Temporal transcription factors and their targets schedule the end of neural proliferation in Drosophila.

The timing mechanisms responsible for terminating cell proliferation toward the end of development remain unclear. In the Drosophila CNS, individual progenitors called neuroblasts are known to express a series of transcription factors endowing daughter neurons with different temporal identities. Here we show that Castor and Seven-Up, members of this temporal series, regulate key events in many different neuroblast lineages during late neurogenesis. First, they schedule a switch in the cell size and identity of neurons involving the targets Chinmo and Broad Complex. Second, they regulate the time at which neuroblasts undergo Prospero-dependent cell-cycle exit or Reaper/Hid/Grim-dependent apoptosis. Both types of progenitor termination require the combined action of a late phase of the temporal series and indirect feedforward via Castor targets such as Grainyhead and Dichaete. These studies identify the timing mechanism ending CNS proliferation and reveal how aging progenitors transduce bursts of transcription factors into long-lasting changes in cell proliferation and cell identity.

Developmental regulation of regenerative potential in Drosophila by ecdysone through a bistable loop of ZBTB transcription factors.

In many organisms, the regenerative capacity of tissues progressively decreases as development progresses. However, the developmental mechanisms that restrict regenerative potential remain unclear. In Drosophila, wing imaginal discs become unable to regenerate upon damage during the third larval stage (L3). Here, we show that production of ecdysone after larvae reach their critical weight (CW) terminates the window of regenerative potential by acting on a bistable loop composed of two antagonistic Broad-complex/Tramtrack/Bric-à-brac Zinc-finger (ZBTB) genes: chinmo and broad (br). Around mid L3, ecdysone signaling silences chinmo and activates br to switch wing epithelial progenitors from a default self-renewing to a differentiation-prone state. Before mid L3, Chinmo promotes a strong regenerative response upon tissue damage. After mid L3, Br installs a nonpermissive state that represses regeneration. Transient down-regulation of ecdysone signaling or Br in late L3 larvae enhances chinmo expression in damaged cells that regain the capacity to regenerate. This work unveils a mechanism that ties the self-renewing and regenerative potential of epithelial progenitors to developmental progression.

Two distinct mechanisms silence chinmo in Drosophila neuroblasts and neuroepithelial cells to limit their self-renewal.

Whether common principles regulate the self-renewing potential of neural stem cells (NSCs) throughout the developing central nervous system is still unclear. In the Drosophila ventral nerve cord and central brain, asymmetrically dividing NSCs, called neuroblasts (NBs), progress through a series of sequentially expressed transcription factors that limits self-renewal by silencing a genetic module involving the transcription factor Chinmo. Here, we find that Chinmo also promotes neuroepithelium growth in the optic lobe during early larval stages by boosting symmetric self-renewing divisions while preventing differentiation. Neuroepithelium differentiation in late larvae requires the transcriptional silencing of chinmo by ecdysone, the main steroid hormone, therefore allowing coordination of neural stem cell self-renewal with organismal growth. In contrast, chinmo silencing in NBs is post-transcriptional and does not require ecdysone. Thus, during Drosophila development, humoral cues or tissue-intrinsic temporal specification programs respectively limit self-renewal in different types of neural progenitors through the transcriptional and post-transcriptional regulation of the same transcription factor.

Serotonin signaling regulates actomyosin contractility during morphogenesis in evolutionarily divergent lineages.

Serotonin is a neurotransmitter that signals through 5-HT receptors to control key functions in the nervous system. Serotonin receptors are also ubiquitously expressed in various organs and have been detected in embryos of different organisms. Potential morphogenetic functions of serotonin signaling have been proposed based on pharmacological studies but a mechanistic understanding is still lacking. Here, we uncover a role of serotonin signaling in axis extension of Drosophila embryos by regulating Myosin II (MyoII) activation, cell contractility and cell intercalation. We find that serotonin and serotonin receptors 5HT2A and 5HT2B form a signaling module that quantitatively regulates the amplitude of planar polarized MyoII contractility specified by Toll receptors and the GPCR Cirl. Remarkably, serotonin signaling also regulates actomyosin contractility at cell junctions, cellular flows and epiblast morphogenesis during chicken gastrulation. This phylogenetically conserved mechanical function of serotonin signaling in regulating actomyosin contractility and tissue flow reveals an ancestral role in morphogenesis of multicellular organisms.

Regulation of developmental hierarchy in Drosophila neural stem cell tumors by COMPASS and Polycomb complexes.

COMPASS and Polycomb complexes are antagonistic chromatin complexes that are frequently inactivated in cancers, but how these events affect the cellular hierarchy, composition, and growth of tumors is unclear. These characteristics can be systematically investigated in Drosophila neuroblast tumors in which cooption of temporal patterning induces a developmental hierarchy that confers cancer stem cell (CSC) properties to a subset of neuroblasts retaining an early larval temporal identity. Here, using single-cell transcriptomics, we reveal that the trithorax/MLL1/2-COMPASS-like complex guides the developmental trajectory at the top of the tumor hierarchy. Consequently, trithorax knockdown drives larval-to-embryonic temporal reversion and the marked expansion of CSCs that remain locked in a spectrum of early temporal states. Unexpectedly, this phenotype is amplified by concomitant inactivation of Polycomb repressive complex 2 genes, unleashing tumor growth. This study illustrates how inactivation of specific COMPASS and Polycomb complexes cooperates to impair tumor hierarchies, inducing CSC plasticity, heterogeneity, and expansion.

Suppression of Polycomb group proteins by JNK signalling induces transdetermination in Drosophila imaginal discs.

During the regeneration of Drosophila imaginal discs, cellular identities can switch fate in a process known as transdetermination. For leg-to-wing transdetermination, the underlying mechanism involves morphogens such as Wingless that, when activated outside their normal context, induce ectopic expression of the wing-specific selector gene vestigial. Polycomb group (PcG) proteins maintain cellular fates by controlling the expression patterns of homeotic genes and other developmental regulators. Here we report that transdetermination events are coupled to PcG regulation. We show that the frequency of transdetermination is enhanced in PcG mutant flies. Downregulation of PcG function, as monitored by the reactivation of a silent PcG-regulated reporter gene, is observed in transdetermined cells. This downregulation is directly controlled by the Jun amino-terminal kinase (JNK) signalling pathway, which is activated in cells undergoing regeneration. Accordingly, transdetermination frequency is reduced in a JNK mutant background. This regulatory interaction also occurs in mammalian cells, indicating that the role of this signalling cascade in remodelling cellular fates may be conserved.

Signaling meets chromatin during tissue regeneration in Drosophila.

As transcription programs become stabilized in fate-determined cells by progressive patterning of chromatin structures, cells lose their plasticity and the ability to freely modify their identity in response to changing developmental cues. By contrast, stem cells maintain this flexibility, enabling them to embark on different determination pathways. However, regeneration of tissue requires an exception because determined cells are forced to switch their transcription programs to reconstruct the missing tissue. In Drosophila, proliferating cells in the regenerating imaginal discs can even switch to a new disc identity. New studies show that the increased plasticity observed during regeneration results from the action of multiple signaling pathways on chromatin malleability, cell-cycle profiles, and expression of 'stemness' genes. Understanding how signaling pathways can integrate to switch determined cells into multipotent cells has a great medical potential, especially in the field of tissue engineering and remodeling.

Temporal patterning in neural progenitors: from Drosophila development to childhood cancers.

The developing central nervous system (CNS) is particularly prone to malignant transformation, but the underlying mechanisms remain unresolved. However, periods of tumor susceptibility appear to correlate with windows of increased proliferation, which are often observed during embryonic and fetal stages and reflect stereotypical changes in the proliferative properties of neural progenitors. The temporal mechanisms underlying these proliferation patterns are still unclear in mammals. In Drosophila, two decades of work have revealed a network of sequentially expressed transcription factors and RNA-binding proteins that compose a neural progenitor-intrinsic temporal patterning system. Temporal patterning controls both the identity of the post-mitotic progeny of neural progenitors, according to the order in which they arose, and the proliferative properties of neural progenitors along development. In addition, in Drosophila, temporal patterning delineates early windows of cancer susceptibility and is aberrantly regulated in developmental tumors to govern cellular hierarchy as well as the metabolic and proliferative heterogeneity of tumor cells. Whereas recent studies have shown that similar genetic programs unfold during both fetal development and pediatric brain tumors, I discuss, in this Review, how the concept of temporal patterning that was pioneered in Drosophila could help to understand the mechanisms of initiation and progression of CNS tumors in children.

Coopted temporal patterning governs cellular hierarchy, heterogeneity and metabolism in Drosophila neuroblast tumors.

It is still unclear what drives progression of childhood tumors. During Drosophila larval development, asymmetrically-dividing neural stem cells, called neuroblasts, progress through an intrinsic temporal patterning program that ensures cessation of divisions before adulthood. We previously showed that temporal patterning also delineates an early developmental window during which neuroblasts are susceptible to tumor initiation (Narbonne-Reveau et al., 2016). Using single-cell transcriptomics, clonal analysis and numerical modeling, we now identify a network of twenty larval temporal patterning genes that are redeployed within neuroblast tumors to trigger a robust hierarchical division scheme that perpetuates growth while inducing predictable cell heterogeneity. Along the hierarchy, temporal patterning genes define a differentiation trajectory that regulates glucose metabolism genes to determine the proliferative properties of tumor cells. Thus, partial redeployment of the temporal patterning program encoded in the cell of origin may govern the hierarchy, heterogeneity and growth properties of neural tumors with a developmental origin.

Brainy but not too brainy: starting and stopping neuroblast divisions in Drosophila.

Drosophila neuroblasts are similar to mammalian neural stem cells in that they self-renew and have the potential to generate many different types of neurons and glia. They have already proved useful for uncovering asymmetric division components and now look set to provide insights into how stem cell divisions are initiated and terminated during neural development. In particular, some of the humoral factors and short-range 'niche' signals that modulate neuroblast activity during postembryonic development have been identified. In addition, recent studies have begun to reveal how the total number of cells generated by a single neuroblast is regulated by spatial and temporal cues from Hox proteins and a transcription-factor series linked to cell cycle progression.

Neural stem cell-encoded temporal patterning delineates an early window of malignant susceptibility in Drosophila.

Pediatric neural tumors are often initiated during early development and can undergo very rapid transformation. However, the molecular basis of this early malignant susceptibility remains unknown. During Drosophila development, neural stem cells (NSCs) divide asymmetrically and generate intermediate progenitors that rapidly differentiate in neurons. Upon gene inactivation, these progeny can dedifferentiate and generate malignant tumors. Here, we find that intermediate progenitors are prone to malignancy only when born during an early window of development while expressing the transcription factor Chinmo, and the mRNA-binding proteins Imp/IGF2BP and Lin-28. These genes compose an oncogenic module that is coopted upon dedifferentiation of early-born intermediate progenitors to drive unlimited tumor growth. In late larvae, temporal transcription factor progression in NSCs silences the module, thereby limiting mitotic potential and terminating the window of malignant susceptibility. Thus, this study identifies the gene regulatory network that confers malignant potential to neural tumors with early developmental origins.

Building a brain under nutritional restriction: insights on sparing and plasticity from Drosophila studies.

While the growth of the developing brain is known to be well-protected compared to other organs in the face of nutrient restriction (NR), careful analysis has revealed a range of structural alterations and long-term neurological defects. Yet, despite intensive studies, little is known about the basic principles that govern brain development under nutrient deprivation. For over 20 years, Drosophila has proved to be a useful model for investigating how a functional nervous system develops from a restricted number of neural stem cells (NSCs). Recently, a few studies have started to uncover molecular mechanisms as well as region-specific adaptive strategies that preserve brain functionality and neuronal repertoire under NR, while modulating neuron numbers. Here, we review the developmental constraints that condition the response of the developing brain to NR. We then analyze the recent Drosophila work to highlight key principles that drive sparing and plasticity in different regions of the central nervous system (CNS). As simple animal models start to build a more integrated picture, understanding how the developing brain copes with NR could help in defining strategies to limit damage and improve brain recovery after birth.

Protection of neuronal diversity at the expense of neuronal numbers during nutrient restriction in the Drosophila visual system.

Systemic signals provided by nutrients and hormones are known to coordinate the growth and proliferation of different organs during development. However, within the brain, it is unclear how these signals influence neural progenitor divisions and neuronal diversity. Here, in the Drosophila visual system, we identify two developmental phases with different sensitivities to dietary nutrients. During early larval stages, nutrients regulate the size of the neural progenitor pool via insulin/PI3K/TOR-dependent symmetric neuroepithelial divisions. During late larval stages, neural proliferation becomes insensitive to dietary nutrients, and the steroid hormone ecdysone acts on Delta/Notch signaling to promote the switch from symmetric mitoses to asymmetric neurogenic divisions. This mechanism accounts for why sustained undernourishment during visual system development restricts neuronal numbers while protecting neuronal diversity. These studies reveal an adaptive mechanism that helps to retain a functional visual system over a range of different brain sizes in the face of suboptimal nutrition.

Temporal specification of neural stem cells: insights from Drosophila neuroblasts.

The mechanisms underlying the temporal specification of neural stem cells (NSCs), a process by which a single progenitor can generate different types of neurons and glia in an invariant order, are still poorly understood in mammals. However, in the past decade, work on Drosophila NSCs, called neuroblasts, has identified a series of sequentially expressed transcription factors that lies at the heart of this phenomenon. Here, I highlight some key findings that illuminate the role of these transcription factors during development and the regulatory principles allowing them not only to promote neuronal diversity but also to control the final number of neurons in the different regions of the nervous system. Ultimately, and given recent evidences of evolutionary conservation, cracking the temporal specification code of Drosophila neuroblasts may provide new perspectives for the safe manipulation of human NSCs and their therapeutic use.

Temporal control of neuronal diversity: common regulatory principles in insects and vertebrates?

It is well established in species as diverse as insects and mammals that different neuronal and glial subtypes are born at distinct times during central nervous system development. In Drosophila, there is now compelling evidence that individual multipotent neuroblasts express a sequence of progenitor transcription factors which, in turn, regulates the postmitotic transcription factors that specify neuronal/glial temporal identities. Here, we examine the hypothesis that the regulatory principles underlying this mode of temporal specification are shared between insects and mammals, even if some of the factors themselves are not. We also propose a general model for birth-order-dependent neural specification and suggest some experiments to test its validity.

A cellular memory module conveys epigenetic inheritance of hedgehog expression during Drosophila wing imaginal disc development.

In Drosophila, the Trithorax-group (trxG) and Polycomb-group (PcG) proteins interact with chromosomal elements, termed Cellular Memory Modules (CMMs). By modifying chromatin, this ensures a stable heritable maintenance of the transcriptional state of developmental regulators, like the homeotic genes, that is defined embryonically. We asked whether such CMMs could also control expression of genes involved in patterning imaginal discs during larval development. Our results demonstrate that expression of the hedgehog gene, once activated, is maintained by a CMM. In addition, our experiments indicate that the switching of such CMMs to an active state during larval stages, in contrast to embryonic stages, may require specific trans-activators. Our results suggest that the patterning of cells in particular developmental fields in the imaginal discs does not only rely on external cues from morphogens, but also depends on the previous history of the cells, as the control by CMMs ensures a preformatted gene expression pattern.