Regulation of astroglial branch morphogenesis during visual circuit assembly in Drosophila

Within our brains, neurons generally exist in a close partnership with different glial subtypes. Astrocytes play a critical role in controlling neural circuit development, function and homeostasis. Although the number and size of astrocytes correlates with brain size and cognitive abilities, they adopt remarkably complex morphologies with veil-like processes that ensheath the cell bodies and axonal and dendritic arborizations of neurons in both vertebrate and invertebrate nervous systems. While all astrocytes have common core physiological properties, they constitute a heterogeneous population with distinct morphologies in different brain regions. Much progress has been made in uncovering the molecular mechanisms that control neuronal morphology and connectivity. However, the fundamental question how astrocytes acquire their distinct shapes has received little attention and only a limited number of determinants has so far been identified to play a role in this important biological process.
The fruit fly Drosophila melanogaster is a powerful model organism for determining the function of known or novel genes in cells and tissues of interest. Considering the high degree of evolutionary conservation of genetic functions, insights gained in Drosophila are highly relevant for studies in mammals, including humans. In this study, we use the visual system of Drosophila to uncover the genes required for correct branch morphogenesis of astrocyte-like glia. Previously, we have identified an astrocyte-like glial subtype with stereotypic fine columnar and layered processes that is well suited for functional genetic studies during development. We have found a novel transmembrane Leucine-rich repeat domain containing cell surface molecule, called Lapsyn, that is essential for glial branch extension into the synaptic neuropil. Outlined research in this proposal seeks to gain insights how Lapsyn functions molecularly to deepen our knowledge of glial branch morphogenesis. Based on our earlier genetic studies we hypothesize that Lapsyn controls branch extension of astrocyte-like glia in response to neuronal membrane-bound or secreted determinants. Preliminary data suggest that in astrocyte-like glia, Lapsyn likely does not function as a receptor but as a co-receptor or structural cell surface proteins which interact with cytoskeletal regulators and components in a complex via other cell surface molecules. We therefore aim to identify the upstream and downstream binding partners of Lapsyn in an unbiased manner and to characterize their function in detail. Furthermore, to enhance our understanding of the significance of correct branch extension of astrocyte-like glia for neuronal connectivity and function, we will examine the link between branch extension and synapse formation and assess their participation in circuit activity. At long term, these insights into normal astrocyte development will advance our understanding of astrocyte-specific genetic contributions to neurodevelopmental and neurodegenerative disorders in humans.

Neural circuits are defined by the connectivity of diverse neuron subtypes and their close association with glia. Astrocytes form a heterogeneous glial population with remarkably complex and distinct shapes. Despite the importance of astrocytes for brain development and function, the mechanisms controlling the formation of their branches during development remain poorly understood.
Using the Drosophila visual system as a genetic model, we have previously found that astrocyte-like medulla neuropil glia acquire stereotypic elongated morphologies with columnar and layered branching patterns in a stepwise fashion. Knockdown and loss-of-function analyses uncovered a novel role for the transmembrane Leucine-rich repeat protein Lapsyn in regulating branch morphogenesis and positioning of this astrocyte subtype. Using these findings as entry point, our proposal has the aim to answer the basic question how astrocytes acquire their distinct morphologies in interactions with neurons. To elucidate the molecular function of Lapsyn, we will first determine the differential requirement of protein domains to gain evidence for a role of this molecule as a receptor, co-receptor or structural cell surface molecule using genetic rescue approaches. Second, using mass spectrometry, we seek to identify candidates as potential in cis and in trans interacting molecules of Lapsyn in astrocytes and neurons, respectively. Third, using expression analyses, advanced genetic and biochemical approaches we will validate identified genes in detail. Utilizing binary expression systems, we will examine the impact of gene knockdown or loss in glia and neurons on glial branch morphogenesis. Finally, we will examine the role of correct astrocyte-like glia branch formation in regulating visual circuit connectivity and function, by examining links to synaptogenesis using electron microscopy techniques and responsiveness to neural activity using Calcium imaging.

The central aim of our outlined research project is to answer a fundamental open scientific question, that is how astrocytes acquire their distinct morphologies during development in interaction with neurons. As a genetic model organism, Drosophila has been instrumental for the discovery of novel genes and their function in vivo. The primary impact of this research project therefore is to advance knowledge relevant for the academic community in the areas of Neuroscience and Developmental Biology seeking to understand how the brain develops and functions. However, considering the significance of astrocytes for brain connectivity and physiology, their stunning abundance in the human brain, and their increasingly established involvement in many neurological diseases, new insights into the genetic control of basic astrocyte biology will ultimately be of wider benefit to human health and thus the general society. The development of therapeutic interventions, to halt the progression of neurodegeneration, for instance in Alzheimer's disease, represents one of the biggest challenges for neurobiological research, yet despite all efforts so far, success has been limited. A better understanding of the cellular and molecular mechanisms regulating astrocyte biology led by basic research therefore contributes to building the foundation for innovative avenues towards the development of effective therapies by clinicians at long term. Because the proposed project takes advantage of the fruit fly Drosophila as a model organism, our research will help to develop complementary strategies in line with the BBSRC's 3Rs remit to reduce the use of animals in research.
The proposed study will develop and utilize a variety of state-of-the art genetic, molecular, mass spectrometry, and light and electron microscopy imaging techniques. Therefore, this research will provide a platform for high-quality training of young researchers at different levels, involved in this project. These newly acquired technical and analytical skills will be applicable to many disciplines beyond Neuroscience and therefore strengthen the scientific endeavour in the UK and other countries, when these experienced researchers move to new positions following their stay in our laboratory. Because the team is strongly committed to outreach and public engagement activities, that convey the excitement of Neuroscience research in general, and the significance of glial biology in particular, the outlined project will also help to inspire young people to choose scientific research as their future career path.