Faculty Bibliography

The adaptable transcriptional response to changes in food availability not only ensures animal survival but also lets embryonic development progress. Interestingly, the CNS is preferentially protected from periods of malnutrition, a phenomenon known as "brain sparing." However, the mechanisms that mediate this response remain poorly understood. To get a better understanding of this, we used Drosophila melanogaster as a model, analyzing the transcriptional response of neural stem cells (neuroblasts) and glia of the blood-brain barrier (BBB) from larvae of both sexes during nutrient restriction using targeted DamID. We found differentially expressed genes in both neuroblasts and glia of the BBB, although the effect of nutrient deficiency was primarily observed in the BBB. We characterized the function of a nutritional sensitive gene expressed in the BBB, the serine protease homolog, scarface (scaf). Scaf is expressed in subperineurial glia in the BBB in response to nutrition. Tissue-specific knockdown of scaf increases subperineurial glia endoreplication and proliferation of perineurial glia in the blood-brain barrier. Furthermore, neuroblast proliferation is diminished on scaf knockdown in subperineurial glia. Interestingly, reexpression of Scaf in subperineurial glia is able to enhance neuroblast proliferation and brain growth of animals in starvation. Finally, we show that loss of scaf in the blood-brain barrier increases sensitivity to drugs in adulthood, suggesting a physiological impairment. We propose that Scaf integrates the nutrient status to modulate the balance between neurogenesis and growth of the BBB, preserving the proper equilibrium between the size of the barrier and the brain.SIGNIFICANCE STATEMENT The Drosophila BBB separates the CNS from the open circulatory system. The BBB glia are not only acting as a physical segregation of tissues but participate in the regulation of the metabolism and neurogenesis during development. Here we analyze the transcriptional response of the BBB glia to nutrient deprivation during larval development, a condition in which protective mechanisms are switched on in the brain. Our findings show that the gene scarface reduces growth in the BBB while promoting the proliferation of neural stem, assuring the balanced growth of the larval brain. Thus, Scarface would link animal nutrition with brain development, coordinating neurogenesis with the growth of the BBB.

Genetic studies of autism have revealed causal roles for chromatin remodeling gene mutations. Chromodomain helicase DNA binding protein 8 (CHD8) encodes a chromatin remodeler with significant de novo mutation rates in sporadic autism. However, relationships between CHD8 genomic function and autism-relevant biology remain poorly elucidated. Published studies utilizing ChIP-seq to map CHD8 protein-DNA interactions have high variability, consistent with technical challenges and limitations associated with this method. Thus, complementary approaches are needed to establish CHD8 genomic targets and regulatory functions in developing brain. We used in utero CHD8 Targeted DamID followed by sequencing (TaDa-seq) to characterize CHD8 binding in embryonic mouse cortex. CHD8 TaDa-seq reproduced interaction patterns observed from ChIP-seq and further highlighted CHD8 distal interactions associated with neuronal loci. This study establishes TaDa-seq as a useful alternative for mapping protein-DNA interactions in vivo and provides insights into the regulatory targets of CHD8 and autism-relevant pathophysiology associated with CHD8 mutations.

Neural stem cells (NSCs) are multipotent progenitors that are responsible for producing all of the neurons and macroglia in the nervous system. In adult mammals, NSCs reside predominantly in a mitotically dormant, quiescent state, but they can proliferate in response to environmental inputs such as feeding or exercise. It is hoped that quiescent NSCs could be activated therapeutically to contribute towards repair in humans. This will require an understanding of quiescent NSC heterogeneities and regulation during normal physiology and following brain injury. Non-mammalian vertebrates (zebrafish and salamanders) and invertebrates (Drosophila) offer insights into brain repair and quiescence regulation that are difficult to obtain using rodent models alone. We review conceptual progress from these various models, a first step towards harnessing quiescent NSCs for therapeutic purposes.

Long-noncoding RNAs (lncRNAs) are emerging as regulators of development and disease. lncRNAs are expressed in exquisitely precise expression patterns in vivo and many interact with chromatin to regulate gene expression. However, the limited sensitivity of RNA-purification techniques has precluded the identification of genomic targets of cell-type specific lncRNAs. RNA-DamID is a powerful new approach to understand the mechanisms by which lncRNAs act in vivo. RNA-DamID is highly sensitive and accurate, and can resolve cell-type-specific chromatin binding patterns without cell isolation. The determinants of RNA-chromatin interactions can be identified with RNA-DamID by analyzing RNA and protein cofactor mutants. Here we describe how to implement RNA-DamID and the design considerations to take into account to accurately identify lncRNA-chromatin interactions in vivo.

Understanding the sequence of events leading to cancer relies in large part upon identifying the tumour cell of origin. Glioblastoma is the most malignant brain cancer but the early stages of disease progression remain elusive. Neural lineages have been implicated as cells of origin, as have glia. Interestingly, high levels of the neural stem cell regulator TLX correlate with poor patient prognosis. Here we show that high levels of the Drosophila TLX homologue, Tailless, initiate tumourigenesis by reverting intermediate neural progenitors to a stem cell state. Strikingly, we could block tumour formation completely by re-expressing Asense (homologue of human ASCL1), which we show is a direct target of Tailless. Our results predict that expression of TLX and ASCL1 should be mutually exclusive in glioblastoma, which was verified in single-cell RNA-seq of human glioblastoma samples. Counteracting high TLX is a potential therapeutic strategy for suppressing tumours originating from intermediate progenitor cells.

Translating advances in cancer research to clinical applications requires better insight into the metabolism of normal cells and tumour cells in vivo. Much effort has focused on understanding how glycolysis and oxidative phosphorylation (OxPhos) support proliferation, while their impact on other aspects of development and tumourigenesis remain largely unexplored. We found that inhibition of OxPhos in neural stem cells (NSCs) or tumours in the Drosophila brain not only decreases proliferation, but also affects many different aspects of stem cell behaviour. In NSCs, OxPhos dysfunction leads to a protracted G₁/S-phase and results in delayed temporal patterning and reduced neuronal diversity. As a consequence, NSCs fail to undergo terminal differentiation, leading to prolonged neurogenesis into adulthood. Similarly, in brain tumours inhibition of OxPhos slows proliferation and prevents differentiation, resulting in reduced tumour heterogeneity. Thus, in vivo, highly proliferative stem cells and tumour cells require OxPhos for efficient growth and generation of diversity.

In living organisms, self-organised waves of signalling activity propagate spatiotemporal information within tissues. During the development of the largest component of the visual processing centre of the Drosophila brain, a travelling wave of proneural gene expression initiates neurogenesis in the larval optic lobe primordium and drives the sequential transition of neuroepithelial cells into neuroblasts. Here, we propose that this 'proneural wave' is driven by an excitable reaction-diffusion system involving epidermal growth factor receptor (EGFR) signalling interacting with the proneural gene l'sc. Within this framework, a propagating transition zone emerges from molecular feedback and diffusion. Ectopic activation of EGFR signalling in clones within the neuroepithelium demonstrates that a transition wave can be excited anywhere in the tissue by inducing signalling activity, consistent with a key prediction of the model. Our model illuminates the physical and molecular underpinnings of proneural wave progression and suggests a generic mechanism for regulating the sequential differentiation of tissues.

Determining the premalignant lesions that develop into malignant tumours remains a daunting task. Brain tumours are frequently characterised by a block in differentiation, implying that normal developmental pathways become hijacked during tumourigenesis. However, the heterogeneity of stem cells and their progenitors in the brain suggests there are many potential routes to tumour initiation. Studies in Drosophila melanogaster have enhanced our understanding of the tumourigenic potential of distinct cell types in the brain. Here we review recent studies that have improved our knowledge of neural stem cell behaviour during development and in brain tumour models.

Following severe or chronic liver injury, adult ductal cells (cholangiocytes) contribute to regeneration by restoring both hepatocytes and cholangiocytes. We recently showed that ductal cells clonally expand as self-renewing liver organoids that retain their differentiation capacity into both hepatocytes and ductal cells. However, the molecular mechanisms by which adult ductal-committed cells acquire cellular plasticity, initiate organoids and regenerate the damaged tissue remain largely unknown. Here, we describe that ductal cells undergo a transient, genome-wide, remodelling of their transcriptome and epigenome during organoid initiation and in vivo following tissue damage. TET1-mediated hydroxymethylation licences differentiated ductal cells to initiate organoids and activate the regenerative programme through the transcriptional regulation of stem-cell genes and regenerative pathways including the YAP-Hippo signalling. Our results argue in favour of the remodelling of genomic methylome/hydroxymethylome landscapes as a general mechanism by which differentiated cells exit a committed state in response to tissue damage.

Chromatin remodeling accompanies differentiation, however, its role in self-renewal is less well understood. We report that in Drosophila, the chromatin remodeler Kismet/CHD7/CHD8 limits intestinal stem cell (ISC) number and proliferation without affecting differentiation. Stem-cell-specific whole-genome profiling of Kismet revealed its enrichment at transcriptionally active regions bound by RNA polymerase II and Brahma, its recruitment to the transcription start site of activated genes and developmental enhancers and its depletion from regions bound by Polycomb, Histone H1, and heterochromatin Protein 1. We demonstrate that the Trithorax-related/MLL3/4 chromatin modifier regulates ISC proliferation, colocalizes extensively with Kismet throughout the ISC genome, and co-regulates genes in ISCs, including Cbl, a negative regulator of Epidermal Growth Factor Receptor (EGFR). Loss of kismet or trr leads to elevated levels of EGFR protein and signaling, thereby promoting ISC self-renewal. We propose that Kismet with Trr establishes a chromatin state that limits EGFR proliferative signaling, preventing tumor-like stem cell overgrowths.