In vivo, genome-wide profiling of endogenously tagged chromatin-binding proteins with spatial and temporal resolution using NanoDam in Drosophila.

NanoDam is a technique for genome-wide profiling of the binding targets of any endogenously tagged chromatin-binding protein in vivo, without the need for antibodies, crosslinking, or immunoprecipitation. Here, we explain the procedure for NanoDam experiments in Drosophila, starting from a genetic cross, to the generation of sequencing libraries and, finally, bioinformatic analysis. This protocol can be readily adapted for use in other model systems after simple modifications. For complete details on the use and execution of this protocol, please refer to Tang et al. (2022).

NanoDam identifies Homeobrain (ARX) and Scarecrow (NKX2.1) as conserved temporal factors in the Drosophila central brain and visual system.

Temporal patterning of neural progenitors is an evolutionarily conserved strategy for generating neuronal diversity. Type II neural stem cells in the Drosophila central brain produce transit-amplifying intermediate neural progenitors (INPs) that exhibit temporal patterning. However, the known temporal factors cannot account for the neuronal diversity in the adult brain. To search for missing factors, we developed NanoDam, which enables rapid genome-wide profiling of endogenously tagged proteins in vivo with a single genetic cross. Mapping the targets of known temporal transcription factors with NanoDam revealed that Homeobrain and Scarecrow (ARX and NKX2.1 orthologs) are also temporal factors. We show that Homeobrain and Scarecrow define middle-aged and late INP temporal windows and play a role in cellular longevity. Strikingly, Homeobrain and Scarecrow have conserved functions as temporal factors in the developing visual system. NanoDam enables rapid cell-type-specific genome-wide profiling with temporal resolution and is easily adapted for use in higher organisms.

Tailless/TLX reverts intermediate neural progenitors to stem cells driving tumourigenesis via repression of asense/ASCL1.

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.

Neural stem cell dynamics: the development of brain tumours.

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.

The proneural wave in the Drosophila optic lobe is driven by an excitable reaction-diffusion mechanism.

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.

A newly discovered neural stem cell population is generated by the optic lobe neuroepithelium during embryogenesis in Drosophila melanogaster.

Neural stem cells must balance symmetric and asymmetric cell divisions to generate a functioning brain of the correct size. In both the developing Drosophila visual system and mammalian cerebral cortex, symmetrically dividing neuroepithelial cells transform gradually into asymmetrically dividing progenitors that generate neurons and glia. As a result, it has been widely accepted that stem cells in these tissues switch from a symmetric, expansive phase of cell divisions to a later neurogenic phase of cell divisions. In the Drosophila optic lobe, this switch is thought to occur during larval development. However, we have found that neuroepithelial cells start to produce neuroblasts during embryonic development, demonstrating a much earlier role for neuroblasts in the developing visual system. These neuroblasts undergo neurogenic divisions, enter quiescence and are retained post-embryonically, together with neuroepithelial cells. Later in development, neuroepithelial cells undergo further cell divisions before transforming into larval neuroblasts. Our results demonstrate that the optic lobe neuroepithelium gives rise to neurons and glia over 60 h earlier than was thought previously.