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Stem cell activity and cell differentiation is robustly influenced by the nutrient availability in the gonads. The signal that connects nutrient availability to gonadal stem cell activity remains largely unknown. In this study, we show that tumor necrosis factor Eiger (Egr) is upregulated in testicular smooth muscles as a response to prolonged protein starvation in Drosophila testis. While Egr is not essential for starvation-induced changes in germline and somatic stem cell numbers, Egr and its receptor Grindelwald influence the recovery dynamics of somatic cyst stem cells (CySCs) upon protein refeeding. Moreover, Egr is also involved in the refeeding-induced, ectopic expression of the CySC self-renewal protein and the accumulation of early germ cells. Egr primarily acts through the Jun N-terminal kinase (JNK) signaling in Drosophila. We show that inhibition of JNK signaling in cyst cells suppresses the refeeding-induced abnormality in both somatic and germ cells. In conclusion, our study reveals both beneficial and detrimental effects of Egr upregulation in the recovery of stem cells and spermatogenesis from prolonged protein starvation.

Transposons are known to participate in tissue aging, but their effects on aged stem cells remain unclear. Here, we report that in the Drosophila ovarian germline stem cell (GSC) niche, aging-related reductions in expression of Piwi (a transposon silencer) derepress retrotransposons and cause GSC loss. Suppression of Piwi expression in the young niche mimics the aged niche, causing retrotransposon depression and coincident activation of Toll-mediated signaling, which promotes Glycogen synthase kinase 3 activity to degrade β-catenin. Disruption of β-catenin-E-cadherin-mediated GSC anchorage then results in GSC loss. Knocking down gypsy (a highly active retrotransposon) or toll, or inhibiting reverse transcription in the piwi-deficient niche, suppresses GSK3 activity and β-catenin degradation, restoring GSC-niche attachment. This retrotransposon-mediated impairment of aged stem cell maintenance may have relevance in many tissues, and could represent a viable therapeutic target for aging-related tissue degeneration., Retrotransposons are known to be involved in aging. Here the authors show that the retrotransposon silencer Piwi contributes to germline stem cell loss during aging in Drosophila.

A key aspect of germ cell development is to establish germline sexual identity and initiate a sex-specific developmental program to promote spermatogenesis or oogenesis. Previously, we have identified the histone reader Plant Homeodomain Finger 7 (PHF7) as an important regulator of male germline identity. To understand how PHF7 directs sexual differentiation of the male germline, we investigated the downstream targets of PHF7 by combining transcriptome analyses, which reveal genes regulated by Phf7, with genomic profiling of histone H3K4me2, the chromatin mark that is bound by PHF7. Through these genomic experiments, we identify a novel spermatocyte factor Receptor Accessory Protein Like 1 (REEPL1) that can promote spermatogenesis and whose expression is kept off by PHF7 in the spermatogonial stage. Loss of Reepl1 significantly rescues the spermatogenesis defects in Phf7 mutants, indicating that regulation of Reepl1 is an essential aspect of PHF7 function. Further, increasing REEPL1 expression facilitates spermatogenic differentiation. These results indicate that PHF7 controls spermatogenesis by regulating the expression patterns of important male germline genes.

Stem cells rely on instructive cues from their environment. Alterations in microenvironments might contribute to tissue dysfunction and disease pathogenesis. Germline stem cells (GSCs) and cyst stem cells (CySC) in Drosophila testes are normally maintained in the apical area by the testicular hub. In this study, we found that reproduction leads to accumulation of early differentiating daughters of CySCs and GSCs in the testes of aged male flies, due to hyperactivation of Jun-N-terminal kinase (JNK) signaling to maintain self-renewal gene expression in the differentiating cyst cells. JNK activity is normally required to maintain CySCs in the apical niche. A muscle sheath surrounds the Drosophila testis to maintain its long coiled structure. Importantly, reproduction triggers accumulation of the tumor necrosis factor (TNF) Eiger in the testis muscle to activate JNK signaling via the TNF receptor Grindelwald in the cyst cells. Reducing Eiger activity in the testis muscle sheath suppressed reproduction-induced differentiation defects, but had little effect on testis homeostasis of unmated males. Our results reveal that reproduction in males provokes a dramatic shift in the testicular microenvironment, which impairs tissue homeostasis and spermatogenesis in the testes.

Synapse formation and growth are tightly controlled processes. How synaptic growth is terminated after reaching proper size remains unclear. Here, we show that Leon, the Drosophila USP5 deubiquitinase, controls postsynaptic growth. In leon mutants, postsynaptic specializations of neuromuscular junctions are dramatically expanded, including the subsynaptic reticulum, the postsynaptic density, and the glutamate receptor cluster. Expansion of these postsynaptic features is caused by a disruption of ubiquitin homeostasis with accumulation of free ubiquitin chains and ubiquitinated substrates in the leon mutant. Accumulation of Ubiquilin (Ubqn), the ubiquitin receptor whose human homolog ubiquilin 2 is associated with familial amyotrophic lateral sclerosis, also contributes to defects in postsynaptic growth and ubiquitin homeostasis. Importantly, accumulations of postsynaptic proteins cause different aspects of postsynaptic overgrowth in leon mutants. Thus, the deubiquitinase Leon maintains ubiquitin homeostasis and proper Ubqn levels, preventing postsynaptic proteins from accumulation to confine postsynaptic growth. DOI: http://dx.doi.org/10.7554/eLife.26886.001

Aging influences stem cells, but the processes involved remain unclear. Insulin signaling, which controls cellular nutrient sensing and organismal aging, regulates the G2 phase of Drosophila female germ line stem cell (GSC) division cycle in response to diet; furthermore, this signaling pathway is attenuated with age. The role of insulin signaling in GSCs as organisms age, however, is also unclear. Here, we report that aging results in the accumulation of tumorous GSCs, accompanied by a decline in GSC number and proliferation rate. Intriguingly, GSC loss with age is hastened by either accelerating (through eliminating expression of Myt1, a cell cycle inhibitory regulator) or delaying (through mutation of insulin receptor (dinR) GSC division, implying that disrupted cell cycle progression and insulin signaling contribute to age-dependent GSC loss. As flies age, DNA damage accumulates in GSCs, and the S phase of the GSC cell cycle is prolonged. In addition, GSC tumors (which escape the normal stem cell regulatory microenvironment, known as the niche) still respond to aging in a similar manner to normal GSCs, suggesting that niche signals are not required for GSCs to sense or respond to aging. Finally, we show that GSCs from mated and unmated females behave similarly, indicating that female GSC-male communication does not affect GSCs with age. Our results indicate the differential effects of aging and diet mediated by insulin signaling on the stem cell division cycle, highlight the complexity of the regulation of stem cell aging, and describe a link between ovarian cancer and aging.

A signature feature of all living organisms is their utilization of proteins to construct molecular machineries that undertake the complex network of cellular activities. The abundance of a protein element is temporally and spatially regulated in two opposing aspects: de novo synthesis to manufacture the required amount of the protein, and destruction of the protein when it is in excess or no longer needed. One major route of protein destruction is coordinated by a set of conserved molecules, the F-box proteins, which promote ubiquitination in the ubiquitin-proteasome pathway. Here we discuss the functions of F-box proteins in several cellular scenarios including cell cycle progression, synapse formation, plant hormone responses, and the circadian clock. We particularly emphasize the mechanisms whereby F-box proteins recruit specific substrates and regulate their abundance in the context of SCF E3 ligases. For some exceptions, we also review how F-box proteins function through non-SCF mechanisms.

A key issue in development is how to specify single isolated precursor cells to adopt a distinct fate from a group of naive cells. Studies on the development of Drosophila external sensory (ES) organs have revealed multiple mechanisms to specify single sensory organ precursors (SOPs) from clusters of cells with equivalent neural potential. Initially single SOPs are selected in part through cell-cell competition from clusters of ectodermal cells that express proneural proteins. To reinforce the singularity, lateral inhibition through the Delta/Notch system and feedback regulations lead to exclusive expression of proneural proteins in SOPs. As transcriptional activators, proneural proteins execute a genetic program in SOP cells for the development of an eventually ES organ. In this article, we will summarize recent advances on how transcriptional regulation, protein degradation, endocytosis and gene silencing by microRNA participate in SOP specification.

Proneural basic helix-loop-helix (bHLH) proteins initiate neurogenesis in both vertebrates and invertebrates. The Drosophila Achaete (Ac) and Scute (Sc) proteins are among the first identified members of the large bHLH proneural protein family. phyllopod ( phyl ), encoding an ubiquitin ligase adaptor, is required for ac - and sc -dependent external sensory (ES) organ development. Expression of phyl is directly activated by Ac and Sc. Forced expression of phyl rescues ES organ formation in ac and sc double mutants. phyl and senseless , encoding a Zn-finger transcriptional factor, depend on each other in ES organ development. Our results provide the first example that bHLH proneural proteins promote neurogenesis through regulation of protein degradation.

During Drosophila external sensory organ development, one sensory organ precursor (SOP) arises from a proneural cluster, and undergoes asymmetrical cell divisions to produce an external sensory (es) organ made up of different types of daughter cells. We show that phyllopod (phyl), previously identified to be essential for R7 photoreceptor differentiation, is required in two stages of es organ development: the formation of SOP cells and cell fate specification of SOP progeny. Loss-of-function mutations in phyl result in failure of SOP formation, which leads to missing bristles in adult flies. At a later stage of es organ development, phyl mutations cause the first cell division of the SOP lineage to generate two identical daughters, leading to the fate transformation of neurons and sheath cells to hair cells and socket cells. Conversely, misexpression of phyl promotes ectopic SOP formation, and causes opposite fate transformation in SOP daughter cells. Thus, phyl functions as a genetic switch in specifying the fate of the SOP cells and their progeny. We further show that seven in absentia (sina), another gene required for R7 cell fate differentiation, is also involved in es organ development. Genetic interactions among phyl, sina and tramtrack (ttk) suggest that phyl and sina function in bristle development by antagonizing ttk activity, and ttk acts downstream of phyl. It has been shown previously that Notch (N) mutations induce formation of supernumerary SOP cells, and transformation from hair and socket cells to neurons. We further demonstrate that phyl acts epistatically to N. phyl is expressed specifically in SOP cells and other neural precursors, and its mRNA level is negatively regulated by N signaling. Thus, these analyses demonstrate that phyl acts downstream of N signaling in controlling cell fates in es organ development.

In the yeast Saccharomyces cerevisiae, Ste12p induces transcription of pheromone-responsive genes by binding to a DNA sequence designated the pheromone response element. We generated a series of hybrid proteins of Ste12p with the DNA-binding and activation domains of the transcriptional activator Gal4p to define a pheromone induction domain of Ste12p sufficient to mediate pheromone-induced transcription by these hybrid proteins. A minimal pheromone induction domain, delineated as residues 301 to 335 of Ste12p, is dependent on the pheromone mitogen-activated protein (MAP) kinase pathway for induction activity. Mutation of the three serine and threonine residues within the minimal pheromone induction domain did not affect transcriptional induction, indicating that the activity of this domain is not directly regulated by MAP kinase phosphorylation. By contrast, mutation of the two tyrosines or their preceding acidic residues led to a high level of transcriptional activity in the absence of pheromone and consequently to the loss of pheromone induction. This constitutively high activity was not affected by mutations in the MAP kinase cascade, suggesting that the function of the pheromone induction domain is normally repressed in the absence of pheromone. By two-hybrid analysis, this minimal domain interacts with two negative regulators, Dig1p and Dig2p (also designated Rst1p and Rst2p), and the interaction is abolished by mutation of the tyrosines. The pheromone induction domain itself has weak and inducible transcriptional activity, and its ability to potentiate transcription depends on the activity of an adjacent activation domain. These results suggest that the pheromone induction domain of Ste12p mediates transcriptional induction via a two-step process: the relief of repression and synergistic transcriptional activation with another activation domain.

Germline stem cells (GSCs) produce gametes throughout the reproductive life of many animals, and intensive studies have revealed critical roles of BMP signaling to maintain GSC self-renewal in Drospophila adult gonads. Here, we show that BMP signaling is downregulated as testes develop and this regulation controls testis growth, stem cell number, and the number of spermatogonia divisions. Phosphorylated Mad (pMad), the activated Drosophila Smad in germ cells, was restricted from anterior germ cells to GSCs and hub-proximal cells during early larval development. pMad levels in GSCs were then dramatically downregulated from early third larval instar (L3) to late L3, and maintained at low levels in pupal and adult GSCs. The spatial restriction and temporal down-regulation of pMad, reflecting the germ cell response to BMP signaling activity, required action in germ cells of E3 ligase activity of HECT domain protein Smurf. Analyses of Smurf mutant testes and dosage-dependent genetic interaction between Smurf and mad indicated that pMad downregulation was required for both the normal decrease in stem cell number during testis maturation in the pupal stage, and for normal limit of four rounds of spermatogonia cell division for control of germ cell numbers and testis size. Smurf protein was expressed at a constant low level in GSCs and spermatogonia during development. Rescue experiments showed that expression of exogenous Smurf protein in early germ cells promoted pMad downregulation in GSCs in a stage-dependent but concentration-independent manner, suggesting that the competence of Smurf to attenuate response to BMP signaling may be regulated during development. Taken together, our work reveals a critical role for differential attenuation of the response to BMP signaling in GSCs and early germ cells for control of germ cell number and gonad growth during development.

Basic helix-loop-helix (bHLH) proneural proteins promote neurogenesis through transcriptional regulation. Although much is known about the tissue-specific regulation of proneural gene expression, how proneural proteins interact with transcriptional machinery to activate downstream target genes is less clear. Drosophila proneural proteins Achaete (Ac) and Scute (Sc) induce external sensory organ formation by activating neural precursor gene expression. Through co-immunoprecipitation and mass spectrometric analyses, we found that nuclear but not cytoplasmic actin associated with the Ac and Sc proteins in Drosophila S2 cells. Daughterless (Da), the common heterodimeric partner of Drosophila bHLH proteins, was observed to associate with nuclear actin through proneural proteins. A yeast two-hybrid assay revealed that the binding specificity between actin and Ac or Sc was conserved in yeast nuclei without the presence of additional Drosophila factors. We further show that actin is required in external sensory organ formation. Reduction in actin gene activity impaired proneural-protein-dependent expression of the neural precursor genes, as well as formation of neural precursors. Furthermore, increased nuclear actin levels, obtained by expression of nucleus-localized actin, elevated Ac–Da-dependent gene transcription as well as Ac-mediated external sensory organ formation. Taken together, our in vivo and in vitro observations suggest a novel link for actin in proneural-protein-mediated transcriptional activation and neural precursor differentiation.

Neurogenesis requires precise control of cell specification and division. In Drosophila, the timing of cell division of the sensory organ precursor (SOP) is under strict temporal control. But how the timing of mitotic entry is determined remains poorly understood. Here, we present evidence that the timing of the G2-M transition is determined by when proneural proteins are degraded from SOPs. This process requires the E3 ubiquitin ligase complex, including the RING protein Sina and the adaptor Phyl. In phyl mutants, proneural proteins accumulate, causing delay or arrest in the G2-M transition. The G2-M defect in phyl mutants is rescued by reducing the ac and sc gene doses. Misexpression of phyl downregulates proneural protein levels in a sina-dependent manner. Phyl directly associates with proneural proteins to act as a bridge between proneural proteins and Sina. As phyl is a direct transcriptional target of Ac and Sc, our data suggest that, in addition to mediating cell cycle arrest, proneural protein initiates a negative-feedback regulation to time the mitotic entry of neural precursors.

The eukaryotic protein degradation pathway has a large number of components, including several E3 ubiquitin ligases that are predicted to have regulatory roles. Control of protein stability by the degradation machinery in a cell-context-dependent manner can be elucidated in the well-defined Drosophila compound eye. During development, the Drosophila eye imaginal disk consists of only a few cell types, and consecutive differentiation stages of these cells can be examined within a single eye disk. Here, we summarize recent advances in the understanding of how E3 ubiquitin ligases control cell proliferation, specification, differentiation and death during Drosophila eye development.

During development, neural competence is conferred and maintained by integrating spatial and temporal regulations. The Drosophila sensory bristles that detect mechanical and chemical stimulations are arranged in stereotypical positions. The anterior wing margin (AWM) is arrayed with neuron-innervated sensory bristles, while posterior wing margin (PWM) bristles are non-innervated. We found that the COP9 signalosome (CSN) suppresses the neural competence of non-innervated bristles at the PWM. In CSN mutants, PWM bristles are transformed into neuron-innervated, which is attributed to sustained expression of the neural-determining factor Senseless (Sens). The CSN suppresses Sens through repression of the ecdysone signaling target gene broad (br) that encodes the BR-Z1 transcription factor to activate sens expression. Strikingly, CSN suppression of BR-Z1 is initiated at the prepupa-to-pupa transition, leading to Sens downregulation, and termination of the neural competence of PWM bristles. The role of ecdysone signaling to repress br after the prepupa-to-pupa transition is distinct from its conventional role in activation, and requires CSN deneddylating activity and multiple cullins, the major substrates of deneddylation. Several CSN subunits physically associate with ecdysone receptors to represses br at the transcriptional level. We propose a model in which nuclear hormone receptors cooperate with the deneddylation machinery to temporally shutdown downstream target gene expression, conferring a spatial restriction on neural competence at the PWM., Author Summary A critical step in building a functional nervous system is to generate neurons at the appropriate locations. Neural competence is acquired at the precursor stage with the expression of specific transcription factors. One such critical factor is Senseless (Sens), as precursors lacking Sens fail to develop to neurons. Here we describe the critical role of protein complex COP9 signalosome (CSN) that regulates Sens expression by integrating temporal and spatial information. This was studied in developing Drosophila wing tissues, in which the anterior wing margin develops neuron-innervated bristles, while the posterior wing margin develops non-innervated bristles. The CSN complex is required for the anterior-posterior difference in spatial patterning of neuron formation, and posterior cells lacking CSN develop innervated bristles like anterior cells. CSN accomplishes this by transforming the temporal hormonal ecdysone signaling from activation to repression of downstream target BR-Z1. As BR-Z1 itself is a transcription activator, repression of BR-Z1 in turn leads to repression of Sens in posterior wing margin, eventually terminating the neural competence. Repression of BR-Z1 expression requires the interaction between the CSN complex and the ecdysone receptors. Our results suggest a novel CSN-mediated regulation that converts temporal hormone signaling to the patterning of neurons at the right place.

The Hedgehog (Hh) morphogen directs distinct cell responses according to its distinct signalling levels. Hh signalling stabilizes transcription factor cubitus interruptus (Ci) by prohibiting SCFSlimb-dependent ubiquitylation and proteolysis of Ci. How graded Hh signalling confers differential SCFSlimb-mediated Ci proteolysis in responding cells remains unclear. Here, we show that in COP9 signalosome (CSN) mutants, in which deneddylation of SCFSlimb is inactivated, Ci is destabilized in low-to-intermediate Hh signalling cells. As a consequence, expression of the low-threshold Hh target gene dpp is disrupted, highlighting the critical role of CSN deneddylation on low-to-intermediate Hh signalling response. The status of Ci phosphorylation and the level of E1 ubiquitin-activating enzyme are tightly coupled to this CSN regulation. We propose that the affinity of substrate–E3 interaction, ligase activity and E1 activity are three major determinants for substrate ubiquitylation and thereby substrate degradation in vivo., Hedgehog signalling gradients are required for proper wing formation in Drosophila, and Hedgehog is known to regulate the cubitus interruptus transcription factor. Here, the authors show that the COP9 signalosome has a critical role in translating a Hedgehog gradient into a cubitus interruptus gradient.

Background The conserved Notch signaling pathway regulates cell fate decisions and maintains stem cells in multicellular organisms. Up-regulation of Notch signaling is observed in several types of cancer and is causally involved in proliferation and survival of cancer cells. Thus, it is of great interest to look for anti-Notch reagents for therapeutic purposes. In model animal Drosophila, Notch signaling restricts selection of sensory organ precursors (SOPs) during external sensory (ES) organ development. To look for novel genes that can suppress Notch signaling, we performed a gain-of-function modifier screen to look for genes that enhance the phenotype of ectopic ES organs induced by overexpression of phyllopod, a gene required for SOP specification. Results From the gain-of-function screen, we discovered that overexpression of polished rice/tarsal-less (pri/tal) increases the numbers of ES organs as well as SOPs. pri/tal is a polycistronic gene that contains four short open reading frames encoding three 11-amino acid and one 32-amino acid peptides. Ectopic expression of the 11 amino-acid peptides recapitulates the pri/tal misexpression phenotype in ectopic ES organ formation. In situ hybridization experiment reveals that pri/tal mRNA is expressed in the SOPs of the chemosensory organs and the stretch-sensing chordotonal organs. In Drosophila wing development, the Notch signaling pathway mediates the formation of the dorsal-ventral (DV) compartmental boundary and the restriction of the vein width from the primordial veins, the proveins. We also found that pri/tal mRNA is expressed in the DV boundary and the longitudinal proveins, and overexpression of Pri/Tal peptides disrupts the DV boundary formation and helps to expand the width of the wing vein. Genetic analyses further show that a Notch loss-of-function allele strongly enhances these two phenotypes. Cut and E(spl)mβ are target genes of the Notch pathway in DV boundary formation and vein specification, respectively. We also found that overexpression of Pri/Tal peptides abolishes Cut expression and co-expression of Pri/Tal peptides with phyl strongly reduces E(spl)mβ expression. Conclusions We show for the first time that the overexpression of Pri/Tal 11-amino acid peptides disrupts multiple Notch-mediated processes and reduces Notch target gene expression in Drosophila, suggesting that these peptides have novel antagonistic activity to the Notch pathway. Thus, our discovery might provide insights into designing new therapeutic reagents for Notch-related diseases.

Summary During development, dendrites arborize in a field several hundred folds of their soma size, a process regulated by intrinsic transcription program and cell adhesion molecule (CAM)-mediated interaction. However, underlying cellular machineries that govern distal higher-order dendrite extension remain largely unknown. Here, we show that Nak, a clathrin adaptor-associated kinase, promotes higher-order dendrite growth through endocytosis. In nak mutants, both the number and length of higher-order dendrites are reduced, which are phenocopied by disruptions of clathrin-mediated endocytosis. Nak interacts genetically with components of the endocytic pathway, colocalizes with clathrin puncta, and is required for dendritic localization of clathrin puncta. More importantly, these Nak-containing clathrin structures preferentially localize to branching points and dendritic tips that are undergoing active growth. We present evidence that the Drosophila L1-CAM homolog Neuroglian is a relevant cargo of Nak-dependent internalization, suggesting that localized clathrin-mediated endocytosis of CAMs facilitates the extension of nearby higher-order dendrites. Video Abstract