Larval and pupal stages (continued)

Dpp and the Genital Disc

The genital disc consists of three primordia: moving from anterior to posterior they are the female genital primordium, the male genital primordium and the anal primordia. Only one of the two genital primordia develops, depending on the individual's sex, whereas the anal primordium develops in both sexes. It is proposed here that the genital disc, which is of ventral origin, is organized in a manner similar to the antennal and leg discs: the expression domains of decapentaplegic and wingless are mostly complementary and abut engrailed expression. An analysis was made of the roles of the genes hedgehog, patched, dpp and wg in the development of the three primordia that form the genital disc. The morphogenetic alterations produced by ectopic expression of hh mimic a lack of ptc function. Both genetic conditions cause derepression of dpp and wg. Ectopic expression of either of these genes causes non-autonomous duplications and/or reductions of genital and anal structures. Some of these alterations are explained by the mutual repression of wg and dpp. In the development of the genital disc, the functional relationships between these genes seem to be analogous to those described for leg and antennal discs: dpp and wg are induced in the anterior compartment by Hh protein blocking the repressive effect of Ptc, and the mutual repression of dpp and wg restrict one another to their respective domains. It may be concluded that dpp and wg act as general organizers for development of the genital disc (Sánchez, 1997).

The development of the genital primordia is based on two processes: cell proliferation and sexual differentiation. Cell proliferation refers to the capacity of each genital primordium to grow or to be kept in the repressed state. Sexual differentiation refers to the type of adult structure formed by each genital primordium. It is proposed that the control of cell proliferation in the male and female genitalia requires the concerted action of Abd-B and doublesex, either directly or indirectly, through the expression of the genes dpp and wingless. Thus, in female genital discs, the repressed male primordium does not express dpp whereas the repressed female primordium of the male genital discs expresses a reduced level of dpp. This reduced level seems to be insufficient to stimulate cell growth. In contrast, when strong dpp levels are obtained in the repressed female primordium of male discs, repressed female primordia overproliferate in mutants for patched or costal-2, as well as in the discs where uniform ectopic expreession of hedgehog is produced. The genes dpp and wg, however, do not participate in the sexual differentiation process, which depends on sexual cytodifferentiation genes. Thus the growth of repressed female primordia of the patched mutant male discs would give rise to no adult female genital structures since the genetic sex is male (Sánchez, 1997 and references).

In both sexes, the Drosophila genital disc comprises three segmental primordia: the female genital primordium derived from segment A8, the male genital primordium derived from segment A9 and the anal primordium derived from segments A10-11. Each segmental primordium has an anterior (A) and a posterior (P) compartment, the P cells of the three segments being contiguous at the lateral edges of the disc. Hedgehog (Hh) expressed in the P compartment differentially signals A cells at the AP compartment border and A cells at the segmental border. As in the wing imaginal disc, cell lineage restriction of the AP compartment border is defined by Hh signalling. There is also a lineage restriction barrier at the segmental borders, even though the P compartment cells of the three segments converge in the lateral areas of the disc. Lineage restriction between segments A9 and A10-11 depends on factors other than the Hh, En and Hox genes. The segmental borders, however, can be permeable to some morphogenetic signals. Furthermore, cell ablation experiments show that the presence of all primordia (either the anal or the genital primordium) during development are required for normal development of genital disc. Collectively, these findings suggest that interaction between segmental primordia is required for the normal development of the genital disc (Gorfinkiel, 2003).

The three segmental primordia of the genital disc are contiguous. This means that the P compartment of one primordium is adjacent to A cells of the corresponding primordium and to A cells of the following primordium. In addition, the P compartment cells of the three segments converge in lateral areas of the genital disc. Hh activates target genes in the receiving cells both behind and in front. These target genes are different on each side of its expression domain. Particularly, Hh at the posterior compartment of the male genital primordium (A9 segment) signals anteriorly, inducing Wg and/or Dpp expression in anterior cells of this primordium, and posteriorly, inducing Ptc expression. Hh also posteriorly signals anterior cells of the anal primordium (segments A10-11), inducing En expression in a narrow band of cells. Interestingly, Cad expression is reduced in these cells. A similar situation has been described in embryonic segments in which Hh activates wg at the AP border and rhomboid at the segmental border. Hh controls Wg and EGF signalling pathways on each side of its expression domain in embryos (Gorfinkiel, 2003).

Hh has a pivotal role in the morphogenesis of all imaginal discs. Ectopic Hh gives rise to duplications of parts of, or whole, appendages in the imaginal discs of the fly. In wild-type genital discs, such as in the leg and antenna, Hh induces the expression of wg and dpp in A compartment cells close to the AP border of each of the three primordia. The ectopic expression of hh in the anal primordium induces complete duplication of the genital disc with the corresponding expression of these genes in their normal expression domains. The repressed male and female primordia also seemed to be duplicated in the female and male genital discs, respectively. These results indicate again that Hh diffuses across the border between the genitalia and analia, although this border acts as a cell lineage restriction barrier. It should be noted that Hh also diffuses across the border between the embryonic segments and between the abdominal segments of adult flies. The results presented here also show that the ectopic expression of either dpp or wg in the analia also affects the development of the male and female genitalia (Gorfinkiel, 2003).

The non-autonomous effect that ectopic Dpp in the analia has on the development of the genitalia is due to diffusion of Dpp itself from the analia to the genitalia, and not to the non-autonomous effect of Dpp downstream genes. By contrast, the same effect on the development of the genitalia is observed when Wg itself or any of the downstream components of the Wg-pathway, Tcf or Arm, are ectopically expressed in the analia. Ectopic expression of Wg in the analia can either recover structures of the genitalia or can prevent the development of both genitalia and analia. These results together with the observation that no Wg protein is detected in the genitalia when it is ectopically expressed in the analia indicate that the non-autonomous effect of ectopic Wg is due to an unknown signal activated by the Wg-pathway (Gorfinkiel, 2003).

Dpp and Oogenesis

Stromal cells are thought to generate specific regulatory microenviroments or 'niches' that control stem cell behavior. Characterizing stem cell niches in vivo remains an important goal that has been difficult to achieve. The individual ovarioles of the Drosophila ovary each contain about two germ line stem cells that maintain oocyte production. Anterior ovariolar somatic cells comprising three cell types act as a germ line stem cell niche. Germ line stem cells lost by normal or induced differentiation are efficiently replaced, and the ability to repopulate the niche increases the functional lifetime of ovarioles in vivo. These studies implicate one of the somatic cell types, the cap cell, as a key niche component (Xie, 2000).

The Drosophila ovary is a tissue where stem cells can be studied at the cellular and molecular level in vivo. Near the beginning of each developing egg string (or ovariole) within the ovary reside about two germ line stem cells (GSCs) whose progeny differentiate into eggs within 8 days as they move at predictable rates along the ovariole. These stem cells are surrounded by three differentiated somatic cell types -- terminal filament, cap, and inner sheath cells -- that help make up an anatomically simple tubular structure known as the germarium. GSCs are easily identified by size, location, and the shape of the fusome, an intracellular structure rich in membrane skeleton proteins. Stem cells usually contain a round fusome, but display a distinctive elongated fusome after division when they remain transiently connected with their daughter cell. Under appropriate conditions, GSCs divide about once per day and are randomly lost by differentiation, with a half-life of 4 to 5 weeks. It has been proposed that the somatic cells at the tip of the ovariole are organized into a niche that maintains and controls GSCs (Xie, 2000).

Ovariolar anatomy is consistent with the existence of a niche at the anterior tip. After stem cell division, the daughter that lies closer to the terminal filament and cap cells remains a stem cell, whereas the daughter that more closely adjoins the inner sheath cells differentiates into a cystoblast. Anatomical asymmetry may ensure that equivalent stem cell daughters receive different fate-determining signals. GSCs require a signal mediated by Dpp, a homolog of human bone morphogenetic proteins 2 and 4, in order to remain as stem cells and to divide at a normal rate. Two other proteins needed to maintain GSCs, Piwi and Fs(1)Yb (Yb), act outside the germ line. However, a requirement for intercellular signals does not by itself indicate the presence of a niche. A true niche should function independently of resident stem cells and be able to reprogram newly introduced cells to become stem cells. Consequently, it was investigated whether the microenvironment at the ovariolar tip can specify cells to become GSCs (Xie, 2000).

Ovarioles normally lose GSCs by differentiation, but the low rate of GSC loss and the possibility that rapid replacement quickly restores the original GSC configuration complicate observing such events. To study germaria with recently lost stem cells, individual GSCs were genetically marked and destabilized. FRT-mediated recombination was used to generate mutant clones of schnurri (shn), a gene that is likely to reduce GSC lifetime by disrupting dpp signaling, under conditions where the mutant cells also lose an armadillo-lacZ marker. Because cystoblasts require 4 to 5 days to exit the germarium, the only remaining lacZ- cells 1 week after transiently activating the hs-FLP transgene by means of a heat shock will be clones consisting of shn mutant stem cells and their progeny of 4 to 5 days. With this marking system, marked shn GSCs that differentiate during the last 4.5 days can be recognized because lacZ- germ cells will remain in the germarium; moreover, the developmental age of the least mature such cell will indicate the elapsed time since GSC loss (Xie, 2000).

The results demonstrate that shn mutant stem cells are lost at an increased rate and are rapidly replaced by wild-type cells. Seventy-nine germaria were found that retained lacZ- germ cells, revealing that a shn stem cell had been recently lost. In every case they contained two wild-type stem cells, indicating that the lost lacZ- stem cell had been replaced by a wild-type stem cell. Even when the stem cell was lost so recently that it remained a cystoblast, two lacZ+ stem cells were present at the tip. These stem cells are connected by an elongated fusome, indicating that they are recently divided sister cells in early interphase; the fusome is oriented in an unusual manner, perpendicular to the anterior/posterior (A/P) axis. These observations suggest a specific model for GSC replacement. After one GSC is lost, its neighboring stem cell divides perpendicular to the A/Paxis, causing a daughter cell to occupy the environment recently vacated by the departed GSC. For this mechanism to work, the environment at the site of the lost GSC must be capable of programming the incoming cell to become a GSC rather than a cystoblast. Observations indicate that it is capable of doing so, and hence that GSCs reside in a true stem cell niche (Xie, 2000).

The ability of the ovariole tip to act as a stem cell niche is likely to be biologically important. Females produce eggs for months, despite the 4- to 5-week half-life of an individual stem cell. To investigate whether stem cell replacement occurs normally, the number of stem cells and somatic niche cells was measured in aging females. During the first 5 weeks of adult life the average number of GSCs per germarium declines from about 2.5 to 2.0, significantly less than the 50% reduction expected in the absence of replacement. Replacement stem cells must function efficiently because the rate of stem cell loss does not increase with age. Some of the ovarioles that did lose a stem cell started with three GSCs, because the number of such ovarioles declined over the same period (Xie, 2000).

One of the three somatic cell types, cap cells, interacts with stem cells in a manner that suggests they play a role in niche function. Over the 36-day period, the number of cap cells and GSCs remained closely correlated at about 2.5 cap cells per GSC. Moreover, GSCs were observed to always make special contacts with cap cells that characteristically align with the A/P axis of the ovariole. The GSC's fusome remains adjacent to the GSC/cap cell interface during most of the cell cycle. In contrast, the behavior of inner sheath cells and terminal filament cells does not correlate closely with GSCs. As germaria age, terminal filament cells decrease in number from an average of 9.2 (3 days) to 5.0 (36 days) and change from a linear to a ball-like arrangement. Likewise, the relative number of inner germarium sheath (IGS) cells and GSCs vary. However, the number of IGS cells is closely correlated with the number of differentiating germ cells. A functional connection between IGS cells and germ cell cysts has been previously suggested, because ovariole tips that develop without germ cells lack IGS cells (Xie, 2000).

To investigate the role of IGS cells in adult germaria females carrying a hs-bam transgene, whose stem cells can be induced to differentiate, were studied. Over the course of several days after heat shock, GSCs are lost and all germ line cysts completed development and left the germarium. Such germaria also lose all IGS cells, further indicating that developing germ cells control IGS cell number. In contrast, terminal filament and cap cells do not change in the absence of germ cells. Somatic cell divisions continue in their vicinity as in germaria that form in the absence of germ cells. Despite their presence near the GSC niche, these dividing somatic cells do not become GSCs (Xie, 2000).

Because the number of cap cells correlates closely with the number of GSCs, whether they might function by preferentially sending a dpp signal was investigated. Suitable antibodies to Dpp are unavailable, so whole-mount in situ hybridization was used to determine which cells at the ovariole tip express dpp mRNA. These experiments detected low levels of dpp mRNA in both cap cells and inner sheath cells, as well as higher levels in prefollicle cells farther posterior in the germarium. No dpp mRNA was seen in terminal filament cells or in any germ line cells, including GSCs. These results show that cap cells are one of several cell types located near the GSCs that express dpp. Moreover, it does not appear to be the absence of contact with a dpp-expressing cell that causes the posterior stem cell daughter to differentiate as a cystoblast (Xie, 2000).

These studies suggest a working model for a GSC niche. It is proposed that cap cells are critical to the formation, maintenance, and regulation of the GSC niche. Cap cells and terminal filament cells form a characteristic structure with sufficient internal surface area to contact two or three GSCs. A special cell-cell junction is likely to form between GSCs and cap cells to explain their intimate juxtaposition throughout adult life. Such a junction likely holds a GSC at the anterior and prevents it from moving away from the ovariolar tip where it might receive differentiation cues. An intercellular signal, possibly dpp, would be needed to maintain this junction and control the rate of GSC division, but need not be spatially graded. Additional signals appear to be involved in niche function as well. Terminal filament cells and/or cap cells express hedgehog, wingless, and armadillo, although roles for these signaling molecules in regulating GSCs remain unclear. Yb and piwi function outside the germ line in maintaining GSCs. The combined action of these genes ensures that precisely one of the GSC daughters loses cap cell contacts and differentiates (Xie, 2000).

These experiments show that a small group of stromal cells located at the tip of the Drosophila ovariole acts as a stem cell niche. Stem cells in many different tissues and organisms may be regulated in a similar manner. In the Drosophila testis, five to seven stem cells are anchored on terminally differentiated somatic hub cells, suggesting that both the ovary and testis could use similar strategies to regulate their stem cells. In Caenorhabditis elegans, distal tip cells have been directly implicated in the maintenance of the GSC population. In the Arabidopsis shoot meristem, an organizing center located nearby is required to maintain meristem stem cells. The reported plasticity of some mammalian stem cells may result from the existence of niches that can reprogram stem cell identity. The studies presented here provide a basis for detailed comparisons between the structure and regulatory properties of niches supporting different stem cells and will assist efforts to elucidate the molecular signals that control stem cell division and differentiation (Xie, 2000).

dpp is expressed during oogenesis in anterior follicle cells. Expression is first detectable at the end of stage 8 in approximately 20 to 30 somatic follicle cells at the anterior tip of the egg chamber. The number of cells expressing dpp increases as they migrate cortically and posteriorly until expression is seen over the anterior third of the egg chamber at the end of stage 9. Border cells do not express dpp. By stage 10A, all follicle cells have completed their posterior movement, and dpp-expressing cells includes approximately 50 thinly stretched nurse cell follicle cells and 20 columnar follicle cells overlying the nurse cell-oocyte boundary. At stage 10B, the cells at the nurse cell-oocyte boundary begin a centripetal migration inward between the oocyte and nurse cells. Here, dpp is expressed in the leading edge (20 cells) of the approximately 150 columnar centripetally migrating follicle cells, which move adjacent to the border cells (Twombly, 1996).

The Drosophila EGF receptor (Torpedo/DER) and its ligand (Gurken) play roles in the determination of anterioposterior and dorsoventral axes of the follicle cells and oocyte. The roles of DER in establishing the polarity of the follicle cells were examined by following the expression of Egfr-target genes. One class of genes (e.g. kekon) is induced by the Egfr pathway at all stages. Broad expression of kekon at the stage in which the follicle cells migrate posteriorly over the oocyte, demonstrates the capacity of the pathway to pattern all follicle cells except the ventral-most rows. This may provide the spatial coordinates for the ventral-most follicle cell fates. A second group of target genes (e.g. rhomboid (rho)) is induced only at later stages of oogenesis, and may require additional inputs by signals emanating from the anterior, stretch follicle cells. A mechanism must exist to prevent rho, and possibly other genes (e.g. bunched), from being triggered by the same pathway at earlier stages. One option is that induction by the DER pathway is not sufficient to trigger rho, and an additional input must be provided by a different group of genes from the stretch follicle cells. Dpp is expressed in these cells and may prove to be a likely candidate. Multiple requirements for triggering rho expression may thus assure that it will normally be induced at a restricted point in space and time, only when the Gurken-induced signal emanating from the oocyte nucleus can be combined with a signal originating from the stretch follicle cells (Sapir, 1998).

short gastrulation (sog) and decapentaplegic (dpp) function antagonistically in the early Drosophila zygote to pattern the dorsoventral (DV) axis of the embryo. This interplay between sog and dpp determines the extent of the neuroectoderm and subdivides the dorsal ectoderm into two territories. Evidence exists that sog and dpp also play opposing roles during oogenesis in patterning the DV axis of the embryo. Maternally produced Dpp increases levels of the IkappaB-related protein Cactus and reduces the magnitude of the nuclear concentration gradient of the NFkappaB-related Dorsal protein, and Sog limits this effect. Evidence is presented suggesting that Dpp signaling increases Cactus levels by reducing a signal-independent component of Cactus degradation. Epistasis experiments reveal that sog and dpp act downstream of, or in parallel to, the Toll receptor to reduce translocation of Dorsal protein into the nucleus. These results broaden the role previously defined for sog and dpp in establishing the embryonic DV axis and reveal a novel form of crossregulation between the NFkappaB and TGFbeta signaling pathways in pattern formation (Araujo, 2000).

In aggregate, the results support models in which Sog and Dpp proteins are produced by the follicle cells and then are delivered to the embryo. These proteins could be deposited in the vitelline membrane or in the oocyte plasma membrane, or might be sequestered in the perivitelline space and remain there protected until early embryogenesis. The fact that sog and dpp are expressed in follicle cells of stage 10 egg chambers, around the time that follicle cells are secreting major structural proteins of the vitelline envelope, is consistent with their products being delivered to the vitelline membrane or perivitelline space. Since sog and dpp are secreted proteins, they could be exported like components of the vitelline membrane to the extracellular compartment between the follicle cells and the oocyte. After stage 13, the vitelline membrane is thought to be an impermeant barrier separating the oocyte from follicle cells making it unlikely that sog and dpp products are transferred after this time. A similar model has been proposed to explain the functions of the dorsal group gene nudel and of the maternal terminal system gene torsolike (tsl). Both of these genes are expressed during midoogenesis, long before their activity is required during early embryogenesis. According to this model, the Sog and Dpp proteins would remain in the perivitelline space until early embryogenesis, when the Tl pathway is activated by Spatzle. In the early embryo, maternal Dpp would decrease the level of Tl-mediated nuclear translocation of Dorsal by decreasing Cactus signal-independent degradation through a pathway acting in parallel to Tl. Presumably, Sog antagonizes the action of Dpp, resulting in maximal nuclear Dorsal translocation (Araujo, 2000).

Consistent with the view that Sog and Dpp proteins are made early (e.g. midoogenesis), but act later in the early embryo, induction of sog expression during midoogenesis by use of a heat-shock sog construct increases levels of a Sog fragment in the early embryo detected by a specific anti-Sog antibody. Thus, Sog protein produced during midoogenesis can be stably stored for a protracted period until the onset of embryogenesis. In contrast to Sog protein, SOG mRNA does not perdure at detectable levels in early pre-blastoderm embryos in these experiments. The fragment of Sog generated in these experiments is the same size (60 kDa) as one that may have activity during pupal development (Araujo, 2000).

There are several unanswered questions regarding how maternal Dpp signaling contributes to embryonic DV patterning. An important remaining question is how maternal Dpp signaling contributes to defining discrete zones of gene expression along the DV axis? Two leading possibilities, which are not necessarily mutually exclusive are: (1) sog and dpp function to determine the relative proportions and positions of the different primary DV domains, and (2) Dpp signaling is necessary to sharpen borders between embryonic DV territories. There is good evidence in support of the first possibility, since the extents of DV expression domains can be altered by increasing maternal Dpp activity. As mentioned above, maternally produced Dpp results in a ventral shift of all DV domains, presumably by lowering the amount of nuclear Dorsal in cells along the entire DV axis. These results also support a role for maternal sog and dpp in refining the normally sharp borders between different territories, since altering the maternal dose of sog or dpp generates overlapping expression of mesodermal and neuroectodermal genes (Araujo, 2000).

Another question is by what mechanism does maternal Sog oppose Dpp in patterning the embryo? Perhaps Sog is necessary to inhibit Dpp signaling through a specific receptor subtype such as the Sax receptor or to restrict Dpp signaling to a specific type of Dpp receptor (e.g. mediated only by Tkv). Alternatively, Sog could be involved in antagonizing another BMP molecule in addition to Dpp, which also functions in embryonic DV patterning. In summary, the results presented in this study indicate that maternal components of Dpp signaling modify elements that converge with signaling downstream of the Tl receptor by regulating Cactus levels and nuclear translocation of Dorsal. This analysis suggests that maternal sog and dpp function to define the relative proportions of embryonic DV domains and may play a role in creating sharp borders between these domains. Further experiments will be necessary to determine the mechanism by which maternal sog and dpp function and how interactions between the Tl and Dpp pathways collaborate to pattern the DV axis of the Drosophila embryo (Araujo. 2000).

Clonal expansion of ovarian germline stem cells during niche formation in Drosophila

Stem cell niches are specific regulatory microenvironments formed by neighboring stromal cells. Owing to difficulties in identifying stem cells and their niches in many systems, mechanisms that control niche formation and stem cell recruitment remain elusive. In the Drosophila ovary, two or three germline stem cells (GSCs) have recently been shown to reside in a niche, in which terminal filaments (TFs) and cap cells are two major components. Signals from newly formed niches promote clonal expansion of GSCs during niche formation in the Drosophila ovary. After the formation of TFs and cap cells, anterior primordial germ cells (PGCs) adjacent to TFs/cap cells can develop into GSCs at the early pupal stage while the rest directly differentiate. The anterior PGCs are very mitotically active and exhibit two division patterns with respect to cap cells. One of these patterns generates two daughters that both contact cap cells and potentially become GSCs. Lineage tracing study confirms that one PGC can generate two or three GSCs to occupy a whole niche ('clonal expansion'). decapentaplegic is expressed in anterior somatic cells of the gonad, including TFs/cap cells. dpp overexpression promotes PGC proliferation and causes the accumulation of more PGCs in the gonad. A single PGC mutant for thick veins, encoding an essential Dpp receptor, loses the ability to clonally populate a niche. Therefore, Dpp is probably one of the mitotic signals that promote the clonal expansion of GSCs in a niche. This study also suggests that signals from newly formed niche cells are important for expanding stem cells and populating niches (Zhu, 2003).

This demonstrates how an adult GSC niche is populated with stem cells in the Drosophila ovary. Before niche formation, all PGCs proliferate as pre-stem cells and are undifferentiated. As niche formation starts, PGCs divide into two distinct subpopulations: anterior PGCs adjacent to cap cells start to acquire stem cell identity, and the remaining PGCs directly proceed to differentiation. GSCs in one niche can come from one PGC. Dpp is likely involved in stimulating clonal expansion of GSCs during niche formation. This study suggests that signals from newly formed niches are important for expanding GSCs and most likely for populating nascent adult GSC niches (Zhu, 2003).

How stem cell identity is established initially remains elusive even in the well-studied stem cell systems -- Drosophila ovary and testis. In the primitive female gonads before the pupal stage, PGCs appear to undergo symmetric division to generate germ cells with the identical pre-stem cell fate. Several studies suggest that GSCs are established at the early pupal stage. At the early pupal stage, there are 136 germ cells on average in each gonad. The adult ovary, which is composed of 12-16 ovarioles with two or three GSCs per ovariole (average of 2.5), contains about 30 to 40 GSCs. Therefore, at the most, 20%-30% of PGCs in the early pupal gonad are recruited to niches and turn into GSCs (Zhu, 2003).

How is a particular germ cell selected and recruited to niches, and how does it become a GSC? Positional information is known to be very important for cell-fate determination in various developmental processes. In this study, a developmental approach was taken to investigate when key niche components form, and how PGCs are subdivided into GSCs and differentiated germ cells. The expression of bam is associated with germ cell differentiation in the adult ovary. Using bam expression as an indicator for germ cell differentiation, it has been shown that no PGCs in late third instar larval gonads have differentiated. In early pupal gonads (about 0-4 hours after pupation), all the PGCs that are not in contact with TFs/cap cells are differentiated; therefore, the PGCs that contact newly formed cap cells remain undifferentiated and become GSCs. Possibly, newly formed TFs/cap cells directly prevent the most anterior PGCs from differentiation when an unknown developmental signal triggers PGC differentiation around the larval-pupal transition stage. This study demonstrates that the stem cell fate of PGCs is determined by their position, i.e. juxtaposition to TFs/cap cells (Zhu, 2003).

The next important question is how these anterior PGCs populate niches. In this study, it has been show that the PGCs in contact with newly formed cap cells at the early pupal stage divide more frequently than the rest of the PGCs. The division patterns are very interesting: one division pattern generates two daughters that are both in contact with cap cells; the other pattern generates only one daughter that is in contact with cap cells. As in the adult ovary, two daughters that are in contact with cap cells can both become GSCs. This is verified by the observation that one marked PGC in the gonad at the late third-instar larval stage can generate two or three GSCs in a niche. The results also indicate that the stem cells in a niche can come from multiple PGCs. Whether GSCs in a niche come from one or multiple PGCs probably depends on whether one or multiple PGCs directly contact cap cells within the developing niche. If only one PGC contacts cap cells, it probably has an opportunity to generate two or three germ cells that contact cap cells and become GSCs. This study shows that newly formed niches do not simply recruit existing PGCs and turn them into GSCs, but also stimulate PGCs to proliferate and produce more GSCs (Zhu, 2003).

The clonal expansion of GSCs in a niche clearly requires the newly established stem cell to divide rapidly and generate a daughter that occupies the same niche, which further prevents other neighboring precursor cells from entering it. Consistent with this prediction, it was observed that the anterior row of germ cells at the early pupal stage is more mitotically active than the rest of the germ cells based on the BrdU incorporation assay. dpp is known to be important for maintaining GSCs and stimulating their division in the adult ovary. dpp is expressed in TFs/cap cells and other anterior somatic cells, and PGCs close to cap cells are capable of responding to dpp. Furthermore, overexpressing dpp promotes PGC proliferation. To demonstrate the necessity of dpp signaling in stimulating GSC clonal expansion, it has been shown that a PGC mutant for tkv, an essential dpp receptor, fails to clonally populate a niche. All these results demonstrate that dpp is probably a signal for stimulating GSC clonal expansion (Zhu, 2003).

As in the adult ovary, hh is also expressed in terminal filaments and cap cells in developing female gonads. Hh has been shown to play a minor role in modulating GSC division. Wingless (Wg) protein is expressed in terminal filaments and cap cells. Its expression in developing female gonads has not been examined. Because wg, dpp and hh often work together to regulate many developmental processes in Drosophila, it is possible that hh and wg could also cooperate with dpp to regulate PGC proliferation and modulate GSC clonal expansion in niches (Zhu, 2003).

PGCs in the gonad do not show any signs of differentiation until the larval-to-pupal transition. At the early pupal stage, only the PGCs in the anterior row remain undifferentiated, but the rest have already differentiated. It seems that a developmental signal(s) starts to appear and then induces the differentiation of PGCs during the transition from larva to pupa. Such a developmental signal could be mediated by a steroid-like hormone ecdysone. Interestingly, during most of the third instar larval stage, the ecdysteroid levels are very low but begin to rise and peak just before pupation. The ecdysteroid peak could be potentially responsible for the initial differentiation of germ cells in the gonad of the larva ready for pupation. It is also possible that the hormone is not a direct signal but controls the production of the signal(s). Somehow, the signals from the anterior somatic cells antagonize the differentiating signals and thus prevent the anterior row of the PGCs from differentiation. One of the signals that prevent PGCs from differentiation could be encoded by dpp. Dpp is known to prevent GSCs from differentiation in the adult ovary. In this study, 2.5% of the marked tkv mutant PGCs and none of the marked mad mutant PGCs before the third instar larval stage were recruited to niches or were maintained as GSCs before adulthood. The failure of tkv and mad mutant GSCs to be maintained in niches could be explained by the role of dpp in preventing PGCs from differentiation. It could also be explained by other possibilities, such as defects in the formation of adherens junctions between cap cells and GSCs. Whether dpp is a signal for maintaining the undifferentiated state of PGCs during early ovarian development remains undetermined. Therefore, the signals that maintain the undifferentiated state of PGCs from TFs/cap cells remain to be identified (Zhu, 2003).

fused regulates germline cyst mitosis and differentiation during Drosophila oogenesis: fu function may be necessary to restrict Dpp signal production in somatic cells

The fused gene encodes a serine-threonine kinase that functions as a positive regulator of Hedgehog signal transduction in Drosophila embryogenesis, wing morphogenesis, and somatic cell development during oogenesis. This study characterizes the germline ovarian tumors present in adult ovaries of fused mutant females, a phenotype not observed upon deregulation of any other component of Hedgehog signaling. In the strongest fused mutant contexts, tumorous ovarian follicles accumulate early spectrosome-containing germ cells corresponding to germline stem cells and/or early cystoblasts as evidenced by activated Dpp signal transduction and transcriptional repression of bag-of-marbles, encoding the cystoblast determination factor. These early germ cells are maintained far from their usual position in a specialized niche of somatic cells in the apical part of the germarium, which appears normal in size in fused mutant ovarioles. Therefore, these results indicate a novel function for fused in downregulation of Dpp signaling which is necessary for de-repression of bag-of-marbles and consequent cystoblast determination. The abnormal accumulation of these early germ cells seems to be due primarily to defects in differentiation, since germline stem cell proliferation in the germarium is not affected. A later block in germline development, at the 16-cell cyst stage before significant nurse cell and oocyte differentiation, is also observed in tumorous follicles when fused function is only partially lowered. Finally, fused mutant ovaries exhibit some germline cysts having undergone a supernumerary fifth mitotic division. Through clonal analysis, evidence is provided that fused regulates these cystocyte divisions cell autonomously, while the tumorous phenotype probably reflects both a somatic and germline requirement of fused for cyst and follicle development (Narbonne-Reveau, 2006).

The fused ovarian tumor phenotype, like that of other ovarian tumor mutants in Drosophila, was discovered several decades ago, but the precise characterization of the associated germline defects has not been carried out until now. The characteristics of fused ovarian tumors, based on past and present studies can be summarized as follows:

This analysis of ovarian tumors from females bearing strong and weaker fused mutant alleles reveals blocks at two points in germline development, cystoblast determination and nurse cell/oocyte differentiation, respectively. In females carrying strong hypomorphic fu alleles (class I or II) over a small deficiency fuZ4 (class 0), the majority of the ovarioles are abnormal and tumorous germaria and follicles are filled with spectrosome-containing germ cells expressing the dad-lacZ Dpp target reporter transgene and not expressing the bamp-Bam:GFP construct, characteristics shared with germline stem cells and early undetermined cystoblasts. Indeed, maintenance of germline stem cells has been shown to require repression of bam transcription via Dpp/BMP signal transduction and consequent phosphorylated-Mad binding to a silencer element in the bam promoter. In wild-type, bam repression occurs only at the apical end of the germarium because of the existence of a somatic cell niche (terminal filament and cap cells), in which dpp/bmp is specifically expressed, while cystoblast differentiation requires bam de-repression as the germ cells move away from the Dpp-expressing niche. In fu tumorous ovarioles, dad-lacZ expression and bam promoter repression is maintained at a considerable distance from the apical end of the germarium, even though the size of the somatic cell niche is not altered as assayed by specific expression of a hh-lacZ reporter construct. In addition, the excess in Dpp-activated early germ cells in fu mutant ovarioles cannot be attributed to elevated mitotic activity of the germline stem cells in the niche. It is not clear, however, whether the Dpp-activated early germ cells in fu mutants are latent or continue dividing with complete cytokinesis to give two spectrosome-containing daughter cells. Nonetheless, the results suggest a novel Hh-independent function for fu in Dpp signaling: fu function may be necessary to restrict Dpp signal production in somatic cells or to downregulate Dpp signal transduction in early germ cells so that bam transcription may be de-repressed allowing consequent cystoblast differentiation to occur. In fu mutants, an elevated level of Dpp signal or of Dpp signal transduction would lead to extended repression of bam and maintenance of the GSC/early cystoblast state beyond the niche. Unlike fu, however, mutations in bam affect both female and male germ cell development in Drosophila. Since germline stem cell maintenance and bam repression in the testis depends primarily on Gbb/BMP signaling, rather than on Dpp signaling as in the ovary, it is possible that fu mutations specifically perturb Dpp signaling and, therefore, only affect oogenesis (Narbonne-Reveau, 2006).

Analysis of weaker fu hypomorphic contexts revealed a different type of tumorous follicle exhibiting a later block in germline development. Females homozygous for different hypomorphic fu alleles (class I and II) present a low to moderately high proportion of tumorous follicles. In these females, tumorous follicles contain dozens of disorganized, immature germline cysts, including numerous 16-cell cysts (and probably 32-cell cysts) blocked before significant nurse cell and oocyte maturation. Although these germline cysts are present in follicles that have separated from the germarium, they exhibit characteristics of cysts normally found only in the germarium, like the presence of branched fusomes and indicators of mitotic activity (bamp-Bam:GFP construct and Phosphohistone H3). Even though oocyte determination does occur in some of the germline cysts in these fu tumorous follicles, as evidenced by oocyte-specific expression of Orb and the synaptonemal complex component C3G, further growth and maturation of nurse cells and oocyte is impeded. It is not possible to affirm, however, whether the block in nurse cell and oocyte development in these follicles reflects a direct role for fu in this specific step of the differentiation program or an indirect effect of the perturbation of interdependent processes. Indeed, one possibility is that inclusion of numerous cysts in one follicle leads to a block in further cyst development due to reduced efficiency of crucial intercellular communication between somatic and germ cells. In favour of this hypothesis, when only a few germline cysts are present in a follicle (2-4 cysts) due to defects in encapsulation by fu mutant prefollicular cells, significant nurse cell and oocyte development is observed (Narbonne-Reveau, 2006).

Finally, analysis of fu mutant ovaries revealed another defect concerning germline cyst development, in particular, the presence of cysts having undergone a supernumerary mitotic division, that is five rounds of mitosis instead of four, producing 31 nurse cells and one oocyte with five ring canals. Significantly, this defect is detected upon induction of fu mutant germline clones, but is not encountered upon induction of fu mutant prefollicular cell clones, thereby indicating that cell autonomous fu function contributes to cell cycle arrest after the fourth round of cystocyte divisions. This mutant phenotype is of particular interest since few factors controlling the number of germline cyst divisions have been identified so far. However, the existence of a ‘counting factor’, whose quantity would be limiting after four rounds of mitosis has long been postulated. The cytoplasmic Bam protein has some characteristics of such a factor since it accumulates specifically in dividing cystocytes. In addition, a genetic interaction has been shown between bam and encore, the latter encoding a gene function specifically required for restriction of germline cyst division. Using a bamp-bam:GFP transgene it was shown that expression of this reporter persists abnormally in 16-cell cysts in fu tumorous follicles. In addition, other markers that are normally expressed only in germline cysts in the germarium, including the fusome, also linger on in germline cysts in fu tumorous follicles indicating severely delayed development of these cysts. These are therefore likely important elements contributing to the disturbance of a counting mechanism and, consequently, occurrence of a fifth mitotic division in some germline cysts in fu mutant ovarioles. How fu function participates, directly or indirectly, to this counting mechanism is not clear, but the results provide a link between fu and cell cycle control (Narbonne-Reveau, 2006).

fused is expressed in both somatic and germline cells of the ovary. Although induction of either fu mutant germline clones or fu mutant prefollicular cell clones, produces cell autonomous defects in cyst development (fifth mitotic division) and cyst encapsulation by prefollicular cells, respectively, this type of analysis did not lead to the production of ovarian tumors. It is possible that the tumorous phenotype in fu mutants reflects both a somatic and germline requirement for fu function. In view of these results, one other attractive hypothesis is that fu function may be necessary in anterior somatic cells of the germarium (difficult to study using clonal analysis) for regulating soma to germline signaling, such as that mediated by Dpp, for germline cyst development (Narbonne-Reveau, 2006).

Germline stem cell number in the ovary is regulated by mechanisms that control Dpp signaling: Bam blocks Dpp signaling downstream of Dpp receptor activation, thus establishing the existence of a negative feedback loop between the action of the two genes

The available experimental data support the hypothesis that the cap cells (CpCs) at the anterior tip of the germarium form an environmental niche for germline stem cells (GSCs) of the Drosophila ovary. Each GSC undergoes an asymmetric self-renewal division that gives rise to both a GSC, which remains associated with the CpCs, and a more posterior located cystoblast (CB). The CB upregulates expression of the novel gene, bag of marbles (bam), which is necessary for germline differentiation. Decapentaplegic (Dpp), a BMP2/4 homolog, has been postulated to act as a highly localized niche signal that maintains a GSC fate solely by repressing bam transcription. The role of Dpp in GSC maintenance has been examined in more detail. In contrast to the above model, it is found that an enhancer trap inserted near the Dpp target gene, Daughters against Dpp (Dad), is expressed in additional somatic cells within the germarium, suggesting that Dpp protein may be distributed throughout the anterior germarium. However, Dad-lacZ expression within the germline is present only in GSCs and to a lower level in CBs, suggesting there are mechanisms that actively restrict Dpp signaling in germ cells. One function of Bam is to block Dpp signaling downstream of Dpp receptor activation, thus establishing the existence of a negative feedback loop between the action of the two genes. Moreover, in females doubly mutant for bam and the ubiquitin protein ligase Smurf, the number of germ cells responsive to Dpp is greatly increased relative to the number observed in either single mutant. These data indicate that there are multiple, genetically redundant mechanisms that act within the germline to downregulate Dpp signaling in the Cb and its descendants, and raise the possibility that a Cb and its descendants must become refractory to Dpp signaling in order for germline differentiation to occur (Casanueva, 2004).

The prevalent model for Dpp action within the ovary is that it is a local niche signal whose activity is permissive for GSC maintenance. In this model, only GSCs within the niche are exposed to Dpp protein and removal of the CB from the niche lessens or eliminates exposure to the ligand. Moreover, the only postulated function of Dpp is to repress the transcription of bam within the GSCs. The data presented in this paper reveal additional aspects of Dpp function in GSC maintenance. The results strongly suggest that Dpp ligand is not restricted to the niche but rather is present throughout the anterior germarium. Data is presented that the observed specificity of Dpp signaling to the GSCs and CBs is due to functionally redundant mechanisms that operate in the germline to actively downregulate Dpp signaling during GSC differentiation. One of these mechanisms is Bam itself, thus establishing a negative feedback loop between the actions of the two genes. These findings indicate GSC differentiation is correlated with downregulation of Dpp signaling, raising the possibility that Dpp signaling plays an active role in GSC maintenance, and that GSC differentiation requires both the presence of Bam and the absence of Dpp signaling (Casanueva, 2004).

If GSCs and CBs are exposed to equivalent amounts of Dpp protein, as is suggested by both the transcription pattern of the Dpp gene and the expression of Dad-lacZ in the CpCs of the niche and the ISCs posterior to the niche, then it is likely that the observed reduction in Dad-lacZ expression between the GSC and the CB results from intracellular modulation of the strength of the Dpp signal. One hallmark of the GSC is its invariant plane of division. It is proposed that the differential Dpp signaling between the GSC and CB sign results from an intracellular modulation of Dpp signal strength between the two daughter cells, either by the asymmetric segregation of one or more cellular components that modulate Dpp signaling, or by loss of a contact-based niche signal that elevates Dpp signaling preferentially within the GSCs. Removal of the CB cell from the niche thus results in partial downregulation of Dpp signaling. A lower level of Dpp signaling in the CB cell results in the transcription of Bam, which plays multiple roles in CB differentiation, one of which is to cause the daughters of the CB cell to become refractory to further Dpp signaling. Thus, sequential regulatory mechanisms cooperate to ensure an irreversible change in the fate of the GSC cell within two generations (Casanueva, 2004).

Loss-of-function mutations in Smurf and gain-of-function mutations in sax increase the number of GSCs, suggesting these genes may perturb the proposed intracellular modulation of Dpp signaling that occurs between the GSC and CB. However, these data are not sufficient to determine whether this proposed modulatory pathway acts through direct regulation of the functions of one or both of these gene products, or whether the proposed pathway acts in parallel to these genes. In the embryo, loss of Smurf activity results in a ligand-dependent elevation of Dpp signaling that has greater, but not indefinite, perdurance (Podos, 2001), suggesting that Dpp signaling in Smurf mutants, and by inference sax mutants, is still responsive both to the amount of ligand and to the presence of other negative regulatory mechanisms. In the ovary, the Dad-lacZ-expressing germ cells in the Smurf and sax mutants fill the region of the anterior germarium that roughly corresponds to the spatial extent of Dad-lacZ expression in the somatic cells of region 1 and 2A of a wild-type germarium, suggesting that potentially all germ cells in region 1 and 2A of the Smurf and sax germaria are equally and fully responsive to the Dpp ligand. It is proposed that GSCs in the Smurf and sax germaria ultimately undergo normal differentiation because in the more posterior regions of the germaria the amount of Dpp ligand may be reduced to a level that allows bam transcription, which further reduces Dpp signaling and causes cyst differentiation (Casanueva, 2004).

The reduction in Dpp signaling between the GSC and the CB releases Bam from Dpp-dependent transcriptional repression, and one, but not the only, function of Bam is to downregulate Dpp signaling downstream of receptor activation prior to overt GSC differentiation. This is the first molecular action ascribed to Bam, and these data could provide an entry point to elucidate the biochemical basis of the function of Bam in CB differentiation. Further work will be necessary to determine whether the action of Bam on the Dpp pathway is direct or indirect, whether Bam action results in the reduction or complete elimination of Dpp signaling in the developing cysts, and which step in the intracellular Dpp signal transduction pathway or expression of Dpp target genes is affected by Bam action. However, it is possible that initial insights into Bam function can be made by comparing the thresholds for Dpp signaling readouts in the developing wing disc of the larva to the data obtained in the germarium. In the wing disc, Dpp diffuses from a limited source to form a gradient throughout the disc that displays different thresholds for multiple signaling readouts. Specifically, Dad-lacZ is transcribed in response to high and intermediate levels of Dpp, but does not respond to the lowest levels of ligand. An antibody exists that recognizes the active phosphorylated form of Mad, pMad. In the wing disc, high level staining with the pMad antibody is present in only a subset of cells that express high levels of Dad-lacZ, suggesting that in this tissue the pMad antibody is less sensitive to Dpp signaling than is Dad-lacZ expression. Intriguingly, in the ovariole pMad staining is visible in the GSCs, CBs and the developing cysts. Because Dad-lacZ expression was never observed in the developing cysts, these results could suggest that the relative sensitivities of these two reagents are reversed within the germline. Alternatively, if the reagents have the same relative sensitivities in the two tissues, the data suggest that Bam could act, probably at a post-transcriptional level, to downregulate Dpp signaling downstream of Mad activation (Casanueva, 2004).

The pattern of Dad-lacZ expression observed in the Smurf; bam and sax; bam double mutant ovarioles is qualitatively different from that observed in any of the single mutant ovarioles. Although Dad-lacZ expression is observed only at the anterior tip of the germarium of each single mutant, many, but not all, of the double mutant ovarioles contain germ cells throughout the ovariole that express high levels of Dad-lacZ. From these data, it is concluded that two redundant pathways downregulate Dpp signaling in the germline, and that in the single mutants, the action of the remaining active pathway is sufficient to constrain Dpp responsiveness to the anterior tip of the germarium. However, not all doubly mutant ovarioles display a spatial expansion of Dpp signaling, and this variability can even be observed in ovarioles from a single female. It is proposed that the observed variability results because the Smurf and sax mutations have modulatory effects on Dpp signaling that are both dependent on the presence of ligand and are sensitive to additional mechanisms that downregulate Dpp signaling. In both the Smurf; bam and sax; bam ovarioles, the germ cells that express Dad-lacZ are observed throughout the ovariole, but are more likely to be near somatic cells. It is possible that the variability in Dad-lacZ expression occurs because of a non-uniform distribution of the Dpp ligand. Nevertheless, there is not a consistent correlation between the domains of Dad-lacZ expression in the somatic and germ cells, suggesting that there may be additional germline intrinsic factors that affect Dpp signaling (Casanueva, 2004).

Gene circuitry controlling a stem cell niche

Many stem cell populations interact with stromal cells via signaling pathways, and understanding these interactions is key for understanding stem cell biology. In Drosophila, germline stem cell (GSC) maintenance requires regulation of several genes, including dpp, piwi, pumilio, and bam. GSCs also maintain continuous contact with cap cells that probably secrete the signaling ligands necessary for controlling expression of these genes. For example, dpp signaling acts by silencing transcription of the differentiation factor, bam, in GSCs. Despite numerous studies, it is not clear what roles piwi, primarily a cap cell factor, and pumilio, a germ cell factor, play in maintaining GSC function. With molecular and genetic experiments, it is shown that piwi maintains GSCs by silencing bam. In contrast, pumilio is not required for bam silencing, indicating that pumilio maintains GSC fate by a mechanism not dependent on bam transcription. Surprisingly, it was found that germ cells can differentiate without bam if they also lack pumilio. These findings suggest a molecular pathway for GSC maintenance. dpp- and piwi-dependent signaling act synergistically in GSCs to silence bam, whereas pumilio represses translation of differentiation-promoting mRNAs. In cystoblasts, accumulating Bam protein antagonizes pumilio, permitting the translation of cystoblast-promoting transcripts (Chen, 2005).

dpp-dependent silencing of bam transcription defines a key -- probably the primary -- mechanism for maintaining GSCs. By repressing bam transcription in the germ cells attached to cap cells, dpp signaling prevents these cells from forming cystoblasts and assigns them as GSCs. It is speculated that all GSC maintenance genes might act by repressing bam transcription and this prediction was tested for piwi and pumilio (Chen, 2005).

Two genetic observations suggested that piwi might negatively regulate bam expression: (1) bam was epistatic to piwi in double mutants, indicating that the piwi GSC-loss phenotype required an active bam gene; (2) Bam coexpression suppressed the formation of extra GSCs induced when piwi was overexpressed. Thus, piwi-dependent GSC formation depends on maintaining low levels of bam expression (Chen, 2005).

Both overexpression and loss-of-function phenotypes could be explained if piwi, like dpp signaling, were necessary to silence bam transcription in GSCs. This possibility was tested by scoring the expression of bam transcriptional reporters in piwi mutant GSCs. Because piwi inactivation causes GSC loss, P{bamP-GFP} reporters were assayed in piwi bgcn (benign gonial cell neoplasm) double mutant flies that preserve GSCs. GSCs lacking bgcn were GFP negative, but GSCs that lacked both piwi and bgcn were GFP positive. Thus, like dpp signaling, piwi+ was necessary to silence bam transcription in GSCs (Chen, 2005).

piwi+ action in somatic, but not germline, cells is critical for GSC maintenance, and, therefore, piwi must act indirectly to repress bam transcription. A putative piwi target (or targets) in GSCs must integrate with dpp signaling because previous work has established that the Mad:Medea binding site in bam is a sufficient silencer element. Two recent findings drew attention to the E3-ligase Dsmurf as a candidate for a germ cell piwi target: (1) Dsmurf inactivation produces extra GSCs, just as does ectopic piwi expression and (2) Dsmurf suppresses dpp signaling by targeting phosphorylated Mad for degradation (Chen, 2005).

If piwi silences bam transcription by repressing Dsmurf in GSCs, then GSCs might be restored in piwi mutants if Dsmurf were simultaneously removed. Therefore ovaries of piwi Dsmurf double mutant females were examined and it was found that most germaria contained supernumerary GSCs and a continuous supply of egg chambers. It was verified that piwi Dsmurf GSC-like cells behaved as GSCs by noting that they did not express BamC protein. In 62/80 double mutant germaria, no cells expressing BamC were detected, whereas BamC-positive germ cells were detected in 18/80 germaria. In those cases, the most apical cells, in the GSC position, were BamC negative (Chen, 2005).

Dpp signaling and piwi act as GSC maintenance factors by repressing bam transcription. pumilio (pum) is a component of an evolutionarily conserved mechanism of translational control and is also essential for ovarian GSCs. The expression of P{bamP-GFP} reporter was examined in pum mutant germ cells to determine if bam transcriptional silencing also depends on pum+. In contrast to piwi, the reporter was properly silenced in pum bam GSCs. For example, GSCs in 84.6% of pumMSC bamBG/pum2003 bamΔ86 germaria were GFP negative. Because pum mutant germ cells are unstable, it was suspected that the few GFP-positive cells in the GSC position had either differentiated or were dying (Chen, 2005).

GSCs required (1) dpp+ and piwi+ signaling to repress cystoblast (CB) differentiation by silencing bam transcription and (2) pum+ to repress CB differentiation by a mechanism that is independent of bam silencing. Because previous work has shown that Pum forms a translational repressor complex with Nanos, it was reasoned that pum+ might maintain GSCs by repressing translation of CB-promoting mRNAs. One candidate target mRNA is bam itself, but, because dpp-dependent transcriptional silencing of bam fully accounts for the absence of bam from GSCs, it is unlikely that Pum sustains GSCs by repressing bam translation (Chen, 2005).

The Pum:Nos repressor complex probably blocks translation of other unidentified target mRNAs that are essential for CB differentiation. Cystoblast formation would then depend on relieving this block, and, because bam is both necessary and sufficient to induce CB differentiation, bam might antagonize or bypass translational repression. The phenotypes of double mutants can distinguish between these possibilities. If bam bypasses translational repression, pum bam germ cells would not form CBs and would resemble bam mutant gonads. If, however, bam antagonizes Pum/Nos-mediated translational repression, pum bam germ cells might differentiate (Chen, 2005).

Ovaries formed in various pum and bam genotypes were compared with several alleles of each gene. Double mutant ovaries produced a complex phenotype that was distinct from either single mutant. Staining nuclei with DNA dyes revealed a mixture of apparently undifferentiated cells and overtly polyploid cells. Indeed, in many cases the polyploid chromosomes were also thick and expanded like nurse cell chromosomes. Most remarkably, these pseudo-nurse cells were occasionally organized within an epithelial layer of follicle cells, like a cyst, although these cysts never contained a full complement of 16 cystocytes. Cells with hallmarks of post-CB differentiation occurred only in the pum bam double mutant ovaries, where they were seen in over half the ovarioles scored (see Table S2) (Chen, 2005).

The appearance of pseudo-nurse cells and even cysts suggested that double mutant germ cells had formed functional cystoblasts, remarkably bypassing the requirement for bam+ expression. To verify that pum bam germ cells were undergoing differentiation, the double mutant cells were examined with several additional markers of differentiation (Chen, 2005).

Orb is expressed in all germ cells, but its levels increase dramatically in the cystocytes of developing cysts. Orb protein levels remain at very low levels in bam mutant cells. Double mutant cells, however, expressed Orb at levels seen in differentiating cysts and well above the levels in bam cells. Orb accumulation revealed that many of the pum bam germ cells that did not yet have obvious pseudo-nurse cell chromosomes had, in fact, progressed well beyond the "pre-CB" stage of bam cells. Pseudo-nurse cells also had high levels of Orb expression, similar to accumulation seen in developing nurse cells (Chen, 2005).

Ring canal formation is a distinctive feature of germ cell cysts, and the multiple cell pum bam cysts contained ring canals. The incidence of these pum bam cysts was modest but reproducible in all double-mutant combinations, including those containing null alleles of bam and very strong or null alleles of pum. It is suspected that the infrequent appearance of multi-nurse cell cysts is due to a second requirement for bam+ to drive cystocyte divisions during cyst formation. This requirement would not have been recognized previously because bam mutations arrested cells as 'pre-CBs' (Chen, 2005).

Although they are not normal, the appearance of these cysts is a striking manifestation that CBs lacking bam could differentiate as long as they also lacked pum. Combined with previous studies showing that ectopic bam expression is sufficient to direct GSC differentiation, the pum bam phenotype strongly suggests that bam acts as a CB-promoting factor by antagonizing, rather than bypassing, pum action. The data suggests further that dpp signaling, which directly regulates bam expression, does not control pum+ expression. A similar conclusion has been reached about the relationship between dpp signaling and nanos expression on the basis of studies of primordial germ cell differentiation (Chen, 2005).

A unifying model is proposed to explain the gene circuitry of GSC and CB fate within the GSC niche. The results suggest that Drosophila ovarian GSCs are retained as stem cells because Pum:Nos complexes repress translation of a pool of mRNAs that induce CB differentiation (Chen, 2005).

In wild-type GSCs that contact cap cells, Pum:Nos translational repression remains active because dpp signaling from stromal cells silences bam transcription and thus blocks the formation of Bam:Bgcn complexes that would antagonize Pum:Nos translational repression. Expression of piwi in stromal cells contributes a key, but unknown, signal that stabilizes or strengthens the Dpp response and bam transcriptional silencing (Chen, 2005).

After the GSC divides, the strength of Dpp signaling falls to levels that can no longer efficiently silence bam transcription in the cell displaced to the posterior and away from cap cells. This could be due to declining Dpp levels or diminished piwi-dependent signals that lead to reduced phospho-Mad levels. As bam transcription increases, Bam:Bgcn complexes antagonize Pum:Nos action and cause derepression of CB-promoting mRNAs, initiating the events of CB differentiation. Because pumilio and nanos are evolutionarily conserved proteins, it will be important to determine if a similar 'multiple-negative' circuitry is at work in mammalian stem cell niches (Chen, 2005).

Notch signaling controls germline stem cell niche formation in the Drosophila ovary; niches located away from their normal location can still sufficiently sustain GSC self-renewal by maintaining high local BMP signaling and repressing bam as in normal GSCs

Stem cells, which can self-renew and generate differentiated cells, have been shown to be controlled by surrounding microenvironments or niches in several adult tissues. However, it remains largely unknown what constitutes a functional niche and how niche formation is controlled. In the Drosophila ovary, germline stem cells (GSCs), which are adjacent to cap cells and two other cell types, have been shown to be maintained in the niche. In this study, Notch signaling is shown to control formation and maintenance of the GSC niche, and cap cells help determine the niche size in the Drosophila ovary. Expanded Notch activation causes the formation of more cap cells and bigger niches, which support more GSCs, whereas compromising Notch signaling during niche formation decreases the cap cell number and niche size and consequently the GSC number. Furthermore, the niches located away from their normal location can still sufficiently sustain GSC self-renewal by maintaining high local BMP signaling and repressing bam as in normal GSCs. Finally, loss of Notch function in adults results in rapid loss of the GSC niche, including cap cells and thus GSCs. These results indicate that Notch signaling is important for formation and maintenance of the GSC niche, and that cap cells help determine niche size and function (Song, 2007).

At the onset of the larval-pupal transition, all of the 16 to 20 terminal filament (TF) stacks have formed and initiate ovariole formation, while another group of somatic cells, cap cells, start to occupy a position between the TFs and the germ cells. The primordial germ cells (PGCs) in direct contact with cap cells are further anchored through E-cadherin and are further expanded through symmetric division and develop into permanent GSCs in the adult ovary. Actin-filament regulator, Cofilin/ADF, and ecdysone signaling, are required for TF formation. However, no studies have been carried out to investigate the formation of cap cells, which are a key component of the GSC niche. In this study, the role of N signaling in controlling cap cell formation was investigated (Song, 2007).

In late third-instar larval female gonads, Dl is expressed in newly formed TFs, while the N receptor is expressed in all the somatic cells, including TFs and cap cells. Consequently, N signaling is active in newly formed TFs and cap cells and its activation is sufficient to induce cap cell formation, suggesting that TF-expressed Dl activates N signaling to induce cap cell formation. To further support the idea that N signaling specifies cap cell fate, reduction of N signaling results in a reduced number of cap cells. Induction of cap cells by N signaling can take place only during the late third instar and early pupal stages, suggesting that active N signaling promotes only cap cell formation along with other factors provided at particular stages. Cap cells can still form without germ cells. This suggests that Dl is unlikely to be required in germ cells for cap cell formation. Therefore, it is concluded that N signaling, activated probably by Dl from newly formed cap cells, specifies cap cell fate through direct induction. In this study, it was also shown that N signaling is required for maintaining the GSC niche in the adult ovary, since loss of N function results in rapid loss of cap cells and GSCs. Taken together, the results of this study demonstrate that N signaling is important for controlling niche formation as well as niche maintenance (Song, 2007).

Although niches have been defined for GSCs in the Drosophila ovary and testis, as well as in several tissue types of the mammalian systems, it remains unclear whether they still function properly for controlling stem cell self-renewal after their location and size are changed. In this study, two pieces of experimental evidence have been provided supporting the idea that expanded niches are functional for controlling GSC self-renewal. First, increased cap cells in the normal location express all known cap cell markers, such as hh-lacZ, wg-lacZ, Lamin C and E-cadherin, and behave like normal cap cells. Second, these expanded cap cells can support self-renewal of extra GSCs, which behave similarly to normal ones based on Dad-lacZ and bam-GFP expression, and their ability to self-renew and generate differentiated germ cells. Even when cap cells cover the anterior half of the germarium, the GSCs associated with the cap cells also appear to be capable of self-renewing and generating differentiated germ cells. These findings show that GSC niche size can be expanded by adding more niche cells (Song, 2007).

This study has also demonstrated that the GSC niche could function in ectopic locations. Ectopic cap cells, which are surrounded by IGS cells or follicle cells, also express known cap cell markers and sufficiently support functional GSCs, supporting the idea that TFs and IGS cells are not essential components of the GSC niche. This is consistent with early published studies showing that the numbers of cap cells and GSCs are closely correlated and that TFs and cap cells express the genes important for GSC self-renewal such as piwi, Yb, hh and dpp/gbb. In light of the recent evidence that ESCs in direct contact with cap cells and GSCs are required for maintaining GSCs, it remains formally possible that some unidentified escort stem cells cells associated with expanded or ectopic cap cells contribute to niche function. In any case, this study demonstrates that the size and location of the GSC niche can be genetically manipulated while it maintains its functions. The ability to manipulate niche location and size will further increase the capacity to investigate how niche formation is controlled and how the niche controls stem cell function in general (Song, 2007).

One of the major unsolved issues for the GSC niche in the Drosophila ovary is how BMPs function as a short-range signal to control GSC self-renewal and allow the GSC daughter adjacent to the GSC to differentiate at the same time. Several previous studies have shown that BMP signaling activity is primarily restricted to the GSC based on Mad phosphorylation and Dad expression, two indicators of BMP signaling. Early work has also shown that dpp is primarily expressed in TF and cap cells, while gbb is expressed in TF and cap cells as well as in IGS cells. In this study BMP signaling activity has been shown to spread two or more cell diameters based on expression of Dad-lacZ and bam-GFP when more cap cells exist. Furthermore, when more cap cells accumulate in ectopic sites, the GSCs associated with the cap cells as well as the germ cells lying two or three cells away are capable of activating BMP signaling and repressing bam expression. One of the explanations for these observations is that cap cells are the source of BMP and more cap cells would produce more BMP to diffuse further away to repress differentiation of germ cells lying two or more cell diameters away. Another explanation is that the ratio of cap cells to ESCs or escort cells increases so that BMP inhibitors, such as Sog, produced by ESCs or escort cells, are diluted or deterred by more cap cells, and consequently more active BMP is available for reaching and activating cells lying more than two cell diameters away. In Xenopus gastrula-stage embryos, an effective BMP-4 activity gradient is established, not by diffusion of BMP-4 protein but by the long-range effects of two BMP-4 inhibitors, Noggin and Chordin. Finally, it is also possible that a combination of both mechanisms contributes to restriction of BMP signaling activity to one cell diameter in the GSC niche. Observations from this study have suggested that a limited amount of active BMP produced by cap cells is probably responsible for its short-range effect on GSC self-renewal in the GSC niche (Song, 2007).

Local BMP receptor activation at adherens junctions in the Drosophila germline stem cell niche

According to the stem cell niche synapse hypothesis postulated for the mammalian haematopoietic system, spatial specificity of niche signals is maximized by subcellularly restricting signalling to cadherin-based adherens junctions between individual niche and stem cells. However, such a synapse has never been observed directly, in part, because tools to detect active growth factor receptors with subcellular resolution were not available. This study describes a novel fluorescence-based reporter that directly visualizes bone morphogenetic protein (BMP) receptor activation and shows that in the Drosophila testis a BMP niche signal is transmitted preferentially at adherens junctions between hub and germline stem cells, resembling the proposed synapse organization. Ligand secretion involves the exocyst complex and the Rap activator Gef26, both of which are also required for Cadherin trafficking towards adherens junctions. It is therefore proposed that local generation of the BMP signal is achieved through shared use of the Cadherin transport machinery (Michel, 2011).

In keeping with the stem cell niche synapse hypothesis, a BMP niche signal in the Drosophila testis is transduced at subcellularly confined sites associated with adherens junctions between hub cells and GSCs. Although BMP ligands are also produced by the somatic CySCs, BMP receptor activation is not detected at the GSC surfaces facing the CySCs. There are several nonexclusive explanations that may contribute to this observation. Either, niche signalling is indeed dominated by the homodimeric Dpp or heterodimeric Dpp/Gbb ligands that are produced preferentially by the hub cells. In support of this idea, Dpp but not Gbb can fully suppress bam transcription upon ectopic expression, and is, at least in the wing, thought to have higher signalling activity. Alternatively, signalling from the CySCs may occur diffusely over the entire GSC surface and thus become diluted below the detection threshold of the reporter. Finally, based on the expression profile of the BMP ligands signalling from the CySCs is presumably dominated by Gbb and may therefore preferentially act through the alternative type I BMP receptor Saxophone, thus avoiding detection by a Tkv-specific reporter (Michel, 2011).

However, without artificial Jak/Stat pathway over-activation in the somatic cells of the testis, the CySC-derived BMP signal is by itself not sufficient to maintain GSC fate. Consequently, GSC detachment form the hub induces Bam derepression6 indicating a loss of BMP pathway activation. The junction-associated BMP signal from the hub to the germline, described in this study, is therefore essential for GSC maintenance (Michel, 2011).

In addition, this study shows that trafficking of both Dpp and DE-Cadherin in the hub cells involves the exocyst complex and the Rab11-positive recycling compartment. It is proposed that the local release of the junctional BMP signal is achieved through this shared use of intracellular machinery. Admittedly, RNAi-mediated inactivation of the exocyst complex is bound to have pleiotropic effects, and it cannot be excluded that the secretion of Upd or other growth factors may not also be affected. Can the observed loss of GSC stemness following exocyst knockdown therefore be directly attributed to a loss of BMP signalling from the hub? This is believed to be the case, because loss of Jak/Stat signalling in the germline would primarily be expected to affect adhesion of the GSCs to the hub. Although this loss of contact secondarily causes Bam derepression, Bam expression was also observed in GSCs still adhering to the hub. As Bam expression indicates a loss of BMP signalling also in the testis, this is attributed directly to the loss of the junction-associated BMP signal that is directly detected using a reporter that detects BMP receptor activation (Michel, 2011).

Future studies are required to address what directs Dpp secretion within the hub cells towards the adherens junctions with the overlying GSCs rather than towards those facing the adjacent hub cells. In addition, how the BMP ligands are confined after secretion to prevent lateral diffusion away from the site of release can now be studied. It is likely that for the latter proteoglycans has an essential role (Michel, 2011).

Finally, it was shown that the exocyst is also required for generation of the Dpp signal in the wing disc, where it forms a long-range morphogen gradient rather than a contact-dependent niche signal. It will be interesting to test whether this reflects a specific requirement of planar transcytosis, with the junctions forming a two dimensional network of signalling synapses. Alternatively, as suggested by zebrafish experiments, subcellularly restricted signal transduction at intercellular junctions may be a more general mechanism operating also in systems where BMP ligands spread through extracellular diffusion (Michel, 2011).

The role of Dpp and its inhibitors during eggshell patterning in Drosophila: Dpp acts together with Grk in a concentration-dependent manner to specify the identity and position of at least two distinct follicle cell types

The Drosophila eggshell is patterned by the combined action of the epidermal growth factor [EGF; Gurken (Grk)] and transforming growth factor ß [TGF-ß; Decapentaplegic (Dpp)] signaling cascades. Although Grk signaling alone can induce asymmetric gene expression within the follicular epithelium, the ability of Grk to induce dorsoventral polarity within the eggshell strictly depends on Dpp. Dpp, however, specifies at least one anterior region of the eggshell in the absence of Grk. Dpp forms an anteriorposterior morphogen gradient within the follicular epithelium and synergizes with the dorsoventral gradient of Grk signaling. High levels of Grk and Dpp signaling induce the operculum, whereas lower levels of both pathways induce the dorsal appendages (DAs). Evidence is presented that the crosstalk between both pathways occurs at least at two levels. First, Dpp appears to directly enhance the levels of EGF pathway activity within the follicular epithelium. Second, Dpp and EGF signaling collaborate in controlling the expression of Dpp inhibitors. One of these inhibitors is Drosophila sno (dSno), a homolog of the Ski/Sno family of vertebrate proto-oncogenes, which synergizes with daughters against dpp and brinker to set the posterior and lateral limits of the region, giving rise to dorsal follicle cells (Shravage, 2007).

The results show that Dpp has Grk-independent and Grk-dependent functions in the follicular epithelium. Even in the absence of Grk, Dpp is required to specify a group of anterior follicle cells that surround the micropyle. All dorsal follicle cells that contribute to a morphologically visible polarization of the eggshell require the combined action of Grk and Dpp. Within the region giving rise to dorsal follicle cells, Dpp acts together with Grk in a concentration-dependent manner to specify the identity and position of at least two distinct follicle cell types (Shravage, 2007).

In the absence of Dpp, Grk can still activate kekkon and repress pipe. Thus, Dpp is not required for Grk signaling per se. It is suggested that Dpp signaling rather activates transcription factors or causes chromatin modifications that allow Grk to induce dorsal target genes involved in follicle cell specification (Shravage, 2007).

Mirror might be such a transcription factor that is activated by Dpp and confers the ability to adopt dorsal fates to a ring of anterior follicle cells. mirror acts downstream of Grk and probably also downstream of Dpp in specifying dorsal follicle cells. However, mirror expression alone leads only to the formation of DA material. Thus, it is likely that mirror only provides the general potential to produce dorsal follicle cells. Additional inputs from Dpp and EGF signaling are needed to produce the full set of dorsal follicle cell fates. This scenario suggests two phases of Dpp signaling. An early phase demarcates the region in which Grk induces dorsal follicle cell fates. This might require only one (low level) threshold of Dpp signaling and is likely to be mediated through activation of mirror. A later phase establishes distinct dorsal follicle cell fates. Here, Dpp acts as a morphogen in combination with EGF signaling (Shravage, 2007).

The results presented in this study suggest that high levels of EGF and Dpp signaling correspond to regions II and III of the operculum, whereas lower levels of both pathways correspond to the DAs. With regard to region III of the operculum that separates the two DAs, the assumption appears to contradict a model based on results that showed that Grk signaling induces the expression of rhomboid (rho), which in turn activates Spitz, a second TGF-α-like molecule. This leads to an amplification of EGF signaling. Highest signaling levels centered at the dorsal midline lead to the induction of the inhibitor argos (aos), which antagonizes Spitz. This in turn lowers the levels of EGF signaling along the dorsal mildline. According to this model, high levels of EGF signaling promote DA, lower levels operculum region III formation. However, the expression patterns of kek, which result from Grk or Dpp overexpression, appear to contradict this model. Indeed, it is believed that the regulatory loop of rho and aos is not required to establish the operculum or DA fates per se. The pattern of BR-C expression is not significantly altered in rho or aos mutant follicle cell clones. However, rho and aos might contribute to patterning processes that are required for the morphogenesis and, as a result of this, for splitting of the DAs. DA extension (tube formation) has been shown to require the collaboration of rho-expressing floor cells and BR-C-expressing roof cells. The rho-expressing floor cells are part of the Fas3 expression domain that separates the BR-C domains. These rho-expressing cells have to form a separate stripe on each side of the dorsal midline to allow the splitting of the DAs. It is suggested that the rho/aos regulatory loop is required to generate two distinct stripes of late-rho expression within the dorsal Fas3 domain. The result is a splitting of the DAs accompanied by the establishment of a region of Fas3 cells that do not express rho, and thus give rise to region III of the operculum (Shravage, 2007).

The establishment of the region giving rise to dorsal follicle cells and its subdivision into operculum and DA-producing cells is an intriguing problem of two-dimensional patterning. The pattern of cell fates depends on the concentration-dependent read-out of two orthogonal signaling gradients (EGF and Dpp). This read-out is complex because the signaling pathways themselves appear to influence each other. (1) There is evidence for a direct influence of Dpp on EGF signaling; (2) the Dpp inhibitors brk and dSno are targets of both pathways, and (3) rho is also a target of both pathways (Shravage, 2007).

Evidence for a direct crosstalk between both pathways is provided by the analysis of kek expression. kek appears to be a primary target gene of EGF signaling, since basal levels of its expression are independent of Dpp. However, an enhancement of kek expression was observed upon dpp overexpression in stage 10A prior to the activation of rho and aos. Moreover, the stage 10B expression patterns of rho and aos do not correlate with the observed changes in kek expression. Thus, these changes cannot be caused by the secondary modulation of the EGF signaling profile. Therefore, a direct crosstalk between both pathways is suggested. This could be because of a Dpp receptor-dependent activation of the ras/MAPK cascade. A TGF-ß receptor-dependent activation of the MAPK cascade has been observed in several vertebrate cell types. One could imagine that the triangular-shaped domain of Fas3 expression, which defines the anterior and dorsal borders of the BR-C domain, is specified by high levels of EGF signaling brought about by a Dpp-dependent enhancement of MAPK signaling. A confirmation of this model would necessitate direct monitoring of MAPK activity upon altered Dpp signaling (Shravage, 2007).

The border between operculum and DAs is also crucially dependent on brk. In brk mutant follicle cell clones, Fas3 expression expands at the expense of the BR-C domains. However, brk expression is upregulated within a broad domain at the dorsal side that also includes the Fas3-expressing region separating the BR-C domains. Although brk represses Fas3 expression in lateral regions allowing BR-C expression, brk is unable to repress Fas3 at the dorsal midline. This suggests that Fas3 expression, which is predominantly dependent on high levels of EGF signaling, cannot be repressed by brk, whereas Fas3 expression in more lateral regions predominantly dependent on Dpp signaling is repressed by brk (Shravage, 2007).

The hemi-circular boundary of the total region giving rise to dorsal chorion fates appears to be defined by a constant value reflecting the sum or the product of EGF and Dpp signaling. The cis-regulatory elements of dSno represent a sensitive sensor for this dual input. At the dorsal midline, lower amounts of Dpp signaling are required to activate dSno than in lateral regions, and the opposite holds true for EGF signaling. During brain development in flies and in several contexts in vertebrates Sno is involved in the control of cell proliferation that has been shown to be crucially dependent on the relative levels of TGF-ß and EGF signaling. It is conceivable that for spatial patterning of the follicular epithelium dSno uses regulatory elements that are derived from a more basic function in the control of cell proliferation in other tissues. The follicle cell expression of dSno might provide a convenient experimental setting to dissect such regulatory elements (Shravage, 2007).

The fact that loss of dSno causes only mild defects is because of redundancy. A combination of three Dpp inhibitors appears to be involved in establishing the border between dorsal follicle cells and the remainder of the mainbody follicular epithelium. brk clones alone have no effect on the position of this border because they cause only a replacement of the DAs by operculum. dad mutant clones seem to lack patterning defects altogether. However, already removing one copy of these inhibitors in a homozygous dSno mutant background leads to an enlargement of operculum and a posterior shift of the DAs. Weak phenotypic effects of dSno have recently been reported for wing vein formation. Wing vein formation, too, represents a developmental context in which several Dpp inhibitors collaborate (Shravage, 2007).

The dSno mutation that was generated deletes a highly conserved protein domain that is responsible for the interaction with Smad proteins in vertebrates and with Medea in flies. The knockout mutations in mice are based on the deletion of this domain. Thus, this dSno mutation should represent a null allele. However, an unusual complexity of the dSno locus has been reported and a deletion is described that suggests that dSno is lethal, in variance to other findings. However, a truncation allele has been described lacking an important part of the conserved Smad interaction domain that, like the currently described allele, is viable. Because the possibility exists that the previously described deletion affects other genes in the chromosomal region of dSno, the question of lethality of dSno requires further analysis (Shravage, 2007).

Loss of dSno in the follicular epithelium does not result in changes in dpp expression or pMAD distribution. Whereas a feedback on dpp expression was not expected, possible changes in pMAD distribution might be below the level of detection of staining protocol. However, there are two other possible explanations. First, in brain development dSno has been shown to be a mediator of Baboon (Activin), rather than Dpp signaling. To investigate whether this also holds true for the follicular epithelium large baboon (Activin type I receptor) mutant follicle cell clones were generated. These clones did not show patterning defects, suggesting that dSno does not act via Baboon signaling with regard to follicle cell patterning. Second, the failure to detect changes in pMAD distribution might follow from the molecular mechanism of Sno action. A core feature of the inhibitory function of Sno proteins results from their ability to bind to the common Smad (Smad4). This binding prevents (or modulates) the interaction with phosphorylated R-Smads required for the transcriptional control of target genes. If this mechanism applies to DSno, the loss of dSno would not change the phosphorylation state of MAD and, if the interaction between DSno and Medea occurred predominantly in the nucleus, there would also be no significant change in the nuclear accumulation of pMAD (Shravage, 2007).

Drosophila eggshell is patterned by sequential action of feedforward and feedback loops

During Drosophila oogenesis, patterning activities of the EGFR and Dpp pathways specify several subpopulations of the follicle cells that give rise to dorsal eggshell structures. The roof of dorsal eggshell appendages is formed by the follicle cells that express Broad (Br), a zinc-finger transcription factor regulated by both pathways. EGFR induces Br in the dorsal follicle cells. This inductive signal is overridden in the dorsal midline cells, which are exposed to high levels of EGFR activation, and in the anterior cells, by Dpp signaling. The resulting changes in the Br pattern affect the expression of Dpp receptor thickveins (tkv), which is essential for Dpp signaling. By controlling tkv, Br controls Dpp signaling in late stages of oogenesis and, as a result, regulates its own repression in a negative-feedback loop. These observations have been synthesized into a model, whereby the dynamics of Br expression are driven by the sequential action of feedforward and feedback loops. The feedforward loop controls the spatial pattern of Br expression, while the feedback loop modulates this pattern in time. This mechanism demonstrates how complex patterns of gene expression can emerge from simple inputs, through the interaction of regulatory network motifs (Yakoby, 2008a).

These results provide new insights into the dynamics and function of the Dpp pathway in oogenesis. First, it was demonstrated that, contrary to the current model of Drosophila eggshell patterning, the pattern of Dpp signaling in oogenesis is not static, and undergoes a clear transition from purely AP to DV pattern in late stages of eggshell patterning. This transition reflects the change in the expression of the type I Dpp receptor and is conserved in fly species separated by more than 40 million years of evolution. Second, it was shown that the early and late patterns of Dpp signaling control the dynamic pattern of br, a transcription factor expressed in the roof of future dorsal appendages. While the AP phase of Dpp signaling represses br in the anterior region of the egg chamber, the DV phase of Dpp signaling limits the duration of br expression in the roof cells. Third, it was established that, in addition to being regulated by Dpp, Br actively controls Dpp signaling, thus regulating its own repression via a negative-feedback loop (Yakoby, 2008a).

The results lead to a new model for the dynamics of Br expression in the roof cells. Within the framework of this model, the rising phase of Br expression is due to an incoherent feedforward loop, a network in which the input activates both the target and its repressor. In this case, the feedforward loop, formed by EGFR, Pointed, and Br, determines the spatial pattern of Br. This pattern is then modulated in time by a negative-feedback, which depends on the Br-mediated increase of tkv expression and Dpp signaling. The feedforward part of the model is supported by the previously published gain- and loss-of-function experiments with Pointed and EGFR signaling, and by the current analysis of Br expression in ras- mosaics. The negative-feedback loop is supported by the correlation of patterns of Br, Tkv, and P-Mad, by published experiments with manipulation of the levels of Dpp, by analysis of Br protein and br transcript in the Dpp pathway loss-of-function experiments, and by the effects of br- clones and Br overexpression on tkv and Dpp signaling (Yakoby, 2008a).

Four phases in the dynamics of the Br pattern are distinguished. (1) Low levels of Br before stage 9 of oogenesis are independent of EGFR signaling and insensitive to repression by Dpp. (2) Following the formation of the DV gradient of EGFR activation, Br is repressed in the midline and in the dorsoanterior cells. The midline repression is due to Pointed, a transcription factor induced by high levels of EGFR activation in the dorsal midline. The dorsoanterior repression is due to the early phase of Dpp signaling, which reflects the anterior secretion of Dpp and uniform expression of Tkv. (3) Levels of Br begin to rise in the roof cells. Changes in the Br pattern have two effects on the spatial pattern of Dpp signaling: higher levels of Br lead to higher levels of tkv in the roof cells. Second, the dorsoanterior and midline repression of Br generates a corresponding repression of tkv. (4) As a result, the anteriorly produced Dpp can diffuse over the 'Tkv-free' area to the roof cells. A combination of the arrival of the anteriorly produced ligand and a higher level of receptor expression leads to a higher level of Dpp signaling in the roof cells and subsequent repression of br. Another layer of regulation is provided by Brk, a transcriptional repressor of Dpp signaling, which is induced by Gurken and repressed by Dpp signaling in the dorsal follicle cells. Brk antagonizes the repressive effect of Dpp in the roof cells until the level of Dpp signaling in the roof cells becomes high enough to repress Brk expression (Yakoby, 2008a).

The network characterized in this study can interact with a number of previously discovered feedback loops. For instance, Argos, which provides negative-feedback control of EGFR signaling in the dorsal midline, is a potential target of Dpp signaling. Future work is required to explore the extent to which this feedback loop, which had been proposed to affect dorsal midline patterning, interacts with the mechanism established in this paper (Yakoby, 2008a).

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).

The glypican Dally is required in the niche for the maintenance of germline stem cells and short-range BMP signaling in the Drosophila ovary

The Drosophila ovary is an excellent system with which to study germline stem cell (GSC) biology. Two or three female GSCs are maintained in a structure called a niche at the anterior tip of the ovary. The somatic niche cells surrounding the GSCs include terminal filament cells, cap cells and escort stem cells. Mounting evidence has demonstrated that BMP-like morphogens are the immediate upstream signals to promote GSC fate by preventing the expression of Bam, a key differentiation factor. In contrast to their morphogenic long-range action in imaginal epithelia, BMP molecules in the ovarian niche specify GSC fate at single-cell resolution. How this steep gradient of BMP response is achieved remains elusive. In this study, it was found that the glypican Dally is essential for maintaining GSC identity. Dally is highly expressed in cap cells. Cell-specific Dally-RNAi, mutant clonal analysis and cell-specific rescue of the GSC-loss phenotype suggest that Dally acts in the cap cells adjacent to the GSCs. It was confirmed that Dally facilitated BMP signaling in GSCs by examining its downstream targets in various dally mutants. Conversely, when Dally was overexpressed in somatic cells outside the niche, the number of GSC-like cells was increased apparently by expanding the pro-GSC microenvironment. Furthermore, in a genetic setting a BMP-sensitivity distinction was revealed between germline and somatic cells, namely that Dally is required for short-range BMP signaling in germline but not in somatic cells. It is proposed that Dally ensures high-level BMP signaling in the ovarian niche and thus female GSC determination (Guo, 2009).

To understand how a steep gradient of BMP response is established and thus determines cell fate at single-cell resolution in the GSC niche of Drosophila ovary, genetic approaches were taken to examine the role of the glypican Dally in the process. Based on the current data, a model is proposed of how the expression pattern of Dally shapes the BMP-signaling range and consequently determines distinct cell fates in the ovarian niche (Guo, 2009).

Female GSC fate requires high BMP signaling, which is provided in the ovarian niche. Dally is highly expressed in the cap cells that contact the GSCs. Cap-cell-localized and membrane-bound Dally either stabilizes/concentrates BMP molecules, or enhances BMP sensitivity to ensure that only the germ cells in contact with cap cells become GSC. Upon removal of Dally, BMP concentration at the niche or BMP sensitivity in the germ cells adjacent to cap cells dissipates, and GSCs cannot be maintained and subsequently differentiate. Conversely, when Dally is ectopically overexpressed in the escort cells posterior to the niche, BMP signaling or sensitivity increases in all germ cells encapsulated by these escort cells, and GSC-like cells accumulate in the germarium (Guo, 2009).

Consistent with this model, the secreted form of Dally expressed from cap cells caused GSC loss, possibly by competing with endogenous membrane-anchored Dally for binding with BMP molecules or by interfering with BMP signaling. Because secreted Dally expressed from somatic cells in addition to cap cells did not cause GSC expansion as the membrane-anchored Dally did, the evidence further supports the idea that Dally's function in the GSC niche depends on the cap-cell-specific expression and membrane anchoring (Guo, 2009).

When somatic cells displace the differentiating germ cells in the niche and become close to or in contact with cap cells where the BMP morphogen is localized, these somatic cells are able to respond to BMP when Dally is lacking. Whether a cellular BMP response is Dally dependent or not distinguishes the germline and somatic cells (Guo, 2009).

It was noticed that it took 15 days for Dally overexpression in somatic cells to make all germ cells GSC-like in the germarium, although C587Gal4 was active since stage larval 3 at the latest. One possible explanation is that Dpp is limited and Dally stabilizes Dpp. This possibility is supported by a recent report demonstrating that Dally and Dpp physically interact with each other in the cultured S2 cells and that Dally stabilizes Dpp on the cell surface in the wing imaginal epithelia. Consistent with their theory of cell-surface-associated stabilization, the secreted Dally, although retaining the ability to bind Dpp, did not have the activity that the full-length Dally possesses in terms of enhancing GSC proliferation. It suggests that Dally can only stabilize Dpp at the cell surface. Additionally, secreted Dally expressed in the same cells in which the endogenous Dally is produced had a weak dominant-negative effect. By contrast, the secreted Dally expressed elsewhere did not have any detectable effect on GSC, suggesting that it did not compete with the endogenous Dally expressed from the cap cells. These results imply that the anterior tip of the germarium contains the main source of BMP molecules, which the secreted Dally from cap cells has a better chance to catch than that from elsewhere (Guo, 2009).

In the imaginal epithelia, glypican Dally and Dlp are essential for Dpp gradient formation but not for short-range Dpp signaling because one to two rows of cells in the glypican double mutant clone are able to respond to the nearby Dpp signals. Similarly, in dally mutant ovary, in which the germarium was emptied due to GSC loss, BMP response was observed in the escort cells getting close to the cap cells, where the BMP source is supposed to be. However, the germ cell surrounded by the BMP-responsive somatic cells was refractory to BMP morphogen in exactly the same circumstances. It appears that when Dally is compromised, the germline is less sensitive to BMP signaling and Dally either recruits more ligands to the adjacent germ cell or somehow enhances its response to BMP. Contrarily, the somatic cells do not require Dally to sense and respond to BMP morphogen in short range. Whether Dlp is essential for germarial somatic cells in BMP response is unclear. What accounts for the distinction in BMP sensitivity between germline and somatic cells remains to be investigated (Guo, 2009).

BMP signaling dynamics in the follicle cells of multiple Drosophila species

The dorsal anterior region of the follicle cells (FCs) in the developing Drosophila egg gives rise to the respiratory eggshell appendages. These tubular structures display a wide range of qualitative and quantitative variations across Drosophila species, providing a remarkable example of a rapidly evolving morphology. In D. melanogaster, the bone morphogenetic protein (BMP) signaling pathway is an important regulator of FCs patterning and dorsal appendages morphology. To explore the mechanisms underlying the diversification of eggshell patterning, BMP signaling was analyzed in the FCs of 16 Drosophila species that span 45 million years of evolution. The spatial patterns of BMP signaling in the FCs were found to be dynamic and exhibit a range of interspecies variation. In most of the species examined, the dynamics of BMP signaling correlate with the expression of the type I BMP receptor thickveins (tkv). This correlation suggests that interspecies variations of tkv expression are responsible for the diversification of BMP signaling during oogenesis. This model was supported by genetic manipulations of tkv expression in the FCs of D. melanogaster that successfully recapitulated the signaling diversities found in the other species. These results suggest that regulation of receptor expression mediates spatial diversification of BMP signaling in Drosophila oogenesis, and they provide insight into a mechanism underlying the evolution of eggshell patterning (Niepielko, 2011).

In FCs of D. melanogaster, dynamics of BMP signaling are regulated by the dynamics of tkv. In most Drosophila species, a correlation between tkv expression and BMP signaling dynamics was found. Remarkably, ectopic expression/depletion of tkv was sufficient to diversify BMP signaling in D. melanogaster. These perturbations successfully transformed the pattern of BMP signaling found in D. melanogaster into the diverse patterns of BMP signaling naturally found across Drosophila species, supporting the claim that tkv plays a major role in specifying the distribution of BMP signaling in the FCs. In D. melanogaster, a similar mechanism restricts the distribution of BMP signaling in wing and haltere imaginal discs (Niepielko, 2011).

In D. melanogaster, the dynamics of tkv are regulated jointly by BR and BMP signaling. Thus, the late patterns of BR, TKV, and P-MAD are observed in a similar group of cells. Surprisingly, in some species, the pattern of P-MAD correlated with the pattern of tkv; however, these patterns did not fully overlap the BR domain. Specifically, in addition to overlapping the BR domain, tkv was expressed in the adjacent cells that lacked BR expression. It is proposed that, in addition to being regulated by BR and BMP signaling, tkv is also regulated by transcription factors that are expressed in the adjacent domain such as Jra and Fos/Kayak (Niepielko, 2011 and references therein).

Of particular interest are species with three DAs' eggshell, due to the absence of tkv from the BR domain. It is suggested that this signaling pattern provides an example for decoupling of the regulation of tkv by BR from its regulation by BMP signaling. A model is proposed by which tkv is regulated by BMP signaling (anterior); however, it lost its regulation by BR. Of note, the mechanism governing tkv patterning is still unknown, and the established tkv enhancer trap in the Drosophila wing failed to recapitulate the patterns of tkv in the FCs. Thus, the proposed modifications in tkv regulation remain to be experimentally validated (Niepielko, 2011).

In D. melanogaster, early BMP signaling appears as an anterior stripe, reflecting the anterior secretion of the ligand DPP that signals through a uniformly expressed tkv receptor. Thus, in species from the virilis repleta groups, the uniform expression of tkv could account for the anterior stripe domain of late BMP signaling. Indeed, ectopic expression of tkv in all FCs prevented the dorsal anterior repression of BMP signaling in D. melanogaster, which was consistent with the pattern found in flies from the virilis repleta groups (Niepielko, 2011).

In the first three patterning classes, spatial modifications in the late patterns of tkv provided a reasonable explanation for the diversity in late patterns of BMP signaling. In the virilis repleta groups, late expression of tkv was uniform in all FCs; at the same time, BMP signaling was patterned. While the possibility cannot be excluded that a second copy of tkv is present in the non-sequenced species, it is proposed that in these species other BMP components have evolved to gain control of BMP signaling dynamics. In D. melanogaster, the disruption of saxophone (sax), a type I BMP receptor, deformed operculum size and DAs' morphologies. Thus, SAX is a potential regulator of the BMP signaling dynamics in the virilis repleta groups. Also, additional mechanisms were shown to regulate BMP signaling across animals including intracellular and extracellular inhibitors, co-receptors, levels of ligand expression, combinations of ligands, and interactions with other signaling pathways. These mechanisms should be studied systematically in order to determine which BMP component controls the DV phase of signaling in these species (Niepielko, 2011).

In D. melanogaster, the early phase of BMP signaling prevents the anterior domain of the follicle cells from acquiring DA cell fate. This mechanism is based on the inhibition of br expression by the anterior BMP signaling. Thus, it is not surprising that disruption of early BMP signaling is associated with eggshell deformations including modifications in the numbers and shapes of DAs'. Due to the high sensitivity of the eggshell's structures to changes in BMP signaling, it is speculated that small differences in early BMP signaling could guide the natural variations in numbers and shapes of DAs found across Drosophila species (Niepielko, 2011).

In D. melanogaster, the late phase of BMP signaling is associated with the repression of br mRNA in the dorsolateral patches; however, its role in eggshell morphology was not explicitly explored. Depletion of BMP signaling from the BR domain did not affect early BR patterning and operculum size; however, this perturbation deformed DAs' morphology possibly due to late migration of BR cells. Interestingly, similar morphologies were found by disrupting Cad74A, a cadherin gene regulated by BMP signaling that was found to be important for proper eggshell morphology. BMP signaling regulates cadherins in the pupal retina of D. melanogaster and in the human renal epithelial cells. It is proposed that late BMP signaling is involved in the morphological processes of DAs' formation by affecting cell adhesion molecules. Other cadherin genes are expressed or repressed in the DAs' forming cells, and it will be interesting to study how their regulation by BMP signaling affects DAs' morphogenesis (Niepielko, 2011).

The niche-dependent feedback loop generates a BMP activity gradient to determine the germline stem cell fate

Stem cells interact with surrounding stromal cells (or niche) via signaling pathways to precisely balance stem cell self-renewal and differentiation. However, little is known about how niche signals are transduced dynamically and differentially to stem cells and their intermediate progeny and how the fate switch of stem cell to differentiating cell is initiated. The Drosophila ovarian germline stem cells (GSCs) have provided a heuristic model for studying the stem cell and niche interaction. Previous studies demonstrated that the niche-dependent BMP signaling is essential for GSC self-renewal via silencing bam transcription in GSCs. It was recently revealed that the Fused (Fu)/Smurf complex degrades the BMP type I receptor Tkv allowing for bam expression in differentiating cystoblasts (CBs). However, how the Fu is differentially regulated in GSCs and CBs remains unclear. This study reports that a niche-dependent feedback loop involving Tkv and Fu produces a steep gradient of BMP activity and determines GSC fate. Importantly, it was shown that Fu and graded BMP activity dynamically develop within an intermediate cell, the precursor of CBs, during GSC-to-CB transition. Mathematic modeling reveals a bistable behavior of the feedback-loop system in controlling the bam transcriptional on/off switch and determining GSC fate (Xia, 1012).

The present data strongly imply that GSC/CB fate finely controlled by Fu protein regulation is important for generating BMP activity gradient between GSCs and CBs. However, the remaining important question is how to understand the mechanism by which the dynamic reciprocal antagonism between Tkv and Fu controls the GSC-to-CB fate switch during GSC division. To clearly answer this question, a mathematical network model was developed based on the experimental evidences with bistable behavior to elucidate how the feedback loop regulation determines the fate specification of GSCs (Xia, 1012).

On the basis of these data, a feedback loop model is proposed to show how the GSC fate is regulated. In the model, the external BMP signal cues stimulate phosphorylation of Tkv protein, the activated Tkv then promotes the synthesis rate of phosphorylated Mad (pMad), and pMad promotes the degradation of Fu protein and represses the transcription of bam. Meanwhile, degradation of the activated Tkv is also controlled by Fu. To assess the dynamic properties of this feedback loop, it was assumed that the transcriptions of genes tkv, mad, and fu are sufficient and that the degradation rate of pMad and the synthesis rate of Fu protein are constants. The network diagram of the feedback loop clearly points out two characteristics of the model: first, the microenvironment-derived BMP ligands serve as a key external signal, the strengths of which are differentially sensed by GSCs, pre-CBs, and CBs, thereby regulating the dynamic expression of the activated Tkv, pMad, and Fu during the asymmetric division of GSCs. Second, although the transcription of the bam gene is regulated negatively by Tkv/pMad, the expressions (and/or regulations) of the activated Tkv, pMad, and Fu are independently of the status of the Bam protein (Xia, 1012).

The dynamic analysis reveals the bistable behavior (i.e., switch behavior) of the system and how the system dynamics respond to the strength of external BMP ligand activity. Specifically, the strong external BMP ligand activity (in GSCs) will lead to a low expression level of Fu as well as high expression levels of the activated Tkv and pMad. Conversely, the weak external BMP ligand activity (in CBs) will lead to a high level of Fu expression (and low levels of the activated Tkv and pMad expression). However, for the transitional stage with intermediate BMP signaling (in pre- CBs), both high and low levels of Fu and pMad expression exist. These theoretical predictions not only exactly match the experimental data, but they also bring an insightful physical interpretation for why the niche dependence of BMP signaling determines the fate of stem cells by precisely balancing of stem cell renewal and differentiation. The current model permits proposal of a comprehensive description of the action of niche signaling that governs the decision between stem cells and differentiating cells (Xia, 1012).

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decapentaplegic: Biological Overview | Evolutionary Homologs | Transcriptional regulation | Targets of activity | Protein Interactions | Post-transcriptional Regulation | Effect of mutation | References

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