Faculty Bibliography

Precise control of neurite guidance during development is essential to ensure proper formation of neuronal networks and correct function of the central nervous system (CNS). How neuronal projections find their targets to generate appropriate synapses is not entirely understood. Although transcription factors are key molecules during neurogenesis, we do not know their entire function during the formation of networks in the CNS. Here, we used the Drosophila melanogaster optic lobe as a model for understanding neurite guidance during development. We assessed the function of Sox102F/SoxD, the unique Drosophila orthologue of the vertebrate SoxD family of transcription factors. SoxD is expressed in immature and mature neurons in the larval and adult lobula plate ganglia (one of the optic lobe neuropils), but is absent from glial cells, neural stem cells and progenitors of the lobula plate. SoxD RNAi knockdown in all neurons results in a reduction of the lobula plate neuropil, without affecting neuronal fate. This morphological defect is associated with an impaired optomotor response of adult flies. Moreover, knocking down SoxD only in T4/T5 neuronal types, which control motion vision, affects proper neurite guidance into the medulla and lobula. Our findings suggest that SoxD regulates neurite guidance, without affecting neuronal fate.

Quiescent stem cells in adult tissues can be activated for homeostasis or repair. Neural stem cells (NSCs) in Drosophila are reactivated from quiescence in response to nutrition by the insulin signaling pathway. It is widely accepted that quiescent stem cells are arrested in G₀ In this study, however, we demonstrate that quiescent NSCs (qNSCs) are arrested in either G₂ or G₀ G₂-G₀ heterogeneity directs NSC behavior: G₂ qNSCs reactivate before G₀ qNSCs. In addition, we show that the evolutionarily conserved pseudokinase Tribbles (Trbl) induces G₂ NSCs to enter quiescence by promoting degradation of Cdc25^String and that it subsequently maintains quiescence by inhibiting Akt activation. Insulin signaling overrides repression of Akt and silences trbl transcription, allowing NSCs to exit quiescence. Our results have implications for identifying and manipulating quiescent stem cells for regenerative purposes.

Successful neurogenesis requires adequate proliferation of neural stem cells (NSCs) and their progeny, followed by neuronal differentiation, maturation and survival. NSCs inhabit a complex cellular microenvironment, the niche, which influences their behaviour. To ensure sustained neurogenesis, niche cells must respond to extrinsic, environmental changes whilst fulfilling the intrinsic requirements of the neurogenic program and adapting their roles accordingly. However, very little is known about how different niche cells adjust their properties to such inputs. Here, we show that nutritional and NSC-derived signals induce the remodelling of Drosophila cortex glia, adapting this glial niche to the evolving needs of NSCs. First, nutrition-induced activation of PI3K/Akt drives the cortex glia to expand their membrane processes. Second, when NSCs emerge from quiescence to resume proliferation, they signal to glia to promote membrane remodelling and the formation of a bespoke structure around each NSC lineage. The remodelled glial niche is essential for newborn neuron survival.

Thousands of long noncoding RNAs (lncRNAs) have been identified in eukaryotic genomes, many of which are expressed in spatially and temporally restricted patterns. Nonetheless, the roles of the majority of these transcripts are still unknown. One of the mechanisms by which lncRNAs function is through the modulation of chromatin states. To assess the functions of lncRNAs, we developed RNA-DamID, a novel approach that detects lncRNA-genome interactions in a cell-type-specific manner in vivo with high sensitivity and accuracy. Identifying the cell-type-specific genome occupancy of lncRNAs is vital to understanding their mechanisms of action in development and disease. We used RNA-DamID to investigate targeting of the lncRNAs in the Drosophila dosage-compensation complex (DCC) and show that initial targeting is cell-type specific.

A key question in developmental biology is how cellular differentiation is controlled during development. While transitions between trithorax-group (TrxG) and polycomb-group (PcG) chromatin states are vital for the differentiation of ES cells to multipotent stem cells, little is known regarding the role of chromatin states during development of the brain. Here we show that large-scale chromatin remodelling occurs during Drosophila neural development. We demonstrate that the majority of genes activated during neuronal differentiation are silent in neural stem cells (NSCs) and occupy black chromatin and a TrxG-repressive state. In neurons, almost all key NSC genes are switched off via HP1-mediated repression. PcG-mediated repression does not play a significant role in regulating these genes, but instead regulates lineage-specific transcription factors that control spatial and temporal patterning in the brain. Combined, our data suggest that forms of chromatin other than canonical PcG/TrxG transitions take over key roles during neural development.

Neural stem cells (NSCs) are multipotent, self-renewing progenitors that generate progeny that differentiate into neurons and glia. NSCs in the adult mammalian brain are generally quiescent. Environmental stimuli such as learning or exercise can activate quiescent NSCs, inducing them to proliferate and produce new neurons and glia. How are these behaviours coordinated? The neurovasculature, the circulatory system of the brain, is a key component of the NSC microenvironment, or 'niche'. Instructive signals from the neurovasculature direct NSC quiescence, proliferation, self-renewal and differentiation. During ageing, a breakdown in the niche accompanies NSC dysfunction and cognitive decline. There is much interest in reversing these changes and enhancing NSC activity by targeting the neurovasculature therapeutically. Here we discuss principles of neurovasculature-NSC crosstalk, and the implications for the design of NSC-based therapies. We also consider the emerging contributions to this field of the model organism Drosophila melanogaster.

The 40,000 neurons of the medulla, the largest visual processing center of the Drosophila brain, derive from a sheet of neuroepithelial cells. During larval development, a wave of differentiation sweeps across the neuroepithelium, converting neuroepithelial cells into neuroblasts that sequentially express transcription factors specifying different neuronal cell fates. The switch from neuroepithelial cells to neuroblasts is controlled by a complex gene regulatory network and is marked by the expression of the proneural gene l'sc. We discovered that microRNA miR-7 is expressed at the transition between neuroepithelial cells and neuroblasts. We showed that miR-7 promotes neuroepithelial cell-to-neuroblast transition by targeting downstream Notch effectors to limit Notch signaling. miR-7 acts as a buffer to ensure that a precise and stereotypical pattern of transition is maintained, even under conditions of environmental stress, echoing the role that miR-7 plays in the eye imaginal disc. This common mechanism reflects the importance of robust visual system development.

This protocol is an extension to: Nat. Protoc. 2, 1467-1478 (2007); doi:10.1038/nprot.2007.148; published online 7 June 2007The ability to profile transcription and chromatin binding in a cell-type-specific manner is a powerful aid to understanding cell-fate specification and cellular function in multicellular organisms. We recently developed targeted DamID (TaDa) to enable genome-wide, cell-type-specific profiling of DNA- and chromatin-binding proteins in vivo without cell isolation. As a protocol extension, this article describes substantial modifications to an existing protocol, and it offers additional applications. TaDa builds upon DamID, a technique for detecting genome-wide DNA-binding profiles of proteins, by coupling it with the GAL4 system in Drosophila to enable both temporal and spatial resolution. TaDa ensures that Dam-fusion proteins are expressed at very low levels, thus avoiding toxicity and potential artifacts from overexpression. The modifications to the core DamID technique presented here also increase the speed of sample processing and throughput, and adapt the method to next-generation sequencing technology. TaDa is robust, reproducible and highly sensitive. Compared with other methods for cell-type-specific profiling, the technique requires no cell-sorting, cross-linking or antisera, and binding profiles can be generated from as few as 10,000 total induced cells. By profiling the genome-wide binding of RNA polymerase II (Pol II), TaDa can also identify transcribed genes in a cell-type-specific manner. Here we describe a detailed protocol for carrying out TaDa experiments and preparing the material for next-generation sequencing. Although we developed TaDa in Drosophila, it should be easily adapted to other organisms with an inducible expression system. Once transgenic animals are obtained, the entire experimental procedure-from collecting tissue samples to generating sequencing libraries-can be accomplished within 5 d.

High-throughput screens allow us to understand how transcription factors trigger developmental processes, including cell specification. A major challenge is identification of their binding sites because feedback loops and homeostatic interactions may mask the direct impact of those factors in transcriptome analyses. Moreover, this approach dissects the downstream signaling cascades and facilitates identification of conserved transcriptional programs. Here we show the results and the validation of a DNA adenine methyltransferase identification (DamID) genome-wide screen that identifies the direct targets of Glide/Gcm, a potent transcription factor that controls glia, hemocyte, and tendon cell differentiation in Drosophila. The screen identifies many genes that had not been previously associated with Glide/Gcm and highlights three major signaling pathways interacting with Glide/Gcm: Notch, Hedgehog, and JAK/STAT, which all involve feedback loops. Furthermore, the screen identifies effector molecules that are necessary for cell-cell interactions during late developmental processes and/or in ontogeny. Typically, immunoglobulin (Ig) domain-containing proteins control cell adhesion and axonal navigation. This shows that early and transiently expressed fate determinants not only control other transcription factors that, in turn, implement a specific developmental program but also directly affect late developmental events and cell function. Finally, while the mammalian genome contains two orthologous Gcm genes, their function has been demonstrated in vertebrate-specific tissues, placenta, and parathyroid glands, begging questions on the evolutionary conservation of the Gcm cascade in higher organisms. Here we provide the first evidence for the conservation of Gcm direct targets in humans. In sum, this work uncovers novel aspects of cell specification and sets the basis for further understanding of the role of conserved Gcm gene regulatory cascades.

Since its introduction in 1993, the GAL4 system has become an essential part of the Drosophila geneticist's toolkit. Widely used to drive gene expression in a multitude of cell- and tissue-specific patterns, the system has been adapted and extended to form the basis of many modern tools for the manipulation of gene expression in Drosophila and other model organisms.