The Interactive Fly

Genes involved in tissue and organ development

Oogenesis and the Oocyte

The process of oogenesis
Growing microtubules push the oocyte nucleus to polarize the Drosophila dorsal-ventral axis
A combinatorial code for pattern formation in Drosophila oogenesis
Long-term live imaging provides new insight into stem cell regulation and germline-soma coordination in the Drosophila ovary
Mei-p26 cooperates with Bam, Bgcn and Sxl to promote early germline development in the Drosophila ovary
Centrosome-dependent asymmetric inheritance of the midbody ring in Drosophila germline stem cell division
ALIX and ESCRT-III coordinately control cytokinetic abscission during germline stem cell division in vivo
Lateral and end-on kinetochore attachments are coordinated to achieve bi-orientation in Drosophila oocytes
mRNA localization and translational control during oogenesis
Independent and coordinate trafficking of single Drosophila germ plasm mRNAs
Sequence-dependent but not sequence-specific piRNA adhesion traps mRNAs to the germ plasm
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
The Drosophila putative histone acetyltransferase Enok maintains female germline stem cells through regulating Bruno and the niche
H3K36 trimethylation-mediated epigenetic regulation is activated by Bam and promotes germ cell differentiation during early oogenesis in Drosophila
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
DNA damage-induced CHK2 activation compromises germline stem cell self-renewal and lineage differentiation
Identification of germline transcriptional regulatory elements in Aedes aegypti
Aging and insulin signaling differentially control normal and tumorous germline stem cells
Protein competition switches the function of COP9 from self-renewal to differentiation
Evidence for chromatin-remodeling complex PBAP-controlled maintenance of the Drosophila ovarian germline stem cells
The NuRD nucleosome remodelling complex and NHK-1 kinase are required for chromosome condensation in oocytes
A role for tuned levels of nucleosome remodeler subunit ACF1 during Drosophila oogenesis
A genetic mosaic screen reveals ecdysone-responsive genes regulating Drosophila oogenesis
Prostaglandins temporally regulate cytoplasmic actin bundle formation during Drosophila oogenesis.
Splice variants of the SWR1-type nucleosome remodeling factor Domino have distinct functions during Drosophila melanogaster oogenesis

Meiosis in females
TORC1 regulators Iml1/GATOR1 and GATOR2 control meiotic entry and oocyte development in Drosophila
A pathway for synapsis initiation during zygotene in Drosophila oocytes
A Balbiani body and the fusome mediate mitochondrial inheritance during oogenesis
A maternal screen for genes regulating Drosophila oocyte polarity uncovers new steps in meiotic progression
Quantitative proteomics reveals the dynamics of protein changes during Drosophila oocyte maturation and the oocyte-to-embryo transition
Developments between gametogenesis and fertilization: Ovulation and female sperm storage
Sperm-storage defects and live birth in Drosophila females lacking spermathecal secretory cells
Mechanical stimulation by osmotic and hydrostatic pressure activates Drosophila oocytes in vitro in a calcium-dependent manner
Calcium waves occur as Drosophila oocytes activate
The coevolutionary period of Wolbachia pipientis infecting Drosophila ananassae and its impact on the evolution of the host germline stem cell regulating genes
The Octopamine receptor Octβ2R regulates ovulation in Drosophila melanogaster
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
Tight coordination of growth and differentiation between germline and soma provides robustness for Drosophila egg development

Populations of follicle cells
Epithelial rotation promotes the global alignment of contractile actin bundles during Drosophila egg chamber
Coupling of Hedgehog and Hippo pathways promotes follicle stem cell maintenance by stimulating proliferation
Development of the dorsal appendages
Regulation of epithelial stem cell replacement and follicle formation in the Drosophila ovary
Gene amplification in follicle cells: Isolation of developmental amplicons
DNA sequence templates adjacent nucleosome and ORC sites at gene amplification origins in Drosophila
Replication fork progression during re-replication requires the DNA damage checkpoint and double-strand break repair
Systematic analysis of the transcriptional switch inducing migration of border cells
The Hippo pathway controls border cell migration through distinct mechanisms in outer border cells and polar cells of the Drosophila ovary
DRP1-dependent mitochondrial fission initiates follicle cell differentiation during Drosophila oogenesis
Lamellipodin and the Scar/WAVE complex cooperate to promote cell migration in vivo
Drosophila eggshell production: identification of new genes and coordination by Pxt
Three-dimensional epithelial morphogenesis in the developing Drosophila egg
A dynamic population of stromal cells contributes to the follicle stem cell niche in the Drosophila ovary
A genome-scale in vivo RNAi analysis of epithelial development in Drosophila identifies new proliferation domains outside of the stem cell niche
Coordinated niche-associated signals promote germline homeostasis in the Drosophila ovary
Stage-specific plasticity in ovary size is regulated by Insulin/Insulin-Like growth factor and Ecdysone signalling in Drosophila
Phantom, a cytochrome P450 enzyme essential for ecdysone biosynthesis, plays a critical role in the control of border cell migration in Drosophila
Ecdysone response gene E78 controls ovarian germline stem cell niche formation and follicle survival in Drosophila.
GTP exchange factor Vav regulates guided cell migration by coupling guidance receptor signalling to local Rac activation
Discs large 5, an essential gene in Drosophila, regulates egg chamber organization
Targeted downregulation of s36 protein unearths its cardinal role in chorion biogenesis and architecture during Drosophila melanogaster oogenesis

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 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).

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).

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 ALIX and the 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).

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).

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).

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).

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).

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. 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).

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)

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).

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).

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.

genes involved in oogenesis

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Yan, D., Neumuller, R. A., Buckner, M., Ayers, K., Li, H., Hu, Y., Yang-Zhou, D., Pan, L., Wang, X., Kelley, C., Vinayagam, A., Binari, R., Randklev, S., Perkins, L. A., Xie, T., Cooley, L. and Perrimon, N. (2014). A regulatory network of Drosophila germline stem cell self-renewal. Dev Cell 28: 459-473. PubMed ID: 24576427

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date revised:  2 January 2016
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