The diversity of cell types and regulatory states in the brain, and how these change during aging, remains largely unknown. We present a single-cell transcriptome atlas of the entire adult Drosophila melanogaster brain sampled across its lifespan. Cell clustering identified 87 initial cell clusters that are further subclustered and validated by targeted cell-sorting. Our data show high granularity and identify a wide range of cell types. Gene network analyses using SCENIC revealed regulatory heterogeneity linked to energy consumption. During aging, RNA content declines exponentially without affecting neuronal identity in old brains. This single-cell brain atlas covers nearly all cells in the normal brain and provides the tools to study cellular diversity alongside other Drosophila and mammalian single-cell datasets in our unique single-cell analysis platform: SCope (http://scope.aertslab.org). These results, together with SCope, allow comprehensive exploration of all transcriptional states of an entire aging brain.
Literature context: opmental Studies Hybridoma Bank RRID:AB_528440), rabbit anti-Rel68 (1:100, gen
Enteroendocrine cells (EEs) are interspersed between enterocytes and stem cells in the Drosophila intestinal epithelium. Like enterocytes, EEs express components of the immune deficiency (IMD) innate immune pathway, which activates transcription of genes encoding antimicrobial peptides. The discovery of large lipid droplets in intestines of IMD pathway mutants prompted us to investigate the role of the IMD pathway in the host metabolic response to its intestinal microbiota. Here we provide evidence that the short-chain fatty acid acetate is a microbial metabolic signal that activates signaling through the enteroendocrine IMD pathway in a PGRP-LC-dependent manner. This, in turn, increases transcription of the gene encoding the endocrine peptide Tachykinin (Tk), which is essential for timely larval development and optimal lipid metabolism and insulin signaling. Our findings suggest innate immune pathways not only provide the first line of defense against infection but also afford the intestinal microbiota control over host development and metabolism.
Literature context: DSHB Cat# MR1A; RRID:AB_528440 Rabbit monoclonal anti-Cleaved
Intestinal infection triggers potent immune responses to combat pathogens and concomitantly drives epithelial renewal to maintain barrier integrity. Current models propose that epithelial renewal is primarily driven by damage caused by reactive oxygen species (ROS). Here we found that in Drosophila, the Imd-NF-κB pathway controlled enterocyte (EC) shedding upon infection, via a mechanism independent of ROS-associated apoptosis. Mechanistically, the Imd pathway synergized with JNK signaling to induce epithelial cell shedding specifically in the context of bacterial infection, requiring also the reduced expression of the transcription factor GATAe. Furthermore, cell-specific NF-κB responses enabled simultaneous production of antimicrobial peptides (AMPs) and epithelial shedding in different EC populations. Thus, the Imd-NF-κB pathway is central to the intestinal antibacterial response by mediating both AMP production and the maintenance of barrier integrity. Considering the similarities between Drosophila Imd signaling and mammalian TNFR pathway, our findings suggest the existence of an evolutionarily conserved genetic program in immunity-induced epithelial shedding.
Literature context: opmental Studies Hybridoma Bank RRID:AB_528440
Understanding how cellular identity naturally interconverts with high efficiency and temporospatial precision is crucial for regenerative medicine. Here, we revealed a natural midgut-to-renal lineage conversion event during Drosophila metamorphosis and identified the evolutionarily-conserved homeodomain protein Cut as a master switch in this process. A steep Wnt/Wingless morphogen gradient intersects with a pulse of steroid hormone ecdysone to induce cut expression in a subset of midgut progenitors and reprogram them into renal progenitors. Molecularly, ecdysone-induced temporal factor Broad physically interacts with cut enhancer-bound Wnt pathway effector TCF/β-catenin and likely bridges the distant enhancer and promoter region of cut through its self-association. Such long-range enhancer-promoter looping could subsequently trigger timely cut transcription. Our results therefore led us to propose an unexpected poising-and-bridging mechanism whereby spatial and temporal cues intersect, likely via chromatin looping, to turn on a master transcription factor and dictate efficient and precise lineage reprogramming.
Literature context: al Studies Hybridoma Bank RRID:AB_528440 Chicken anti-GFP Abcam Cat# ab1
Adenomatous polyposis coli (APC) mutations cause Wnt pathway activation in human cancers. Current models for APC action emphasize its role in promoting β-catenin degradation downstream of Wnt receptors. Unexpectedly, we find that blocking Wnt receptor activity in APC-deficient cells inhibits Wnt signaling independently of Wnt ligand. We also show that inducible loss of APC is rapidly followed by Wnt receptor activation and increased β-catenin levels. In contrast, APC2 loss does not promote receptor activation. We show that APC exists in a complex with clathrin and that Wnt pathway activation in APC-deficient cells requires clathrin-mediated endocytosis. Finally, we demonstrate conservation of this mechanism in Drosophila intestinal stem cells. We propose a model in which APC and APC2 function to promote β-catenin degradation, and APC also acts as a molecular "gatekeeper" to block receptor activation via the clathrin pathway.
Literature context: mouse anti-Pros DSHB RRID:AB_528440 rabbit anti-PH3 Abcam RRID:AB_2
In vertebrates and invertebrates, neurons and glia are generated in a stereotyped manner from neural stem cells, but the purpose of invariant lineages is not understood. We show that two stem cells that produce leg motor neurons in Drosophila also generate neuropil glia, which wrap and send processes into the neuropil where motor neuron dendrites arborize. The development of the neuropil glia and leg motor neurons is highly coordinated. However, although motor neurons have a stereotyped birth order and transcription factor code, the number and individual morphologies of the glia born from these lineages are highly plastic, yet the final structure they contribute to is highly stereotyped. We suggest that the shared lineages of these two cell types facilitate the assembly of complex neural circuits and that the two birth order strategies-hardwired for motor neurons and flexible for glia-are important for robust nervous system development, homeostasis, and evolution.
Literature context: 1A; RRID:AB_528440 Rabbit anti-pdm1 Laboratory of
The balance between self-renewal and differentiation ensures long-term maintenance of stem cell (SC) pools in regenerating epithelial tissues. This balance is challenged during periods of high regenerative pressure and is often compromised in aged animals. Here, we show that target of rapamycin (TOR) signaling is a key regulator of SC loss during repeated regenerative episodes. In response to regenerative stimuli, SCs in the intestinal epithelium of the fly and in the tracheal epithelium of mice exhibit transient activation of TOR signaling. Although this activation is required for SCs to rapidly proliferate in response to damage, repeated rounds of damage lead to SC loss. Consistently, age-related SC loss in the mouse trachea and in muscle can be prevented by pharmacologic or genetic inhibition, respectively, of mammalian target of rapamycin complex 1 (mTORC1) signaling. These findings highlight an evolutionarily conserved role of TOR signaling in SC function and identify repeated rounds of mTORC1 activation as a driver of age-related SC decline.
Literature context: D:RRID:AB_528440), anti-Atg9 (1:100) (Tang et al
Autophagy is essential for maintaining cellular homeostasis and survival under various stress conditions. Autophagy-related gene 9 (Atg9) encodes a multipass transmembrane protein thought to act as a membrane carrier for forming autophagosomes. However, the molecular regulation and physiological importance of Atg9 in animal development remain largely unclear. Here, we generated Atg9 null mutant flies and found that loss of Atg9 led to shortened lifespan, locomotor defects, and increased susceptibility to stress. Atg9 loss also resulted in aberrant adult midgut morphology with dramatically enlarged enterocytes. Interestingly, inhibiting the TOR signaling pathway rescued the midgut defects of the Atg9 mutants. In addition, Atg9 interacted with PALS1-associated tight junction protein (Patj), which associates with TSC2 to regulate TOR activity. Depletion of Atg9 caused a marked decrease in TSC2 levels. Our findings revealed an antagonistic relationship between Atg9 and TOR signaling in the regulation of cell growth and tissue homeostasis.
Literature context: , Oregon), mouse anti-Prospero (RRID:AB_528440) (1:10, DSHB, Iowa City, Iowa),
Drosophila color vision is achieved by comparing outputs from two types of color-sensitive photoreceptors, R7 and R8. Ommatidia (unit eyes) are classified into two subtypes, known as 'pale' or 'yellow', depending on Rhodopsin expression in R7 and R8. Subtype specification is controlled by a stochastic decision in R7 and instructed to the underlying R8. We find that the Activin receptor Baboon is required in R8 to receive non-redundant signaling from the three Activin ligands, activating the transcription factor dSmad2. Concomitantly, two BMP ligands activate their receptor, Thickveins, and the transcriptional effector, Mad. The Amon TGFβ processing factor appears to regulate components of the TGFβ pathway specifically in pale R7. Mad and dSmad2 cooperate to modulate the Hippo pathway kinase Warts and the growth regulator Melted; two opposing factors of a bi-stable loop regulating R8 Rhodopsin expression. Therefore, TGFβ and growth pathways interact in postmitotic cells to precisely coordinate cell-specific output.
Literature context: at# MR1A; RRID:AB_528440 Rabbit pol
Organ fitness depends on appropriate maintenance of stem cell populations, and aberrations in functional stem cell numbers are associated with malignancies and aging. Symmetrical division is the best characterized mechanism of stem cell replacement, but other mechanisms could also be deployed, particularly in situations of high stress. Here, we show that after severe depletion, intestinal stem cells (ISCs) in the Drosophila midgut are replaced by spindle-independent ploidy reduction of cells in the enterocyte lineage through a process known as amitosis. Amitosis is also induced by the functional loss of ISCs coupled with tissue demand and in aging flies, underscoring the generality of this mechanism. However, we also found that random homologous chromosome segregation during ploidy reduction can expose deleterious mutations through loss of heterozygosity. Together, our results highlight amitosis as an unappreciated mechanism for restoring stem cell homeostasis, but one with some associated risk in animals carrying mutations.
Literature context: Cat#MR1A; RRID:AB_528440 Rat anti-M
Asymmetric stem cell division establishes an initial difference between a stem cell and its differentiating sibling, critical for maintaining homeostasis and preventing carcinogenesis. Yet the mechanisms that consolidate and lock in such initial fate bias remain obscure. Here, we use Drosophila neuroblasts to demonstrate that the super elongation complex (SEC) acts as an intrinsic amplifier to drive cell fate commitment. SEC is highly expressed in neuroblasts, where it promotes self-renewal by physically associating with Notch transcription activation complex and enhancing HES (hairy and E(spl)) transcription. HES in turn upregulates SEC activity, forming an unexpected self-reinforcing feedback loop with SEC. SEC inactivation leads to neuroblast loss, whereas its forced activation results in neural progenitor dedifferentiation and tumorigenesis. Our studies unveil an SEC-mediated intracellular amplifier mechanism in ensuring robustness and precision in stem cell fate commitment and provide mechanistic explanation for the highly frequent association of SEC overactivation with human cancers.
Sex differences in physiology and disease susceptibility are commonly attributed to developmental and/or hormonal factors, but there is increasing realization that cell-intrinsic mechanisms play important and persistent roles. Here we use the Drosophila melanogaster intestine to investigate the nature and importance of cellular sex in an adult somatic organ in vivo. We find that the adult intestinal epithelium is a cellular mosaic of different sex differentiation pathways, and displays extensive sex differences in expression of genes with roles in growth and metabolism. Cell-specific reversals of the sexual identity of adult intestinal stem cells uncovers the key role this identity has in controlling organ size, reproductive plasticity and response to genetically induced tumours. Unlike previous examples of sexually dimorphic somatic stem cell activity, the sex differences in intestinal stem cell behaviour arise from intrinsic mechanisms that control cell cycle duration and involve a new doublesex- and fruitless-independent branch of the sex differentiation pathway downstream of transformer. Together, our findings indicate that the plasticity of an adult somatic organ is reversibly controlled by its sexual identity, imparted by a new mechanism that may be active in more tissues than previously recognized.
Stem cells possess the capacity to generate two cells of distinct fate upon division: one cell retaining stem cell identity and the other cell destined to differentiate. These cell fates are established by cell-type-specific genetic networks. To comprehensively identify components of these networks, we performed a large-scale RNAi screen in Drosophila female germline stem cells (GSCs) covering ∼25% of the genome. The screen identified 366 genes that affect GSC maintenance, differentiation, or other processes involved in oogenesis. Comparison of GSC regulators with neural stem cell self-renewal factors identifies common and cell-type-specific self-renewal genes. Importantly, we identify the histone methyltransferase Set1 as a GSC-specific self-renewal factor. Loss of Set1 in neural stem cells does not affect cell fate decisions, suggesting a differential requirement of H3K4me3 in different stem cell lineages. Altogether, our study provides a resource that will help to further dissect the networks underlying stem cell self-renewal.
Stem cell self-renewal and differentiation must be carefully controlled during development and tissue homeostasis. In the Drosophila optic lobe, neuroepithelial cells first divide symmetrically to expand the stem cell population and then transform into asymmetrically dividing neuroblasts, which generate medulla neurons. The mechanisms underlying this cell fate transition are not well understood. Here, we show a crucial role of some cell cycle regulators in this transition. We find that loss of function in replication protein A (RPA), which consists of three highly conserved protein subunits and functions in DNA replication, leads to disintegration of the optic lobe neuroepithelium and premature differentiation of neuroepithelial cells into medulla neuroblasts. Clonal analyses of RPA loss-of-function alleles indicate that RPA is required to prevent neuroepithelial cells from differentiating into medulla neuroblasts. Inactivation of the core cell cycle regulators, including the G1/S regulators E2F1, Cyclin E, Cdk2, and PCNA, and the G2/M regulators Cyclin A, Cyclin B, and Cdk1, mimic RPA loss-of-function phenotypes, suggesting that cell cycle progression is required for both maintaining neuroepithelial cell identity and suppressing neuroblast formation. We further find that RPA or E2F1 inactivation in the neuroepithelial cells correlates with downregulation of Notch signaling activity, which appears to result from Numb mislocalization. Thus, we have shown that the transition from neuroepithelial cells to neuroblasts is directly regulated by cell cycle regulators and propose a model in which the inhibition of neuroepithelial cell cycle progression downregulates Notch signaling activity through Numb, which leads to the onset of neurogenesis.
Asymmetric stem cell division balances maintenance of the stem cell pool and generation of diverse cell types by simultaneously allowing one daughter progeny to maintain a stem cell fate and its sibling to acquire a progenitor cell identity. A progenitor cell possesses restricted developmental potential, and defects in the regulation of progenitor cell potential can directly impinge on the maintenance of homeostasis and contribute to tumor initiation. Despite their importance, the molecular mechanisms underlying the precise regulation of restricted developmental potential in progenitor cells remain largely unknown. We used the type II neural stem cell (neuroblast) lineage in Drosophila larval brain as a genetic model system to investigate how an intermediate neural progenitor (INP) cell acquires restricted developmental potential. We identify the transcription factor Klumpfuss (Klu) as distinguishing a type II neuroblast from an INP in larval brains. klu functions to maintain the identity of type II neuroblasts, and klu mutant larval brains show progressive loss of type II neuroblasts due to premature differentiation. Consistently, Klu protein is detected in type II neuroblasts but is undetectable in immature INPs. Misexpression of klu triggers immature INPs to revert to type II neuroblasts. In larval brains lacking brain tumor function or exhibiting constitutively activated Notch signaling, removal of klu function prevents the reversion of immature INPs. These results led us to propose that multiple mechanisms converge to exert precise control of klu and distinguish a progenitor cell from its sibling stem cell during asymmetric neuroblast division.
Cancer stem cells (CSCs) are postulated to be a small subset of tumor cells with tumor-initiating ability that shares features with normal tissue-specific stem cells. The origin of CSCs and the mechanisms underlying their genesis are poorly understood, and it is uncertain whether it is possible to obliterate CSCs without inadvertently damaging normal stem cells. Here we show that a functional reduction of eukaryotic translation initiation factor 4E (eIF4E) in Drosophila specifically eliminates CSC-like cells in the brain and ovary without having discernable effects on normal stem cells. Brain CSC-like cells can arise from dedifferentiation of transit-amplifying progenitors upon Notch hyperactivation. eIF4E is up-regulated in these dedifferentiating progenitors, where it forms a feedback regulatory loop with the growth regulator dMyc to promote cell growth, particularly nucleolar growth, and subsequent ectopic neural stem cell (NSC) formation. Cell growth regulation is also a critical component of the mechanism by which Notch signaling regulates the self-renewal of normal NSCs. Our findings highlight the importance of Notch-regulated cell growth in stem cell maintenance and reveal a stronger dependence on eIF4E function and cell growth by CSCs, which might be exploited therapeutically.
Notch signaling mediates multiple developmental decisions in Drosophila. In this study, we have examined the role of Notch signaling in Drosophila larval optic lobe development. Loss of function in Notch or its ligand Delta leads to loss of the lamina and a smaller medulla. The neuroepithelial cells in the optic lobe in Notch or Delta mutant brains do not expand but instead differentiate prematurely into medulla neuroblasts, which lead to premature neurogenesis in the medulla. Clonal analyses of loss-of-function alleles for the pathway components, including N, Dl, Su(H), and E(spl)-C, indicate that the Delta/Notch/Su(H) pathway is required for both maintaining the neuroepithelial stem cells and inhibiting medulla neuroblast formation while E(spl)-C is only required for some aspects of the inhibition of medulla neuroblast formation. Conversely, Notch pathway overactivation promotes neuroepithelial cell expansion while suppressing medulla neuroblast formation and neurogenesis; numb loss of function mimics Notch overactivation, suggesting that Numb may inhibit Notch signaling activity in the optic lobe neuroepithelial cells. Thus, our results show that Notch signaling plays a dual role in optic lobe development, by maintaining the neuroepithelial stem cells and promoting their expansion while inhibiting their differentiation into medulla neuroblasts. These roles of Notch signaling are strikingly similar to those of the JAK/STAT pathway in optic lobe development, raising the possibility that these pathways may collaborate to control neuroepithelial stem cell maintenance and expansion, and their differentiation into the progenitor cells.
bHLH-O proteins are a subfamily of the basic-helix-loop-helix transcription factors characterized by an 'Orange' protein-protein interaction domain. Typical members are the Hairy/E(spl), or Hes, proteins, well studied in their ability, among others, to suppress neuronal differentiation in both invertebrates and vertebrates. Hes proteins are often effectors of Notch signalling. In vertebrates, another bHLH-O protein group, the Hey proteins, have also been shown to be Notch targets and to interact with Hes. We have studied the single Drosophila Hey orthologue. We show that it is primarily expressed in a subset of newly born neurons, which receive Notch signalling during their birth. Unlike in vertebrates, however, Hey is not expressed in precursor cells and does not block neuronal differentiation. It rather promotes one of two alternative fates that sibling neurons adopt at birth. Although in the majority of cases Hey is a Notch target, it is also expressed independently of Notch in some lineages, most notably the larval mushroom body. The availability of Hey as a Notch readout has allowed us to study Notch signalling during the genesis of secondary neurons in the larval central nervous system.
In Drosophila subperineurial glia (SPG) ensheath and insulate the nerve. SPG is under strict cell cycle and survival control because cell division or death of such a cell type would compromise the integrity of the blood-nerve barrier. The mechanisms underlying the survival of SPG remain unknown. Here, we show that the embryonic peripheral glia expresses the Zfh1 transcription factor, and in zfh1 mutants a particular SPG subtype, ePG10, undergoes apoptosis. Our findings show that in ePG10, Zfh1 represses the pro-apoptotic RHG-motif gene reaper in a cell-autonomous manner. Zfh1 also blocks the activation of the Jun N-terminal kinase (JNK) pathway, and reducing or enhancing JNK signalling in zfh1 mutants prevents or promotes ePG10 apoptosis. Our study shows a novel function for Zfh1 as an anti-apoptotic molecule and uncovers a cryptic JNK-dependent apoptotic programme in ePG10, which is normally blocked by Zfh1. We propose that, in cells such as SPG that do not undergo self-renewal and survive long periods, transcriptional control of RHG-motif gene expression together with fine tuning of JNK signalling is crucial for cell survival.
The Notch and Epidermal Growth Factor Receptor (EGFR) signaling pathways interact cooperatively and antagonistically to regulate many aspects of Drosophila development, including the eye. How output from these two signaling networks is fine-tuned to achieve the precise balance needed for specific inductive interactions and patterning events remains an open and important question. Previously, we reported that the gene split ends (spen) functions within or parallel to the EGFR pathway during midline glial cell development in the embryonic central nervous system. Here, we report that the cellular defects caused by loss of spen function in the developing eye imaginal disc place spen as both an antagonist of the Notch pathway and a positive contributor to EGFR signaling during retinal cell differentiation. Specifically, loss of spen results in broadened expression of Scabrous, ectopic activation of Notch signaling, and a corresponding reduction in Atonal expression at the morphogenetic furrow. Consistent with Spen's role in antagonizing Notch signaling, reduction of spen levels is sufficient to suppress Notch-dependent phenotypes. At least in part due to loss of Spen-dependent down-regulation of Notch signaling, loss of spen also dampens EGFR signaling as evidenced by reduced activity of MAP kinase (MAPK). This reduced MAPK activity in turn leads to a failure to limit expression of the EGFR pathway antagonist and the ETS-domain transcriptional repressor Yan and to a corresponding loss of cell fate specification in spen mutant ommatidia. We propose that Spen plays a role in modulating output from the Notch and EGFR pathways to ensure appropriate patterning during eye development.
Neuralized (neur) is a neurogenic mutant of Drosophila in which many signaling events mediated by the Notch (N) receptor are disrupted. Here, we analyze the role of neur during eye development. Neur is required in a cell-autonomous fashion to restrict R8 and other photoreceptor fates and is involved in lateral inhibition of interommatidial bristles but is not required for induction of the cone cell fate. The latter contrasts with the absolute requirement for Suppressor of Hairless and the Enhancer of split-Complex for cone cell induction. Using gain-of-function experiments, we further demonstrate that ectopic wild-type and truncated Neur proteins can interfere with multiple N-controlled aspects of eye development, including both neur-dependent and neur-independent processes.
Subcellular distribution of the Prospero protein is dynamically regulated during Drosophila embryonic nervous system development. Prospero is first detected in neuroblasts where it becomes cortically localized and tethered by the adapter protein, Miranda. After division, Prospero enters the nucleus of daughter ganglion mother cells where it functions as a transcription factor. We have isolated a mutation that removes the C-terminal 30 amino acids from the highly conserved 100 amino acid Prospero domain. Molecular dissection of the homeo- and Prospero domains, and expression of chimeric Prospero proteins in mammalian and insect cultured cells indicates that Prospero contains a nuclear export signal that is masked by the Prospero domain. Nuclear export of Prospero, which is sensitive to the drug leptomycin B, is mediated by Exportin. Mutation of the nuclear export signal-mask in Drosophila embryos prevents Prospero nuclear localization in ganglion mother cells. We propose that a combination of cortical tethering and regulated nuclear export controls Prospero subcellular distribution and function in all higher eukaryotes.
The bristle mechanosensory organs of the adult fly are composed of four different cells that originate from a single precursor cell, pI, via two rounds of asymmetric cell division. Here, we have examined the pattern of cell divisions in this lineage by time-lapse confocal microscopy using GFP imaging and by immunostaining analysis. pI divided within the plane of the epithelium and along the anteroposterior axis to give rise to an anterior cell, pIIb, and a posterior cell, pIIa. pIIb divided prior to pIIa to generate a small subepithelial cell and a larger daughter cell, named pIIIb. This unequal division, oriented perpendicularly to the epithelium plane, has not been described previously. pIIa divided after pIIb, within the plane of the epithelium and along the AP axis, to produce a posterior socket cell and an anterior shaft cell. Then pIIIb divided perpendicularly to the epithelium plane to generate a basal neurone and an apical sheath cell. The small subepithelial pIIb daughter cell was identified as a sense organ glial cell: it expressed glial cell missing, a selector gene for the glial fate and migrated away from the sensory cluster along extending axons. We propose that mechanosensory organ glial cells, the origin of which was until now unknown, are generated by the asymmetric division of pIIb cells. Both Numb and Prospero segregated specifically into the basal glial and neuronal cells during the pIIb and pIIIb divisions, respectively. This revised description of the sense organ lineage provides the basis for future studies on how polarity and fate are regulated in asymmetrically dividing cells.
We report the identification of RK2, a glial-specific homeodomain protein. RK2 is localized to the nucleus of virtually all embryonic and imaginal glial cells, with the exception of midline glia. Embryos mutant for the gene encoding RK2 are embryonic lethal but normal for early gliogenesis (birth, initial divisions and migration of glia) and axonogenesis (neuronal pathfinding and fasciculation). However, later in development, there are significantly fewer longitudinal glia that are spatially disorganized; in addition, there is a slight disorganization of axon fascicles and a defective nerve cord condensation. This suggests that RK2 is not required for early glial determination, but rather for aspects of glial differentiation or function that are required for embryonic viability.