The Interactive Fly
Genes involved in tissue and organ development
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
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).
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).
The Drosophila egg is an intricately patterned structure with distinct specializations and polarities. These features are critical to subsequent embryonic development because the polarities of the egg are transmitted to the embryo, establishing the initial pattern in a developing zygote. The pattern of the mature egg is established by complex cellular interactions among and between both somatic follicle cells and germline cells. Each egg begins as a 16-cell germline cyst, from which one cell will become the oocyte and the remainder will become the supporting nurse cells. In the germarium, the anterior structure in which oogenesis is initiated, the germline cyst, is surrounded by a monolayer of somatic follicle cell precursors. As the encapsulated cyst exits from the germarium, approximately 10-14 of the somatic cells cease proliferation and differentiate. This group of cells forms two distinct populations: two polar cells at the anterior and posterior poles of each chamber and approximately seven stalk cells that form a bridge between the consecutive cysts. As the cyst exits the germarium, the other somatic cells covering each chamber, the epithelial follicle cells, remain undifferentiated (Xi, 2003 and references therein).
After pinching off from the germarium, each germline cyst grows, while the epithelial follicle cells proliferate. During this time, the anterior-posterior polarity that will ultimately determine all of the epithelial follicular fates is established. Elegant experiments have shown that the underlying prepattern of the follicular epithelium displays mirror image symmetry at the termini in the anterior-posterior (A/P) axis. Cells adopt one of three anterior terminal fates [border, stretched, and centripetal cells (terminal to central)], depending on proximity to the poles. In the intervening region between the terminal domains, cells will adopt a default 'main body' identity, and the posterior terminal cells form nearest the posterior pole. The symmetry of the A/P pattern is broken by EGFR signaling at the posterior. Secreted Grk from the posteriorly localized oocyte activates EGFR on the overlying follicle cells, establishing posterior terminal fate. In the absence of EGFR signaling, the anterior pattern is repeated at the posterior (Xi, 2003 and references therein).
By stage 7, the epithelial follicle cells cease proliferation and enter an endocycle. Afterward, these cells begin to show morphological and molecular signs of differentiation into the five epithelial fates: border, stretched, centripetal, posterior, and main body cells. Each of these subpopulations of follicle cells has a specific function with respect to the production of a mature egg, such that the correct number and position of each type is critical to ultimate egg morphology. These functions inluence the production of structures that are essential to the egg, such as the dorsal respiratory appendages and the micropyle. These functions are also critical for proper anterior-posterior organization of the oocyte and, therefore, also for the resulting embryo (Xi, 2003 and references therein).
Though much has been learned about the process of ovarian follicle maturation through studies of oogenesis in both vertebrate and invertebrate systems, less is known about how follicles form initially. In Drosophila, two somatic follicle stem cells (FSCs) in each ovariole give rise to all polar cells, stalk cells, and main body cells needed to form each follicle. One daughter from each FSC founds most follicles but that cell type specification is independent of cell lineage, in contrast to previous claims of an early polar/stalk lineage restriction. Instead, key intercellular signals begin early and guide cell behavior. An initial Notch signal from germ cells is required for FSC daughters to migrate across the ovariole and on occasion to replace the opposite stem cell. Both anterior and posterior polar cells arise in region 2b at a time when approximately 16 cells surround the cyst. Later, during budding, stalk cells and additional polar cells are specified in a process that frequently transfers posterior follicle cells onto the anterior surface of the next older follicle. These studies provide new insight into the mechanisms that underlie stem cell replacement and follicle formation during Drosophila oogenesis (Nystul, 2010).
The Drosophila ovary is a highly favorable system for studying epithelial cell differentiation downstream from a stem cell. New follicles consisting of 16 interconnected germ cells surrounded by an epithelial (follicle cell) monolayer are continuously produced during adult life and develop sequentially within ovarioles (see Prefollicle cells associate with cysts in an ordered fashion downstream from follicle stem cells). Follicle formation begins in the germarium, a structure at the tip of each ovariole that houses 2-3 germline stem cells (GSCs) and 2 follicle stem cells (FSCs) within stable niches. Successive GSC daughters known as cystoblasts are enclosed by a thin covering of squamous escort cells and divide asymmetrically four times in sucession to produce 16-cell germline cysts, comprising 15 presumptive nurse cells and a presumptive oocyte. At the junction between region 2a and region 2b, cysts are forced into single file as they encounter the FSCs, lose their escort cell covering, and begin to acquire a follicular layer. Follicle cells derived from both FSCs soon mold them into a 'lens shape' characteristic of region 2b. Under the influence of continued somatic cell growth, cysts and their surrounding cells round up, enter region 3 (also known as stage 1), and bud from the germarium as new follicles that remain connected to their neighbors by short cellular stalks (Nystul, 2010).
A complex sequence of signaling and adhesive interactions between follicular and germline cells is required for follicle budding, oocyte development, and patterning. However, the mechanisms orchestrating the initial association between follicle cells and cysts within the germarium are less well understood. While lineage analysis indicates the presence of two FSCs, low fasciclin III (FasIII) expression has been claimed to specifically mark FSCs, leading to the conclusion that more FSCs are present under some conditions (Nystul, 2010).
The differentiation of polar cells at both their anterior and posterior ends is required for normal follicle production, and depends on Notch signals received from the germline. Subsequently, anterior polar cells send JAK-STAT and Notch signals that specify stalk cells. While the source of these signals and their effects are clear, the timing of polar cell specification and its dependence on cell lineage are not. Some anterior and posterior polar cells (but not stalk cells) were inferred by lineage analysis to arise and cease division within region 2b. In contrast, on the basis of marker gene expression it was concluded that anterior polar cells are specified later, in stage 1, and posterior polar cells in stage 2. Up to four polar cells may eventually form, but apoptosis reduces their number to a single pair at each end by stage 5. Moreover, polar and stalk are believed to arise exclusively from 'polar/stalk' precursors that separate from the rest of the FSC lineage and these cells were proposed to invade between the last region 2b cyst to affect follicle budding (Nystul, 2010).
This study analyzes the detailed behavior of FSCs and their daughters in the germarium. No evidence of polar/stalk precursors was observed, and it was shown that the first anterior and posterior polar cells are specified in region 2b, prior to the previously accepted time of follicle cell specialization. Additional polar cells are also formed later during stages 1 and 2. Follicle cell differentiation appears to be independent of cell lineage, but is orchestrated by sequential cell interactions, and in particular by Notch signaling. These results reveal the sophisticated, self-correcting behavior of an epithelial stem cell lineage at close to single-cell resolution (Nystul, 2010).
The data provide a much clearer picture of the follicle stem cell lineage than was previously available. They suggest that key aspects of FSC regulation depend on mechanisms that move cysts into a single file and program the loss of their escort cells precisely as they encounter FSCs and enter region 2b. Contact with incoming region 2a cysts likely induces FSC divisions, ensuring that cysts acquire a daughter cell from each stem cell as they stretch out to span the width of the germarium at the region 2a/2b junction. The asymmetry in cyst organization exposes FSC daughter cells to different cyst faces and, therefore, potentially to different signals. The FSC and daughter located on the same side as the entering cyst are exposed to the posterior face of the cyst while it is still in region 2a, covered by escort cells. In contrast, the opposite FSC and daughter contact the anterior face of the cyst as it migrates into 2b, at a time when the cyst is shedding its escort cell layer and exposing the Delta signal on the germ cell surface. Since region 2a cysts tend to interdigitate in forming a single file, cyst entry will usually alternate sides as successive cysts pass, causing FSC daughters arising from the same side to alternate migration paths. An advantage to this system may be its flexibility, allowing follicles to form normally even if multiple cysts enter from the same side in succession (Nystul, 2010).
Notch signaling in early FSC daughters promotes a 'prefollicle' state by blocking follicle cell differentiation. Consistent with this, it was observed that FSCs and their early daughters have much lower levels of differentiation markers such as FasIII and IMP-GFP. This developmental delay may prevent prefollicle cells from immediately incorporating into the differentiated follicular epithelium, allowing them to instead retain a more mesenchymal character conducive to cross-migration, and may also contribute to their ability to compete with the resident FSC for niche occupancy. Notch mutant daughters did not replace wild-type FSCs, most likely because they were unable to migrate into proximity. A role for Notch in suppressing differentiation downstream from the FSC might also explain why cells expressing activated Notch failed to migrate posteriorly (Nystul, 2010).
Follicle cell fates are specified by intercellular signals rather than lineage: The two FSC daughters and their descendants, with few exceptions, continue to associate with the cyst they first contact at the 2a/2b boundary throughout subsequent development. Their division rate increases briefly, because daughters divide four times in the time it takes to generate three new cysts. Despite their growing number, however, all the cells retain the ability to produce main body, stalk, and polar cells for at least the first two to three divisions (8- to 16-cell stage). In contrast to previous reports, no evidence was found that polar and stalk cells derive from a lineage-restricted polar/stalk precursor population. Claims of a polar/stalk fate were based on experiments using higher rates of clone induction than in the experiments reported in this study. While many clones were also observed in these studies that contained both polar and follicle cells or both stalk and follicle cells, they were discounted as double clones (Nystul, 2010).
By examining clones induced at low frequency (more than threefold lower than in previous studies) it was possible to minimize the need for statistical correction for double clones. Furthermore, by studying clones induced at multiple times downstream from the FSC, overweighting small clones induced just as the polar and stalk cell fates are being determined by signaling within small cell groups was avoided. This has the effect of increasing the proportion of clones containing only one or two cell types even in the absence of any lineage restriction. At early, intermediate, and late times in somatic cell development in the germarium, clones that included all combinations of cell fates were always observed, indicating that follicle cells are multipotent prior to polar or stalk specification. This fits well with recent studies showing that many additional cells in the germarium can be induced to take on a polar cell fate by strong Notch signaling, while high levels of JAK-STAT signaling can induce more stalk cells. In contrast, no mechanism, time, or location where putative polar/stalk precursor cells are specified has ever been documented. Previous models also did not explain how these cells would preferentially arrive in the zone of cells separating regions 2b and 3 or what would become of the many extra cells that can sometimes be found in this region beyond the number needed for these fates (Nystul, 2010).
The finding that polar cells are initially specified in region 2b suggests that more spatial information is available within region 2b follicles than has been detected in earlier experiments. It was found that the first anterior and posterior polar cells are specified when cysts are associated with 8- to 16-cell follicle cells, in mid-to-late region 2b. This agrees closely with previous studies, which found that polar cells were first specified at the 14-cell stage. The early polar cells are detected by lineage because they cease dividing; however, no gene expression markers specific for these cells have been identified. Consequently, it remains uncertain where they are located at the time of induction or whether they function while remaining in region 2b. Since evidence was observed of Notch signal reception within individual follicle cells located at the anterior and posterior regions of stage 2b cysts, the simplest model is that these polar cells are induced by Delta signaling from the germline in a normal anterior/posterior (A/P) orientation. Although no Upd expression was detected at this time, these cells may nonetheless signal to the surrounding somatic cells to establish the graded levels of cadherin that define the initial anterior/posterior axis of the cyst (Nystul, 2010).
Where does the information come from that allows a small number of polar cells to be specified at this time? One possibility is a 'signal relay' from more posterior follicles. Highly accurate timing of polar cell formation relative to the signaling events during follicle budding might help to further test this model. However, the observation of localized Notch signal reception and polar cell specification in region 2b follicles suggests that the germline at this stage is already sufficiently polarized to signal in a limited manner along the future A/P axis. Some of this information may come from the inherent asymmetry within the germline cyst whose cells differ systematically in their fusome content, organelle content, and microtubule organization. The future oocyte and its sister four-ring canal cell are always located in the center of the region 2b cysts and hence might be one source for this inductive signal. Alternatively, there may be additional differences within this region of the germarium that have yet to be detected and that may contribute (Nystul, 2010).
These studies confirm previous conclusions that additional polar cells are formed during the process of budding and provide new insight into the budding process itself. Anterior-biased clones were almost always confined to a single follicle, but a significant fraction of posterior-biased clones (~33%) extended onto the next older follicle where they encompassed both an anterior polar cell and 2-30 anterior follicle cells. This suggests that cells at the posterior of the nascent follicle outgrow their cyst as it rounds up and are forced into the space between the posterior 2b cyst and the budding cyst. The origin of these cells has long been a mystery. A fraction of the interstitial cells likely contact and move onto the anterior of the downstream cyst where those that happen to lie adjacent to the existing polar cells are induced as new polar cells and stalk cells. Any remaining interstitial cells likely rejoin the main body of follicle cells as budding is completed or are eliminated by apoptosis as the stalk resolves to its final size (Nystul, 2010).
This study of early follicle cell development provides a rare opportunity to analyze how epithelial cells behave downstream from a stem cell. Most characterized Drosophila stem cell daughters receive information asymmetrically from their mother stem cell and differentiate rapidly. Germline stem cells and their niches ensure that cystoblasts receive an asymmetric fusome segment as well as differential environmental signals that program exactly four stereotyped divisions prior to entering meiosis. Under nonstressed conditions, intestinal stem cells utilize Notch signals to specify their daughters as either enterocytes or enteroendocrine cells and to terminate subsequent division. Neuroblasts program a stereotyped sequence of daughter cell fates by differential division and signaling. In contrast, FSC daughters undergo eight to nine divisions and differentiate independently of lineage over the course of several divisions and are capable of producing normal follicles even when the usual pattern of cellular interactions is altered. The increased resolution of follicle cell behavior afforded by these studies provides a valuable opportunity to study how epithelial cells are able to robustly bring about defined outcomes in the absence of the precise early programming (Nystul, 2010).
Several mechanisms are likely to contribute to successful follicle formation. First, genes characteristic of a polarized epithelium turn on slowly downstream of the FSC. The cross-migrating cell and several other cells frequently lacked such gene expression, but instead expressed genes characteristic of escort cells, suggesting that follicles are able to maintain some germline-soma interactions while completely replacing their somatic coverings. The early differentiation of polar cells may help guide subsequent cell behavior. In conjunction with the intrinsic asymmetric structure of germline cysts, differential adhesive interactions between germ and somatic cells across the follicle, differential pressures resulting from cell growth, and the resistive forces of the ovariolar wall, signals from these cells may be sufficient to ensure that the oocyte moves to the posterior and that cysts begin to round (Nystul, 2010).
These characteristics of the FSC lineage, although unique among well-studied stem cells in Drosophila, may be closer to those governing the epithelial lineages within many mammalian tissues. Thus, the mechanisms that give FSCs and their daughters their developmental flexibility and robustness are likely to be both widespread and medically relevant (Nystul, 2010).
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).
Gene amplification is known to be critical for upregulating gene expression in a few cases, but the extent to which amplification is utilized in the development of diverse organisms remains unknown. By quantifying genomic DNA hybridization to microarrays to assay gene copy number, two additional developmental amplicons, termed DAFC (Drosophila Amplicon in Follicle Cells)-30B and -62D were identified in the follicle cells of the Drosophila ovary. Both amplicons contain genes which, following their amplification, are expressed in the follicle cells, and the expression of three of these genes becomes restricted to specialized follicle cells late in differentiation. Genetic analysis establishes that at least one of these genes, yellow-g, is critical for follicle cell function, because mutations in yellow-g disrupt eggshell integrity. Thus, during follicle cell differentiation the entire genome is overreplicated as the cells become polyploid, and subsequently specific genomic intervals are overreplicated to facilitate gene expression (Claycomb, 2004).
The maximally amplified genes in DAFC-62D, yellow-g and yellow-g2, are members of the yellow gene family that are predicted to encode secreted proteins. The family shares homology with the Major Royal Jelly Protein Family in honeybees (Apis mellifera), involved in the specification of the queen bee. The founding member of the Yellow family, Yellow-y, is known to play a role in mating behavior and in the melanization and hardening of the adult cuticle. Other Yellow family members have been shown to act as dopachrome-conversion enzymes that catalyze a key reaction in the melanization process. Interestingly, a similar process is used in the hardening of the egg chorion in mosquitoes and suggests that Yellow-g and Yellow-g2 may play a catalytic role in the crosslinking of the chorion and/or underlying vitelline membrane proteins in Drosophila (Claycomb, 2004).
A second group of genes encodes proteins with chitin binding motifs that could function in egg production. Genes of this type are present in both amplicons, with DAFC-62D containing two such genes and DAFC-30B containing one. Chitin binding domains serve an antimicrobial function in a variety of plants and marine invertebrates. Homologs of marine invertebrate proteins, such as tachycitin, could provide the egg with protection against microbes. Alternatively, chitin, a structural polysaccharide found in many organisms, could also be a component of the eggshell, and interaction with the chitin binding proteins might contribute to eggshell integrity (Claycomb, 2004).
In both DAFC-30B and 62D, there are also a number of genes whose role in follicle cells is not yet clear. These include both genes encoding proteins without known sequence motifs and genes whose products are predicted to have the enzymatic activities of adenylate cyclases, membrane transporters, calcium-transporting ATPases, GTP dissociation inhibitors, and others (Claycomb, 2004).
The yellow-g gene is essential for a rigid eggshell, and the predicted gene products of the yellow-g and yellow-g2 genes suggest a molecular explanation for these mutant defects. The eggshell is composed of several layers, including the outermost exochorion, the endochorion, the inner chorion layer, and the vitelline membrane, which is the innermost structure that also contacts the oocyte. The collapsed embryos and disrupted vitelline membranes that result from mutation of yellow-g indicate that yellow-g is necessary for the structural integrity of the eggshell. At the level of the light microscope, the exochorion of embryos laid by mutant mothers appears normal. The collapsed embryos are reminiscent of vitelline membrane defects, leading to the hypothesis that yellow-g is necessary for proper vitelline membrane formation (Claycomb, 2004).
It is proposed that Yellow-g and Yellow-g2 act to crosslink the vitelline membrane, or perhaps the inner chorion layer. The Yellow family members, Yellow-f and Yellow-f2, are capable of catalyzing the conversion of dopachrome to dihydroxyindole, a limiting step in the melanization pathway, during larval, pupal, and adult stages. The enzymatic events leading to the crosslinking of the vitelline membrane are not well understood, but seem to involve one phase of disulfide bond formation and a subsequent disulfide bond-independent phase. Additionally, the α methyl dopa resistant (amd) gene product, which acts in the conversion of dopamine during the polymerization of the adult cuticle, is required in the follicle cells for proper vitelline membrane crosslinking. This suggests that a similar set of dopamine conversion reactions catalyzed by Yellow-g and Yellow-g2 may be necessary for the crosslinking of the vitelline membrane just prior to egg laying. Consistent with this hypothesis, it is observed that eggs laid by homozygous yellow-g mutant females are highly sensitive to sodium hypochlorite (bleach), and the majority of these embryos burst upon brief exposure. Of the remaining, intact embryos, 100% were permeable to the dye neutral red, which has been used to assay vitelline membrane defects. These results are indicative of a failure to crosslink the vitelline membrane and further implicate yellow-g in the crosslinking process. However, this hypothesis does not explain the specific expression of the yellow-g and yellow-g2 genes in the follicle cells producing the micropyle late in egg chamber development. It is possible that crosslinking of the vitelline membrane or inner chorion layer within this specialized structure requires distinct regulation or timing. A more detailed analysis of the eggshell defect and biochemical studies of Yellow-g and Yellow-g2 will help gain a better understanding of the steps necessary for vitelline membrane crosslinking and will uncover any specialized micropyle functions (Claycomb, 2004).
DAFC-30B and DAFC-62D provide insights into the use of amplification as a developmental strategy. All of the previously characterized amplified genes play a purely structural role in eggshell formation; no enzymes necessary for proper eggshell formation have been examined. None of the genes of DAFC-30B and DAFC-62D encode known structural components of the eggshell. However, several of the amplified genes that are highly expressed in follicle cells, including CG18419 and the yellow-g genes, encode products predicted to possess enzymatic, signal transduction, or transporting activities. Furthermore, at least yellow-g is essential for proper egg formation, thus revealing an additional function of amplification: to increase the levels of enzymes needed to catalyze developmentally important reactions. Thus the identification of additional amplicons highlights genes likely to be crucial in developmental events and opens the possibility that other tissues employ amplification to maximize gene expression during differentiation. It is surprising that a 4- to 6-fold increase in gene copy number would affect gene product levels in a developmentally significant manner. It is possible, however, that copy number increases are considerably higher in subsets of follicle cells, or that the replication process itself facilitates transcription (Claycomb, 2004).
The follicle cell amplicons serve as superb model metazoan replicons, permitting delineation of cis-regulatory elements, identification of replication proteins, and clarifying the developmental control of the initiation and elongation. Developmental distinctions between DAFC-62D and the previously studied DAFCs provide clues into how origin firing can be linked to developmental signals. It has been shown by real-time PCR that replication initiates at DAFC-66D and -7F, coupled with replication fork movement, during egg chamber stages 10B and 11. Subsequently (stages 12 and 13), origins cease firing and only existing replication forks move bidirectionally to produce a gradient of copy number that extends over 100 kb. Furthermore, the replication initiation factor ORC2 localizes to amplification origins only during the initiation phase and dissociates at the onset of the elongation phase. Replication factors involved in multiple steps of DNA replication, such as MCM2-7 and PCNA, colocalize with BrdU throughout amplification (Claycomb, 2004).
DAFC-62D behaves differently from these amplicons and from DAFC-30B. There is a final increase in copy number at a very precise region of the amplicon, about 1.5 kb downstream of yellow-g2, during stage 13. As it is the peak of amplification, this region is likely to possess a replication origin. Understanding how DAFC-62D can undergo a final initiation hours after ORC is no longer detectable at origins by immunofluorescence will provide insights into the control of replication initiation. The additional replication in stage 13 may occur in only subsets of follicle cells, and ORC could persist specifically at DAFC-62D in these cells. For example, additional gene copies could permit optimal levels of expression of the yellow-g genes in the follicle cells building the micropyle (Claycomb, 2004).
These studies were initiated to devise a systematic approach for finding developmental amplicons. The microarray assay is sensitive and can detect low levels of gene amplification, and amplification levels as low as 4-fold can be developmentally important. Thus, this approach will be invaluable in surveying for gene amplification in a number of tissues and in a variety of organisms where amplification has not been detected. Not only has the microarray strategy identified additional amplicons, but when coupled with the power of a genetic organism, it has proven to be a functional genomics approach for highlighting genes involved in specific developmental pathways (Claycomb, 2004).
Dorsal appendage morphogenesis in Drosophila oogenesis has been used as a model system for studying the relationship between patterning and morphogenesis. Each of the two dorsal respiratory appendages of the Drosophila egg chamber is formed by secretion of eggshell proteins into a tube of follicle cells. This tube is generated by cell shape changes and rearrangements within an epithelial sheet. Dorsal appendage formation is therefore similar to more complicated examples of organogenesis. In addition, the study of dorsal appendage formation provides several advantages that make it an excellent system for investigating the regulation of epithelial morphogenesis. For example, the signaling events that determine two populations of dorsal follicle cells are well understood. This understanding facilitates an ability to uncouple effects on patterning from morphogenesis. Further, powerful genetic tools, including mutations that disrupt dorsal appendage formation, have allowed for an unraveling of the genetic circuitry underlying the regulation of epithelial morphogenesis (French, 2003).
The Drosophila egg chamber contains 16 interconnected germline cells, consisting of 1 oocyte nourished by 15 highly polyploid nurse cells; these germline cells are surrounded by a monolayer of ~1000 somatic follicle cells. The follicle cells secrete the chorion that makes up the three layers of the eggshell: the vitelline envelope, the endochorion, and the exochorion. A subset of these follicle cells undergoes morphogenesis to generate the dorsal appendages, specialized structures that facilitate gas exchange in the developing embryo (French, 2003).
At stage 10 of oogenesis, the oocyte occupies the posterior half of the egg chamber, the nurse cells the anterior half, and the oocyte nucleus is positioned at the dorsal anterior corner of the oocyte. The majority of follicle cells forms a columnar layer over the oocyte, while a few follicle cells are stretched out over the nurse cells. During stage 10B, those follicle cells closest to the nurse cell/oocyte boundary begin to migrate centripetally, between nurse cells and oocyte. The centripetal cells secrete the operculum (a thin layer of chorion that functions as an escape hatch for the larva), the collar (a hinge on which the operculum swings), and the micropyle, a coneshaped structure through which the sperm enters (French, 2003).
Shortly after centripetal migration (stage 10B), the nurse cells rapidly transfer their contents into the oocyte (stage 11) then begin to degenerate and undergo apoptosis (stages 12-14). At the same time, two groups of approximately 65-80 anterior, dorsal follicle cells, one on each side of the dorsal midline of the egg chamber, migrate over the nurse cells, laying down the chorion of the two dorsal appendages. Extensive studies have defined the signaling events that determine two populations of dorsal follicle cells. Dorsal follicle-cell fate determination begins when transcripts encoding the TGFalpha-like signaling molecule Gurken (Grk) become localized in a cap above the oocyte nucleus. Grk signals via the epidermal growth factor receptor homolog (Egfr) to the follicle cells, activating a signal transduction cascade involving the Ras/Raf/MAPK pathway. This initial signaling event defines a set of dorsal anterior follicle cells and induces a second signaling cascade involving three additional Egfr ligands. This second cascade amplifies and refines the initial Grk signal, leading to the definition of two separate populations of dorsal follicle cells. These events are required for the production of two separate dorsal appendages. Disruptions of this process result in dorsalization or ventralization of the follicular epithelium and the eggshell. Partial ventralization generally results in failure to determine two separate populations of cells, leading to the production of a single dorsal appendage at the dorsal midline. Complete ventralization results in the absence of dorsal cell fates and the concomitant loss of dorsal appendages (French, 2003).
Information along the anterior-posterior axis also contributes to cell-fate determination within the dorsal appendage primordia. The BMP2/4 homolog encoded by dpp is expressed in the stretch cells and a single row of centripetally migrating cells. This morphogen radiates posteriorly and alters columnar cell fates. High levels of Dpp repress dorsal identities and specify operculum; moderate levels synergize with Grk to define dorsal, while low levels of Dpp are insufficient to allow cells to respond to Egfr signaling. Thus, loss-of-function mutations generate short, often paddleless appendages, while overexpression either expands the operculum at the expense of appendage material or creates multiple, often antler-shaped dorsal structures. The subsequent events underlying dorsal appendage morphogenesis are only beginning to be understood. Analyses of cultured wild-type egg chambers have revealed several phases of dorsal appendage morphogenesis. From stages 10B to 12, two groups of dorsal anterior follicle cells move out from the follicular epithelium to form short tubes. Each tube extends forward over the nurse cells, secreting chorion proteins that make up the cylindrical stalk of the dorsal appendage. Cells at the anterior end of the tube change shape to produce the flattened paddle of the distal dorsal appendage. Finally, upon oviposition, the entire follicular epithelium sloughs off, leaving behind the chorionic structures (French, 2003).
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).
Additional information about mRNA localization in Drosophila eggs can be found at the following Interactive Fly sites: Gurken, Homeless, Hunchback, Orb, Pumillo, Staufen, and Vasa.
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date revised: 5 April 2013Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
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