations to produce a high quality dataset of gene expression kinetics in the brain. Extending beyond our own experiments, genome-wide measurements of RNA stability have largely been limited to cell culture systems rather than whole tissue and have never been applied to the brain. Hence, our dataset provides a rich resource for the community and will lead to new hypotheses about gene regulation in the brain.

This project has had a favourable economic impact on the host lab and the wider research community. Firstly, we pushed the technological limits of nascent RNA purification and developed optimizations that allow high quality nascent RNA sequencing data to be collected from small tissue samples. We have additionally developed a reagent to enable higher coverage sequencing from fly RNA samples that was not available commercially. These advances will allow our lab and others to feasibly perform experiments that were too difficult or expensive previously. We have presented these technological advances at conferences and will further disseminate them in our upcoming publications. We also used data from this project to successfully obtain funding for a spin-off project about RNA stability regulation in the context of learning and memory.

Finally, we believe that our research into fundamental mechanisms of brain development is of key societal importance, and we worked to share the importance of our work via outreach activities during the funding period. Basic research on the mechanisms of brain development will shed light on the causes of developmental brain disorders and paediatric brain tumours. We hope that our findings will ultimately provide inspiration for novel therapeutics to treat these disorders. To communicate the importance of our research to the public, I presented the project in two formats. During Oxford Science Festival (2018), I volunteered as a researcher in a month long ‘Ask a scientist’ interactive web chat with secondary students that culminated in a presentation at the Weston library ‘Living library’ event in which I showed interested members of the public a movie of live fruit fly brains growing under the microscope and explained my research interests and broader motivation. Later, I organized an exhibit at the Oxford Museum of Natural History in which several members of my lab and I presented videos and demonstrations related to brain development and RNA biology during Cells Day, a day for secondary students to listen to scientific talks and tour the museum.