The Interactive Fly

Genes involved in tissue and organ development

Oogenesis and the Oocyte

(Part 1 | Part2)

Oogenesis - Stem cells and the niche

  • Long-term live imaging provides new insight into stem cell regulation and germline-soma coordination in the Drosophila ovary
  • The Drosophila putative histone acetyltransferase Enok maintains female germline stem cells through regulating Bruno and the niche
  • Defining gene networks controlling the maintenance and function of the differentiation niche by an in vivo systematic RNAi screen
  • gone early, a novel germline factor, ensures the proper size of the stem cell precursor pool in the Drosophila ovary
  • Regulation of ribosome biogenesis and protein synthesis controls germline stem cell differentiation
  • Aging and insulin signaling differentially control normal and tumorous germline stem cells
  • The neuropeptide allatostatin C from clock-associated DN1p neurons generates the circadian rhythm for oogenesis
  • H3K36 trimethylation-mediated epigenetic regulation is activated by Bam and promotes germ cell differentiation during early oogenesis in Drosophila
  • An AMPK phosphoregulated RhoGEF feedback loop tunes cortical flow-driven amoeboid migration in vivo
  • Insulin signaling acts in adult adipocytes via GSK-3beta and independently of FOXO to control Drosophila female germline stem cell numbers
  • Evidence for chromatin-remodeling complex PBAP-controlled maintenance of the Drosophila ovarian germline stem cells
  • Specification and spatial arrangement of cells in the germline stem cell niche of the Drosophila ovary depend on the Maf transcription factor Traffic jam
  • Quantitative microscopy of the Drosophila ovary shows multiple niche signals specify progenitor cell fate
  • Division-independent differentiation mandates proliferative competition among stem cells
  • Spaghetti, a homolog of human RPAP3 (RNA polymerase II-associated protein 3), determines the fate of germline stem cells in Drosophila ovary
  • ALIX and ESCRT-III coordinately control cytokinetic abscission during germline stem cell division in vivo
  • Aubergine controls germline stem cell self-renewal and progeny differentiation via distinct mechanisms
  • Diet regulates membrane extension and survival of niche escort cells for germline homeostasis via insulin signaling
  • Survival of Drosophila germline stem cells requires the chromatin binding protein Barrier-to-autointegration factor
  • Aging shifts mitochondrial dynamics toward fission to promote germline stem cell loss
  • Neuronal octopamine signaling regulates mating-induced germline stem cell increase in female Drosophila melanogaster
  • Hormone receptor 4 is required in muscles and distinct ovarian cell types to regulate specific steps of Drosophila oogenesis
  • Drosophila CG2469 encodes a homolog of human CTR9 and is essential for development
  • Drosophila female germline stem cells undergo mitosis without nuclear breakdown
  • Novel intrinsic factor Yun maintains female germline stem cell fate through Thickveins
  • DIP1 modulates stem cell homeostasis in Drosophila through regulation of sisR-1
  • Cyclin B3 deficiency impairs germline stem cell maintenance and its overexpression delays cystoblast differentiation in Drosophila ovary
  • Quantitative microscopy of the Drosophila ovary shows multiple niche signals specify progenitor cell fate
  • A targeted RNAi screen reveals Drosophila female-sterile genes that control the size of germline stem cell niche during development
  • Lmx1a is required for the development of the ovarian stem cell niche in Drosophila
  • Adipocyte amino acid sensing controls adult germline stem cell number via the amino acid response pathway and independently of Target of Rapamycin signaling in Drosophila
  • Target of Rapamycin Drives Unequal Responses to Essential Amino Acid Depletion for Egg Laying in Drosophila melanogaster
  • Ecdysone response gene E78 controls ovarian germline stem cell niche formation and follicle survival in Drosophila
  • WD40 protein Wuho controls germline homeostasis via TRIM-NHL tumor suppressor Mei-p26 in Drosophila
  • Drosophila anion exchanger 2 is required for proper ovary development and oogenesis
  • Ballchen is required for self-renewal of germline stem cells in Drosophila melanogaster
  • Piwi reduction in the aged niche eliminates germline stem cells via Toll-GSK3 signaling
  • CTP synthase polymerization in germline cells of the developing Drosophila egg supports egg production
  • Differentiating Drosophila female germ cells initiate Polycomb silencing by regulating PRC2-interacting proteins
  • The Bric-a-Brac BTB/POZ transcription factors are necessary in niche cells for germline stem cells establishment and homeostasis through control of BMP/DPP signaling in the Drosophila melanogaster ovary
  • Mapping parameter spaces of biological switches
  • The Osa-Containing SWI/SNF Chromatin-Remodeling Complex Is Required in the Germline Differentiation Niche for Germline Stem Cell Progeny Differentiation
  • Mad dephosphorylation at the nuclear pore is essential for asymmetric stem cell division
  • Multiple niche compartments orchestrate stepwise germline stem cell progeny differentiation
  • Live imaging of the Drosophila ovarian niche shows spectrosome and centrosome dynamics during asymmetric germline stem cell division
  • Heparan sulfate proteoglycan molecules, syndecan and perlecan, have distinct roles in the maintenance of Drosophila germline stem cells
  • Chemical Genetic Screen in Drosophila Germline Uncovers Small Molecule Drugs That Sensitize Stem Cells to Insult-Induced Apoptosis
  • brinker levels regulated by a promoter proximal element support germ cell homeostasis
  • Checkpoint activation drives global gene expression changes in Drosophila nuclear lamina mutants
  • Frazzled/Dcc acts independently of Netrin to promote germline survival during Drosophila oogenesis
  • Regulation of Mating-Induced Increase in Female Germline Stem Cells in the Fruit Fly Drosophila melanogaster
  • Clonal dominance in excitable cell networks
  • Warm and cold temperatures have distinct germline stem cell lineage effects during Drosophila oogenesis
  • A dual role of lola in Drosophila ovary development: regulating stem cell niche establishment and repressing apoptosis
  • Canonical Wnt Signaling Promotes Formation of Somatic Permeability Barrier for Proper Germ Cell Differentiation

  • Process of oogenesis

  • The process of oogenesis
  • A combinatorial code for pattern formation in Drosophila oogenesis
  • Mei-p26 cooperates with Bam, Bgcn and Sxl to promote early germline development in the Drosophila ovary
  • mRNA localization and translational control during oogenesis
  • Independent and coordinate trafficking of single Drosophila germ plasm mRNAs
  • Novel roles for RNA binding proteins squid, hephaesteus, and Hrb27C in Drosophila oogenesis
  • Drosophila protein kinase N (Pkn) is a negative regulator of actin-myosin activity during oogenesis
  • Evidence for the mechanosensor function of filamin in tissue development
  • Identification of germline transcriptional regulatory elements in Aedes aegypti
  • Increased levels of superoxide dismutase suppress meiotic segregation errors in aging oocytes
  • Prolonged ovarian storage of mature Drosophila oocytes dramatically increases meiotic spindle instability
  • Protein competition switches the function of COP9 from self-renewal to differentiation
  • Pleiotropic functions of the chromodomain-containing protein Hat-trick during oogenesis in Drosophila melanogaster
  • The Drosophila LIN54 homolog Mip120 controls two aspects of oogenesis
  • The NuRD nucleosome remodelling complex and NHK-1 kinase are required for chromosome condensation in oocytes
  • Impact of Germline Depletion of Bonus on Chromatin State in Drosophila Ovaries
  • A genetic mosaic screen reveals ecdysone-responsive genes regulating Drosophila oogenesis
  • Novel cis-regulatory regions in ecdysone responsive genes are sufficient to promote gene expression in Drosophila ovarian cells
  • RNA methyltransferase BCDIN3D is crucial for female fertility and miRNA and mRNA profiles in Drosophila ovaries
  • Splice variants of the SWR1-type nucleosome remodeling factor Domino have distinct functions during Drosophila melanogaster oogenesis
  • Proximity labeling reveals novel interactomes in live Drosophila tissue
  • Makorin 1 is required for Drosophila oogenesis by regulating insulin/Tor signaling
  • A role for tuned levels of nucleosome remodeler subunit ACF1 during Drosophila oogenesis
  • Prostaglandins temporally regulate cytoplasmic actin bundle formation during Drosophila oogenesis.
  • Transfer of Dorsoventral and terminal information from the ovary to the embryo by a common group of eggshell proteins in Drosophila
  • Growing microtubules push the oocyte nucleus to polarize the Drosophila dorsal-ventral axis
  • A single-cell atlas and lineage analysis of the adult Drosophila ovary
  • A single-cell atlas of adult Drosophila ovary identifies transcriptional programs and somatic cell lineage regulating oogenesis
  • A single-cell atlas reveals unanticipated cell type complexity in Drosophila ovaries
  • A visual screen for diet-regulated proteins in the Drosophila ovary using GFP protein trap lines
  • The genetic architecture of ovariole number in Drosophila melanogaster: Genes with major, quantitative, and pleiotropic effects
  • Long Oskar controls mitochondrial inheritance in Drosophila melanogaster
  • Liquid-to-solid phase transition of oskar ribonucleoprotein granules is essential for their function in Drosophila embryonic development
  • Receptor-mediated yolk uptake is required for oskar mRNA localization and cortical anchorage of germ plasm components in the Drosophila oocyte
  • Localization of oskar mRNA by agglomeration in ribonucleoprotein granules
  • Knock down analysis reveals critical phases for specific oskar noncoding RNA functions during Drosophila oogenesis
  • Cup is essential for oskar mRNA translational repression during early Drosophila oogenesis
  • Stonewall and Brickwall: Two partially redundant determinants required for the maintenance of female germline in Drosophila
  • GFP-Forked, a genetic reporter for studying Drosophila oocyte polarity
  • Transposon silencing in the Drosophila female germline is essential for genome stability in progeny embryos>
  • Maternal Nanos inhibits Importin-alpha2/Pendulin-dependent nuclear import to prevent somatic gene expression in the Drosophila germline
  • A ribosomal protein S5 isoform is essential for oogenesis and interacts with distinct RNAs in Drosophila melanogaster
  • Nuclear pores assemble from Nucleoporin condensates during oogenesis
  • Optical flow analysis reveals that Kinesin-mediated advection impacts on the orientation of microtubules in the Drosophila oocyte
  • The Drosophila spectraplakin Short stop regulates focal adhesion dynamics by cross-linking microtubules and actin
  • Comparative Proteomics Reveal Me31B's Interactome Dynamics, Expression Regulation, and Assembly Mechanism into Germ Granules during Drosophila Germline Development
  • An interaction network of RNA-binding proteins involved in Drosophila oogenesis
  • LOTUS domain protein MARF1 binds CCR4-NOT deadenylase complex to post-transcriptionally regulate gene expression in oocytes
  • Drosophila MARF1 ensures proper oocyte maturation by regulating nanos expression
  • Cellular metabolic reprogramming controls sugar appetite in Drosophila
  • Fitness trade-offs incurred by ovary-to-gut steroid signalling in Drosophila
  • Systemic Regulation of Host Energy and Oogenesis by Microbiome-Derived Mitochondrial Coenzymes
  • Mitochondrial Dynamics in the Drosophila Ovary Regulates Germ Stem Cell Number, Cell Fate, and Female Fertility
  • Drosophila oocyte proteome composition covaries with female mating status
  • An RNA-interference screen in Drosophila to identify ZAD-containing C2H2 zinc finger genes that function in female germ cells
  • RNA degradation is required for the germ-cell to maternal transition in Drosophila
  • dHNF4 regulates lipid homeostasis and oogenesis in Drosophila melanogaster
  • Drosophila female reproductive tract gene expression reveals coordinated mating responses and rapidly evolving tissue-specific genes
  • Precise levels of the Drosophila adaptor protein Dreadlocks maintain the size and stability of germline ring canals
  • A progressive somatic cell niche regulates germline cyst differentiation in the Drosophila ovary
  • Quantitative models for building and growing fated small cell networks
  • Sequestration to lipid droplets promotes histone availability by preventing turnover of excess histones
  • Dynein light chain-dependent dimerization of Egalitarian is essential for maintaining oocyte fate in Drosophila
  • Histone acetyltransferase Enok regulates oocyte polarization by promoting expression of the actin nucleation factor spire
  • Hecw controls oogenesis and neuronal homeostasis by promoting the liquid state of ribonucleoprotein particles
  • The SAGA core module is critical during Drosophila oogenesis and is broadly recruited to promoters
  • The Drosophila ribosome protein S5 paralog RpS5b promotes germ cell and follicle cell differentiation during oogenesis
  • Orcokinin neuropeptides regulate reproduction in the fruit fly, Drosophila melanogaster
  • The R-SNARE Ykt6 is required for multiple events during oogenesis in Drosophila Cells
  • The IRM cell adhesion molecules Hibris, Kin of irre and Roughest control egg morphology by modulating ovarian muscle contraction in Drosophila
  • Redundant functions of the SLC5A transporters Rumpel, Bumpel, and Kumpel in ensheathing glial cells
  • Distinct gene expression dynamics in germ line and somatic tissue during ovariole morphogenesis in Drosophila melanogaster
  • The Drosophila anterior-posterior axis is polarized by asymmetric myosin activation
  • Maintenance of quiescent oocytes by noradrenergic signals
  • Biosynthesis of S-adenosyl-methionine enhances aging-related defects in Drosophila oogenesis
  • Essential functions of mosquito ecdysone importers in development and reproduction
  • The phospho-docking protein 14-3-3 regulates microtubule-associated proteins in oocytes including the chromosomal passenger Borealin
  • Cross-species incompatibility between a DNA satellite and the Drosophila Spartan homolog poisons germline genome integrity
  • A translation control module coordinates germline stem cell differentiation with ribosome biogenesis during Drosophila oogenesis
  • Tropomyosin 1-I/C coordinates kinesin-1 and dynein motors during oskar mRNA transport

  • Meiosis
    see Oogenesis Part2

    Nurse Cells
    see Oogenesis Part2

    Follicle cells
    see Oogenesis Part2

    see Oogenesis Part2

    Egg activation
    see Oogensis Part2

    Genes involved in oogenesis

    The process of oogenesis

    Development in the fly begins well before the moment of conception. Hundreds of genes participate in oogenesis, the building of the egg. The egg is a highly structured chamber. Its RNA, DNA and proteins do not roam freely, but rather are directed and controlled by the egg's dorsoventral polarity and anterior-posterior axis. Neither is the egg cell an independently created entity. It receives input from two other cell sources: nurse cells and follicle cells.

    Follicle cells envelop the maturing, pre-fertilized egg. Information is exchanged between oocyte and follicle cells, conditioning and preparing all parties for the rapid changes that will ensue after fertilization (See Gurken). Nurse cells provide nutrients the embryo will require, storing them in the yolk, as though filling pantry shelves in anticipation of future need.

    Given this simplified picture of oocyte development in a pre-fertilized state, one may ask not only what happens next, but working backward, where does the egg comes from in the first place? The fly needs to build not only eggs, but an organ system "factory" for their production and delivery. Soon after fertilization, pole cells bud from the posterior end of the blastula [Images], the earliest cellular phase of embryonic development. Budding from this terminal or polar region gives them their name. They are the germ line for the next generation, and also referred to as such. These germ line stem cells, that will eventually locate to the adult ovary, are the precursors of the eggs (or in males, the sperm) that will produce the next generation.

    Pole cells are carried dorsally, enter the posterior midgut rudiment, move between the endodermal cells and split into two groups, one for each ovary-to-be. The pole/germ line stem cells are then enveloped by somatic mesodermal cells. In effect, the ovary structure is built around these germ line stem cells. Oviducts, accessory glands, uterus, vagina and external genitalia are all of ectodermal and mesodermal (non germ cell) lineage, derived from the genital imaginal disc. The separation of the pole cells from the rest of the developing embryo is one of the first events in morphogenesis. They will be held in reserve, locate to the adult gonads that will be assembled during the third instar larva stage. The maturation of the oocyte with which this discussion began takes place inside a germarium surrounded by follicle cells.

    The germarium is an assembly line for new egg chambers. Each ovary contains more than a dozen germaria. At the start of the assembly line in any single germarium are two germ line stem cells. At the end of the line, new single stage-1 egg chambers roll off, like kits for building model flies; packed, boxed, and ready to go. One can imagine the various work stations along the way: the stem cells alternate in producing one cystoblast at a time, (the precursor cell of the egg); next, the cystoblast passes through three successive stages of cysts in formation; further along, 6 cysts, each composed of 16 cells, mature sequentially, each yielding a single stage-1 egg chamber that will exit the factory seven days from the time its originating cystoblast was formed.

    A more detailed look at stem cell division reveals the two resulting cells are asymmetric in both function and size. What had been a single stem cell is now two new cells: a cystoblast and a daughter stem cell (from which a new daughter stem cell and a new cystoblast will soon arise). The cystoblast continues along the line, undergoing four cycles of cell division to form a 16 cell cyst. During this process, a single cystocyte develops a microtubule organizing center (MTOC) and forms a polarized microtubule network that extends into all 16 cells. Ring canals maintain the connection among cells in the dividing cyst. The single cell of cyst that contains the MTOC develops into the oocyte. Only the oocyte and will continue through meiosis, while the remainder will differentiate to become nurse cells for the presumptive oocyte. The nurse cell genome becomes polytene, a process in which DNA is replicated without cell division. Nurse cells grow and accumulate yolk material in a process termed vitellogenesis (oogenesis stages 8 and 9).

    To understand oocyte determination, one needs to ask how a particular cystocyte is singled out to acquire an active microtubule organizing center. Germ cells contain a spherical cytoplasmic structure called the spectrosome, which contains components of the sub-membrane cytoskeleton. At the first cystoblast division, the spectrosome is inherited by only one of the two daughter cells. During the following divisions, the spectrosome grows from this cystocyte into the other cells to form a branched structure called the fusome, which interacts with one spindle pole at each division, before disappearing after the 4th division. These observations have led to the suggestion that the asymmetric inheritance of the spectrosome determines which of the two daughter cells will give rise to the cell that becomes the oocyte. How this happens is unclear, but one possibility is that the position of the spectrosome polarizes the fusome, which in turn directs the formation of an active MTOC in a single cell.

    During stage 9, follicle cells, not of germ cell origin, migrate over the surface of the egg chamber, the majority drawn to the oocyte, leaving only a few follicle cells over the nurse cells. A small group of border cells, similarly non-germ cell in origin, that have lagged behind at the anterior end of the developing cystoblast-becoming-oocyte, pass through three nurse cell junctions to arrive at the anterior end of the oocyte. The border cells are implicated in the terminal system, which creates and maintains anterior-posterior polarity in the egg. Follicle cells at the posterior end of the oocyte develop a posterior cell fate. This induction requires gurken function in the oocyte and Epidermal growth factor receptor (Torpedo) in the follicle cell layer. The posterior follicle cells send an unidentified signal back to the oocyte to repolarize the the anterior-posterior axis. Protein kinase A is involved in signalling that results in a disassembly and reassembly of the MTOC.

    By stage 10, the follicle cells have matured and begun secretion of a vitelline membrane around the oocyte. The mature nurse cells cytoplasmic contents flow in bulk into the oocyte by means of a cytoskeletal-based mechanism, and having completed their task, the nurse cells are then broken down (stage 11). The follicle cells that earlier produced the vitelline membrane, now add a shell over that membrane. Membrane and shell are both external to the egg, and covered by follicle cells. Specialized follicle cells also make the micropyle, the egg's terminal structure through which sperm pass into the egg.

    Mature eggs ovulate one at a time and pass into the uterus. Once mated, females are able to release stored sperm from their seminal receptacles. Sperm enter the micropyle while the egg is still in the uterus. The first meiotic division has begun and is still in progress when fertilization and ovulation occur. Males contribute more than just DNA to the zygote. The zygotic centrosomes, essential for cell division, are derived from the male.

    For information about the establishment of oocyte axes, messenger RNA localization in the oocyte, and the basis of polarity in the developing embryo, see posterior group genes, anterior group genes, dorsal group genes and terminal genes

    Growing microtubules push the oocyte nucleus to polarize the Drosophila dorsal-ventral axis

    The Drosophila dorsal-ventral (DV) axis is polarized when the oocyte nucleus migrates from the posterior to the anterior margin of the oocyte. Prior work suggested that dynein pulls the nucleus to the anterior side along a polarized microtubule cytoskeleton, but this mechanism has not been tested. By imaging live oocytes, this study found that the nucleus migrates with a posterior indentation that correlates with its direction of movement. Furthermore, both nuclear movement and the indentation depend on microtubule polymerization from centrosomes behind the nucleus. Thus, the nucleus is not pulled to the anterior but is pushed by the force exerted by growing microtubules. Nuclear migration and DV axis formation therefore depend on centrosome positioning early in oogenesis and are independent of anterior-posterior axis formation (Zhao, 2012).

    The correct positioning of the nucleus is important for several developmental processes, such as cell migration, formation of the neuromuscular junction, and asymmetric cell divisions, whereas nuclear mislocalization is a feature of neurological disorders, such as lissencephaly. Positioning of the nucleus plays an essential role in Drosophila axis formation, as the movement of the nucleus from the posterior of the oocyte to a point at its anterior circumference breaks radial symmetry to polarize the DV axis. At stage 7 of oogenesis, an unknown signal from the posterior follicle cells induces a major reorganization of the oocyte microtubule cytoskeleton. The posterior microtubule organizing center (MTOC) is disassembled, and microtubules are nucleated from the anterior-lateral cortex, resulting in an anterior-posterior (AP) gradient of microtubules that defines the AP axis. It is believed that dynein subsequently uses this polarized microtubule cytoskeleton to pull the nucleus to the oocyte anterior, making polarization of the DV axis dependent on the prior polarization of the AP axis (Zhao, 2012).

    To investigate the mechanism of nuclear positioning directly, the movement of the nucleus in living oocytes was imaged. The nucleus migrates at a speed of 4.0 ± 0.7 μm/hour, and takes 2-3 hours to move across the oocyte. The trajectory of the nucleus is variable: sometimes it moves around the cortex of the oocyte directly to an anterior corner, but it often migrates up the center of the oocyte and then turns to move along the anterior cortex, confirming the random nature of this symmetry-breaking event. This study observed that all migrating nuclei have large posterior indentations, suggesting that they are being pushed rather than pulled toward the anterior. This could reflect an intrinsic reorganization of the nuclear architecture, or a deformation induced by an external force to the nucleus. In support of the latter view, the direction of the indentation correlates with the direction of migration, suggesting that the same force creates the indentation and moves the nucleus. This indentation is not an artefact of long-term imaging in oil, as egg chambers dissected directly into strong fixative have identical indentations (Zhao, 2012).

    This idea that the nucleus is pulled to the anterior by dynein is based on the finding that mutations in the dynein accessory factors, Lis1 and Bic-D, as well as disruption of the dynactin complex result in mislocalized nuclei at stage 10. This is not compatible with the pushing model of nuclear movement, as motor proteins can only pull their cargoes. Therefore this study re-examined the role of the dynein complex by imaging the nucleus in Lis1 mutant egg chambers. Lis1 mutant oocytes are much smaller than normal because dynein is required for transport from the nurse cells into the oocyte. Nevertheless, the oocyte nucleus migrates normally with a prominent posterior indentation. Thus, dynein is presumably required for the anchoring of the nucleus once it has reached the anterior, rather than for its migration. Consistent with this, the nuclei are only rarely mispositioned in Lis1 and Bic-D mutant oocytes at stage 9, but are mislocalized much more frequently at later stages (Zhao, 2012).

    Both actin and microtubule polymerization can generate pushing forces that lead to cellular or organelle deformations. Two lines of evidence suggest that microtubules are responsible for the nuclear indentation: First, depolymerization of actin with latrunculin A or B does not affect nuclear positioning, whereas the microtubule-depolymerizing drug, colcemid, induces mislocalized nuclei. Secondly, several microtubule-associated proteins become enriched on the posterior nuclear envelope during migration, including the Dynein light intermediate chain (Dlic), Calmodulin (Cam), and the Drosophila NuMA homolog Mushroom body defect, Mud (Zhao, 2012).

    To test the role of microtubules in the formation of the indentation, colcemid was added to living egg chambers expressing the +TIP protein, EB1-GFP (end binding-1), which forms a 'comet' on the growing plus ends of microtubules. Colcemid takes 3.5 min to diffuse into the oocyte, as monitored by a decrease in the number of EB1 comets on growing microtubule plus ends. As soon as microtubule growth starts to decrease, the indentation diminishes in size. A focus of EB1-GFP persists posterior to the nucleus for several minutes, and as this disappears, the nucleus relaxes completely and becomes spherical. Thus, the nuclear indentation depends on microtubule polymerization and its size is proportional to the number of growing microtubules (Zhao, 2012).

    Using EB1-GFP to track the growing microtubule plus ends in time-lapse movies of nuclear migration revealed several strong foci of EB1-GFP behind the indentation, with growing microtubules emanating from them in all directions. This indicates that microtubules are nucleated from MTOCs behind the nucleus. These MTOCs resemble the centrosomes, which migrate from the nurse cells into the oocyte during early oogenesis in a dynein-dependent manner, and localize to the posterior cortex as a result of the initial oocyte polarity. Indeed, the centriolar markers Sas4 and PACT, as well as a marker for pericentriolar material (PCM), Centrosomin (Cnn), localize to the foci behind the nuclear indentation at the onset of migration. The centrosomes behave rather dynamically during migration and change reversibly from a dense cluster to a more dispersed distribution. Upon completion of nuclear migration, the centrosomes are recruited to the anterior-dorsal cortex of the oocyte, presumably as a consequence of the activation of the dynein-dependent anchoring mechanism that retains the nucleus in this position. Active centrosomes are therefore positioned behind the nucleus before and during migration (Zhao, 2012).

    To test the role of the centrosomes in creating the nuclear indentation, they were inactivated by laser ablation. Upon ablation of the entire cluster of centrosomes, the indentation disappears and the nucleus becomes spherical within 1 min. This nuclear relaxation may occur more rapidly, as centrosome ablation causes local bleaching of the nuclear envelope, making it impossible to monitor nuclear shape during the first minute. Local laser ablation of the nuclear envelope at the site of the indentation has no effect, however, excluding the possibility that the disappearance of the indentation is a consequence of bleaching of the nuclear membrane. Furthermore, ablation of the anterior of the nucleus does not affect the indentation, arguing against any pulling force from the anterior. As described above, centrosomes are sometimes scattered behind the nucleus, causing multiple indentations. Ablating one cluster of centrosomes abolishes only the indentation facing them. The non-ablated centrosomes remain active and induce an indentation on the adjacent side of the nucleus. Thus, the nucleus is not a rigid structure and the growing microtubules from the centrosomes exert force on the nuclear envelope to induce its deformation (Zhao, 2012).

    The centrosomes are dispensable for oogenesis. Therefore nuclear migration was examined in DSas-4 mutant ovaries that lack centrosomes. Consistent with the previous study, all nuclei migrate to the anterior-dorsal corner and show a posterior indentation during migration. GFP-Cnn is still localized in foci behind the nucleus and EB1-GFP tracks reveal active posterior MTOCs. Thus, acentrosomal MTOCs form in the absence of centrosomes and can provide the pushing force for nuclear migration (Zhao, 2012).

    As a further test of the idea that the centrosomal microtubules push the nucleus to the anterior, par-1 hypomorphs, in which some centrosomes fail to migrate to the posterior of the oocyte, were examined. These anterior centrosomes induce anterior nuclear indentations, leading to dumbbell-shaped nuclei, confirming the role of centrosomal microtubules in pushing the nucleus. These ectopic centrosomes eventually fuse with the posterior centrosomes to move the nucleus to the anterior-dorsal corner. This explains why the nucleus migrates normally in par-1 mutants, even though the anterior-posterior axis is not polarized. Consistent with this, the nucleus in wild-type oocytes can migrate to the anterior before the anterior-to-posterior microtubule gradient is established (Zhao, 2012).

    Another documented example of nuclear positioning by microtubule pushing comes from S. pombe, where microtubule bundles push against the cell ends to maintain the nucleus in the cell center. The oocyte nucleus moves a much greater distance, however, and appears to be pushed by the force exerted by single growing microtubules. To test the feasibility of this mechanism, Stoke's law (F = 6πηrv) to estimate the drag force (F) exerted on the nucleus. Assuming a cytoplasmic viscosity (η) &asymp 100 Pas and the measured values of the nuclear radius (r) ∼ 5 &muν;m and the velocity of migration (v) ∼ 4 μm/hour yields a drag force ∼ 10 pN. It is expected that the actual drag force is lower, because nuclear migration is so slow (1 nm/s) that the cytoplasmic actin mesh will turn over ahead of the nucleus, decreasing the effective viscosity. The longest microtubules can reach ∼ 10 μm between the posterior of the nucleus and the posterior oocyte cortex, resulting in a critical buckling force Fc ≈ 5 pN. This value is probably an underestimate, as microtubules embedded within an elastic cytoplasm in vivo have been reported to bear compressive loads a hundred times higher than in vitro. Each microtubule can therefore generate a pushing force of at least 5 pN. Thus, the force of only two microtubules pushing at any time should be sufficient to move the nucleus to the oocyte anterior (Zhao, 2012).

    The number of microtubules pushing the nucleus was measured using EB1-GFP. 15.3 ± 1.6 microtubules hit the nuclear indentation per minute in one z-plane (n = 10, 2 oocytes), and they continue growing and presumably exerting force on the nucleus for 2.77 ±. 0.14 s. Given the thickness of a confocal section (0.8 μm) and the radius of the indentation [4.3 ± 0.2 μm (n = 10)], an average of 5.9 ± 0.7 microtubules are pushing the nucleus at any given time. Microtubule polymerization can therefore provide sufficient pushing force to drive nuclear migration (Zhao, 2012).

    The migration of the nucleus is triggered by an unknown signal from the posterior follicle cells, which could act either by activating the centrosomes or by releasing the nucleus from a posterior tether. To address this question, when the indentation appears during oogenesis was examined. Active centrosomes are already localized behind the nucleus at stage 5 of oogenesis and induce a posterior indentation. This suggests that the centrosomes continually exert a pushing force on the nucleus, which is tethered to the posterior until it receives a signal for migration. The nucleus remains at the posterior in gurken (grk) mutants, which block follicle cell signaling to the oocyte (39% penetrance). These posterior nuclei still maintain a posterior indentation later in oogenesis, suggesting that they fail to migrate because they are not released from the posterior tether. Indeed, microtubules growing from active centrosomes probably always exert a pushing force on the nucleus that must be countered by an opposing pulling force or anchor to keep the nucleus in place. For example, a clear nuclear indentation is still visible adjacent to the centrosomes after the nucleus is anchored at the anterior (Zhao, 2012).

    The results lead to a revised model for how the oocyte nucleus moves to break radial symmetry and polarize the Drosophila dorsal-ventral axis. This model explains the failure to recover mutants that specifically disrupt nuclear migration, since the driving force is provided solely by microtubule polymerization. Furthermore, the results imply that migration is triggered by the release of the nucleus from a posterior anchor, rather than by microtubule reorganization. Thus, polarization of the dorsal-ventral axis is independent of the formation of the microtubule array that defines the anterior-posterior axis, as previously proposed (Zhao, 2012).

    A single-cell atlas and lineage analysis of the adult Drosophila ovary

    The Drosophila ovary is a widely used model for germ cell and somatic tissue biology. This study used single-cell RNA-sequencing (scRNA-seq) to build a comprehensive cell atlas of the adult Drosophila ovary that contains transcriptional profiles for every major cell type in the ovary, including the germline stem cells and their niche cells, follicle stem cells, and previously undescribed subpopulations of escort cells. In addition, Gal4 lines were identified with specific expression patterns, and lineage tracing of subpopulations of escort cells and follicle cells was performed. A distinct subpopulation of escort cells was capable of converting to follicle stem cells in response to starvation or upon genetic manipulation, including knockdown of escargot, or overactivation of mTor or Toll signalling (Rust, 2020).

    In summary, this study has generated a detailed atlas of the cells in the adult Drosophila ovary. This atlas consists of 26 clusters that each correspond to a distinct population in the ovary. Through experimental validation and referencing well-characterized markers in the literature, the identity of each cluster was identified, and all of the major cell types in the ovariole were found to be represented. Several transcriptionally distinct subpopulations were identified within these major cell types, such as the anterior, central, and posterior Escort cell (EC) populations. Both the GSCs and the FSCs were identified in the dataset, which revealed several genes that are predicted to be specific for each of these stem cell populations. In addition, several Gal4 drivers, including Pdk1-Gal4, fax-Gal4, and stl-Gal4, were identified with unique expression patterns that make it possible to target transgene expression to the subsets of cells marked by these drivers. Lastly, although this study primarily focused on the most uniquely expressed genes for each cluster in this study, the transcriptional profile of each cluster is a rich dataset that can be mined to identify populations of cells that are relevant for a topic of interest. For example, gene expression profile of each cluster was compared to a list of human disease genes that are well-suited for analysis in Drosophila. It was found that germ cells are enriched for cells expressing major drivers of cancer, and ECs and follicle cells are enriched for genes involved in cardiac dysfunction, suggesting that these cell types may be good starting points for studies into the genetic interactions that underlie these human diseases (Rust, 2020).

    This study also demonstrates the utility of using CellFindR9 in combination with monocle320 to identify unique populations of cells within a dataset. Because CellFindR produces clusters in a structured, iterative fashion, it was possible to construct a hierarchical tree that corresponds to a transcriptome relationship between clusters, and this outperformed other clustering methods. The tree built by CellFindR aligns well with expectations and provides some interesting new insights. For example, it was expected that germ cells would cluster apart from somatic cells in Tier 1 because these populations are substantially different from each other, arising at different times during development and from completely different lineages. However, it was surprising that the FSC, pFCs, polar cells, and stalk cells clustered more closely to escort cells than to the follicle cells of budded follicles. This suggests that many cell types in the germarium, which are often studied separately, have biologically relevant similarities (Rust, 2020).

    The use of G-TRACE to assess the lineage potential of somatic cells in the germarium led to the surprising finding that ECs can convert to FSCs under starvation conditions. Recent studies have described similar forms of cellular plasticity in other tissues, suggesting that the ability of non-stem cells to convert to stem cells may be a more general feature of adult stem cell niches. However, this aspect of tissue homeostasis remains poorly understood. The finding that the conversion of ECs to FSCs can be induced by perturbations of mTor or Toll signaling is consistent with a role for these pathways in responding to starvation and cellular stress in other tissues, and provides a new opportunity to investigate the mechanisms of cellular responses to physiological stress in an adult stem cell niche (Rust, 2020).

    Overall, this study provides a resource that will be valuable for a wide range of studies that use the Drosophila ovary as an experimental model. Additional scRNA-seq datasets provided by other studies will further increase the accuracy and resolution of the ovary cell atlas, and it will be important to follow up on the predictions of the atlas with detailed studies that focus on specific populations of cells. Collectively, these efforts will help drive discovery forward by providing a deeper understanding of the cellular composition of the Drosophila ovary (Rust, 2020).

    A combinatorial code for pattern formation in Drosophila oogenesis

    Two-dimensional patterning of the follicular epithelium in Drosophila oogenesis is required for the formation of three-dimensional eggshell structures. Analysis of a large number of published gene expression patterns in the follicle cells suggests that they follow a simple combinatorial code based on six spatial building blocks and the operations of union, difference, intersection, and addition. The building blocks are related to the distribution of inductive signals, provided by the highly conserved epidermal growth factor receptor and bone morphogenetic protein signaling pathways. The validity of the code is demonstrated by testing it against a set of patterns obtained in a large-scale transcriptional profiling experiment. Using the proposed code, 36 distinct patterns were distinguished for 81 genes expressed in the follicular epithelium, and their joint dynamics were characterize over four stages of oogenesis. The proposed combinatorial framework allows systematic analysis of the diversity and dynamics of two-dimensional transcriptional patterns and guides future studies of gene regulation (Yakoby, 2008b).

    Drosophila eggshell is a highly patterned three-dimensional structure that is derived from the follicular epithelium in the developing egg chamber. The dorsal-anterior structures of the eggshell, including the dorsal appendages and operculum, are formed by the region of the follicular epithelium, which is patterned by the highly conserved epidermal growth factor receptor (EGFR) and bone morphogenetic protein (BMP) signaling pathways. The EGFR pathway is activated by Gurken (GRK), a transforming growth factor α-like ligand secreted by the oocyte. The BMP pathway is activated by Decapentaplegic (DPP), a BMP2/4-type ligand secreted by the follicle cells stretched over the nurse cells (Yakoby, 2008b).

    Acting through their uniformly expressed receptors, these ligands establish the dorsoventral and anteroposterior gradients of EGFR and DPP signaling and control the expression of multiple genes in the follicular epithelium. Under their action, the expression of a Zn finger transcription factor, Broad (BR), evolves into a pattern with two patches on either side of the dorsal midline. The BR-expressing cells form the roof (upper part) of the dorsal appendages. Adjacent to the BR-expressing cells are two stripes of cells that express rhomboid (rho), a gene that is directly repressed by BR and encodes ligand-processing protease in the EGFR pathway. These cells form the floor (lower part) of the appendages (Yakoby, 2008b).

    The patterns of genes expressed during the stages of egg development that correspond to appendage morphogenesis are very diverse. At the same time, inspection of a large number of published patterns suggests that they can be 'constructed' from a small number of building blocks. For instance, the T-shaped pattern of CG3074 is similar to the domain 'missing' in the early pattern of br, while the two patches in the late pattern of br appear to correspond to the two 'holes' in the expression of 18w. Based on a number of similar observations, it was hypothesized that all of the published patterns could be constructed from just six basic shapes, or primitives, which reflect the anatomy of the egg chamber and the spatial structure of the patterning signals (Yakoby, 2008b).

    In computer graphics, representation of geometrical objects in terms of a small number of building blocks is known under the name of constructive solid geometry, which provides a way to describe complex shapes in terms of just a few parameters -- the types of the building blocks, such as cylinders, spheres, and cubes, their sizes, and operations, such as difference, union, and intersection. Thus, information about a large number of structures can be stored in a compact form of statements that contain information about the types of the building blocks and the operations from which these structures were assembled. This study describes a similar approach for two-dimensional patterns and demonstrate how it enables the synthesis, comparison, and analysis of gene expression at the tissue scale (Yakoby, 2008b).

    The six building blocks used in the annotation system can be related to the structure of the egg chamber and the spatial distribution of the EGFR and DPP signals. The first primitive, M (for 'midline'), is related to the EGFR signal. It reflects high levels of EGFR activation and has a concave boundary, which can be related to the spatial pattern of GRK secretion from the oocyte. The second primitive, denoted by D (for 'dorsal'), reflects the intermediate levels of EGFR signaling during the early phase of EGFR activation by GRK, and is defined as a region of the follicular epithelium that is bounded by a level set (line of constant value) of the dorsoventral (DV) profile of EGFR activation. The boundary of this shape is convex and can be extracted from the experimentally validated computational model of the GRK gradient. The third primitive, denoted by A (for 'anterior'), is an anterior stripe which is obtained from a level set of the early pattern of DPP signaling in the follicular epithelium. This pattern is uniform along the DV axis, as visualized by the spatial pattern of phosphorylated MAD (P-MAD). Thus, the D, M, and A primitives represent the spatial distribution of the inductive signals at the stage of eggshell patterning when the EGFR and DPP pathways act as independent AP and DV gradients (Yakoby, 2008b).

    Each of the next two primitives, denoted by R (for 'roof') and F (for 'floor'), is composed of two identical regions, shaped as the respective expression domains of br and rho, and reflect spatial and temporal integration of the EGFR and DPP pathways in later stages of eggshell patterning. The mechanisms responsible for the emergence of the F and R domains are not fully understood. It has been shown that the R domain is established as a result of sequential action of the feedforward and feedback loops within the EGFR and DPP pathways. The formation of the F domain requires the activating EGFR signal and repressive BR signal, expressed in the R domain. Thus, at the current level of understanding, the R and F domains should be viewed as just two of the shapes that are commonly seen in the two-dimensional expression patterns in the follicular epithelium. The sixth primitive, U (for 'uniform'), is spatially uniform and will be used in combination with other primitives to generate more complex patterns (Yakoby, 2008b).

    While a number of patterns, such as those of jar and Dad, can be described with just a single primitive, more complex patterns are constructed combinatorially, using the operations of intersection (∩), difference ( ), and union (∪) For example, the dorsal anterior stripe of argos expression is obtained as an intersection of the A and D primitives (A∩D). The ventral pattern of pip is obtained as a difference of the U and D primitives (U D). The pattern of 18w is constructed from the A, D, and R primitives, joined by the operations of union and difference (A∪D R). For a small number of published patterns, the annotations reflect the experimentally demonstrated regulatory connections. For example, the U D annotation for pip reflects that actual repression of pip by the dorsal gradient of EGFR activation. For a majority of genes, the annotations should be viewed as a way to schematically represent a two-dimensional pattern and as a hypothetical description of regulation (Yakoby, 2008b).

    The geometric operations of intersection, difference, and union can be implemented by the Boolean operations performed at the regulatory regions of individual genes. Boolean operations evaluate expression at each point and assign a value of 0 (off) or 1 (on). As an example, consider a regulatory module, hypothesized for argos, that performs a logical AND operation on two inputs: the output of the module is 1 only when both inputs are present. When both of the inputs are spatially distributed, the output is nonzero only in those regions of space where both inputs are present, leading to an output that corresponds to the intersection of the two inputs. Similarly, a spatial difference of the two inputs can be realized by a regulatory module that performs the ANDN (ANDNOT) operation. This is the case for pip, repressed by the DV gradient of GRK signaling and activated by a still unknown uniform signal. Finally, a regulatory module that performs an OR operation is nonzero when at least one of the inputs is nonzero. When the inputs are spatially distributed, the output is their spatial union (Yakoby, 2008b).

    Boolean operations on primitives lead to patterns with just two levels of expression (the gene is either expressed or not). In addition to Boolean logic, developmental cis-regulatory modules and systems for posttranscriptional control of gene expression can perform analog operations, leading to multiple nonzero levels of output. Consider a module that adds the two binary inputs, shaped as the primitives. The output is nonzero in the domain shaped as the union of the two primitives, but is characterized by two nonzero levels of expression. This type of annotation is reserved only for those cases where the application of Boolean operations would lead to a loss of the spatial structure of the pattern (such as the A + U expression pattern of mia at stage 11 of oogenesis. For example, the union of the A and U primitives is a U primitive, whereas the sum of these primitives is an anterior band superimposed on top of a spatially uniform background (Yakoby, 2008b).

    Signaling pathways guide organogenesis through the spatial and temporal control of gene expression. While the identities of genes controlled by any given signal can be identified using a combination of genetic and transcriptional profiling techniques, systematic analysis of the diversity of induced patterns requires a formal approach for pattern quantification, categorization, and comparison. Multiplex detection of gene expression, which has a potential to convert images of the spatial distribution of transcripts into a vector format preferred by a majority of statistical methods, is currently feasible only for a small number of genes and systems with simple anatomies. This paper presents an alternative approach based on the combinatorial construction of patterns from simple building blocks (Yakoby, 2008b).

    In general, the building blocks can be identified as shapes that are overrepresented in a large set of experimentally collected gene expression patterns. This approach can be potentially pursued in systems where mechanisms of pattern formation are yet to be explored. At the same time, in well-studied systems, the building blocks can be linked to identified patterning mechanisms. This study chose six primitives based on the features that are commonly observed in real patterns and related to the structure of the tissue as well as the spatial distribution of the inductive signals. A similar approach will be useful whenever a two-dimensional cellular layer is patterned by a small number of signals, when cells can convert smoothly varying signals into spatial patterns with sharp boundaries, and when the regulatory regions of target genes have the ability to combinatorially process the inductive signals. One system in which this approach could be feasible is the wing imaginal disc, which is patterned by the spatially orthogonal wingless and DPP morphogens (Yakoby, 2008b).

    The six primitives are sufficient to describe the experimentally observed patterns during stages 10-12 of oogenesis. A natural question is whether it is possible to accomplish this with a smaller number of primitives. Two of the primitives, R and F, could be potentially constructed from the D, M, and A primitives, which are related to the patterns EGFR and DPP activation during the earlier stages of eggshell patterning. Specifically, recent studies of br regulation suggest that the R domain is formed as a difference of the D, A, and M patterns (Yakoby, 2008a). Furthermore, the formation of the F domain requires repressive action in the adjacent R domain. With the R and F domains related to the other four primitives, the size of the spatial alphabet will be reduced even further (from six to four), but at the expense of increasing the complexity of the expressions used to describe various spatial patterns (Yakoby, 2008b).

    Previously, the question of the diversity of the spatial patterns has been addressed only in one-dimensional systems. For example, transcriptional responses to the Dorsal morphogen gradient in the early Drosophila embryo give rise to three types of patterns in the form of the dorsal, lateral, and ventral bands. This work provides an attempt to characterize the diversity and dynamics of two-dimensional patterns. Thirty-six qualitatively different patterns were constructed, and it is proposed that each of them can be constructed using a compact combinatorial code. The sizes of the data sets from the literature and from transcriptional profiling experiments are approximately the same (117 and 96 patterns, respectively. Based on this observation, it is expected that discovered patterns will be readily described using this annotation system (Yakoby, 2008b).

    A gene expressed in more than one stage of oogenesis is more likely to appear in different patterns, and it was found that groups of genes sharing the same pattern at one time point are more likely to scatter in the future than to stay together. More detailed understanding of the dynamics of the spatial patterns of the EGFR and DPP pathway activation is crucial for explaining these trends and the two observed scenarios for the emergence of complex patterns. A gene that makes its first appearance as a complex pattern, such as the A∩D pattern of argos at stage 10B, can be a direct target of the EGFR and DPP signal integration. In contrast, a gene such as Cct1, which changes from the A to the R pattern, can be a dedicated target of DPP signaling alone, and changes as a consequence of change in the spatial pattern of DPP signaling. Future tests of such hypotheses require analysis of cis-regulatory modules responsible for gene regulation in the follicular epithelium. While only a few enhancers have been identified at this time, this categorization of patterns should accelerate the identification of enhancers for a large number of genes (Yakoby, 2008b).

    Proposed for the spatial patterns of transcripts, these annotations can also describe patterns of protein expression, modification, and subcellular localization. For example, the stage 10A patterns of MAD phosphorylation and Capicua nuclear localization can be accurately described using the A and U D annotations, respectively. The ultimate challenge is to use the information about the patterning of the follicular epithelium to explore how it is transformed into the three-dimensional eggshell. A number of genes in the assembled database encode cytoskeleton and cell adhesion molecules, suggesting that they provide a link between patterning and morphogenesis. It is hypothesized that the highly correlated expression patterns of these genes give rise to the spatial patterns of force generation and mechanical properties of cells that eventually transform the follicular epithelium into a three-dimensional eggshell (Yakoby, 2008b).

    Transfer of Dorsoventral and terminal information from the ovary to the embryo by a common group of eggshell proteins in Drosophila

    The Drosophila eggshell is an extracellular matrix that confers protection to the egg and also plays a role in transferring positional information from the ovary to pattern the embryo. Among the constituents of the Drosophila eggshell, Nasrat, Polehole and Closca form a group of proteins related by sequence, secreted by the oocyte and mutually required for their incorporation into the eggshell. Besides their role in eggshell integrity, Nasrat, Polehole and Closca are also required for embryonic terminal patterning by anchoring or stabilizing Torso-like at the eggshell. This study shows that they are also required for dorsoventral patterning, thereby unveiling that the dorsoventral and terminal systems, hitherto considered independent, share a common extracellular step. Furthermore, Nasrat, Polehole and Closca are required for proper activity of Nudel, a protease acting both in embryonic dorsoventral patterning and eggshell integrity, thus providing a means to account for the role of Nasrat, Polehole and Closca. It is proposed that a Nasrat/Polehole/Closca complex acts as a multifunctional hub to anchor various proteins synthesized at oogenesis, ensuring their spatial and temporal restricted function (Mineo, 2017).

    Terminal and dorsoventral signaling rely on initial spatial cues, which originate in the follicle cells surrounding the oocyte, that induce pattern formation in embryogenesis. Since follicle cells degenerate long before the cues perform their action in embryogenesis, all the information necessary for embryonic patterning has to be retained in the egg. In this scenario, the role of Nasrat, Polehole, and Closca in the localization of Tsl and Ndl suggests that a Nasrat/Polehole/Closca complex acts as a multifunctional hub at the vitelline membrane to anchor various proteins synthesized at oogenesis and with later functions in the eggshell and/or in triggering embryonic patterning (Mineo, 2017).

    Although the mechanism responsible for eggshell integrity is not fully understood, Ndl, and in particular its protease activity, Nasrat, Polehole, and Closca clearly participate in this process. The current results now identify Ndl as an effector of Nasrat, Polehole, and Closca both in eggshell integrity and in their so far unknown role in dorsoventral patterning. In this regard, it is worth mentioning that, in spite of the many analyses of Ndl activity, it remains an open question as to whether its function in dorsoventral axis specification and eggshell integrity are independent of each other. Besides, LeMosy and collaborators have proposed an additional role for the nonprotease regions of Ndl in eggshell integrity (LeMosy, 2000; Mineo, 2017 and references therein).

    Likewise, it is difficult to establish whether the diverse roles of Nasrat, Polehole, and Closca imply specific and independent functional protein domains. Although Nasrat, Polehole, and Closca belong to a common group of proteins, they show only moderate similarity, and no functional domains have been identified in any of them. The observation that a point mutation impairs the terminal functions of Clos proteins, as well as dorsoventral patterning and eggshell integrity, suggests a lack of clear independent domains responsible for each individual function. However, fs(1)N211 and fs(1)ph1901 mutants are thought to specifically impair the terminal function of Nasrat and Polehole proteins, respectively, which suggests that these might be modular proteins with different functional domains. Similarly, the fs(1)NA1038 mutation supports the notion of independent functional domains. In particular, all eggs from homozygous fs(1)NA1038 females collapse due to eggshell integrity defects; the same phenotype is observed in hemizygous fs(1)NA1038 females and in transheterozygote females of fs(1)NA1038 over a null fs(1)N mutant allele. However, eggs from transheterozygous females of fs(1)NA1038 over the fs(1)N211 terminal allele give rise to wild-type larvae and adults. This intra-allelic complementation suggests that separate domains specifically affect the integrity and the terminal functions of the Nasrat protein. To further characterize these putative protein domains, this study mapped the molecular lesion in the fs(1)NA1038 mutation and found it to correspond to an E to V transition at residue 350. This observation suggests that the domain of the Nasrat protein encompassing this residue is required for eggshell integrity but has no effect on embryonic patterning (Mineo, 2017).

    In conclusion, this study has found that terminal and dorsoventral signaling, hitherto considered independent in their extracellular pathways, have Nasrat, Polehole, and Closca as common mediators. It is proposed that a complex of these proteins constitutes a multifunctional hub to ensure the proper temporal localization/stabilization and activity of proteins synthesized at oogenesis and required at egg activation, thus guaranteeing the coordination of the hardening of the eggshell with the trigger of early embryonic patterning (Mineo, 2017).

    Long-term live imaging provides new insight into stem cell regulation and germline-soma coordination in the Drosophila ovary

    The Drosophila ovariole tip produces new ovarian follicles on a 12-hour cycle by controlling niche-based germline and follicle stem cell divisions and nurturing their developing daughters. Static images provide a thumbnail view of folliculogenesis but imperfectly capture the dynamic cellular interactions that underlie follicle production. This study describes a live-imaging culture system that supports normal ovarian stem cell activity, cyst movement and intercellular interaction over 14 hours, which is long enough to visualize all the steps of follicle generation. The results show that live imaging has unique potential to address diverse aspects of stem cell biology and gametogenesis. Stem cells in cultured tissue respond to insulin and orient their mitotic spindles. Somatic escort cells, the glial-like partners of early germ cells, do not adhere to and migrate along with germline stem cell daughters as previously proposed. Instead, dynamic, microtubule-rich cell membranes pass cysts from one escort cell to the next. Additionally, escort cells are not replenished by the regular division of escort stem cells as previously suggested. Rather, escort cells remain quiescent and divide only to maintain a constant germ cell:escort cell ratio (Morris, 2011).

    Drosophila germaria support a remarkably complex and diverse array of multicellular processes within a confined space: stem cells divide asymmetrically, daughters migrate and differentiate, cysts form, enter meiosis, choose an oocyte and form new follicles. The results show that even these complex, interdependent, hormonally regulated events can proceed with remarkable fidelity during in vitro culture and in the presence of regular illumination. The ability to image cells under these conditions has revised and clarified several important aspects of understanding of stem cell and escort cell behavior (Morris, 2011).

    Live imaging has provided, for the first time, the ability to visualize active pairs of GSCs within their niche. The system allows the direction of division to be unambiguously determined based on the relative location of the daughter nuclei immediately after nuclear membranes reform. The results confirm that divisions are oriented with respect to the a/p axis. GSC divisions are typically observed at ~35° off axis, which is very similar to those in published images of GSC mitosis, although a higher level of variation in spindle direction (±24°) was observed than previously reported. Given that no differences in the shape of the GSC niche region or in any other aspect of GSC division were observed in vitro, it is unlikely that GSC divisions in vitro differ in orientation from those in vivo (Morris, 2011).

    Drosophila female GSCs reside in a well-characterized niche, the operational mechanisms of which allow stem cell activity to respond to levels of environmental resources. The results indicate that at least one important aspect of environmental regulation, namely insulin-mediated control of GSC division, takes place in cultured germaria. Previous studies have demonstrated that the movement of GSCs through the G2 phase of the cell cycle is controlled by both insulin and the TOR pathway. In vivo, under normal conditions, each GSC divides approximately once per 24 hours. Without added insulin a rate of GSC division was observed in vitro that was consistent with this expectation. However, in the presence of supplemental insulin, nearly every GSC underwent division within 12-14 hours, confirming that exogenous insulin removes constraints that normally limit G2 progression. In line with this observation, a substantial increase was observed in the rate of GSC division, which peaked just 2-4 hours after the onset of incubation, indicating that many GSCs were arrested within 2-4 hours of M phase at the onset of the experiment. Additionally, after incubation in culture for 2 hours the proportion of cells in G2 decreased, as would be expected if G2-arrested cells were activated by insulin treatment. Consequently, GSCs respond to exogenous insulin during in vitro culture in the same manner as in living animals. The ability to study the response of stem cells to insulin (and very likely to other serum factors) within germaria developing in vitro provides many experimental advantages that can be exploited toward a better understanding of this crucial and widespread aspect of stem cell control (Morris, 2011).

    Previously, it was proposed that escort cells are maintained by six to eight anterior escort stem cells that divide to provide an average of two new escort cells for each new cystoblast. Similar stem cells, the CPCs, do exist in close association with the germline stem cells of the Drosophila testis and their daughters accompany male gametes throughout their subsequent development. However, the experiments reported in this study rule out this model in the case of the ovariole. Since escort cells do not move along with cysts or turn over regularly at the 2a/2b junction, the need for new escort cells is much lower than the need for new germ and follicle cells. New escort cells are sometimes required, since it was confirmed that a low level of escort cell apoptosis takes place even under optimal nutritional conditions. Furthermore, as reported previously, escort cell numbers maintain a fixed relationship to the number of stem cells, cystoblasts and cysts. However, parity is not maintained by upregulating anterior escort stem cells but rather by inducing differentiated escort cells at multiple positions within the germarium to divide. In this regard, escort cells resemble the differentiated cells of many mammalian tissues, such as liver and pancreas, which retain the ability to divide following injury (Morris, 2011).

    These experiments provide new insight into the longstanding issue of what mechanism moves cysts towards the posterior of the germarium. The division of stem cells and cysts at the anterior is not required for movement, as all the existing cysts continue to mature and exit the germarium over several days following the forced differentiation of stem cells by Bam expression. Each ovariole is surrounded by a muscular epithelial sheath and this tissue might play a role in posterior germline cyst movement; indeed, waves of contraction are observed in sheath-covered ovarioles cultured in vitro. However, germaria appeared to be fully functional when transplanted into hosts in the absence of the muscle sheath and germline cyst migration was observed in vitro after the muscle sheath had been removed. Consequently, it is proposed that the dynamic activity of the escort cells provides the force that moves cysts through the germarium (Morris, 2011).

    Finally, the results also have important implications for the nature of the FSC niche. Each germarium contains two FSCs that occupy separate niches on opposite lateral walls near the region 2a/2b border. Although much has been learned about these cells, like most adult mammalian stem cells they have remained difficult to study at the single-cell level. The finding that escort cells function in a fixed location suggests that one or more specific escort cells at the 2a/2b junction act to define and maintain a specialized microenvironment at the niche sites. Such a candidate cell was followed, and it was shown to remain at the 2a/2b junction despite cyst passage. The ability to study follicle cell production in living germaria cultured in vitro should make it possible to gain a more detailed understanding of the role of specific escort cells in this model epithelial stem cell niche (Morris, 2011).

    Spaghetti, a homolog of human RPAP3 (RNA polymerase II-associated protein 3), determines the fate of germline stem cells in Drosophila ovary

    The Drosophila ovary provides an attractive model for studying the extrinsic or intrinsic factors that regulate the fate of germline stem cells (GSCs). Using this model, this study identified a new role for Drosophila spaghetti (spag), encoding a homolog of human RNA polymerase II-associated protein 3 (RPAP3), in regulating the fate of ovarian GSCs. Results from spag knockdown and genetic mosaic studies suggest that spag functions as an intrinsic factor for GSCs maintenance. Loss of Spag by, either spag RNAi or null mutation failed to trigger apoptosis in ovarian GSCs. Overexpression of spag led to negligible increases in the number of GSC/Cystoblast (CB) cells, suggesting that an excess of Spag is not sufficient to accelerate the proliferation of GSCs or delay CBs' differentiation. This study provides evidence supporting that spag is involved in adult stem cells maintenance. In addition, the RNAi screen results showed that knockdown of Hsp90, one of known Spag interacting partners, led to loss of ovarian GSCs in Drosophila. Heterozygous mutations in hsp90 (hsp90/+) dramatically accelerated the GSC loss in spag RNAi ovaries, suggesting that the Spag-contained complex possibly plays an essential role in controlling the GSCs fate (Chen, 2017).

    Mei-p26 cooperates with Bam, Bgcn and Sxl to promote early germline development in the Drosophila ovary

    In the Drosophila female germline, spatially and temporally specific translation of mRNAs governs both stem cell maintenance and the differentiation of their progeny. However, the mechanisms that control and coordinate different modes of translational repression within this lineage remain incompletely understood. This study presents data showing that Mei-P26 associates with Bam, Bgcn and Sxl and nanos mRNA during early cyst development, suggesting that this protein helps to repress the translation of nanos mRNA. Together with recently published studies, these data suggest that Mei-P26 mediates both GSC self-renewal and germline differentiation through distinct modes of translational repression depending on the presence of Bam (Li, 2013).

    This study presents data that Mei-P26 cooperates with Bam, Bgcn and Sxl to control the translation of nanos mRNA in the Drosophila female germline. Co-immunoprecipitation experiments indicate Mei-P26 physically associates with the differentiation factors Bam, Bgcn and Sxl and yeast 2-hybrid assays suggest the interaction between Mei-P26 and Bgcn may be direct. Disruption of mei-P26, or snf, which disrupts sxl expression in the germline, results in the upregulation of Nanos protein expression in early differentiating cysts. Both Mei-P26 and Sxl protein associate with nanos mRNA (Chau, 2012). In light of the recently published study that shows mutating Sxl binding sites within the 3′UTR of nanos mRNA leads to mis-regulation of the gene (Chau, 2012), these results suggest that Mei-P26 may be part of a Sxl, Bgcn and Bam complex that serves to promote cyst development by directly repressing the expression of Nanos. However, despite repeated attempts, direct interactions between Bam and Bgcn with nanos mRNA could not be detected. While various technical issues may prevent the detection of these specific interactions, the inability to observe direct association between Bam/Bgcn and nanos mRNA leaves open the possibility that interactions between the components of the Mei-P26, Sxl, Bam and Bgcn complex and its target mRNAs may be dynamic in nature. For instance, Bam and Bgcn may help to prepare Sxl and Mei-P26 for mRNA binding but do not themselves directly interact or only transiently interact with these targets. Further experiments will be needed to clarify the more specific molecular mechanisms that underlie Bam/Bgcn function with respect to the translational repression of nanos mRNA (Li, 2013).

    Two other recent studies investigated the role of mei-P26 during germline development. Liu (2009) showed that the RNA helicase Vasa directly regulates the translation of mei-P26 mRNA through poly (U) elements within its 3' UTR. Mutations in each gene strongly enhance the phenotype of the other, resulting in the formation of cystic germline tumors. Neumuller (2008) focused on the function of Mei-P26, showing that it negatively regulates the activity of the miRNA pathway. It is now proposed that Mei-P26 functions in both GSCs and early differentiating germ cells. Within GSCs, Mei-P26 is in a complex with miRISC proteins and enhances miRNA-mediated silencing. In addition, Mei-P26 associates with Nanos protein and promotes BMP signaling within GSCs by repressing the expression of the negative regulator Brat. GSC daughters displaced away from the cap cell niche experience less BMP signaling, allowing for the expression of Bam (Li, 2013).

    It is speculated that upon Bam expression, Mei-P26 switches its activity and/or its mRNA targets. This switch allows Mei-P26 to promote germline differentiation by both negatively regulating the miRNA pathway and cooperating with Bam, Bgcn and Sxl to repress the translation of specific mRNAs such as nanos. However the complex functional relationships between Mei-P26, Sxl, Bam and Bgcn remain incompletely understood. While evidence is provided that these factors can physically associate with each other under certain conditions, disruption of these genes results in two discrete phenotypes. mei-P26 and snf mutants exhibit a cystic tumorous phenotype marked by the accumulation of undifferentiated cysts that do not express A2BP1, a molecular marker present in 4-, 8- and 16-cell cysts in wild-type samples. In contrast, disruption of bam or bgcn results in the formation of single cell germ cell tumors. These phenotypic differences suggest that Bam and Bgcn carry out additional functions independent of Mei-P26 and Sxl. A more complete characterization of the regulatory networks that govern the very early steps of germline cyst differentiation will have to await a better biochemical characterization of Bam and Bgcn function (Li, 2013).

    Together these data suggest that Mei-P26 has a variety of molecular functions inside and outside of the germline. It remains unclear whether Mei-P26 exhibits the same biochemical activity when complexed with different proteins or whether its function completely changes depending on context. Based on the presence of a RING domain, Mei-P26 may act as an ubiquitin ligase. However this specific enzymatic activity has not been demonstrated nor have any direct in vivo substrates been identified. In regards to the translational repression of specific mRNAs, a model is favored in which Mei-P26 exhibits the same molecular activity within GSCs and their early differentiating daughters. It is further speculated that association of Mei-P26 with different mRNA binding proteins modulates its targeting of specific mRNAs, and/or the degree to which these different targets are repressed. The expression of Bam correlates with changes in the development role of Mei-P26 but the manner in which Bam alters the composition or activity of the Mei-P26 complex remains unknown. Regardless, the findings that Bam can associate with Mei-P26 and Sxl provide further support for the hypothesis that Bam regulates the translation of specific mRNAs to promote the early steps of differentiation within the Drosophila female germline (Li, 2013).

    Centrosome-dependent asymmetric inheritance of the midbody ring in Drosophila germline stem cell division

    Many stem cells, including Drosophila germline stem cells (GSCs), divide asymmetrically, producing one stem cell and one differentiating daughter. Cytokinesis is often asymmetric, in that only one daughter cell inherits the midbody ring (MR) upon completion of abscission even in apparently symmetrically dividing cells. However, whether the asymmetry in cytokinesis correlates with cell fate or has functional relevance has been poorly explored. This study shows that the MR is asymmetrically segregated during GSC divisions in a centrosome age-dependent manner: male GSCs, which inherit the mother centrosome, exclude the MR, whereas female GSCs, which is shown in this study to inherit the daughter centrosome, inherit the MR. It is further shown that stem cell identity correlates with the mode of MR inheritance. Together these data suggest that the MR does not inherently dictate stem cell identity, although its stereotypical inheritance is under the control of stemness and potentially provides a platform for asymmetric segregation of certain factors (Salzmann, 2014).

    This the MR is inherited asymmetrically during GSC divisions in the Drosophila germline and that this correlates with centrosome age and depends on a functional centrosome. Interestingly, inheritance of the MR by the cell containing the daughter centrosome is opposite to a recent observation in mammalian cells (Kuo, 2011). Further studies are required to determine whether the asymmetrically inherited MR, or factors associated with it, regulates stem cell behavior, and whether this regulation occurs in a species- or cell type-dependent manner. Importantly, mutations that randomize MR inheritance (cnn and dsas-4) do not drastically modulate stem cell identity, and cnn and dsas-4 mutants show apparently normal progression of differentiation regarding the cell fate. Furthermore, the MR is inherited by the differentiating daughter in the male germline, whereas it is inherited by the stem cell in the female germline. Therefore, it is unlikely that the MR harbors an inherent fate determinant. However, it is tempting to speculate that certain fate determinants 'hitchhike' the MR in certain cell types, taking advantage of its stereotypical inheritance. Additionally, it is possible that the MR regulates an aspect of stem cell behavior rather than identity per se; for example, the MR could regulate the rate of stem cell division. The fact that multiple MRs are never seen in a single cell (GSC or CySC) may indicate that removal of the MR is a prerequisite of cell cycle progression into the next cell cycle. Moreover, the MR that is transferred from the GB to the CySC/CC might function as a messenger to coordinate the division frequency between GSCs and CySCs (Salzmann, 2014).

    The reports by Kuo and Ettinger are seemingly contradictory in that Kuo reported that stem cells are characterized by the accumulation of MRs, whereas Ettinger reported that they are characterized by the high capacity for MR release into the extracellular space (Ettinger, 2011; Kuo, 2011). This study using male and female GSCs demonstrates that MR fates are highly stereotypical yet strikingly distinct depending on the cell type. This finding indicates that each cell type handles MRs with its own elaborate cellular program. The reason why MR must be handled in such an elaborate manner awaits future investigation. Nonetheless, this study reveals that a basic cellular asymmetry such as MR inheritance correlates with asymmetry during stem cell division (Salzmann, 2014).

    ALIX and ESCRT-III coordinately control cytokinetic abscission during germline stem cell division in vivo

    Abscission is the final step of cytokinesis that involves the cleavage of the intercellular bridge connecting the two daughter cells. Recent studies have given novel insight into the spatiotemporal regulation and molecular mechanisms controlling abscission in cultured yeast and human cells. The mechanisms of abscission in living metazoan tissues are however not well understood. This study shows that ESCRT-III component Shrub are required for completion of abscission during Drosophila female germline stem cell (fGSC) division. Loss of ALIX or Shrub function in fGSCs leads to delayed abscission and the consequent formation of stem cysts in which chains of daughter cells remain interconnected to the fGSC via midbody rings and fusome. ALIX and Shrub interact and that they co-localize at midbody rings and midbodies during cytokinetic abscission in fGSCs. Mechanistically, this study shows that the direct interaction between ALIX and Shrub is required to ensure cytokinesis completion with normal kinetics in fGSCs. It is concluded that ALIX and ESCRT-III coordinately control abscission in Drosophila fGSCs and that their complex formation is required for accurate abscission timing in GSCs in vivo (Eikenes, 2015).

    Cytokinesis is the final step of cell division that leads to the physical separation of the two daughter cells. It is tightly controlled in space and time and proceeds in multiple steps via sequential specification of the cleavage plane, assembly and constriction of the actomyosin-based contractile ring (CR), formation of a thin intercellular bridge and finally abscission that separates the two daughter cells. Studies in a variety of model organisms and systems have elucidated key machineries and signals governing early events of cytokinesis. However, the mechanisms of the final abscission step of cytokinesis are less understood, especially in vivo in the context of different cell types in a multi-cellular organism (Eikenes, 2015).

    During the recent years key insights into the molecular mechanisms and spatiotemporal control of abscission have been gained using a combination of advanced molecular biological and imaging technologies. At late stages of cytokinesis the spindle midzone transforms to densely packed anti-parallel microtubules (MTs) that make up the midbody (MB) and the CR transforms into the midbody ring (MR, diameter of ~1-2 μm). The MR is located at the site of MT overlap and retains several CR components including Anillin, septins (Septins 1, 2 and Peanut in Drosophila melanogaster), myosin-II, Citron kinase (Sticky in Drosophila) and RhoA (Rho1 in Drosophila) and eventually also acquires the centralspindlin component MKLP1 (Pavarotti in Drosophila). In C. elegans embryos the MR plays an important role in scaffolding the abscission machinery even in the absence of MB MTs (Eikenes, 2015).

    Studies in human cell lines, predominantly in HeLa and MDCK cells, have shown that components of the endosomal sorting complex required for transport (ESCRT) machinery and associated proteins play important roles in mediating abscission. Abscission occurs at the thin membrane neck that forms at the constriction zone located adjacent to the MR. An important signal for initiation of abscission is the degradation of the mitotic kinase PLK1 (Polo-like kinase 1) that triggers the targeting of CEP55 (centrosomal protein of 55 kDa) to the MR. CEP55 interacts directly with GPP(3x)Y motifs in the ESCRT-associated protein ALIX (ALG-2-interacting protein X) and in the ESCRT-I component TSG101, thereby recruiting them to the MR. ALIX and TSG101 in turn recruit the ESCRT-III component CHMP4B, which is followed by ESCRT-III polymerization into helical filaments that spiral/slide to the site of abscission. The VPS4 ATPase is thought to promote ESCRT-III redistribution toward the abscission site. Prior to abscission ESCRT-III/CHMP1B recruits Spastin that mediates MT depolymerization at the abscission site. ESCRT-III then facilitates membrane scission of the thin membrane neck, thereby mediating abscission (Eikenes, 2015).

    Cytokinesis is tightly controlled by the activation and inactivation of mitotic kinases at several steps to ensure its faithful spatiotemporal progression. Cytokinesis conventionally proceeds to completion via abscission, but is differentially controlled depending on the cell type during the development of metazoan tissues. For example, germ cells in species ranging from insects to humans undergo incomplete cytokinesis leading to the formation of germline cysts in which cells are interconnected via stable intercellular bridges. How cytokinesis is modified to achieve different abscission timing in different cell types is not well understood, but molecular understanding of the regulation of the abscission machinery has started giving some mechanistic insight (Eikenes, 2015).

    The Drosophila female germline represents a powerful system to address mechanisms controlling cytokinesis and abscission in vivo. Each Drosophila female germline stem cell (fGSC) divides asymmetrically with complete cytokinesis to give rise to another fGSC and a daughter cell cystoblast (CB). Cytokinesis during fGSC division is delayed so that abscission takes place during the G2 phase of the following cell cycle (about 24 hours later). The CB in turn undergoes four mitotic divisions with incomplete cytokinesis giving rise to a 16-cell cyst in which the cells remain interconnected by stable intercellular bridges called ring canals (RCs). One of the 16 cells with four RCs will become specified as the oocyte and the cyst becomes encapsulated by a single layer follicle cell epithelium to form an egg chamber. Drosophila male GSCs (mGSCs) also divide asymmetrically with complete cytokinesis to give rise to another mGSC and a daughter cell gonialblast (GB). Anillin, Pavarotti, Cindr, Cyclin B and Orbit are known factors localizing at RCs/MRs and/or MBs during complete cytokinesis in fGSCs and/or mGSCs. It has been recently reported that Aurora B delays abscission and that Cyclin B promotes abscission in Drosophila germ cells and that mutual inhibitions between Aurora B and Cyclin B/Cdk-1 control the timing of abscission in Drosophila fGSCs and germline cysts. However, little is known about further molecular mechanisms controlling cytokinesis and abscission in Drosophila fGSCs (Eikenes, 2015).

    This study has characterize the roles of ALIX and the ESCRT-III component Shrub during cytokinesis in Drosophila fGSCs. ALIX and Shrub are required for completion of abscission in fGSCs. They co-localize during this process, and their direct interaction is required for abscission with normal kinetics. This study thus shows that a complex between ALIX and Shrub is required for abscission in fGSCs and provide evidence of an evolutionarily conserved functional role of the ALIX/ESCRT-III pathway in mediating cytokinetic abscission in the context of a multi-cellular organism (Eikenes, 2015).

    Loss of ALIX or/and Shrub function or inhibition of their interaction delays abscission in fGSCs leading to the formation of stem cysts in which the fGSC remains interconnected to chains of daughter cells via MRs. As abscission eventually takes place a cyst of e.g. 2 germ cells may pinch off and subsequently undergo four mitotic divisions to give rise to a germline cyst with 32 germ cells. Consistently, loss of ALIX or/and Shrub or interference with their interaction caused a high frequency of egg chambers with 32 germ cells during Drosophila oogenesis. It was also found that ALIX controls cytokinetic abscission in both fGSCs and mGSCs and thus that ALIX plays a universal role in cytokinesis during asymmetric GSC division in Drosophila. Taken together this study provides evidence that the ALIX/ESCRT-III pathway is required for normal abscission timing in a living metazoan tissue (Eikenes, 2015).

    The results together with findings in other models underline the evolutionary conservation of the ESCRT system and associated proteins in cytokinetic abscission. Specifically, ESCRT-I or ESCRT-III have been implicated in abscission in a subset of Archaea (ESCRT-III), in A. thaliana (elch/tsg101/ESCRT-I) and in C. elegans (tsg101/ESCRT-I). In S. cerevisiae, Bro1 (ALIX) and Snf7 (CHMP4/ESCRT-III) have also been suggested to facilitate cytokinesis. In cultured Drosophila cells, Shrub/ESCRT-III mediates abscission and in human cells in culture ALIX, TSG101/ESCRT-I and CHMP4B/ESCRT-III promote abscission. ALIX and the ESCRT system thus act in an ancient pathway to mediate cytokinetic abscission (Eikenes, 2015).

    Despite the fact that an essential role of ALIX in promoting cytokinetic abscission during asymmetric GSC division was found in the Drosophila female and male germlines, strong bi-nucleation directly attributed to cytokinesis failure was found in Drosophila alix mutants in the somatic cell types that were examined. This might have multiple explanations. One possibility is that maternally contributed alix mRNA may support normal cytokinesis and development. Whereas ALIX and CHMP4B depletion in cultured mammalian cells causes a high frequency of bi- and multi-nucleation it is also possible that cells do not readily become bi-nucleate upon failure of the final step of cytokinetic abscission in the context of a multi-cellular organism. Consistent with the observations of a high frequency of stem cysts upon loss of ALIX and Shrub in the germline, Shrub depletion in cultured Drosophila cells resulted in chains of cells interconnected via intercellular bridges/MRs due to multiple rounds of cell division with failed abscission. Moreover, loss of ESCRT-I/tsg101 function in the C. elegans embryo did not cause furrow regression. These and the current observations suggest that ALIX- and Shrub/ESCRT-depleted cells can halt and are stable at the MR stage for long periods of time and from which cleavage furrows may not easily regress, at least not in these cell types and in the context of a multi-cellular organism. It is also possible that redundant mechanisms contribute to abscission during symmetric cytokinesis in somatic Drosophila cells. Further studies should address the general involvement of ALIX and ESCRT-III in cytokinetic abscission in somatic cells in vivo (Eikenes, 2015).

    Different cell types display different abscission timing, intercellular bridge morphologies and spatiotemporal control of cytokinesis. In fGSCs it was found that ALIX and Shrub co-localize throughout late stages of cytokinesis and abscission. In human cells ALIX localizes in the central region of the MB, whereas CHMP4B at first localizes at two cortical ring-like structures adjacent to the central MB region and then progressively distributes also at the constriction zone where it promotes abscission. ALIX and CHMP4B are thus found at discrete locations within the intercellular bridge as cells approach abscission in human cultured cells. In contrast, ESCRT-III localizes to a ring-like structure during cytokinesis in Archaea, resembling the Shrub localization at MRs was observed in Drosophila fGSCs. Moreover, ALIX and Shrub are present at MRs for a much longer time (from G1/S) prior to abscission (in G2) in fGSCs than in human cultured cells. Here, ALIX and CHMP4B are increasingly recruited about an hour before abscission and then CHMP4B acutely increases at the constriction zones shortly (~30 min) before the abscission event (Eikenes, 2015).

    How may ALIX and Shrub be recruited to the MR/MB in Drosophila cells in the absence of CEP55 that is a major recruiter of ALIX and ultimately CHMP4/ESCRT-III in human cells? Curiously, a GPP(3x)Y consensus motif was detected within the Drosophila ALIX sequence (GPPPGHY, aa 808-814) resembling the CEP55-interacting motif in human ALIX (GPPYPTY, aa 800-806). Whether Drosophila ALIX is recruited to the MR/MB via a protein(s) interacting with this motif or other domains is presently uncharacterized. Accordingly, alternative pathways of ALIX and ESCRT recruitment have been reported, as well as suggested in C. elegans, where CEP55 is also missing. Further studies are needed to elucidate mechanisms of recruitment and spatiotemporal control of ALIX and ESCRT-III during cytokinesis in fGSCs and different cell types in vivo (Eikenes, 2015).

    This study found that the direct interaction between ALIX and Shrub is required for completion of abscission with normal kinetics in fGSCs. This is consistent with findings in human cells in which loss of the interaction between ALIX and CHMP4B causes abnormal midbody morphology and multi-nucleation. Following ALIX-mediated recruitment of CHMP4B/ESCRT-III to cortical rings adjacent to the MR in human cells, ESCRT-III extends in spiral-like filaments to promote membrane scission. Due to the discrete localizations of ALIX and CHMP4B during abscission in human cells ALIX has been proposed to contribute to ESCRT-III filament nucleation. In vitro studies have shown that the interaction between ALIX and CHMP4B may release autoinhibitory intermolecular interactions within both proteins and promote CHMP4B polymerization. Specifically, ALIX dimers can bundle pairs of CHMP4B filaments in vitro [65]. Moreover, in yeast, the interaction of the ALIX homologue Bro1 with Snf7 (CHMP4 homologue) enhances the stability of ESCRT-III polymers. There is a high degree of evolutionary conservation of ALIX and ESCRT-III proteins and because ALIX and Shrub co-localize and interact to promote abscission in fGSCs it is possible that ALIX can facilitate Shrub filament nucleation and/or polymerization during this process (Eikenes, 2015).

    The current findings indicate that accurate control of the levels and interaction of ALIX and Shrub ensure proper abscission timing in fGSCs. Their reduced levels or interfering with their complex formation caused delayed abscission kinetics. How cytokinesis is modified to achieve a delay in abscission in Drosophila fGSCs and incomplete cytokinesis in germline cysts is not well understood. Aurora B plays an important role in controlling abscission timing both in human cells and the Drosophila female germline. During Drosophila germ cell development Aurora B contributes to mediating a delay of abscission in fGSCs and a block in cytokinesis in germline cysts. Bam expression has also been proposed to block abscission in germline cysts. It will be interesting to investigate mechanisms regulating the levels, activity and complex assembly of ALIX and Shrub and other abscission regulators at MRs/MBs to gain insight into how the abscission machinery is modified to control abscission timing in fGSCs (Eikenes, 2015).

    Intercellular bridge MTs in fGSC-CB pairs were degraded in G1/S when the fusome adopted bar morphology. Abscission in G2 thus appears to occur independently of intercellular bridge MTs in Drosophila fGSCs. This has also been described in C. elegans embryonic cells where the MR scaffolds the abscission machinery as well as in Archaea that lack the MT cytoskeleton]. In mammalian and Drosophila S2 cells in culture, on the other hand, intercellular bridge MTs are present until just prior to abscission (Eikenes, 2015).

    It is interesting to note a resemblance of the stem cysts that appeared upon loss of ALIX and Shrub function to germline cysts in that the MRs remained open for long periods of time similar to RCs. Some modification of ALIX and Shrub levels/recruitment may thus contribute to incomplete cytokinesis in Drosophila germline cysts under normal conditions. Because stem cysts were detected in the case when ALIX weakly interacted with Shrub it is also possible that inhibition of their complex assembly/activity may contribute to incomplete cytokinesis in germline cysts. Abscission factors, such as ALIX and Shrub, may thus be modified and/or inhibited during incomplete cytokinesis in germline cysts. Such a scenario has been shown in the mouse male germline where abscission is blocked by inhibition of CEP55-mediated recruitment of the abscission machinery, including ALIX, to stable intercellular bridges. Altogether these data thus suggest that ALIX and Shrub are essential components of the abscission machinery in Drosophila GSCs, and it is speculated that their absence or inactivation may contribute to incomplete cytokinesis. More insight into molecular mechanisms controlling abscission timing and how the abscission machinery is modified in different cellular contexts will give valuable information about mechanisms controlling complete versus incomplete cytokinesis in vivo (Eikenes, 2015).

    In summary, this study reports that a complex between ALIX and Shrub is required for completion of cytokinetic abscission with normal kinetics during asymmetric Drosophila GSC division, giving molecular insight into the mechanics of abscission in a developing tissue in vivo (Eikenes, 2015).

    Lateral and end-on kinetochore attachments are coordinated to achieve bi-orientation in Drosophila oocytes

    In oocytes, where centrosomes are absent, the chromosomes direct the assembly of a bipolar spindle. Interactions between chromosomes and microtubules are essential for both spindle formation and chromosome segregation. This study examined oocytes lacking two kinetochore proteins, NDC80 and SPC105R, and a centromere-associated motor protein, CENP-E, to characterize the impact of kinetochore-microtubule attachments on spindle assembly and chromosome segregation in Drosophila oocytes. The initiation of spindle assembly was shown to result from chromosome-microtubule interactions that are kinetochore-independent. Stabilization of the spindle, however, depends on both central spindle and kinetochore components. This stabilization coincides with changes in kinetochore-microtubule attachments and bi-orientation of homologs. It is proposed that the bi-orientation process begins with the kinetochores moving laterally along central spindle microtubules towards their minus ends. This movement depends on SPC105R, can occur in the absence of NDC80, and is antagonized by plus-end directed forces from the CENP-E motor. End-on kinetochore-microtubule attachments that depend on NDC80 are required to stabilize bi-orientation of homologs. A surprising finding was that SPC105R but not NDC80 is required for co-orientation of sister centromeres at meiosis I. Together, these results demonstrate that, in oocytes, kinetochore-dependent and -independent chromosome-microtubule attachments work together to promote the accurate segregation of chromosomes (Radford, 2015).

    It is well established that oocyte spindle assembly in many organisms occurs in the absence of centrosomes. Instead, chromatin-based mechanisms play an important role in spindle assembly. The interactions between chromosomes and microtubules are paramount in oocytes, necessary for both the assembly of the spindle and the forces required for chromosome segregation. Less well understood, however, is the nature of the functional connections between chromosomes and microtubules in these cells. The role of the kinetochores, the primary site of interaction between chromosomes and microtubules, is poorly understood in acentrosomal systems. For example, spindles will assemble and chromatin will move without kinetochores in both Caenorhabditis elegans and mouse oocytes. In addition, both C. elegans and mouse oocytes experience a prolonged period during which chromosomes have aligned but end-on kinetochore-microtubule attachments have not formed. Previously shown that the central spindle, composed of antiparallel microtubules that assemble adjacent to the chromosomes, is important for spindle bipolarity and homolog bi-orientation. These studies suggest that lateral interactions between the chromosomes and microtubules drive homolog bi-orientation, but whether these interactions are kinetochore-based is not clear (Radford, 2015).

    There have been few studies directly analyzing kinetochore function in oocyte spindle assembly and chromosome segregation. Assembling a functional spindle requires the initiation of microtubule accumulation around the chromatin, the organization of microtubules into a bipolar structure, and the maturation of the spindle from promoting chromosome alignment to promoting segregation. Whether the kinetochores are required for spindle assembly or the series of regulated and directed movements chromosomes undergo to ensure their proper partitioning into daughter cells is not known. In Drosophila, the chromosomes begin the process within a single compact structure called the karyosome. Within the karyosome, centromeres are clustered prior to nuclear envelope breakdown (NEB). This arrangement, which is established early in prophase and maintained throughout diplotene/diakinesis, is also found in many other cell types. It is possible that the function of centromere clustering is to influence the orientation of the centromeres on the spindle independent of chiasmata. Following NEB, the centromeres separate. In Drosophila oocytes, centromere separation depends on the chromosomal passenger complex (CPC). Whether this movement depends on interactions between chromosomes and microtubules remains to be established (Radford, 2015).

    Following centromere separation, homologous centromeres move towards opposite spindle poles. During this time in Drosophila oocytes, the karyosome elongates and achiasmate chromosomes may approach the poles, separating from the main chromosome mass. As prometaphase progresses, the chromosomes once again contract into a round karyosome. These chromosome movements appear analogous to the congression of chromosomes to the metaphase plate that ultimately results in the stable bi-orientation of chromosomes. In mitotic cells, congression depends on lateral interactions between kinetochores and microtubules, and bi-orientation depends on the formation of end-on kinetochore-microtubule attachments. In oocytes, lateral chromosome-microtubule interactions have been suggested to be especially important, but how lateral and end-on kinetochore-microtubule attachments are coordinated to generate homolog bi-orientation has not been studied (Radford, 2015).

    To investigate the roles of lateral and end-on kinetochore-microtubule attachments in spindle assembly and prometaphase chromosome movements of acentrosomal oocytes, this study characterized Drosophila oocytes lacking kinetochore components. The KNL1/Mis12/Ndc80 (KMN) complex is at the core of the kinetochore, providing a link between centromeric DNA and microtubules. Both KNL1 and NDC80 bind to microtubules in vitro, but NDC80 is required specifically for end-on kinetochore-microtubule attachments. Therefore, this study examined oocytes lacking either NDC80 to eliminate end-on attachments or the Drosophila homolog of KNL1, SPC105R, to eliminate all kinetochore-microtubule interactions. Oocytes lacking the centromere-associated kinesin motor CENP-E because CENP-E promotes the movement of chromosomes along lateral kinetochore-microtubule attachments in a variety of cell types (Radford, 2015).

    This work has identified three distinct functions of kinetochores that lead to the correct orientation of homologs at meiosis I. First, SPC105R is required for the co-orientation of sister centromeres at meiosis I. This is a unique process that fuses sister centromeres, ensuring they attach to microtubules from the same pole at meiosis I. Second, lateral kinetochore-microtubule attachments are sufficient for prometaphase chromosome movements, which may be required for each pair of homologous centromeres to establish connections with microtubules from opposite poles. Third, end-on attachments are dispensable for prometaphase movement but are essential to stabilize homologous chromosome bi-orientation. Surprisingly, it was found that although Drosophila oocytes do not undergo traditional congression of chromosomes to the metaphase plate, CENP-E is required to prevent chromosomes from becoming un-aligned and to promote the correct bi-orientation of homologous chromosomes. It was also shown that the initiation of acentrosomal chromatin-based spindle assembly does not depend on kinetochores, suggesting the presence of important additional interaction sites between chromosomes and microtubules. The stability of the oocyte spindle, however, becomes progressively more dependent on kinetochores as the spindle transitions from prometaphase to metaphase. Overall, this work shows that oocytes integrate several chromosome-microtubule connections to promote spindle formation and the different types of chromosome movements that ensure the proper segregation of homologous chromosomes during meiosis (Radford, 2015).

    In acentrosomal oocytes, spindle assembly depends on the chromosomes. How the chromosomes can organize a bipolar spindle that then feeds back and drives processes like bi-orientation of homologous centromeres has been unclear. Previous studies have demonstrated that the central spindle is required for homolog bi-orientation. This study found that several types of functional chromosome-microtubule interactions exist in oocytes, and that each type participates in unique aspects of chromosome orientation and spindle assembly. A model for chromosome-based spindle assembly and chromosome movements in oocytes highlights the multiple and unappreciated roles played by kinetochore proteins such as SPC105R and NDC80, with implications for how homologous chromosomes bi-orient during meiosis I (Radford, 2015).

    While the spindle is assembling and becoming organized, the evidence suggests that the chromosomes undergo a series of movements that ultimately result in the bi-orientation of homologous chromosomes. The separation of clustered centromeres is CPC-dependent (Radford, 2012), but not kinetochore-dependent. One possibility is that the CPC-dependent interaction of microtubules with non-kinetochore chromatin drives centromere separation. An alternative is that CPC activity may result in a release of the factors that hold centromeres together in a cluster prior to NEB. A candidate for this factor is condensin, a known target of the CPC, that has been shown to promote the 'unpairing' of chromosomes in the Drosophila germline (Radford, 2015).

    Following separation of clustered centromeres, each pair of homologous centromeres bi-orients by separating from each other towards opposite poles. How bi-orientation is established in acentrosomal oocytes is poorly understood. Previous studies in C. elegans and mouse oocytes have suggested a combination of kinetochore-dependent and kinetochore-independent (e.g. involving chromokinesins and chromosome arms) microtubule interactions drive chromosome alignment and segregation. This study found that kinetochores play multiple roles, and the process of chromosome bi-orientation can be broken down into a series of chromosome movements that depend mostly on the kinetochores. First, the centromeres make an attempt at bi-orientation. In Drosophila oocytes, this results in the directed poleward movement of centromeres toward the edge of the karyosome and is accompanied by a stretching of the karyosome. Lateral kinetochore-microtubule attachments mediated by SPC105R are sufficient for this initial attempt at bi-orientation. End-on kinetochore-microtubule attachments via NDC80, however, are essential to maintain the bi-orientation of centromeres. Maintenance of centromere bi-orientation is associated with the stable positioning of the centromeres at the edges facing the poles (Radford, 2015).

    The lateral-based chromosome movements required for chromosome orientation are probably mediated by the meiotic central spindle, which have been shown to essential for chromosome segregation. In addition, recent reports in both mitotic and meiotic cells suggest that the initial orientation of chromosomes depends on the formation of a 'prometaphase belt' that likely brings centromeres into the vicinity of the central spindle. Therefore, it is proposed that the initial attempt at bi-orientation occurs during the period when both kinetochores and the central spindle are required for spindle stability. Then, as the oocyte progresses toward metaphase, and the central spindle decreases in importance, this reflects a trend toward the formation of stable end-on kinetochore-microtubule attachments that, in turn, stabilize the bipolar spindle. This model is also corroborated by evidence from mouse oocytes that stable end-on kinetochore-microtubule attachments form after a prolonged prometaphase (Radford, 2015).

    The data demonstrate that some chromosome movements, critical for bi-orientation, are dependent on lateral kinetochore-microtubule attachments. The kinetochore-associated kinesin motor CENP-E is thought to be responsible for chromosome movement along lateral kinetochore-microtubule attachments, resulting in chromosome alignment on the metaphase plate. However, because Drosophila meiotic chromosomes are compacted into a karyosome prior to NEB, they do not need to migrate in a plus-end-directed manner to achieve congression and alignment. Instead, centromeres must move toward the poles, perhaps in a minus-end directed manner, to achieve bi-orientation. Interestingly, this study found that CENP-E opposes this minus-end directed movement because in the absence of CENP-E, the karyosome split via lateral kinetochore-microtubule attachments. It is not yet clear what mediates the minus-end-directed movement, but the motors Dynein and NCD (the Drosophila kinesin-14 homolog) or microtubule flux are prime candidates (Radford, 2015).

    This study also observed that CMET (CENP-E) is required for the correct bi-orientation of homologous chromosomes. The function proposed in opposing minus-end directed movement may be required for making the correct attachments. As the centromere moves to the edge of the karyosome, CENP-E may not only prevent its separation from the karyosome, but could also force it back towards the opposite pole in cases where the homologs are not bi-oriented. A similar idea has been proposed for CENP-E in mouse oocytes. Alternatively, CENP-E has a second function in tracking microtubule plus-ends and regulating kinetochore-microtubule attachments. In fact, this study found that end-on kinetochore-microtubule stability is affected in the absence of CENP-E. Regulating the stability of microtubule plus-end attachments with kinetochores is critical for establishing correct bi-orientation of homologs. Therefore, both functions of CENP-E could contribute to the correct bi-orientation of centromeres in Drosophila oocytes (Radford, 2015).

    Loss of SPC105R has a more severe phenotype than loss of either NDC80 or CENP-E, consistent with a role as a scaffold. It recruits additional microtubule interacting proteins like NDC80 and CENP-E and also recruits checkpoint proteins such as ROD. In analyzing oocytes lacking SPC105R, another class of factors it may recruit was discovered: proteins required for co-orientation of sister centromeres during meiosis I. Co-orientation is a process that fuses the core centromeres and is important to ensure that two sister kinetochores attach to microtubules that are attached to the same spindle pole. Co-orientation could involve a direct linkage between sister kinetochores, as may be the case with budding yeast Monopolin or in maize, where a MIS12-NDC80 linkage may bridge sister kinetochores at meiosis I [56]. In contrast, in fission yeast meiosis I, cohesins are required for co-orientation. Cohesion is stably maintained at the core centromeres during meiosis I but not mitosis, and this depends on the meiosis-specific proteins Moa1 and Rec8. There is also evidence that Rec8 is required for co-orientation in Arabidopsis and this study found that loss of ORD, which is required for meiotic cohesion, also results in a loss of centromere co-orientation. Further studies, however, are necessary to determine if cohesins are required for co-orientation in Drosophila. Indeed, the proteins and mechanism that mediate this process in animals has not been known. Recently, however, the vertebrate protein MEIKIN has been found to provide a similar function to Moa1. Interestingly, both Moa1 and MEIKIN depend on interaction with CENP-C, but do not show sequence homology. Thus, Drosophila may have a Moa1/MEIKIN ortholog that has not yet been identified. In the future, it will be important to identify the proteins recruited by SPC105R and their targets in maintaining centromere co-orientation and how these interact with proteins recruited by CENP-C. The mechanism may involve the known activity of SPC105R in recruiting PP1, because PP1 has been shown to have a role in maintaining cohesion in meiosis I of C. elegans (Radford, 2015).

    This study's model for spindle assembly and chromosome orientation raises several important questions for future consideration. The CPC is required for spindle assembly in Drosophila oocytes and the current results highlight the importance of two CPC targets in homolog bi-orientation. One target is central spindle proteins, possibly through the CPC-dependent recruitment of spindle organization factors such as Subito. The CPC is also required for kinetochore assembly, similar to what has been shown in yeast, human cells, and Xenopus and consistent with the finding in human cells that Aurora B promotes recruitment of the KMN complex to CENP-C. It will be important to identify targets of the CPC that drive the initiation of spindle assembly, centromere separation, and bi-orientation. In addition, while this study has found that the CPC is required for kinetochore assembly, it is not known if the CPC promotes error correction by destabilizing kinetochore-microtubule attachments. The CPC may not promote kinetochore-microtubule detachment during meiosis because of the different spatial arrangement of sister centromeres during meiosis I. Indeed, it is not known what is responsible for correcting incorrect attachments at meiosis I or how they are differentiated from correct attachments (Radford, 2015).

    In prometaphase, the central spindle and kinetochores contribute to spindle stability. The current data suggests that the kinetochores increase in importance as the oocyte progresses to metaphase, perhaps as a result of the stabilization of end-on kinetochore-microtubule attachments as homologous chromosomes become bi-oriented. However, lateral kinetochore-microtubule interactions demonstrated some resistance to colchicine and allow bivalents to stretch in mouse oocytes. Thus, further studies are necessary to determine if lateral kinetochore-microtubule interactions also confer some stability. The current model also proposes that the transition from prometaphase to metaphase involves a switch from dynamic lateral kinetochore-microtubule interactions to stable end-on kinetochore-microtubule attachments. This transition involves the loss of central spindle microtubules, which occurs regardless of microtubule attachment status. Further studies will be necessary to determine if the prometaphase-to-metaphase transition is developmentally regulated rather than being controlled by the spindle assembly checkpoint. As proposed in mouse oocytes, this may contribute to the propensity for chromosome segregation errors in acentrosomal oocytes by closing the window of opportunity for error correction after key developmental milestones have been passed. Finally, one of the most poorly understood features of meiosis is co-orientation of sister centromeres at meiosis I. What SPC105R interacts with to mediate co-orientation will provide the first insights into the mechanism and regulation of this process in Drosophila (Radford, 2015).

    Multiple pools of PP2A regulate spindle assembly, kinetochore attachments, and cohesion in Drosophila oocytes

    Meiosis in female oocytes lacks centrosomes, the microtubule-organizing center. In Drosophila oocytes, meiotic spindle assembly depends on the chromosomal passenger complex (CPC). To investigate the mechanisms that regulate Aurora B activity, the role of Protein Phosphatase 2A (PP2A) in oocyte meiosis was examined. Both forms of PP2A, B55 and B56, antagonize the Aurora B spindle assembly function, suggesting that a balance between Aurora B and PP2A activity maintains the oocyte spindle during meiosis I. PP2A-B56, which is encoded by two partially redundant paralogs, wdb and wrd, is also required for maintaining sister chromatid cohesion, establishing end-on microtubule attachments, and the metaphase I arrest in oocytes. WDB recruitment to the centromeres depends on BUBR1, MEI-S332, and kinetochore protein SPC105R. While BUBR1 stabilizes microtubule attachments in Drosophila oocytes, it is not required for cohesion maintenance during meiosis I. It is proposed that at least three populations of PP2A-B56 regulate meiosis, two of which depend on SPC105R and a third that is associated with the spindle (Jang, 2021).

    A single-cell atlas of adult Drosophila ovary identifies transcriptional programs and somatic cell lineage regulating oogenesis

    Oogenesis is a complex developmental process that involves spatiotemporally regulated coordination between the germline and supporting, somatic cell populations. This process has been modeled extensively using the Drosophila ovary. Although different ovarian cell types have been identified through traditional means, the large-scale expression profiles underlying each cell type remain unknown. Using single-cell RNA sequencing technology, this study has built a transcriptomic data set for the adult Drosophila ovary and connected tissues. Using this data set, the transcriptional trajectory was identified of the entire follicle-cell population over the course of their development from stem cells to the oogenesis-to-ovulation transition. This study further identified expression patterns during essential developmental events that take place in somatic and germline cell types such as differentiation, cell-cycle switching, migration, symmetry breaking, nurse-cell engulfment, egg-shell formation, and corpus luteum signaling. Extensive experimental validation of unique expression patterns in both ovarian and nearby, nonovarian cells also led to the identification of many new cell type-and stage-specific markers. The inclusion of several nearby tissue types in this data set also led to identification of functional convergence in expression between distantly related cell types such as the immune-related genes that were similarly expressed in immune cells (hemocytes) and ovarian somatic cells (stretched cells) during their brief phagocytic role in nurse-cell engulfment. Taken together, these findings provide new insight into the temporal regulation of genes in a cell-type specific manner during oogenesis and begin to reveal the relatedness in expression between cell and tissues types (Jevitt, 2020).

    A single-cell atlas reveals unanticipated cell type complexity in Drosophila ovaries

    Organ function relies on the spatial organization and functional coordination of numerous cell types. The Drosophila ovary is a widely used model system to study the cellular activities underlying organ function, including stem cell regulation, cell signaling and epithelial morphogenesis. However, the relative paucity of cell type-specific reagents hinders investigation of molecular functions at the appropriate cellular resolution. This study used single-cell RNA sequencing to characterize all cell types of the stem cell compartment and early follicles of the Drosophila ovary. Transcriptional signatures were computed, and specific markers were identified for nine states of germ cell differentiation, and 23 somatic cell types and subtypes. An unanticipated diversity was uncovered of escort cells, the somatic cells that directly interact with differentiating germline cysts. Three escort cell subtypes reside in discrete anatomical positions, and they express distinct sets of secreted and transmembrane proteins, suggesting that diverse micro-environments support the progressive differentiation of germ cells. Finally, 17 follicle cell subtypes were uncovered, and their transcriptional profiles were characterized. Altogether, this study provides a comprehensive resource of gene expression, cell type-specific markers, spatial coordinates and functional predictions for 34 ovarian cell types and subtypes (Slaidina, 2021).

    A single-cell atlas was gemerated of the stem cell compartment and early differentiating egg chambers of adult ovaries of Drosophila melanogaster. Cell type-specific transcriptional signatures were characterized and novel markers were identified. In only a few cases does a single marker gene uniquely identify a specific cell type, but in combination and by intensity of gene expression, these genes can be used as cell type markers that distinguish between related cell types and developmental transitions. With these transcriptional profiles, functional predictions were generated for 34 cell types and subtypes-nine states of GC differentiation, and 25 somatic cell types including GSC niche cells, three escort cell (EC) subtypes, FSC/pre-FCs, three clusters corresponding to polar and stalk cell lineages, and 17 epithelial FC subtypes. This extensive annotation will bolster future studies, for example, by capitalizing on cluster-specific markers to develop cell type-specific genetic tools and for the analysis of stage and cell type-specific functions (Slaidina, 2021).

    It was not possible to distinguish GSCs and FSCs from their daughters by clustering. This is likely due to the relatively low numbers of stem cells in the data set, but it also reflects the high similarity between the transcriptomes of stem cells and their daughters. This perdurance of the stem cell transcriptome might have a functional relevance. Indeed, CBs and germline cysts can dedifferentiate and compete with GSCs for niche occupancy. Likewise, FSC daughters migrate across the germarium and possibly compete for niche occupancy with other FSCs. Thus, stem cell daughters initially retain the ability to revert to a stem cell state, possibly because they do not extensively remodel their transcriptome immediately after the asymmetric division (Slaidina, 2021).

    ECs have a dual role-they promote GSC self-renewal and GC differentiation forming a domain termed the differentiation niche. Three EC subtypes-anterior, central, and posterior ECs. Each EC subtype interacts with GCs of a particular differentiation state and likely sends and receives distinct signals. Likewise, EC morphologies differ between locations; cEC and pEC protrusions are longer than aEC protrusions, as they interact with increasingly larger germline cysts. A number of secreted proteins, adhesion molecules, and ECM components are differentially expressed between EC subtypes, suggesting that each subtype creates a distinct microenvironment. Thus, as GCs progress through differentiation and move posteriorly along the germarium, their immediate microenvironment changes. These observations open the possibility that the spatial organization of distinct EC microenvironments supports progressive GC differentiation and that maturing GC may feed-back on their microenvironment to define and stabilize its pattern (Slaidina, 2021).

    Highly granular clustering of GC transcriptomes and precise cluster identity assignments led to identification of transcription factors that are dynamically expressed over the course of differentiation. A number of Notch signaling responsive transcription factors (Enhancer of split Complex) are enriched in four- and eight-cell cysts, raising a possibility that Notch signaling regulates early steps of GC differentiation (Slaidina, 2021).

    Two recent studies have generated similar adult ovary atlases. Two additional studies have focused mainly on escort cells. Each study had a distinct focus and approach (a detailed comparative analysis of these studies and discussion is presented in supplemental materials). Overall, these studies and those of produced similar results. The majority of markers identified showed the expected expression in the current data set. The exact cluster boundaries occasionally differ between the studies-for example, for EC subtypes. This study provides a more granular subclustering of ovarian cell types and precise cluster mapping to cell types and differentiation stages by direct visualization of mRNAs in situ. Thus, this study delivers a comprehensive resource of gene expression profiles and markers for each cluster and provides gene class annotations for transcriptional signatures and functional predictions (Slaidina, 2021).

    Despite being one of the most extensively studied adult organs, this analysis of the Drosophila ovary reveals higher cell type diversity than previously anticipated. These findings suggest that numerous, yet unidentified, cell subpopulations with distinct functions exist even in the most thoroughly studied organs. The ongoing Human Cell Atlas and similar projects in model organisms will start revealing this complexity, although focused studies are needed to uncover the interplay between the subpopulations and their functions, finally leading to a full comprehensive description of organ function in homeostasis and disease (Slaidina, 2021).

    mRNA localization and translational control during oogenesis

    RNA localization is often combined with local translational control. This allows simultaneous spatial and temporal control of protein synthesis within a particular region of the cell. In the case of neurons it is speculated that localized mRNAs may be regulated translationally in response to synaptic activity. The combination of translational control with RNA localization can also serve to restrict protein activities to defined regions in the cytoplasm, thereby preventing deleterious interactions from occurring elsewhere in the cell. This seems to be the case for myelin basic protein, which causes membranes to compact, and for developmental determinants whose activities specify the basic body axes and early differentiation of the embryo (Gunkel, 1998 and references).

    Most of the current knowledge of how localized mRNAs are controlled translationally has come from studies in Drosophila of the determinants of embryonic polarity encoded by bicoid (bcd), nanos (nos), and oskar (osk). In all three examples, the mRNA is made in nurse cells, transported into the adjacent oocyte, and subsequently localized within the cell. Translation of these mRNAs is silenced transiently during their transport and until the protein is required. The importance of controlling translation during mRNA transport is underlined by the fact that premature or ectopic translation leads to severe developmental defects (Gunkel, 1998 and references). All three of these proteins have additional roles in localization of egg components or in regulation of mRNA translation. Besides acting as a transcriptional regulator, Bicoid functions as a translational regulator of Caudal mRNA. Nanos is an RNA binding protein that binds to and inhibits translation of Bicoid and Hunchback mRNAs. Oscar is a novel protein that functions in the assembly of the germ plasm (Gunkel, 1998 and references).

    In a wide range of organisms, including Drosophila, Xenopus, and mouse, many mRNAs appear to be silenced by underadenylation, and their translation is activated by the cytoplasmic elongation of their poly(A) tails. In Drosophila, the poly(A) tail of anteriorly localized bcd mRNA increases from ~50 nucleotides in oocytes, where it is translationally silent, to 150 nucleotides in early embryos, coincident with its activation. Experiments with injected Bcd mRNAs show that a poly(A) tail of 150 nucleotides rescues the bcd phenotype of embryos, whereas a mere 50 nucleotides, as present in the oocyte, do not suffice (Gunkel, 1998 and references).

    For NOS, OSK, and very likely a number of other localized mRNAs, translational regulation does not involve modulation of the length of the poly(A) tail. In contrast to Bcd, localization of OSK and NOS mRNAs is required for their translation. Upon fertilization, NOS mRNA is activated only if it resides at the posterior pole. Similarly, OSK mRNA remains repressed translationally in mutants that prevent OSK RNA localization to the posterior pole. Localization and translational repression of NOS and OSK transcripts require regulatory sequences in the 3' UTR. RNA-binding proteins that mediate repression have been identified. A 130-kD protein named Smaug is thought to prevent translation of nos transcripts that have failed to become localized. Smaug repression is mediated by multiple sites, namely the SREs or TCE (Smaug response elements or translation control element) within the Nos 3' UTR. The TCE mediates localization and activation of Nos mRNA, indicating that these aspects of Nos translational regulation are tightly linked and perhaps interdependent. In the case of OSK, premature translation is prevented by Bruno, a 68-kD protein encoded by the arrest (aret) locus. Bruno recognizes a repeated conserved sequence (BRE, for Bruno response element) in the OSK 3' UTR, and colocalizes with OSK mRNA to the posterior pole. The aret mutant phenotype and the colocalization of the protein with other mRNAs in the oocyte suggest that Bruno-mediated repression is not limited to OSK mRNA (Gunkel, 1998 and references).

    Translation of localized OSK mRNA is activated specifically through a discrete element situated at the 5' end of the transcript. This element is only active at the posterior pole and is only required when the transcript is repressed through the BRE, suggesting that it functions as a derepressor rather than as a simple enhancer of translation. There is a direct correlation between translational derepression and the binding of a 50-kD (p50) and a 68-kD protein (p68) to this element. One of the 5' binding proteins, p50, also interacts with the BRE in the 3' UTR, and this binding appears to be required for full translation repression. It is concluded that translational activation of localized OSK mRNA is caused not by the local inactivation of repressor, but rather by an active and specific derepression event mediated by a prelocalized machinery. These findings add the notion of "derepressor element", in addition to repressor removal and poly(A) tail lengthening, as means to achieve translational activation (Gunkel, 1998 and references).

    Drosophila protein kinase N (Pkn) is a negative regulator of actin-myosin activity during oogenesis

    Nurse cell dumping is an actin-myosin based process, where 15 nurse cells of any given egg chamber contract and transfer their cytoplasmic content through the ring canals into the growing oocyte. This study isolated two mutant alleles of protein kinase N (pkn) and showed that Pkn negatively-regulates activation of the actin-myosin cytoskeleton during the onset of dumping. Using live-cell imaging analysis it was observed that nurse cell dumping rates sharply increase during the onset of fast dumping. Such rate increase was severely impaired in pkn mutant nurse cells due to excessive nurse cell actin-myosin activity and/or loss of tissue integrity. This work demonstrates that the transition between slow and fast dumping is a discrete event, with at least a five to six-fold dumping rate increase. Pkn was shown to negatively regulates nurse cell actin-myosin activity. This is likely to be important for directional cytoplasmic flow. It is proposed that Pkn provides a negative feedback loop to help avoid excessive contractility after local activation of Rho GTPase (Ferreira, 2014).

    Evidence for the mechanosensor function of filamin in tissue development

    Cells integrate mechanical properties of their surroundings to form multicellular, three-dimensional tissues of appropriate size and spatial organisation. Actin cytoskeleton-linked proteins such as talin, vinculin and filamin (Cheerio in Drosophila) function as mechanosensors in cells, but it has yet to be tested whether the mechanosensitivity is important for their function in intact tissues. This study tested how filamin mechanosensing contributes to oogenesis in Drosophila. Mutations that require more or less force to open the mechanosensor region demonstrate that filamin mechanosensitivity is important for the maturation of actin-rich ring canals that are essential for Drosophila egg development. The open mutant was more tightly bound to the ring canal structure while the closed mutant dissociated more frequently. Thus, these results show that an appropriate level of mechanical sensitivity is required for filamins' function and dynamics during Drosophila egg growth and support the structure-based model in which the opening and closing of the mechanosensor region regulates filamin binding to cellular components (Huelsmann, 2016).

    The concept of mechanical regulation of tissue development, mechanotransduction, has been proven in several experimental settings. In adult mesenchymal stem cells, the elastic modulus of the culture substrate regulates cell differentiation along different lineages. In muscles, mechanical stretching generates various responses that affect cell proliferation and differentiation. Different sensor molecules, including many cytoskeleton-linked proteins, elicit these cellular responses according to the mechanical cues they perceive. For instance, the actin filament - plasma membrane linkers talin and vinculin regulate the mechanical tuning of cell adhesion strength by sensing the ECM rigidity and the sarcomeric ruler titin modulate the mechanical signaling in muscles. Filamins cross-link actin filaments and anchor them to membranes, and pulling forces regulate protein-interaction sites within their C-terminal immunoglobulin-like domains. The C-terminal mechanosensor region (MSR) of filamin has two protein interaction sites that are masked by neighbouring sequences (closed conformation, and masking is released by small forces of 2-5 pN. In cell culture models, the cytoplasmic tails of integrin adhesion receptors preferentially bind to open filamins as indicated by reduction of the interaction decay time when myosin is active. Rare mutations in humans and animal models demonstrate that filamins are involved in three-dimensional tissue morphogenesis and the maintenance of muscle integrity. This study tests whether the mechanosensor function of filamins has a role within tissues (Huelsmann, 2016).

    This paper shows that filamin mechanosensing-altering mutations disrupt ring canal development in the Drosophila ovary and lead to small, partially sterile eggs. Of note, changing the spring properties of the mechanosensor site in either direction: releasing (open MSR mutation) or tightening (closed MSR), caused similar ring canal phenotypes, yet distinct dynamics of filamin in the ring canal. These data fit with a model in which filamins have a mechanosensory function during animal tissue development (Huelsmann, 2016).

    Definite mutations that alter spring properties of a cytoskeletal proteins have not been tested rigorously in animal models. For example, deletions in titin M-band region cause muscle development and regeneration defects in mouse, but it is unclear to what extent altered mechanosensitivity contributes to these phenotypes. The unique structure of filamin MSR allowed mutations to be made that do not change the interaction sites themselves, but change how they are exposed. Previous structural and functional information allowed engineering of mutations that either require less or more force for opening than the normal structure. The same mutations that were made in this study in the Drosophila filamin Cheerio have been earlier used in human Filamin A for biochemical studies and single molecule studies. SAXS analysis suggested that the Cheerio MSR has similar shape parameters as the human Filamin A and that the open MSR mutation had changed the structure as expected. In the absence of external force the overall structure of the closed MSR mutant fragment was similar as WT (Huelsmann, 2016).

    For the in vivo experiments, the open and closed MSR substitution mutations were made n the genomic locus of cheerio. This allowed study of the effects of mutations at normal gene copy number without interference from the WT gene. Although it has been previously shown that some mutations disrupting the structure filamins cause aggregation of the mutant protein in skeletal or cardiac muscle and that filamin unfolding may trigger chaperone-assisted autophagocytosis in muscle, the MSR mutations used here did not cause filamin aggregates or decreased the protein amount. In contrast, immunoblotting experiments showed that all mutants were expressed at similar protein levels as the WT (Huelsmann, 2016).

    In addition to the finding that mechanosensing modifying filamin mutants disrupted the maturation of ring canals, the second main finding in the current study was that the mutants showed altered dynamics in the ring canal: open MSR mutant had markedly reduced recovery rate, whereas the closed MSR or ΔMSR mutant recovered faster than the WT filamin. This is consistent with the hypothesis that the open MSR interacts more strongly with ring canal components, whereas the interaction of closed MSR and ΔMSR mutants is mainly mediated by other regions of filamins, presumably the actin binding domain. Unfortunately, it was not possible to analyse the dynamics of the MSR mutants in the ring canals at late developmental stages, as the ring canals disintegrate in the MSR mutants (Huelsmann, 2016).

    How can the apparently conflicting results that the open and closed MSR mutants cause similar ring canal phenotypes, but yet have dramatically different levels of exchange at their site of function? These observations do not fit with a strictly structural role of the filamin MSR in the ring canal. If that were the case, it would be expected that the MSR interaction- stabilizing mutant would also abnormally stabilize the ring canal structure. This was not observed. Instead, a model is favored in which different domains of filamin coordinate structural and mechanosensory roles during oogenesis. The C-terminal MSR region regulates the dynamics of filamins and is essential for maintaining the protein at ring canals. In contrast, the N-terminal actin-binding domain is not essential for localisation, but for ring canals growth: filamins with a non-functional actin binding domain localise to ring canals, but ring canals stay small. Furthermore, the results suggest that to be fully functional the MSR must oscillate between open and closed conformations during ring canal maturation and growth: both, open and closed MSR mutants, destabilised the structure at the stage when it was supposed to grow. Thus, the data fit with a regulatory, mechanosensory function of filamin in ring canals. It would be predicted that growth of the ring canal would therefore involve membrane tension driven oscillations in the amount force within the structure. When under high tension filamin would bind tightly and perhaps also recruit new components to expand the structure. As the ring canal enlarges the tension would reduce, allowing filamin to redistribute within the expanded structure (Huelsmann, 2016).

    In mammalian model systems and in human patients, mutations in FLNA, FLNB and FLNC genes have been associated with developmental or regeneration abnormalities in vasculature, cartilage, bone and muscle. The current study suggests that some of these filaminopathies with either truncations of the C-terminal parts of filamins or point mutations at or near the MSR, may be caused by defects in mechanosensor function. For instance, missense mutations in the MSR of FLNC have been recently linked hypertrophic cardiomyopathy (Huelsmann, 2016).

    In conclusion, the current results suggest that normal filamin function requires mechanically regulated conformational changes within developing intact tissues. This fits with the model that filamin has a mechanosensing function during tissue growth that is be conserved from flies to mammals (Huelsmann, 2016).

    The Drosophila putative histone acetyltransferase Enok maintains female germline stem cells through regulating Bruno and the niche

    Maintenance of adult stem cells is largely dependent on the balance between their self-renewal and differentiation. The Drosophila ovarian germline stem cells (GSCs) provide a powerful in vivo system for studying stem cell fate regulation. It has been shown that maintaining the GSC population involves both genetic and epigenetic mechanisms. Although the role of epigenetic regulation in this process is evident, the underlying mechanisms remain to be further explored. This study found that Enoki mushroom (Enok), a Drosophila putative MYST family histone acetyltransferase controls GSC maintenance in the ovary at multiple levels. Removal or knockdown of Enok in the germline causes a GSC maintenance defect. Further studies show that the cell-autonomous role of Enok in maintaining GSCs is not dependent on the BMP/Bam pathway. Interestingly, molecular studies reveal an ectopic expression of Bruno, an RNA binding protein, in the GSCs and their differentiating daughter cells elicited by the germline Enok deficiency. Misexpression of Bruno in GSCs and their immediate descendants results in a GSC loss that can be exacerbated by incorporating one copy of enok mutant allele. These data suggest a role for Bruno in Enok-controlled GSC maintenance. In addition, it was observed that Enok is required for maintaining GSCs non-autonomously, functioning in cap cells. Compromised expression of enok in the niche (cap) cells (CpC) impairs the niche maintenance and BMP signal output, thereby causing defective GSC maintenance. This is the first demonstration that the niche size control requires an epigenetic mechanism. Taken together, studies in this paper provide new insights into the GSC fate regulation (Xin, 2013).

    As a Drosophila putative histone acetyltransferase of the MYST family, Enok has been shown to be essential for neuroblast proliferation in the mushroom body (Scott, 2001). This paper presents evidence that Enok is required intrinsically and extrinsically for maintaining GSCs in the ovary. In the case of intrinsic mechanisms, Bruno was identified as an intermediate factor for Enok- controlled GSC maintenance. Molecular and genetic studies revealed that enok mutations in the germline lead to ectopic expression of Bruno in the GSCs, thereby inducing GSC loss probably via promoting cell differentiation. Meanwhile, Enok was also shown a having a non-cell autonomous role in controlling GSC self-renewal throughregulating the niche maintenance and niche-derived BMP signaling output. Thus, this study unraveled a novel regulatory mechanism governing the GSC maintenance mediated by a putative epigenetic regulator in Drosophila. Since Moz and Qkf, the mammalian homologs of Enok, are involved in controlling self-renewal of adult stem cells such as hematopoietic and neural stem cells, the new findings in this paper will help to address how the adult stem cell fate regulation occurs in higher organisms (Xin, 2013).

    Numerous studies have shown that GSC maintenance in the Drosophila ovary depends on at least three intrinsic machineries: the BMP/Bam pathway, the Nos/Pum complex and the miRNA pathway. The present study observed that Enok in the germline controls GSC self-renewal independently of BMP/Bam pathway. In the meantime, it was found that loss of enok function does not intrinsically alter the expression pattern of either Nos or Pum in the GSCs, and that enok displays no genetic interactions with either nos or pum in GSCs maintenance. Hence, the results exclude the possibility that the Nos/Pum complex is implicated in Enok-controlled GSC maintenance. Intriguingly, the molecular studies identified Bruno as a potential target of Enok involved in the GSC maintenance. Further genetic analyses suggest that increased expression of Bruno in the GSCs mutant for enok contributes to the GSC loss. bruno encodes an RNA-Recognition-Motifs-containing RNA binding protein which targets a number of mRNAs for their translational repression in the ovary and early embryo. Early on, Bruno was shown to function in patterning the embryo along the AP and DV axis by regulating the translation of oskar and gurken mRNA during late oogenesis. Later, it was reported that Bruno plays a pivotal role in CB differentiation and germline cyst formation at early oogenesis via targeting the Sex-lethal (Sxl) gene. This study has defined a novel function for Bruno in mediating the intrinsic requirements of Enok for maintaining GSCs (Xin, 2013).

    Misexpression of Bruno in the germline causes a derepression of PGC differentiation in the gonads from the late third instar larvae. This precocious differentiation phenotype further suggests that bruno gain-of-function in the enok mutants promotes GSC differentiation, thereby eliciting a stem cell loss (Xin, 2013).

    To better understand how enok mutation-induced ectopic expression of Bruno promotes the GSC differentiation, it is necessary to identify the potential mRNA target(s) of this RNA-binding protein in the GSCs and their immediate descendants that may function as the differentiation-inhibiting factor in this context. Of all known target genes of Bruno, only Sxl is dynamically expressed in early germ cells including GSCs and CBs, and essential for the GSC/CB fate switch. Preliminary data show that the expression pattern of Sxl remains unchanged in the mutant GSC or CB clones homozygous for the enok allele, ruling out a possible role of Sxl in Enok/Bruno-mediated differentiation control process. Given that the Bruno Response Element (BRE) consensus sequences located in the 3'UTR of the target mRNAs is important for Bruno binding, target candidates from the ovarian mRNAs that contain putative BRE sequences will be sought, based on bioinformatics approaches. However, it is noteworthy that Bruno can also regulate the expression of its target mRNA in a BRE-independent manner. Thus, high-throughput screens such as microarray analysis for differentially expressed genes in the enok mutant ovaries may give more clues for unraveling the mystery (Xin, 2013).

    It has been shown that mammalian Moz can acetylate histones H3 and H4 at a number of specific lysine residues. In particular, this MYST family histone acetyltransferase is required for H3K9 acetylation at Hox gene clusters, thus for correct body segment patterning in mice. As the Drosophila homolog of Moz, Enok possesses a conserved MYST histone acetyltransferase (HAT) domain, as well as two PHD fingers and a shared N-terminal domain. Previous studies showed that a point mutation in the MYST HAT domain of Enok causes an arrest in neuroblast proliferation of mushroom body as a null allele (Scott, 2001). Combined with the observation in this paper that the same mutation (enok2) gives defective GSC maintenance phenotype, it is proposed that the HAT activity is implicated in Enok's function during the indicated developmental processes. To further test this scenario, studies will attempt to determine whether the expression of Bruno in the early germ cells could be under the epigenetic control of Enok by examining a possible binding of Enok to bruno gene using chromatin immunoprecipitation (ChIP). In this case, high-throughput screens based on a combination of ChIP-seq and microarray analysis may lead to identification of more target genes of Enok that could mediate the GSC fate regulation controlled by this putative epigenetic factor (Xin, 2013).

    The GSC niche plays a key role in controlling GSC self-renewal in the ovary. Although the niche regulation itself is less understood, recent studies showed that systemic factors such as insulin signaling control the niche size, and consequently GSC maintenance at adulthood. Specifically, systemic insulin-like signals maintain the cap cell (CpC) population via modulating Notch signaling. The present study provides the first evidence that the niche maintenance also requires a putative epigenetic factor, and that decrease in the CpC number induced by enok knockdown in the niche is attributable to impaired Notch signaling. Thus, identification and functional characterization of the targets of Enok in controlling the niche size would provide more insights towards understanding how the niche is maintained. Given that insulin signaling is required for controlling the normal decline of both CpCs and GSCs in the aging process, and that epigenetic regulation is important for aging stem cells in mammals, it is assumed that Enok-mediated niche maintenance via Notch signaling has implications in both niche and GSC aging. If this is the case, Enok activity in the niche should display an age-dependent decline. Furthermore, increasing Enok activity could significantly attenuate the age-dependent decrease in the number of both CpCs and GSCs (Xin, 2013).

    In conclusion this paper shows that Enok controls GSC maintenance in the Drosophila ovary at multiple levels. In the case of a cell-autonomous control of GSC self-renewal, Enok acts in a BMP/ Bam-independent manner. Instead, activation of Bruno expression in the GSCs and their differentiating progeny links enok mutations in the germline to the GSC loss. In parallel, Enok plays a non-autonomous role in maintaining the GSC population via regulating the niche size and niche-derived BMP signal output from cap cells. Collectively, these results reveal a novel mechanism underlying a putative epigenetic factor-controlled GSC fate regulation (Xin, 2013).

    Defining gene networks controlling the maintenance and function of the differentiation niche by an in vivo systematic RNAi screen

    In the Drosophila ovary, escort cells (ECs) extrinsically control germline stem cell (GSC) maintenance and progeny differentiation. However, the underlying mechanisms remain poorly understood. This study identified 173 EC genes for their roles in controlling GSC maintenance and progeny differentiation by using an in vivo systematic RNAi approach. Of the identified genes, 10 and 163 are required in ECs to promote GSC maintenance and progeny differentiation, respectively. The genes required for progeny differentiation fall into different functional categories, including transcription, mRNA splicing, protein degradation, signal transduction and cytoskeleton regulation. In addition, the GSC progeny differentiation defects caused by defective ECs are often associated with BMP signaling elevation, indicating that preventing BMP signaling is a general functional feature of the differentiation niche. Lastly, exon junction complex (EJC) components, which are essential for mRNA splicing, are required in ECs to promote GSC progeny differentiation by maintaining ECs and preventing BMP signaling. Therefore, this study has identified the major regulators of the differentiation niche, which provides important insights into how stem cell progeny differentiation is extrinsically controlled (Gao, 2019).

    Independent and coordinate trafficking of single Drosophila germ plasm mRNAs

    Messenger RNA localization is a conserved mechanism for spatial control of protein synthesis, with key roles in generating cellular and developmental asymmetry. Whereas different transcripts may be targeted to the same subcellular domain, the extent to which their localization is coordinated is unclear. Using quantitative single-molecule imaging, this study analysed the assembly of Drosophila germ plasm mRNA granules inherited by nascent germ cells. The germ-cell-destined transcripts nanos, cyclin B and polar granule component travel within the oocyte as ribonucleoprotein particles containing single mRNA molecules but co-assemble into multi-copy heterogeneous granules selectively at the posterior of the oocyte. The stoichiometry and dynamics of assembly indicate a defined stepwise sequence. The data suggest that co-packaging of these transcripts ensures their effective segregation to germ cells. In contrast, compartmentalization of the germline determinant oskar mRNA into different granules limits its entry into germ cells. This exclusion is required for proper germline development (Little, 2015).

    Messenger RNA localization is a conserved mechanism for spatial control of protein synthesis, with key roles in generating cellular and developmental asymmetry. Whereas different transcripts may be targeted to the same subcellular domain, the extent to which their localization is coordinated is unclear. Using quantitative single-molecule imaging, this study analysed the assembly of Drosophila germ plasm mRNA granules inherited by nascent germ cells. The germ-cell-destined transcripts nanos, cyclin B and polar granule component travel within the oocyte as ribonucleoprotein particles containing single mRNA molecules but co-assemble into multi-copy heterogeneous granules selectively at the posterior of the oocyte. The stoichiometry and dynamics of assembly indicate a defined stepwise sequence. The data suggest that co-packaging of these transcripts ensures their effective segregation to germ cells. In contrast, compartmentalization of the germline determinant oskar mRNA into different granules limits its entry into germ cells. This exclusion is required for proper germline development (Little, 2015).

    Quantitative analysis of germ plasm-localized mRNAs has revealed several intriguing features about the localization process and the coordinate regulation of their integration into the pole cells. nos, pgc and cycB mRNAs are transported within the oocyte as single mRNAs and are co-packaged into granules specifically at the posterior cortex concomitant with localization. Thus, localization serves not only to concentrate these transcripts at the posterior but also generates large, multi-copy polar granules to coordinate the efficient incorporation of these transcripts into the pole cells (Little, 2015).

    Polar granules are heterogeneous with respect to both the amount of a particular mRNA and the combination of different mRNAs. Although nos mRNA content of polar granules varies over a large range, there is a tendency towards higher values fitting a log-normal distribution. Log-normal distributions are often associated with exponential growth processes, whereby the rate at which an object grows is proportional to the size of the object. Thus, a log-normal distribution suggests that large granules grow at faster rates compared with small ones as assembly is accelerated through positive feedback. In addition, this study found that for granules containing both nos and pgc or nos and cycB, the quantities of the two different mRNAs are correlated, and there is a greater fraction of granules completely lacking one species of mRNA entirely than granules containing just a few copies of that mRNA. Together, these data suggest that for each type of mRNA, cooperative interactions generate homotypic RNPs that then 1) accelerate the recruitment of additional mRNAs of the same type, and 2) facilitate granule assembly by promoting interactions with similarly sized homotypic clusters of other mRNAs. These results also predict the existence of dedicated molecular pathways, one to form homotypic clusters, and another to assemble homotypic clusters of many different transcripts into higher-order granules. These higher-order granules may form by fusion of smaller homotypic granules and indeed fusion of granules labelled with GFP-Vas is observed by live imaging. Alternatively, clusters of different mRNAs may grow alongside each other on a predefined granule scaffold. The localization of Caenorhabditis elegans germ granules -- P granules -- occurs through a phase transition in which soluble RNP components condense at the posterior of the embryo. It is interesting to consider whether formation of homotypic clusters occurs by a condensation of single transcript RNPs mediated by RNA-binding proteins (Little, 2015).

    In contrast to other posteriorly localized RNAs, which travel as single molecules, osk forms oligomeric complexes beginning in the nurse cells. Previous studies indicated that reporters containing the osk 3'UTR can hitchhike on wild-type osk mRNA by 3'UTR-mediated dimerization. Hitchhiking is not required for osk transport, but co-packaging may be important for translational repression of osk before localization. Consistent with this, multi-copy RNPs are competent for localization by both kinesin-dependent transport during mid-oogenesis and diffusion/entrapment during late stages of oogenesis (Little, 2015).

    Previous ISH-immuno-electron microscopy analysis of stage 10 oocytes showed co-localization of osk with Stau, but not with Osk protein in polar granules and the results indicate that osk/Stau RNPs are continuously segregated from the germ plasm granules. This physical separation has functional consequences. Whereas co-packaging of nos, pgc and cycB coordinates their transport to posterior nuclei and consequent segregation to the pole cells, osk is specifically excluded. Although it is not known why targeting of Osk to polar granules seems to alter their function, it is clearly detrimental to germline development. It will be interesting to determine whether osk/ Stau granules contain other mRNAs whose functions are required specifically during oogenesis or early embryogenesis but must be excluded from pole cells (Little, 2015).

    Novel roles for RNA binding proteins squid, hephaesteus, and Hrb27C in Drosophila oogenesis

    Reproductive capacity in many organisms is maintained by germline stem cells (GSCs). A complex regulatory network influences stem cell fate, including intrinsic factors, local signals, and hormonal and nutritional cues. Posttranscriptional regulatory mechanisms ensure proper cell fate transitions, promoting germ cell differentiation to oocytes. As essential RNA binding proteins with constitutive functions in RNA metabolism, heterogeneous nuclear ribonucleoproteins (hnRNPs) have been implicated in GSC function and axis specification during oocyte development. HnRNPs support biogenesis, localization, maturation, and translation of nascent transcripts. Whether and individual hnRNPs specifically regulate GSC function has yet to be explored. This study demonstratea that hnRNPs are expressed in distinct patterns in the Drosophila germarium. Three hnRNPs, squid, hephaestus, and Hrb27C are cell-autonomously required in GSCs for their maintenance. Although these hnRNPs do not impact adhesion of GSCs to adjacent cap cells, squid and hephaestus (but not Hrb27C) are necessary for proper bone morphogenetic protein signaling in GSCs. Moreover, Hrb27C promotes proper GSC proliferation, whereas hephaestus promotes cyst division. It is concluded find that hnRNPs are independently and intrinsically required in GSCs for their maintenance in adults. These results support the model that hnRNPs play unique roles in stem cells essential for their self-renewal and proliferation (Finger, 2022).

    Sequence-dependent but not sequence-specific piRNA adhesion traps mRNAs to the germ plasm

    The conserved Piwi family of proteins and piwi-interacting RNAs (piRNAs) have a central role in genomic stability, which is inextricably linked to germ-cell formation, by forming Piwi ribonucleoproteins (piRNPs) that silence transposable elements. In Drosophila melanogaster and other animals, primordial germ-cell specification in the developing embryo is driven by maternal messenger RNAs and proteins that assemble into specialized messenger ribonucleoproteins (mRNPs) localized in the germ (pole) plasm at the posterior of the oogenesis. Maternal piRNPs, especially those loaded on the Piwi protein Aubergine (Aub), are transmitted to the germ plasm to initiate transposon silencing in the offspring germ line. The transport of mRNAs to the oocyte by midoogenesis is an active, microtubule-dependent process; mRNAs necessary for primordial germ-cell formation are enriched in the germ plasm at late oogenesis via a diffusion and entrapment mechanism, the molecular identity of which remains unknown. Aub is a central component of germ granule RNPs, which house mRNAs in the germ plasm, and interactions between Aub and Tudor are essential for the formation of germ granules. This study shows that Aub-loaded piRNAs use partial base-pairing characteristics of Argonaute RNPs to bind mRNAs randomly in Drosophila, acting as an adhesive trap that captures mRNAs in the germ plasm, in a Tudor-dependent manner. Notably, germ plasm mRNAs in drosophilids are generally longer and more abundant than other mRNAs, suggesting that they provide more target sites for piRNAs to promote their preferential tethering in germ granules. Thus, complexes containing Tudor, Aub piRNPs and mRNAs couple piRNA inheritance with germline specification. These findings reveal an unexpected function for piRNP complexes in mRNA trapping that may be generally relevant to the function of animal germ granules (Vourekas, 2016).

    Aubergine controls germline stem cell self-renewal and progeny differentiation via distinct mechanisms

    Piwi family protein Aubergine (Aub) maintains genome integrity in late germ cells of the Drosophila ovary through Piwi-associated RNA-mediated repression of transposon activities. Although it is highly expressed in germline stem cells (GSCs) and early progeny, it remains unclear whether it plays any roles in early GSC lineage development. This study reports that Aub promotes GSC self-renewal and GSC progeny differentiation. RNA-iCLIP results show that Aub binds the mRNAs encoding self-renewal and differentiation factors in cultured GSCs. Aub controls GSC self-renewal by preventing DNA-damage-induced Chk2 activation and by translationally controlling the expression of self-renewal factors. It promotes GSC progeny differentiation by translationally controlling the expression of differentiation factors, including Bam. Therefore, this study reveals a function of Aub in GSCs and their progeny, which promotes translation of self-renewal and differentiation factors by directly binding to its target mRNAs and interacting with translational initiation factors (Ma, 2017).

    Aub is an essential piRNA pathway component known to be important for silencing TEs to ensure normal late germ cell development in the Drosophila ovary. This study demonstrates that Aub is required intrinsically to maintain GSC self-renewal and promote their progeny differentiation in the Drosophila ovary. aub is required intrinsically in GSCs to maintain self-renewal by preventing DNA-damage-evoked Chk2 activation. Aub promotes cystoblast (CB) differentiation by maintaining Bam expression. In addition, Aub directly binds over 1,100 mRNAs in GSCs, some of which are known to be important for GSC maintenance and differentiation, suggesting that Aub can control GSC maintenance and progeny differentiation also by directly regulating gene expression. Aub directly binds bam, dnc, and Rm62 mRNAs, and regulates their expression at the translation level via 3' UTR, which can also help mechanistically explain how Aub controls GSC maintenance and progeny differentiation. Finally, Aub was shown to physically associated with the translation initiation eIF4 complex and pAbp, suggesting that it promotes gene expression via regulation of translation initiation. Therefore, this study has revealed new roles of Aub in promoting GSC self-renewal and GSC progeny differentiation through different mechanisms (Ma, 2017).

    Although Aub represses TEs by controlling the 'ping-pong' piRNA amplification cycle in late germ cells of the Drosophila ovary, its importance in GSCs, CBs, and mitotic cysts has not been previously reported. THE genetic results show that Aub intrinsically maintains GSC self-renewal partly by preventing DNA-damage-evoked checkpoint activation. First, aub mutant ovaries have two GSCs at eclosion, but gradually lose their GSCs within 25 days. Second, marked aub mutant GSC clones are lost from the niche faster than the marked control GSCs. Third, aub mutant GSCs accumulate DNA damage recognized by γ-H2AvD, indicating that Aub is also important to produce piRNAs for repressing TEs, thereby preventing TE-induced DNA damage. Fourth, the GSC loss caused by aub mutations can also be partially and significantly rescued by Chk2 inactivation, indicating that DNA damage is one of the major causes for the loss of the aub mutant GSCs. This interpretation is supported by a recent study showing that Chk2 inactivation can drastically and significantly rescue the DNA-damage-induced GSC loss. This is also consistent with the previous finding that the dorsal-ventral polarity defect of aub mutant egg chambers can be drastically rescued by Chk2 inactivation. Finally, the loss of aub mutant GSCs could be caused primarily by differentiation, but might also be contributed by apoptosis. Taken together, these results demonstrate that Aub intrinsically controls GSC self-renewal partly by preventing DNA-damage-induced Chk2 activation, and have also confirmed that piRNAs are important in GSCs to repress TEs and thus prevent DNA damage. In addition, Aub regulates nanos (nos) mRNA localization in the oocyte and also controls the decay of maternal mRNAs in the early Drosophila embryo, including nos, in cooperation with piRNAs. This study has adopted the iCLIP experimental procedure to the in vitro cultured GSCs for the identification of potential mRNA targets of Aub in GSCs. These cultured GSCs can self-renew, and, more importantly, can also be induced to differentiate into 16-cell cysts upon Bam induction. iCLIP results show that Aub can bind more than 1,100 mRNAs in GSCs primarily via 5' UTR, 3' UTR, or both, 59 of which encode known GSC self-renewal factors, including BMP pathway components, E-cadherin, and chromatin remodeling factors (Ma, 2017).

    This study has revealed another role of Aub in promoting GSC progeny differentiation by regulating the expression of differentiation factors via direct binding. First, Aub is required intrinsically to promote GSC progeny differentiation. The aub mutant ovaries accumulate excess undifferentiated CBs, whereas the marked mutant aub GSCs also produce excess undifferentiated CBs. Second, Aub sustains Bam protein expression in mitotic germ cells. Bam is a master regulator for driving GSC progeny differentiation. Genetically, aub interacts with bam to promote germ cell differentiation. Molecularly, Aub is required in mitotic cysts to sustain Bam protein expression, and its binding to the bam 3' UTR is important for regulating the levels of its protein but not mRNA, suggesting that Aub can regulate Bam expression at the translational level. In addition, bam transcription and mRNA levels are significantly lower in aub mutant ovaries than in the control ones, indicating that Aub can also regulate Bam at the transcriptional level. Thus, Aub can regulate Bam expression in early germ cells at the transcriptional and translational levels. Third, Aub is also required to maintain Rm62 expression in CBs and mitotic cysts via its 3' UTR. The deletion of the Aub-binding site from the Rm62 3' UTR significantly decreases its expression in CBs and mitotic cysts, indicating that Aub binding is critical for Rm62 protein expression in CBs and mitotic cysts. This study has confirmed the previously reported role of Rm62 in promoting GSC progeny differentiation. Taken together, this study demonstrates that Aub promotes GSC progeny differentiation at least in part by controlling the expression of differentiation factors at the translational level (Ma, 2017).

    Previous studies have shown that Aub can regulate mRNA stability and localization in a piRNA-dependent manner. By examining the existence of the binding sites for known germline-specific piRNAs over the 3' UTRs of 1,189 mRNAs, no significant enrichment was seen of piRNA-binding sites in the Aub-binding regions of these Aub target mRNAs, indicating that Aub binding to its target mRNAs in GSCs is likely independent of piRNAs. More importantly, Aub promotes the expression of its target mRNAs at least partly at the translation level via direct 3' UTR binding in GSCs and their progeny, thereby promoting GSC maintenance, progeny differentiation, or both. Interestingly, co-immunoprecipitation experimental results show that Aub is physically associated with the translation initiation complex eIF4 and the poly(A)-binding protein pAbp in S2 cells. The eIF4 complex and pAbp are known to be able to interact with each other to bring the 5' UTR and 3' UTR in close proximity, facilitating the loading of eIF3 and the 40S ribosome subunit to initiate translation. However, the current findings do not contradict the previous finding that Aub can promote mRNA degradation in different developmental and cellular contexts. Many independent studies have shown that translation regulators can also control mRNA stability, and mRNA stability factors can also regulate translation. Based on these findings, it is proposed that Aub can regulate mRNA translation at the initiation step by facilitating the interaction between the eIF4 complex and pAbp in GSCs and their early progeny (Ma, 2017).

    H3K36 trimethylation-mediated epigenetic regulation is activated by Bam and promotes germ cell differentiation during early oogenesis in Drosophila

    Epigenetic silencing is critical for maintaining germline stem cells in Drosophila ovaries. However, it remains unclear how the differentiation factor, Bag-of-marbles (Bam), counteracts transcriptional silencing. This study found that the trimethylation of lysine 36 on histone H3 (H3K36me3), a modification that is associated with gene activation, is enhanced in Bam-expressing cells. H3K36me3 levels were reduced in flies deficient in Bam. Inactivation of the Set2 methyltransferase, which confers the H3K36me3 modification, in germline cells markedly reduced H3K36me3 and impaired differentiation. Genetic analyses revealed that Set2 acts downstream of Bam. Furthermore, orb expression, which is required for germ cell differentiation, was activated by Set2, probably through direct H3K36me3 modification of the orb locus. These data indicate that H3K36me3-mediated epigenetic regulation is activated by bam, and that this modification facilitates germ cell differentiation, probably through transcriptional activation. This work provides a novel link between Bam and epigenetic transcriptional control (Mukai, 2015).

    To examine histone modifications in differentiating germ cells, wild-type ovaries were stained using monoclonal antibodies specific for histone modifications. The H3K36me3 histone modification, associated with active genes, accumulated in differentiating cystoblasts. H3K36me3 signals were increased in the differentiating cystoblasts that expressed the bam reporter gene (bam-GFP). By contrast, the H3K27me3 modification associated with gene repression accumulated in early germ cells, and its signals decreased as the cells differentiated. These results suggest that the H3K36me3 levels were upregulated in differentiating cystoblasts. Next, H3K36me3 levels were examined in the ovaries of the third instar larvae and bam86 mutant adult females, both of which contain undifferentiated germ cells. Although H3K27me3 signals were detected in these undifferentiated germ cells, strong H3K36me3 signals were not detected. Taken together, these data supported the idea that H3K36me3-mediated epigenetic regulation may be involved in germ cell differentiation. (Mukai, 2015).

    Set2 methyltransferase is responsible for the H3K36me3 modification. Immunostaining revealed that, in the germarium region, Set2 was expressed in most of the germline cells, and that nuclear Set2 levels increased in differentiating cystoblasts. To determine whether Set2 participates in H3K36me3 accumulation and differentiation, Set2 expression was inhibited by using an UAS-Set2.IR line. Set2 levels in germ cells were reduced by the expression of Set2 RNAi. Specifically, while Set2 signals in differentiating cystoblasts were detected in 100% of control (nanos-Gal4/+) germaria, the Set2 signals in the cystoblasts were significantly reduced in 57% of the germaria, when Set2 RNAi was expressed in germ cells under the control of the nanos-Gal4 driver. Next, H3K36me3 levels were investigated in the ovaries expressing Set2 RNAi. As expected, H3K36me3 levels were reduced as a consequence of Set2 RNAi treatment. In control ovaries, H3K36me3 signals in differentiating cystoblasts were detected in 97% of germaria. By contrast, when Set2 RNAi was expressed in germ cells under the control of the nanos-Gal4 driver, H3K36me3 signals in cystoblasts were severely reduced in 41% of the germaria. Moreover, germ cell differentiation was impaired because of the expression of Set2 RNAi. In 96% of the control germaria, cysts with branched fusomes were observed. However, fragmented fusomes were detected in 34% of the germaria expressing Set2 RNAi. These results indicate that Set2 was required for both H3K36me3 accumulation and cyst formation. Mosaic analysis was performed by using a Set2 null allele Set21. Strong H3K36me3 signals were observed in 80% of the control germline clones. By contrast, H3K36me3 levels were considerably reduced in 74% of the Set2- cystoblasts. Furthermore, a differentiation defect was observed that was similar to that induced by Set2 RNAi treatment in 84% of Set2- mutant cysts. These results suggest that Set2 is intrinsically required both for H3K36me3 accumulation in cystoblasts and for differentiation (Mukai, 2015).

    To investigate the potential regulatory link between Set2 and Bam, their genetic interaction was analyzed. Reduction in Set2 activity by introduction of a single copy of Set21 dominantly increased the number of germaria with the differentiation defect in bam86/+ flies. Fragmented fusomes were observed in 26% of germaria from the Set21/+; bam86/+ females , as compared to 5% in bam86/+ and 3% in Set21/+ females. These results indicated that Set2 cooperates with bam to promote cyst formation. To determine whether bam expression requires Set2 activity, Bam expression in Set2- germline clones by immunostaining. Indeed, Set2 activity in germ cells was dispensable for bam expression. Conversely, nuclear Set2 expression in the germ cells was significantly reduced by bam mutation, suggesting that bam is involved in the regulation of Set2 in these cells. This result is consistent with the observation that H3K36me3 levels were reduced by bam mutation. Moreover, reducing of bam activity by introducing of a single copy of bam86 dominantly increased the number of germaria with weaker H3K36me3 signals in Set21/+ flies. Decreased H3K36me3 signals in the cystoblasts were observed in 29% of germaria from the Set21/+; bam86/+ females, as compared to 3% in Set21/+ and 2% in bam86/+ females. These data prompted an exploration of the mechanism of regulation of Set2 activity by bam (Mukai, 2015).

    To address whether bambam is sufficient for H3K36me3 accumulation, H3K36me3 levels were examined in the ovaries carrying the hs-bam transgene, which is used to ectopically express bam+ by heat shock treatment (Ohlstein and McKearin, 1997). No GSCs with a strong H3K36me3 signal were observed in germaria from wild-type females 1 hour post-heat shock (PHS; n = 42). However, H3K36me3 levels in the GSCs were significantly increased in 51% of the germaria from hs-bam females 1 hour PHS (n = 65), indicating that ectopic bam expression is sufficient for H3K36me3 accumulation. Because Set2 is responsible for H3K36me3, it is speculated that bam may regulate Set2 activity to control H3K36me3 accumulation and GSC differentiation. To determine whether Set2 activity is required for these bam-mediated processes, the effect was studied of a reduction in Set2 activity on the GSC differentiation induced by bam. When bam+ was ectopically expressed by heat shock, GSC differentiation was induced as previously reported. In 71% of ovaries from hs-bam flies dissected 24 hours PHS, it was found that differentiating cysts, instead of GSCs, occupied the tip of germaria. By contrast, when both bam and Set2 RNAi were ectopically expressed, GSC loss was significantly suppressed. These data suggest that Set2 activity is regulated by Bam, and that Set2 acts downstream of bam and promotes differentiation (Mukai, 2015).

    Nuclear Set2 levels were increased in differentiating cystoblasts. Furthermore, nuclear Set2 levels in germ cells were reduced by bam mutation. It is speculated that bam may regulate Set2 nuclear localization. Therefore, whether bam expression is sufficient for Set2 nuclear accumulation was examined. The subcellular localization of Set2 was examined in hs-bam flies cultured at 30°C. First, H3K36me3 levels were examined in the GSCs. H3K36me3 levels in GSCs were increased in 36% of the germaria from the hs-bam females, as compared to 6% in wild-type females. This result suggests that the ectopic expression of bam is sufficient for H3K36me3 accumulation. Next, Set2 subcellular localization was examined in GSCs of hs-bam females cultured at 30°C. Nuclear Set2 levels in GSCs were increased in 54% of the germaria from the hs-bam females, as compared to 12% in wild-type females. These results suggest that bam promotes the nuclear accumulation of Set2 (Mukai, 2015).

    To understand the mechanism by which Set2 regulates germ cell differentiation, the genetic interaction between Set2 and the differentiation genes A2BP1 and orb, both of which are required for cyst differentiation, were examined. Reduction of Set2 activity by introduction of a single dose of Set21 dominantly increased the number of germaria exhibiting a differentiation defect in orbdec/+ flies. In 24% of germaria from the Set21/+; orbdec/+ females, fragmented fusomes were observed, as compared with 4% in orbdec/+ and 7% in Set21/+ females. By contrast, the reduction of Set2 activity did not significantly affect cyst formation in A2BP1KG06463/+ ovaries). These results implied that Set2 function is required to specifically regulate orb expression and promote cyst formation. To confirm this, orb expression was examined in Set2- cyst clones. Deletion of Set2 led to the delayed activation of orb. Although 74% of the control cyst clones located at the boundary of germarium regions 1 and 2a initiated orb expression, only 31% of Set2- cyst clones expressed orb. Most (61%) of the Set2- cyst clones in germarium region 2b recovered orb expression. These observations suggest that Set2 was required for the proper activation of orb in differentiating cysts. Next, the H3K36me3 state of the orb locus was investigated in the ovaries. ChIP assays demonstrated that the H3K36me3 enrichment in the 3'-UTR region of orb was significantly higher than in the 5'-UTR region. It has been reported that the H3K36me3 modification exhibits a 3'-bias, such that H3K36me3 is preferentially enriched at the 3' regions of actively transcribed genes. These results support the idea that orb expression in differentiating cysts is controlled in part by H3K36me3-mediated epigenetic regulation (Mukai, 2015).

    Next, the H3K36me3 status was investigated in the orb gene in bam86 mutant ovaries. ChIP assays showed that bam mutation reduced the amount of H3K36me3 in the 3'-UTR region of the orb gene. The H3K36me3 modification is linked to transcriptional elongation. Therefore, the results suggested that bam activates orb expression through the epigenetic control. Additionally, H3K4me3 and RNA polymerase II levels in the 5'-UTR region of the orb gene were also reduced by bam mutation, implying a role for bam in transcriptional initiation. To investigate this possibility, further investigation will be needed in order to identify the enzymes responsible for H3K4me3 and exploring the interactions between bam and those enzymes (Mukai, 2015).

    These results have shown that H3K36me3 levels are regulated by bam. As a cytoplasmic protein, Bam may indirectly regulate Set2 nuclear localization. Set2 exerts its functions through the interactions with cofactors. Understanding the mechanism by which Bam regulates Set2 will require the identification of the cofactors that mediate the nuclear transport of Set2. These data suggest a link between Bam and epigenetic transcriptional control. Bam may counteract epigenetic silencing in GSCs through H3K36me3-mediated epigenetic regulation. This study showed that orb expression is activated by epigenetic regulation. Because orb encodes a cytoplasmic polyadenylation element-binding protein, Orb may control translation in differentiating cysts in a polyadenylation-associated manner. Bam antagonizes the Nanos/Pumilio complex, which suppresses the translation of target mRNAs that encode differentiation factors . However, the ientity of the target mRNAs and the mechanisms for transcriptional activation have not yet been elucidated. Because Set2 is required for bam-induced GSC differentiation, studies focused on identifying the genes marked by H3K36me3 and on their epigenetic regulation will aid in the identification of the differentiation genes. Because Set2 is linked to transcriptional elongation, differentiation genes in GSCs might be poised for expression, but may be kept awaiting bam expression for full activation. It is anticipated that these results will facilitate a better understanding of the epigenetic mechanisms that regulate gametogenesis (Mukai, 2015).

    Aging and insulin signaling differentially control normal and tumorous germline stem cells

    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. This study reports 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, GSCs from mated and unmated females behave similarly, indicating that female GSC-male communication does not affect GSCs with age. These 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 (Kao, 2014).

    Although aging results in a decline in stem cell proliferation, relatively few studies have addressed how stem cell cycle progression is altered by aging. DNA damage is mainly induced by by-products of cellular metabolism, such as reactive oxygen species (ROS) and environmentally induced lesions upon irradiation. Accumulation of irreversible genomic DNA damage has been implicated as a prominent cause of aging, both at the organismal and at the cellular levels. Cells respond to DNA damage by activating checkpoint pathways, which delay cell cycle progression and allow for repair of the defects. This study observed that aged GSCs exhibit accumulation of DNA damage and a prolonged S phase, suggesting that the former may be responsible for the latter in GSCs during aging. (Kao, 2015).

    DNA breaks result in activation of ATM/ATR kinases (ataxia-telangiectasia mutated and Rad3 related), which phosphorylate a variant of histone H2A (H2AX); this histone variant is a critical factor in facilitating the assembly of specific DNA-repair complexes on damaged DNA. ATM/ATR kinase-mediated signaling is part of the intra-S phase checkpoint pathway, and its activation is often associated with a delay in S phase progression. However, ATR heterozygous mutant (mei-41D3/+) GSCs still exhibited a similar degree of S phase delay compared to wild-type, suggesting that ATR may be dispensable for age-induced S phase delay, although it is possible that disruption of one copy of ATR may not be sufficient to block the intra-S check point pathway (Kao, 2015).

    Surprisingly, it was observed that there was a 65% increase of aged tufeatm-8/+ (atm heterozygous mutant) GSCs in S phase (1.98-fold increase relative to young tufeatm-8/+ GSCs), as compared to its sibling controls at the same age (1.33-fold increase relative to young control GSCs). Coincidently, a recent publication on Drosophila reported that ATM functions in DNA damage repair and exerts negative feedback control over the level of programmed double strand breaks (DSBs) during meiosis, and thus the number of H2AX foci (a marker of DNA damage) is dramatically increased in tufeatm-8 mutant germ cells. It was speculated that tufeatm-8/+ GSCs may induce more DNA damage via feedback regulation, thereby causing more severe S phase delay. However, in mice, Atm-/- undifferentiated spermatogonia are not maintained in the testis due to DNA damage-induced cell cycle G1 arrest, suggesting that ATM may function in the G1 phase in response to DNA damage. Nevertheless, it remains to be elucidated whether ATM mediates different cell cycle regulators in different cell contexts or in response to different types of stress-induced DNA damage (Kao, 2015).

    With age, cells may accumulate DNA mutations that allow them to escape normal regulatory processes and become tumor cells. Although tumorigenesis is harmful to health in the long term, it may also serve as a survival and protective mechanism when the body is highly threatened. While the germarium normally houses differentiating 8- or 16-germ cell cysts interconnected with branched fusomes, this study found that the middle portion of the aged germarium was occupied by tumor-like GSCs, which express pMad (a Dpp signaling effector) and possess rounded fusomes. This result recalls an earlier report that forced stemness Dpp signaling causes differentiating germ cell cysts to revert into functional stem cells in Drosophila ovaries, through the induction of ring canal closure and fusome scission (Kao, 2015).

    It has also been reported that aged human epidermal cells can dedifferentiate into stem cell-like cells via Wnt/β-catenin signaling, and injury can drive the dedifferentiation of epidermal cells via the β-integrin-mediated signaling pathway; these findings suggest that dedifferentiation is a process by which organisms address aging or tissue damage. Given that GSCs play a fundamental role in producing the next generation, this study suspects that these tumor-like GSCs may be derived from germ cell cysts through a dedifferentiation process triggered by aging; however, the possibility that these tumor-like GSCs are derived from the transformation of normal GSCs could not be ruled out (Kao, 2015).

    In invertebrates, including C. elegans and Drosophila, mating is detrimental to the lifespan of females, to increase progeny production. In Drosophila, mating females die earlier than unmated females, and sex peptides, produced from the male accessory gland, may be responsible for this effect. In C. elegans, females shrink and die after mating, and this is partially due to the stimulation of GSC proliferation by sperm. This study, however, did not observe differences in GSC proliferation rates between mated and unmated females at any age, suggesting that the promotion of GSC proliferation by mating may be specific to C. elegans. In addition, the results also indicate that sex peptides do not affect GSCs, at least at the level of proliferation. Moreover, similar rates of aging-induced GSC loss were observed in mated and unmated females, suggesting that mating does not affect the physiological status of GSCs (Kao, 2015).

    The neuropeptide allatostatin C from clock-associated DN1p neurons generates the circadian rhythm for oogenesis

    The link between the biological clock and reproduction is evident in most metazoans. The fruit fly Drosophila melanogaster, a key model organism in the field of chronobiology because of its well-defined networks of molecular clock genes and pacemaker neurons in the brain, shows a pronounced diurnal rhythmicity in oogenesis. Still, it is unclear how the circadian clock generates this reproductive rhythm. A subset of the group of neurons designated "posterior dorsal neuron 1" (DN1p), which are among the ~150 pacemaker neurons in the fly brain, produces the neuropeptide allatostatin C (AstC-DN1p). This study reports that six pairs of AstC-DN1p neurons send inhibitory inputs to the brain insulin-producing cells, which express two AstC receptors, star1 and AICR2. Consistent with the roles of insulin/insulin-like signaling in oogenesis, activation of AstC-DN1p suppresses oogenesis through the insulin-producing cells. This study shows evidence that AstC-DN1p activity plays a role in generating an oogenesis rhythm by regulating juvenile hormone and vitellogenesis indirectly via insulin/insulin-like signaling. AstC is orthologous to the vertebrate neuropeptide somatostatin (SST). Like AstC, SST inhibits gonadotrophin secretion indirectly through gonadotropin-releasing hormone neurons in the hypothalamus. The functional and structural conservation linking the AstC and SST systems suggest an ancient origin for the neural substrates that generate reproductive rhythms (Zhang, 2021).

    Six pairs of DN1p neurons were discovered that are part of the circadian pacemaker neuron network in the brain and make functional inhibitory connections to the brain IPCs. The IPCs are endocrine sensors that link the organism's nutritional status with anabolic processes, such as those associated with growth in developmental stages and with reproduction in adults. In juvenile stages, activation of insulin and insulin-like growth factor (IGF) signaling (IIS) through the InR results in larger flies, whereas inhibition of this pathway produces smaller flies. Consistent with this, it was also found that forced activation of the AstC-DN1p (i.e., CNMa-Gal4/UAS-NaChBac) during development resulted in 12% smaller adults, confirming their role as a negative regulator of the IPCs. In adults, the IPCs are associated with many physiological and behavioral processes, such as feeding, glycaemic homeostasis, sleep, lifespan, and stress resistance. As such, the IPCs receive a variety of modulatory inputs from both central and peripheral sources, such as sNPF, corazonin, tachykinins, limostatin, allatostatin A, adipokinetic hormone, GABA, serotonin, and octopamine. Regarding reproduction, IIS directed by the IPCs stimulates GSC proliferation and vitellogenesis. The results also indicate that AstC from AstC-DN1p suppresses the secretory activity of the IPCs and juvenile hormone (JH)-dependent oocyte development (i.e., vitellogenesis). Indeed, it was found that the JH mimic methoprene can rescue the suppression of oogenesis induced by AstC-DN1p activation. From these results it is concluded that IPCs are inhibited by AstC released by AstC-DN1p. A similar link between IIS and the circadian clock has also been reported in mammals, but the mechanism remains unclear (Zhang, 2021).

    Although the genetic evidence supporting the inhibitory action of AstC-DN1p on IPCs is compelling, it is also puzzling because a previous study found forced activation of 8 to 10 pairs of DN1p neurons (i.e., Clk4.1-LexA+ neurons) induced Ca2+ transients in IPCs. This study also found that, under LD 12:12 conditions, the IPCs showed electrical activity early in the morning when DN1p neurons are also active. The same study, however, reported that, under DD conditions, the IPCs showed no bursting activity in the morning (i.e., CT0 to -4). Instead, they showed bursting activity in the late afternoon (i.e., CT8 to -12) when DN1 activity falls. Furthermore, DN1p activation evokes varying levels of Ca2+ transients from individual IPCs, some of which produce no detectable Ca2+ transient. Thus, like mammalian pancreatic β-cells, the IPCs in Drosophila seem to comprise a heterogeneous cell population. It is noted that individual IPCs show highly variable AstC-R1 expression, which would also lead to individual IPCs showing variable responses to AstC (Zhang, 2021).

    In D. melanogaster, the LD cycle generates an egg-laying rhythm by influencing oogenesis and oviposition. Oviposition depends on light cues, whereas oogenesis cycles with the circadian rhythm that itself continues to run in DD conditions. In live-brain Ca2+ imaging experiments, DN1 neurons show a circadian Ca2+ activity rhythm that peaks around CT19 and reaches its lowest point between CT6 and CT8. This DN1 activity rhythm correlates well with the rhythm of vitellogenesis initiation observed in this study. In this model, the lowest point in DN1 Ca2+ activity between CT6 and CT8 leads to a significant attenuation of AstC secretion. This leads to a derepression of IPC activity, which eventually induces JH biosynthesis and vitellogenesis initiation. The 6-h delay required for previtellogenic stage 7 follicles to develop into vitellogenic stage 8 follicles would result in a peak in the number of stage 8 follicles between CT12 and CT14. Notably, the ovaries of the AstC-deficient mutant showed similar numbers of stage 8 oocytes at all examined circadian time points, indicating that any other JH- or vitellogenesis-regulating factors play only minor roles in producing the circadian vitellogenesis rhythm (Zhang, 2021).

    Like the IPCs, the DN1p cluster is also heterogeneous. A subset of the DN1p neurons is most active at dawn and promotes wakefulness. Another subset of the DN1p cluster (also known as, spl-gDN1) promotes sleep. The DN1p cluster comprises two morphologically distinct subpopulations, a-DN1p and vc-DN1p. The a-DN1p subcluster promotes wakefulness by inhibiting sleep promoting neurons, whereas the vc-DN1p subcluster resembles the sleep-promoting spl-gDN1. These results indicate AstC-DN1p are a-DN1p neurons that project to the anterior optic tubercle (AOTU. Although the possibility cannot be ruled out that AstC-DN1p is also heterogeneous and includes some vc-DN1p neurons, the wake-promoting role of a-DN1p aligns well with the circadian vitellogenesis rhythm that requires the secretory activity of AstC-DN1p to be lowest in the afternoon and highest at dawn. Furthermore, AstC-DN1p neurons express Dh31. Dh31-expressing DN1 clock neurons are intrinsically wake-promoting and Dh31-DN1p activity in the late night or early morning suppresses sleep. Again, this is consistent with the observation that AstC-DN1p are also wake-promoting a-DN1p. It is speculated Dh31 plays a limited role in oogenesis regulation, because unlike AstC, RNAi-mediated knockdown of Dh31 had a negligible impact on female fecundity (Zhang, 2021).

    Besides AstC-DN1p, the female brain has many additional AstC neurons. However, it seems unlikely that other AstC neurons contribute to the circadian vitellogenesis rhythm. This is because restoring AstC expression specifically in AstC-DN1p almost completely restored the vitellogenesis rhythm in AstC-deficient mutants. It is feasible, however, that other AstC neurons contribute to different aspects of female reproduction. Indeed, a sizable difference was noted in the final oogenesis outcome between AstC-Gal4 neuron activation and brain-specific AstC-Gal4 neuron activation. This suggests AstC cells outside of the brain also regulate oogenesis probably in other physiological contexts, such as the postmating responses (Zhang, 2021).

    AstC receptors are orthologous to mammalian SST receptors (sstr1-5). SST is a brain neuropeptide that was originally identified as an inhibitor of growth hormone (GH) secretion in the anterior pituitary. Thus, the observation that AstC inhibits IIS from IPCs, a major endocrine signal that promotes growth in Drosophila, suggests remarkable structural and functional conservation between the invertebrate AstC and vertebrate SST systems. In addition, SST inhibits the hypothalamic neuropeptide GnRH, which stimulates the anterior pituitary's production of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). FSH stimulates clutches of immature follicles to initiate follicular development, while LH stimulates ovulation. Thus, both AstC and SST regulate the secretion of gonadotropins (JH in insects, FSH and LH in mammals) indirectly through the IPCs in insects and through the hypothalamic GnRH neurons in mammals. This functional conservation between AstC and SST is also evident in the immune system. AstC inhibits the innate immune system in insects, while SST inhibits inflammation in mammals (Zhang, 2021).

    In many seasonal breeders, the changing photoperiod as the seasons progress acts as an environmental cue for the biological clock system, which would then direct any necessary physiological changes. During the winter, Drosophila females enter a form of reproductive dormancy characterized by a pronounced suppression of vitellogenesis. A winter-like condition (i.e., short-day length, low temperature, and food shortage) down-regulates neural activity in the IPCs. But the IPCs are not equipped with a cell-autonomous clock, so they must receive seasonal information from the brain clock neuron network. Indeed, two clock related neuropeptides~pigment dispersing factor and short neuropeptide F~from circadian morning pacemaker or M-cells have been implicated in regulating reproductive dormancy. Intriguingly, AstC-DN1p neurons are the DN1p subset that receives pigment dispersing factor signals from these M-cells. Furthermore, DN1p can process light and temperature information for the circadian regulation of behavior. Finally, the finding that AstC-DN1p generates the circadian vitellogenesis rhythm via the IPCs makes AstC-DN1p neurons the prime candidates for integrating the seasonal cues that control the entrance, maintenance, or exit from reproductive dormancy. Considering the functional and structural conservation between the AstC and SST systems, the SST system may also link the brain clock, GnRH, and/or its downstream reproductive pathways in controlling seasonal reproductive patterns in vertebrates (Zhang, 2021).

    Msl3 promotes germline stem cell differentiation in female Drosophila

    Gamete formation from germline stem cells (GSCs) is essential for sexual reproduction. However, the regulation of GSC differentiation is incompletely understood. Set2, which deposits H3K36me3 modifications, is required for GSC differentiation during Drosophila oogenesis. The H3K36me3 reader Male-specific lethal 3 (Msl3) and histone acetyltransferase complex Ada2a-containing (ATAC) cooperate with Set2 to regulate GSC differentiation in female Drosophila. Msl3, acting independently of the rest of the male-specific lethal complex, promotes transcription of genes, including a germline-enriched ribosomal protein S19 paralog RpS19b. RpS19b upregulation is required for translation of RNA-binding Fox protein 1 (Rbfox1), a known meiotic cell cycle entry factor. Thus, Msl3 regulates GSC differentiation by modulating translation of a key factor that promotes transition to an oocyte fate (McCarthy, 2022).

    Increased levels of superoxide dismutase suppress meiotic segregation errors in aging oocytes

    The risk of meiotic segregation errors increases dramatically during a woman's thirties, a phenomenon known as the maternal age effect. In addition, several lines of evidence indicate that meiotic cohesion deteriorates as oocytes age. One mechanism that may contribute to age-induced loss of cohesion is oxidative damage. In support of this model, it has been reported that the knockdown of the reactive oxygen species (ROS)-scavenging enzyme, superoxide dismutase (SOD), during meiotic prophase causes premature loss of arm cohesion and segregation errors in Drosophila oocytes. If age-dependent oxidative damage causes meiotic segregation errors, then the expression of extra SOD1 (cytosolic/nuclear) or SOD2 (mitochondrial) in oocytes may attenuate this effect. To test this hypothesis, flies were generatee that contain a UAS-controlled EMPTY, SOD1, or SOD2 cassette, and expression was induced using a Gal4 driver that turns on during meiotic prophase. The fidelity of chromosome segregation was compared in aged and non-aged Drosophila oocytes for all three genotypes. As expected, p{EMPTY} oocytes subjected to aging exhibited a significant increase in nondisjunction (NDJ) compared with non-aged oocytes. In contrast, the magnitude of age-dependent NDJ was significantly reduced when expression of extra SOD1 or SOD2 was induced during prophase. These findings support the hypothesis that a major factor underlying the maternal age effect in humans is age-induced oxidative damage that results in premature loss of meiotic cohesion. Moreover, this work raises the exciting possibility that antioxidant supplementation may provide a preventative strategy to reduce the risk of meiotic segregation errors in older women (Perkins, 2019).

    Insulin signaling acts in adult adipocytes via GSK-3beta and independently of FOXO to control Drosophila female germline stem cell numbers

    Tissue-specific stem cells are tied to the nutritional and physiological environment of adult organisms. Adipocytes have key endocrine and nutrient-sensing roles and have emerged as major players in relaying dietary information to regulate other organs. For example, previous studies in Drosophila melanogaster revealed that amino acid sensing as well as diet-dependent metabolic pathways function in adipocytes to influence the maintenance of female germline stem cells (GSCs). How nutrient-sensing pathways acting within adipocytes influence adult stem cell lineages, however, is just beginning to be elucidated. This study reports that insulin/insulin-like growth factor signaling in adipocytes promotes GSC maintenance, early germline cyst survival, and vitellogenesis. Further, adipocytes use distinct mechanisms downstream of insulin receptor activation to control these aspects of oogenesis, all of which are independent of FOXO. GSC maintenance is modulated by Akt1 through GSK-3beta, early germline cyst survival is downstream of adipocyte Akt1 but independent of GSK-3beta, and vitellogenesis is regulated through an Akt1-independent pathway in adipocytes. These results indicate that, in addition to employing different types of nutrient sensing, adipocytes can use distinct axes of a single nutrient-sensing pathway to regulate multiple stages of the GSC lineage in the ovary (Armstrong, 2018).

    Makorin 1 is required for Drosophila oogenesis by regulating insulin/Tor signaling

    Reproduction is a process that is extremely sensitive to changes in nutritional status. The nutritional control of oogenesis via insulin signaling has been reported; however, the mechanism underlying its sensitivity and tissue specificity has not been elucidated. This study determined that Drosophila Makorin RING finger protein 1 gene (Mkrn1) functions in the metabolic regulation of oogenesis. Mkrn1 was endogenously expressed at high levels in ovaries and Mkrn1 knockout resulted in female sterility. Mkrn1-null egg chambers were previtellogenic without egg production. FLP-FRT mosaic analysis revealed that Mkrn1 is essential in germline cells, but not follicle cells, for ovarian function. As well, AKT phosphorylation via insulin signaling was greatly reduced in the germline cells, but not the follicle cells, of the mutant clones in the ovaries. Furthermore, protein-rich diet elevated Mkrn1 protein levels, without increased mRNA levels. The p-AKT and p-S6K levels, downstream targets of insulin/Tor signaling, were significantly increased by a nutrient-rich diet in wild-type ovaries whereas those were low in Mkrn1exS compared to wild-type ovaries. Taken together, these results suggest that nutrient availability upregulates the Mkrn1 protein, which acts as a positive regulator of insulin signaling to confer sensitivity and tissue specificity in the ovaries for proper oogenesis based on nutritional status (Jeong, 2019).

    gone early, a novel germline factor, ensures the proper size of the stem cell precursor pool in the Drosophila ovary

    In order to sustain lifelong production of gametes, many animals have evolved a stem cell-based gametogenic program. In the Drosophila ovary, germline stem cells (GSCs) arise from a pool of primordial germ cells (PGCs) that remain undifferentiated even after gametogenesis has initiated. The decision of PGCs to differentiate or remain undifferentiated is regulated by somatic stromal cells: specifically, epidermal growth factor receptor (EGFR) signaling activated in the stromal cells determines the fraction of germ cells that remain undifferentiated by shaping a Decapentaplegic (Dpp) gradient that represses PGC differentiation. However, little is known about the contribution of germ cells to this process. This study shows that a novel germline factor, Gone early (Goe; CG9634), limits the fraction of PGCs that initiate gametogenesis. goe encodes a non-peptidase homologue of the Neprilysin family metalloendopeptidases (see Neprilysin 4). At the onset of gametogenesis, Goe was localized on the germ cell membrane in the ovary, suggesting that it functions in a peptidase-independent manner in cell-cell communication at the cell surface. Overexpression of Goe in the germline decreased the number of PGCs that enter the gametogenic pathway, thereby increasing the proportion of undifferentiated PGCs. Inversely, depletion of Goe increased the number of PGCs initiating differentiation. Excess PGC differentiation in the goe mutant was augmented by halving the dose of argos, a somatically expressed inhibitor of EGFR signaling. This increase in PGC differentiation resulted in a massive decrease in the number of undifferentiated PGCs, and ultimately led to insufficient formation of GSCs. Thus, acting cooperatively with a somatic regulator of EGFR signaling, the germline factor goe plays a critical role in securing the proper size of the GSC precursor pool. Because goe can suppress EGFR signaling activity and is expressed in EGF-producing cells in various tissues, goe may function by attenuating EGFR signaling, and thereby affecting the stromal environment (Matsuoka, 2014: PubMed).

    Regulation of ribosome biogenesis and protein synthesis controls germline stem cell differentiation

    Complex regulatory networks regulate stem cell behavior and contributions to tissue growth, repair, and homeostasis. A full understanding of the networks controlling stem cell self-renewal and differentiation, however, has not yet been realized. To systematically dissect these networks and identify their components, this study performed an unbiased, transcriptome-wide in vivo RNAi screen in female Drosophila germline stem cells (GSCs). Based on characterized cellular defects, 646 identified genes were classified into phenotypic and functional groups, and a comprehensive set was unveiled of networks regulating GSC maintenance, survival, and differentiation. This analysis revealed an unexpected role for ribosomal assembly factors in controlling stem cell cytokinesis. Moreover, the data show that the transition from self-renewal to differentiation relies on enhanced ribosome biogenesis accompanied by increased protein synthesis. Collectively, these results detail the extensive genetic networks that control stem cell homeostasis and highlight the intricate regulation of protein synthesis during differentiation (Sanchez, 2015).

    DNA damage-induced CHK2 activation compromises germline stem cell self-renewal and lineage differentiation

    This study used germline stem cells (GSCs) in the Drosophila ovary to show that DNA damage retards stem cell self-renewal and lineage differentiation in a CHK2 kinase-dependent manner. Both heatshock-inducible endonuclease I-CreI expression and X-ray irradiation can efficiently introduce double-strand breaks in GSCs and their progeny, resulting in a rapid GSC loss and an accumulation of ill-differentiated GSC progeny. Elimination of CHK2 or its kinase activity can almost fully rescue the GSC loss and the progeny differentiation defect caused by DNA damage induced by I-CreI or X-ray. Surprisingly, checkpoint kinases ATM and ATR have distinct functions from CHK2 in GSCs in response to DNA damage. The reduction in BMP signaling and E-cadherin only makes limited contribution to DNA damage-induced GSC loss. Finally, DNA damage also decreases the expression of the master differentiation factor Bam in a CHK2-dependent manner, which helps explain the GSC progeny differentiation defect. Therefore, this study demonstrates, for the first time in vivo, that CHK2 kinase activation is required for the DNA damage-mediated disruption of adult stem cell self-renewal and lineage differentiation, and might also offer novel insight into how DNA damage causes tissue aging and cancer formation. It also demonstrates that inducible I-CreI is a convenient genetic system for studying DNA damage responses in stem cells (Ma, 2016).

    Stem cells in adult tissues are responsible for generating new cells to combat against aging, and could also be cellular targets for tumor formation. Although aged stem cells have been shown to accumulate DNA damage, it remains largely unclear how DNA damage affects stem cell self-renewal and differentiation. A previous study has reported that upon weak irradiation apoptotic differentiated GSC progeny can prevent GSC loss by activating Tie-2 receptor tyrosine kinase signaling (Xing, 2015). This study shows that temporally introduced DNA double-stranded breaks cause premature GSC loss and slow down GSC progeny differentiation. Mechanistically, DNA damage causes GSC loss at least via two independent mechanisms, down-regulation of BMP signaling and E-cadherin-mediated GSC-niche adhesion as well as CHK2 activation- dependent GSC loss. In addition, CHK2 activation also decreases Bam protein expression by affecting its gene transcription and translation, slowing down CB differentiation into mitotic cysts and thus causing the accumulation of CB-like cells. Surprisingly, unlike in many somatic cell types, ATM, ATR, CHK1 and p53 do not work with CHK2 in DNA damage checkpoint control in Drosophila ovarian GSCs. Therefore, this study demonstrates that DNA damage-induced CHK2 activation causes premature GSC loss and also retards GSC progeny differentiation. The findings could also offer insight into how DNA damage affects stem cell-based tissue regeneration. In addition, this study also shows that the inducible I-CreI system is a convenient method for studying stem cell responses to transient DNA damage because it does not require any expensive irradiation equipment as the X-ray radiation does (Ma, 2016).

    DNA damage normally leads to cell apoptosis to eliminate potential cancer- forming cells. This study, shows that transient DNA damage causes GSC loss not through apoptosis based on twopieces of experimental evidence: first, DNA-damaged GSCs are not positive for the cleaved Caspase-3, a widely used apoptosis marker; Second, forced expression of a known apoptosis inhibitor p35 does not show any rescue effect on DNA damage-induced GSC loss. Thus, DNA damage-induced GSC loss is likely due to self-renewal defects though the possibility could not be ruled out that other forms of cell death are responsible. p53 is known to be required for DNA damage-induced apoptosis from flies to humans. This study, however, demonstrates that p53 prevents the DNA damage-induced GSC loss. Vacating DNA-damaged GSCs from the niche via differentiation might allow their timely replacement and restoration of normal stem cell function. Therefore, the findings argue strongly that DNA damage primarily compromises self-renewal, thus causing GSC loss. Both niche-activated BMP signaling and E-cadherin-mediated cell adhesion are essential for GSC self-renewal. Consistent with the idea that DNA damage compromises GSC self-renewal, it significantly decreases BMP signaling activity and apical accumulation of E-cadherin in GSCs. Since constitutively active BMP signaling alone or in combination with E-cadherin overexpression can only moderately rescue GSC loss caused by DNA damage, it is concluded that decreased BMP signaling and apical E-cadherin accumulation might partly contribute to the DNA damage-induced GSC loss. Therefore, the findings suggest that DNA damage-mediated down-regulation of BMP signaling and E-cadherin-mediated adhesion only moderately contributes to the GSC loss (Ma, 2016).

    DNA damage leads to checkpoint activation and cell cycle slowdown, thus giving more time for repairing DNA damage. In various cell types, ATM-CHK2 and ATR-CHK1 kinase pathways are responsible for DNA damage-induced checkpoint activation. During Drosophila meiosis, ATR, but not ATM, is required for checkpoint activity, indicating that ATM and ATR could have different functions in germ cells. Both ATR and CHK2 have been shown to be required for DNA damage-evoked checkpoint control in Drosophilagerm cells and embryonic cells, while CHK1 can control the entry into the anaphase of cell cycle in response to DNA damage, the G2-M checkpoint activation as well as the Drosophila midblastula transition (Ma, 2016).

    This study has shown that these four checkpoint kinases function differently in GSCs. First, CHK2 is required for DNA damage-induced GSC loss, but is dispensable for normal GSC maintenance. Particularly, inactivation of its kinase activity can almost fully rescue DNA damage-induced GSC loss. Interestingly, inactivation of CHK2 function can also rescue the female germ cell defect caused by DNA damage in the mouse ovary, indicating that CHK2 function in DNA damage checkpoint activation is conserved at least in female germ cells. However, it remains unclear if CHK2 behaves similarly in mammalian stem cells in response to DNA damage. Second, ATM promotes GSC maintenance in the absence and presence of DNA damage. This is consistent with the finding that ATM is required for the maintenance of mouse male germline stem cells and hematopoietic stem cells. It will be interesting to investigate if ATM also prevents the oxidative stress in Drosophila GSCs as in mouse hematopoietic stem cells. Third, ATR is dispensable for normal GSC maintenance, but it protects GSCs in the presence of DNA damage. Although CHK2 and ATR behave similarly in DNA damage checkpoint control during meiosis and late germ cell development, they behave in an opposite way in GSCs in response to DNA damage. Finally, CHK1 is dispensable for GSC self-renewal in the absence and presence of DNA damage. Consistent with the current findings, the females homozygous for grp, encoding CHK1 in Drosophila, can still normally lay eggs, but those eggs could not develop normally. It will be of great interest in the future to figure out how CHK2 inactivation prevents DNA damage-induced GSC loss and how ATM and ATR inactivation promotes DNA damage-induced GSC loss at the molecular level. A further understanding of the functions of CHK2, ATM and ATR in stem cell response to DNA damage will help preserve aged stem cells and prevent their transformation into CSCs. DNA damage-evoked CHK2 activation retards GSC progeny differentiation by decreasing Bam expression at least at two levels This study has also revealed a novel mechanism of how DNA damage affects stem cell differentiation. Bam is a master differentiation regulator controlling GSC- CB and CB-cyst switches in the Drosophila ovary: CB-like single germ cells accumulate in bam mutant ovaries, whereas forced Bam expression sufficiently drives GSC differentiation. This study shows that DNA damage causes the accumulation of CB-like cells in a CHK2- dependent manner because CHK2 inactivation can fully rescue the germ cell differentiation defect caused by DNA damage. In addition, a heterozygous bam mutation can drastically enhance, and forced bam expression can completely repress, the DNA damage-induced germ cell differentiation defect, indicating that DNA damage disrupts Bam-dependent differentiation pathways. Consistently, Bam protein expression is significantly decreased in DNA damaged mitotic cysts in comparison with control ones. Interestingly, CHK2 inactivation can also fully restore Bam protein expression levels in the DNA-damaged mitotic cysts. Taken together, CHK2 activation is largely responsible forBam down-regulation in DNA damaged mitotic cysts, which can mechanistically explain the DNA damage-induced germ cell differentiation defect. It was further shown that DNA damage decreases Bam protein expression at least at two different levels. First, the bam transcription reporter bam-gfp was used to show that DNA damage decreases bamtranscription in CBs and mitotic cysts. Second, the posttranscriptional reporter Pnos-GFP-bam 3'UTR was generated to show that DNA damage decreases Bam protein expression via its 3'UTR in CBs and mitotic cysts at the level of translation. Although the detailed molecular mechanisms underlying regulation of Bam protein expression by DNA damage await future investigation, these findings demonstrate that DNA damage causes the GSC progeny differentiation defect by decreasing Bam protein expression at transcriptional and translational levels (Ma, 2016).

    Taken together, these findings from Drosophila ovarian GSCs could offer important insight into how DNA damage affects stem cell-based tissue regeneration, and have also established Drosophila ovarian GSCs as a new paradigm for studying how DNA damage affects stem cell behavior at the molecular level. Because many stem cell regulatory strategies are conserved from Drosophilato mammals, what has been learned from this study should help understand how mammalian adult stem cells respond to DNA damage (Ma, 2016).

    Identification of germline transcriptional regulatory elements in Aedes aegypti

    The mosquito Aedes aegypti is the principal vector for the yellow fever and dengue viruses, and is also responsible for recent outbreaks of the alphavirus chikungunya. Vector control strategies utilizing engineered gene drive systems are being developed as a means of replacing wild, pathogen transmitting mosquitoes with individuals refractory to disease transmission, or bringing about population suppression. Several of these systems, including Medea, UDMEL, and site-specific nucleases, which can be used to drive genes into populations or bring about population suppression, utilize transcriptional regulatory elements that drive germline-specific expression. This study report the identification of multiple regulatory elements able to drive gene expression specifically in the female germline, or in the male and female germline, in the mosquito Aedes aegypti. These elements can also be used as tools with which to probe the roles of specific genes in germline function and in the early embryo, through overexpression or RNA interference (Akbari, 2014).

    Protein competition switches the function of COP9 from self-renewal to differentiation

    The balance between stem cell self-renewal and differentiation is controlled by intrinsic factors and niche signals. In the Drosophila melanogaster ovary, some intrinsic factors promote germline stem cell (GSC) self-renewal, whereas others stimulate differentiation. However, it remains poorly understood how the balance between self-renewal and differentiation is controlled. This study used D. melanogaster ovarian GSCs to demonstrate that the differentiation factor Bam controls the functional switch of the COP9 complex (see CSN5) from self-renewal to differentiation via protein competition. The COP9 complex is composed of eight Csn subunits, Csn1-8, and removes Nedd8 modifications from target proteins. Genetic results indicated that the COP9 complex is required intrinsically for GSC self-renewal, whereas other Csn proteins, with the exception of Csn4, were also required for GSC progeny differentiation. Bam-mediated Csn4 sequestration from the COP9 complex via protein competition inactivated the self-renewing function of COP9 and allowed other Csn proteins to promote GSC differentiation. Therefore, this study reveals a protein-competition-based mechanism for controlling the balance between stem cell self-renewal and differentiation. Because numerous self-renewal factors are ubiquitously expressed throughout the stem cell lineage in various systems, protein competition may function as an important mechanism for controlling the self-renewal-to-differentiation switch (Pan, 2014).

    Evidence for chromatin-remodeling complex PBAP-controlled maintenance of the Drosophila ovarian germline stem cells

    In the Drosophila oogenesis, germline stem cells (GSCs) continuously self-renew and differentiate into daughter cells for consecutive germline lineage commitment. This developmental process has become an in vivo working platform for studying adult stem cell fate regulation. An increasing number of studies have shown that while concerted actions of extrinsic signals from the niche and intrinsic regulatory machineries control GSC self-renewal and germline differentiation, epigenetic regulation is implicated in the process. This study reports that Brahma (Brm), the ATPase subunit of the Drosophila SWI/SNF chromatin-remodeling complexes, is required for maintaining GSC fate. Removal or knockdown of Brm function in either germline or niche cells causes a GSC loss, but does not disrupt normal germline differentiation within the germarium evidenced at the molecular and morphological levels. There are two Drosophila SWI/SNF complexes: the Brm-associated protein (BAP) complex and the polybromo-containing BAP (PBAP) complex. More genetic studies reveal that mutations in polybromo/bap180, rather than gene encoding Osa, the BAP complex-specific subunit, elicit a defect in GSC maintenance reminiscent of the brm mutant phenotype. Further genetic interaction test suggests a functional association between brm and polybromo in controlling GSC self-renewal. Taken together, studies in this paper provide the first demonstration that Brm in the form of the PBAP complex functions in the GSC fate regulation (He, 2014. PubMed ID: 25068272).

    Pleiotropic functions of the chromodomain-containing protein Hat-trick during oogenesis in Drosophila melanogaster

    Chromatin-remodeling proteins play a profound role in the transcriptional regulation of gene expression during development. This study shows that the chromodomain-containing protein Hat-trick is predominantly expressed within the oocyte nucleus, specifically within the heterochromatinized karyosome and that a mild expression is observed in follicle cells. Co-localization of Hat-trick with Heterochromatin Protein 1 and a Synaptonemal Complex component- C(3)G along with the diffused karyosome after hat-trick down-regulation shows the role of this protein in heterochromatin clustering and karyosome maintenance. Germline mosaic analysis reveals that hat-trick is required for maintaining the dorso-ventral patterning of eggs by regulating the expression of Gurken. The increased incidence of Double strand breaks (DSBs), delayed DSB repair, defects in karyosome formation, altered Vasa mobility and consequently misexpression and altered localization of Gurken in hat-trick mutant egg chambers, clearly suggest a putative involvement of Hat-trick in the early stages of oogenesis. In addition, based on phenotypic observations in hat-trick mutant egg chambers, it is speculated that hat-trick plays a substantial role in cystoblast proliferation, oocyte determination, nurse cell endoreplication, germ cell positioning, cyst encapsulation, and nurse cell migration. These results demonstrate that hat-trick has profound pleiotropic functions during oogenesis in Drosophila melanogaster (Singh, 2018).

    The Drosophila LIN54 homolog Mip120 controls two aspects of oogenesis

    The conserved multi-protein MuvB core associates with the Myb oncoproteins and with the RB-E2F-DP tumor suppressor proteins in complexes that regulate cell proliferation, differentiation, and apoptosis. Drosophila Mip120, a homolog of LIN54, is a sequence-specific DNA-binding protein within the MuvB core. A mutant of Drosophila mip120 was previously shown to cause female and male sterility. This study shows that Mip120 regulates two different aspects of oogenesis. First, in the absence of the Mip120 protein, egg chambers arrest during the transition from stage 7 to 8 with a failure of the normal program of chromosomal dynamics in the ovarian nurse cells. Specifically, the decondensation, disassembly and dispersion of the endoreplicated polytene chromosomes fail to occur without Mip120. The conserved carboxy-terminal DNA-binding and protein-protein interaction domains of Mip120 are necessary but are not sufficient for this process. Second, a lack of Mip120 was shown to cause a dramatic increase in the expression of benign gonial cell neoplasm (bgcn), a gene that is normally expressed in only a small number of cells within the ovary including the germline stem cells (Cheng, 2017).

    Specification and spatial arrangement of cells in the germline stem cell niche of the Drosophila ovary depend on the Maf transcription factor Traffic jam

    Germline stem cells in the Drosophila ovary are maintained by a somatic niche. The niche is structurally and functionally complex and contains four cell types, the escort, cap, and terminal filament cells and the newly identified transition cell. The large Maf transcription factor Traffic jam (Tj) is essential for determining niche cell fates and architecture, enabling each niche in the ovary to support a normal complement of 2-3 germline stem cells. In particular, this study focused on the question of how cap cells form. Cap cells express Tj and are considered the key component of a mature germline stem cell niche. It is concluded that Tj controls the specification of cap cells, as the complete loss of Tj function caused the development of additional terminal filament cells at the expense of cap cells, and terminal filament cells developed cap cell characteristics when induced to express Tj. Further, it is proposed that Tj controls the morphogenetic behavior of cap cells as they adopted the shape and spatial organization of terminal filament cells but otherwise appeared to retain their fate when Tj expression was only partially reduced. The data indicate that Tj contributes to the establishment of germline stem cells by promoting the cap cell fate, and controls the stem cell-carrying capacity of the niche by regulating niche architecture. Analysis of the interactions between Tj and the Notch (N) pathway indicates that Tj and N have distinct functions in the cap cell specification program. It is proposed that formation of cap cells depends on the combined activities of Tj and the N pathway, with Tj promoting the cap cell fate by blocking the terminal filament cell fate, and N supporting cap cells by preventing the escort cell fate and/or controlling the number of cap cell precursors (Panchal, 2017).

    Stem cells retain the capacity for development in differentiated organisms, which is important for tissue growth, homeostasis and regeneration, and for long-term reproductive capability. Stem cells are often associated with a specialized microenvironment, a niche that is essential for the formation, maintenance, and self-renewal of stem cells by preventing cell differentiation and controlling rate and mode of cell division. The niche for the germline stem cells (GSCs) in Drosophila serves as an important model for the analysis of interactions between niche and stem cells. The astounding fecundity of Drosophila females that can lay dozens of eggs per day over several weeks depends on approximately 100 GSCs that are sustained by 40 stem cell niches. To understand the formation and maintenance of these GSCs, it is important to understand how stem cell niches form and how they function (Panchal, 2017).

    The GSC niche of the Drosophila ovary consists of three somatic cell types: cap cells, escort cells, and terminal filament (TF) cells. GSCs are anchored to cap cells by DE-cadherin-mediated adhesion and require close proximity to cap cells to retain stem cell character. Cap cells secrete the BMP homolog Decapentaplegic (Dpp), activating the TGFβ signaling pathway in adjacent GSCs, which leads to the repression of the germline differentiation factor Bag-of-Marbles (Bam). Through Hedgehog (Hh) signaling, cap cells also appear to stimulate escort cells to secrete Dpp. The combined pool of Dpp from cap and escort cells, together with mechanisms that concentrate Dpp in the extracellular space around GSCs, promotes the maintenance of 2-3 GSCs, whereas the adjacent GSC daughter cells that have lost the contact to cap cells will enter differentiation as cystoblasts. In contrast, TFs are not in direct contact with GSCs but serve important functions in the development and probably also in the maintenance and function of GSC niches (Panchal, 2017).

    Formation of GSC niches begins with the progressive assembly of TFs by cell intercalation during the 3rd larval instar. The process of TF cell specification is not understood but might start in 2nd instar when the first TF precursor cells appear to leave the cell cycle. TF morphogenesis depends on the Bric à brac transcriptional regulators that control the differentiation of TF cells and their ability to form cell stacks, and involves the Ecdysone Receptor (EcR), Engrailed, Cofilin, and Ran-binding protein M (RanBPM). The number of TFs that form at the larval stage determine the number of GSC niches at the adult stage, and are regulated by several signaling pathways that control cell division and timing of cell differentiation in the larval ovary, including the EcR, Hippo and Jak/Stat, Insulin and Activin pathways. Despite the recent advance in elucidating mechanisms that control the number of GSC niches and the temporal window in which they form, relatively little is known about the origin and specification of the somatic cell types of the GSC niche (Panchal, 2017).

    Notably, the origin and specification of cap cells, the main component of an active GSC niche is little understood. Cap cells (also called germarial tip cells) are first seen at the base of completed TFs at the transition from the 3rd larval instar to prepupal stage. They appear to derive from the interstitial cells (also called intermingled cells) of the larval ovary that are maintained by Hh signaling from TFs. The formation of cap cells is accompanied by the establishment of GSCs. The N pathway contributes to the development of cap cells. A strongly increased number of functionally active cap cells per niche form in response to overexpression of the N ligand Delta (Dl) in germline or somatic cells, or the constitutive activation of N in somatic gonadal cells. The ability of N to induce additional cap cells seems to depend on EcR signaling. Loss of Dl or N in the germline had no effect on cap cells. However, loss of N in cap cell progenitors or Dl in TF cells caused a decrease in the number of cap cells. A current model suggests that Dl signaling from basal-most TF cells to adjacent somatic cells together with Dl signaling between cap cells allows for a full complement of cap cells to form. Furthermore, N protects cap cells from age-dependent loss as long as its activity is maintained by the Insulin receptor. The Jak/Stat pathway, which operates downstream or in parallel to the N pathway in the niche, is not required for cap cell formation. As cap cells were reduced in number but never completely missing when the N pathway components were compromised, the question remains whether N signaling is the only factor that is important for cap cell formation. Furthermore, no factor that operates downstream of N has been identified that is crucial for cap cell formation (Panchal, 2017).

    This study finds that Traffic jam (Tj) is both required for cap cell specification and for the morphogenetic behavior of cap cells, enabling them to form a properly organized niche that can accommodate 2-3 GSCs. Tj is a large Maf transcription factor that belongs to the bZip protein family. Its four mammalian homologs control differentiation of several cell types and are associated with various forms of cancer. Tj is essential for normal ovary and testis development, and is only expressed in somatic cells of the gonad. Interestingly, Tj is present in cap cells and escort cells but not in TFs. This study shows that Tj is essential for the formation of the GSC niche. First, Tj regulates the behavior of cap cells, enabling them to form a cell cluster instead of a cell stack, which appears to be important for the formation of a normal-sized GSC niche with the capacity to support more than one GSC. Second, cap cells adopt the fate of TF cells in the absence of Tj function, and TF cells develop cap cell-like features when forced to express Tj, indicating that Tj specifies the cap cell fate. Genetic interactions suggest that Tj and N are required together for cap cell formation, but have different functions in this process. For somatic gonadal cells to adopt the cap cell fate, it is proposed that Tj has to be present to inhibit the TF cell fate and N has to be present to prevent the escort cell fate and/or produce the correct number of cap cell precursors (Panchal, 2017).

    Loss of Tj has a profound negative effect on the establishment, number, and maintenance of GSCs. Effects of Tj on the germline were previously shown to be indirect as Tj is neither expressed nor cell-autonomously required in the germline. Therefore, it is proposed that the dramatic change in the structure of the somatic niche affects GSCs when Tj function is compromised. An inverse causal relationship, where a reduced number of GSCs would trigger the somatic niche defects was ruled out by showing that cap cells can still look and behave normally in the absence of any germ cells. It is concluded that Tj controls GSCs indirectly by controlling somatic cell fate and cell arrangement in the stem cell niche (Panchal, 2017).

    By controlling the morphology and behavior of the cap cells, Tj regulates the GSC-carrying capacity of the niche. When Tj expression was moderately reduced, the number of GSCs per niche was reduced, with the remaining GSC properly maintained over several weeks. The decrease of GSCs per niche correlated with a decrease of cap cells in the germarium. Two cap cells were on average required to sustain one GSC, similar to what has been proposed for a wild-type ovary. The data indicate that the reduced niche capacity is due to a reduction in the available contact surface between cap cells and GSCs. Tj-depleted cap cells that convert from forming a cluster inside the germarium to forming a stalk outside the germarium minimize their availability for GSC attachment. A connection between the GSC-cap cell contact area and niche capacity is similarly reflected in the increased number of GSCs that accompanies an increase in cap cell size due to loss of RanBPM. This study shows that the spatial arrangement of the cap cells has a crucial impact on the number of stem cells per niche (Panchal, 2017).

    When Tj function was completely abolished, the number of GSCs was drastically reduced, as expected in the absence of cap cells. The very few pMad-positive GSC-like cells in tj mutant prepupal ovaries were always associated with a TF, suggesting that TFs might temporarily provide enough Dpp to activate Mad in a few germline cells, consistent with the finding that Dpp is expressed in TFs at the late larval stage\. This is not sufficient, however, to maintain GSCs and adult ovaries rarely contain pMad-positive germline cells. This is in agreement with the finding that Dpp is not detected in adult TFs, and corroborates that cap cells are required for GSC maintenance. In addition, the rapid loss of the entire germ cell pool in Tj-depleted ovaries during the pupal stage might be precipitated by loss or defects in escort cells. Escort cell precursors are not properly intermingled with germ cells at the larval stage and differentiated escort cells appear to be missing in adult ovaries that lack Tj. As escort cells are crucial for germ cell differentiation, the defect in escort cell differentiation could be responsible for the demise of the germline in tj mutants (Panchal, 2017).

    GSCs have broad cellular protrusions, which they use to reach and tightly ensheath the accessible surface of cap cells. In wild type, relatively short protrusions are sufficient to make extensive contact with more than one cap cell. However, when cap cells formed a stalk, GSCs were often observed to produce unusually long extensions that allowed them not only to contact the immediate cap cell neighbor but also a more distantly located cap cell. This suggests that GSCs respond to a chemotactic signal from cap cells and send protrusions toward this signal. It remains to be investigated whether this is a response to Dpp signaling or signaling through another pathway. The importance of cellular protrusions in signaling events in the stem cell niche has recently come to light with the discovery of nanotubes that mediate Dpp signaling between GSCs and hub cells in the Drosophila testes, and cytonemes that contribute to Hh signaling from cap to escort cells in the ovary (Panchal, 2017).

    This analysis shows that Tj is required for the specification of cap cells. In the absence of Tj function, additional TF cells form at the expense of cap cells, resulting in unusually long TFs while the cap cell fate is not established. Whereas the formation of cap cell precursors appears not to require Tj, this transcription factor is essential for the ability of these precursors to take on the cap cell fate and to prevent the TF cell fate that is otherwise adopted as a default state. The following findings support this conclusion: (1) In the absence of Tj function, cap cells were missing while additional cells that displayed TF cell-characteristic morphology, behavior and marker expression were integrated into the TF. The number of additional TF cells was comparable to the normal number of cap cells. (2) Prospective cap cells cell-autonomously adopted a TF-specific morphology and behavior in the absence of functional Tj. (3) A hypomorphic tj mutant provided direct evidence for the incorporation of cap cells into TFs, forming the basal portion of these stalks. (4) Ectopic expression of Tj in TF cells caused a change toward cap cell-typical marker expression and morphology. Together, these data demonstrate that Tj promotes cap cell specification (Panchal, 2017).

    The expression pattern of Tj supports the notion that Tj has a function in cap cells but not in TF cells. Tj is continuously expressed in cap cells. Tj is also present in the anterior interstitial cells of the larval ovary, which are thought to develop into cap cells. In contrast, Tj is neither detected in the cell population that gives rise to TFs during 3rd larval instar, nor in differentiated TFs. Interestingly, even in the absence of Tj function, the tj gene remains differentially expressed in the anterior niche, being inactive in regular TF cells but active in the additional TF cells, which form the apical and basal portion of a TF, respectively. This differential expression of Tj indicates that a regionally or temporally regulated mechanism operates upstream of Tj that initiates differences in anterior niche cells. Although it is conspicuous that Tj expression from 3rd instar onwards is restricted to cells that are in direct contact with germline cells, which includes cap cells but excludes TF cells, it has previously been shown that Tj expression is not dependent on the germline. This suggests that a soma-specific mechanism is responsible for the differential expression of Tj in anterior niche cells. Interestingly, a recent study uncovered the importance of Hh signaling from TFs to neighboring interstitial cells in the larval ovary and proposes that tj is a direct target of the Hh signaling pathway (Panchal, 2017).

    The current findings suggest the presence of a new cell type in the GSC niche that has been named 'transition cell' as it is located between the cap cell cluster and the TF, connecting these two structures of the niche. Notably, the one or occasionally two transition cells have the morphology of TF cells and align with neighboring TF cells despite displaying a cap cell-like marker profile that includes the expression of Tj—although Tj expression is substantially lower than in cap cells. Interestingly, cap cells from ovaries with reduced Tj expression (tjhypo) similarly displayed a TF cell-like morphology and behavior while their expression profile remained cap cell-like. A similar, although weaker effect was noted in a tj hemizygous condition, suggesting that Tj function is haplo-insufficient in cap cells. Thus, when Tj levels are reduced, cap cells adopt very similar molecular and morphogenetic properties as the transition cell in a wild-type niche, and might have adopted this cell fate (Panchal, 2017).

    Together, the current findings indicate that Tj has an important role in the establishment of three cell types in the GSC niche: TF cells, transition cells, and cap cells. As lack of Tj function seems to cause a transformation of cap and transition cells into TF cells, and a mild reduction of Tj a cap to transition cell transformation, it is proposed that different Tj expression levels establish different cell fates and morphogenetic traits. It is proposed that a high concentration of Tj leads to the formation of cap cells and a lower concentration to the formation of the transition cell, whereas absence of Tj is required for the formation of TF cells. This model implies that different levels of Tj have different effects on target genes. It is predicted that Tj has at least one target gene that only responds to high levels of Tj and that specifically controls the morphogenetic behavior of cap cells, allowing them to adopt a round morphology and organize into a cell cluster. Whether this relates to an effect of Tj on the expression of adhesion molecules as observed in other gonadal tissues awaits further analysis (Panchal, 2017).

    This study identifies Tj as essential for cap cell formation. In addition, this process depends on the N pathway. Therefore, it was asked how the functions of Tj and N in cap cell formation relate to each other. A comparison between the loss and gain-of-function phenotypes suggests that Tj and N have different functions in the establishment of cap cells. In the absence of Tj function, cap cell precursor cells are present but take on the fate of TF cells, whereas depletion of N leads to a loss of cap cells but does not cause the formation of additional TF cells. Ectopic activation of N can induce a strong increase in the number of cap cells, whereas overexpression of Tj did not appear to affect the number of cap cells. Therefore, both factors are important for cap cell formation but contribute differently to this process. The questions then are: What is the respective contribution of Tj and N to cap cell formation, and how are their functions related (Panchal, 2017)?

    The function of N in cap cell formation is still not fully understood. The observation that depletion of N reduces the number of cap cells confirms previous findings. However, neither in this nor any previously published experiments were cap cells lost completely when the N pathway was compromised, and it remains therefore unclear whether N is de facto essential for cap cell formation or primarily functions in regulating the size of the cap cell pool. Interestingly, evidence amounts to a function of the N pathway in a decision between the cap cell and escort cell fate: First, Dl signal from TF cells activates the N pathway in adjacent interstitial cells, inducing them as cap cells, whereas the remaining interstitial cells are thought to develop into escort cells. Second, escort cells expressing activated N can develop into cap cells. Third, when tj-Gal4 was used to express active N in interstitial cells, the number of cap cells dramatically increased while the escort cell region became smaller, and some germaria seemed to lack escort cells all together. These germaria also lacked germline cells, although a larger pool of cap cells was expected to increase the number of GSCs. However, the absence of germline cells is consistent with an absence of escort cells, as escort cells have been shown to be important for maintaining the germline. Together, these observations support the hypothesis that N is involved in a cap cell versus escort cell fate decision, and suggest that the N pathway might promote the formation of cap cells by inhibiting the escort cell fate (Panchal, 2017).

    To determine how the functions of Tj and N depend on each other, genetic interactions were examined. The N pathway seems to be still functional in tj mutants. First, the expression of N and Dl appeared unaffected and E(spl) was activated in the additional TF cells (= transformed cap cells) similarly to normal cap cells. Second, the formation of additional TF cells in the absence of Tj depended on the presence of N, as only very few additional TF cells formed in a N compromised background. These findings indicate that the N pathway is still active in cap cell precursors when Tj function is abolished. This together with the observation that constitutively active N cannot suppress the tj mutant phenotype suggests that Tj does not act upstream of N in regulating cap cell fate (Panchal, 2017).

    Therefore, it was asked whether Tj might operate downstream of N. Loss of Tj was not detected upon N depletion, and this together with the finding that Tj is expressed in all interstitial cells, and not only in those that receive Dl signaling argues against a requirement of N signaling for tj expression. If at all, one would expect tj to be negatively regulated by N as cap cells express a lower level of Tj than escort cells. The maintenance of somatic cell types in N mutant ovaries that are lost in tj mutant ovaries, including the escort cells is also not consistent with a linear relationship. Nevertheless, the ability of Tj to promote the formation of cap cells appears to depend on the activity of the N pathway in cap cell precursors. Again, this is suggested by the finding that when N and Tj were both compromised, the number of additional TF cells were much smaller than when N was fully active. Therefore, it is proposed that N activity sets aside a pool of percursor cells that in the presence of Tj take on the cap cell fate, and in its absence the TF fate (Panchal, 2017).

    Similar to the ovary, N is important for the formation of the GSC niche (= hub) in the Drosophila testis. Interestingly, N contributes to hub cell specification by downregulating the expression level of Tj. Not only is the hub still present in tj mutant testes but additionally, ectopic hub cells form in the absence of Tj. Thus, Tj seems to have opposing functions in testes and ovaries, suppressing the niche cell fate in the testis, while promoting it in the ovary (Panchal, 2017).

    The interplay between Tj and N seems not restricted to the cap cell fate in the ovary. Whereas neither factor alone is required for TF cell formation, as TF cells formed normally in the absence of either Tj or N, the combined loss of Tj and N led to a strong reduction in the number of TFs and number of TF cells within stalks. This suggests that their combined action is already required at an earlier stage of ovary development, when Tj is still expressed in all somatic cells of the ovary. Moreover, Tj knockdown combined with expression of activated N caused TF cells to be the only cell type remaining of the ovary, indicating that several cell types in the ovary require proper input from both factors. Taken together, the findings support a model, in which both Tj and N operate together to promote the cap cell fate but have separate functions. It is proposed that Tj and N promote the cap cell fate by blocking the TF cell fate and escort cell fate, respectively, and that the combined actions of Tj and the N pathway are required to establish the cap cell fate (Panchal, 2017).

    The NuRD nucleosome remodelling complex and NHK-1 kinase are required for chromosome condensation in oocytes>

    Chromosome condensation during condensin I subunits or topoisomerase II in oocytes only mildly affected chromosome condensation. In contrast, severe undercondensation of chromosomes after depletion of the Mi-2 containing NuRD nucleosome remodelling complex or the protein kinase NHK-1/ballchen. The further phenotypic analysis suggests that Mi-2 and NHK-1 are involved in different pathways in chromosome condensation. The main role of NHK-1 in chromosome condensation is to phosphorylate BAF and suppress its activity in linking chromosomes to nuclear envelope proteins. Further this study showed that NHK-1 is important for chromosome condensation in mitosis as well as in oocytes (Nikalayevich, 2014).

    During cell division, chromosomes undergo morphological changes from a cloud-like interphase morphology into rod-like structures. This transformation is referred to as chromosome condensation. Chromosome condensation is important for faithful chromosome segregation during cell division. The organisation of condensed metaphase chromosomes has been a focus of debate for a long time, and various models have been proposed. One model is that there is a hierarchical organisation, starting from nucleosomes folded into a 30-nm fibre, which form larger and larger loops. Another long-standing, and not mutually exclusive, model is that there is a chromosome scaffold, which has been observed after removal of DNA and most of the chromosome proteins from the metaphase chromosomes. However, the existence and the biological role of this scaffold are subjects of continuous discussion. The most recently proposed model is a polymer model based on data from a chromosome conformation capture method. This proposes that there is a compressed linear array of loops without hierarchical organisation (Nikalayevich, 2014).

    Among thousands of proteins found in metaphase chromosomes, condensin complexes and topoisomerase II have been studied most extensively for their involvement in chromosome condensation during cell division. The condensin complex was originally found as the main chromosome condensation factor in Xenopus extract. The involvement of condensin complexes in this process has been demonstrated in many systems. Higher eukaryotes have two condensin complexes, condensin I and II. The two complexes appear to have different localisations and functions. The exact molecular mechanism by which condensin functions has not been established, but it has an ability to positively supercoil DNA (Nikalayevich, 2014).

    It has been demonstrated in several systems that topoisomerase II is required for chromosome structure as well as correct chromosome segregation in mitosis and meiosis. Topoisomerase II is present on chromosomes in mitosis and meiosis and is also enriched on centromeres and pericentromeric regions during meiosis. Topoisomerase II decatenates supercoiled DNA by introducing temporary double-strand DNA breaks, and it has been suggested and demonstrated that topoisomerase II acts in opposition to condensin and KIF4A. Both condensin and topoisomerase II are required for the correct chromatin structure of the centromere (Nikalayevich, 2014).

    Despite extensive research on the roles of condensin and topoisomerase II in chromosome condensation, some evidence casts doubts on whether these proteins are the only major factors involved in chromosome condensation. In some systems, chromosomes are still able to condense, with various abnormalities, after depletion of condensin subunits. Depletion of condensin does not prevent condensation of chromosomes until the initiation of anaphase, but causes chromosomes to decondense prematurely during anaphase. This has led to a proposal that there is a 'regulator of chromosome architecture' (RCA), an as yet unidentified factor, which acts redundantly with condensin to condense metaphase chromosomes (Nikalayevich, 2014).

    Evidence suggests that there are crucial chromosome condensation factors other than condensin and topoisomerase II. Recently, attempts have been made to find new chromosome condensation factors. For example, a chromosome condensation assay allowed high-throughput analysis of genes required for chromosome condensation in fission yeast. In that study, eight new conditional condensin alleles were discovered, together with a new role for DNA polymerase ε (pol ε) and F-box DNA helicase I in chromosome condensation. In addition, a very recent study has identified mutations in several genes that cause chromosome segregation defects similar to those induced by depletion of condensin. Four out of five of these genes encode components of the nucleosome-remodelling complexes (Nikalayevich, 2014)

    This report describes the first use of Drosophila oocytes to study chromosome condensation. RNA interference (RNAi)-mediated depletion of a set of chromosomal proteins revealed that depletion of the nucleosome-remodelling protein Mi-2 and the protein kinase NHK-1 (Nucleosomal histone kinase-1, also known as Ballchen in Drosophila) resulted in much more severe defects than depletion of well-known chromosome condensation factors. The condensation defects of Mi-2 and NHK-1 depletion were distinct from each other, suggesting that these proteins function in different pathways. This study found that the main NHK-1 action in chromosome condensation is to suppress Barrier-to-autointergration factor (BAF) activity, which functions to link nuclear envelope proteins to chromosomes (Nikalayevich, 2014)

    Therefore, knockdown of potential chromosomal proteins or regulators by RNAi in oocytes has identified new factors promoting chromosome condensation (the NuRD complex and NHK-1) as well as known factors (condensin I, topoisomerase II and Aurora B). Depletion of the protein kinase NHK-1 and the NuRD nucleosome remodelling complex containing Mi-2 caused severe chromosome condensation defects that were distinct from each other. Further study revealed that BAF is the main substrate of NHK-1 for its chromosome condensation function and that NHK-1 promotes chromosome condensation by suppressing the linker activity of BAF between nuclear envelope proteins and DNA. Finally, it was shown that NHK-1 is also important for chromosome condensation in mitosis (Nikalayevich, 2014)

    The Drosophila oocyte combined with RNAi is an excellent system for research of chromosome condensation, which complements commonly used mitotic systems. Firstly, Drosophila oocytes grow enormously in volume between completion of pre-meiotic mitosis and recombination and chromosome condensation. Small hairpin RNA (shRNA) expression can be induced after the protein executes its role in the previous mitosis and/or recombination but prior to oocyte growth. Even if the target protein is stable, it becomes sufficiently diluted before chromosome condensation in oocytes. This is in contrast to mitotic cycles where cells only double in size between divisions. Secondly, Drosophila oocytes arrest in metaphase of the first meiotic division. This allows chromosome defects to be studied in the first division after the target protein is depleted, rather than as a mixture of defects accumulated through multiple divisions caused by a gradual decrease of the protein. Finally, as oocytes are large, the condensation state of chromosomes can be clearly observed without mechanical treatment such as squashing or spreading. Therefore, RNAi in Drosophila oocytes could be a powerful system to study chromosome condensation, although negative results should be interpreted with caution as they might be caused by insufficient depletion, genetic redundancy or cell-type-specific function (Nikalayevich, 2014)

    Indeed, in this study, a small-scale survey of chromosomal proteins, new chromosome condensation factors were identified in addition to well-known ones, demonstrating the effectiveness of Drosophila oocytes as a research system. Well-known factors, including condensin I subunits, topoisomerase II and Aurora B, showed milder chromosome condensation defects. Knockdown of topoisomerase II or condensin I showed similar condensation defects, and appeared to affect mainly centromeric and/or pericentromeric regions. The previous reports in mitosis are consistent with these result, suggesting that these two factors are not the main condensation factors in mitosis or in meiosis (Nikalayevich, 2014)

    A previous study of Mi-2 in Drosophila suggested that it promotes decondensation of chromosomes because overexpression of wild-type Mi-2 results in chromosome decondensation in polytene or mitotic cells and overexpression of dominant-negative Mi-2 results in overcondensation. In the current study, Mi-2 RNAi in oocytes showed chromosome decondensation, whereas in a preliminary study in neuroblasts Mi-2 RNAi did not show chromosome decondensation. The difference from the previous study might be due to the method of disrupting the Mi-2 function or cell types used for the studies. It is argued that the phenotype caused by RNAi in oocytes is a better reflection of the in vivo function. RNAi of other NuRD subunits indicated that the NuRD complex is important for chromosome condensation (Nikalayevich, 2014)

    How does the NuRD complex promote chromosome condensation? It is possible that nucleosome remodelling is directly required during chromosome condensation. For example, proper positioning of nucleosomes might be important for full chromosome condensation. Indeed, other nucleosome remodelling complexes have been suggested to be involved in chromosome condensation in fission yeast. Alternatively, histone deacetylase acivity of the NuRD complex might be important for chromosome condensation, as histone modifications are a major way to regulate chromosome structure. The possibility that NuRD acts through transcription of other chromosome condensation factors cannot be excluded, as it is known to regulate gene transcription. Further studies using more sophisticated mutations would help to distinguish these possibilities (Nikalayevich, 2014)

    Knockdown of NHK-1 resulted in severe chromosome condensation defects in nearly all oocytes. Previously, involvement of NHK-1 or its orthologues in metaphase chromosome condensation has not been reported, although overexpression of the human orthologue disrupts chromatin organisation in interphase. None of the three female sterile nhk-1 mutants showed chromosome condensation defects in metaphase I in oocytes. This might be because the minimal NHK-1 activity required for producing viable adults is sufficient to allow chromosome condensation in oocytes. Female-germline-specific RNAi is likely to have achieved greater depletion of NHK-1 in oocytes. This study showed that phosphorylation of BAF, thus inactivating its linking of DNA to LEM-domain-containing inner nuclear membrane proteins, is the major role of NHK-1 in chromosome condensation in oocytes. However, NHK-1 might regulate multiple pathways during condensation, for example, it has been shown that it is required for histone 2A phosphorylation and condensin recruitment in prophase I oocytes (Nikalayevich, 2014)

    A crucial question is whether the chromosome condensation defect is a direct consequence of NHK-1 loss or a secondary consequence of a karyosome defect in prophase I oocytes. Evidence indicates that the compact karyosome in the prophase I nucleus and chromosome condensation in metaphase I are at least partly independent. In female-sterile hypomophic nhk-1 mutants, chromatin organisation in prophase I oocytes is defective, but metaphase I chromosomes are properly condensed in mature oocytes. By contrast, in Mi-2 RNAi oocytes, the karyosome is normal in prophase I, but chromosomes become undercondensed after nuclear envelope breakdown in some metaphase I oocytes. Furthermore, as chromosome condensation in mitosis is also defective in nhk-1 mutants, the role for NHK-1 in chromosome condensation must be at least partly independent from meiosis-specific chromatin organisation. Therefore, release of LEM-containing nuclear envelope proteins from chromosomes might be a prerequisite for proper chromosome condensation (Nikalayevich, 2014)

    In conclusion, this targeted survey using RNAi in Drosophila oocytes has already identified new factors required for chromosome condensation. Further analysis provided new insights into the molecular mechanism of condensation including the release of nuclear envelope proteins from chromosomes and nucleosome remodelling and/or histone deacetylation as essential steps for condensation. In future, a larger scale screen of putative chromosomal proteins might prove to be fruitful (Nikalayevich, 2014).

    Impact of Germline Depletion of Bonus on Chromatin State in Drosophila Ovaries

    Gene expression is controlled via complex regulatory mechanisms involving transcription factors, chromatin modifications, and chromatin regulatory factors. Histone modifications, such as H3K27me3, H3K9ac, and H3K27ac, play an important role in controlling chromatin accessibility and transcriptional output. In vertebrates, the Transcriptional Intermediary Factor 1 (TIF1) family of proteins play essential roles in transcription, cell differentiation, DNA repair, and mitosis. This study focused on Bonus, the sole member of the TIF1 family in Drosophila, to investigate its role in organizing epigenetic modifications. The findings demonstrated that depleting Bonus in ovaries leads to a mild reduction in the H3K27me3 level over transposon regions and alters the distribution of active H3K9ac marks on specific protein-coding genes. Additionally, through mass spectrometry analysis, novel interacting partners of Bonus in ovaries were identified, such as PolQ, providing a comprehensive understanding of the associated molecular pathways. Furthermore, this research revealed Bonus's interactions with the Polycomb Repressive Complex 2 and its co-purification with select histone acetyltransferases, shedding light on the underlying mechanisms behind these changes in chromatin modifications (Godneeva, 2023).

    TIF1 family members play an important role in remodeling chromatin and the modulation of underlying transcriptional mechanisms. Bon is the only member of the TIF1 subfamily of TRIM/RBCC proteins in Drosophila, setting it apart from its mammalian counterparts, where four distinct members exhibit diverse functions and mechanisms of action. A genome-wide analysis was performed of several activating and silencing histone modifications in germline Bon-depleted ovaries. Bon’s depletion did not significantly alter the genome-wide abundance of H3K27me3, although subtle reductions in H3K27me3 were observed in certain transposon-associated regions. Previous research indicated that Bon is not involved in transposon repression. This can be explained that despite the mild reduction in H3K27me3, transposon expression remained largely unchanged due to the persistent presence of the repressive H3K9me3 mark. While it has been well-established that H3K9me3 primarily marks transposable elements and satellite sequences, several studies have revealed the coexistence both the H3K9me3 and H3K27me3 marks on the same subset of genes, and in some the H3K27me3 mark is primarily responsible for the repression of transposons. In mammals, TIF1β/KAP-1 is known to repress endogenous retroviruses in an H3K9me3-dependent way. Although relatively few studies have showed the connection between the TIF1 family proteins and the H3K27me3 mark, one study did reveal that KAP-1 depletion resulted in a decreased level of H3K27me3 on the HIV-1 promoter. Therefore, it is proposed that in Drosophila, Bon participates in the repression of transposons through its association with the H3K27me3 mark, indicating a new role within the TIF1 subfamily (Godneeva, 2023).

    Additionally, the association between Bon and the PRC2 complex, revealed through co-immunoprecipitation assays, raises intriguing questions regarding the functional consequences of this interaction. Bon has not previously been described in connection with Polycomb complexes, apart from a potential interaction between Bon and unknown members of a Polycomb group that was mentioned in the discussion of an early work. Interestingly, in mammals, KAP-1 was shown to interact with EZH2/E(z) in a PRC2-independent manner to coordinately regulate genes with roles in breast stem cell maintenance. In another study, KAP-1 associates with the PRC1 complex to repress differentiation-inducible genes in embryonic stem cells. Therefore, Bon may be involved in facilitating the recruitment of the PRC2 complex to specific genomic loci or can also associate with the PRC1 complex to repress the subsets of several genes (Godneeva, 2023).

    The members of the TIF1 family are involved in both transcriptional activation and repression. Analysis of the active marks H3K9ac and H3K27ac indicates that Bon is directly regulating some of its targets by facilitating their H3K9 acetylation. However, the levels of these activation marks remained unchanged following Bon depletion on most of its targets, suggesting the involvement of other activation marks. Indeed, the Gcn5 histone acetyltransferase responsible for the deposition of the H3K9ac mark was not identified in mass-spec analysis for Bon interactors. The presence of HATs MOF, nej, and Taf1 in the Bon-interacting complex raises the possibility that Bon’s modulation of gene expression may be accomplished through diverse acetylation marks. Additionally, indirect regulation through other transcriptional regulators remains a plausible mechanism. Notably, due to their C-terminal PHD/bromodomain, TIF1 proteins can function as 'readers' of histone modifications. Thus, mammalian TIF1γ/TRIM33 specifically recognizes an H3 tail that is acetylated at lysines K18 and K23, and TIF1α/TRIM24 was shown to recognize acetylated H3K23. Since the current results could not pinpoint a specific acetylation mark involved in Bon-dependent regulation, future studies will be necessary to precisely identify the acetylation mark associated with this process (Godneeva, 2023).

    The search for interacting partners of Bon revealed an interesting interaction with PolQ, a specialized DNA polymerase (Pol θ) participating in DNA repair, which suggests Bon’s potential involvement in end-joining repair of DSBs. In mammals, TIF1 members are known to play a major role in DSB repair mechanisms. DSBs are serious threats to cell survival and genome stability; therefore, in the future, it will be interesting to determine whether Bon is required for the repair activities or whether it can enhance the efficiency and accuracy of this repair process, ensuring the preservation of genomic integrity (Godneeva, 2023).

    In summary, this study expands understanding of Bon’s roles in the epigenetic landscape and offers a valuable resource of Bon’s interactors in Drosophila ovaries. Although this study has demonstrated Bon’s influence on the distribution of activating marks (H3K9ac and H3K27ac) and a repressive mark (H3K27me3), it is essential to acknowledge the complexity of chromatin regulation. Therefore, it will be necessary to study other histone modifications and effector molecules to better understand the mechanisms underlying Bon’s impact on gene expression that controls the function of chromatin (Godneeva, 2023).

    In conclusion, this study of the specific role of Bon has provided significant insights into its diverse contributions to the epigenetic landscape. Depletion of Bon affects the distribution of histone marks H3K27me3, H3K9ac, and H3K27ac, revealing its complex involvement in the regulation of chromatin states. The interaction with the PRC2 complex suggests potential involvement in Polycomb-mediated gene regulation, raising questions about the functional consequences of this cooperation. This study reveals previously unknown interactions between Bon and chromatin-modifying complexes, including histone acetyltransferases. Furthermore, the dual role of Bon in transcriptional activation and repression suggests the existence of a complex regulatory network. While this study provides valuable insights into Bon’s interactions with key chromatin-modifying complexes and its role in DNA repair, the complex nature of chromatin regulation requires further exploration of additional histone modifications and effector molecules. Future studies, with a focus on identifying specific acetylation marks associated with Bon-dependent regulation and elucidating Bon’s role in DNA repair mechanisms, will enhance understanding of Bon's influence on gene expression and genome stability (Godneeva, 2023).

    A role for tuned levels of nucleosome remodeler subunit ACF1 during Drosophila oogenesis

    The Chromatin Accessibility Complex (CHRAC) consists of the ATPase ISWI, the large ACF1 subunit and a pair of small histone-like proteins, CHRAC-14 and CHRAC-16. CHRAC is a prototypical nucleosome sliding factor that mobilizes nucleosomes to improve the regularity and integrity of the chromatin fiber. This may facilitate the formation of repressive chromatin. This study explored roles for ACF1 during Drosophila oogenesis. ACF1 is expressed in somatic and germline cells, with notable enrichment in germline stem cells and oocytes. The asymmetrical localization of ACF1 to these cells depends on the transport of the Acf1 mRNA by the Bicaudal-D/Egalitarian complex. Loss of ACF1 function in the novel Acf17 allele leads to defective egg chambers and their elimination through apoptosis. In addition, a variety of unusual 16-cell cyst packaging phenotypes were found in the previously known Acf11 allele, with a striking prevalence of egg chambers with two functional oocytes at opposite poles. Surprisingly, Acf11 deletion - despite disruption of the Acf1 reading frame - expresses low levels of a PHD-bromodomain module from the C-terminus of ACF1 that becomes enriched in oocytes. Expression of this module from the Acf1 genomic locus leads to packaging defects in the absence of functional ACF1, suggesting competitive interactions with unknown target molecules. Remarkably, a two-fold overexpression of CHRAC (ACF1 and CHRAC-16) leads to increased apoptosis and packaging defects. Evidently, finely tuned CHRAC levels are required for proper oogenesis (Börner, 2016a).

    A genetic mosaic screen reveals ecdysone-responsive genes regulating Drosophila oogenesis

    Multiple aspects of Drosophila oogenesis, including germline stem cell activity, germ cell differentiation, and follicle survival are regulated by the steroid hormone ecdysone. While the transcriptional targets of ecdysone signaling have been studied extensively during development, targets in the ovary remain largely unknown. Early studies of salivary gland polytene chromosomes led to the model that ecdysone stimulates a hierarchical transcriptional cascade, wherein a core group of ecdysone-sensitive transcription factors induce tissue-specific responses by activating secondary branches of transcriptional targets. More recently, genome-wide approaches have identified hundreds of putative ecdysone-responsive targets. Determining whether these putative targets represent bona fide targets in vivo, however, requires that they be tested via traditional mutant analysis in a cell-type specific fashion. To investigate the molecular mechanisms whereby ecdysone signaling regulates oogenesis, this study used genetic mosaic analysis to screen putative ecdysone-responsive genes for novel roles in the control of the earliest steps of oogenesis. A cohort of genes required for stem cell maintenance, stem and progenitor cell proliferation, and follicle encapsulation, growth, and survival were identified. These genes encode transcription factors, chromatin modulators, and factors required for RNA transport, stability, and ribosome biogenesis, suggesting that ecdysone might control a wide range of molecular processes during oogenesis. Results suggest that although ecdysone target genes are known to have cell type-specific roles, many ecdysone response genes that control larval or pupal cell types at developmental transitions are reiteratively used in the adult ovary. These results provide novel insight into the molecular mechanisms by which ecdysone signaling controls oogenesis, laying new ground for future studies (Ables, 2016).

    Prostaglandins temporally regulate cytoplasmic actin bundle formation during Drosophila oogenesis.

    While Prostaglandins (PGs), lipid signals produced downstream of cyclooxygenase (COX) enzymes, regulate actin dynamics in cell culture and platelets, their roles during development are largely unknown. This study definee a new role for Pxt, the Drosophila COX-like enzyme, in regulating the actin cytoskeleton-temporal restriction of actin remodeling during oogenesis. PGs are required for actin filament bundle formation during stage 10B (S10B). Additionally, loss of Pxt results in early actin remodeling, including extensive actin filaments and aggregates, within the posterior nurse cells of stage 9 (S9) follicles; wild-type follicles exhibit similar structures at a low frequency. Hu li tai shao (Hts), the homolog of Adducin, and Villin (Quail), an actin bundler, localize to all early actin structures, while Enabled (Ena), an actin elongation factor, preferentially localizes to those in pxt mutants. Reduced Ena levels strongly suppress early actin remodeling in pxt mutants. Furthermore, loss of Pxt results in reduced Ena localization to the sites of bundle formation during S10B. Together these data lead to the model that PGs temporally regulate actin remodeling during Drosophila oogenesis by controlling Ena localization/activity, such that in S9, PG signaling inhibits, while at S10B, it promotes Ena-dependent actin remodeling (Spracklen, 2013).

    Proximity labeling reveals novel interactomes in live Drosophila tissue

    Gametogenesis is dependent on intercellular communication facilitated by stable intercellular bridges connecting developing germ cells. During Drosophila oogenesis, intercellular bridges (referred to as ring canals) have a dynamic actin cytoskeleton that drives their expansion to a diameter of 10mum. While multiple proteins have been identified as components of ring canals (RCs), a basic understanding of how RC proteins interact together to form and regulate the RC cytoskeleton is lacking. This study optimized a procedure for proximity-dependent biotinylation in live tissue using the APEX enzyme to interrogate the RC interactome. APEX was fused to four different RC components (RC-APEX baits) and 55 unique high-confidence preys were identified. The RC-APEX baits produced almost entirely distinct interactomes that included both known RC proteins as well as uncharacterized proteins. The proximity ligation assay was used to validate close-proximity interactions between the RC-APEX baits and their respective preys. Further, an RNAi screen revealed functional roles for several high-confidence prey genes in RC biology. These findings highlight the utility of enzyme-catalyzed proximity labeling for protein interactome analysis in live tissue and expand understanding of RC biology (Mannix, 2019).

    Splice variants of the SWR1-type nucleosome remodeling factor Domino have distinct functions during Drosophila melanogaster oogenesis

    SWR1-type nucleosome remodeling factors replace histone H2A by variants to endow chromatin locally with specialized functionality. In Drosophila melanogaster a single H2A variant, H2A.V, combines functions of mammalian H2A.Z and H2A.X in transcription regulation and the DNA damage response. A major role in H2A.V incorporation for the only SWR1-like enzyme in flies, Domino, is assumed but not well documented in vivo. It is also unclear whether the two alternatively spliced isoforms, DOM-A and DOM-B, have redundant or specialized functions. Loss of both DOM isoforms compromises oogenesis, causing female sterility. This study systematically explored roles of the two DOM isoforms during oogenesis using a cell type-specific knockdown approach. Despite their ubiquitous expression, DOM-A and DOM-B have non-redundant functions in germline and soma for egg formation. It was shown that chromatin incorporation of H2A.V in germline and somatic cells depends on DOM-B, whereas global incorporation in endoreplicating germline nurse cells appears to be independent of DOM. By contrast, DOM-A promotes the removal of H2A.V from stage 5 nurse cells. Remarkably, therefore, the two DOM isoforms have distinct functions in cell type-specific development and H2A.V exchange (Börner, 2016b).

    In D. melanogaster the properties of the two ancient, ubiquitous histone H2A variants H2A.X and H2A.Z are combined in a single molecule, H2A.V. Given that H2A.V carries out functions as a DNA damage sensor and architectural element of active promoters, as well as having further roles in heterochromatin formation, this histone appears loaded with regulatory potential. Accordingly, placement of the variant, either randomly along with canonical H2A during replication or more specifically through nucleosome remodeling factors, becomes a crucial determinant in its function. Mechanistic detail for replacement of H2A-H2B dimers with variants comes from the analysis of the yeast SWR1 complex, which incorporates H2A.Z in a stepwise manner at strategic positions next to promoters (Börner, 2016b).

    So far, the published phenotypes associated with dom mutant alleles have not been systematically complemented. The comprehensive complementation analysis of this study shows that dom mutant phenotypes are indeed due to defects in the dom gene. Remarkably, dom lethality and sterility can be partially rescued by complementation with the orthologous human SRCAP gene, providing an impressive example of functional conservation of SWR1-like remodelers. The contributions of the two splice variants DOM-A and DOM-B had not been assessed. This study now demonstrates that both isoforms are essential for development, suggesting non-redundant functions. The DOM-A isoform contains a SANT domain followed by several poly-Q stretches, which are widely found in transcriptional regulators, where they may modulate protein interactions. By contrast, SANT domains are thought to function as histone tail interaction modules that couple binding to enzyme catalysis. Therefore, the SANT domain in DOM-A could mediate specificity towards H2A.V eviction depending on particular functional contexts. These features are also present in the C-terminus of p400 (EP400), the second human SWR1 ortholog, but are absent in either DOM-B or SRCAP. Remarkably, p400 interacts directly with TIP60 and the SANT domain of p400 inhibits TIP60 catalytic activity providing an interesting lead for further investigation of DOM isoforms and TIP60 interactions (Börner, 2016b).

    It is speculated that distinct functions of p400 and SRCAP in humans might be accommodated to some extent by the two DOM isoforms in flies. Accordingly, it will be interesting to explore whether the two isoforms reside in distinct complexes. Previous affinity purification of a TIP60-containing complex using a tagged pontin subunit apparently only identified DOM-A, but not DOM-B. Following up on the initial observation of early defects in GSCs and cyst differentiation upon loss of DOM (Yan, 2014), this study now finds that this phenotype is exclusively caused by loss of DOM-A. Interestingly, studies with human embryonic stem cells show that p400/TIP60 (KAT5) integrates pluripotency signals to regulate gene expression, suggesting similar roles for DOM-A in GSCs. This is in contrast to requirements for both isoforms for germline development outside of the germarium, highlighting a developmental specialization of the two DOM remodelers (Börner, 2016b).

    DOM is also involved in the differentiation and function of SSCs in the germarium. The data now document non-redundant requirements of both DOM isoforms in somatic cells for proper coordination of follicle cell proliferation with cyst differentiation. Failure to adjust these two processes leads to 16-cell cyst packaging defects that manifest as compound egg chambers. These rare phenotypes had previously only been described upon perturbation of some signaling pathways, such as Notch, or Polycomb regulation (Börner, 2016b).

    Because the phenotypes of DOM depletions resemble those of H2A.V depletion, the idea was favored that many of the cell-specification defects are due to compromised H2A.V incorporation, depriving key promoters of the H2A.Z-related architectural function. Alternatively, scaffolding activities might partially explain some roles of chromatin remodelers, as suggested for SRCAP. So far, knowledge of the mechanisms of H2A.V incorporation has been anecdotal. This comprehensive analysis revealed a specific involvement of DOM-B for the incorporation of H2A.V into chromatin at the global level. The N-termini of SWR1 and DOM-B harbor the HSA and ATPase spacer domains, with interaction surfaces for further complex subunits, and an additional H2A.Z-H2B dimer binding site. Given the requirement for both isoforms for cell specification during oogenesis, it is speculated that DOM-B might serve to incorporate bulk H2A.V into chromatin similar to SWR1, whereas DOM-A would be more involved in the regulatory refinement of location (Börner, 2016b).

    Although the failures in cell specification and egg morphogenesis are likely to be explained by loss of the H2A.Z-related features of H2A.V, ablation of DOM might also compromise the DNA damage response, which involves phosphorylation of H2A.V (γH2A.V). Conceivably, the role of γH2A.V as a DNA damage sensor might be best fulfilled by a broad distribution of H2A.V throughout the chromatin. Such an untargeted incorporation may be achieved by stochastic, chaperone-mediated incorporation during replication or by an untargeted activity of DOM-B. DOM-independent incorporation in endoreplicating polyploid nurse cells of stage 3 egg chambers is observed, where global H2A.V and γH2A.V signals did not depend on DOM. Immunofluorescence microscopy may lack the sensitivity to detect DOM-dependent incorporation of H2A.V at some specific sites. Nevertheless, DOM-independent incorporation of H2A.V might serve to cope with many naturally occurring DNA double-strand breaks during the massive endoreplication of nurse cells (Börner, 2016b).

    There is some evidence that nucleosome remodelers not only incorporate H2A variants but can also remove them. In yeast, the genome-wide distribution of H2A.Z appears to be established by the antagonistic functions of the SWR1 and Ino80 remodeling complexes, where Ino80 replaces stray H2A.Z-H2B with canonical H2A-H2B dimers. A recent study identified the vertebrate-specific histone chaperone ANP32E as part of a TIP60/p400 complex that facilitates the eviction of H2A.Z-H2B dimers from chromatin. Remarkably, in D. melanogaster a TIP60/DOM-A complex is involved in a similar reaction. The TIP60/DOM-A complex acetylates γH2A.V at lysine 5 to facilitate exchange of γH2A.V by unmodified H2A.V during the DNA damage response. Furthermore, it has been speculated that H2A.V and γH2A.V could be actively removed from nurse cells, since corresponding signals are absent from stage 5 onwards. This study now demonstrates that depletion of DOM-A and TIP60 leads to the persistence of H2A.V and γH2A.V in nurse cells of late egg chambers, clearly documenting the ability of the remodeler to remove bulk H2A.V and variants modified during DNA damage induction (Börner, 2016b).

    These findings highlight the specific requirements of DOM splice variants for the incorporation and removal of H2A.V during D. melanogaster oogenesis. It remains an interesting and challenging question how DOM-A and DOM-B complexes are targeted genome-wide and function in vivo to establish specific H2A.V patterns in different cell types during development (Börner, 2016b).

    Oxidative stress in oocytes during midprophase induces premature loss of cohesion and chromosome segregation errors

    One hallmark of aging cells is an increase in oxidative damage caused by reactive oxygen species (ROS). Increased oxidative damage in older oocytes may be one of the factors that leads to premature loss of chromosome cohesion and segregation errors. To test this hypothesis, an RNAi strategy was used to induce oxidative stress in Drosophila oocytes, and the fidelity of chromosome segregation was measured during meiosis. Knockdown of either the cytoplasmic SOD or mitochondrial ROS scavenger superoxide dismutase (SOD) caused a significant increase in segregation errors, and heterozygosity for an smc1 deletion enhanced this phenotype. FISH analysis indicated that SOD knockdown moderately increased the percentage of oocytes with arm cohesion defects. Consistent with premature loss of arm cohesion and destabilization of chiasmata, the frequency at which recombinant homologs missegregate during meiosis I is significantly greater in SOD knockdown oocytes than in controls. Together these results provide an in vivo demonstration that oxidative stress during meiotic prophase induces chromosome segregation errors and support the model that accelerated loss of cohesion in aging human oocytes is caused, at least in part, by oxidative damage (Perkins, 2016).

    Stress-induced reproductive arrest in Drosophila occurs through ETH deficiency-mediated suppression of oogenesis and ovulation

    Environmental stressors induce changes in endocrine state, leading to energy re-allocation from reproduction to survival. Female Drosophila melanogaster respond to thermal and nutrient stressors by arresting egg production through elevation of the steroid hormone ecdysone. However, the mechanisms through which this reproductive arrest occurs are not well understood. This study reports that stress-induced elevation of ecdysone is accompanied by decreased levels of ecdysis triggering hormone (ETH). Depressed levels of circulating ETH lead to attenuated activity of its targets, including juvenile hormone-producing corpus allatum and, as described in this study for the first time, octopaminergic neurons of the oviduct. Elevation of steroid thereby results in arrested oogenesis, reduced octopaminergic input to the reproductive tract, and consequent suppression of ovulation. ETH mitigates heat or nutritional stress-induced attenuation of fecundity, which suggests that its deficiency is critical to reproductive adaptability. These findings indicate that, as a dual regulator of octopamine and juvenile hormone release, ETH provides a link between stress-induced elevation of ecdysone levels and consequent reduction in fecundity (Meiselman, 2018).

    Evidence presented in this study establishes a new paradigm for Drosophila reproduction, wherein stressful conditions arrest egg production via a hormonal cascade involving reciprocal ecdysone and ETH signaling. As steroid levels fluctuate in response to stress, so too does ETH, a consequence of steroid-regulated changes in Inka cell secretory competence. ETH activates two downstream targets: the JH-producing corpus allatum and modulatory OA neurons innervating the ovary and oviducts. This study characterized the nature of ETH dependence, and assigned function and context to a newly recognized hormonal axis governing reproductive responses to stress (Meiselman, 2018).

    Previous report showed that ETH is an obligatory allatotropin, promoting oogenesis and fecundity through JH production; consequently, ETH deficiency results in low JH levels and arrested oogenesis (Meiselman, 2017). The present work demonstrates that ovulation of stage 14 oocytes depends upon ETH activation of OA neurons innervating the ovary and oviduct. A comprehensive explanation is offered for the change in distribution of vitellogenic oocytes reported in EcR mutants or under conditions of high or low ecdysone, depending on stress levels. ETH deficiency or ETHR knockdown results in accumulation of stage 14 oocytes in the ovary due to ovulation block, and a mechanistic link between altered endocrine state and ovulation is provided (Meiselman, 2018).

    ETH promotes ovulation through activation OA neurons to induce contractions in the ovary and relaxation of the oviducts. It is interesting that ETH triggers calcium dynamics in vitro on distal axonal projections, suggesting ETH-stimulated OA release results from direct action of ETH on axons and/or nerve terminals. While ovary contractions in response to ETH exposure occur in both virgin and mated females, this study chose virgin females for analysis due to higher spontaneous contractile activity in mated females. This is likely due to actions of ovulin after insemination, which stimulate outgrowth of octopaminergic neurons innervating the oviduct. In virgin females, concentration-dependent ETH actions on the ovary are in the range predicted for activation of ETHR-A receptors (Meiselman, 2018).

    Acting through OA neurons, ETH mobilizes calcium in the epithelium enveloping the ovary, initiating bursts of contractions in the peritoneal sheath at the base of the ovary associated with ovulation. Although bath-applied ETH and OA are both sufficient to induce calcium mobilization in the oviduct epithelium, they induce distinctive response patterns. OA causes a rapid, sustained calcium wave with a slowly waning plateau following the peak response. ETH actions occur with longer latency and induce oscillatory calcium dynamics, which could be a consequence of periodic synaptic reuptake of OA by nerve terminals. No changes in intensity were observed between treatments or at different doses, suggesting a possible threshold effect. It is also interesting to note that calcium waves spread through the epithelial layer, suggesting that the epithelium is a functional syncytium, which undoubtedly aids in coordination of relaxation (Meiselman, 2018).

    Injection of mated females with either ETH or OA induces ovulation in vivo, whereas injected virgin females respond much more weakly. In order for ovulation to occur, OA causes follicle rupture inside the ovaries, a process requiring one to several hours ex vivo. It is hypothesized that mated females are in the proper endocrine state for ovulation, and thus follicle rupture may already be in progress before application of ETH or OA. As follicle rupture is the critical first step for egg-laying, this limiting factor would explain the length of time (up to 60 min) elapsed after physiological levels of ETH/OA are reached for in vivo ovulation to occur, given that ovary contraction and oviduct relaxation occur within seconds (Meiselman, 2018).

    Agents previously implicated in oviduct contractions were also examined, including tyramine, glutamate, and proctolin. While the ineffectiveness of tyramine and glutamate is not surprising, the negative result with proctolin is at variance with prior literature. Examination of proctolin-induced contractions revealed that they are localized to the distal tip (germaria) of the ovaries. Moreover, proctolin does not stimulate ovulation in vitro. It appears that the role of proctolin in Drosophila ovaries is more limited than in the well-studied locust oviduct (Meiselman, 2018).

    This study has shown that elevated ecdysone levels in response to heat and nutritional stress are associated with a drop in circulating ETH levels. It was previously hypothesized that the Inka cell secretory competence model governing ecdysis signaling during developmental stages may persist into adulthood (Meiselman, 2017). The results presented in this study support this hypothesis (Meiselman, 2018).

    Both stress and ETH deficiency have similar consequences for reproduction, namely arrested oogenesis and reduced ovulation, resulting in increased stage 14 egg retention and lower egg production. Progression of mid-oogenetic oocytes is directly correlated with JH levels, while OA release from reproductive tract neurons is necessary for ovulation. This study shows that arrested oogenesis and ovulation contributing to the ovariole profile observed in heat-stressed flies can be explained by ETH deficiency, which has a dual role in regulating JH levels and activity of OA neurons innervating ovaries and oviducts. Indeed, arrest of both oogenesis and ovulation deficiencies can be rescued by ETH, either through TRPA1 activation of Inka cells or direct injection of ETH1 (Meiselman, 2018).

    The mechanism through which elevated ecdysone leads to ETH deficiency was examined by performing rescue experiments designed to (1) suppress steroid signaling in Inka cells and (2) express the transcription factor βFTZ-F1, which confers secretory competence of Inka cells and is suppressed by high ecdysone levels. Although somewhat variable in their effectiveness, these manipulations resulted in clear rescue of oogenesis and ovulation in heat-stressed females, confirming that the thermal stress response operates through the influence of ecdysone on Inka cell secretion (Meiselman, 2018).

    Methoprene treatment increases progression of oogenesis but does not increase oviposition in stressed animals. In fact, this study observed a significant increase in eggs retained after methoprene treatment, suggesting that synthesis of mature eggs resumes with JH treatment, but ovulation remains impaired under conditions of elevated ecdysone and ETH deficiency. This suggests that ovulation provides a gating mechanism under stressful conditions, limiting egg production while conditions are suboptimal. A recent report suggested that normal ecdysone levels stimulate follicle rupture and ovulation, but that elevated levels inhibit follicle rupture (Knapp, 2017). The present work provides an additional mechanism for suppression of ovulation associated with elevated ecdysone levels: repression of ETH release leading to reduced OA neuron activity (Meiselman, 2018).

    It is interesting to note that wet starvation reduces ecdysone levels and increases ETH levels, whereas sugar starvation increases ecdysone levels and, as is shown in this study, increases ETH levels. Wet-starved females were precisely synchronized in mating on day 4, and began starvation (no nutrient source, wet KimWipe) 24 h later for an additional 24 h. mino acid-deprived females were group-raised until day 3, and groups were placed on agar plus 10% sucrose for 24 h. Mating was not controlled in sugar-starved females, though it is known to influence ecdysone levels dramatically in the short term. Arguably the most interesting result is that ecdysone decrease led to elevated circulating ETH. This adds credence to the hypothesis that ETH and ecdysone levels are generally inversely correlated (Meiselman, 2018).

    Unique stresses may garner different endocrine responses because different types of cues require differential behavioral adaptation. The ability of a hormone to coordinate a wide variety of target tissues to change in state makes it a perfect tool for stress adaptation. As an organism encounters a new type of stress, they may adapt a new endocrine state to coordinate a tissue-wide response. Many hormones in closely related insects play markedly different roles, which evolve as rapidly as behavioral niches, but an endocrine core in E-ETH-JH is highly conserved, similar to the hypothalamic-pituitary-gonadal (HPG) axis among vertebrates. A hormonal network with competence to adjust reproductive output in response to environmental changes is undoubtedly a common phenomenon among multicellular organisms. The discovery of a stress response hormonal axis and, more aptly, a peptide hormone with the potential to alleviate stress-induced deficits in reproduction could be of particular relevance to the honey bee Apis mellifera. In recent years, Apis reproductives have been producing fewer progeny due to a variety of stressors, including temperature extrema. While proctolin has already been found to be a short-term reproductive stimulant in Apis queens, ETH is attractive as it can alter JH levels, which in turn may rescue poor pheromone production, the proximal cause of supersedure (Meiselman, 2018).

    Quantitative microscopy of the Drosophila ovary shows multiple niche signals specify progenitor cell fate

    Adult stem cells commonly give rise to transit-amplifying progenitors, whose progeny differentiate into distinct cell types. It is unclear if stem cell niche signals coordinate fate decisions within the progenitor pool. This study used quantitative analysis of Wnt, Hh, and Notch signalling reporters and the cell fate markers Eyes Absent (Eya) and Castor (Cas) to study the effects of hyper-activation and loss of niche signals on progenitor development in the Drosophila ovary. Follicle stem cell (FSC) progeny adopt distinct polar, stalk, and main body cell fates. Wnt signalling transiently inhibits expression of the main body cell fate determinant Eya, and Wnt hyperactivity strongly biases cells towards polar and stalk fates. Hh signalling independently controls the proliferation to differentiation transition. Notch is permissive but not instructive for differentiation of multiple cell types. These findings reveal that multiple niche signals coordinate cell fates and differentiation of progenitor cells (Dai, 2017).

    Diet regulates membrane extension and survival of niche escort cells for germline homeostasis via insulin signaling

    Diet is an important regulator of stem cell homeostasis, however, the underlying mechanisms of this regulation are not fully known. This study reports that insulin signaling mediates dietary maintenance of Drosophila ovarian germline stem cells (GSCs) by promoting the extension of niche escort cell (EC) membranes to wrap around GSCs. This wrapping may facilitate the delivery of BMP stemness factors from ECs in the niche to GSCs. In addition to the effects on GSCs, insulin signaling-mediated regulation of EC number and protrusions controls the division and growth of GSC progeny. The effects of insulin signaling on EC membrane extension are, at least in part, driven by enhanced translation of Failed axon connections (Fax) via Ribosomal protein S6 kinase. Fax is a membrane protein that may participate in Abl-regulated cytoskeletal dynamics and is known to be involved in axon bundle formation. Therefore, it is concluded that dietary cues stimulate insulin signaling in the niche to regulate EC cellular structure, probably via Fax-dependent cytoskeleton remodeling. This mechanism enhances intercellular contact and facilitates homeostatic interactions between somatic and germline cells in response to diet (Su, 2018).

    Survival of Drosophila germline stem cells requires the chromatin binding protein Barrier-to-autointegration factor

    The nuclear lamina (NL) is an extensive protein network that underlies the inner nuclear envelope. This network includes LAP2-emerin-MAN1-domain (LEM-D) proteins that associate with the chromatin and DNA binding protein Barrier-to-autointegration factor (BAF). this study investigated the partnership between three NL Drosophila LEM-D proteins and BAF. In most tissues, only D-emerin/Otefin is required for NL enrichment of BAF, revealing an unexpected dependence on a single LEM-D protein. Prompted by these observations, BAF contributions were studied in the ovary, a tissue where D-emerin/Otefin function is essential. Germ cell-specific BAF knockdown causes phenotypes that mirror d-emerin/otefin mutants. Loss of BAF disrupts NL structure, blocks differentiation and promotes germ cell loss, phenotypes that are partially rescued by inactivation of the ATR and Chk2 kinases. These data suggest that similar to d-emerin/otefin mutants, BAF depletion activates the NL checkpoint that causes germ cell loss. Taken together, these findings provide evidence for a prominent NL partnership between the LEM-D protein D-emerin/Otefin and BAF, revealing that BAF functions with this partner in the maintenance of an adult stem cell population (Duan, 2020).

    The nuclear lamina (NL) is an extensive protein network that underlies the inner nuclear membrane. Comprising lamins and hundreds of associated proteins, the NL builds contacts with the genome to regulate transcription, replication and DNA repair. The NL also connects the nucleus with the cytoskeleton, facilitating transduction of regulatory information between cellular compartments. The composition of the NL is cell-type specific, providing a diverse platform for the integration of developmental regulatory signals. Changes in NL structure occur during physiological aging and disease, suggesting that maintenance of NL function is crucial for cellular health and longevity (Duan, 2020).

    One prominent family of NL proteins are LEM domain (LEM-D) proteins, named after the founding human members: LAP2, emerin and MAN1. The defining feature of this conserved family is the LEM domain (LEM-D), an ∼40 amino acid domain that directly interacts with the metazoan chromatin-binding protein Barrier-to-autointegration factor (BAF, sometimes referred to as BANF1). Purified human BAF directly binds double-stranded DNA, the A-type lamin and histones in vitro, suggesting that BAF also promotes chromatin-NL connections using non-LEM-D-dependent mechanisms. In dividing metazoan cells, regulated formation of complexes between LEM-D proteins, BAF and lamin controls mitotic spindle assembly and positioning, as well as the reformation of the nucleus. In non-dividing metazoan cells, LEM-D proteins and BAF cooperate to tether the genome to the nuclear periphery and form repressed chromatin. These properties highlight central connections between LEM-D proteins and BAF in NL function (Duan, 2020).

    Studies in Drosophila melanogaster have begun to define the role of LEM-D proteins and BAF in development. Drosophila has three NL LEM-D proteins that bind BAF, including two emerin orthologues (Emerin/Otefin and Emerin2/Bocksbeutel) and MAN1. Each LEM-D protein is globally expressed during development. Even so, loss of individual NL LEM-D proteins causes different, non-overlapping defects in the several tissues, including the ovaries, testes, wings and the nervous system. These restricted mutant phenotypes reflect functional redundancy among the Drosophila LEM-D proteins, as loss of any two proteins is lethal. Strikingly, phenotypes of the emerin double mutants (otefin-/-; bocksbeutel-/-) phenocopy baf null mutants (Furukawa, 2003). Both baf and the emerin double mutants die before pupation, resulting from decreased mitosis and increased apoptosis of imaginal discs (Barton, 2014; Furukawa, 2003). In contrast, emerin/otefin; MAN1 or emerin2/bocksbeutel; MAN1 die during pupal development, without associated defects in mitosis or apoptosis (Barton, 2014). Together, genetic studies indicate that the Drosophila emerin orthologues and BAF are important partners (Duan, 2020).

    This study extend investigations of the Drosophila NL LEM-D and BAF protein partnership. Using a CRISPR generated gfp-baf allele, this study confirmed that BAF is a globally expressed nuclear protein that shows strong enrichment at the NL in diploid cells. Strikingly, this NL enrichment largely depends upon one LEM-D protein, Emerin/Otefin. Prompted by these observations, BAF contributions were studied in the ovary, a tissue where Emerin/Otefin function is essential. In germline stem cells (GSCs), loss of Emerin/Otefin causes a thickening of the NL and reorganization of heterochromatin. These structural nuclear defects are linked to activation of two kinases of the DNA damage response pathway: Ataxia Telangiectasia and Rad3-related (ATR) and Checkpoint kinase 2 (Chk2). Although oogenesis in emerin/otefin mutants is rescued by loss of these DDR kinases, canonical triggers are not responsible for pathway activation. Instead, ATR and Chk2 activation is linked to defects in NL structure itself (Barton, 2018). Given the roles of BAF in mitotic nuclear envelope formation and repair (Halfmann, 2019; Samwer, 2017; Mehsen, 2018), it was reasoned that checkpoint activation in emerin/otefin mutants might result from altered BAF function. This prediction was tested using germ cell-specific RNA interference (RNAi) to knockdown BAF. This study shows that BAF depletion disrupts NL structure, blocks differentiation and promotes GSC loss, mutant phenotypes that mirror Emerin/Otefin loss. Additionally, mutation of atr or chk2 partially restores germ cell differentiation in the baf mutant background, supporting the possibility that BAF depletion activates the NL checkpoint. Taken together, these findings suggest that Emerin/Otefin plays a dominant role in the enrichment of BAF to the NL and provide evidence that BAF functions with this prominent partner in the maintenance of an adult stem cell population (Duan, 2020).

    This study extended in vivo studies of the BAF and LEM-D partnership. Capitalizing on a newly generated gfp-baf allele, this study shows that NL localization of BAF largely depends upon a single LEM-D protein, Emerin/Otefin. Loss of Emerin/Otefin is sufficient to disperse BAF in cells that express the A- and B-type lamins, Emerin2/Bocksbeutel and MAN1 in the NL. These data establish the in vivo existence of a prominent NL partnership between one LEM-D protein and BAF (Duan, 2020).

    The basis for the unexpected reliance on Emerin/Otefin is unknown. One possibility is that LEM-Ds have different affinities for BAF. Pairwise alignment of amino acid residues within LEM-Ds shows the highest conservation between Drosophila emerin orthologues (70% similarity; Barton, 2014). Nonetheless, all LEM-Ds are strongly conserved in BAF-binding residues (42% identical, 67% similar). A second possibility is that the interaction of LEM-D proteins with BAF depends upon how a given LEM-D protein assembles into the NL network. Self-association of emerin influences both BAF and lamin binding. Finally, post-translational modifications (PTMs) of LEM-D proteins might impact BAF partnerships. As an example, O-GlcNAcylation modification of emerin affects BAF association, representing a regulated PTM that has the potential to alter NL function in response to nutrient availability. However, such signal-dependent PTMs are likely to be tissue specific, predicting a tissue-restricted, not global, effect on the NL enrichment of BAF. Further studies are needed to resolve the basis for the strong partnership between Emerin/Otefin and BAF (Duan, 2020).

    BAF is essential for viability, with dying baf null larvae exhibiting a typical mitotic mutant phenotype that is associated with high levels of apoptosis (Furukawa, 2007). Several observations suggest that loss of NL BAF is not equivalent to complete loss of BAF. First, emerin/otefin null animals are viable, even though there is a global loss of NL BAF. Second, emerin/otefin null animals have lower levels of apoptosis in larval tissues than baf animals, without effects on the development of adult structures. Third, emerin/otefin mutant imaginal disc cells display an unchanged nuclear shape and chromatin architecture (Barton, 2018), whereas these cells are affected in baf mutants (Furukawa, 2003). Based on these data, it is suggested that BAF function at the NL during interphase is not essential. It is predicted that the essential BAF function relates to its contributions in mitosis and depends upon both Drosophila emerin orthologues, as these double mutant animals die with a mitotic mutant phenotype (Duan, 2020).

    Effects of mislocalized BAF share features resulting from BAF overexpression in other systems. In emerin/otefin mutant germ cells, BAF dispersal contributes to the aggregation of heterochromatin. Defects in HP1a distribution have also been found in human cells overexpressing BAF or expressing a BAF mutant defective in interacting with NL components. Furthermore, several diseases affecting expression and processing of lamin A alter the distribution of BAF and resemble a BAF overexpression phenotype. Together, these findings support a model in which BAF contributes to the deleterious effects resulting from lamin or LEM-D mutations (Duan, 2020).

    BAF is required for maintenance of Drosophila GSCs. Germ cell-specific BAF knockdown caused GSC loss, with remaining GSCs displaying a thickened and irregular NL structure, a phenotype shared with emerin/otefin mutants. These data support a model in which Emerin/Otefin and BAF function together to build NL structure in this cell type. Such a dependence on Emerin/Otefin for NL structure is consistent with limiting levels of the second Drosophila Emerin ortholog, Emerin2/Bocksbeutel (Barton, 2014). It is predicted that, in GSCs, the Emerin/Otefin and BAF might have a shared function in nuclear reformation at the end of mitosis (Duan, 2020).

    Activation of the NL checkpoint is linked to NL deformation (Barton, 2018). Strikingly, baf mutant phenotypes are partially suppressed in nos>bafRNAi animals, with double mutant ovaries showing increased germ cell survival and differentiation. Yet cell death remained in the double mutant backgrounds. Based on these observations, it is predicted that BAF loss in germ cells has multiple consequences. First, NL structure is affected. Second, loss of nuclear BAF might affect transcriptional networks required for GSC maintenance, suggested from studies showing BAF is an epigenetic regulator (Montes de Oca, 2011). Notably, the maintenance of mammalian stem cells also depends on BAF. Knockdown of BAF in either mouse or human embryonic stem cells promoted premature differentiation and reduced survival, phenotypes associated with an altered cell cycle. It remains possible that loss of Drosophila BAF in GSCs perturbs mitosis, which might induce apoptosis. Additional studies are needed to elucidate cell cycle contributions of BAF in GSCs (Duan, 2020).

    These studies emphasize the important role of BAF within the NL network. Evidence is presented for consequences of BAF dispersal and loss during development, showing BAF dysfunction causes cell-type specific responses. Further definition of the developmental contributions of BAF will advance understanding of laminopathies, including the Nestor-Guillermo syndrome: a rare hereditary progeroid disorder caused by a missense mutation in BAF/BANF1 (Duan, 2020).

    Novel intrinsic factor Yun maintains female germline stem cell fate through Thickveins

    Germline stem cells (GSCs) are critical for the reproduction of an organism. The self-renewal and differentiation of GSCs must be tightly controlled to avoid uncontrolled stem cell proliferation or premature stem cell differentiation. However, how the self-renewal and differentiation of GSCs are properly controlled is not fully understood. This study finds that the novel intrinsic factor Yun is required for female GSC maintenance in Drosophila. GSCs undergo precocious differentiation due to de-repression of differentiation factor Bam by defective BMP/Dpp signaling in the absence of yun. Mechanistically, Yun associates with and stabilizes Thickveins (Tkv), the type I receptor of Dpp/BMP signaling. Finally, ectopic expression of a constitutively active Tkv (Tkv(QD)) completely suppresses GSC loss caused by yun depletion. Collectively, these data demonstrate that Yun functions through Tkv to maintain GSC fate. These results provide new insight into the regulatory mechanisms of how stem cell maintenance is properly controlled (Zhao, 2022).

    Aging shifts mitochondrial dynamics toward fission to promote germline stem cell loss

    Changes in mitochondrial dynamics (fusion and fission) are known to occur during stem cell differentiation; however, the role of this phenomenon in tissue aging remains unclear. This study reports that mitochondrial dynamics are shifted toward fission during aging of Drosophila ovarian germline stem cells (GSCs), and this shift contributes to aging-related GSC loss. As GSCs age, mitochondrial fragmentation and expression of the mitochondrial fission regulator, Dynamin-related protein (Drp1), are both increased, while mitochondrial membrane potential is reduced. Moreover, preventing mitochondrial fusion in GSCs results in highly fragmented depolarized mitochondria, decreased BMP stemness signaling, impaired fatty acid metabolism, and GSC loss. Conversely, forcing mitochondrial elongation promotes GSC attachment to the niche. Importantly, maintenance of aging GSCs can be enhanced by suppressing Drp1 expression to prevent mitochondrial fission or treating with rapamycin, which is known to promote autophagy via TOR inhibition. Overall, these results show that mitochondrial dynamics are altered during physiological aging, affecting stem cell homeostasis via coordinated changes in stemness signaling, niche contact, and cellular metabolism. Such effects may also be highly relevant to other stem cell types and aging-induced tissue degeneration (Amartuvshin, 2020).

    Neuronal octopamine signaling regulates mating-induced germline stem cell increase in female Drosophila melanogaster

    Stem cells fuel the development and maintenance of tissues. Many studies have addressed how local signals from neighboring niche cells regulate stem cell identity and their proliferative potential. However, the regulation of stem cells by tissue-extrinsic signals in response to environmental cues remains poorly understood. This study reports that efferent octopaminergic neurons projecting to the ovary are essential for germline stem cell (GSC) increase in response to mating in female Drosophila. The neuronal activity of the octopaminergic neurons is required for mating-induced GSC increase as they relay the mating signal from sex peptide receptor-positive cholinergic neurons. Octopamine and its receptor Oamb are also required for mating-induced GSC increase via intracellular Ca(2+) signaling. Moreover, Matrix metalloproteinase-2 was identified as a downstream component of the octopamine-Ca(2+) signaling to induce GSC increase. This study provides a mechanism describing how neuronal system couples stem cell behavior to environmental cues through stem cell niche signaling (Yoshinari, 2020).

    This study reports that the mating-induced GSC increase in female D. melanogaster is regulated by octopaminergic neurons directly projecting to the ovary. From in vivo and ex vivo experiments, the following model is proposed to explain the mating-induced GSC increase. After mating, the male seminal fluid SP is transferred into the female uterus, stimulating SPR-positive neurons. As the liganded SPR silences the neuronal activity of SPR-positive sensory neurons (SPSNs), the acetylcholine released from SPSNs is suppressed. As SPSNs and dsx+ Tdc2+ neurons are directly connected, this suppression directly modulates dsx+ Tdc2+ neuronal activity. Because this study has shown that nAChRs in dsx+ Tdc2+ neurons exhibit an inhibitory effect with an unknown mechanism, the inactivation of nAChRs in the absence of acetylcholine results in the activation of dsx+ Tdc2+ neurons in mated females. As a consequence, octopamine is released from dsx+ Tdc2+ neurons, received by Oamb, induces [Ca2+]i in the escort cells, and finally activates the Mmp2 enzymatic activity. The activity of Mmp2 positively regulates the Dpp-mediated niche signaling, thereby leading to mating-induced GSC increase. (Yoshinari, 2020).

    The proposed model is that the OA from dsx+ Tdc2+ neurons is directly received by the escort and follicle cells in the germarium. This model is supported by two observations. First, mating-induced GSC increase is impaired by Oamb RNAi using a GAL4 driver that is active specifically in the germarium cells but not mature follicle cells. Second, OA treatment evokes [Ca2+]i elevation in these germarium cells in an Oamb-dependent manner. However, this study did not address whether the escort cells and/or the follicle cells in the germarium express Oamb, as no clear GAL4 expression was observed in two independent Oamb-T2A-GAL4 drivers. It is surmised that this may be due to lower amounts of Oamb transcript in the germarium (Yoshinari, 2020).

    It was shown that the activation of the ovary-projecting dsx+ Tdc2+ neurons is necessary and sufficient to induce GSC increase. However, from an anatomical point of view, the dsx+ Tdc2+ neurons project to the distal half of the ovary but not to the germarium. Considering the model described above, this disagreement can be attributed to the characteristic volume transmission of monoamine neurotransmitters. In other words, neurotransmitters act at a distance well beyond their release sites from cells or synapses. Therefore, the OA secreted from the terminals of dsx+ Tdc2+ neurons could reach the germarium located at the most proximal part of the ovary (Yoshinari, 2020).

    Several previous studies have revealed that OA signaling has a pivotal role in reproductive tissues other than germarium, such as mature follicle cells, oviduct, and ovarian muscle, to promote ovulation, oviduct remodeling, and ovarian-muscle contraction, respectively. Therefore, it is likely that the dsx+ Tdc2+ neurons orchestrate multiple different events during oogenesis in response to mating stimulus. Because a mated female needs to activate oogenesis to continuously produce eggs in concert with sperm availability, it is reasonable that the ovary-projecting neurons switch on the activity of the entire process of reproduction (Yoshinari, 2020).

    Based on the present study and several previous studies, the OA-Oamb-Ca2+-Mmp2 axis is required for GSC increase and follicle rupture, both of which are induced by mating stimuli in D. melanogaster. In both cases, Mmp2 enzymatic activity is likely to be essential, as the overexpression of Timp encoding a protein inhibitor of Mmp2 suppresses GSC increase, as well as follicle rupture. Mmp2 in mature follicle cells cleaves and downregulates Viking/collagen VI. In fact, several previous studies have revealed that Viking/collagen VI is required for GSC maintenance in female D. melanogaster. However, no significant change was observed in Viking/Collagen VI levels in the germarium between the control and Mmp2 RNAi flies. Therefore, it is concluded that Viking/collagen VI is not a substrate of Mmp2 in the regulation of mating-induced GSC increase. Besides Viking/Collagen VI, Dally-like (Dlp) is another basement membrane protein associated with extracellular matrix and known as the Mmp2 substrate. Interestingly, dlp is expressed in the escort cells. Moreover, Dlp controls the distribution of Dpp and Wnts, both of which significantly affect GSC self-renewal and differentiation. Future research should decipher the exact substrate by which Mmp2 controls Dpp and/or Wnts to modulate GSC behavior in response to mating stimulus (Yoshinari, 2020).

    Another remaining question to be addressed is how Mmp2 function is regulated in GSC increase. Ecdysteroid biosynthesis and signaling in the ovary are necessary but not sufficient for the OA-Oamb-Ca2+-mediated GSC increase and follicle rupture. This study found that in the regulation of mating-induced GSC increase, ecdysteroid signaling acts downstream of Ca2+ signaling. On the other hand, in the follicle rupture process, ecdysteroid signaling either acts downstream, upstream, or both, of Ca2+ signaling. Further, the precise action of ecdysteroid has yet to be elucidated. The Mmp2-GFP fusion protein level in the follicle cells is not changed in the loss-of-Ecdysone receptor-function flies, implying that ecdysteroid signaling might regulate Mmp2 enzymatic activity by an unknown mechanism. Considering the involvement of both the OA-Oamb-Ca2+-Mmp2 axis and ecdysteroid biosynthesis, it is very likely that the Mmp2 enzymatic activity is also regulated by the same, unknown mechanism not only in the mature follicle cells to control follicle rupture, but also in the germarium to control mating-induced GSC increase (Yoshinari, 2020).

    In many animals, reproduction involves significant behavioral and physiological shifts in response to mating. In female D. melanogaster, several post-mating responses are coordinated by SPSNs and their downstream afferent neuronal circuit, including Stato-Acoustic Ganglion neurons, the ventral abdominal lateral Myoinhibitory peptide neurons, and the efferent dsx+ Tdc2+ neurons. GRASP analysis indicates a direct synaptic connection between cholinergic SPSNs and OAergic neurons. Moreover, this study demonstrated that nAChRs in dsx+ Tdc2+ neurons are responsible for the suppression of their neuronal activity in virgin females. However, nAChRs are the cation channels leading to depolarization upon acetylcholine binding, and therefore usually activate neurons. How is the opposite role of nAChRs in dsx+ Tdc2+ neuronal activity achieved? One possibility is that acetylcholine-nAChR signaling does not evoke a simple depolarization but rather generates a virgin-specific temporal spike pattern in dsx+ Tdc2+ neurons. Interestingly, recent studies demonstrated that the pattern, instead of the frequency, of neuronal firing is significant in adjusting the neuronal activity of clock neurons in D. melanogaster. The firing pattern relies on control of ionic flux by the modulation of Ca2+-activated potassium channel and Na+/K+ ATPase activity. Because whether mating changes the firing pattern of dsx+ Tdc2+ neurons remains to be examined, the neuronal activity in SPSNs and the dsx+ Tdc2+ neuronal circuit between virgin and mated females are future research areas (Yoshinari, 2020).

    In the last decades, there is growing evidence that GSCs and their niche are influenced by multiple humoral factors. Based on the data from the current study and previous studies, there are at least four crucial humoral factors for regulating the increase and/or maintenance of D. melanogaster female GSCs, including DILPs, ecdysteroids, Neuropeptide F (NPF), and OA. Notably, all of these come from different sources: DILPs are from the insulin-producing cells located in the pars intercerebralis of the central brain; ecdysteroids from the ovary; NPF from the midgut; and OA from the neurons located in the abdominal ganglion. In addition to these identified humoral factors, recent studies also imply that adiponectin and unknown adipocyte-derived factor(s) are essential for GSC maintenance. These data clearly indicate that D. melanogaster female GSCs are systemically regulated by interorgan communication involving multiple organs. The additional interorgan communication mechanisms that ensure the faithful coupling of the increase and maintenance of GSC to the organism's external and physiological environments are essential to be investigated in future studies (Yoshinari, 2020).

    To modulate the increase and maintenance of GSC, ecdysteroids are received by both GSCs and niche cells, whereas DILPs, NPF, and OA are received by niche cells. A major signal transduction mechanism of each of these humoral factors have been well characterized, namely phosphoinositide 3-kinase pathway for DILPs-InR signaling, EcR/Ultraspiracle-mediated pathway for ecdysteroid signaling, cAMP pathway for NPF-NPFR signaling, and Ca2+ pathway for OA-Oamb signaling. However, it remains unclear whether and how each of these signaling pathways control the production and secretion of the niche signal, as well as its distribution and transduction. In addition, it is important to understand whether and how the multiple system signals are integrated to control the mating-induced increase and maintenance of GSCs (Yoshinari, 2020).

    In recent years, many studies have revealed that not only local niche signals but also systemic and neuronal factors play indispensable roles in regulating GSC behavior. In D. melanogaster, ecdysteroid signaling is essential for the proliferation and maintenance of GSCs and neural stem cells. This study has identified the ovary-projecting OAergic neurons as new regulators of stem cell homeostasis. Both steroid hormones and OA-like monoamines, such as noradrenaline, are also involved in stem cell regulation in mammals. For example, the mammalian steroid hormone, estrogen, is important in regulating cell division and/or maintenance of hematopoietic stem cells, mammary stem cell, neural stem cells, and hematopoietic stem cells. Moreover, noradrenergic neurons, which directly project to the bone marrow, regulate the remodeling of hematopoietic stem cells niche. Therefore, the steroid hormone- and noradrenergic nerve-dependent control of stem cell homeostasis are likely conserved across animal species. In this regard, the D. melanogaster reproductive system will further serve as a powerful model to unravel the conserved systemic and neuronal regulatory mechanisms for stem cell homeostasis in animals (Yoshinari, 2020).

    Hormone receptor 4 is required in muscles and distinct ovarian cell types to regulate specific steps of Drosophila oogenesis

    The conserved nuclear receptor superfamily has crucial roles in many processes, including reproduction. Nuclear receptors with known roles in oogenesis have been studied mostly in the context of their ovary-intrinsic requirement. Recent studies in Drosophila, however, have begun to reveal new roles of nuclear receptor signaling in peripheral tissues in controlling reproduction. This study identified Hormone receptor 4 (Hr4) as an oogenesis regulator required in the ovary and muscles. Global Hr4 knockdown leads to increased germline stem cell (GSC) loss, reduced GSC proliferation, early germline cyst death, slowed follicle growth and vitellogenic follicle degeneration. Tissue-specific knockdown experiments uncovered ovary-intrinsic and peripheral tissue requirements for Hr4. In the ovary, Hr4 is required in the niche for GSC proliferation and in the germline for GSC maintenance. Hr4 functions in muscles to promote GSC maintenance and follicle growth. The specific tissues that require Hr4 for survival of early germline cysts and vitellogenic follicles remain unidentified. These results add to the few examples of muscles controlling gametogenesis and expand our understanding of the complexity of nuclear receptor regulation of various aspects of oogenesis (Weaver, 2021).

    Drosophila CG2469 encodes a homolog of human CTR9 and is essential for development

    Conserved from yeast to humans, the Paf1 complex participates in a number of diverse processes including transcriptional initiation and polyadenylation. This complex typically includes 5 proteins: Paf1, Rtf1, Cdc73, Leo1 and Ctr9. Previous efforts have identified clear Drosophila homologs of Paf1, Rtf1 and Cdc73 based on sequence similarity. Further work has showed that these proteins help to regulate gene expression and are required for viability. To date, a Drosophila homolog of Ctr9 has remained uncharacterized. This study shows that the gene CG2469 encodes a functional Drosophila Ctr9 homolog. Both human and Drosophila Ctr9 localize to the nuclei of Drosophila cells and appear enriched in histone locus bodies. RNAi knock-down of Drosophila Ctr9 results in a germline stem cell loss phenotype marked by defects in the morphology of germ cell nuclei. A molecular null mutation of Drosophila Ctr9 results in lethality and a human cDNA Ctr9 transgene rescues this phenotype. Clonal analysis in the ovary using this null allele reveals that loss of Drosophila Ctr9 results in a reduction of global levels of histone H3 trimethylation of lysine 4 (H3K4me3) but does not compromise the maintenance of stem cells in ovaries. Given the differences between the null mutant and RNAi knockdown phenotypes, the germ cell defects caused by RNAi likely result from the combined loss of Drosophila Ctr9 and other unidentified genes. These data provide further evidence that the function of this Paf1 complex component is conserved across species (Chaturvedi, 2016).

    Drosophila female germline stem cells undergo mitosis without nuclear breakdown

    Stem cell homeostasis requires nuclear lamina (NL) integrity. In Drosophila germ cells, compromised NL integrity activates the ataxia telangiectasia and Rad3-related (ATR) and checkpoint kinase 2 (Chk2) checkpoint kinases, blocking germ cell differentiation and causing germline stem cell (GSC) loss. Checkpoint activation occurs upon loss of either the NL protein emerin or its partner barrier-to-autointegration factor, two proteins required for nuclear reassembly at the end of mitosis. This study examined how mitosis contributes to NL structural defects linked to checkpoint activation. These analyses led to the unexpected discovery that wild-type female GSCs utilize a non-canonical mode of mitosis, one that retains a permeable but intact nuclear envelope and NL. The interphase NL is remodeled during mitosis for insertion of centrosomes that nucleate the mitotic spindle within the confines of the nucleus. Depletion or loss of NL components causes mitotic defects, including compromised chromosome segregation associated with altered centrosome positioning and structure. Further, in emerin mutant GSCs, centrosomes remain embedded in the interphase NL. Notably, these embedded centrosomes carry large amounts of pericentriolar material and nucleate astral microtubules, revealing a role for emerin in the regulation of centrosome structure. Epistasis studies demonstrate that defects in centrosome structure are upstream of checkpoint activation, suggesting that these centrosome defects might trigger checkpoint activation and GSC loss. Connections between NL proteins and centrosome function have implications for mechanisms associated with NL dysfunction in other stem cell populations, including NL-associated diseases, such as laminopathies (Duan, 2021).

    Nuclear structure is shaped by proteins resident in the nuclear lamina (NL). This extensive network is composed of lamins and hundreds of lamin-associated proteins that line the inner nuclear membrane. The NL confers nuclear rigidity and contributes to chromatin organization important for regulation of transcription, replication, and DNA repair. Additionally, NL proteins transmit regulatory information between cellular compartments through connections that link the nucleoskeleton with the cytoskeleton. Nuclear structure correlates with cell-type-specific changes in NL composition, differences that impact genome organization and function during development (Duan, 2021).

    The LAP2-emerin-MAN1 domain (LEM-D) protein family has a prominent role in the NL. These proteins share an ~40-amino-acid domain (LEM-D) that interacts with Barrier-to-autointegration factor (BAF) (sometimes referred to as BANF1), a conserved chromatin protein. In non-dividing cells, interactions between LEM-D proteins and BAF link the genome with the nuclear periphery. In dividing cells, these interactions control mitotic spindle assembly and positioning, as well as nuclear reassembly at the end of mitosis. These properties highlight mechanisms wherein the LEM-D and BAF partnership contributes to nuclear architecture (Duan, 2021).

    Physiological aging and many diseases are associated with changes in nuclear structure. Indeed, misshapen and lobulated nuclei are common features of laminopathies, diseases that result from mutations in genes encoding NL proteins. Laminopathies affect some cell types more than others, with primary defects found in skeletal muscle, skin, fat, and bone. Age-associated worsening of laminopathic diseases has been linked to failures in stem cell maintenance suggesting that the NL plays an important role in balancing stem cell proliferation with differentiation. Although contributions of the NL to stem cell function are being investigated, mechanisms that preserve healthy stem cell populations and promote tissue homeostasis remain poorly understood (Duan, 2021).

    Studies in Drosophila melanogaster have identified roles for LEM-D proteins and BAF in adult stem cell maintenance. Drosophila encodes three NL LEM-D proteins that bind BAF, including two emerin orthologs (emerin also known as otefin and emerin2 also known as Bocksbeutel) and MAN1. Notably, loss of emerin compromises homeostasis of adult stem cells in the female and male germlines, blocking germ cell differentiation and causing germline stem cell (GSC) loss. These defects are coupled with GSC-restricted deformation of the NL and accumulation of DNA damage, phenotypes that mirror those found in laminopathic cells. Strikingly, gametogenesis of Drosophila emerin mutant germ cells is rescued by mutation of two DNA damage response (DDR) kinases, the responder kinase ataxia telangiectasia and Rad3-related (ATR) or the transducer kinase checkpoint kinase 2 (Chk2). Although emerin mutant GSCs carry DNA damage, genetic and molecular analyses suggest that ATR and Chk2 activation occurs independently of canonical DNA damage triggers and is linked to the NL structural deformation. Germ-cell-specific BAF depletion also causes NL deformation and GSC loss that is partially rescued by chk2 mutation. These findings indicate that emerin and BAF contribute to shared NL functions needed for GSC maintenance (Duan, 2021).

    This study extends an investigations of events associated with activation of the NL checkpoint. Prompted by shared requirements for emerin and BAF in nuclear reassembly, this study examined whether defects in mitosis were responsible for NL deformation and checkpoint activation. To this end, the structure of the NL was followed throughout female GSC (fGSC) mitosis. These analyses led to the unexpected discovery that wild-type fGSCs use a non-canonical mode of mitosis, wherein a permeable but intact nuclear envelope (NE) and remodeled NL remain throughout mitosis. This mode of mitosis imposes requirements for NL components, evidenced by observations that depletion or loss of NL components causes defects in centrosome positioning and spindle structure. In emerin mutant fGSCs, centrosomes remain embedded in the interphase NL and retain large amounts of pericentriolar material (PCM) that nucleates astral microtubules. These observations reveal a role for emerin in the regulation of centrosome structure. Epistasis studies demonstrate that PCM retention in emerin mutant GSCs is upstream of Chk2 activation, indicating that the altered structure of the interphase centrosome is linked with NL checkpoint activation. Based on these data, it is proposed that other stem cells might employ distinct modes of mitosis that sensitize these cells to defects in the NL. These findings have implications for mechanisms associated with NL dysfunction in other systems, including laminopathies (Duan, 2021).

    The NL has a central role in establishing structures important for the homeostasis of diverse stem cells. Indeed, survival of Drosophila GSCs depends upon the integrity of the NL, wherein NL deformation is linked to activation of the ATR and Chk2 kinases that leads to GSC loss. This studu investigated mechanisms leading to NL deformation in fGSCs, examining the role of mitosis in shaping the NL (Duan, 2021).

    Two main modes of mitosis exist, open and closed. In open mitosis, the NE and NL break down, enabling spindle microtubules nucleated from cytoplasmic centrosomes to capture and segregate chromosomes. In closed mitosis, mitotic spindles are nucleated by spindle pole bodies (centrosome equivalents) that are embedded in a retained NE. It is generally assumed that metazoan cells use open mitosis, whereas fungi use closed mitosis. This assumption is linked with metazoan-limited expression of lamin and BAF, two proteins that are required for nuclear reassembly at the end of open mitosis. However, several exceptions to this rule exist, indicating that open and closed mitoses represent extremes of a continuum of mitotic strategies. A classic example of an exceptional mode of mitosis occurs in the Drosophila early embryo. In these early divisions, limited nuclear breakdown occurs, wherein local NE and NL breakdown occurs near centrosomes, with large portions of the NE and NL remaining until metaphase. This NL has a stabilizing function on spindle microtubules, as disruption of the lamin-B delays prometaphase spindle assembly. However, progression to anaphase requires NL and NE dispersal, as increased stabilization of the lamin-B prevents spindle elongation. These mitotic events indicate that the regulation of NE and NL remodeling optimizes progression through mitosis (Duan, 2021).

    Mitotic divisions of Drosophila female germ cells also deviate from open mitosis. In contrast to somatic cells in the ovary that show universal NL dispersal, germ cells change the mode of mitosis depending on developmental stage. In larval PCGs, nuclear breakdown begins in prometaphase, forming large, broken patches of NL, whereas in adult fGSCs, the NL remains intact, even into anaphase. As a result, spindle microtubules form within the mitotic fGSC nucleus, emanating from centrosomes embedded in the NL. Notably, features of this non-canonical mitosis are shared with fungi. For example, in Aspergillus nidulans, the never-in-mitosis kinase partially disassembles NPCs, increasing permeability of an otherwise intact mitotic NE to allow mitotic regulators access to the prophase nucleoplasm. Similarly, it was found that NPCs in mitotic fGSCs are partially disassembled, allowing for exchange of nuclear and cytoplasmic components. However, other features differ between fGCS and fungal mitosis. For example, Saccharomyces cerevisiae carry a centriole-less spindle pole body that is embedded in the NE, which allows nucleation of spindle and astral microtubules within the nuclear compartment. In contrast, the centriole-containing centrosomes of fGCSs move into the NE and NL to promote spindle microtubule assembly within the nucleoplasm. fGCS centrosomes are inserted into a cup-like structure composed of lamin-B and emerin, suggesting that localized remodeling of the NE and NL occurs. Based on these comparisons with fungi, it is proposed that fGSCs employ an intermediate form of mitosis, one that is not completely closed because the NE becomes permeable or completely open because the NE and NL remain intact. The current data add additional evidence that metazoans do not solely employ an open mode of mitosis. Indeed, in Drosophila, modes of mitosis are cell type and developmental stage specific (Duan, 2021).

    Genetic studies suggest that NL proteins are required for execution of fGSC mitosis. Loss of emerin or depletion of lamin-B alters the structure and positioning of mitotic spindles. Both NL mutants increase the frequency of lagging chromosomes in anaphase, suggesting that the quality of fGSC mitosis is compromised upon NL dysfunction. Defects in the mitotic spindle might result from disruption of mitotic spindle assembly or mitotic matrix formation, as mammalian lamin B is a structural component of the spindle matrix that promotes microtubule organization in mitosis. Alternatively, mitotic spindle defects might result from an altered distribution of nuclear pores, as centrosome separation in mitotic prophase is linked to nuclear pore distribution. Further studies are needed to address these possibilities (Duan, 2021).

    The significance of the developmental switch between PGCs and fGSCs mitosis is unclear. Notably, this switch correlates with the transition from symmetric to asymmetric division, suggesting that retention of a mitotic NL might contribute to acquisition of distinct cell fates that occur within a single cell division. Indeed, fGSC homeostasis is linked to the asymmetric inheritance of two nuclear factors that regulate rRNA transcription and maturation, Wicked/U3 small nucleolar RNA (snoRNA)-associated protein 18 and UnderdevelopedTAF1 (TATA-Box Binding Protein Associated Factor 1). The data indicate that asymmetric trafficking of these proteins occurs within an intact mitotic NL, which might help establish the distinct distribution of these proteins and possibly others. For example, microtubules direct the asymmetric distribution of pSmad to one centrosome for its degradation in cultured human embryonic stem cells. Although niche signaling has a dominant role in fGSC maintenance, it is suggested that retention of a mitotic NL might amplify mechanisms used in asymmetric division (Duan, 2021).

    Although emerin and lamin-B are required for fGSC mitosis, only loss of emerin leads to fGSC death. Indeed, oogenesis in nos > lam RNAi females occurs without evidence of Chk2 activation (Duan, 2021).

    Although the low levels of lamin-B that remain in nos > lam RNAi fGSCs might be sufficient to guide mitosis, it is also possible that emerin and lamin-B make distinct contributions. Observations that the structure of the interphase centrosome differs in the two mutant backgrounds provide support for the latter possibility. In emerin mutant fGSCs, centrosomes remain embedded in the NL, and these centrosomes retain increased amounts of PCM, defects not observed in nos>RNAi mutants. Embedded centrosomes might contribute to the extensive structural deformation of the NL found in these emerin mutant fGSCs, because the expanded PCM retains γ-tubulin, the major microtubule nucleating component of the PCM.82 As a result, emerin, but not nos > lam RNAi, mutants nucleate astral microtubules in interphase fGSCs. Epistasis studies demonstrate that both PCM expansion and microtubule nucleation remain in chk2, emerin double mutants, implying that these features are independent or upstream of checkpoint activation. It is predicted that differences in interphase centrosome structure are connected to NL checkpoint activation (Duan, 2021).

    Mechanisms responsible for expanded PCM in emerin mutants are unknown. Centrosome maturation and disassembly involve regulated activities of kinases that promote PCM expansion and phosphatases that reverse phosphorylation of PCM proteins. Structural defects of the interphase emerin mutant centrosome might originate from incomplete or partial PCM disassembly or from premature recruitment of PCM. Effects on centrosome structure might be direct, as emerin is a component of mitotic centrosomes in Drosophila embryos. This association appears to be conserved, as human emerin is also found in mitotic centrosomes. Further, phosphorylation of Drosophila emerin by Aurora-A kinase is required for mitotic exit in SL2 cells, indicating that emerin has a regulatory function at the centrosome. Alternatively, effects of loss of emerin might be indirect, resulting from gene expression changes that alter levels of mitotic regulators. Regardless of mechanism, the current data suggest that emerin has a role in PCM regulation. Further investigations are needed to test contributions of emerin to the centrosome cycle in fGSC mitosis (Duan, 2021).

    ATR and Chk2 kinases localize to mitotic centrosomes. Studies in human cells indicate that ATR associates with γ-tubulin and influences the kinetics of microtubule formation at centrosomes. Localization of ATR to the centrosome provides a link between mitosis and the DDR. However, DDR proteins localize to centrosomes even in the absence of DNA damage, raising the possibility that ATR is a general sensor of structure and function at centrosomes. Building from these observations, it is predicted that the structurally defective centrosome in emerin mutant fGSCs might be responsible for transmitting signals to ATR and Chk2 kinases, ultimately leading to fGSC loss (Duan, 2021).

    Mutations in NL LEM-D proteins cause diseases linked to compromised stem cell homeostasis. This study links centrosome dysfunction with failures in stem cell homeostasis due to mutation of the Drosophila NL protein Emerin. These studies align with observations in human fibroblasts that emerin anchors interphase centrosomes to the nucleus through direct interactions with microtubules and that expression of mutant forms of emerin in HeLa cells causes aberrant nuclear shape and mislocalization of tubulin and centrosomes. Taken together, these observations reinforce connections between emerin and centrosomes. As mechanisms of stem cell homeostasis are shared between cell types and organisms, it is possible that other stem cell populations used non-canonical modes of mitosis to ensure robustness of the asymmetric division, which might sensitize division of these cells to defects in the NL composition (Duan, 2021).

    RNA methyltransferase BCDIN3D is crucial for female fertility and miRNA and mRNA profiles in Drosophila ovaries

    RNA methyltransferases post-transcriptionally add methyl groups to RNAs, which can regulate their fates and functions. Human BCDIN3D (Bicoid interacting 3 domain containing RNA methyltransferase) has been reported to specifically methylate the 5'-monophosphates of pre-miR-145 and cytoplasmic tRNAHis. Methylation of the 5'-monophosphate of pre-miR-145 blocks its cleavage by the miRNA generating enzyme Dicer, preventing generation of miR-145. Elevated expression of BCDIN3D has been associated with poor prognosis in breast cancer. However, the biological functions of BCDIN3D and its orthologs remain unknown. This study examined the biological and molecular functions of CG1239, a Drosophila ortholog of BCDIN3D. Ovary-specific knockdown of Drosophila BCDIN3D causes female sterility. High-throughput sequencing revealed that miRNA and mRNA profiles are dysregulated in BCDIN3D knockdown ovaries. Pathway analysis showed that many of the dysregulated genes are involved in metabolic processes, ribonucleoprotein complex regulation, and translational control. These results reveal BCDIN3D's biological role in female fertility and its molecular role in defining miRNA and mRNA profiles in ovaries (Zhu, 2019).

    Division-independent differentiation mandates proliferative competition among stem cells

    Cancer-initiating gatekeeper mutations that arise in stem cells would be especially potent if they stabilize and expand an affected stem cell lineage. It is therefore important to understand how different stem cell organization strategies promote or prevent variant stem cell amplification in response to different types of mutation, including those that activate proliferation. Stem cell numbers can be maintained constant while producing differentiated products through individually asymmetrical division outcomes or by population asymmetry strategies in which individual stem cell lineages necessarily compete for niche space. This study considers alternative mechanisms underlying population asymmetry and used quantitative modeling to predict starkly different consequences of altering proliferation rate: A variant, faster proliferating mutant stem cell should compete better only when stem cell division and differentiation are independent processes. For most types of stem cells, it has not been possible to ascertain experimentally whether division and differentiation are coupled. However, Drosophila follicle stem cells (FSCs) provided a favorable system with which to investigate population asymmetry mechanisms and also for measuring the impact of altered proliferation on competition. Detailed cell lineage studies that division and differentiation of an individual FSC were found to be uncoupled. FSC representation, reflecting maintenance and amplification, was highly responsive to genetic changes that altered only the rate of FSC proliferation. The FSC paradigm therefore provides definitive experimental evidence for the general principle that relative proliferation rate will always be a major determinant of competition among stem cells specifically when stem cell division and differentiation are independent (Reilein, 2018).

    Canonical Wnt Signaling Promotes Formation of Somatic Permeability Barrier for Proper Germ Cell Differentiation

    Morphogen-mediated signaling is critical for proper organ development and stem cell function, and well-characterized mechanisms spatiotemporally limit the expression of ligands, receptors, and ligand-binding cell-surface glypicans. This study shows that in the developing Drosophila ovary, canonical Wnt signaling promotes the formation of somatic escort cells (ECs) and their protrusions, which establish a physical permeability barrier to define morphogen territories for proper germ cell differentiation. The protrusions shield germ cells from Dpp and Wingless morphogens produced by the germline stem cell (GSC) niche and normally only received by GSCs. Genetic disruption of EC protrusions allows GSC progeny to also receive Dpp and Wingless, which subsequently disrupt germ cell differentiation. These results reveal a role for canonical Wnt signaling in specifying the ovarian somatic cells necessary for germ cell differentiation. Additionally, it was demonstrated the morphogen-limiting function of this physical permeability barrier, which may be a common mechanism in other organs across species (Chen, 2022).

    Essential functions of mosquito ecdysone importers in development and reproduction

    The primary insect steroid hormone ecdysone requires a membrane transporter to enter its target cells. Although an organic anion-transporting polypeptide (OATP) named Ecdysone Importer (EcI) serves this role in the fruit fly Drosophila melanogaster and most likely in other arthropod species, this highly conserved transporter is apparently missing in mosquitoes. This study reports three additional OATPs that facilitate cellular incorporation of ecdysone in Drosophila and the yellow fever mosquito Aedes aegypti. These additional ecdysone importers (EcI-2, -3, and -4) are dispensable for development and reproduction in Drosophila, consistent with the predominant role of EcI. In contrast, in Aedes, EcI-2 is indispensable for ecdysone-mediated development, whereas EcI-4 is critical for vitellogenesis induced by ecdysone in adult females. Altogether, these results indicate unique and essential functions of these additional ecdysone importers in mosquito development and reproduction, making them attractive molecular targets for species- and stage-specific control of ecdysone signaling in mosquitoes (Hun, 2022).

    The phospho-docking protein 14-3-3 regulates microtubule-associated proteins in oocytes including the chromosomal passenger Borealin

    Global regulation of spindle-associated proteins is crucial in oocytes due to the absence of centrosomes and their very large cytoplasmic volume, but little is known about how this is achieved beyond involvement of the Ran-importin pathway. Previous work has uncovered a novel regulatory mechanism in Drosophila oocytes, in which the phospho-docking protein 14-3-3 suppresses microtubule binding of Kinesin-14/Ncd away from chromosomes. This paper reports systematic identification of microtubule-associated proteins regulated by 14-3-3 from Drosophila oocytes. Proteins from ovary extract were co-sedimented with microtubules in the presence or absence of a 14-3-3 inhibitor. Through quantitative mass-spectrometry, proteins or complexes were identified whose ability to bind microtubules is suppressed by 14-3-3, including the chromosomal passenger complex (CPC), the centralspindlin complex and Kinesin-14/Ncd. 14-3-3 binds to the disordered region of Borealin, and this binding is regulated differentially by two phosphorylations on Borealin. Mutations at these two phospho-sites compromised normal Borealin localisation and centromere bi-orientation in oocytes, showing that phospho-regulation of 14-3-3 binding is important for Borealin localisation and function (Repton, 2022).

    Cross-species incompatibility between a DNA satellite and the Drosophila Spartan homolog poisons germline genome integrity

    Satellite-rich genomic regions mediate strictly conserved, essential processes such as chromosome segregation and nuclear structure. A leading resolution to this paradox posits that satellite DNA and satellite-associated chromosomal proteins coevolve to preserve these essential functions. This study experimentally test this model of intragenomic coevolution by conducting the first evolution-guided manipulation of both chromosomal protein and DNA satellite. The 359bp satellite spans an 11 Mb array in Drosophila melanogaster that is absent from its sister species, Drosophila simulans. This species-specific DNA satellite colocalizes with the adaptively evolving, ovary-enriched protein, Maternal haploid (MH), the Drosophila homolog of Spartan. To determine if MH and 359bp coevolve, the D. simulans version of MH ("MH[sim]") was swapped into D. melanogaster. MH[sim] triggers ovarian cell death, reduced ovary size, and loss of mature eggs. Surprisingly, the D. melanogaster mh-null mutant has no such ovary phenotypes, suggesting that MH[sim] is toxic in a D. melanogaster background. Using both cell biology and genetics, it was discovered that MH[sim] poisons oogenesis through a DNA-damage pathway. Remarkably, deleting the D. melanogaster-specific 359bp satellite array completely restores mh[sim] germline genome integrity and fertility, consistent with a history of coevolution between these two fast-evolving loci. Germline genome integrity and fertility are also restored by overexpressing topoisomerase II (Top2), suggesting that MH[sim] interferes with Top2-mediated processing of 359bp. The observed 359bp-MH[sim] cross-species incompatibility supports a model under which seemingly inert repetitive DNA and essential chromosomal proteins must coevolve to preserve germline genome integrity (Brand, 2022).

    A translation control module coordinates germline stem cell differentiation with ribosome biogenesis during Drosophila oogenesis

    Ribosomal defects perturb stem cell differentiation, and this is the cause of ribosomopathies. How ribosome levels control stem cell differentiation is not fully known. This study discovered that three DExD/H-box proteins govern ribosome biogenesis (RiBi) and Drosophila oogenesis. Loss of these DExD/H-box proteins, which were named Aramis, Athos, and Porthos, aberrantly stabilizes p53, arrests the cell cycle, and stalls germline stem cell (GSC) differentiation. Aramis controls cell-cycle progression by regulating translation of mRNAs that contain a terminal oligo pyrimidine (TOP) motif in their 5' UTRs. TOP motifs confer sensitivity to ribosome levels that are mediated by La-related protein (Larp). One such TOP-containing mRNA codes for novel nucleolar protein 1 (Non1), a conserved p53 destabilizing protein. Upon a sufficient ribosome concentration, Non1 is expressed, and it promotes GSC cell-cycle progression via p53 degradation. Thus, a previously unappreciated TOP motif in Drosophila responds to reduced RiBi to co-regulate the translation of ribosomal proteins and a p53 repressor, coupling RiBi to GSC differentiation (Martin, 2022).

    All life depends on the ability of ribosomes to translate mRNAs into proteins. Despite this universal requirement, perturbations in ribosome biogenesis (RiBi) affect some cell types more than others. Stem cells, a cell type that underlies the generation and expansion of tissues, have an increased ribosomal requirement. Ribosome production is dynamically regulated to maintain higher amounts in stem cells. Reduction of ribosome levels in several stem cell systems can cause differentiation defects. In Drosophila, perturbations that reduce ribosome levels in the germline stem cells (GSCs) result in differentiation defects, causing infertility. Similarly, humans with impaired RiBi are afflicted with clinically distinct diseases known as ribosomopathies, such as Diamond-Blackfan anemia, that often result from loss of proper differentiation of tissue-specific progenitor cells. However, the mechanisms by which RiBi is coupled to proper stem cell differentiation remain incompletely understood (Martin, 2022).

    RiBi requires the transcription of ribosomal RNAs (rRNAs) and of mRNAs encoding ribosomal proteins (RPs) . Hundreds of factors, including DExD/H-box proteins, transiently associate with maturing rRNAs to facilitate rRNA processing, modification, and folding. RPs are imported into the nucleus, where they assemble with rRNAs in the nucleolus to form precursors to the 40S and 60S ribosomal subunits, which are then exported to the cytoplasm (Martin, 2022).

    In mammals, mRNAs that encode the RPs contain a terminal oligo pyrimidine (TOP) motif within their 5' untranslated region (UTR), which regulates their translation in response to nutrient levels. Under growth-limiting conditions, La-related protein 1 (Larp1) binds to the TOP sequences and to mRNA caps to inhibit translation of RPs. When growth conditions are suitable, Larp1 is phosphorylated by the mammalian target of rapamycin complex 1 (mTORC1) and does not efficiently bind the TOP sequence, allowing for translation of RPs. Whether TOP motifs exist in Drosophila to coordinate RP synthesis is unclear. The Drosophila ortholog of Larp1, Larp is required for proper cytokinesis and meiosis in Drosophila testis, as well as for female fertility, but its targets remain undetermined (Martin, 2022).

    Germline depletion of RiBi factors results in a stereotypical GSC differentiation defect during Drosophila oogenesis. Female Drosophila maintain 2-3 GSCs in the germarium. Asymmetric cell division of GSCs produces a self-renewing daughter GSC and a differentiating daughter, called the cystoblast (CB). This asymmetric division is unusual: following mitosis, the abscission of the GSC and CB is not completed until the following G2 phase. The GSC is marked by a round structure called the spectrosome, which elongates and eventually bridges the GSC and CB, similar to the fusomes that connect differentiated cysts. During abscission, the extended spectrosome structure is severed and a round spectrosome is established in the GSC and the CB. RiBi defects result in failed GSC-CB abscission, causing cells to accumulate as interconnected cysts called the 'stem cysts' that are marked by a fusome-like structure. In contrast with differentiated cysts, these stem cysts do not express the differentiation factor bag of marbles (Bam), do not differentiate, and typically die, resulting in sterility. How proper RiBi promotes GSC abscission and differentiation is not known (Martin, 2022).

    During Drosophila oogenesis, efficient RiBi is required in the germline for proper GSC cytokinesis and differentiation. The outstanding questions that needed to be addressed were: (1) Why does disrupted RiBi impair GSC abscission? And (2) How does the GSC monitor and couple RiBi to differentiation? The results suggest that a germline RiBi defect stalls the cell cycle, resulting a loss of differentiation and the formation of stem cysts. Proper RiBi was found to be monitored through a translation control module that allows for co-regulation of RPs and a p53 repressor. Ais, Ath, and Pths support RiBi and allowing for translation of a p53 repressor, preventing p53 stabilization, cell-cycle arrest, and loss of stem cell differentiation (Martin, 2022).

    The developmental upregulation of p53 during GSC differentiation concomitant with reduced RiBi parallels observations in disease states, such as ribosomopathies. This study found that p53 levels in GSCs are regulated by the conserved p53 regulator Non1. Although Non1 has been shown to directly interact with p53, how it regulates p53 levels in both humans and Drosophila is not known (Martin, 2022).

    TOP-containing mRNAs are known to be coregulated to coordinate ribosome production in response to environmental cues. Surprisingly, the observation that loss of ais reduces translation, albeit indirectly via regulation of RiBi, of a cohort of TOP-containing mRNAs, including Non1, suggests that the TOP motif also sensitizes their translation to lowered levels of RiBi. This notion is supported by TOP reporter assays demonstrating that reduced translation upon loss of ais requires the TOP motif. It is hypothesized that limiting TOP mRNA translation lowers RP production to maintain a balance with reduced rRNA production. This feedback mechanism would prevent the production of excess RPs that cannot be integrated into ribosomes and the ensuing harmful aggregates (Martin, 2022).

    The translation and stability of TOP-containing mRNAs are mediated by Larp1 and its phosphorylation. This study found that perturbing rRNA production and thus RiBi, without directly targeting RPs, also results in dysregulation of TOP mRNAs. The current data show that Drosophila Larp binds the RpL30 and Non1 5' UTR in a TOP-dependent manner in vitro and to 97% of the translation targets identified in vivo. Together, these data suggest that rRNA production regulates TOP mRNAs via Larp albeit indirectly. Furthermore, the cytokinesis defect caused by OE of Larp-DM15 in the germline suggests that Larp regulation could maintain the homeostasis of RiBi by balancing the expression of RP production with the rate of other aspects of RiBi, such as rRNA processing, during development (Martin, 2022).

    Ribosomopathies arise from RiBi defects. The underlying mechanisms of tissue specificity remain unresolved. This study demonstrates that loss of proteins involved in rRNA processing lead to cell-cycle arrest. Given that Drosophila GSCs undergo an atypical cell cycle as a normal part of their development it may be that this underlying cellular program in the germline leads to the tissue-specific phenotype of stem-cyst formation (Sanchez et al., 2016). This model implies that other tissues would likewise exhibit tissue-specific manifestations of ribosomopathies due to their underlying cell state. These data suggest two other sources of potential tissue specificity: (1) tissues express different cohorts of mRNAs, such as Non1, which are sensitive to ribosome levels (2). p53 activation, as previously described, is differentially tolerated in different tissues . Together, these mechanisms could begin to explain the tissue-specific nature of ribosomopathies and their link to differentiation (Martin, 2022).

    The exact processing steps that Ais, Ath, and Pths promote in Drosophila RiBi remain unknown; it is hypothesized that the processing step they act on the rRNA would be similar to what has been reported in yeast and mammals. Lack of a full rescue from ais, ath, and pths GKD in p53 mutants suggest that multiple genes likely influence the cell-cycle arrest. Finally, it is possible that the roles of Ais, Ath, and Pths in indirectly promoting Non1 translation does not represent a general effect of RiBi defects and is specific to these three proteins. However, this is thout unlikely as nearly all genes involved in RiBi outside of RPs share the same phenotype when depleted during Drosophila oogenesis (Martin, 2022).

    Tropomyosin 1-I/C coordinates kinesin-1 and dynein motors during oskar mRNA transport

    Dynein and kinesin motors mediate long-range intracellular transport, translocating towards microtubule minus and plus ends, respectively. Cargoes often undergo bidirectional transport by binding to both motors simultaneously. However, it is not known how motor activities are coordinated in such circumstances. In the Drosophila female germline, sequential activities of the dynein-dynactin-BicD-Egalitarian (DDBE) complex and of kinesin-1 deliver oskar messenger RNA from nurse cells to the oocyte, and within the oocyte to the posterior pole. This study shows through in vitro reconstitution that Tm1-I/C, a tropomyosin-1 isoform, links kinesin-1 in a strongly inhibited state to DDBE-associated oskar mRNA. Nuclear magnetic resonance spectroscopy, small-angle X-ray scattering and structural modeling indicate that Tm1-I/C suppresses kinesin-1 activity by stabilizing its autoinhibited conformation, thus preventing competition with dynein until kinesin-1 is activated in the oocyte. Thus work reveals a new strategy for ensuring sequential activity of microtubule motors (Heber, 2024).

    oskar (osk) mRNA localization in the Drosophila egg chamber is an attractive system for studying dual motor transport. Delivery of osk to the posterior pole of the developing oocyte, which drives abdominal patterning and germline formation in the embryo, is driven by the successive activities of dynein and kinesin-1. In early oogenesis, osk mRNA that is synthesized in the nurse cells is transported into the interconnected oocyte by dynein in complex with dynactin and the activating adaptor Bicaudal D (BicD), which is linked to double-stranded mRNA localization signals by the RNA-binding protein Egalitarian (Egl). Association of Egl with BicD and consequent dynein activation are enhanced by binding of Egl to RNA, indicating a role for the cargo in promoting dynein activity. In early oogenesis, microtubule minus ends are nucleated in the oocyte, consistent with the dynein-based delivery of mRNAs into this cell. During mid-oogenesis, the polarity of the microtubule network shifts dramatically, with plus ends pointing towards the oocyte posterior. At this stage, Khc translocates osk to the posterior pole. This process is independent of Klc, raising the question of how Khc is linked to osk and how its motor activity is regulated (Heber, 2024).

    Transport of osk RNA by Khc requires the unique I/C isoform of tropomyosin-1, Tm1-I/C (hereafter Tm1). Tm1 binds to a noncanonical but conserved cargo-binding region in the Khc tail and stabilizes interaction of the motor with RNA, suggesting a function as an adaptor. Both Khc and Tm1 are loaded onto osk ribonucleoprotein particles (RNPs) shortly after their export from the nurse cell nuclei, although the motor only appears to become active in the mid-oogenesis oocyte. Similarly, dynein remains associated with osk RNPs during Khc-mediated transport within the oocyte, but is inactivated by displacement of Egl by Staufen. How the two motors are linked simultaneously to osk RNPs, and how Khc is inhibited during dynein-mediated transport into the oocyte, is not known (Heber, 2024).

    This study shows that Tm1 inhibits Khc by stabilizing its autoinhibited conformation through a new mechanism involving the motor’s regulatory tail domain and stalk. Tm1 also links Khc to the dynein-transported osk RNP, thereby allowing cotransport of inactive Khc on osk RNA by dynein. In vivo, such a mechanism would avoid competition between the two motors during delivery of osk RNPs to the oocyte by dynein, while ensuring that Khc is available on these structures to mediate their delivery to the oocyte posterior in mid-oogenesis. With its cargo-binding and motor regulatory functions, it is proposed that Tm1 is a noncanonical light chain for kinesin-1 (Heber, 2024).

    Tm1 was recently implicated as an RNA adaptor for kinesin-1. The current study reveals a previously unknown role of Tm1 in osk transport. Tm1 is shown to negatively regulates Khc activity, which is proposed to occurs a conformational change in the Khc stalk that stabilizes the Khc motor-tail interaction and thereby enhances autoinhibition (Heber, 2024).

    With its functions in cargo binding and motor regulation, it is speculated that Tm1 is an alternative Klc in the Drosophila female germline. Because precise osk RNA localization during oogenesis is critical for development, positive regulators of Khc function such as PAT1 and negative regulators such as Tm1 may have replaced Klc to provide nuanced control of Khc activity. If this hypothesis is correct, Klc should also be dispensable for Tm1-dependent RNA localization in somatic tissues (Heber, 2024).

    Tm1 also stimulates association of Khc in a strongly inhibited state with dynein-associated osk RNPs. This mechanism would allow dynein-mediated transport of osk RNPs from the nurse cells to the oocyte to proceed without competition with Khc, while positioning the plus-end directed motor on the RNPs for their posteriorward transport within the ooplasm in mid-oogenesis (Heber, 2024).

    Kinesin-1 autoinhibition is not fully understood, in part because of a paucity of structural information for the full-length molecule. It has been proposed that interaction of the tail’s IAK motif with the motor domain plays a key role in kinesin-1 autoinhibition. Consistent with this notion, strongly enhanced motility was obserced of Drosophila Khc when the IAK motif was deleted. However, it was recently shown that Klc inhibits Khc activity independently of the IAK-motor interaction. It was also found that the IAK motif is not needed for inhibition of Khc by Tm1. Instead, it was found that Tm1-mediated inhibition occurs via the Khc AMB domain, a region adjacent to the IAK motif that is essential for osk localization (Heber, 2024).

    Structural analyses suggest that Tm1 stabilizes the autoinhibited folded conformation of Khc by inducing rearrangement of the Khc coiled-coil stalk. Therefore, a model is proposed in which both the motor-IAK interaction and interactions within the Tm1-bound stalk that include the AMB domain act synergistically to achieve the stable inhibited conformation of Khc (Heber, 2024).

    Two recent studies have proposed compact structural arrangements for mammalian kinesin-1. Those studies used chemical crosslinking and cryo-EM, which are likely to enrich for a homogeneous population of compact conformations of Khc and Khc–Klc tetramers, to provide detailed static structural information. By contrast, in the current study of Drosophila Khc and Khc–Tm1 complexes, NMR and SAXS were employed providing insight into the different conformational states, and thus flexibility, of kinesin-1 by enabling analysis of structures in solution. A previous study observed the compact, inhibited conformation in the isolated Khc, showing that Klc-binding does not induce a new fold of Khc but rather stabilizes the inhibited conformation. This is in agreement with the current model, in which Tm1, a putative alternative Klc, shifts the structural equilibrium of Khc towards the autoinhibited state by stabilizing its compact conformation (Heber, 2024).

    Although it is known that many cargo types are transported by the concerted action of dynein and kinesins, the underlying regulatory mechanisms have been elusive. This study has identified one of very few examples of a factor that not only links dynein and kinesin-mediated transport, but also modulates transport through differential motor regulation. Linkage of dynein and kinesin-3 by the dynein-activating adaptor Hook3 has been demonstrated, but the cellular events for which this is relevant are still emerging. Recently, reconstituted coupling of dynein and kinesin-1 by TRAK1 and TRAK2 has provided insight into how the motors are recruited and regulated for mitochondrial transport. However, unlike this study's integration of DDBE and Khc in reconstituted osk RNPs, these systems lacked intact native cargoes and thereby excluded potential cargo-directed positioning and modulation of motor complexes (Heber, 2024).

    Several studies have reported that active dynein and kinesin motors engage in a tug-of-war when artificially coupled. This study also observe motor opposition in reconstituted RNPs containing DDBE and Khc, presumably because of the stochastic engagement of autoinhibited Khc with microtubules. However, the observation that Tm1 supports efficient dynein-mediated RNA transport through robust inhibition of Khc highlights the importance of regulatory factors in addition to mechanical coupling in native transport complexes. Supporting the in vivo relevance of negative regulation of Khc during bidirectional transport, kinesin-1-activating IAK mutations were recently shown to impair dynein-mediated transport processes in Aspergillus nidulans Collectively, these observations point to complex interplay between opposite-polarity motors that are bound simultaneously to cargoes. Further reconstitutions of dual motor systems on native cargoes should reveal generalities of dynein-kinesin crosstalk, as well as any cargo-specific regulatory mechanisms (Heber, 2024).

    This study provides mechanistic insight into two critical aspects of osk mRNA transport—assembly of the dual motor complex and how Khc activity is suppressed during dynein-mediated delivery of the transcript from the nurse cells to the oocyte. However, it is not understood how Khc takes over from dynein after osk RNPs arrive in the oocyte. Although recent work has shown that inactivation of dynein by the RNA-binding protein Stau is part of this process, how Tm1- and IAK-mediated inhibition of Khc is alleviated to allow delivery of the mRNA to the oocyte posterior is an open question. One candidate to fulfill this role is Ensconsin, which is required for posterior osk localization and is enriched in the oocyte relative to the nurse cells. Strikingly, the human counterpart of Ensconsin (MAP7) was recently shown to stimulate activity of mammalian kinesin-1 in vitro. Other candidate Khc activators include the exon junction complex, which, together with the SOLE RNA structure, is essential for transport of osk to the oocyte posterior. Because Tm1 needs to remain bound to the osk RNP throughout its posterior translocation, it is likely that the activating factor(s) induces a conformational change in the Khc–Tm1 complex rather than dissociation of Tm1. Future investigations of these regulatory mechanisms are likely to elucidate how kinesin-1 activity is orchestrated in other systems (Heber, 2024).

    Additional information about mRNA localization in Drosophila eggs can be found at the following Interactive Fly sites: Gurken, Homeless, Hunchback, Orb, Pumillo, Staufen, and Vasa.

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