Effects of Mutation or Deletion

Table of contents

Notch and tracheal development

The Drosophila tracheal system consists of a stereotyped network of epithelial tubes formed by several tracheal cell types. By the end of embryogenesis, when the general branching pattern is established, some specialized tracheal cells then mediate branch fusion, while others extend fine terminal branches. Evidence is presented that the Notch signaling pathway acts directly in the tracheal cells to distinguish individual fates within groups of equivalent cells. Notch helps to single out those tracheal cells that mediate branch fusion by blocking their neighbours from adopting the same fate. This function of Notch would require the restricted activation of the pathway in specific cells. In addition, and probably later, Notch also acts in the selection of those tracheal cells that extend the terminal branches. Both the localized expression and the mutant phenotypes of Delta, coding for a known ligand for Notch, suggest that Delta may activate Notch to specify cell fates at the tips of the developing tracheal branches (Llimargas, 1999).

Zygotic N mutant embryos show abnormalities in most of the ectodermal derivatives, including the tracheal system, where some of the tracheal cells are converted into neuroblasts; the embryos exhibit only rudimentary branches, most probably due to the loss of tracheal cells. In addition, abnormalities in fusion and terminal branching are also detected when luminal or cellular markers are used. These phenotypes could be due to early requirements for the N pathway before tracheal fate is allocated. To detect late N requirements, Nts mutant embryos (Nts over a null N allele) were shifted to the restrictive temperature at stage 11. The terminal branching and fusion phenotypes produced resemble those observed in zygotic null N mutants, yet the defects in the primary branching pattern are milder. The phenotypes observed both in Nts combinations and in null N mutants suggest that, in addition to its early role in tracheal specification, N acts later in both fusion and terminal branching programs (Llimargas, 1999).

To analyse the requirements of N in fusion and terminal branching, the identity of the tracheal cells was studied using the expression of several tracheal markers. In wild-type embryos, tracheal cells that mediate branch fusion express the fusion markers; a single fusion cell is found at the tips of the following branches: DB, lateral trunk anterior (LTa), lateral trunk posterior (LTp), dorsal trunk anterior (DTa) and dorsal trunk posterior (DTp). Conversely, the antifusion markers are generally expressed in all other tracheal cells. Finally, the terminal markers are expressed in about 20 cells per tracheal metamere. Those are the cells, either clustered or isolated, that will extend terminal branches from most primary branches except the DT (Llimargas, 1999).

Zygotic null mutants of N show abnormal patterns for these molecular markers. In the mutants, Fusion-1 (escargot), Fusion-2 and Fusion-3 (two unidentified enhancer traps used as tracheal markers) are ectopically expressed in almost all the cells forming the rudimentary DB, DT and LT. The expression of Terminal-1 (pruned) is found mainly in some cells along the transverse connective (TC) and the visceral branches (VB), but almost never in the DB. Antifusion-1 and Antifusion-2 (two unidentified tracheal markers) are consistently expressed only in the TC. Similarly, the overexpression of Hairless (H), a Notch signaling antagonist, in the tracheal cells also causes changes in the expression of these tracheal markers. In most embryos, there is an increase in the number of cells expressing fusion markers: DBs contain up to four cells expressing Fusion-1 and Fusion-2 as compared to one cell in the wild type; and LT and DT contain up to four fusion cells, as compared to two cells in normal conditions. Conversely, there is a decrease in the number of cells expressing Terminal-1, mainly in the DBs and in the GBs, which in wild-type embryos contain a single cell expressing the terminal markers. The pattern of expression of Antifusion-1 is similar to wild type, although there is one fewer cell on average in DBs expressing the marker. Cell counts in the DBs of wild type and of embryos overexpressing H indicate that there is no change in the number of cells when N function is reduced. This suggests that the extra fusion cells observed may arise by a transformation of presumptive terminal or antifusion cells into fusion cells. The formation of ectopic terminal branches observed sporadically when overexpressing H correlates with the expression of Terminal-1 in more than one cell in some DBs and GBs. The presence of extra terminal branches suggests a second N role in selecting the correct number of terminal cells. Engineered forms of the N protein that eliminate the extracellular domain are active in the absence of ligand. If N is required for the execution of terminal branching and fusion programs, a constitutively activated form of N could cause a transformation that is the opposite of the loss of N function, and indeed this was found to be true (Llimargas, 1999).

Cells expressing fusion and terminal markers arise from the tips of the primary branches. At early stages of tracheal morphogenesis, broad domains at the tips of each budding primary branch express the pantip markers. Their pattern of expression suggests that the tip cells are apparently equivalent before adopting individual fates. The N pathway affects the early diversification of the tip cells. pointed (pnt, Pantip-1) and sprouty (spry, Pantip-2) are expressed in each DB in about 3 to 4 cells at stages 12 and 13; at stage 13/14, high levels are found in the two tip cells and at later stages expression is restricted to the cell expressing the terminal markers. Under N loss-of-function conditions, the expression of pnt and spry in the DBs is frequently lost from stage 13 while under N gain-of-function conditions high levels of pnt and spry are maintained in more than one cell at stage 16. This suggests that the activity of N allows the tip cells to diversify. This also suggests a possible repression of these pantip markers by the fusion genes, since their expression decays in the cells expressing the fusion factors (Llimargas, 1999).

How is the activity of N regulated in the tracheal system? Dl has been shown to act as a ligand for N. Zygotic null N mutants have a weaker tracheal phenotype than that of null Dl mutants, possibly because the maternal N product rescues the zygotically mutant embryos. The tracheal branches in Dl mutants are so truncated that it is not possible to determine effects on fusion or terminal branching. The overexpression of Dl driven by the breathless-Gal4 line affects fusion and terminal branching in ways similar to both the gain and loss of N function. Thus, ectopic fusions and unfused branches are observed with variable frequency, as well as missing or extra terminal branches. These phenotypes correlate with both the lack and the excess of cells expressing fusion and terminal markers. The expression pattern of Dl in the tracheal cells was studied in detail using both in situ hybridization and antibody staining to further clarify Dl function. The Dl protein accumulates in vesicles at higher levels in the tip cells of the primary branches from stage 12 to 13. The localized accumulation of the protein is coextensive with a localized expression of the gene in the tip cells. At later stages, large amounts of transcripts and protein are found in the DT. The early accumulation of Dl depends on N activity. Dl accumulates at low levels at the tips of the branches in embryos with constitutively active N, while in null N mutants, there is a broader accumulation of Dl protein. The coincidence of the early Dl expression with the activity of the N pathway and the phenotypes of Dl overexpression, suggests that Dl activates N to diversify the tip cells of the primary branches (Llimargas, 1999).

A model is presented for the establishment of tracheal fates at the tips of the branches. bnl, coding for the FGF ligand, is expressed in clusters of cells outside the tracheal system and, in addition to guiding branch migration, it is necessary to pattern the tips of the primary branches. Bnl activates the Breathless receptor in a gradient, leading to the expression of the pantip, terminal and fusion markers. Two pieces of evidence indicate that the tip cells are initially equivalent: (1) they all express the pantip markers in response to bnl and, (2) they can all behave in the same way in different mutant backgrounds. The expression of the pantip markers becomes restricted to those cells at the leading ends of the branches, since the Breathless (Btl) activity is higher there due to the proximity of the Bnl source. Among these tip cells, some become fusion cells while others can differentiate as terminal cells, even though they all receive similar amounts of Bnl. Therefore, the bnl/btl pathway is not sufficient to account for the diversification of the tip cells, and it is proposed that the N signaling acts to achieve this. The pnt gene (Pantip-1) has been shown to repress the fusion markers, yet even though the tip cells all express pnt, only one of them acquires the fusion fate. A possible resolution of this paradox might be that there is a balance between the bnl/btl pathway, which promotes the fusion fate, and the N signal and pnt, which represses it. The N pathway shifts this balance: its presumed inactivity in one cell would allow that cell to overcome the repression by pnt, while its activity in the remaining cells would allow them to overcome the activation by bnl/btl. It is possible that biasing differences in the tip cells are also required to shift the balance. Once the fusion cell is specified, the expression of some pantip markers decays in that cell, suggesting that the fusion factors repress the pantip genes. A piece of evidence supports this: in N mutants, where all the cells express the fusion markers, Pantip-1 and Pantip-2 expressions are extinguished. Simultaneously, the fusion cell seems to signal via Dl to its neighbors to repress the fusion genes, allowing them to express the antifusion or the terminal genes. The data suggest that the fusion genes repress the antifusion ones, because in the wild type the antifusion and the fusion genes are expressed in complementary patterns and, in null N mutants (where all the cells express the fusion markers), the antifusion genes are repressed. Similarly, the specification of the terminal fate also depends on a balance between inductive signals, mediated by bnl/btl and the pantip genes, and repressive signals, mediated by N, spry and hdc. Cells closest to the Bnl source that also express high levels of pantip markers and that do not acquire the fusion fate, can become terminal cells. The remaining cells receive negative signals from the already specified terminal cell (mediated by Dl and spry) and from the also specified fusion cell (mediated by the novel protein Headcase, the Fusion 6 marker) to repress the terminal fate. Thus, the acquisition of fates among a group of cells located at the tips of each primary branch depends on the integration of positive and negative effects from different signaling pathways (Llimargas, 1999 and references).

The patterned branching in the Drosophila tracheal system is triggered by the FGF-like ligand Branchless (Bnl) that activates a receptor tyrosine kinase Breathless (Btl) and the MAP kinase pathway. A single fusion cell at the tip of each fusion branch expresses the zinc-finger gene escargot, leads branch migration in a stereotypical pattern and contacts with another fusion cell to mediate fusion of the branches. A high level of MAP kinase activation is also limited to the tips of the branches. Restriction of such cell specialization events to the tip is essential for tracheal tubulogenesis. Notch signaling plays crucial roles in the singling out process of the fusion cell. Notch is activated in tracheal cells by Branchless signaling through stimulation of Delta (Dl) expression at the tips of tracheal branches and activated Notch represses the fate of the fusion cell. In addition, Notch is required to restrict activation of MAP kinase to the tips of the branches, in part through the negative regulation of Branchless expression. Notch-mediated lateral inhibition in sending and receiving cells is thus essential to restrict the inductive influence of Branchless on the tracheal tubulogenesis (Ikeya, 1999).

Six primary branches form in the tracheal primordia, among which the dorsal branch (DB), anterior and posterior dorsal trunk (DTa, DTp), and anterior and posterior lateral trunk (LTa, LTp) migrate along a stereotyped path to be connected with other branches from adjacent primordia. These fusion branches are capped with fusion cells that express Esg. The remaining visceral branch (VB) migrates to reach the internal organs. Terminal cells expressing Drosophila serum response factor (DSRF) are formed in each primary branch except in DTs and later differentiate multiple tracheoles. High expression of DL mRNA and protein is expressed in the DT of stage-15 embryos. Cells in the tracheal primordium just after invagination expressed Dl uniformly. At early stage 11, Dl expression started to be elevated in 2-3 cells at the tip of the branches in which outgrowth had begun and the number of the Dl-expressing cells was reduced to one at late stage 11. At stage 14, high Dl expression remains only in the DT, which has completed fusion. Ser protein also accumulates at the apical side of the DT cells at the same stage. In the case of the trachea, the level of N protein expression remains uniform, suggesting that the expression level of Dl, or its potentiation, must be crucial for N activation. An esg-lacZ reporter is initially expressed in 2-3 cells at the tip of the fusion branches in mid stage 11 embryos, and is downregulated to be maintained in only a single cell at the tip of each fusion branch at late stage 11. These cells also express a high level of Dl. Therefore, localized elevation of Dl expression in stage 11 correlates well with the selection process of a single fusion competent cell (Ikeya, 1999).

When tracheal cells became unresponsive to Bnl due to btl mutation, no sign of primary branching and Dl upregulation is observed. On the contrary, when Btl is hyperactivated by overexpression of Bnl in all the tracheal cells, primary branching is severely inhibited and Dl expression is elevated. These results suggest that elevation of Dl expression is triggered by the external signal Bnl. The results also suggest that the N/Dl pathway may mediate the Bnl signal to control cell migration and cell fate decisions (Ikeya, 1999).

In Nts1 embryos grown under non-permissive condiditons a misrouting defect was observed in DB, which normally elongates to the dorsal midline where it meets its counterpart from the other side of the metamere. DBs are often curved in the anteroposterior direction and make contact with the tip of DB from the same side. The misrouted DBs accumulate a luminal component detectable by 2A12 antibody at the ectopic contact sites, but do not appear to fuse properly. Cell migration defect is also observed in DB. DB consists of a total of 5-7 cells in the case of Tr5, of which two specialized cells are located at the tip. One is the terminal cell, from which a thin terminal branch sprouts: the other is the fusion cell. The remaining stalk cells are located between the tip and DT at regular intervals. In Nts1 mutants, the number of cells at the tip is increased with a corresponding decrease in the number of stalk cells, the latter having become unusually elongated. The total number of cell nuclei do not change compared to the controls, so no additional mitosis occurs. This cell migration phenotype suggests that stalk cells and terminal cells acquire a property of fusion cells to become localized at the tip of DB. Since Esg represses DSRF expression and terminal branching, the loss of terminal branching in Nts1 embryos may be the consequence of ectopic Esg expression. Similar defects in Esg and DSRF expression are also observed in null N mutants. These results suggest that in N minus embryos, several terminal and stalk cells are recruited to the fate of fusion cells (Ikeya, 1999).

A three-step model is presented for tubule formation in fusion branches. During induction, exogenously supplied Bnl activates its receptor Btl in equivalent tracheal cells where N is inactive. The signal is transduced by activation of MAPK to stimulate Dl expression. The expression of Bnl is also regulated negatively by N signaling. During lateral inhibition, induced Dl activates N in neighboring cells, which in turn, represses esg transcription. N may also repress Dl expression. However, a high level of Dl inhibits N signaling in a cell autonomous manner, allowing activation of esg and MAPK. A small difference in the response to Bnl within equivalent tracheal cells is amplified to select out a single fusion cell with a high level of Dl and esg expression. During tubule formation, the fusion cell becomes the only cell that responds to Bnl and becomes motile. Maintenance of Btl activity by Bnl would limit the migration toward the source of Bnl. Other tracheal cells follow fusion cells to become stalk cells (Ikeya, 1999).

Decapentaplegic (Dpp) signaling determines the number of cells that migrate dorsally to form the dorsal primary branch during tracheal development. Dpp is expressed in dorsolateral epidermal clusters located near the tips of the outgrowing dorsal branches. The Dpp receptor, Thick veins, is expressed in all tracheal cells during embryogenesis and is required for in dorsal branch outgrowth ectopic activation of fusion markers in cells of the dorsal branch. Dpp signaling is required for the differentiation of one of three different cell types in the dorsal branches, the fusion cell. In Mad mutant embryos or in embryos expressing dominant negative constructs of the two type I Dpp receptors in the trachea the number of cells expressing fusion cell-specific marker genes is reduced and fusion of the dorsal branches is defective. Ectopic expression of Dpp or the activated form of the Dpp receptor Tkv in all tracheal cells induces ectopic fusions of the tracheal lumen and ectopic expression of fusion gene markers in all tracheal branches. Delta is among the fusion marker genes that are activated in the trachea in response to ectopic Dpp signaling. In conditional Notch loss of function mutants, additional tracheal cells adopt the fusion cell fate. Ectopic expression of an activated form of the Notch receptor in fusion cells results in suppression of fusion cell markers and disruption of the branch fusion. The number of cells that express the fusion cell markers in response to ectopic Dpp signaling is increased in Notch ts1 mutants, suggesting that the two signaling pathways have opposing effects in the selection of the fusion cells in the dorsal branches (Steneberg, 1999).

Many organs are composed of tubular networks that arise by branching morphogenesis in which cells bud from an epithelium and organize into a tube. Fibroblast growth factors (FGFs) and other signalling molecules have been shown to guide branch budding and outgrowth, but it is not known how epithelial cells coordinate their movements and morphogenesis. Genetic mosaic analysis has been used in Drosophila to show that there are two functionally distinct classes of cells in budding tracheal branches: cells at the tip that respond directly to Branchless FGF and lead branch outgrowth, and trailing cells that receive a secondary signal to follow the lead cells and form a tube. These roles are not pre-specified; rather, there is competition between cells such that those with the highest FGF receptor activity take the lead positions, whereas those with less FGF receptor activity assume subsidiary positions and form the branch stalk. Competition appears to involve Notch-mediated lateral inhibition that prevents extra cells from assuming the lead. There may also be cooperation between budding cells, because in a mosaic epithelium, cells that cannot respond to the chemoattractant, or respond only poorly, allow other cells in the epithelium to move ahead of them (Ghabrial, 2006).

Because small differences in btl dosage or activity affect a cell's ability to compete for the lead, whether lateral inhibitory mechanisms that amplify small differences in signalling might be operative was investigated. Data suggest that the Notch pathway, a lateral signalling pathway implicated in cell specification events including cell fate determination at tracheal branch tips, also affects cell arrangement. Nts embryos shifted to the restrictive temperature during budding formed branches in which most DB cells behaved like lead cells, resulting in large clusters of cells congregated at the lead position, whereas expression of constitutively active NACT throughout the tracheal epithelium had the opposite effect, arresting outgrowth and stalling cells near the base of the branch. It is proposed that Notch-mediated lateral inhibition among tracheal cells prevents extra cells from assuming the lead position (Ghabrial, 2006).

These results provide evidence for social stratification and dynamic social interactions between epithelial cells during branching morphogenesis. First, the results show that budding cells are functionally specialized. A cell at the branch tip requires btl and leads outgrowth towards the Bnl signalling center. Trailing cells do not require btl but nevertheless follow the lead cell towards the Bnl source. Because tracheal cells do not migrate or form tubes in btl-/- animals, trailing cells must receive a secondary signal generated by the lead cell that induces them to migrate and also activates their tubulogenesis program. This could be a secreted molecule or physical stimulus such as pulling or stretching the trailing cells (Ghabrial, 2006).

Second, these roles are not pre-specified. Rather, there is competition between cells such that those with high Btl FGFR activity become lead cells whereas those with less or no btl FGFR activity become trailing cells and form the branch stalk. Competition appears to involve Notch-mediated lateral inhibitory signalling between tracheal cells, and it may also be influenced by positive feedback mechanisms such as increased activation and expression of Btl as cells approach the Bnl source. Third, there may be cooperation between cells, because in a genetically mosaic epithelium, tracheal cells with less Btl activity allow those with more activity to move ahead of them (Ghabrial, 2006).

There may be similar social interactions between budding cells in other branching organs. Studies of other branching processes have identified genes selectively expressed in tip cells of budding branches, and in some cases these cells display morphological specializations indicating that they might actively lead outgrowth. However, because most budding branches contain hundreds or thousands of cells, it is difficult to track and manipulate individual cells to investigate social behaviours like those described here. Recent analyses of chimaeric Ret+/Ret- mouse renal ureteric buds in culture and btl mosaic air sacs reveal that cells lacking these receptor tyrosine kinases are excluded from branch tips, indicating that RTK-dependent interactions similar to those described here might be operative in more complex branching events (Ghabrial, 2006).

Notch and hindgut development

The Drosophila hindgut develops three morphologically distinct regions along its anteroposterior axis: small intestine, large intestine and rectum. Single-cell rings of 'boundary cells' delimit the large intestine from the small intestine at the anterior, and the rectum at the posterior. The large intestine also forms distinct dorsal and ventral regions; these are separated by two single-cell rows of boundary cells. Boundary cells are distinguished by their elongated morphology, high level of both apical and cytoplasmic Crb protein, and gene expression program. During embryogenesis, the boundary cell rows arise at the juxtaposition of a domain of Engrailed- plus Invected-expressing cells with a domain of Delta (Dl)-expressing cells. Analysis of loss-of-function and ectopic expression phenotypes shows that the domain of Dl-expressing cells is defined by En/Inv repression. Further, Notch pathway signaling, specifically the juxtaposition of Dl-expressing and Dl-non-expressing cells, is required to specify the rows of boundary cells. This Notch-induced cell specification is distinguished by the fact that it does not appear to utilize the ligand Serrate and the modulator Fringe (Iwaki, 2002).

At its anterior, the hindgut joins the posterior midgut; at its posterior, it forms the anus. Along this AP axis, the hindgut of the mature embryo consists of three morphologically distinct domains: the wide, looping small intestine, the long and narrow large intestine, and the tapered rectum. Beginning at stage 13, these domains are demarcated at their junctions by rings of unusually high accumulation of the apical surface protein Crumbs (Crb). The ring at the small intestine/large intestine junction is designated the anterior boundary cell ring, and the ring at the large intestine/rectum junction is designated the posterior boundary cell ring (Iwaki, 2002).

Patterning of the hindgut in the DV axis is detected at stage 10 (germ band extension) when the hindgut develops an interiorly directed (dorsal) convexity. The side of the hindgut closest to the interior of the embryo is dorsal and expresses both En and Inv; that closest to the exterior is ventral and expresses dpp. By the completion of germ band retraction, the convexity at the anterior of the hindgut has shifted toward the left side of the embryo. Thus at the anterior of the hindgut, the initially dorsal, En- and Inv-expressing side comes to lie on the outer (left-facing) curve, while the initially ventral, Dpp-expressing side of the hindgut comes to lie on the inner (right-facing) curve; the DV relationship is retained at the posterior connection to the rectum. These initially DV patterned domains of the large intestine persist to the end of embryogenesis and into the larval stages; they are referred to as large intestine dorsal (li-d) and large intestine ventral (li-v). At each of the two boundaries between li-d and li-v, there is a single row of cells with high levels of Crb expression running the length of the large intestine, from the anterior boundary cell ring to the posterior boundary cell ring. These are designated the 'boundary cell rows'. In addition to their high level of Crb expression, the boundary cell rows and rings express the nuclear protein Dead ringer (Dri). Double antibody staining reveals that boundary cell rows at the border of the En/Inv-expressing li-d domain and the Dpp-expressing li-v domain express Dri in their nuclei and have strong Crb expression at their apical surfaces (Iwaki, 2002).

In addition to expressing Dpp, the li-v domain expresses the Notch ligand Delta (Dl); Dl is also expressed in the anterior of both the rectum and the small intestine. Fringe (Fng), a modulator of Notch signaling, is expressed opposite Dl in the Drosophila wing and eye; in the hindgut, Fng is expressed in li-d and the boundary cell rows, opposite the domain of Dl expression in li-d (Iwaki, 2002).

The boundary cell rows form at the junction of the li-d and li-v domains, which express different genes. To investigate whether the spatially restricted gene expression observed in these domains is essential for establishment of boundary cell rows, embryos homozygous for loss-of-function alleles of en, inv, dpp, dri, Dl, Ser, Notch, or fng were examined. The presence or absence of boundary cells was assessed by anti-Crb staining, since this delineates their characteristic morphology, and also detects one of their unique differentiated features (i.e. the cytoplasmic accumulation of Crb) (Iwaki, 2002).

Embryos lacking Dl function are extremely deformed and do not always have a recognizable hindgut, indicating that function of Dl early in embryogenesis is required to establish and/or maintain the hindgut. Since Dl encodes a ligand for Notch, embryos lacking the zygotic contribution of Notch were examined. Strikingly, Notch mutant hindguts completely lack both boundary cell rows and rings, revealing that Notch signaling is required to establish the boundary cells. The data demonstrate that formation of the boundary cell rows at the border of Dl expression requires the Notch receptor; however, Fng does not appear to be required for this process (Iwaki, 2002).

To further investigate the required role of Dl in establishing the boundary cells, a dominant-negative form of Dl was expressed throughout the hindgut. bynGal4:UAS-Dl.DN embryos show a complete absence of boundary cell rows and rings; this phenotype closely resembles that seen in Notch loss-of-function embryos. Expression of a dominant negative Notch receptor throughout the hindgut results in a similar absence of boundary cell rows and rings. Furthermore, bynGal4 driven expression of UAS-Hairless, which acts to suppress activity of Su(H) also results in an absence of boundary cells. This last result indicates that the Notch signaling required to establish the boundary cells must act through Su(H). In summary, the above results demonstrate required roles in boundary cell specification of the following Notch pathway components: the ligand Dl, the receptor Notch, and the downstream transcription factor Su(H). It is therefore concluded that the Notch signaling pathway is required for boundary cell induction (Iwaki, 2002).

An intriguing observation, given the demonstrated role of the LIN-12/Notch signaling pathway in generation of left¯right asymmetry in the Caenorhabditis elegans intestine is that a large portion of 455.2Gal4:UAS¯Su(H)VP16 hindguts display a reversal of left¯right looping (Iwaki, 2002).

Ectopic expression experiments, taken together with the loss-of-function experiments, demonstrate that establishment of the boundary cell rows requires the juxtaposition of Dl-expressing and Dl-non-expressing cells and signaling via Notch and Su(H). In addition to Notch and spatially restricted Dl, establishment of the anterior ring requires localized activity of Dpp; the posterior ring requires En/Inv activity (which does not need to be localized) and the localized activity of Dl (Iwaki, 2002).

Since the experiments described in the preceding sections show that both spatially localized En/Inv and a boundary of Dl expression are required to establish the boundary cells, it was asked whether En/Inv might control the boundary of Dl expression. In Df(enE) embryos, Dl is not restricted to li-v, but rather is uniform in the hindgut circumference, indicating that en/inv is required to repress Dl. In the large intestine, uniform expression of En/Inv results in an absence of Dl expression. Expression of En/Inv in li-d is thus both necessary and sufficient to restrict Dl expression to li-d. While it represses Dl throughout the large intestine, ectopic En/Inv does not affect Dl expression in the rectum. Embryos with ectopic En/Inv not only express Dl at the anterior of the rectum, they also form the posterior boundary cell ring. Thus a boundary of Dl-expressing with Dl-non-expressing cells is required not only to establish the boundary cell rows but also likely to establish the posterior ring; the posterior ring also requires En/Inv activity, but this activity does not need to be localized (Iwaki, 2002).

Consistent with observations that En and Inv are repressors with the same targets, the data presented in this study demonstrate that Dl expression in the large intestine is restricted to the li-v domain by the repressive activity of En/Inv in li-d (Iwaki, 2002).

The data presented here support the following model. En/Inv is expressed in li-d and represses Dl in that domain; Dl expression is thereby restricted to the li-v domain. At the li-v/li-d transition, the Dl-expressing cells induce, by Notch signaling, a row of Dl-non-expressing cells to become a boundary cell row. Since En/Inv is not detected in differentiated boundary cells, Notch activation likely represses En/Inv expression. Notch activation also leads to Dri expression and an upregulation of Crb expression. While all of these transcriptional changes could be mediated by Su(H), they could also be further downstream (Iwaki, 2002).

In summary, three steps in the establishment of the Drosophila hindgut boundary cell rows are similar to steps characterized in other Notch dependent boundary-forming systems. (1) A homeodomain transcription factor (En/Inv in the case of the boundary cells) is expressed on one side of the forming boundary; (2) this transcription factor defines two domains, one which expresses Dl and one which does not; (3) Notch activation in the Dl-non-expressing cells that confront Dl-expressing cells leads to a unique cell fate (Iwaki, 2002).

Notch and myogenesis

Drosophila muscles originate from the fusion of two types of myoblasts -- founder cells (FCs) and fusion-competent myoblasts (FCMs). To better understand muscle diversity and morphogenesis, a large-scale gene expression analysis was performed to identify genes differentially expressed in FCs and FCMs. Embryos derived from Toll10b mutants were employed to obtain primarily muscle-forming mesoderm, and activated forms of Ras or Notch were expressed to induce FC or FCM fate, respectively. The transcripts present in embryos of each genotype were compared by hybridization to cDNA microarrays. Among the 83 genes differentially expressed, genes known to be enriched in FCs or FCMs, such as heartless or hibris, previously characterized genes with unknown roles in muscle development, and predicted genes of unknown function, were found. These studies of newly identified genes revealed new patterns of gene expression restricted to one of the two types of myoblasts, and also striking muscle phenotypes. Whereas genes such as phyllopod play a crucial role during specification of particular muscles, others such as tartan are necessary for normal muscle morphogenesis (Artero, 2003).

The Toll10b mutation gives rise to embryos composed primarily of somatic mesoderm. In these embryos FCs and FCMs are readily detected, and they respond to the Ras and Notch signaling pathways in the same way as their wild-type counterparts. Advantage was taken of this fact to enrich Toll10b mutant embryos for FCs or FCMs, which allowed a concentration on the transcription in these two specific cell types within the context of the entire embryo. Genes known to be expressed and regulated in FCs or FCMs emerged from the screen in the proper categories. Not all known FC/FCM genes were detected in the screen for several reasons: the high stringency set for interpretation of the array data; the presence of only about one-third of the genome on the arrays; the loss of Dpp in the Toll10b background, and the specific window of myogenesis (5- to 9-hours) that was the focus of this investigation. However, a plethora of potential new muscle regulators were uncovered, including known genes with no previously recognized function in the mesoderm (such as phyl and asteroid), and genes predicted from the Drosophila genome sequence but not previously analyzed (Artero, 2003).

Various tests were applied to ascertain the validity of the results. Available databases were analyzed to find evidence that the known and predicted genes were expressed at the correct time and place. In addition, Northern analysis with eleven genes tested the reliability of the microarray detection and selection criteria; the results from all genes tested agreed with the array data (Artero, 2003).

A Toll10b sample on the Northern blots allowed ascertainment of why a gene is enriched in a particular condition. For example, in the case of FC enriched genes, the signal in the Ras and Notch lanes can be compared with Toll10b alone to determine whether the Ras/Notch ratio for a gene is due to activation by Ras or repression by Notch. Those genes that are 'enriched under Notch conditions', for example, could reflect a variety of transcription mechanisms that would result in a ratio of less than 0.6. By Northern analysis, many of the 'Notch-regulated' genes, and hence the predicted FCM genes, were found to be repressed by Ras signaling and slightly activated by Notch. As a case in point, hibris is induced by Notch (2-fold) and repressed by Ras (10-fold), both by Northern analysis and by in situ hybridization in embryos (Artero, 2003).

A combination of in situ hybridization, immunostaining and confocal microscopy was used to verify that the differential expression changes that were observed in these overexpression embryos reflected true differential expression in the wild-type situation. The expression of nine genes from different functional categories was analyzed. For seven of these, expression was detected in the predicted type of myoblast. For two, asteroid (ast) and cadmus, no specific staining in embryos was detected by in situ hybridization. For those genes that fell into the category of 'specific role in muscle development uncertain', in situ hybridization of several (28%) showed expression in tissues other than somatic mesoderm that are present in the Toll10b background. These genes changed their expression levels in response to Ras or Notch, and may be Ras and Notch targets in non-mesodermal tissues (Artero, 2003).

The most stringent test, mutational analysis, was applied to a set of genes for which mutations are available. Preliminary analyses of another four FCM-enriched genes was carried out: Elongation factor Tu mitochondrial (EfTuM), Glutamine synthetase 1, cadmus and parcas. All four mutants have muscle defects, including muscle losses and aberrant muscle morphologies. Thus all the genes tested show some muscle defect, supporting the usefulness of the genetic and genomic approach (Artero, 2003).

Taken together, these data suggest that the majority of genes detected play important roles in FCs or FCMs during muscle development. Some of these genes might not have been found in traditional forward genetic screens because of partial or complete genetic redundancy. The data complement traditional forward genetic approaches for finding genes crucial for muscle morphogenesis (Artero, 2003).

Each of the thirty FCs per abdominal hemisegment is hypothesized to produce its own unique combination of transcriptional regulators, though the evidence for this is limited. In turn the combination of regulators would control the morphology of the final muscle. Although several transcriptional regulators have been linked to FC identity, the molecular description is far from complete. This screen contributed two more FC-specific genes. Previously known markers, such as slouch or eve, once induced in the muscle FC, are maintained throughout the remainder of development. Ubx, which emerged from this screen, is a similarly simple case, as its transcripts are steadily present in most FCs. By contrast, more complex patterns of gene expression have been identified in FCs, such as the transient transcription of asense in a subset of FCs. The subsequent transcriptional inactivation of asense may underlie temporal changes in cell properties (Artero, 2003).

Even less is known about transcriptional regulators controlling FCM differentiation. Only one gene, lame duck, has been shown to have a role in FCMs. This screen has uncovered three more potential players: delilah, E(spl)mß and CG4136, confirming that FCMs follow their own, distinct, myogenic program. Discovering what aspects of FCM biology are controlled by these transcriptional regulators awaits analysis of the loss-of-function phenotypes (Artero, 2003).

Notch and Ras signaling pathways interact during muscle progenitor segregation. The results suggest that phyl and polychaetoid (pyd) may be additional links between the two signaling pathways in FCs. phyl and pyd both interact genetically with Notch and Delta. The transcription of phyl, which promotes neural differentiation, is negatively regulated by Notch signaling during specification of SOPs and their progeny. This study shows a similar regulation in muscle cells, where Notch signaling represses phyl expression and Ras signaling increases phyl expression. Likewise, in the nervous system, the segregation of SOPs requires pyd, a Ras target gene, to negatively regulate ac-sc complex expression. Similarly, Pyd may restrict the muscle progenitor fate to a single cell, perhaps by regulating lethal of scute transcription. Thus, Pyd would collaborate with Notch signaling to restrict muscle progenitor fate to one cell (Artero, 2003).

FCMs appear to integrate Ras and Notch signaling differently. Two genes whose transcripts were enriched under activated Notch conditions, parcas and asteroid (ast), have been implicated in Ras signaling in other tissues, directly (ast) or indirectly (parcas). These data are suggestive of a role for Ras signaling in the FCMs, in addition to its role in FC specification. In addition, Notch signaling to FCMs may prime cells for subsequent Ras signaling during muscle morphogenesis, much as occurs in FCs where Ras signaling primes the cell for subsequent Notch signaling during asymmetric division of the muscle progenitor (Artero, 2003).

Embryos that lack or ectopically express phyl have morphological defects in specific muscles, for example, in LL1 and DO4 in response to diminished phyl function, and in DT1 and LT4 in response to increased phyl function. The morphological defects in the loss-of-function embryos appear to be due to a failure to specify particular FCs, a conclusion that is based upon missing or abnormal production of the FC marker Kr. In eye development and SOP specification, Phyl directs degradation of the transcriptional repressor Tramtrack. In a subset of the primordial muscle cells, Phyl may work similarly, targeting Tramtrack for degradation. The presence of Tramtrack would contribute to the specific identity program of the muscle. Since Tramtrack is expressed in the mesoderm, this possibility is likely. Alternatively, Phyl may be required for targeted degradation of some other protein in a subset of FCs. The molecular partner for Phyl during muscle differentiation is unknown, although preliminary data suggest that sina is also expressed in somatic mesoderm and thus may be its partner. These studies have identified a new role for Phyl in muscle progenitor specification and suggest the importance of targeted ubiquitination for proper muscle patterning (Artero, 2003).

A role for ubiquitination in muscle differentiation is further reinforced by the identification of the RING finger-containing protein Goliath (Gol), induced by activated Notch conditions, and CG17492, induced by activated Ras conditions. Several RING-containing proteins function as E3 ubiquitin ligases, with the ligase activity mapping to the RING motif itself. Ligase function has been experimentally confirmed for the Gol ortholog GREUL1 in Xenopus. Thus, targeted protein degradation during muscle morphogenesis could serve a host of crucial functions, such as protein turnover, vesicle sorting, transcription factor activation and signal degradation (Artero, 2003).

The simplest view of the 'founder cell' hypothesis is that each FC contains all the information for the development of a particular muscle. By contrast, FCMs have been seen as a naïve group of myoblasts, entrained to a particular muscle program upon fusion to the FC. This work indicates that these two groups of myoblasts have distinct transcriptional profiles. These data raise the possibility of a greater role for FCMs in determining the final morphology of the muscle and emphasize a need to characterize fully those FCM genes. For example, this screen identified a protein kinase of the SR splice site selector factors (SRPK) whose transcripts are enriched in FCMs, suggesting that regulation of the splicing machinery is important for muscle morphogenesis. The Mhc gene undergoes spatially and temporally regulated alternative splicing in body wall muscles conferring different physiological properties on these muscles. This FCM-specific expression of SRPK may indicate that the production of a particular Mhc isoform is regulated by the FCMs that contribute to that muscle, rather than by the particular FC that seeds the muscle. In addition, a number of observations suggest that FCMs may be a diverse population of myoblasts, with different subsets having different potential to contribute to the final muscle pattern. For example, hbs expression suggests that only a subset of FCMs express the gene, and twist expression in lame duck mutant embryos persists in a subset of FCMs. This study provides additional genes for exploring whether FCMs are a heterogeneous population of myoblasts as well as determining the nature of FCM contribution to the final muscle (Artero, 2003).

The molecular events underlying complex morphological changes, such as migration, cell fusion, cell shape changes or changes in the physiology of a cell, require a rich and dynamic program of transcription changes. This study has described approximately one-third of this transcriptional profile. The FC- or FCM-specific transcription of seven genes, and the mutant phenotype of four selected genes, allowed the definition of new muscle mutations that specifically affect the morphological traits of a subset of muscles (Artero, 2003).

The mechanisms underlying the setting of myotubes and choice of myotube number in adult Drosophila were examined. The pattern of adult myotubes is prefigured by a pattern of dumbfounded (duf) as determined by examining duf-lacZ-expressing myoblasts at appropriate locations. Selective expression of duf-lacZ in single myoblasts emerges from generalized, low-level expression in all adult myoblasts during the third larval instar. The number of founders, thus chosen, corresponds to the number of fibers in a muscle. In contrast to the embryo, the selection of individual adult founder cells during myogenesis does not depend on Notch-mediated lateral inhibition. These results suggest a general mechanism by which multi-fiber muscles can be patterned (Dutta, 2004).

In the Drosophila embryo, the diversification of muscle forming mesoderm into founders and fusion-competent cells occurs through a process of lateral inhibition mediated by Notch. Since single duf-lacZ-expressing cells are selected and appear to act as founder myoblasts during adult myogenesis, it is important to show whether, as in the embryo, a Notch-dependent lateral inhibition pathway mediates this selection process. To test whether Notch has a function in selecting specific myoblasts for duf-lacZ expression, a dominant-negative and a constitutively active form of Notch was used. It was reasoned that if lateral inhibition were involved, then overexpression of a dominant-negative form of Notch (dnNotch) in adult myoblasts would lead to an increase in the number of duf-lacZ-expressing founders, whereas overexpression of the active form (Nintra) should suppress duf-lacZ expression altogether (Dutta, 2004).

In fact, the results of these experiments appear to be contradictory: thus, expression of UAS-dnNotch caused no change in the number of DVM founders and flies of the genotype 1151GAL4;UAS-dnNotch had the correct number of DLM and DVM fibers. This conclusion was verified by reducing Notch function in two additional ways. Function in the Notch signalling pathway in myoblasts was reduced by overexpressing truncated forms of the protein Mastermind (Mam), an essential component of the Notch signalling pathway. Mam interacts with the intracellular domain of Notch and with Suppressor of Hairless, and forms a transcriptional activation complex. Two truncated versions of Mam, MamH and MamN, when overexpressed by the GAL4-UAS system behave as dominant-negative proteins and elicit Notch loss-of-function phenotypes. Overexpression of either UASMamN or UAS-MamH in myoblasts using 1151-GAL4 had no effect on the number of DVM founders. The role of Notch was further examined by using a conditional allele, Nts1. Because of the close proximity of the duf and Notch loci, duf-lacZ, Nts recombinants could not be generated and hence 22C10 was used as the marker for founder cells in the abdomen. The earliest time at which myoblasts expressing high levels of duf-lacZ are also labeled with 22C10 is at 24 hours APF. Notch function was removed for different periods (2 hours, 4 hours, 6 hours and 8 hours) before this stage by raising Nts animals to the non-permissive temperature, and the number of 22C10-stained cells associated with the abdominal nerves was determined. The numbers of 22C10-expressing cells in the dorsal or lateral segments of the abdomen were determined and shown to be unaffected in these experiments. It is known that all three approaches -- whether using the dominant-negative Notch or mastermind constructs, or using Nts animals -- are effective, since they all can reduce the levels of Twist expression in adult myoblasts, a known consequence of Notch reduction in adult myoblasts (Dutta, 2004).

Taken together, these results suggest that Notch is not required for the selection of duf-lacZ-expressing myoblasts. However, in the converse experiment, expression of Nintra in the myoblasts does suppress the formation of founders, as would be expected for a selection mechanism based on lateral inhibition. How can these apparently contradictory findings be reconciled? Earlier studies have shownthat Notch acts as a positive regulator of Twist in the myoblast population. Thus, Nintra expression in adult myoblasts leads to maintained expression of Twist in these cells and to a failure of muscle differentiation. If the absence of founders that was observe is a consequence of this sustained expression of Twist in the myoblasts, then it would be expected that simply expressing Twist constitutively in the myoblasts would mimic the activated-Notch phenotype. Using 1151-GAL4 to drive Twist expression in the adult myoblasts, it was found that at 12 hours APF there were no DVM founders, suggesting that a decline in Twist expression, which is antagonized by the action of Nintra, is a requirement for elevated duf-lacZ expression. Indeed, founders are the first cells in the myoblast pool to show declining levels of Twist expression, with the result that the duf-lacZ-expressing founders and Twist-expressing myoblasts are mutually exclusive cell populations (Dutta, 2004).

Notch and 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).

Previous studies have suggested that Notch activity controls two distinct processes during the specification of cardiac cell fates. First, it is required to single initial progenitors out of a field of competence by supporting the selection and inhibiting surrounding cells from adopting the same fate. Subsequent to the progenitor specification, Notch is required again for the specification of alternative cell fates of sibling cells produced during asymmetric cell divisions. In this study, the cell autonomy of Notch was examined, by using eme-Gal4 (which confers expression in the mesodermal Eve lineage) to drive activated forms of Notch and Su(H) exclusively in the mesodermal Eve lineages. Conditional ubiquitous expression of activated Notch was used to examine its lineage-specific function in other cardiac lineages. Notch was found to be required for specification of a non-myogenic fate in both the Eve and the Svp lineages of the cardiac mesoderm. By contrast, activation or inhibition of the Notch pathway does not affect cell fate decisions within the symmetric lineages. This suggests a mechanism by which cell type diversity may be increased during evolution by co-opting the Notch pathway during cell division to distinguish between alternative fates of the daughter cells. The inability of activated Su(H) to autonomously influence cell fates in symmetric cardiac lineages further suggests that other factors or activities, not present in symmetric lineages, are crucial for the asymmetric lineage-specific functions of Notch and Su(H) (Han, 2003).

Interestingly, this influence of the Notch pathway on cell fate decision in asymmetric cardiac Eve and Svp lineages is not altered when cell division is arrested. Thus, cell division is not essential to distinguish between alternative cell fates. The data also suggest that the default cell fate of an asymmetrically dividing cardiac precursor in Drosophila is determined to assume a myogenic fate, owing to Numb-mediated inhibition of Notch, unless that fate is switched by the activation of target genes downstream of Su(H). Moreover, in a double mutant of Notch and numb one would expect to observe the same lineage phenotype as of Notch alone, i.e., a myogenic cell fate, since the primary role of Numb is to inhibit Notch signaling. Unfortunately, analysis of such double mutants is complicated by the earlier role of Notch in lateral inhibition (Han, 2003).

Another unresolved issue is the source of the Notch ligand that activates signal transduction within asymmetric cardiac lineages. If the myogenic cell were to produce the ligand for Notch activation in its pericardial sibling, then the undivided progenitor would have to secrete its own Notch ligand. This is unlikely, since production of the ligand is usually inhibited within the cell that experiences Notch signaling. In the asymmetric MP2 lineage of the Drosophila CNS, for example, ligand production appears to be required in cells outside the MP2 lineage. A similar scenario may be operating in the asymmetric cardiac lineages (Han, 2003).

Within the Eve lineages, Notch activation is mimicked by Su(H) fused to the VP16, a potent transcriptional activation domain. Recent studies suggest that in the absence of Notch activity, DNA-bound Su(H) prevents activators from promoting transcription. When Notch ligands, such as Delta, bind to its receptor, Notch is cleaved to produce an intracellular domain fragment, N(icd), which is thought to enter the nucleus and interact directly with Su(H) to recruit transcriptional co-activators and alleviate Hairless-mediated repression, thus promoting transcription. In support of this model, it has been found that Su(H) overexpression can mimic Notch activation only when linked directly to a transcriptional activator, but not in its wild-type form when it presumably associates with co-repressors, such as Hairless and Groucho, that prevent Su(H)-dependent transcriptional activation in the absence of Notch signaling (Han, 2003).

The role of the PTB-containing, membrane-associated protein Numb in preventing Notch activation in the nervous system is well established. To explore at which level Notch signaling is inhibited by Numb in the cardiac lineages, numb was overexpressed simultaneously with N(icd) or Su(H)vp16 within the mesodermal Eve lineages. Excess Numb was able to counteract activated Notch but not activated Su(H) function, suggesting that Numb can inhibit Notch activity after Notch has been cleaved, possibly by preventing its nuclear translocation, but is unlikely to prevent the transcriptional activator function of Su(H) directly. Recent data suggest that Numb is involved in stimulating endocytosis of Notch, thus removing it from the cell surface and inhibiting its function. It is not clear, however, if this inhibition by endocytosis is at the level of the entire Notch receptor, or (also) at the level of N(icd) after it is cleaved off. Experiments described here provide strong evidence that Numb can indeed interfere with N(icd) function, but it remains to be determined if endocytosis is an obligatory intermediate in this inhibition of activated Notch (Han, 2003).

The Notch pathway may also have a role in vertebrates in specifying pericardial and other non-myogenic cell fates within the dorsolateral cardiogenic region of the anterolateral plate mesoderm. As in the Eve and Svp lineage of the Drosophila heart, activation of the Notch pathway decreases myocardial gene expression and increased expression of a pericardial marker, whereas inhibition of Notch signaling resulted in an increase of cardiac myogenesis. Similar results were obtained with an activated form of RBP-J [a vertebrate homolog of Drosophila Su(H) fused to vp16, as in this study]. These data indicate that the Notch pathway may play a role in the specification of myocardial versus pericardial cell fates in both Drosophila and vertebrates. This raises the question of whether the mechanism of Notch mediated cell identity determination is also conserved between vertebrates and flies. Because it is not yet known if (Numb-controlled) asymmetric cell divisions are also involved in vertebrate heart development, the answer awaits future studies. However, recent studies on the role of Numb during cortical development suggest that it is likely to have a similar control function in cell fate specification in vertebrates as it does in flies (Han, 2003 and references therein).

Notch controls lineage specification during larval hematopoiesis

Drosophila larval hemocytes originate from hematopoietic organs called lymph glands. These are composed of paired lobes located along the dorsal vessel. Two mature blood cell populations are found in the circulating hemolymph: the macrophage-like plasmatocytes, and the crystal cells that contain enzymes of the immune-related melanization process. A third class of cells, called lamellocytes, are normally absent in larvae but differentiate after infection by parasites too large to be phagocytosed. Evidence is presented that the Notch signaling pathway plays an instructive role in the differentiation of crystal cells. Loss-of-function mutations in Notch result in severely decreased crystal cell numbers, whereas overexpression of Notch provokes the differentiation of high numbers of these cells. In this process, Serrate, not Delta, is the Notch ligand. In addition, Notch function is necessary for lamellocyte proliferation upon parasitization, although Notch overexpression does not result in lamellocyte production. Finally, Notch does not appear to play a role in the differentiation of the plasmatocyte lineage. This study underlines the existence of parallels in the genetic control of hematopoiesis in Drosophila and in mammals (Duvic, 2002).

Drosophila hematopoiesis occurs in two distinct phases; one occurs during embryonic development and a second occurs in larvae. In embryos, a population of blood cells (hemocytes) differentiates in the head mesoderm then migrates to colonize the whole organism. These cells exhibit macrophagic activity and are called plasmatocytes. A second population of hemocytes, the crystal cells, differentiate simultaneously in the region of the anterior midgut. In larvae, hematopoiesis takes place essentially in so-called lymph glands, which are composed of a variable number (2-6) of paired lobes distributed along the dorsal vessel. The circulating hemolymph of larvae contain three fully differentiated hemocyte types: plasmatocytes, the professional phagocytes, represent the majority of the cells; crystal cells, which contain the enzymes necessary for immune-related melanization reactions and represent less than 5% of the cells; and lamellocytes, which are essentially devoted to encapsulation of large-sized invading parasites and are produced upon parasitization. At metamorphosis, the lymph glands degenerate, and, in adults, only the plasmatocyte population persists (Duvic, 2002).

A genetic dissection of hematopoiesis has shown that hemocyte differentiation in embryos is dependent on several transcription factors: blood cell fate is determined by the GATA factor Serpent (encoded by srp), plasmatocyte identity is specified by the zinc-finger transactivator Glial Cells Missing (gcm), and crystal cell identity is specified by the Runt domain AML1-related Lozenge transactivator (lz). The Friend-of-Gata homolog, U-shaped (ush), antagonizes crystal cell development. In larvae, a similar approach has shown that gcm and lz, respectively, control plasmatocyte and crystal cell differentiation. srp is expressed early in larval lymph gland cells, and it is assumed, although not proven, that it has the same function as in embryonic hematopoiesis (Duvic, 2002).

The potential role of N in Drosophila hematopoiesis was addressed by taking advantage of the temperature-sensitive Nts1 allele. At a permissive temperature, Nts1 larvae have a wild-type number of circulating plasmatocytes and crystal cells. However, when second instar larvae were shifted to the restrictive temperature (29°C), it was observed that (at the wandering stage) the mutant larvae exhibited a strong reduction (up to 60%) in the number of crystal cells. Plasmatocyte numbers are not affected. This analysis was shifted by using a transgenic line in which a cDNA sequence encoding a ligand-independent constitutively active form of N (Nic) was placed under the control of UAS elements. Overexpression of Nic with an ubiquitous Gal4 driver (hsp-Gal4) results in a dramatic increase in the number of crystal cells in larvae (up to 7-fold). Similar high numbers of crystal cells were recorded in larvae carrying the gain-of-function allele of Notch NMcd8. Altogether, these results indicate that the function of N is mandatory for crystal cell differentiation in larval development. Since plasmatocyte numbers remain wild-type in these experiments, the observed phenotypes do not reflect a generalized effect on blood cell proliferation, but they are specific to one lineage, i.e., the crystal cells (Duvic, 2002).

Two well-established downstream effectors of the Notch signaling pathway, Suppressor of Hairless [Su(H)] and Deltex (Dx), were examined for their involvement in hematopoiesis. A larval viable Su(H) interallelic combination was generated and mutant third instar larvae were observed to be almost totally devoid of crystal cells; however, they had wild-type plasmatocyte counts. In contrast, Dx (in Dx1 and DxP strong hypomorphic alleles) mutant blood cell counts were normal for both cell types, suggesting that the effect of N on crystal cell production is Dx independent (Duvic, 2002).

Crystal cells differentiate within the larval hematopoietic organ, the lymph glands, and the effect of the various mutations on differentiation in situ were examined. For this, an antibody raised against dipteran prophenoloxidase (proPO), a zymogen required for melanization reactions, was examined. In Drosophila, proPO is produced and stored in crystal cells and is released during host defense reactions. It was first noted that, in wild-type larvae, proPO is synthesized during the differentiation of crystal cells within the hematopoietic organ. Interestingly, a gradient of differentiation is apparent within the successive lymph gland lobes along the anteroposterior axis: the anteriormost lobes contained numerous proPO-positive cells, whereas the adjacent, more posterior lobes contain no or few positive cells and the most posterior lobes are totally devoid of differentiating crystal cells. In Nts1 mutants placed at a restrictive temperature, the number of proPO-positive cells is significantly reduced in the lymph glands, and they were occasionally totally absent. A similar phenotype was observed in a Su(H) and in a Ser mutant context. Conversely, activation of the Notch pathway in NMcd8 larvae and in larvae carrying UAS-Nic, UAS-Ser, or UAS-Dl transgenes driven by hsp-Gal4 dramatically increased the number of proPO-positive cells in lymph glands. Not only are the anteriormost lobes packed with such cells, but more posterior lobes are often found to contain large numbers of differentiating crystal cells. As expected, similar phenotypes are obtained when the e33C-Gal4 driver is used; this driver is strongly expressed in the lymph glands (Duvic, 2002).

The GATA factor Srp determines the hemocyte fate in Drosophila. Since srp is expressed in the larval lymph glands, it was asked whether this expression was affected in an N mutant context. Nts1 larvae raised at the restrictive temperature were examined. The expression of srp is wild-type, indicating that the effect of Notch signaling on crystal cell differentiation occurs downstream of srp function (Duvic, 2002).

In Drosophila larvae, an additional blood cell type, the lamellocyte, is normally not present in healthy individuals. Lamellocyte differentiation is triggered by immune conditions such as infestation by parasitic wasps that lay eggs in second instar larvae. Lamellocytes, together with crystal cells, participate in the encapsulation of wasp eggs, which are eventually killed within the melanized capsules. Lamellocyte production is initiated a few hours after wasp egg laying, and 48 hr later, lamellocytes represent an important proportion of circulating hemocytes. Cellular reactions to wasp parasitization were examined in Nts1 larvae placed at the restrictive temperature. Although the production of lamellocytes was not totally abolished in the mutant larvae, it was significantly reduced compared to wild-type larvae or to Nts1 larvae that had been maintained at the permissive temperature. Thus, the absence of N function prevents normal lamellocyte differentiation in response to wasp parasitization. However, overexpression of Nic does not result in lamellocyte production, in contrast to crystal cells (Duvic, 2002).

These data indicate that the Notch signaling pathway plays an instructive role in the specification of the crystal cell lineage in Drosophila larvae. This role is distinct from that reported for the lz gene. Indeed, although lz function is mandatory for crystal cell differentiation, its overexpression per se has no phenotype with regard to crystal cells. Notch signaling has been extensively analyzed in the context of mammalian hematopoiesis. In particular, Notch1 prevents the differentiation of pluripotent stem cells through stimulating expression of the GATA-2 transactivator. This picture somewhat contrasts with that observed in Drosophila, in which N is clearly not involved in maintaining pools of undifferentiated blood cell precursors and does not regulate the expression of the GATA factor srp. At later stages of mammalian hematopoiesis, Notch1 controls lymphoid cell fate specification, namely, by favoring T cell commitment over that of B cells. This conceptually parallels the role of N in Drosophila hematopoiesis, in which it favors crystal cell commitment and differentiation (Duvic, 2002).

It was also found that N plays a role in the differentiation of lamellocytes following wasp infection. In this case, N is not instructive, since its overexpression does not lead to lamellocyte production. However, a wild-type function of N is clearly necessary for massive differentiation of lamellocytes. Finally, there is no evidence for a function of N in plasmatocyte differentiation (Duvic, 2002).

An unexpected link between Notch signaling and ROS in restricting the differentiation of hematopoietic progenitors in Drosophila

A fundamental question in hematopoietic development is how multipotent progenitors achieve precise identities, while the progenitors themselves maintain quiescence. In Drosophila larvae, multipotent hematopoietic progenitors support the production of three lineages, exhibit quiescence in response to cues from a niche, and from their differentiated progeny. Infection by parasitic wasps alters the course of hematopoiesis. This study addresses the role of Notch (N) signaling in lamellocyte differentiation in response to wasp infection. Notch activity is moderately high and ubiquitous in all cells of the lymph gland lobes, with crystal cells exhibiting the highest levels. Wasp infection reduces Notch activity, which results in fewer crystal cells and more lamellocytes. Robust lamellocyte differentiation is induced even in N mutants. Using RNA interference-knockdown of N, Serrate, and Neuralized, and twin clone analysis of a N null allele, this study shows that all three genes inhibit lamellocyte differentiation. However, unlike its cell-autonomous function in crystal cell development, Notch's inhibitory influence on lamellocyte differentiation is not cell-autonomous. High levels of reactive oxygen species in the lymph gland lobes, but not in the niche, accompany NRNAi-induced lamellocyte differentiation and lobe dispersal. These results define a novel dual role for Notch signaling in maintaining competence for basal hematopoiesis: while crystal cell development is encouraged, lamellocytic fate remains repressed. Repression of Notch signaling in fly hematopoiesis is important for host defense against natural parasitic wasp infections. These findings can serve as a model to understand how reactive oxygen species and Notch signals are integrated and interpreted in vivo (Small, 2013).

BTB-zinc finger oncogenes are required for Ras and Notch-driven tumorigenesis in Drosophila

During tumorigenesis, pathways that promote the epithelial-to-mesenchymal transition (EMT) can both facilitate metastasis and endow tumor cells with cancer stem cell properties. To gain a greater understanding of how these properties are interlinked in cancers, Drosophila epithelial tumor models were used, that are driven by orthologues of human oncogenes (activated alleles of Ras and Notch) in cooperation with the loss of the cell polarity regulator, scribbled (scrib). Within these tumors, both invasive, mesenchymal-like cell morphology and continual tumor overgrowth, are dependent upon Jun N-terminal kinase (JNK) activity. To identify JNK-dependent changes within the tumors a comparative microarray analysis was used to define a JNK gene signature common to both Ras and Notch-driven tumors. Amongst the JNK-dependent changes was a significant enrichment for BTB-Zinc Finger (ZF) domain genes, including chronologically inappropriate morphogenesis (chinmo). chinmo was upregulated by JNK within the tumors, and overexpression of chinmo with either RasV12 or Nintra was sufficient to promote JNK-independent epithelial tumor formation in the eye/antennal disc, and, in cooperation with RasV12, promote tumor formation in the adult midgut epithelium. Chinmo primes cells for oncogene-mediated transformation through blocking differentiation in the eye disc, and promoting an escargot-expressing stem or enteroblast cell state in the adult midgut. BTB-ZF genes are also required for Ras and Notch-driven overgrowth of scrib mutant tissue, since, although loss of chinmo alone did not significantly impede tumor development, when loss of chinmo was combined with loss of a functionally related BTB-ZF gene, abrupt, tumor overgrowth was significantly reduced. abrupt is not a JNK-induced gene, however, Abrupt is present in JNK-positive tumor cells, consistent with a JNK-associated oncogenic role. As some mammalian BTB-ZF proteins are also highly oncogenic, this work suggests that EMT-promoting signals in human cancers could similarly utilize networks of these proteins to promote cancer stem cell states (Doggett, 2015).

This report has defined the transcriptional changes induced by JNK signaling within both scrib>RasACT and scrib>NACT tumors by carrying out comparative microarray expression arrays. This analysis that JNK exerts a profound effect upon the transcriptional profile of both Ras and Notch-driven tumor types. The expression of nearly 1000 genes was altered by the expression of bskDN in either Ras or Notch-driven tumors, and less than half of these changes were shared between the two tumor types, indicating that JNK signaling elicits unique tumorigenic expression profiles depending upon the cooperating oncogenic signal. Nevertheless, of the 399 JNK-regulated probe sets shared between Ras and Notch-driven tumors, it is hypothesized that these had the potential to provide key insights into JNK's oncogenic activity, and to prioritize these targets, it was considered that the expression of the critical oncogenic regulators would not just be altered by bskDN, but would be normalized to close to wild type levels. This subset of the 399 probe set was identified by comparing the expression profile of each genotype back to control tissue, thereby producing a more focussed JNK signature of 103 genes. Notably, this included previously characterized targets of JNK in the tumors, such as Mmp1,cherand Pax, thereby providing validation of the approach. Also amongst these candidates were 4 BTB-ZF genes; two of which were upregulated by JNK in the tumors (chinmo and fru), and two downregulated (br and ttk) (Doggett, 2015).

Focussing upon chinmo, chinmo overexpression was shown to be sufficient to prime epithelial cells for cooperation with RasACT in both the eye antennal disc and in the adult midgut epithelium, and that chinmo is required for cooperative RasACTor NACT-driven tumor overgrowth, although its function was only exposed when its knockdown was combined with knockdown of a functionally similar BTB-ZF transcription factor, abrupt. This family of proteins is highly oncogenic in Drosophila, since previous work has shown that ab overexpression can cooperate with loss of scrib to promote neoplastic overgrowth, and in these studies, it was also shown that overexpression of a fru isoform normally expressed in the eye disc is capable of promoting cooperation with RasACT and NACT in the eye-antennal disc, in a similar manner to chinmo overexpression. Thus, whether fru also plays a role in driving Ras or Notch-driven tumorigenesis warrants further investigation. Indeed, a deeper understanding of the oncogenic activity of these genes is likely to be highly relevant to human tumors, since of the over 40 human BTB-ZF family members, many are implicated in both haematopoietic and epithelial cancers, functioning as either oncogenes (eg., Bcl6, BTB7) or tumor suppressors (eg., PLZF, HIC1). Furthermore, over-expression of BTB7, can also cooperate with activated Ras in transforming primary cells, and its loss makes MEFs refractory to transformation by various key oncogenes such as Myc, H-rasV12 and T-Ag, suggesting that cooperating mechanisms between BTB-ZF proteins and additional oncogenic stimuli might be conserved (Doggett, 2015).

JNK signaling in Drosophila tumors is known to promote tumor overgrowth through both the STAT and Hippo pathways. Deregulation of the STAT pathway was evident in the arrays through the upregulation of Upd ligands by JNK in both Ras and Notch-driven tumors. In contrast, although cher was identified in the arrays as being upregulated in both tumor types and previous studies have shown that cher is partly required for the deregulation of the Hippo pathway in scrib>RasACT tumors, more direct evidence for Hippo pathway deregulation amongst the JNK signature genes was lacking. In part, this could be due to JNK regulating the pathway through post-transcriptional mechanisms involving direct phosphorylation of pathway components. However, the failure to identify known Hippo pathway target genes, and proliferation response genes in general, may simply highlight limitations in the sensitivity of the array assay and the cut-offs used for determining significance, despite its obvious success in correctly identifying many known JNK targets (Doggett, 2015).

Whether tumor overgrowth through STAT and Yki activity is somehow associated with a stem cell or progenitor-like state remains uncertain. Although imaginal discs exhibit developmental plasticity and regeneration potential, and JNK signaling is required for both of these stem-like properties, there is no positive evidence for the existence of a population of asymmetrically dividing stem cells within imaginal discs. Instead, symmetrical divisions of progenitor cells may be the means by which imaginal discs can rapidly generate enough cells to form the differentiated structures of the adult fly. To date, progenitor cells have only been characterized in the eye disc neuroepithelium. These cells have a pseudostratified columnar epithelial morphology and express the MEIS family transcription factor, Hth, which is downregulated as cells initiate differentiation and begin expressing Dac and Eya. Interestingly, they also require Yki for their proliferation, and can be induced to overproliferate in response to increased STAT activity. However, analysis of cell fate markers indicated that tumor overgrowth was not likley to be solely due to the overproliferation of these undifferentiated progenitor cells. Although scrib>RasACT/NACT tumors, were characterized by the failure to transition to Dac/Eya expression in the eye disc, blocking JNK in scrib > RasACT/NACT tumors did not restore tumor cell differentiation, despite overgrowth being curtailed, and Hth expression was not maintained in the tumors in a JNK-dependent manner. Nevertheless, a JNK-induced gene such as chinmo is likely to be associated with promoting a progenitor-like state, since it is a potential STAT target gene required for adult eye development that is expressed in eye disc progenitor cells in response to increased Upd activity and its overexpression alone is sufficient to block Dac/Eya expression. Furthermore, chinmo is also required for cyst stem cell maintenance in the Drosophila testis, and the current work has shown that chinmo overexpression promotes increased numbers of esgGFP expressing stem cells or enteroblasts in the adult midgut. As the BTB-ZF protein Ab is also highly oncogenic and expressed in the eye disc progenitor cells, it is hypothesize that the JNK-induced expression ofchinmo in scrib>RasACT/NACT tumors could cooperate with Ab to maintain a progenitor-like cell state in the eye disc, and that this is required for scrib->RasACT/NACT tumor overgrowth. However, although Ab was expressed in chinmo-expressing, JNK positive tumor cells, Ab does not appear to be a JNK-induced gene. What JNK-independent mechanisms control ab expression will therefore require further analysis (Doggett, 2015).

Interestingly, previous studies have observed that ab overexpression in eye disc clones upregulates chinmo expression and although the effect of chinmo expression upon ab is yet to be described, the data at least suggest that the control of their expression is interlinked in a yet to be defined manner (Doggett, 2015).

Consistent with Chinmo being important for scrib->RasACT/NACTv tumor overgrowth, chinmo overexpression itself is also highly oncogenic. Over-expression of chinmo with RasACT or NACT drives tumorigenesis in the eye-antennal disc, and also resulted in enlarged brain lobes, presumably due to the generation of overexpressing clones within the neuroepithelium of the optic lobes. In the adult midgut, the overexpression of chinmo with RasACT in the stem cell and its immediate progeny, the enteroblast, promoted massive tumor overgrowth, resulting in esgGFP expressing cells completely filling the lumen of the gut, and eventual host lethality. The luminal filling of esgGFP cells is reminiscent of the effects of RasACT expression in larval adult midgut progenitor cells. Together with the data linking Chinmo function to stem or progenitor cells, these data reinforce the idea that epithelial tumorigenesis can be primed by signals, such as chinmo over-expression, that promote a stem or progenitor cell state (Doggett, 2015).

The function of some Drosophila BTB-ZF proteins including Chinmo and Ab, has also been linked to heterochronic roles involving the conserved let-7 miRNA pathway and hormone signals, to regulate the timing of differentiation. Indeed, Ab can directly bind to the steroid hormone receptor co-activator Taiman (Tai or AIB1/SRC3 in humans), to represses the transcriptional response to ecdysone signaling. Thus, the capacity of BTB-ZF proteins to influence the timing of developmental transitions, particularly if they impede developmental transitions within stem or progenitor cells, could help account for their potent oncogenic activity. Indeed, ecdysone-response genes were repressed by JNK in the tumorigenic state, consistent with the failure of the larvae to pupate and a delay in developmental timing. Whether repressing the ecdysone response cell autonomously might contribute to tumor overgrowth and/or invasion will be an interesting area of future investigation, given the complex role of hormone signaling in mammalian stem cell biology and cancers (Doggett, 2015).

Previous studies have suggested that JNK-dependent tumor cell invasion is developmentally similar to the JNK-induced EMT-like events occurring during imaginal disc eversion. Thus the capacity of JNK to also promote tumor overgrowth is reminiscent of how EMT inducers such as Twist (Twi) and Snail (Sna) are associated with the acquisition of cancer stem cell properties. In Drosophila, however, twi and snawere not induced by JNK in the tumors, although transcription factors involved in mesoderm specification, including the NF-kappaB homologue, dl (a member of the 103 JNK signature), and Mef2 (a member of the 399 JNK signature), were amongst the up-regulated JNK targets. Mesoderm specification is not necessarily associated with a mesenchymal-like cell morphology, however, dl is involved in the induction of EMT during embryonic development, and both dl and Mef2 act with Twi and Sna to coordinate mesoderm formation. Interestingly, recent studies have identified dl in an overexpression screen for genes capable of cooperating with scrib > in Drosophila tumorigenesis, and Mef2 has been identified as a cooperating oncogene in Drosophila, and possibly also in humans, where a correlation exists between the expression of Notch and Mef2 paralogues in human breast tumor samples. It is therefore possible that dl and Mef2 either act in combination with Twi or Sna, or independently of them but in a similar oncogenic capacity, to promote a mesodermal cell fate in scrib > RasACT/NACT tumors. The potential relevance of this to the mesenchymal cell morphology associated with tumor cell invasion, as well as the acquisition of progenitor states is worthy of further investigation (Doggett, 2015).

In mef2-driven tumors both overgrowth and invasion depend upon activation of JNK signaling, suggesting that Mef2 is not capable of promoting invasive capabilities independent of JNK. In contrast, chinmo+RasACT/NACT tumors appeared non-invasive and retained epithelial morphology despite the massive overgrowth, although closer examination of cell polarity markers will be required to confirm this. Furthermore, the overgrowth of chinmo+RasACT/NACT tumors was not dependent upon JNK signaling, suggesting that the maintenance of a progenitor-like state could be uncoupled from JNK-induced EMT-effectors associated with invasion. Whether clear divisions between mesenchymal behaviour and progenitor states in tumors can be clearly separated in this manner is not yet clear, however, overall, it is likely that multiple JNK-regulated genes will participate in both promoting tumor overgrowth as well as migration/invasion. Although this study used the 103 JNK signature as a means to focus upon potential key candidates, an analysis of the 399 JNK-regulated probe sets common to both Ras and Notch-driven tumours has the potential to provide deeper insights into the multiple effectors of JNK signaling during tumorigenesis. Whilst the individual role of these genes can be probed with knockdowns, the complexity of the response, potentially with multiple redundancies and cross-talk, will ultimately need a network level of understanding to more fully expose key nodes participating in overgrowth and invasion. This approach has considerable potential to further expose core principles and mechanisms that drive human tumorigenesis, since it is clear that many fundamental commonalities underlie the development of tumors in Drosophila and mammals (Doggett, 2015).

Frequent somatic mutation in adult intestinal stem cells drives neoplasia and genetic mosaicism during aging

Adult stem cells may acquire mutations that modify cellular behavior, leading to functional declines in homeostasis or providing a competitive advantage resulting in premalignancy. However, the frequency, phenotypic impact, and mechanisms underlying spontaneous mutagenesis during aging are unclear. This study reports two mechanisms of genome instability in adult Drosophila intestinal stem cells (ISCs) that cause phenotypic alterations in the aging intestine. First, frequent loss of heterozygosity was found arising from mitotic homologous recombination in ISCs that results in genetic mosaicism. Second, somatic deletion of DNA sequences and large structural rearrangements, resembling those described in cancers and congenital diseases, frequently result in gene inactivation. Such modifications induced somatic inactivation of the X-linked tumor suppressor Notch in ISCs, leading to spontaneous neoplasias in wild-type males. Together, these findings reveal frequent genomic modification in adult stem cells and show that somatic genetic mosaicism has important functional consequences on aging tissues (Siudeja, 2015).

Notch and long term memory

To gain insights into the functions of Notch signaling in the adult brain, the involvement of N in Drosophila olfactory learning and memory was examined. Long-term memory (LTM) was disrupted by blocking N signaling in conditional mutants or by acutely induced expression of a dominant-negative N transgene. In contrast, neither learning nor early memory were affected. Furthermore, induced overexpression of a wild-type (normal) N transgene specifically enhances LTM formation. These experiments demonstrate that N signaling contributes to LTM formation in the Drosophila adult brain (Ge, 2004).

Consistent with the idea that N may affect LTM, it was found that alteration of N activity does not affect learning or short-term memory. A temperature-sensitive N allele, Nts2, is viable at permissive temperature but embryonically lethal at restrictive temperature. As adults, these flies are behaviorally normal (shock reactivity and olfactory acuity) and show normal learning scores after 2 days of incubation at the restrictive temperature (30°C). Furthermore, learning is also not affected in transgenic flies that express dominant-negative N, hs-N Deltacdc10rpts. Expression of hs-N Deltacdc10rpts was induced acutely via heat-shock treatment (30 min at 37°C, with 3 h of recovery before training). The N Deltacdc10rpts protein lacks the intracellular domain that diffuses into the nucleus after N activation. Without the N intracellular domain, the N Deltacdc10rpts protein binds ligands normally but is unable to regulate gene expression. All mutants tested were outcrossed to control lines to remove potential modifiers (Ge, 2004).

In contrast, reduction of N activity disrupts 1-day memory. One-day memory after spaced training (10 training sessions with a 15-min rest interval between each) normally is composed of roughly equal amounts of anesthesia-resistant memory (ARM) and LTM, whereas 1-day memory after massed training (10 training sessions with no rest interval) is composed only of ARM. One-day memory is significantly reduced in Nts2 mutants at a restrictive temperature (30°C) when compared with the permissive temperature (18°C). There was also a slight but statistically insignificant reduction in memory scores in the control group when comparing the higher temperature with the lower temperature. Induced expression of dominant-negative N in hs-N Deltacdc10rpts flies also significantly reduces 1-day memory after spaced training. Flies were given a 30-min heat shock and subjected to spaced training 3 h afterward. To determine which components (i.e., ARM or LTM) were affected, 1-day memory was examined after massed training in hs-N Deltacdc10rpts transgenic flies. There was no difference for 1-day memory after massed training, indicating disruption of LTM but not ARM. The heat-shock treatment did not exert any detectable effect on control flies. Thus, reduced N activity, resulting either from a conditional mutation or induced expression of a dominant-negative N, specifically blocked the formation of LTM without affecting early memory or ARM (Ge, 2004).

The effects were evaluated of overexpression of wild-type N+ in hs-N+ transgenic flies. Flies were given a 30-min heat shock and trained and tested 3 h afterward. Overexpression of N+ had no significant effect on learning, consistent with observations that disruption of N does not affect learning. Overexpression of N+ after heat-shock treatment was confirmed by RT-PCR of adult head mRNA. Acutely induced overexpression of N+ also did not affect 1-day memory for flies subjected to the normal spaced training procedure (10 training cycles). However, 1-day memory was enhanced significantly for flies overexpressing N+ that had only one or two spaced training sessions. To characterize the nature of this enhancement in 1-day memory, it was shown that enhanced memory elicited by one or two spaced training sessions in flies overexpressing N+ could be blocked by feeding of cycloheximide, a drug blocking protein synthesis, to flies. It has been shown that feeding cycloheximide to flies can block formation of protein-synthesis-dependent LTM. There was no significant difference for sensory modalities necessary for performing the learning task. Thus, overexpression of the N+ gene facilitates LTM formation (Ge, 2004).

Thus one function for N in the adult brain is to mediate the formation of LTM. Three different lines of evidence support this conclusion. (1) 1-day memory was reduced in a temperature-sensitive N mutant at the restrictive temperature (30°C) at which N receptor function is supposedly defective; (2) 1-day memory was diminished in transgenic flies that express a dominant-negative N Deltacdc10rpts; (3) 1-day memory formation was facilitated by overexpression of N+. Although known for its crucial role in development of the nervous system, the use of temperature-sensitive mutations and of transgenic flies carrying inducible genes through acute treatment have allowed the role of N in adult physiology to be disassociated from its role in development. For all experiments that led to the observations noted above, flies were allowed to develop under relatively normal conditions and N function was perturbed acutely at the adult stage before the training. Moreover, it was also shown that temporary disruption of N function at the adult stage exerted no significant effects on sensorimotor responses as well as on learning. Thus, the observed effects can be attributed specifically to memory, confirming that 1-day memory can be affected by manipulation of N function. This finding is corroborated by an independent observation in which 1-day memory is reduced by disturbing N function through the use of RNA interference and Nts1 loss of function in adult flies (Ge, 2004).

One-day memory consists of both ARM and LTM phases. A specific effect of manipulation of N function on LTM is suggested. One-day memory elicited by massed training (ARM only) is not affected by expression of dominant-negative N Deltacdc10rpts, whereas 1-day memory after spaced training (ARM and LTM) is reduced significantly. Consistent with this observation, enhanced memory induced by one or two spaced training sessions was blocked by a drug that inhibits protein synthesis. Such drugs are known to block LTM specifically (Ge, 2004).

It is notable that in flies with overexpression of N+, only one training session was required to elicit LTM instead of the usual 10 spaced training cycles. This enhancement is very similar to that reported for CREB. Induced expression of hs-dCREb2-activator also reduced the number of spaced training sessions, required to yield the maximal level of LTM in normal flies, from 10 sessions to 1 session. Future experiments may examine specific roles for the CREB and N pathways in the formation of LTM. Involvement of the CREB pathway in LTM formation is a mechanism observed in a wide range of organisms including Drosophila, Aplysia, and vertebrates. The N signaling pathway is also highly conserved evolutionarily. In fact, a recent report shows that learning and memory are defective in a heterozygous Notch1+/- mouse knockout, although a developmental etiology could not be ruled out for such knockout mice. The current results extend this observation and the evolutionary implications thereof by revealing an acute role for N signaling during memory formation (Ge, 2004).

Notch signaling modulates sleep homeostasis and learning after sleep deprivation in Drosophila

The role of the transmembrane receptor Notch in the adult brain is poorly understood. This study provides evidence that bunched, a negative regulator of Notch, is involved in sleep homeostasis. Genetic evidence indicates that interfering with bunched activity in the mushroom bodies (MBs) abolishes sleep homeostasis. Combining bunched and Delta loss-of-function mutations rescues normal homeostasis, suggesting that Notch signaling may be involved in regulating sensitivity to sleep loss. Preventing the downregulation of Delta by overexpressing a wild-type transgene in MBs reduces sleep homeostasis and, importantly, prevents learning impairments induced by sleep deprivation. Similar resistance to sleep loss is observed with Notchspl-1 gain-of-function mutants. Immunohistochemistry reveals that the Notch receptor is expressed in glia, whereas Delta is localized in neurons. Importantly, the expression in glia of the intracellular domain of Notch, a dominant activated form of the receptor, is sufficient to prevent learning deficits after sleep deprivation. Together, these results identify a novel neuron-glia signaling pathway dependent on Notch and regulated by bunched. These data highlight the emerging role of neuron-glia interactions in regulating both sleep and learning impairments associated with sleep loss (Seugnet, 2011).

The evidence presented suggests that Notch signaling controls factors that reduce the negative consequences of waking as measured by an attenuated sleep rebound and intact learning following 12 hr of sleep deprivation. Although it is tempting to speculate that the intact learning seen following sleep loss is simply due to the flies not being sleepy, previous studies have shown that sleepiness does not result in performance impairments in aversive phototaxic suppression (APS; Seugnet, 2008). Thus, Notch activation may preserve learning by preventing neuronal overstimulation during extended waking. Reducing neuronal stimulation may also prevent the buildup of sleep debt and thus explain the lack of sleep rebound. Canonical Notch signaling leads to Su(H)-dependent changes in transcription, but several other downstream pathways have been identified; thus, further work is required to determine which pathway downstream of the receptor is effectively involved in this context. The results suggest that Notch is mediating a neuron-glia signaling mechanism. These data provide additional support to recent work showing an involvement of glia in sleep homeostasis and cognitive impairments. In mammals, adenosine released by glia appears to play a critical role (Halassa, 2009). Given that mutants for the only known Drosophila adenosine receptor have normal sleep homeostasis (Wu, 2009), other factors are likely to be involved. It is interesting to note in this context that expression of the cell adhesion molecule Klingon, required for long-term memory and controlled by Notch in the adult brain, has been reported to be expressed in the glia. It should be noted that Notch localization and activation in glia may seem at odds with reports showing a requirement for Notch as well as the downstream effector Su(H) in MB neurons for memory consolidation. The data do not exclude a low level of Notch expression in neurons. In fact, it would not be surprising if Notch is expressed in both cell types and mediates two-way signaling between adjacent cells, given that it occurs commonly during developmental processes (Seugnet, 2011).

Table of contents

Notch: Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions | Post-transcriptional regulation of Notch mRNA | Developmental Biology | References

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