In blastoderm embryos, DAP mRNA is detected at the posterior pole of the embryo. It is here that pole cells form, the precursors of the germ line. At the onset of gastrulation, DAP mRNA is first observed in the amnioserosa and in regions of the cephalic and ventral furrows. Following germ-band elongation, dap is expressed in both dorsal and ventral cells of the epidermis. After germ band retraction, DAP mRNA is detected in cells of the central nervous system and the peripheral nervous system. Subsequent to dorsal closure, dap is expressed in a small subset of cells in the ventral nerve cord and in the brain. In each of these situations, DAP mRNA expression coincides with cell cycle arrest during development (de Nooij, 1996).


Later in development, dap is expressed in the imaginal discs. In the eye imaginal disc, a strip of DAP mRNA is detected in the morphogenetic furrow and only weak mRNA expression is detected posterior to this strip of expression. Expression levels of DAP mRNA in the eye imaginal disc appear to be relatively low. Expression is absent anterior to the furrow, but is found in postmitotic differentiating cells posterior to the furrow (de Nooij, 1996).

Global programmed switch in neural daughter cell proliferation mode triggered by a temporal gene cascade

During central nervous system (CNS) development, progenitors typically divide asymmetrically, renewing themselves while budding off daughter cells with more limited proliferative potential. Variation in daughter cell proliferation has a profound impact on CNS development and evolution, but the underlying mechanisms remain poorly understood. This study found that Drosophila embryonic neural progenitors (neuroblasts) undergo a programmed daughter proliferation mode switch, from generating daughters that divide once (type I) to generating neurons directly (type 0). This typeI> 0 switch is triggered by activation of Dacapo (mammalian p21CIP1/p27KIP1/p57Kip2) expression in neuroblasts. In the thoracic region, Dacapo expression is activated by the temporal cascade (castor) and the Hox gene Antennapedia. In addition, castor, Antennapedia, and the late temporal gene grainyhead act combinatorially to control the precise timing of neuroblast cell-cycle exit by repressing Cyclin E and E2f. This reveals a logical principle underlying progenitor and daughter cell proliferation control in the Drosophila CNS (Baumgardt, 2014).

Proliferation analysis of the developing Drosophila VNC reveals that most, if not all, lateral NBs initially divide in the type I proliferation mode, generating daughters that divide once. Three specific lineages, as well as many other NBs, subsequently switch to generating daughters that do not divide (type 0 mode). The full extent of the typeI>0 switch is currently difficult to precisely assess for several reasons. One such complicating issue pertains to possible developmental changes in daughter cell-cycle length over time. On this note, however, no obvious change was found in NB5-6T daughter divisions prior to the switch. In addition, if the accelerated decline in daughter division was indeed caused by a lengthening of the cell cycle rather than a typeI>0 switch, a long 'tail' of daughter divisions would be expected, perduring into St16-17. This is not the case; rather, daughter proliferation drops down to almost zero by St16-17. Similarly, no evidence was found for changes in NB cell-cycle length over time in the three specific NB lineages. Another complicating issue pertains to the fact that NBs differ in their time point of delamination, number of division rounds, and time point of switching, so that even if all NBs switched, only a fraction of the NBs would be in their type 0 window at the same time. However, these complications are more likely to lead to under- rather than overappreciation of the extent of the typeI>0 switch, and it is tempting to speculate that it may indeed involve the vast majority of NBs (Baumgardt, 2014).

The typeI>0 switch is triggered by the onset of Dap expression in NBs at precise stages of lineage progression. The mammalian Dap orthologs, p21CIP1/p27KIP1/p57Kip2, can act as inhibitors of the CycE/Cdk2 complex. By analogy, the mechanism behind the typeI>0 switch is, presumably, that type 0 daughters are prevented from entering the cell cycle by the presence of Dap at the G1/S checkpoint. The onset of Dap expression already in the NB suggests that Dap needs to be present at an early stage in newborn daughters to block their entry into S phase. These findings are also in line with the emerging role for the Cip/KIP family and cell-cycle exit in the mammalian CNS, although there has been no report of a connection to changes in daughter proliferation mode (Baumgardt, 2014).

No evidence was found for a role of pros in the type 0 mode, and, conversely, no evidence was found for a role of dap in the type I mode. The distinct roles of pros and dap in control of the type I versus 0 modes is further underscored by the expression of E2f, CycE, and Dap. In type I daughters (GMCs), E2f and CycE are rapidly repressed, by pros, and Dap is only weakly expressed at a later stage, around the time point of mitosis. The short window of E2f and CycE expression is still sufficient for the GMC to enter another cell cycle, since Dap expression is absent. As each GMC divides, the postmitotic cells (neurons/glia) are prevented from entering the cell cycle by the lack of E2f and CycE. In type 0 daughters, on the other hand, E2f and CycE expression is robust, but daughters still fail to enter the cell cycle due to the presence of high levels of Dap. These findings point to strikingly different strategies in daughter proliferation control: pros repression of E2f/CycE in type I, and Dap overriding E2f/CycE/Cdk2 in type 0 daughters (Baumgardt, 2014).

Changes in daughter cell proliferation could perhaps have been envisioned to merely result from a gradual loss of the proliferative potential of each progenitor, as a result of its undergoing many rapid cell cycles. If so, typeI>0 switches could have been predicted to occur somewhat stochastically toward the end stage of each lineage, perhaps loosely linked to the last NB division. In contrast to such simplified models, this study found that the typeI>0 switch can occur many divisions prior to NB exit and that it is programmed to occur at a precise stage during each lineage development. In the thorax, it was found that the precise timing of typeI>0 switches is controlled by the temporal gene cas and the Hox gene Antp, which are expressed at a late stage within NBs. Remarkably, in cas mutants, most, if not all, thoracic typeI>0 lineages fail to enter the type 0 mode. The primary mechanism by which cas and Antp control the switch appears to be by activating the expression of Dap, evident by the reduction of Dap in cas and Antp mutants; by the finding that cas-Antp co-misexpression triggers ectopic Dap expression; and by the finding that cas can be cross-rescued by elav>dap (Baumgardt, 2014).

The finding that the timing of the typeI>0 switch is scheduled by a temporal gene cascade points to an intriguing regulatory model where daughter cell proliferation mode switches are executed at stereotyped positions within the lineage tree by the activity of specific temporal genes. Since temporal genes also control the progression of NB competence, evident by their roles in cell fate specification, the temporal cascade can act to simultaneously control both cell fate and cell number, thereby ensuring that precise number of each neural cell subtype is produced (Baumgardt, 2014).

After a stereotyped number of divisions, each NB subtype stops proliferating. This study found that, for many NBs, this is a G1/S decision influenced by the activities of E2f, CycE, and dap. The nuclear localization of Pros was previously identified to be associated with cell-cycle exit in postembryonic NBs. However, previous studies of NB5-6T, and the current study on NB7-3A, do not indicate a general role for pros in NB cell-cycle exit in the embryonic CNS. Instead, in the thorax, the expression levels of E2f, CycE, and Dap are gradually modulated during lineage progression, by the temporal genes cas and grh as well as Antp. Because Cas, Grh, and Antp are progressively activated in thoracic NBs, this brings into view a logical model for timely NB cell-cycle exit where sequential activation of temporal and Hox genes act combinatorially to push E2f, CycE, and/or Dap to limiting levels after a determined number of divisions (Baumgardt, 2014).

For the majority of NBs in the thorax, cell-cycle exit is followed by quiescence until larval stages. In contrast, for the majority of abdominal NBs, cell-cycle exit is followed by apoptosis. However, for some NBs, such as NB7-3A, apoptosis is the functional exit mechanism. Thus, three general strategies for lineage stop are emerging: (1) cell-cycle exit > quiescence (most thoracic NBs), (2) cell-cycle exit > apoptosis (NB5-6T), and (3) lineage stop by apoptosis (NB7-3A). The balance of E2f, CycE, and Dap is involved in the first two strategies, while the balance of apoptosis gene expression presumably is at the core of the latter strategy (Baumgardt, 2014).

In addition to the type I and type 0 daughter proliferation modes described here in the embryo, recent studies of Drosophila larval CNS development have identified a third, more prolific, proliferation mode: the type II mode, identified in a small number of larval brain. Type II NBs divide asymmetrically, renewing themselves while budding of daughters that, in turn, undergo multiple rounds of proliferation before finally differentiating. This allows for the generation of very large lineages (some 500 cells) from each individual type II NB (Baumgardt, 2014).

In mammals, the most obvious equivalent of Drosophila NBs is the radial glia cell (RG), which divides asymmetrically to generate neurons and. During these RG asymmetric divisions, studies have identified several different division modes; RGs dividing asymmetrically to bud off a neuron, to bud off a daughter cell that divides once to generate two neurons, or to bud off daughter cells that themselves divide multiple times before generating neurons. Although mammalian CNS development likely will involve more complex and more elaborate lineage variations, there is, nevertheless, a striking similarity between these alternate mammalian daughter proliferation modes and the type 0, I and II modes now identified in Drosophila. Intriguingly, in line with these analogies between Drosophila and mammals, recent time-lapse studies on the developing primate cortex have revealed a global temporal switch in the proliferation profiles of daughter cells (Betizeau, 2013). It will be interesting to learn if such temporal proliferation changes are intrinsically controlled and if they are stereotypically linked to changes in neural subtype specification also in mammals (Baumgardt, 2014).

Integrins regulate epithelial cell differentiation by modulating Notch activity

Coordinating exit from the cell cycle with differentiation is critical for proper development and tissue homeostasis. Failure to do so can lead to aberrant organogenesis and tumorigenesis. However, little is known about the developmental signals that regulate the cell cycle exit-to-differentiation switch. Signals downstream of two key developmental pathways, Notch and Salvador-Warts-Hippo (SWH), and of myosin activity regulate this switch during the development of the follicle cell epithelium of the Drosophila ovary. This study identified a fourth player, the integrin signaling pathway. Elimination of integrin function blocks mitosis-to-endocycle switch and differentiation in posterior follicle cells (PFCs), via regulation of the CDK inhibitor Dacapo. In addition, integrin mutant PFCs show defective Notch signalling and endocytosis. Furthermore, integrins act in PFCs by modulating the activity of the Notch pathway, as reducing the amount of Hairless, the major antagonist of Notch, or misexpressing Notch intracellular domain rescues the cell cycle and differentiation defects. Altogether, these findings reveal a direct involvement of integrin signalling on the spatial and temporal regulation of epithelial cell differentiation during development (Gomez-Lamarca, 2014).

Effects of Mutation or Deletion

Each of the three P-element insertions in dap results in lethality for a significant part of the mutant population early in development. Cells in several tissues fail to stop dividing at the appropriate time in dap mutant embryos. In wild-type embryos, most cells in the epidermis complete three mitotic divisions after the blastoderm stage and arrest in the G1 stage of cycle 17. In contrast, dap mutant embryos fail to arrest their cell cycles at this precise stage, and most epidermal cells seem to progress through another complete cell cycle. Some mutant embryos have as many as 70% more cells than wild-type embryos, indicating that, in most cases, the majority of cells in the epidemis have completed an additional division. In mutant embryos there is once again an additional S phase in the peripheral nervous system. It should be noted that loss of dap function does not result in unrestricted proliferation. In dap mutant embryos, most cells in the epidermis and some PNS precursors appear to undergo only a single additional cycle. Thus, after the single additional cycle, cells in dap mutant embryos are able to exit from the cell cycle permanently, since there is no further indication of additional cell cycle initiation or progression. This permanent exit must occur using mechanisms that do not require dap function (de Nooij, 1996).

Animal oocytes undergo a highly conserved developmental arrest in prophase of meiosis I. Often this marks a period of rapid growth for the oocyte and is necessary to coordinate meiotic progression with the developmental events of oogenesis. In Drosophila, the oocyte develops within a 16-cell germline cyst. Throughout much of oogenesis, the oocyte remains in prophase of meiosis I. By contrast, its 15 mitotic sisters enter the endocycle and become polyploid in preparation for their role as nurse cells. How germline cysts establish and maintain these two independent cell cycles is unknown. This study demonstrates a role for the p21CIP/p27Kip1/p57Kip2-like cyclin-dependent kinase inhibitor (cki) dacapo in the maintenance of the meiotic cycle in Drosophila oocytes. The data indicate that it is through the differential regulation of the cki Dacapo that two modes of cell-cycle regulation are independently maintained within the common cytoplasm of ovarian cysts (Hong, 2003).

In order to determine the precise onset of the differential regulation of Dap in the nurse cells versus the oocyte, wild-type ovaries were stained with an antibody against Dap. Dap levels oscillate during the four mitotic cyst divisions in region 1 of the germarium. In region 2, a meiotic gradient forms that emanates from the pro-oocytes, the two cells with four ring canals. Although there is a clear gradient with respect to meiosis, this is not reflected in the distribution of the Dap protein. In young post-mitotic cysts, in region 2a and early 2b, Dap is at uniformly low levels throughout the cyst. However, just prior to when the nurse cells enter the endocycle, in late region 2b/early region 3 (stage 1), Dap levels rise dramatically throughout the cyst. Thus, uniformly high levels of Dap are observed in all cyst nuclei in early stage 1 egg chambers, which contain a single oocyte in late prophase of meiosis I and 15 nurse cells that are poised to enter the first endocycle S phase (Hong, 2003).

The differential behavior of Dap in the nurse cells versus the oocyte is first observed in late stage 1 egg chambers. Although high levels of Dap persist in the oocyte, the levels of Dap begin to oscillate in the nurse cells as they asynchronously enter the endocycle. Dap levels continue to oscillate in the nurse cells until stage 10 of oogenesis when the nurse cells stop replicating their DNA. By contrast, Dap levels remain high in the oocyte until well after the nurse cells exit the endocycle. Dap inhibits DNA replication in both mitotic and endocycling cells. Thus, it is likely that the cycling of Dap protein in the nurse cells allows cyclinE-Cdk2 kinase activity to rise high enough to trigger the endocycle S phases. Consistent with this proposal, S phase in the nurse cells occurs when Dap levels are low. Therefore, as the nurse cells enter the endocycle in stage 1 there is a clear correlation between the differential regulation of Dap and the distinct cell cycles of the nurse cells and oocyte (Hong, 2003).

Does the cki Dap prevent the oocyte from entering the endocycle with the nurse cells and thus preserve the prophase I meiotic arrest? To answer this question, homozygous germline clones of the dap null allele dap4 were generated. dap4 contains a deletion of the conserved CDK-binding domain and acts as a complete loss-of-function allele. In greater than 80% of the egg chambers that contain dap germline clones, the oocyte enters the endocycle and becomes polyploid. The extent of polyploidy in these oocytes is variable. 53±9% of dap cysts have 16-polyploid nurse cells and no oocyte. In this phenotypic class, all cells in the cyst have similar DNA contents, indicating all 16 cells have undergone approximately the same number of endocycles. These data suggest that in greater than 50% of the egg chambers with dap germline clones, the oocyte enters the endocycle at the same time as the nurse cells. In 28±7% of the germline clones the oocyte is polyploid, but can be distinguished from the adjacent nurse cells by its lower DNA content and posterior position (Hong, 2003).

In wild-type egg chambers the oocyte DNA condenses into a compact karyosome in stage 3 of oogenesis. In the 10±6% of dap clones in which the oocyte is not obviously polyploid, karyosome formation is often aberrant. For example, the oocyte DNA is present in one or more elongated masses within the oocyte nucleus or in a thin rim near the nuclear envelope. The ~9% of dap clones that contained greater than 16 nurse cells were not included in this analysis. These data indicate that dap regulates entry into or is required for the maintenance of the meiotic cycle in the oocyte (Hong, 2003).

What accounts for the observed variability of the dap phenotype? Because the clonal progeny were examined from germline stem cell clones that had undergone numerous divisions, it is not believed the observed phenotypic variability results from the perdurance of the Dap protein. In support of this conclusion, immunocytochemistry using an antibody against Dap indicated that clonal mutant egg chambers contain no Dap protein. In addition, a similar phenotypic distribution was observed when examining the ovaries from dap4 homozygous escapers. Finally, dap germline cysts surrounded by dap4 and dap+ follicle cells had similarly variable phenotypes, indicating that the genotype of the follicle cells is not the source of variability. Protecting the oocyte from inappropriate entry into the endocycle is critical to the production of a functional gamete. Therefore, it is predicted that additional factors act in concert with Dap to inhibit DNA replication during meiosis (Hong, 2003).

In stage 1 egg chambers the cell-cycle environment within the cyst changes dramatically as the nurse cells asynchronously enter the S phase of the first endocycle. In wild-type stage 1 egg chambers, the oocyte remains safely arrested in prophase of meiosis I. However, in the majority of dap mutant cysts, the oocyte enters the endocycle with the nurse cells. The data indicate that inappropriate entry into the endocycle disrupts oocyte differentiation. Beginning at stage 1, a decrease, relative to similarly aged wild-type egg chambers, is observed in the preferential accumulation of the BicD and Orb proteins in dap oocytes. As oogenesis progresses, there is a strong inverse correlation between the degree of polyploidization in dap oocytes and the preferential accumulation of BicD and Orb. dap cysts with highly polyploid oocytes have little to no preferential accumulation of BicD. By contrast, dap cysts in which the oocyte has undergone limited polyploidization frequently have BicD and Orb levels indistinguishable from wild type. As is observed in other mutants that disrupt oocyte differentiation, egg chambers that contain dap clones rarely develop beyond stage 6. In Drosophila vitellogenesis begins in stage 7 when the oocyte begins to take up large quantities of yolk. The small percentage of dap oocytes that progress far enough to take up yolk, invariably have undergone little to no polyploidization. These data demonstrate that dap is required for oocyte differentiation. In addition, they indicate that the loss of oocyte identity observed in dap clones is a direct consequence of the oocyte entering the endocycle (Hong, 2003).

To explore further the apparent loss of oocyte identity that accompanies inappropriate entry into the endocycle, the distribution of microtubules was examined in dap cysts. The preferential accumulation of Orb and BicD in the oocyte is dependent on a polarized network of microtubules that directs these, and other oocyte-specific factors, from the nurse cells to the oocyte. The disruption of this network by microtubule depolymerizing agents leads to the production of egg chambers with 16 polyploid nurse cells. In wild-type cysts, the asymmetric distribution of microtubules within the germline cyst can be visualized as a preferential accumulation of alpha-tubulin in the single oocyte, which contains the microtubule-organizing center. In dap cysts, this focus of alpha-tubulin staining is present in nonpolyploid oocytes but absent in polyploid oocytes. These data suggest that entry into the endocycle may disrupt the polarized microtubule network, which in turn blocks oocyte differentiation. However, the exact relationship between entry into the endocycle and the disruption of the microtubule network remains undefined (Hong, 2003).

In females homozygous for the hypomorphic mutation cycE01672, a fraction of egg chambers contain two cells that have oocyte-like nuclear features, such as low ploidy values, an endobody and a small DNA mass in a very large nucleus. Egg chambers that contain two oocyte nuclei have only 14 polyploid nurse cells, indicating that a cell that was destined to develop as a nurse cell has been partially transformed towards the oocyte fate. The extra oocyte nucleus (which can be distinguished from the true oocyte by its presence in a small cell that lacks signs of cytoplasmic oocyte differentiation), almost invariably is the other four-ring canal cell in the cyst. Interestingly, these transformed nuclei accumulate persistently high levels of Dap protein in a manner similar to the true oocyte. Cells with persistently high levels of Dap have low ploidy values, indicating they have either not entered the endocycle or have prematurely exited the cycle. By contrast, in wild-type egg chambers the other four-ring canal cell develops as a highly polyploid posterior nurse cell in which Dap levels oscillate. Thus, mutations in dap result in germline cysts in which all 16 cells enter the endocycle and develop as nurse cells, while a mutation in cycE has the opposite effect, resulting in two or more cells that have persistently high levels of Dap that cannot enter and/or maintain the endocycle (Hong, 2003).

To examine the relationship between cycE and dap in the regulation of the cell-cycle program of ovarian cysts, whether mutations in dap could dominantly modify the cycE01672 two oocyte phenotype was examined. Reducing the dose of the dap gene by half, results in ~2.5 fold suppression of the cycE01672 two oocyte phenotype. In wild-type egg chambers the four posterior nurse cells, which are connected to the oocyte via ring canals, have the highest ploidy values in the cyst. In cycE01672 females 37±7% of egg chambers contain a cell adjacent to the true oocyte with inappropriately low ploidy values. When a single copy of the null allele dap4 was placed in the cycE01672 background, fewer than 14±7% of egg chambers had a posterior nurse cell with a reduced DNA content. These data indicate that whether a cyst cell enters and/or maintains the endocycle is at least partially determined by the balance of CycE and Dap. In addition, they strongly suggest that, as is observed during embryogenesis, the primary target of Dap in the ovary is the CycE/Cdk2 complex (Hong, 2003).

These observations suggest a model for how the meiotic cycle and the endocycle are independently maintained within Drosophila ovarian cysts. It is proposed that the presence of high levels of the cki Dap in the oocyte (throughout the time the nurse cells are in the endocycle) persistently inhibits cyclinE-Cdk2 kinase activity and prevents inappropriate DNA replication during meiosis. Without the inhibition of cyclinE-Cdk2 kinase activity provided by high levels of Dap, the majority of dap mutant oocytes abandon the meiotic cycle and enter the endocycle with the nurse cells. Importantly, these data indicate that, as has recently been observed in mice, oocytes in prophase of meiosis I are competent to replicate their DNA. In the mouse oocyte, the inhibition of DNA replication during prophase of meiosis I may be accomplished through the downregulation of the G1 cyclins and Cdk2. In the Drosophila oocyte, the inhibition of cyclinE-Cdk2 activity by Dap achieves the same aim. In contrast to the oocyte, the nurse cells require a period when Dap levels are low to allow cyclinE-Cdk2 kinase activity to rise high enough to trigger each endocycle S phase. These low points occur during the oscillations of the Dap protein. The data indicate that it is through the differential regulation of Dap that two apparently incompatible cell cycles are stably maintained within the common cytoplasm of the ovarian cyst (Hong, 2003).

The regulatory relationship between cyclinE-Cdk2 activity and the Dap ortholog p27 suggest a feedback loop that may account for the long-term stabilization of Dap in post germarial oocytes. In mammalian cells, phosphorylation by cyclinE-Cdk2 targets the p27 protein for destruction by the proteasome. Similarly, the Dap protein contains a CDK phosphorylation consensus site (Ser205) and can be phosphorylated by mammalian cyclinE-Cdk2 in vitro. It is proposed that in early stage 1 egg chambers, the balance of cyclinE-Cdk2 activity and Dap protein is slightly different in the 15-nurse cells versus the single oocyte. In the oocyte, the balance is tipped towards the inhibitor Dap, resulting in diminished cyclinE-Cdk2 activity. Lower cyclinE-Cdk2 activity leads to a reduced rate of Dap phosphorylation and proteolysis, thereby increasing the concentration of the Dap protein. The stabilization of the Dap protein ultimately results in the permanent inhibition of cyclinE-Cdk2 activity in the oocyte. In contrast to the oocyte, in stage 1 nurse cells cyclinE-Cdk2 kinase activity reaches high enough levels to trigger the phosphorylation and subsequent destruction of the Dap protein, thus allowing endocycle progression. The above model predicts that additional proteins that are targeted for destruction by cyclinE-Cdk2 phosphorylation should be stabilized in the oocyte but not in the nurse cells. Like p27, the proteolytic destruction of CycE itself is dependent on phosphorylation by the cyclinE-Cdk2 complex. As predicted by the model, CycE is stabilized in the oocyte and accumulates to high levels as oogenesis progresses. This model allows the amplification of a slight difference in the balance of cyclinE-Cdk2 activity and Dap early in oogenesis, resulting in the two cell types of the germline cyst permanently adopting dramatically different cell cycles (Hong, 2003).

Myogenic cells, asymmetric lineages, the Notch pathway and the effects of blocking cell division in heart development

During the formation of the Drosophila heart, a combinatorial network that integrates signaling pathways and tissue-specific transcription factors specifies cardiac progenitors, which then undergo symmetric or asymmetric cell divisions to generate the final population of diversified cardiac cell types. Much has been learned concerning the combinatorial genetic network that initiates cardiogenesis, whereas little is known about how exactly these cardiac progenitors divide and generate the diverse population of cardiac cells. In this study, the cell lineages and cell fate determination in the heart have been examined by using various cell cycle modifications. By arresting the cardiac progenitor cell divisions at different developing stages, the exact cell lineages for most cardiac cell types have been determined. Once cardiac progenitors are specified, they can differentiate without further divisions. Interestingly, the progenitors of asymmetric cell lineages adopt a myocardial cell fate as opposed to a pericardial fate when they are unable to divide. These progenitors adopt a pericardial cell fate, however, when cell division is blocked in numb mutants or in embryos with constitutive Notch activity. These results suggest that a numb/Notch-dependent cell fate decision can take place even in undivided progenitors of asymmetric cell divisions. By contrast, in symmetric lineages, which give rise to a single type of myocardial-only or pericardial-only progeny, repression or constitutive activation of the Notch pathway has no apparent effect on progenitor or progeny fate. Thus, inhibition of Notch activity is crucial for specifying a myogenic cell fate only in asymmetric lineages. In addition, evidence is provided that whether or not Suppressor-of-Hairless can become a transcriptional activator is the key switch for the Numb/Notch activity in determining a myocardial versus pericardial cell fate (Han, 2003).

All mesodermal cells go through three postblastoderm cell cycles (cycle 14-16). The mesodermal cells enter the first postblastoderm division (mitosis 14) at 210 minutes of development as domain 10 (see Mitotic domains). They are the first embryonic cells to go through the second postblastoderm division at about 250 minutes. The mesodermal cells are thought to divide in an approximately synchronous fashion in the first two postblastoderm cell divisions. The third division (mitosis 16) takes place during late stage 10 to early stage 11 between 280-300 minutes. During this division, a continuous longitudinal zone that may generate the heart precursors and part of the visceral mesoderm appears as a subdomain of mitosis 16. It is likely that tinman is involved in generating this subdomain because it is specifically expressed in such a continuous longitudinal zone at stage 10 (Han, 2003).

The asymmetric cell divisions of the Eve lineage that generate the Eve paracardial cell (EPC) progenitors and muscle founders DO2 are arrested in CycA or Rca1 mutants, in which cell cycle 16 is blocked, but not in twi>dap (dacapo driven by twiGAL4) embryos, in which the subsequent division of the EPC progenitor is inhibited (cycle 17). Based on the effects of these genes in the ectoderm, it is likely that CycA mutant blocks the progression of cell cycle also in the mesoderm, but ectopic dacapo induces early exit from the cell cycle. Therefore, in CycA mutants mitosis may be blocked at a certain time point during development (such as at G2/M transition of mitosis 16), but ectopic dap may induce skipping of the last division. It has been shown that the Numb crescent in the precursor P2, which generates the DO2 founder and the EPC progenitor, appears at late stage 10 and the division happens between late stage 10 and early stage 11, consistent with this being mitosis 16 of the mesodermal cells. Therefore, it seems that many cells of the cardiac mesoderm go through three postblastoderm cell divisions (mitosis 14-16), but some of them (such as the EPC lineage) undergo an additional division (mitosis 17) (Han, 2003).

Two different models have been proposed for the mesodermal Eve lineages. One model suggests that each EPC share a progenitor with a muscle founder. The other model suggests that the two EPCs per hemisegment share a progenitor, which in turn share a progenitor with one of the muscle founder cells (DO2), whereas the other DA1 muscle founder derives from the second progenitor. The data presented in this paper strongly support the latter model. The most direct evidence derives from pan-mesodermal dap overexpression, which results in the formation of a single EPC, probably because of a block in the last division. By contrast, the first model predicts formation of either no EPC or two EPCs, clearly not what is observed. These conclusions are also supported by recent lineage tracing experiments (Han, 2003).

Recent studies have suggested that cardiac specification along the anterior-posterior axis is under the control of homeotic genes. For example, Svp myocardial cells are only present in the abdominal segments of the heart, but not in the thoracic segments, and is probably under the control of Antennapedia, which is active in these segments. In this study it was found that in addition to cell identity differences between the anterior two and the posterior heart segments, the lineage of the T3-A1 myocardial cells is also distinct. In cycle 16, blocking CycA or Rca1 mutants, the last cycle of myocardial divisions in T3-A1 is not arrested unlike the case for the posterior myocardial cells. The anterior myocardial progenitors may either undergo the last division during cycle 15 or they may be less susceptible to a loss-of-CycA-function. The first possibility is consistent with the observation that embryos with overexpression of the last division inhibitor, dap (but not cycle 16 arrested CycA mutants), exhibit a reduction of Tinman myocardial cells (TMCs) in T3-A1. Alternatively, it is possible that all T3-A1 myocardial lineages are asymmetric (as are the more posterior Svp lineages), except they all express tinman, due to the lack of svp in these two segments. Thus, blocking division in CycA mutants would not alter the number of TMC in these two segments. Recent lineage tracing data are consistent with this view, but further experiments are needed to elucidate these lineages (Han, 2003).

The second postblastoderm cell division of the mesodermal cells (mitosis 15) seems to be arrested if both CycA and CycB functions are lost. In CycA;CycB double mutants, only two of the normally six myocardial cells are formed in A2-A7 segments, one exhibiting TMC and the other Seven up myocardial cell (SMC) characteristics. Therefore, it is proposed that in each hemisegment two myocardial superprogenitors are specified: the TMC superprogenitor (TSP) divides twice symmetrically, whereas the SMC superprogenitor (SSP) first divides symmetrically and then asymmetrically. Present and previous studies suggest there are probably five progenitors in each hemisegment that give rise to 14 heart-associated cells (six myocardial and eight pericardial): the TSP gives rise to four myocardial cells (two of which are Labial mycardial cells); the SSP generates two myocardial and two SOPC (Svp and Odd co-expressing pericardial cells); the Eve-expressing pericardial cell progenitor gives rise to two EPCs; the remaining four pericardial cells, to two OPC and two LPC, probably derived from two symmetrically dividing precursors, although their lineage is not as well understood (Han, 2003).

Asymmetric divisions have been studied in the context of cell cycle progression in the Drosophila PNS and CNS. In the PNS, Notch activity is required for specification of a type I versus type II neuronal fate. When sensory organ progenitor cell division is blocked in stg- mutants, the undivided precursor adopts a type II neuronal fate, whereas in numb;stg double mutants, a type I fate is chosen. In the CNS, Notch is required for specification of the sib cell fate versus the RP2 cell fate of the GMC1 asymmetric cell division. In Rca1 mutants, the undivided GMC1 adopts a RP2 fate, whereas in numb;Rca1 double mutants, the undivided GMC1 often adopts the sib cell fate. Both experimental outcomes are analogous to what is observed in CycA mutants: the undivided P2 progenitor adopts a pericardial fate in the absence of numb function instead of a myogenic fate in a wild-type background. Taken together, these observations suggest that arrest of an asymmetric cell division leads the undivided progenitor to adopt the fate of the daughter cell that inherits Numb, and in the absence of Numb the alternative fate is chosen (Han, 2003).

Extracellular signals responsible for spatially regulated proliferation in the differentiating Drosophila eye

Spatially and temporally choreographed cell cycles accompany the differentiation of the Drosophila retina. The extracellular signals that control these patterns have been identified through mosaic analysis of mutations in signal transduction pathways. All cells arrest in G1 prior to the start of neurogenesis. Arrest depends on Dpp and Hh, acting redundantly. Most cells then go through a synchronous round of cell division before fate specification and terminal cell cycle exit. Cell cycle entry is induced by Notch signaling and opposed in subsets of cells by EGF receptor activity. Unusually, Cyclin E levels are not limiting for retinal cell cycles. Rbf/E2F and the Cyclin E antagonist Dacapo are important, however. All retinal cells, including the postmitotic photoreceptor neurons, continue dividing when rbf and dacapo are mutated simultaneously. These studies identify the specific extracellular signals that pattern the retinal cell cycles and show how differentiation can be uncoupled from cell cycle exit (Firth, 2005).

The EGFR holds R2-R5 cells in G1 phase and promotes G2/M progression of other cells during the second mitotic wave (SMW). Earlier regulation is now found to depend on longer-range signaling by the Hh, DPP, and N signals already known to drive the progression of the morphogenetic furrow. These studies exclude other models that show that Hh, Dpp, or N act indirectly by releasing other, cell cycle-specific signals from differentiating cells, or that patterned cell cycle withdrawal or reentry occur independent of extracellular signals, such as by synchronized growth. Instead, specific signals are necessary or sufficient for each aspect of cell cycle patterning (Firth, 2005).

G1 arrest ahead of the morphogenetic furrow depends on posterior-to-anterior spread of Hh and Dpp. Hh is secreted from differentiating cells, starting at column 0 in the morphogenetic furrow. Dpp is transcribed in ~6 ommatidial columns in the morphogenetic furrow in response to Hh. Cells accumulate in G1 about 16-17 cell diameters anterior to column 0, suggesting an effective range of ~13-17 cells for Hh and Dpp (Firth, 2005).

The contribution of Dpp to this cell cycle arrest is known already, but that of Hh was not suspected. Both Dpp and Hh signaling can promote proliferation in other developmental contexts (Firth, 2005).

S phase entry in the SMW depends on another signal, N. Expression of the N ligand Dl begins at the anterior of the morphogenetic furrow. The first S phase cells are detected 6-8 cell diameters more posteriorly, just behind column 0. The transmembrane protein Dl must act more locally or more slowly than the secreted Hh and Dpp proteins, to explain gaps between S phases (Firth, 2005).

Although N activity has been associated with growth through indirect mechanisms involving the release of other secreted growth factors and also regulates endocycles, this appears to be the first report of a specific role of N in G1/S in diploid Drosophila cells. Notably, deregulated N signaling contributes to at least two human cancers and is oncogenic in mice (Firth, 2005).

At the same time that N promotes S phase entry in the SMW, EGFR activity ensures that R2-R5 cells remain in G1. N is still required in the absence of EGFR, so N activity is a positive signal and is not required only to counteract EGFR activity. Instead, EGFR activity interferes with S phase entry in response to N (Firth, 2005).

Ligands for the EGF receptor are thought to be released from R8 precursor cells, although EGFR-dependent MAPK phosphorylation is detected one ommatidial column before the column where R8 precursor cells can be identified, which is in column 0. This means that EGFR activation begins after Dl expression but before S phase DNA synthesis starts. Later, ligands released from differentiating precluster cells activate EGFR in surrounding cells to permit SMW mitosis around columns 3-5 (Firth, 2005).

Hh and Dpp together promote expression of Dl and of EGFR ligands; in part, this occurs indirectly through Atonal and the onset of differentiation. EGF receptor activity also promotes Dl expression (Firth, 2005).

At least three genetic mechanisms arrest distinct retinal cells in G1. Arrest ahead of the morphogenetic furrow depends on Dpp and Hh. During the SMW, R2-R5 cells are held in G1 by EGFR, which counteracts the SMW-promoting N activity. In addition, R8 cells, which are defined by the proneural gene atonal, remain in G1 independent of EGFR. After the SMW, all cells remain in G1 indefinitely, independent of EGFR. Although cell cycle withdrawal roughly correlates with differentiation, many of the cells that arrest after the SMW are still unspecified (Firth, 2005).

Loss of rbf and dap together overcome all cell cycle blocks, even though cell differentiation continues. This redundancy indicates that Cyclin E/Cdk2 targets other than Rbf are needed for proliferation, consistent with many other studies. Dap may be regulated by EGFR in R2-R5 cells. If rbf regulates the normal SMW, where Cyclin E expression seems not to be limiting, then other E2F targets may be involved. Some cell cycle arrest can also be overridden by forced expression of Cyclin E, E2F/DP, dRef, and ORC1, or by mutation of the Cyclin A antagonist rux (Firth, 2005).

The results show that mechanisms that assure both short- and long-term arrest of retinal cells must operate upstream of (or parallel to) Rbf and Cyclin E activities. They might resemble the barriers to transformation and regeneration that exist in mammals (Firth, 2005).

Cell cycle genes regulate vestigial and scalloped to ensure normal proliferation in the wing disc of Drosophila

In Drosophila, the Vestigial-Scalloped (VG-SD) dimeric transcription factor is required for wing cell identity and proliferation. Previous results have shown that VG-SD controls expression of the cell cycle positive regulator dE2F1 during wing development. Since wing disc growth is a homeostatic process, the possibility was investigated that genes involved in cell cycle progression regulate vg and sd expression in feedback loops. The experiments focused on two major regulators of cell cycle progression: dE2F1 and the antagonist Dacapo (Dap). The results reinforce the idea that VG/SD stoichiometry is critical for correct development and that an excess in SD over VG disrupts wing growth. Transcriptional activity of VG-SD and the VG/SD ratio are both modulated by down-expression of cell cycle genes. A dap-induced sd up-regulation was detected that disrupts wing growth. Moreover, a rescue was observed of a vg hypomorphic mutant phenotype by dE2F1 that is concomitant with vg and sd induction. This regulation of the VG-SD activity by dE2F1 is dependent on the vg genetic background. The results support the hypothesis that cell cycle genes fine-tune wing growth and cell proliferation, in part, through control of the VG/SD stoichiometry and activity. This points to a homeostatic feedback regulation between proliferation regulators and the VG-SD wing selector (Legent, 2006).

Cell proliferation relies on the tight control of cell cycle genes, and, in the wing pouch, VG-SD is also critically required. Accordingly, vg up-regulates dE2F1 expression and antagonizes the CKI dap. This study investigated the effects of these two antagonistic proliferation regulators in the wing pouch of the disc, and tested the hypothesis that cell cycle genes fine-tune proliferation, through regulation of the respective expressions of vg and sd and VG-SD dimer activity, thereby providing a feedback control (Legent, 2006).

Combined loss and gain of function experiments has ascertained the requirement of a precise VG/SD ratio for normal wing development and has shown that an excess in SD disrupts VG-SD function in wing growth, and probably acts as a dominant-negative through titration of functional VG-SD dimers. Therefore, sd induction may efficiently restrain VG-SD function in vivo, and a similar effect may also be physiologically achieved down-regulating vg. Moreover, since SD DNA target selectivity is modified upon binding of VG to SD in vitro, the hypothesis cannot be discarded that, in vivo too, VG-SD targets might be different from the targets of SD alone. This could explain to some extent the phenotypes observed when sd is induced (Legent, 2006).

The results show that the CKI member DAP, homogeneously expressed in the wing disc, regulates VG-SD function. dap heterozygotes display a wild type wing phenotype, reduced levels of both vg and sd transcripts, but an almost normal vg/sd ratio, thus VG-SD activity is normal. Consistently, no abnormal wing phenotype could be detected. Therefore, the relative vg/sd stoichiometry, rather than absolute vg and sd expression levels, determines wing growth. Interestingly, it had been observed that dap homozygous mutant adult escapers display duplication of the wing margin, indicating a role of DAP at the D/V boundary. This phenotype could be linked to an enhanced proliferation due to the absence of CKI function. Moreover, D/V-specific over-expression of dap alters wing margin structures. This dap over-expression triggers both ectopic expression of sd and subsequent impairment of VG-SD activity associated with a proliferation decrease.The associated wing phenotype is clearly enhanced in vg heterozygous flies, providing evidence that dap over-expression affects VG/SD stoichiometry and represses VG-SD activity in wing development. This reveals a model in which, in the wing pouch, cell proliferation down-regulation through cyclin/CDK inhibition by DAP, may be enhanced by an additive reduction of VG-SD proliferation function. Such a mechanism probably participates in vivo in the control of balanced wing growth (Legent, 2006).

The results also demonstrate that dE2F1-DP regulates VG-SD: the dE2F1 heterozygote displays a reduced vg/sd ratio due to a decrease in vg and an increase in sd transcripts, associated with reduced dimer activity, comparable to the vgnull/+ context. Thus, dE2F1 is required for vg normal expression. This supports the hypothesis that the slower proliferation observed in these contexts is linked to an imbalance in the dimer ratio (Legent, 2006).

Conversely, over-expressing dE2F1-DP-P35, in a vg83b27 hypomorphic mutant context, rescues expression of both vg and sd and normal VG-SD function, wing appendage specification and growth. This is not observed in vgnull flies implying the necessity for vg sequences, but the second intron, missing in the vg83b27 mutant. In addition, it was ascertained that not all the genes triggering cell cycle progression or cell proliferation can induce vg expression. Neither ectopic expression of CYC E, which promotes dE2F1-induced G1/S cell cycle transition, nor the growth regulator Insulin receptor (InR) is sufficient to elicit VG expression and wing growth in the vg83b27 mutant. These results demonstrate that vg induction is a prerequisite for vg83b27 wing pouch growth in response to dE2F1 activity (Legent, 2006).

In a vg+ genetic background, dE2F1 over-expression induces only sd, disrupting VG/SD stoichiometry. Consistently, at the D/V boundary, wing notching was observed. Therefore, although dE2F1 basically displays a positive role in proliferation, this sd induction in response to dE2F1 over-expression is clearly associated with wing growth impairment. This effect is significantly weaker in a vg heterozygote background, and a rescue of the wing phenotype was observed, supporting the hypothesis that VG/SD stoichiometry is restored. Therefore, sd induction by dE2F1 depends on the vg genetic context. This indicates that the effects of over-expressing dE2F1 differ depending on the growth-state of the wing pouch, which is tightly linked with the vg genotype (Legent, 2006).

Clearly, feedback regulations rule the growth of the wing disc. Regulation has been noted in three different vg genetic contexts that can be analyzed in the light of a homeostasis hypothesis. In the vg83b27under-proliferative wing pouch, ectopic dE2F1 expression coordinately increases vg and sd expression in a positive feedback loop. This triggers VG-SD activity, and induces both cell proliferation and wing specification in the mutant. Conversely, no such crosstalk occurs in a correctly grown vg+ disc, where over-growth should be prevented. In this latter case, sd induction (VG/SD decrease) probably restrains the proliferation function of dE2F1. Consistently, wings were not overgrown, but notches were observed. This phenotype was partially suppressed in a vg heterozygote background. As a whole, these results support the hypothesis that VG-SD/dE2F1 coordination tends to ensure normal wing growth and that the dimer does not trigger unrestricted cell proliferation in a vg+ context, since an excess in dE2F1 attenuates VG-SD function in a negative feedback loop. Thus, molecular interactions between dE2F1, vg and sd, display a clear plasticity depending on the vg genetic context (Legent, 2006).

Establishing and maintaining homeostasis is critical during development. This is achieved in part through a balance between cell proliferation and death. In mammals E2F1 and p21, the dacapo homolog, play a key role in this process. In the wing disc compensatory proliferation induced by cell death has been observed. However, the role of cell cycle genes in this process has not yet been established. How patterns of cell proliferation are generated during development is still unclear. It seems nevertheless likely that the gene responsible for regulating differentiation also regulates proliferation and growth. For instance, Hedgehog (HH) induces the expression of Cyclins D and E. This mediates the ability of HH to drive growth and proliferation. In the same way, other data support a direct regulation of dE2F1 by the Caudal homeodomain protein required for anterio-posterior axis formation and gut development. Wingless (WG) also displays both patterning and a cell cycle regulator function during Drosophila development (Legent, 2006).

Growth control in the wing pouch seems to be achieved through both positive and negative feedback regulations linking dE2F1 and VG-SD, but also via additive impairment of VG-SD by DAP. In fact, in a vg+ background, over-expression of both dap and dE2F1 induces sd, impairs VG-SD and alters wing development. Nevertheless, clear opposite behaviors are observed in vgnull/+ flies where dap-induced nicks are enhanced, while those of dE2F1 are partially rescued. This highlights the functional discrepancy between these two types of feedback regulation. It is suggested that dap expression inhibits cell proliferation through a process involving both Cyclin-CDK inhibition and VG-SD impairment in the wing pouch. In contrast, it is proposed that dE2F1 over-expression triggers a homeostatic response. It will either induce vg and sd to ensure proliferation (in a vg83b27 genotype), or decrease the VG/SD ratio in a vg+ context. In this latter genotype, down-regulation probably counteracts fundamental proliferative properties of dE2F1 and governs homeostatic wing disc growth (Legent, 2006).

At late third instar, wing discs display a Zone of Non-proliferating Cells (ZNC) along the wing pouch D/V boundary. It has been shown that, although dE2F1-DP is expressed in this area, its proliferative function is inactivated late, because of RBF1-induced G1 arrest. Accordingly, although expression of vg and sd presents a peak at the D/V boundary, in late third instar, VG-SD activity is decreased in D/V cells, and it was suggested to result from an excess of SD. Therefore, the ZNC setting may also reflect a VG-SD/dE2F1 coordinated dialogue that triggers a decrease in proliferation signals in this area (Legent, 2006).

Previous studies of homeostatic control of cell proliferation in the wing reported that, to some extent, over-expression of positive or negative cell cycle regulators only weakly affects the overall division rate. For instance, although dap over-expression alters dE2F1 function in G1-S cell cycle transition, it also promotes dE2F1 expression and function in G2-M transition, preventing a decrease in the overall rate of cell division. Strikingly, the cells seemed able to monitor each phase length and maintain cell cycle duration and normal proliferation in the wing pouch of the disc. Therefore, dE2F1 is a central component that enables cells to ensure normal proliferation in the wing disc and prevents imbalance in the process. The fact that dE2F1 triggers quite different or opposite responses in vg+ or vg hypomorphic contexts suggests that the VG-SD/dE2F1 crosstalk plays a role in the same sort of homeostatic process that ensures entire wing growth (Legent, 2006).

Such regulations are likely to reveal a precise physiological fine-tuning of vg and sd by cell cycle effectors, promoting an exquisite control of wing growth. Feedback loops between the developmental selector VG-SD and cell cycle effectors may stand for a control mechanism to guarantee that the tissue can sustain balanced growth and a reproducible size. Such a subtle mechanism, on a local scale, would correct the alterations in cell proliferation that may occur during development (Legent, 2006).

dacapo: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | References

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