Faculty Bibliography

Homeostasis of the nervous system is maintained by a population of resident neural stem cells (NSCs) retained in a state of reversible cell-cycle arrest called quiescence. Quiescent NSCs can resume proliferation in response to different physiological stimuli. Reactivation requires changes in gene expression, much of which is regulated at the epigenomic level. We mapped epigenomic changes in NSC chromatin during stem cell quiescence and reactivation in Drosophila in vivo. Contrary to expectations, chromatin accessibility is increased in quiescent NSCs. Surprisingly, genes crucial for cell-cycle progression are repressed while remaining within permissive H3K36me3-bound euchromatin. At the same time, genes necessary for cell-cell communication are derepressed by eviction of histone H1 and transition to an SWI/SNF-enriched active state. Our results reveal global expansion of accessible chromatin in quiescent NSCs without concomitant transcriptional activation. Strikingly, this process reverses upon reactivation, indicating that opening of chromatin is a quiescence-specific event.

Understanding the molecular mechanisms driving lineage decisions and differentiation during development is challenging in complex systems with a diverse progenitor pool, such as the mammalian cerebellum. Importantly, how different transcription factors cooperate to generate neural diversity and the gene regulatory mechanisms that drive neuron production, especially during the late stages of cerebellum development, are poorly understood. We used single cell RNA-sequencing (scRNA-seq) to investigate the developmental trajectories of Nestin-expressing progenitors (NEPs) in the neonatal mouse cerebellum. We identified FOXO1 as a key regulator of NEP-to-inhibitory neuron differentiation, acting directly downstream of ASCL1. Genome occupancy and functional experiments using primary NEP cultures showed that both ASCL1 and FOXO1 regulate neurogenesis genes during differentiation while independently regulating proliferation and survival, respectively. Furthermore, we demonstrated that WNT signalling promotes the transition from an ASCL1+ to a FOXO1+ cellular state. Finally, the role of WNT signalling in promoting neuron production via FOXO1 is conserved in primary human NEP cultures. By resolving how cerebellar inhibitory neurons differentiate, our findings could have implications for cerebellar disorders such as spinocerebellar ataxia, where these cells are overproduced.

Histone modifications play a key role in regulating gene expression and cell fate during development and disease. Current methods for cell-type-specific genome-wide profiling of histone modifications require dissociation and isolation of cells and are not compatible with all tissue types. Here we adapt Targeted DamID (TaDa) to recognize specific histone marks, by fusing chromatin-binding proteins or single-chain antibodies to Dam, an Escherichia coli DNA adenine methylase. When combined with TaDa, this enables cell-type-specific chromatin profiling in intact tissues or organisms. We first profiled H3K4me3, H3K9ac, H3K27me3 and H4K20me1 in vivo in neural stem cells of the developing Drosophila brain. Next, we mapped cell-type-specific H3K4me3, H3K9ac and H4K20me1 distributions in the developing mouse brain. Finally, we injected RNA encoding DamID constructs into 1-cell stage Xenopus embryos to profile H3K4me3 distribution during gastrulation and neurulation. These results illustrate the versatility of TaDa to profile cell-type-specific histone marks throughout the genome in diverse model systems.

The Notch signalling pathway is a master regulator of cell fate transitions in development and disease. In the brain, Notch promotes neural stem cell (NSC) proliferation, regulates neuronal migration and maturation and can act as an oncogene or tumour suppressor. How NOTCH and its transcription factor RBPJ activate distinct gene regulatory networks in closely related cell types in vivo remains to be determined. Here we use Targeted DamID (TaDa), requiring only thousands of cells, to identify NOTCH and RBPJ binding in NSCs and their progeny in the mouse embryonic cerebral cortex in vivo. We find that NOTCH and RBPJ associate with a broad network of NSC genes. Repression of NSC-specific Notch target genes in intermediate progenitors and neurons correlates with decreased chromatin accessibility, suggesting that chromatin compaction may contribute to restricting NOTCH-mediated transactivation.

Adult stem cells coordinate intrinsic and extrinsic, local and systemic, cues to maintain the proper balance between self-renewal and differentiation. However, the precise mechanisms stem cells use to integrate these signals remain elusive. Here, we show that Escargot (Esg), a member of the Snail family of transcription factors, regulates the maintenance of somatic cyst stem cells (CySCs) in the Drosophila testis by attenuating the activity of the pro-differentiation insulin receptor (InR) pathway. Esg positively regulates the expression of an antagonist of insulin signaling, ImpL2, while also attenuating the expression of InR. Furthermore, Esg-mediated repression of the InR pathway is required to suppress CySC loss in response to starvation. Given the conservation of Snail-family transcription factors, characterizing the mechanisms by which Esg regulates cell-fate decisions during homeostasis and a decline in nutrient availability is likely to provide insight into the metabolic regulation of stem cell behavior in other tissues and organisms.

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.

The discovery of human obesity-associated genes can reveal new mechanisms to target for weight loss therapy. Genetic studies of obese individuals and the analysis of rare genetic variants can identify novel obesity-associated genes. However, establishing a functional relationship between these candidate genes and adiposity remains a significant challenge. We uncovered a large number of rare homozygous gene variants by exome sequencing of severely obese children, including those from consanguineous families. By assessing the function of these genes in vivo in Drosophila, we identified 4 genes, not previously linked to human obesity, that regulate adiposity (itpr, dachsous, calpA, and sdk). Dachsous is a transmembrane protein upstream of the Hippo signalling pathway. We found that 3 further members of the Hippo pathway, fat, four-jointed, and hippo, also regulate adiposity and that they act in neurons, rather than in adipose tissue (fat body). Screening Hippo pathway genes in larger human cohorts revealed rare variants in TAOK2 associated with human obesity. Knockdown of Drosophila tao increased adiposity in vivo demonstrating the strength of our approach in predicting novel human obesity genes and signalling pathways and their site of action.

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.

Stem cells reside in specialized microenvironments or niches that balance stem cell proliferation and differentiation.¹

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.