escargot


DEVELOPMENTAL BIOLOGY

Gene expression is primarily ectodermal, occuring approximately at cell cycle 11 in the syncytial blastoderm stage. Transcripts are detected along the dorsal surface, just anterior to the region where the cephalic furrow will form. Prior to gastrulation, dorsal expression disappears and a grid-like pattern is found in the neurogenic region of the ventral ectoderm and in the head region. Later transcripts are found in the ventral midline. After germ band shortening, transcription is seen in leg, wing, haltere, and genital imaginal discs. Strong expression is also seen in the anterior region, including cells outlining the site of head involution [Images], and the dorsal ridge and frontal sac. histoblast cells of the abdominal segments also express escargot (Whiteley, 1992).

Expression of escargot in imaginal tissues is also found in third instar larvae (Hayashi, 1993).

Cellular interaction between the proximal and distal domains of the limb plays key roles in proximal-distal patterning. In Drosophila, these domains are established in the embryonic leg imaginal disc as a proximal domain expressing escargot, surrounding the Distal-less expressing distal domain in a circular pattern. The leg imaginal disc is derived from the limb primordium that also gives rise to the wing imaginal disc. Essential roles of Wingless in patterning the leg imaginal disc are described. (1) Wingless signaling is essential for the recruitment of dorsal-proximal, distal, and ventral-proximal leg cells. Wingless requirement in the proximal leg domain appears to be unique to the embryo, since it has previously been shown that Wingless signal transduction is not active in the proximal leg domain in larvae. (2) Downregulation of Wingless signaling in wing disc is essential for its development, suggesting that Wg activity must be downregulated to separate wing and leg discs. In addition, evidence is provided that Dll restricts expression of a proximal leg-specific gene expression. It is proposed that those embryo-specific functions of Wingless signaling reflect its multiple roles in restricting competence of ectodermal cells to adopt the fate of thoracic appendages (Kubota, 2003).

At embryonic stage 11, the early expression of D11 expression marks the entire limb primordium that gives rise to both wing and leg discs. After separation of wing and leg discs at stage 12, Dll expression becomes restricted to the center of the leg disc. Double labeling of stage 15 leg discs reveals that there is still a significant number of cells that coexpress Dll and the proximal leg marker Esg, suggesting that expression of Dll and Esg is not a strictly exclusive event. Rather, the result suggests that those marker genes respond differentially to inductive signals in the leg primordium (Kubota, 2003).

In the leg disc, Hth defines the trunk and proximal cell identities, and its expression is excluded in the distal leg domain in the larval stage. Double labeling with antibodies against Esg and Hth reveal that the Esg expression overlaps with Hth expression. Esg is used as a marker uniquely labeling the distinct cell identity of the proximal leg domain in the trunk region (Kubota, 2003).

The expression domain of wg and the position of wing and leg primordia were compared. Wg expression in the trunk ectoderm starts as stripes along the anterior side of the compartment boundaries. At early stage 11, most of the limb primordia marked with Dll protein expression overlap with wg stripes, as revealed by the wg-lacZ reporter. At late stage 11, wg-lacZ stripes break up into dorsal patches and ventromedial stripes. By late stage 12, expression of Dll protein becomes limited to a group of cells partially overlapping the dorsal edge of ventromedial wg stripes. The ventromedial wg stripe also overlaps with proximal leg cells that are labeled with anti-Esg at stage 15. The ventral half of proximal leg cells is nearly completely included within the ventral wg stripes. The dorsal half of leg cells is also located adjacent to, but not included in, the dorsal edge of the wg stripes. On the other hand, a reciprocal relationship between wg expression and wing primordia was observed. When wing primordia are first recognizable at stage 12 as cells expressing Vestigial (Vg), they do not overlap with the stripe of wg. Dorsal cell migration further separates wing primordia from the source of Wg at stage 15. The absence of Wg expression near wing primordia suggests that Wg does not play a positive role in wing disc development (Kubota, 2003).

To confirm whether Wg signaling is required cell autonomously for leg disc development, the dominant-negative forms of Drosophila TCF (DTCFdeltaN) or Drosophila axin (Daxin) were expressed in the limb primordia. The Dll-Gal4 driver, which is turned on in the limb primordium at stage 11 and continues to be active in leg and wing discs, was used. In Dll-GAL4 embryos carrying UAS-DTCFdeltaN or UAS-Daxin, the overall size of leg discs was reduced. Expression of Esg was preferentially reduced in the dorsal side. The drastic reduction of Dll mRNA and protein in distal leg cells in the armH8.6 mutants as well as in DTCFdeltaN- and Daxin-expressing embryos demonstrate that Wg signaling is required for both proximal and distal leg cells. In arm mutants, Hth-expressing cells expand to the distal domain. This observation suggests that, upon loss of Wg signaling, prospective leg disc cells lose their identity and adopt the fate of trunk ectoderm. However, disc-specific reduction of Wg signaling does not affect wing disc formation, although the Dll-Gal4 driver is active in the wing primordium. It is concluded that late function of Wg signaling promotes formation of the leg disc with a higher requirement in the proximal domain, but is dispensable for wing disc formation (Kubota, 2003).

Cell fate maintenance of proximal leg requires continuous signaling by Wg. The requirement for arm and wg is higher in the proximal leg domain. arm mutations nearly eliminate all Esg expression, but leave some Dll-positive cells. wgts is a hypomorph at the restrictive temperature and leaves distal leg cells nearly intact while significantly affecting proximal leg cells, especially those at the dorsal side of the disc. Dorsal cells are far from the source of Wg and are first to lose identity upon reduction of Wg activity. Since Esg expression in ventral proximal cells overlaps with the wg stripe, it is proposed that the localized expression of Wg and its range of diffusion are major determinants of the site of proximal cell formation. It is likely that dorsal-proximal cells require a higher level of Wg to be produced to reach their position (Kubota, 2003).

Dll expression is initially found in the entire limb primordia and becomes restricted to the edge of the Wg stripe that becomes the center of the leg disc. One candidate for an additional factor that places Dll in this position is Dpp, is expressed in stripes abutting the Wg stripe; Dpp is known to be required for distal leg development (Kubota, 2003).

Finally, the center of the embryonic leg disc is devoid of the expression of proximal cell markers Esg and Hth, marking the distal leg domain. Separation of the proximal and distal leg domains is a slow process, taking several hours to complete. One model for the mechanism regulating this separation process is that proximal gene expression is downregulated by a distal gene, as shown by ectopic Dll expression repressing Esg expression. Since expression of Esg is also regulated by positive input from Wg signaling, Esg expression does not necessary mirror the absence of Dll. In support of this idea, Dll is known to repress proximal genes in larval leg discs. The second possibility is a restriction of proximal cell movement into the distal domain. Cells in the Hth-expressing proximal domain in the larval leg disc have distinct cell-adhesive properties from those in the Dll-expressing distal domain, and by extension, cells with high levels of Dll or Hth may not mix well in the embryo as well. Since Hth is widely expressed in the embryonic ectoderm, Dll-expressing cells may be forced to localize at the center of the leg disc (Kubota, 2003).

Although the analogous pattern of Wg and Dpp expression plays essential roles in PD patterning in embryonic and larval leg development, significant differences are noted. In embryonic leg discs, expression of both proximal and distal leg markers is lost in mutants of Wg signaling or Dpp signaling. Therefore, Wg and Dpp contribute to both proximal and distal leg development in the embryo. In the larvae, reduction of Wg and Dpp expression due to the loss of hh function causes a loss of the distal domain, but no effect on the proximal gene expression was observed, suggesting that Wg and Dpp play little or no role in the development of proximal domain. The inability of Wg or Dpp to participate in the proximal leg patterning in the larvae is due to, at least in part, the function of Hth to block activation of target genes for Wg and Dpp. In the embryo, however, Hth does not block expression of esg, a target gene for Wg, as demonstrated by coexpression of Esg and Hth. Therefore, proximal domains of embryonic and larval leg discs are different in the way Hth regulates target genes for Wg. This difference may reflect distinct stages of leg development in the embryo, where proximal leg and epidermal cells are continuous, as defined by Hth expression, and in the larvae, where they are separated by the peripodial membrane (Kubota, 2003).

The complementary pattern of Wg and Dpp expression in the larval leg disc is maintained by mutual repression. No evidence for mutual repression of Wg and Dpp was observed in embryonic leg discs. Perhaps the complementary expression pattern of Wg and Dpp in the embryonic leg disc is under the control of the mechanism regulating the global dorsoventral pattern of the embryo (Kubota, 2003).

Thus, in Drosophila, specific mechanisms are involved in embryonic development as opposed to larval leg development. This finding gives rise to the question as to which of the mechanisms is used in other primitive hemimetabolous insects, where the specification and growth of the leg occur simultaneously (Kubota, 2003).

Evidence that stem cells reside in the adult Drosophila midgut epithelium: expression of escargot in dividing cells

Adult stem cells maintain organ systems throughout the course of life and facilitate repair after injury or disease. A fundamental property of stem and progenitor cell division is the capacity to retain a proliferative state or generate differentiated daughter cells; however, little is currently known about signals that regulate the balance between these processes. A proliferating cellular compartment has been characterized in the adult Drosophila midgut. Using genetic mosaic analysis it has been demonstrated that differentiated cells in the epithelium arise from a common lineage. Furthermore, reduction of Notch signalling leads to an increase in the number of midgut progenitor cells, whereas activation of the Notch pathway leads to a decrease in proliferation. Thus, the midgut progenitor's default state is proliferation, which is inhibited through the Notch signalling pathway. The ability to identify, manipulate and genetically trace cell lineages in the midgut should lead to the discovery of additional genes that regulate stem and progenitor cell biology in the gastrointestinal tract (Micchelli, 2006).

The adult Drosophila midgut can be identified on the basis of two anatomical landmarks along the anterior-posterior axis of the gastrointestinal tract: the cardia and pylorus. The inner surface of the midgut is lined with a layer of cells that project into the gut lumen. These cells exhibit apical-basal polarity; staining for F-actin reveals the presence of a distinct striated border on their lumenal surface. This observation is consistent with the suggestion that the midgut is lined by a cellular epithelium (Micchelli, 2006).

Wild-type midguts were stained with 4,6-diamidino-2-phenylindole (DAPI) to reveal the distribution of cell nuclei within the tissue. Nuclei of the midgut display a distinct distribution and fall into two main categories. The most prominent cells lining the midgut contain large oval nuclei that stain strongly with DAPI. These cells exhibit a region of the nucleus that does not stain with DAPI, giving the nucleus a hollow appearance. This unstained region may correspond to the large nucleolus characteristic of differentiated cells. A second population of cells containing small nuclei can be detected at a basal position within the tissue. The small nuclei are distant from the gut lumen and often lie in close apposition to the two layers of overlying visceral muscle that surround the gut. On the basis of nuclear size, position and morphology two general populations of midgut cells can, therefore, be distinguished (Micchelli, 2006).

Previous studies in Drosophila have led to conflicting views over the existence of cell proliferation in the adult gastrointestinal tract. Early reports suggested that somatic stem cells were present in the adult because of morphological similarity to certain larval cells and by analogy to different insect species. In contrast, 3H-thymidine labelling experiments detected DNA synthesis in the adult Drosophila midgut, but no mitotic figures were observed in a large sample analysed. On the basis of these observations, it was concluded that no somatic cell division occurs during the lifetime of Drosophila. To distinguish between these possibilities, a series of three independent assays was used to test whether cell proliferation can be detected in the adult midgut. In the first assay genetically marked wild-type cell lineages were used to identify dividing cells. The production of marked clones after mitotic recombination depends upon subsequent cell division and is, therefore, a direct means to assay proliferation. In these experiments, wild-type lineages were positively marked in adult flies using the MARCM system. Mitotic recombination was induced by heat shock and green fluorescent protein (GFP)-marked clones could be detected in the midgut. Similar results were obtained when adults were heat shocked up to 10 days after eclosion. This suggests that the ability to generate clones is not transient, and probably persists throughout the entire life of the animal (Micchelli, 2006).

Under the experimental conditions used, the MARCM system produced some background GFP signal that could be detected in control animals. To quantify the background signal, the number of GFP-labelled cells was compared in control and experimental animals. A greater than sixfold increase in the number of GFP-labelled cells was detected after heat shock. A second independent clone marking method was used that did not rely on either Gal4 or Gal80. In these experiments, clones were marked by the loss of a ubiquitously expressed GFP and similar results were observed. It is concluded that a population of actively dividing somatic cells is present in the adult Drosophila midgut (Micchelli, 2006).

To extend these findings, 5-bromodeoxyuridine (BrdU) incorporation studies were constructed. Both large and small BrdU-labelled midgut cells were detected. Large nuclei adjacent to each other can be differentially labelled, suggesting asynchrony in the timing or extent of DNA synthesis over the course of the labelling period. This is consistent with the notion that the large nuclei are endoreplicating. However, both endoreplication and the canonical cell cycle require new DNA synthesis. To distinguish endoreplicating from dividing cells in the midgut the tissue was stained with an antibody raised against phospho-histone H3. Careful examination revealed that very low levels of phospho-histone H3 staining could be detected in all cells. However, double staining with DAPI revealed that elevated levels of phospho-histone H3 indicative of mitosis could be detected only among the population of cells with small nuclei. Thus, cells in the midgut seem to have two distinct cell cycles; whereas both large and small nuclei undergo DNA synthesis, only the cells with small nuclei undergo cell division (Micchelli, 2006).

In order to characterize further the small cell population, an expression screen was conducted to identify cell-specific molecular markers. Three markers expressed in small cells were identified: escargot (esg), a transcription factor that belongs to the conserved Snail/Slug family; prospero (pros), a conserved homodomain transcription factor, and Su(H)GBE-lacZ, a transcriptional reporter of the Notch signalling. Simultaneous detection of esg expression (esg-Gal4, UAS-GFP), anti-Pros, Su(H)GBE-lacZ expression and DAPI has demonstrated that small cells can be subdivided into the following classes on the basis of differential gene expression: esg-positive (esg+), pros-positive (pros+), esg-negative pros-negative (esg- pros-), esg-positive Su(H)GBE-lacZ-positive [esg+ Su(H)GBE-lacZ+] and esg-positive Su(H)GBE-lacZ-negative [esg+ Su(H)GBE-lacZ-]. esg+ and pros+ expression define distinct cell populations, whereas Su(H)GBE-lacZ expression subdivides the esg+ class into esg+ Su(H)GBE-lacZ+ and esg+ Su(H)GBE-lacZ- subpopulations. Quantification reveals that each cell type is present in the midgut in different proportions. The ability to distinguish different cell types using molecular markers enabled determination of the cell lineage relationships in this tissue. If the large and small nuclei are lineally distinct then marked clones should be restricted to one or the other cell type. However, if a common stem cell progenitor exists in the adult midgut, then marked lineages should contain both large and small nuclei within a clone. To distinguish between these possibilities positively marked MARCM clones were generated and nuclei were labeled using DAPI. Lineage analysis shows that marked clones generated in the adult contain both large and small nuclei. In addition, both esg expression and anti-Pros-labelled cells could be detected within the clones. These lineage-tracing experiments suggest that a stem cell progenitor exists and is sufficient to generate the distinct cell types of the adult midgut. This cell is referred to as the adult intestinal stem cell (ISC) (Micchelli, 2006).

esg expression in diploid cells has been shown to be necessary for the maintenance of diploidy. In addition, the distribution of esg messenger RNA has been used as a marker for male germline stem cells. Together, these observations raise the hypothesis that esg expression may also mark a population of progenitors in the midgut. It was therefore asked whether esg expression correlates with markers of cell proliferation. Simultaneous staining with anti-BrdU and DAPI reveals that esg-expressing cells are among the population of cells that are also positively labelled by BrdU. To ask whether esg-expressing cells also undergo cell division, the midgut was double stained to detect both esg expression and phospho-histone H3. High levels of phospho-histone H3 can be detected specifically in esg-expressing cells. These results demonstrate that esg expression marks a population of proliferating progenitor cells in the midgut (Micchelli, 2006).

However, the esg+ cell population can be divided on the basis of Su(H)GBE-lacZ expression. To distinguish functionally the two esg+ populations, the consequences of altering Notch signalling in the adult midgut were examined. The effect of globally reducing Notch signalling was tested using the conditional Notch temperature-sensitive (Nts) mutant. Nts flies were first crossed to an allelic series that included N55e11, N264.47, Nts1 and Nnd.1. The strongest loss of function combinations (Nts/N55e11 and Nts/N264.47) failed to generate viable adult flies even at the permissive temperature, often dying as pharate adults. Nts/Nts flies produced viable adults at the permissive temperature with midguts similar to wild type. Nts/Nts flies shifted to the non-permissive temperature led to a mild increase in the number of small cells. The weakest allelic combination, Nts/Nnd.1, also produced viable adults at the permissive temperature but showed no detectable phenotype when shifted to the non-permissive temperature (Micchelli, 2006).

The requirement of N only in esg+ progenitor cells was tested. To obtain both spatial and temporal control over transgene expression in esg-expressing cells, the temperature-sensitive Gal80 inhibitor, Gal80ts was combined with the esg-Gal4 transcriptional activator. To verify that the Gal80ts transgene functions in the midgut, the temporal and spatial induction of a UAS-GFP transgene was characterized. Adult esg-Gal4,UAS-GFP, tub-Gal80ts flies grown at the permissive temperature showed no detectable GFP expression in their midguts In contrast, when these flies were shifted to the non-permissive temperature they showed high levels of GFP expression that were detectable after 1 day and maximal by 2 days (Micchelli, 2006).

The requirement of Notch was then tested in esg+ cells using a UAS-NRNAi transgene, to reduce Notch signalling. In control experiments, UAS-NRNAi; esg-Gal4,UAS-GFP, tub-Gal80ts flies grown at the permissive temperature appear to have wild-type midguts and show no detectable GFP expression, suggesting that under these conditions UAS transgenes are efficiently suppressed. In contrast, UAS-NRNAi; esg-Gal4,UAS-GFP, tub-Gal80ts flies shifted to the non-permissive temperature show an increase in the number of small cells (19 out of 20 midguts). Notably, the presence of esg-Gal4, UAS-GFP in this experiment enabled a determination that the increased number of small cells were also esg+. When these guts were co-stained with anti-Pros antibody ectopic small cells were observed that also expressed pros, and these cells were often associated with lower levels of esg expression. Taken together these experiments suggest that Notch signalling in esg+ cells is necessary to restrict proliferation (Micchelli, 2006).

The effect of Notch activation was tested in esg+ cells using Nintra, a constitutively active form of Notch. In control experiments, esg-Gal4,UAS-GFP, tub-Gal80ts; UAS-Nintra flies grown at the permissive temperature appear to have wild-type midguts and show no detectable GFP expression. In contrast, esg-Gal4,UAS-GFP, tub-Gal80ts; UAS-Nintra flies shifted to the non-permissive temperature showed a decrease in phospho-histone H3 staining compared to controls that were not shifted. In addition, although some esg+ cells appear to be wild type, a region-specific decrease was observed in the levels of esg expression and a concomitant increase in nuclear size similar to that of midgut epithelial cells. These observations demonstrate that Notch activation is sufficient to limit proliferation of esg+ cells and suggests that Notch may also be sufficient to promote early steps of epithelial cell differentiation (Micchelli, 2006).

This characterization of the adult Drosophila midgut suggests that a population of adult stem cells resides within this tissue. This analysis of the Notch signalling pathway in esg+ cells suggests that esg+ Su(H)GBE-lacZ- cells mark a population of dividing progenitors and that Notch is necessary and sufficient to regulate proliferation. A model is proposed in which esg+ Su(H)GBE-lacZ- progenitors generate at least two different types of daughter cells depending on the level of Notch activation. Under conditions of reduced Notch function an expansion of both esg+ progenitor cells and pros+ cells is observed. These observations suggest that esg+ cells give rise to pros+ cells in a Notch-independent manner. Under conditions of Notch activation a decrease is observed in the proliferation and promotion of epithelial cell fate differentiation, while the number of pros+ cells remains unaffected (Micchelli, 2006).

Several lines of evidence suggest that pros+ cells correspond to gut enteroendocrine cells. Previous studies show that prox1, the vertebrate pros homologue, is associated with post-mitotic cells and early steps of differentiation in the central nervous system. Furthermore, in Drosophila, pros is thought to be a pan-neural selector gene that is both necessary and sufficient to terminate cell proliferation. Finally, although vertebrate enteroendocrine cells arise from endodermal origins they are known to express neural-specific markers. Therefore, pros+ cells probably define a population of enteroendocrine cells in the midgut (Micchelli, 2006).

Studies of stem cell compartments in Drosophila have led to the characterization of two types of progenitor cells in the germ line. The first is referred to as the germline stem cell and is sufficient to give rise to the respective cells of either the male or female germ line. The second type of progenitor cell described is called the cystoblast in female germ line and gonialblast in the male germ line. Although the cystoblast and gonialblast both have the capacity to generate the differentiated cells of their respective tissues, they are thought to be more restricted in their fate than the germline stem cells. On this basis, it is suggested that an analogous progenitor may also exist in the adult Drosophila midgut; this cell is referred to as the enteroblast (EB). The population of esg+ Su(H)GBE-lacZ- progenitor cells, which has been described, displays characteristics of both the ISC and the EB; therefore, additional experiments will be necessary to distinguish unambiguously these alternatives (Micchelli, 2006).

EGFR signaling regulates the proliferation of Drosophila adult midgut progenitors

In holometabolous insects, the adult appendages and internal organs form anew from larval progenitor cells during metamorphosis. The adult Drosophila midgut, including intestinal stem cells (ISCs), develops from adult midgut progenitor cells (AMPs) that proliferate during larval development in two phases. Dividing AMPs, as visualized using esgGal4-driven GFP expression, first disperse, but later proliferate within distinct islands, forming large cell clusters that eventually fuse during metamorphosis to make the adult midgut epithelium. Signaling through the EGFR/RAS/MAPK pathway is necessary and limiting for AMP proliferation. Midgut visceral muscle produces a weak EGFR ligand, Vein, which is required for early AMP proliferation. Two stronger EGFR ligands, Spitz and Keren, are expressed by the AMPs themselves and provide an additional, autocrine mitogenic stimulus to the AMPs during late larval stages (Jiang, 2009).

Drosophila AMPs were previously thought to be relatively quiescent during larval development, dividing just once or twice, and not initiating rapid proliferation until the onset of metamorphosis. This is the case for several other larval progenitor/imaginal cell types, such as the abdominal histoblasts and cells in the salivary gland, foregut and hindgut imaginal rings. Studies have suggested that AMP proliferation might precede the onset of metamorphosis. However, the extensive proliferation of the AMPs that is seen in this study has not been reported and the early larval proliferative phase when the AMPs divide and disperse has not been reported. The extensive proliferation of the AMPs is similar to that of the larval imaginal disc cells, which also proliferate throughout larval development, dividing about ten times (Jiang, 2009).

AMPs occurs in two distinct phases. In early larvae, the AMPs divide and disperse throughout the midgut to form individual islets. During later larval development, the AMPs continue to divide but do so within these islets, forming large cell clusters. It is speculated that in the early larva, secretion of Vn from the midgut visceral muscle (VM) cells results in low-level activation of EGFR signaling in the AMPs, which is sufficient for their proliferation and might also promote their dispersal. No proliferation defects were seen in AMPs defective in shot function, suggesting that the mechanism of EGFR activation used by tendon cells during muscle/tendon development is probably not the same as in the larval midgut. Specifically, it is unlikely that the Shot-mediated concentration of Vn on AMPs activates EGFR signaling in the AMPs during early larval development. Consistent with this, dpERK staining is only seen in AMP clusters and not in the isolated AMPs present at early larval stages (Jiang, 2009).

The mechanisms that regulate the transition between these two proliferation phases remain unclear. Fewer AMP clusters are seen when sSpi, sKrn, lambdaTOP (activated Egfr) or RasV12 were induced in the AMPs starting from early larval stages, suggesting that EGFR signaling, in addition to its crucial role as an AMP mitogen, might also play a role in AMP cluster formation. In the late larval midgut (96-120 hours AED), high-level EGFR activation, resulting from expression of spi and Krn in the AMPs themselves, might not only promote AMP proliferation, but might also suppress AMP dispersal and thus promote formation of the AMP clusters. How the timing and location of Spi- or Krn-mediated EGFR activation are regulated during larval development is also unclear. It is noted, however, that the pro-ligand form of Krn acted similarly to sKrn, and that no functions were uncovered for the Rho-like gene products that regulate Spi and Krn function by proteolytic cleavage in other tissues. This suggests that the localized expression of these ligands in the AMP clusters might be the critical parameter that controls their effects. Consistent with this, Rho-independent cleavage and function of Krn have been documented (Reich, 2002; Jiang, 2009).

In the developing Drosophila wing, EGFR/RAS/MAPK signaling promotes the expression and controls the localization of the cell adhesion molecule Shotgun (Shg, Drosophila DE-cadherin). RasV12-expressing clones generated in the wing imaginal disc are round, much like the AMP clusters described in this study, owing to increased adhesive junctions. In developing Drosophila trachea, EGFR activity upregulates shg expression to maintain epithelial integrity in the elongating tracheal tubes. In the eye, EGFR activity leads to increased levels of Shg and adhesion between photoreceptors. Given these precedents, it seems reasonable to suggest that high-level EGFR activity in the AMP islets upregulates Shg and promotes the homotypic adhesion of the AMPs. Alternatively, changes in the differentiated cells of the midgut epithelium might promote AMP clustering. In either case, the dispersal of early AMPs and subsequent formation of late AMP clusters facilitate the formation of the adult midgut epithelium during metamorphosis (Jiang, 2009).

This study confirms previous reports that Drosophila AMPs replace larval midgut epithelial cells to form the adult midgut epithelium during metamorphosis. Furthermore, it was shown that the majority of AMPs lose esgGal4-driven GFP expression as they differentiate to form the new adult midgut epithelium. These cells lacked Prospero, which marks enteroendocrine cells in both the larval and adult midgut. They went through several rounds of endoreplication during late pupal development, and thus probably all differentiated into adult enterocytes (ECs). During early metamorphosis, some cells in the new midgut epithelium remained small and diploid and maintained strong esgGal4 expression. For several reasons, it is thought that these esg-positive cells are the future adult intestinal stem cells (ISCs). (1) esgGal4 expression marks AMPs, including adult ISCs and enteroblasts. (2) Mitoses in the adult midgut are only observed in ISCs, and this study observed mitoses only in the esg-positive cells during metamorphosis. (3) esg-positive cells migrated to the basal side of the midgut epithelium, the location of adult ISCs. (4) AMP clones generated during early larval development contained just a few esg-positive cells when the new adult midgut first formed (24 hours APF), but when such clones were scored in newly eclosed adults, they contained large numbers of ECs, as well as cells positive for the enteroendocrine marker Prospero and the ISC marker Delta. This suggests that a small fraction of AMPs differentiate into adult ISCs. However, esg-positive cells in the new pupal midgut lacked Delta expression until eclosion, suggesting that they are probably not mature adult ISCs (Jiang, 2009).

How a small fraction of AMPs are selected to become adult ISCs in the newly formed pupal midgut epithelium is not known. One possibility is that the adult ISCs are determined during larval development, long before the formation of the adult midgut. Another is that they are specified during early metamorphosis. This second hypothesis is preferred for several reasons. First, in the lineage analysis, it was found that all AMP clones induced during early larval stages formed multiple clusters. This suggests that there are no quiescent AMPs in the larval midgut. Second, when AMP clones were induced at mid-third instar, the mosaic clusters always contained multiple GFP-positive cells, suggesting that all AMPs in the mid-third instar midgut remain equally proliferative. Third, during larval development, differentiation of the AMPs were never observed, as judged by their ploidy (diploid) and lack of expression of the enteroendocrine marker Prospero. Fourth, all AMPs appeared to express esgGal4 throughout larval development. Given the crucial role that Notch signaling plays in regulating AMPs during embryonic midgut development and ISCs in adult midgut homeostasis, it is edexpect that Notch might also function to specify adult ISCs during metamorphosis (Jiang, 2009).

EGFR signaling is both required and sufficient to promote AMP proliferation. Hyperactivation of EGFR signaling, such as by expression of activated Ras (RasV12), promoted massive AMP overproliferation and generated hyperplastic midguts that were clearly dysfunctional. In contrast, inhibiting EGFR/RAS/MAPK signaling dramatically reduced AMP proliferation. Furthermore, the ability of EGFR signaling to induce ectopic AMP proliferation is almost unique. With the exception of larval hemocytes, activated EGFR signaling does not promote cell proliferation in the imaginal discs, salivary gland imaginal rings, abdominal histoblasts, foregut and hindgut imaginal rings. This suggests that the regulation of AMP proliferation is different from that in other imaginal cells (Jiang, 2009).

Despite the obvious differences between adult ISCs and their larval progenitors, the AMPs, there are also similarities. (1) When the new adult midgut epithelium forms, larval AMPs give rise to the new adult midgut including the adult ISCs. Many genes, such as esg, that are specifically expressed in the larval AMPs are also expressed in the adult ISCs. (2) The structure of the midgut epithelium with basal AMPs or ISCs is similar in larval and adult stages. (3) vn expression in larval VM persists in the adult midgut, suggesting that Vn from the adult VM might also regulate the ISCs (Jiang, 2009).

In two Drosophila stem cell models, the testis and ovary, stem cells reside in special niches comprising other supporting cell types. These niches maintain the stem cells and provide them with proliferative cues. For example, in the testis, germ stem cells attach to the niche that comprises cap cells. The cap cells release Jak/Stat and BMP ligands [Upd (Os) and Gbb/Dpp], which maintain the stem cells and induce their proliferation. Whether Drosophila ISCs utilize supporting cells that constitute a niche remains unclear. This study shows that multiple EGFR ligands are involved in the regulation of Drosophila AMP proliferation. During early larval development, the midgut VM expresses the EGFR ligand vn, which is required for AMP proliferation. Thus, the early AMPs might be considered to require a niche comprising non-epithelial VM. Later in larval development, however, the AMPs express two other EGFR ligands, spi and Krn, which are capable of autonomously promoting their proliferation and may render vn dispensable. This study found, however, that depleting spi and Krn in the AMPs did not affect AMP proliferation, suggesting that vn or another trigger of EGFR/RAS/MAPK activity might complement spi and Krn in late-stage larvae (Jiang, 2009).

Multipotent somatic stem cells contribute to the stem cell niche in the Drosophila testis

Adult stem cells reside in specialized microenvironments, or niches, that have an important role in regulating stem cell behaviour. Therefore, tight control of niche number, size and function is necessary to ensure the proper balance between stem cells and progenitor cells available for tissue homeostasis and wound repair. The stem cell niche in the Drosophila male gonad is located at the tip of the testis where germline and somatic stem cells surround the apical hub, a cluster of approximately 10-15 somatic cells that is required for stem cell self-renewal and maintenance. Somatic stem cells in the Drosophila testis contribute to both the apical hub and the somatic cyst cell lineage. The Drosophila orthologue of epithelial cadherin (DE-cadherin) is required for somatic stem cell maintenance and, consequently, the apical hub. Furthermore, the data indicate that the transcriptional repressor escargot regulates the ability of somatic cells to assume and/or maintain hub cell identity. These data highlight the dynamic relationship between stem cells and the niche and provide insight into genetic programmes that regulate niche size and function to support normal tissue homeostasis and organ regeneration throughout life (Voog, 2008).

Many stem cell niches include support cells that influence stem cell behaviour through secretion of diffusible molecules. Physical contact between stem cells and support cells and/or the extracellular matrix holds stem cells within the niche and close to self-renewal signals. Furthermore, niches provide spatial and mechanical cues that influence the fate of stem cell daughters. Therefore, the stem cell niche has an important role in regulating stem cell maintenance, self-renewal and survival. However, little is known about the factors that regulate niche maintenance or size (Voog, 2008).

Approximately ten somatic cells, called the hub, are found at the apical tip of the Drosophila testis. Germline stem cells (GSCs) and somatic stem cells (SSCs) surround and are in contact with hub cells. Whereas GSCs sustain spermatogenesis, SSCs produce cyst cells that encapsulate the maturing germ cells and ensure differentiation. Hub cells secrete the growth factor Unpaired (Upd), which activates the JAK-STAT signal transduction pathway in adjacent stem cells. JAK-STAT signalling is necessary for stem cell maintenance and is sufficient to specify self-renewal of both GSCs and SSCs in the testis (Voog, 2008).

The apical hub is typically described as a post-mitotic, static structure. However, in agametic flies, SSCs proliferate and express hub markers, leading to an apparent expansion of the apical hub. Furthermore, recent studies have noted that hub cell number and function decrease with age, indicating that the stem cell niche in the testis is dynamic. Hub cells and SSCs share numerous features, including similar gene expression patterns and close association with GSCs and each other; however, the precise relationship between SSCs and hub cells has not been explored (Voog, 2008).

It has been proposed that SSCs may serve as a source of cells that contribute to the apical hub and, consequently, the stem cell niche. To address whether SSCs give rise to hub cells, positively marked beta-galactosidase-expressing (beta-gal+) SSCs were generated using mitotic recombination, a technique typically used for lineage tracing analyses. Labelled SSCs were generated by heat-shocking flies of the appropriate genotype to initiate FLP-mediated recombination, resulting in reconstitution of the alpha-tubulin promoter upstream of the lacZ gene1. Hub cells were identified by immunolabelling with antibodies to Fasciclin III (FasIII)14 or DE-cadherin, cell-surface proteins concentrated at hub cell junctions. SSCs and early cyst cells were identified by immunolabelling with antibodies to Traffic Jam (TJ), a transcription factor that is strongly expressed in early cyst cell nuclei and weakly expressed in hub cells (Voog, 2008).

Wild-type SSC clones were identified as beta-gal+ cells adjacent to the apical hub and surrounding germ cells. A series of heat shocks after eclosion (hatching) led to at least one beta-gal+ SSC in 57% of testes from 3-day-old males analysed 1 day after heat shock. At 5 days after heat shock, 28% of testes contained at least one beta-gal+ SSC, and this frequency decreased to 10%, 10% and 4% at 10, 15 and 30 days after heat shock, respectively (Voog, 2008).

In addition to beta-gal+ SSCs, beta-gal+ hub cells were also observed that co-labelled with DE-cadherin and FasIII. In fact, beta-gal+ cells were found within the hub in 34%, 47% and 60% of testes from males at 1, 5 and 15 days after heat shock, respectively. No beta-gal+ cells were observed in flies not exposed to the heat-shock protocol (Voog, 2008).

Previous reports concluded that hub cells are post-mitotic; however, it is possible that hub cells undergo rare divisions to become marked during recombination. To test whether hub cells are mitotic, dividing cells in the testis were labelled with 5'-bromo-2-deoxyuridine (BrdU), which is incorporated into newly synthesized DNA during S phase. Flies were fed ('pulsed') BrdU for 30 min and aged ('chased') for up to 15 days. Subsequently, labelled testes were co-stained with antibodies to BrdU, as well as to the hub marker FasIII. No BrdU-positive (BrdU+) hub cells were detected after a 1-day chase, although cells adjacent to the hub were clearly labelled (Voog, 2008).

However, BrdU+ hub cells were observed 3-10 days after labelling. BrdU+ hub cells were present in 4%, 8% and 3% of testes assayed at 5, 7-8 and 10 days after labelling. Moreover, these BrdU+ cells co-expressed FasIII and an upd reporter, indicating that these cells function as hub cells. These data are consistent with previous results indicating that hub cells are post-mitotic and support the hypothesis that mitotically active SSCs act as a source of cells that can contribute to the apical hub (Voog, 2008).

SSCs are reported to be the only dividing somatic cells in the testis; however, two distinct populations of somatic cells were observed dividing near the testis tip. One group, which constituted 69% of phospho-histone-H3-positive somatic cells, appeared to be immediately adjacent to the hub, similar to GSCs. Somatic cells were also observed that were dividing 1-2 cell distances away from the hub. Several scenarios could explain these observations: there are two SSC populations, one which gives rise to hub cells and another that sustains the cyst cell lineage, or there are SSCs that produce both hub cells and a transient amplifying somatic cell population (Voog, 2008).

To distinguish between these possibilities, the heat-shock regime was adjusted to label, on average, only one beta-gal+ somatic cell, and the clones derived from these marked cells were analysed. Thirteen per cent of testes examined contained marked somatic cells adjacent to the hub at 1 day after heat shock, which decreased to 9.5%, 3.3% and 2.8% of testes at 5, 10 and 15 days after heat shock, indicating a half-life for SSCs between 5-10 days. Single, marked somatic cells displaced away from the hub were found initially in 11.3% of testes examined at 1 day after heat shock, but this number decreased to 0.5% by 5 days after heat shock, which is consistent with these cells being transient non-stem-cell clones. In contrast, the number of testes that contained marked hub cells increased from 11.9% at 1 day after heat shock to 25.8%, 24.5% and 18.1% at 5, 10 and 15 days after heat shock, respectively. Clones containing all three cell types were observed in 22% and 14% of testes that contained marked SSCs at 5 and 10 days after heat shock, respectively. From these data it is concluded that multipotent SSCs self-renew and contribute to both hub and cyst cell lineages, whereas dividing cyst cells, which are called cyst progenitor cells (CPCs), expand the pool of cells capable of encapsulating newly divided gonialblasts and maturing spermatogonia to ensure terminal differentiation (Voog, 2008).

To identify factors required for incorporation of cells into the apical hub, SSCs were generated that were mutant for genes expressed in both cell types: DE-cadherin, which is encoded by the shotgun (shg) gene, and the transcriptional repressor Escargot (Esg). DE-cadherin is expressed in cyst cells and is strongly enriched in the hub. SSC clones were generated that were homozygous mutant for either the loss-of-function shgIG29 or amorphic shgIH allele. SSC maintenance and frequency of marked hub cells were assayed at various time points. In this experiment, marked cells and their progeny subsequently become permanently labelled by ubiquitous green fluorescent protein (GFP) expression (Voog, 2008).

Heat-shocked wild-type testes possessed GFP+ GSCs and SSCs, as well as GFP+ hub cells that co-stained with FasIII and DE-cadherin. In contrast to wild type, shg mutant GSC and SSC clones were not maintained, indicating that DE-cadherin has a role in stem cell maintenance in the testis, similar to its role in the ovary, presumably by holding stem cells within the niche and close to self-renewal signals (Voog, 2008).

Marked hub cells were observed in 14%, 35% and 65% of wild-type testes examined at 5, 10 and 15 days after heat shock, respectively. Notably, progeny of DE-cadherin mutant SSCs contributed to the apical hub at a frequency similar to progeny from wild-type SSCs. These data indicate that although DE-cadherin is required for SSC maintenance, it is not absolutely required for mediating the contribution of SSC progeny to the hub (Voog, 2008).

To confirm that shg is not required in hub cells for maintaining the apical hub, RNAi-mediated knockdown of shg expression was carried out in hub cells. A FasIII+ apical hub was detected in 100% of testes from 1-day-old, 10-day-old and 20-day-old males, despite a reduction in DE-cadherin expression in hub cells. Testes collected at 20 days also displayed normal expression of a upd reporter (98%) and contained TJ+ (100%) cells near the apical tip. These data support the findings that DE-cadherin is not absolutely required in hub cells to maintain a functional stem cell niche (Voog, 2008).

However, shg is required in SSCs and early cyst cells for maintaining the apical hub. Knockdown of shg in all SSCs and early cyst cells resulted in a decrease in the number of TJ+ cells in 1-day-old males, consistent with a role for shg in SSC maintenance. Surprisingly, decreased levels of DE-cadherin were also observed in hub cells. In 29% of 15-day-old and 44% of 20-day-old males, the apical hub was severely diminished or lost, as determined by FasIII expression. These data support the model that SSCs act as a source of cells to maintain the apical hub (Voog, 2008).

The transcriptional repressor Escargot is expressed in many tissues, including GSCs, early cyst cells and hub cells in the testis. Males carrying a viable, hypomorphic allele of esg, called shutoff, exhibit loss of apical hub cells during development. Therefore, it is hypothesized that esg may be required for regulating the contribution of SSCs to the hub. Mutant labelled SSCs were generated using two amorphic esg alleles (Voog, 2008).

Unlike progeny from shg mutant SSCs, progeny from esg mutant SSCs did not contribute to the hub at the same frequency as wild-type controls: esgL2 mutant GFP+ hub cells were observed in 5%, 3%, 0% and 4% of testes examined at 1, 5, 10 and 15 days after heat shock, respectively. In instances when esg mutant GFP+ hub cells were observed, normal hub morphology was often severely disrupted. These data suggest that esg regulates either the contribution of SSC progeny to the hub, perhaps by facilitating the cell fate transition between SSC and hub cell, or maintenance of hub cell fate (Voog, 2008).

To explore Esg function further, the agametic oskar (osk) mutant phenotype was used, in which SSCs proliferate and express hub markers, resulting in an apparent expansion of the apical hub. If Esg is required for mediating the transition of somatic cyst cells to the apical hub, it was predicted that the expansion of FasIII+ cells would be blocked in an esg;osk double mutant background. In contrast to the expansion of FasIII+ cells in 82% of osk mutant testes, only 22% of testes from esgshof;osk mutant males showed expansion of FasIII, despite there being clearly more TJ+ somatic cyst cells. These data support previous results and indicate that esg is required for the ability of somatic cells to assume and/or maintain hub cell fate (Voog, 2008).

These findings demonstrating that SSCs can adopt a hub cell fate highlight the dynamic nature of the stem cell-niche relationship and provide a mechanism to regulate the size and function of the stem cell niche in the Drosophila testis. In this model, as somatic cells are displaced from the hub, there is a decline in self-renewal and proliferation potential, which could be reinforced by encapsulation of differentiating germ cells. Interestingly, expansion of the somatic cyst cells as a consequence of germline loss suggests that germ cells exert an anti-proliferative influence that must be overcome in SSCs (Voog, 2008).

A better understanding of how stem cell niches are established and regulated in mammalian systems could facilitate modulation of the niche to enhance transplantation of stem cells in regenerative medicine. Conversely, if an expanded or modified niche accompanies tumour progression or metastasis, then blocking niche maintenance programmes (niche ablation) could be used as an important anti-cancer therapeutic (Voog, 2008).

A region-specific neurogenesis mode requires migratory progenitors in the Drosophila visual system

Brain areas each generate specific neuron subtypes during development. However, underlying regional variations in neurogenesis strategies and regulatory mechanisms remain poorly understood. In Drosophila, neurons in four optic lobe ganglia originate from two neuroepithelia, the outer (OPC) and inner (IPC) proliferation centers. Using genetic manipulations, this study found that one IPC neuroepithelial domain progressively transformed into migratory progenitors that matured into neural stem cells (neuroblasts) in a second domain. Progenitors emerged by an epithelial-mesenchymal transition-like mechanism that required the Snail-family member Escargot and, in subdomains, Decapentaplegic signaling. The proneural factors Lethal of scute and Asense differentially controlled progenitor supply and maturation into neuroblasts. These switched expression from Asense to a third proneural protein, Atonal. Dichaete and Tailless mediated this transition, which was essential for generating two neuron populations at defined positions. It is proposed that this neurogenesis mode is central for setting up a new proliferative zone to facilitate spatio-temporal matching of neurogenesis and connectivity across ganglia. (Apitz, 2014).

Recent studies have distinguished three neurogenesis modes in the Drosophila CNS. First, type I neuroblasts arise from neuroepithelia and generate GMCs, which produce neuronal and glial progeny. Second, Dpn+ type II neuroblasts in the dorsomedial central brain go through a transit-amplifying Dpn+, Ase+ population, called intermediate neural precursors, which generate GMCs and postmitotic offspring. Third, lateral OPC neuroepithelial cells bypass the neuroblast stage and generate lamina precursor cells (LPCs) that divide once to produce lamina neurons. The current results provide evidence for a fourth strategy: p-IPC neuroepithelial cells give rise to progenitors that migrate to a second neurogenic domain, where they mature into type I neuroblasts. These progenitors are distinct, as they originate from the neuroepithelium, do not express markers for neuroblasts, intermediate neural precursors, GMCs or postmitotic neurons, and acquire NSC properties after completing their migration (Apitz, 2014).

Migratory progenitors arise from the p-IPC by a mechanism that shares cellular and molecular characteristics with EMT. On the basis of data on gastrulation and neural crest formation, EMT is commonly associated with cells adopting a mesenchymal state, enabling them to leave their epithelial tissue and migrate through the extracellular matrix to new locations. A recent study also reported an EMT-like process in the mammalian neocortex, whereby newborn neurons and intermediate progenitors delaminate from the ventricular neuroepithelium and radially migrate to the pial surface. This study observed that neuroepithelial cells at the p-IPC margins and migratory progenitors upregulated the Snail homolog Esg, whereas E-cad levels were decreased. Moreover, esg knockdown caused the formation of ectopic E-cad-expressing clusters adjacent to the p-IPC. Although this is a previously uncharacterized role of Drosophila esg, these findings are consistent with the requirement of two Snail transcription factors, Scratch1 and 2, and downregulation of E-cad in cortical EMT migration (Apitz, 2014).

Although TGFβ signaling is well known to induce EMT, it was unclear whether it could have such a role in the brain. Two lines of evidence are consistent with a requirement of the Drosophila family member Dpp. First, it is expressed and downstream signaling is activated in dorsal and ventral p-IPC subdomains and emerging cell streams. Second, tkv mutant cells form small neuroepithelial clusters in p-IPC vicinity. Similar to the neural crest, where distinct molecular cascades control delamination in the head and trunk, region-specific regulators may also be required in p-IPC subdomains. Because neuroblasts derived from Dpp-dependent cell streams map to defined areas in the d-IPC, this pathway could potentially couple EMT and neuron subtype specification (Apitz, 2014).

Cell migration is an essential feature of vertebrate brain development. Commonly, postmitotic immature neurons migrate from their proliferation zones to distant regions, where they further differentiate and integrate into local circuits. Examples include the radial migration of projection neurons and tangential migration of interneurons in the embryonic cortex, as well as migration of interneuron precursors in the rostral migratory stream to the olfactory bulb in adults. In contrast, IPC progenitors develop into NSCs (neuroblasts) after they migrated. A recent study found that NSCs relocating from the embryonic ventral hippocampus to the dentate gyrus act as source for adult NSCs in the subgranular zone. In addition, cerebellar granule cell precursors migrate from the rhombic lip to the external granule layer, where they proliferate during early postnatal development. The migration of neural cell types that become proliferative in a new niche could therefore constitute a more general strategy. IPC progenitors form streams of elongated, closely associated cells. Despite their different developmental state, their organization is notably similar to the neuronal chain network in the lateral walls of the subventricular zone and the rostral migratory stream in mammals, or of migratory trunk neural crest cells in chick. Further studies will need to identify the determinants directing migratory progenitors into the d-IPC (Apitz, 2014).

Several constraints could shape a neurogenesis mode that requires migratory progenitors in the larval optic lobe. The OPC is located superficially and the IPC is positioned centrally. If medulla and lobula neurons arose by neuroepithelial duplications, these new populations would need to be integrated into an ancestral visual circuit consisting of lamina and lobula plate neurons. Cellular migration may therefore be a derived feature and serve as an essential spatial adjustment of the IPC to the newly added medulla. In principle, the migratory population could consist of immature neurons. However, migratory progenitors help to establish a new superficial proliferative niche, and to align OPC and d-IPC neuroblast positions. This in turn enables the OPC and IPC to use spatially matching birth order-driven neurogenesis patterns for establishing functionally coherent connections across ganglia (Apitz, 2014).

IPC progenitors were primed to mature into neuroblasts, but were prevented to do so in cell streams. Consistently, progenitors showed weak cytoplasmic Mira expression and prematurely differentiated into neuroblasts following loss of Pcl. Although Dichaete has been shown to repress ase to maintain embryonic neuroectodermal cells in an undifferentiated state, this study did not identify such a role in the IPC. Future studies are therefore required to distinguish whether this block in neuroblast maturation is released in the d-IPC by cell-intrinsic mechanisms or locally acting signals (Apitz, 2014).

The p-IPC and d-IPC consecutively expressed three proneural factors. esg-positive p-IPC neuroepithelial cells transiently expressed L'sc as they converted into progenitors. Following arrival in the d-IPC, progenitors matured into neuroblasts, which switched bHLH protein expression from Ase to Ato. This correlated with a change in cell division orientations from toward the lamina to the optic lobe surface and the generation of two lineages, distal cells and lobula plate neurons. The progression of neuroblasts through two stages is supported by the observations that progenitors solely entered the lower d-IPC, all neuroblasts were labeled with Ase in this area, and idpp reporter gene expression in a progenitor subset persisted in both lower and upper d-IPC neuroblasts and their progeny (Apitz, 2014).

Late l'sc knockdown reduced the number of d-IPC neuroblasts and both neuron classes, whereas p-IPC formation and EMT of progenitors appeared to be unaffected. This supports the idea that l'sc promotes neuroblast formation by controlling the rate of conversion and the progenitor supply. In contrast, ase loss severely decreased the amount of lower d-IPC neuroblasts and distal cells. This revealed a central role in the maturation of progenitors into neuroblasts, endowing them with the potential to proliferate and generate a specific lineage. Although these functions are the opposite of those observed in the OPC, they align with the role of a murine Ase homolog, Achaete-scute homolog 1 (Ascl1), in the embryonic telencephalon. Ase- neuroblasts with type I proliferation patterns have not previously been described. Further underscoring the context-dependent activities of proneural bHLH factors, ato does not have the equivalent role of ase in conferring neurogenic properties to upper d-IPC neuroblasts, but acts upstream of differentiation programs controlling the projections of lobula plate neurons (Apitz, 2014).

Although Ase and Ato each regulated distinct aspects of d-IPC development, they were not required for either the transition or the extent of their expression domains. These functions were fulfilled by Dichaete and tll, whose cross-regulatory interactions were essential for the transition from Ase+ to Ato+, Dac+ expression. To link birth order and fate, temporal identity transcription factors are sequentially expressed by neuroblasts and inherited by GMCs and their progeny born during a given developmental window. Acting as the final two members of the OPC-specific series of temporal identity factors, Dichaete is required for Tll expression, whereas tll is sufficient, but not required, to inhibit Dichaete Although OPC and d-IPC neuroblasts shared the sequential expression of Dichaete and Tll, key differences include the fact that d-IPC progeny did not maintain Dichaete, that Tll was transiently expressed in newborn progeny of the upper d-IPC and was not maintained in older lineages, that Dichaete in the lower d-IPC was not required in its own expression domain for neurogenesis, and that Dichaete was required to activate tll, and tll to repress Dichaete and ase, as well as to independently upregulate Ato and Dac. Although the mechanisms that trigger the timing of the switch require further analysis, these observations support the notion that, in the d-IPC, Dichaete and tll do not function as temporal identity factors, but as switching factors between two sequential neuroblast stages. The vertebrate homologs of Dichaete and tll, Sox2 and Tlx, are essential for adult NSC maintenance and Sox2 positively regulates Tlx expression, suiggesting that core regulatory interactions between Dichaete and tll family members may be conserved (Apitz, 2014).

These studies uncovered molecular signatures for generating a migratory neural population by EMT and subsequent NSC development that are in part shared between the fly optic lobe and vertebrate cortical neurogenesis. The unexpected parallels suggest that ancestral gene regulatory cassettes imparting specific cellular properties may have been re-employed during vertebrate brain development. Analysis of p-IPC and d-IPC neurogenesis in the Drosophila optic lobe therefore opens new possibilities for systematically identifying genes regulating EMT, cell migration and sequential NSC specification (Apitz, 2014).

The adult Drosophila malpighian tubules are maintained by multipotent stem cells

All animals must excrete the waste products of metabolism. Excretion is performed by the kidney in vertebrates and by the Malpighian tubules in Drosophila. The mammalian kidney has an inherent ability for recovery and regeneration after ischemic injury. Stem cells and progenitor cells have been proposed to be responsible for repair and regeneration of injured renal tissue. In Drosophila, the Malpighian tubules are thought to be very stable and no stem cells have been identified. This study has identified multipotent stem cells in the region of lower tubules and ureters of the Malpighian tubules. Using lineage tracing and molecular marker labeling, it was demonstrated that several differentiated cells in the Malpighian tubules arise from the stem cells and an autocrine JAK-STAT signaling regulates the stem cells' self-renewal. Identifying adult kidney stem cells in Drosophila may provide important clues for understanding mammalian kidney repair and regeneration during injury (Singh, 2008).

The regenerating renal cells may come from one of the three possible sources, based on previous studies. First, the circulating blood contains bone marrow-derived stem cells able to differentiate into non-haematopoietic cells, such as cells of the kidney. Second, the differentiated glomerular and tubular cells may also be able to dedifferentiate into stem-like cells to repair the damaged tissues. Third, large numbers of slowly cycling cells have recently been identified in the mouse renal papilla region; these cells may be adult kidney stem cells and may participate in renal regeneration after ischemic injury. Further, the ureter and the renal collecting ducts were formed from the epithelium originating from the ureteric bud, and the nephrons and glomeruli were formed from the metanephric mesoderm-derived portion during kidney development. Two distinguished stem cell types have been proposed as responsible for repairing the renal collecting tubules and the nephrons. This study identified a type of pluripotent stem cells (RNSCs) in the Drosophila renal organ. The stem cells are able to generate all cell types of the adult fly MTs. In the region of lower tubules and ureters, autocrine JAK-STAT signaling regulates the stem cell self-renewal. Weak JAK-STAT signaling may convert an RNSC into a renalblast (RB), which will differentiate into an RC in the region of lower tubules and ureters, and a type I or type II cell in the upper tubules. These data indicate that only one type of stem cell may be responsible for repair and regeneration of the whole damaged tissues in mammalian kidney (Singh, 2008).

The Drosophila RNSCs represent a unique model to study the molecular mechanisms that regulate stem cell or cancer stem cell behavior. In most of the stem cell systems that has been well characterized to date, stem cells always reside in a specialized microenvironment, called a niche. A niche is a subset of neighboring stromal cells and has a fixed anatomical location. The stromal cells often secrete growth factors to regulate stem cell behavior. The stem cell niche plays an essential role in maintaining stem cells, and stem cells will lose stem cell status once they are detached from the niche. The niche often provides the balanced (proliferation-inhibiting and proliferation-stimulating) signals that keep the stem cells dividing slowly. The inhibitory signals keep the stem cell quiescent most of the time while the stimulating signals promote stem cell division, to replenish lost differentiated cells. Maintaining the balance between proliferation-inhibiting and proliferation-stimulating signals is the key to maintaining tissue homeostasis (Singh, 2008).

Drosophila RNSCs are controlled differently. This study has demonstrated that the JAK-STAT signaling regulates the stem cell self-renewal. Both the ligand Upd and the receptor Dome are expressed in the RNSCs and the autocrine JAK-STAT signaling regulates the stem cell self-renewal; thus, the self-sufficient stem cells control their self-renewal or differentiation and do not need to constrained to a fixed niche. However, the RNSCs are still confined to the region of lower tubules and ureters even in the Upd overexpressed flies, suggesting that some other factors besides the JAK-STAT signaling may restrict the RNSCs to the region of the lower tubules and ureters (Singh, 2008).

Recent studies also suggest that tumors may arise from small populations of so-called cancer stem cells (CSCs). The CSCs probably have arisen from mutations that dysregulate normal stem cell self-renewal. For example, mutations that block the proliferation-inhibiting signals or promote the proliferation-stimulating signals can convert the normal stem cells into CSCs. This study demonstrates that amplifying the JAK-STAT signaling by overexpressing its ligand Upd stimulates the RNSCs to proliferate and also to differentiate into RC, which results in tumorous overgrowth in the MT. Therefore, the Drosophila RNSC system may also be a valuable in vivo system in which to study CSC regulation (Singh, 2008).

The RNSCs are located in the region of the lower tubules and ureter of the MTs, while ISCs are located at the posterior midgut. The MTs' ureters connect to the posterior midgut. The two types of stem cells are at close anatomical locations in the adult fly digestion system and also share some properties. For example, both of them are small nuclear cells, Arm-positive, and express esg. However, RNSCs and ISCs produce distinctly different progenies. ISCs produce progenies that include either Su(H)GBE-lacZ- or Pros-positive cells, which are not among the progenies of RNSCs because Su(H)GBE-lacZ and Pros are not expressed in the MTs. RNSCs produce progenies that include Cut- or TSH-positive cells, which are not among the progenies of ISCs because Cut and TSH are not expressed in the posterior midgut. One possibility for this difference is that, although RNSCs and ISCs originate from the same stem cell pool, their particular environments restrict their differentiation patterns. Future experiments, such as transferring RNSCs to the posterior midgut and vice versa, should be able to test this model (Singh, 2008).

The JAK-STAT signaling regulates self-renewal of the male germline, the male somatic, female escort stem cells in fly. The signaling also regulates self-renewal and maintenance of mammalian embryonic stem cells. This study reports that the JAK-STAT signaling regulates self-renewal of RNSCs. The JAK-STAT signaling may be a general stem cell signaling and also regulate stem cell self-renewal in other, un-characterized stem cell systems (Singh, 2008).

esg has been used as a marker of both male germline stem cells. This study has demonstrated that the esg-Gal4. UAS-GFP transgene is specifically expressed in RNSCs. The function of the esg gene is to maintain cells as diploid in Drosophila imaginal cells. Stem cells may have to be diploid, and esg may be a general stem cell factor. Identifying a stem cell signaling pathway (such as the JAK-STAT signal transduction pathway) and a stem cell factor (such as esg) will provide useful tools for identifying stem cells in other systems and for understanding stem cell regulation in general (Singh, 2008).

Robust intestinal homeostasis relies on cellular plasticity in enteroblasts mediated by miR-8-Escargot switch

This study used tracing methods that allow simultaneously capturing the dynamics of intestinal stem and committed progenitor cells (called enteroblasts) and intestinal cell turnover with spatiotemporal resolution. Intestinal stem cells (ISCs) divide 'ahead' of demand during Drosophila midgut homeostasis. Their newborn enteroblasts, on the other hand, take on a highly polarized shape, acquire invasive properties and motility. They extend long membrane protrusions that make cell-cell contact with mature cells, while exercising a capacity to delay their final differentiation until a local demand materializes. This cellular plasticity is mechanistically linked to the epithelial-mesenchymal transition (EMT) programme mediated by escargot, a snail family gene. Activation of the conserved microRNA miR-8/miR-200 in 'pausing' enteroblasts in response to a local cell loss promotes timely terminal differentiation via a reverse EMT by antagonizing escargot. These findings unveil that robust intestinal renewal relies on hitherto unrecognized plasticity in enteroblasts and reveal their active role in sensing and/or responding to local demand (Antonello, 2015).

The robustness of intestinal cell renewal relies on cellular plasticity in committed progenitor cells and a rather loose regulation of ISCs proliferation. One key finding is that stem cells divide continually and generate a 'stock' of committed progenitor cells that do not terminally differentiate right away but postpone their final differentiation for long time intervals in the absence of a local epithelial cell loss. Accordingly, one noticeable change in newborn progenitor cells after their (enterocyte) fate commitment is their transformation from rounded cells to spindle-shaped cells that appear to actively monitor their surroundings by extending long membrane actin-rich protrusions that make cell-cell contact with mature epithelial cells and their mother ISCs. Timely terminal differentiation with epithelial cell loss is orchestrated by activation of a conserved pro-epithelial microRNA, in turn, directly repressing the repressors of differentiation. A microRNA-induced repression of the repressors of differentiation provides a faster mechanism than one involving a transcriptional regulator since synthesizing a miRNA likely requires less time than synthesizing a protein. Importantly, mutual antagonism between the microRNA (MiR-8/miR-200) and its targets (Escargot/Snail2 and Zfh1/ZEB) may serve to slow down the mesenchymal-to-epithelial process inside individual mesenchymal/progenitor cells until they are successfully integrated in the epithelium. Consistently, abrupt transition as in mir-8 overexpressing midguts results in erroneous tissue repair (Antonello, 2015).

Supply and demand in business production involves frequently two alternative solutions called 'make-to-stock' and 'make-to-order'. In 'make-to-stock' or MTS, production is continuous so that response to customers can be supplied immediately. However, as production is not based on actual demand, the MTS solution is not robust against fluctuations in demand and errors in forecasting can result in shortages (if there is insufficient residual stock) or overproduction. In 'make-to-order', or MTO, production only starts upon receiving a customer's order, thereby precisely matching production to demand. However, the MTO generates a delay in the response and can be less efficient and competitive than the MTS paradigm. The dynamics of stem cells and committed progenitor cells in the midgut suggests a hybrid solution between MTS and MTO -- reminiscent to the business solution known as delayed differentiation. Thus, in basal homeostasis, production of new cells to replace cell loss occurs in two stages: (1) a 'make-to-stock' stage where committed progenitor cells are continually generated and 'stocked' in an 'undifferentiated' state; and (2) a 'make-to-order' stage where terminal differentiation takes place only in response to a local demand. In mice and humans, the rapid turnover that occurs in the small intestinal epithelium is thought to be the result of continual shedding of superficial cells balanced by the continual stem cell production. The mechanism described in this study may be more general than expected and could account for how murine cells after fate commitment like the secretory-committed cells defer for long periods their terminal differentiation (Buczacki et al, 2013; Antonello, 2015).

Escargot/Snail2 sustains the undifferentiated state and self-renewing divisions of midgut intestinal stem cells. However, the committed progenitor cells also express escargot and apparently at higher levels than the stem cells. It is hypothesized that below a certain threshold level, Escargot maintains stemness and a partial EMT that may facilitate regular cell division and a topologically confined position at the base of the intestinal epithelium. Conversely, when Escargot surpasses a certain threshold level, it promotes a full EMT that confers invasive properties and motility for the successful response and integration of the newly differentiated cells in the preexisting epithelium. Intriguingly, the enteroendocrine cells appear to escape from this block in terminal differentiation and differentiate at the normal rate in the absence of escargot. There is as yet no explanation for the behaviour of these progenitor cells (Antonello, 2015).

Mechanistically, the different levels of escargot could be achieved via Notch signalling pathway, which is prominently activated in enterocyte-committed progenitors. Notch signalling activates directly zfh1 gene and Zfh1, a homolog of the mammalian stemness and EMT-determinant Zeb1,2, and binds to the escargot promoter region, and this study shows that Zfh1 acts genetically upstream of escargot. Thus, progenitor cells receiving Notch signalling might enhance escargot transcriptional levels via Notch-induced zfh1 transcription. Such regulatory mechanism would explain, for example, that loss of Notch results in stem-like/round cells (Antonello, 2015).

In mammalian cell culture, the EMT process has been linked to the acquisition of stem-like nature via an interplay between the ZEB1,2 and Snail transcription factors and the microRNAs of the miR-200 family. Moreover, EMT determinants often regulate each other to promote EMT. Thus, the interactions between Escargot/Snail2, zfh1/Zeb and miR-8/miR-200 that were identified in this study exemplify the conservation of the regulatory mechanisms involved in EMT/MET and stemness in an in vivo context and a normal physiology of an adult organism. However, this study shows that escargot-zfh1 promotes stemness and full EMT/invasive properties in distinct cell populations and likely at different concentration levels, highlighting the utility of Drosophila midgut as a model to dissect out mechanisms linking physiological EMT to cellular plasticity and stemness as well as provide novel insights linking polyploidy and EMT towards stemness (Antonello, 2015).

Although midgut mesenchymal/progenitor cells have motility, most of them maintain their own local area as clearly defined by Flybow clonal analysis. This situation is similar to the leading edge mesenchymal cells during collective cell migration. Midgut enteroblasts retain contact via E-cadherin with their mother ISC, a process that might be regulated by escargot as in tracheal cells. Cell-cell contact is crucial to sustain Notch signalling in committed progenitor cells and likely to help to stabilize polarity of enteroblasts and their membrane protrusions that contact mature cells. Through these protrusions, mesenchymal/enteroblasts might actively monitor their surroundings. When a protrusion detects changes in tension and mechanical forces generated during the elimination of a dying cells, a positive input might be created that triggers the activation of expression of the microRNA mir-8 in the particular progenitor cell which, in turn, promotes the epithelial state and integration of the newly differentiated cell in the epithelium. Adhesion via E-cadherin could facilitate communication between an epithelial cells and a mesenchymal/progenitor cell in its vicinity so that a single, newly differentiated cell fills the gap left by the cleared cell (Antonello, 2015).

Dynamic pseudopodia in migrating cells have been proposed as a mechanism for temporal and spatial sensing during cell migration. Direction sensing is also consistent with time-lapse data showing individual progenitor cells re-adjusting position in the homeostatic midguts. Transduction of mechanical cues via YAP and TAZ (called Yorkie in flies) is functionally involved in differentiation of mesenchymal stem cells. Hence, Drosophila Hippo/Yorkie-YAP in mature enterocytes is a primary candidate pathway for a potential transduction of mechanical cues activating mir-8 in response to cell death (Antonello, 2015).

In summary, the miR-8-escargot-zfh1 axis and the EMT/MET programme provides a conceptual shift of the current stem cell-centred view of tissue renewal and offers a starting point for investigating how mature cells speak with neighbouring committed progenitor cells to ensure that epithelial cell loss and cell addition are kept in balance (Antonello, 2015).

Effects of Mutation or Deletion

Escargot regulates tracheal branch fusion in Drosophila. During development of tubular networks such as the mammalian vascular system, the kidney and the Drosophila tracheal system, epithelial tubes must fuse to each other to form a continuous network. Little is known of the cellular mechanisms or molecular control of epithelial tube fusion. A tracheal cell located at the developing fusion point expresses a sequence of specific markers as it grows out and contacts a similar cell from another tube; the two cells adhere and form an intercellular junction, and they become doughnut-shaped cells with the lumen passing through them. The early fusion marker Fusion-1 is identified as the escargot gene. It lies near the top of the regulatory hierarchy, activating the expression of later fusion markers and repressing genes that promote branching. Ectopic expression of escargot activates the fusion process and suppresses branching throughout the tracheal system, leading to ectopic tracheal connections that resemble certain arteriovenous malformations in humans. This establishes a simple genetic system to study fusion of epithelial tubes (Samakovlis, 1996).

The mutant phenotypes of cdc2 are similar to those of escargot: many diploid cells in imaginal discs, salivary glands and the central nervous system enter an endocycle, characterized by DNA replication without a subsequent mitotic phase. Such endocycling cells are often polytene, possessing thick chromosomes with DNA replicated many times over. When escargot function is eliminated, diploid imaginal cells that were arrested in G2 lose Cyclin A, a regulatory subunit of G2/M cdk, and entered endocycle. escargot genetically interacts with cdc2, suggesting an intimate biological interaction. Since mitotically quiescent abdominal histoblasts still require cdc2 to remain diploid, the inhibitory activity of Cdc2 on DNA replication appears to be separable from its activity as the mitosis promoting factor. These results suggest that in G2, escargot is required to maintain a high level of G2/M cdk, which actively inhibits the entry into S phase. Expression of Cyclin A is lost in escargot mutants, suggesting that Cdc2 activity (dependent on its regulatory subunit Cyclin A), indirectly depends on escargot (Hayashi, 1996).

In some escargot mutants, abdominal histoblasts become polypoid. It has been suggested that one role of esg is to maintain diploidy of imaginal cells (Fuse, 1994 and Hayashi, 1993).

The mesoderm determinant Snail collaborates with related zinc-finger proteins to control Drosophila neurogenesis

By the time of neuroblast delamination, Sna is present in most of the neuroblasts that have segregated from the ectoderm. Despite the extensive expression in the neuroblasts, prior to this study, Sna had no known function in the developing nervous system. The neuroblast pattern of sna resembles that of a group of genes called pan-neural genes. One of these genes, scratch (scrt), encodes a protein that has sequence similarity to Sna in the zinc-finger domain. Mutations of scrt have no obvious phenotype except that viable escapers have morphological defects in the eyes. Furthermore, no nervous system defect can be seen in sna scrt double mutants. However, the scrt dpn double mutants exhibit some defects in nervous system development. deadpan (dpn) is another pan-neural gene that encodes a basic helix-loop-helix protein. Therefore, scrt does have a function in the central nervous system (CNS), but the function of sna, if any, in the nervous system does not overlap with that of scrt (Ashraf, 1999 and references therein).

Escargot (Esg) is another protein that contains five zinc fingers with sequences highly homologous to those of Sna. The expression of esg is rather dynamic during embryonic development. The gene is expressed in the epidermis, neuroectoderm and imaginal precursor cells. The Esg protein probably acts through the cdc2 kinase to maintain the proper cell cycle in larval imaginal disc cells; in esg mutant larvae the imaginal disc cells lose their diploidy as they re-enter the S phase without going through mitosis. Moreover, esg and sna are both expressed in the embryonic wing imaginal disc primodia and the two genes have redundant functions in this tissue; the vestigial marker gene expression in the disc is lost in esg sna double mutants. Despite a clear demonstration of the redundant requirements of sna and esg in the wing disc, the double mutant has been reported to have no significant embryonic CNS phenotype. Thus, the function of sna in nervous system development has remained a mystery (Ashraf, 1999 and references therein).

Evidence is provided that CNS expression of Snail is required for nervous system development. The neural function of snail is masked by two closely linked genes, escargot and worniu. worniu (pronounced war-niu, Chinese for 'snail') encodes a protein with a zinc-finger domain highly homologous to those of Sna and Esg; it has been identified from the Berkeley Drosophila Genome Project database. RNA in situ hybridization reveals extensive expression of worniu in the developing nervous system. wor is located between esg and sna, ~100 kb apart, in the 35D region of the second chromosome. Although not affecting expression of early neuroblast markers, the deletion of the region containing all three genes correlates with loss of expression of CNS determinants including fushi tarazu, pdm-2 and even-skipped. Transgenic expression of each of the three Snail family proteins efficiently rescues the fushi tarazu defects, and partially rescues the pdm-2 and even-skipped CNS patterns. These results demonstrate that the Snail family proteins have essential functions during embryonic CNS development, around the time of ganglion mother cell formation (Ashraf, 1999).

The putative Wor protein sequence contains a C-terminal domain with six zinc fingers that are very similar to those of Sna and Esg, even though those proteins contain only five fingers. The N-terminal halves of these proteins have rather divergent sequences, except that they all contain a conserved basic motif very close to the N-termini. The function of this motif is not known. Moreover, the proteins contain two P-DLS-K motifs. The P-DLS-K domains in Sna have been shown to interact with the Drosophila C-terminal binding protein (dCtBP) and to play important roles in transcriptional repression. Since all three Sna family proteins contain highly homologous corepressor-interacting and DNA-binding domains, and can bind to similar DNA sequences, it is possible that they bind to promoters of overlapping sets of target genes and repress transcription (Ashraf, 1999 and references therein).

While there is no maternal RNA deposition of wor, zygotic expression can be detected first at the onset of neurogenesis. At a late stage 8, WOR transcript can be observed in two small patches of cells in the dorsal head region anterior to the cephalic furrow, representing precursor cells of the developing brain. At stage 9 wor expresses in the first wave of delaminating neuroblasts along either side of the midline, as well as in cells in the head region. Later in the germ band-extended embryo, most of the neuroblasts contain WOR mRNA. This pattern greatly resembles that of sna at this stage of development, except that sna expression in some of the centrally located neuroblasts in each hemisegment is at lower levels. In later stages, wor continues to express in the brain and part of the ventral nerve cord. No expression of wor is detected in any other embryonic tissue (Ashraf, 1999).

There is no extensive expression of esg in the neuroblasts similar to that shown for wor or sna. However, it has been demonstrated that esg RNA is expressed in the ventral neuroectoderm. Careful examination of the expression reveals that esg transcript is probably present in the CNS, albeit at variable levels. Based on the expression analyses, it is hypothesized that the newly identified wor might serve a redundant function with that of sna or esg during neural development. This would explain why neither single nor double mutants of sna and esg show severe defects in the nervous system (Ashraf, 1999).

To test the hypothesis that the Sna family proteins function redundantly in the developing nervous system, the neural phenotype associated with a deletion that uncovers all three genes was examined. wor is located between esg and sna, ~100 kb apart, in the 35D region of the second chromosome. Advantage of the close proximity of these genes and the phenotypes of a deficiency mutant are examined. Since high levels of Sna and Wor are present in the neuroblasts, the expression of the proneural gene achaete, which marks a subset of early delaminating neuroblasts was examined. This expression is not affected in the osp29 deficiency mutants. The expression patterns of additional neuroblast markers including hunchback, dpn, scrt and lethal of scute also are similar in wild-type and mutant embryos. Therefore, the early waves of neuroblast delamination are normal in the absence of the Snail family proteins (Ashraf, 1999).

The CNS patterns of GMC markers ftz, pdm-2 and eve were examined. ftz is expressed in a number of midline precursor cells and extensively in GMC. In contrast to the neuroblast markers, the ftz expression is almost abolished in the mutant embryo. The pdm-2 gene is also expressed in some neuroblasts and GMC. The early neuroblast expression of pdm-2 in the mutant is nearly normal, while the expression in later staged embryos is highly defective. eve gene products are present in a number of GMC and postmitotic neurons during normal development. All the eve CNS expression is absent in homozygous osp29 deletion mutant embryos. Taken together, the deletion mutant that uncovers the three sna family genes shows severe defects in CNS development (Ashraf, 1999).

To confirm the function of these three proteins in neural development, transgenic rescue plasmids were constructed in which individual genes (esg, wor or sna) were placed under the control of a sna promoter, containing an enhancer element that directs expression in the neuroblasts. The transgenic flies obtained were then crossed with the osp29 strain and analyzed for CNS development. In the presence of any one of the three constructs the ftz expression is restored significantly. Analysis of the rescued pattern under higher magnification reveals that part of the ftz staining is clearly absent. However, more detailed analysis is required to pinpoint the exact cell lineages that are missing. Nevertheless, the results demonstrate that each of the three sna family genes can perform essential functions in the CNS in the absence of the other two. The rescue by the transgenes of the expression of pdm-2 and eve, both of which are defective in the osp29 mutant, was also examined. While all three sna family genes clearly can rescue the expression of pdm-2 , the effect is not as extensive compared with that of ftz. For eve RNA, the transgenes rescue the expression in a significant number of cells when compared with the total loss of expression in the parental osp29 mutant. The rescue of eve, again, is not as extensive as that of ftz. Later stage CNS morphology in the rescued embryos was also monitored by BP102 staining. The embryos carrying the transgenes have slightly better overall CNS axonal morphology, but they are still highly abnormal when compared with the wild type (Ashraf, 1999).

Pairwise recombination of the transgenes were constructed and a test was performed to see whether they could achieve better rescue. By staining embryos obtained from stable lines that are homozygous for two transgenes, the constructs were found to give slightly direct the expression of ftz slightly better. Meanwhile, the eve and BP102 antigen expression in the presence of two transgenes reveals only minor improvement of the axonal morphology. These results suggest that the three proteins may have some collaborative function. It is also possible that the promoter used has some limitation in driving the rescue transgenes or that there are additional genes involved for the severe CNS phenotype (Ashraf, 1999).

Increasing numbers of sna-related genes have been identified in diverse species. These proteins have been assigned to the Sna family based mostly on the similarity of the sequences in the zinc-finger domains. The expression patterns and some functional studies of the vertebrate proteins suggest a role in regulating cell movement. However, gene knock-out experiments have demonstrated that mutating a mouse Slug homolog does not lead to a detectable cell movement defect. Such a result suggests a possible redundant function provided by other genes, similar to this report. If the vertebrate homologs do have a function in controlling cell movement, it would be reminiscent of the control of cell movement during gastrulation by Drosophila Sna. However, the expression of vertebrate Sna proteins in developing CNS has not been demonstrated. A careful examination of the expression and function in the CNS is needed to reveal the importance of Sna expression. The analysis of the functions of Sna, Esg and Wor in Drosophila CNS development will certainly provide a foundation for similar analysis in other species (Ashraf, 1999 and references therein).

A family of Snail-related zinc finger proteins regulates two distinct and parallel mechanisms that mediate Drosophila neuroblast asymmetric divisions

Three snail family genes -- snail, escargot and worniu -- encode related zinc finger transcription factors that mediate Drosophila central nervous system (CNS) development. Simultaneous removal of all three genes causes defective neuroblast asymmetric divisions; inscuteable transcription/translation is delayed/suppressed in the segmented CNS. Furthermore, defects in localization of cell fate determinants and orientation of the mitotic spindle in dividing neuroblasts are much stronger than those associated with inscuteable loss of function. In inscuteable neuroblasts, cell fate determinants are mislocalized during prophase and metaphase, yet during anaphase and telophase the great majority of mutant neuroblasts localize these determinants as cortical crescents overlying one of the spindle poles. This phenomenon, known as 'telophase rescue', does not occur in the absence of the snail family genes; moreover, in contrast to inscuteable mutants, mitotic spindle orientation is completely randomized. These data provide further evidence for the existence of two distinct asymmetry-controlling mechanisms in neuroblasts both of which require snail family gene function: an inscuteable-dependent mechanism that functions throughout mitosis and an inscuteable-independent mechanism that acts during anaphase/telophase (Cai, 2001).

CNS development is abnormal in Df(2L)osp29 embryos due to deletion of Sna family proteins. Both Sna and Wor are expressed strongly in all NBs, including those in the procephalic region, during early neurogenesis. The expression of Esg is also seen in NBs and other tissues, as visualized with anti-Esg immunostaining. Expression of Esg can be detected in the midline cells as well as GMCs during embryonic development. The functions of these three genes are overlapping; the early CNS defects are detected only when all three genes are removed simultaneously. In order to test whether the defects of localization of Mir/Pros and Pon/Numb seen in Df(2L)TE35BC-3 embryos are due to the absence of the three sna family genes, the localization of Mir/Pros and Pon/Numb was examined in embryos single mutant for sna, esg or wor, a double mutant for sna/esg and deletions that removed sna/wor or esg/wor, as well as embryos double mutant for sna/esg and further subjected to wor double-stranded RNA (RNAi) treatment. In single and double mutant embryos, both Mir/Pros and Pon/Numb form normal basal crescents in mitotic NBs. Only the sna/esg double mutant embryos that have been injected with wor RNAi reproduce the phenotype found in Df(2L)TE35BC-3 embryos (Cai, 2001).

In wild-type embryos, NBs are located between the ectoderm and mesoderm. The Df(2L)TE35BC-3 embryos lack mesoderm. Therefore, it is possible that correct NB asymmetry requires signal(s) from the mesoderm, and the asymmetry defects seen in Df(2L)TE35BC-3 could be due simply to the absence of mesoderm in these embryos. This is unlikely since NB asymmetry is intact in sna embryos, which lack mesoderm and share the abnormal morphology of Df(2L)TE35BC-3 embryos. Furthermore, the partial rescue of mesoderm in Df(2L)TE35BC-3 embryos by ectopic expression of the Sna protein driven by twist-gal4 does not reverse the asymmetry defects. Thus, it is concluded that mislocalization of Mir/Pros and Pon/Numb in Df(2L)TE35BC-3 embryos is due to the absence of all three sna family genes. Based on this conclusion, Df(2L)TE35BC-3 is referred to as sna/esg/wor deficient and was used in subsequent studies (Cai, 2001).

In wild-type embryos, Baz, Insc and Pins form a complex that is localized to the apical cortex of the dividing NBs. The apical complex is required for the asymmetric distribution of cell fate determinants such as Pros and Numb to the basal cortex of NBs and coordinates the orientation of the mitotic spindle along the apical-basal axis of the NB. In embryos deficient for the sna family genes, Mir/Pros and Pon/Numb are no longer concentrated to the basal cortex of mitotic NBs, indicating defects in NB asymmetry. It is possible that the asymmetry defects seen in sna/esg/wor-deficient NBs are due to the alteration of Insc expression. Anti-Insc staining indicates that Insc protein is indeed undetectable in the segmented CNS of sna/esg/wor-deficient embryos. Although the signal intensity in the procephalic region is comparable to that in the wild-type controls, the number of cells with anti-Insc staining appears to be decreased. This altered expression of Insc in the mutant embryos suggests that the mislocalization of Mir/Pros and Pon/Numb in sna/esg/wor-deficient embryos is, at least in part, due to a lack of Insc protein expression in dividing NBs. As expected, Baz protein levels are low and undetectable in the great majority of mutant NBs. The lack of easily detectable Baz in NBs is probably due to the instability of the protein when Insc is absent since the baz mRNA levels remain unchanged in sna/esg/wor NBs. Pins protein localization is also affected in sna/esg/wor-deficient embryos (Cai, 2001).

The down-regulation of Insc protein in NBs is also dependent on the simultaneous loss of sna, esg and wor functions. Insc expression in double mutant embryos of sna/esg was similar to that of wild-type embryos. In sna/esg double mutant embryos, further removal of the third member of sna gene family, wor, with RNAi leads to the total loss of Insc protein expression. Moreover, ectopic expression of any one of the sna family genes under the control of an early neural driver sca-gal4 in sna family gene mutant embryos largely restores the Insc expression in NBs (sna 79%; esg 64% and wor 44%), further indicating that Insc expression is indeed regulated by the Sna family proteins (Cai, 2001).

insc transcript levels were examined in the sna/esg/wor-deficient embryos. In wild-type stage 9-10 embryos, insc RNA is expressed prominently in NBs of the segmented CNS and in the procephalic region. The transcript level is maintained in the segmented CNS and procephalic NBs throughout embryogenesis. In sna/esg/wor-deficient embryos, RNA in situ hybridization data indicate that the insc RNA is absent in the segmented CNS at stages 9-10 but is detectable in the procephalic NBs. This suppression of insc RNA transcription in the segmented CNS of sna/esg/wor-deficient embryos provides evidence that the Sna family proteins are essential for insc mRNA transcription during early neurogenesis. The suppression of insc transcription in the segmented CNS is transient and insc RNA can be detected, at a lower level, in late stage 11 embryos. However, Insc protein in the segmented CNS of sna/esg/wor-deficient embryos remains undetectable at late stage 11 when the insc RNA levels partially recover by an unknown mechanism. It is obvious that translation of insc RNA in late stage 11 embryos is inhibited in the segmented CNS of embryos deficient for sna/esg/wor. Although the inhibition mechanism is unknown, it is believed that the insc 5'- and/or 3'-untranslated regions (UTRs) are involved since Insc protein can be ectopically expressed in sna/esg/wor-deficient embryos from a uas-insc transgene in which the 5'- and 3'-UTRs have been partially removed. Considering that the Sna family proteins are localized to nuclei, it is unlikely that they interact directly with 5'- and/or 3'-UTRs of insc RNA. Presumably other genes regulated by the Sna family proteins mediate the observed translational effect (Cai, 2001).

The observation of delayed and decreased insc mRNA transcription and the inhibition of Insc protein synthesis in the segmented CNS of sna/esg/wor-deficient embryos suggests the dual regulation of insc expression by the Sna family proteins at both transcriptional (stage 9-10) and translational (stage 11 onwards) levels. This dual regulation mechanism is prominent in the segmented CNS but insc RNA and protein expression in the procephalic region is only partially affected in sna/esg/wor-deficient embryos. The mechanism that enables the partial restoration of insc transcription in NBs of the segmented CNS at late stage 11 in the absence of sna family gene function remains to be identified (Cai, 2001).

In insc22 mutant NBs, in which the apical complex required for correct asymmetric division is abolished, basal components such as Mir/Pros and Pon/Numb often form random crescents, sometimes broad and loose, from prophase to metaphase; however, Pros/Mir and Pon/Numb can eventually be redistributed to the 'budding site' of the future GMCs, although sometimes not as exclusively as seen in wild-type embryos, at anaphase and telophase even when the spindle is misorientated. Consequently, the great majority of all GMCs inherit, at least in part, cell fate determinants such as Pros and adopt correct GMC fate. This phenomenon, referred to as 'telophase rescue', does not occur in NBs lacking the three sna family genes. For example, in sna/esg/wor-deficient NBs, basal proteins Mir/Pros and Pon/Numb form a randomly localized crescent in dividing NBs but, unlike in insc embryos, these proteins are not redistributed at anaphase/telophase to the region of the cortex that gives rise to the GMC. Consequently, the great majority of the GMCs do not inherit the basal proteins Mir/Pros and Pon/Numb and thus lose their GMC identities. This finding explains why GMCs are not specified correctly in Df(2L)osp29 embryos (Cai, 2001).

Furthermore, it is known that the mitotic spindle in NBs rotates 90° during metaphase so that it is realigned along the apical-basal (A/B) axis of the embryos; in insc mutants, this spindle rotation during metaphase occurs only in a small proportion (~20%) of NBs; nevertheless, even some of these NBs are able to reorient spindles late in mitosis. The NB spindle orientation during anaphase or telophase was measured in wild-type and mutant embryos and they were catagorized into four equal quadrants depending on the angle that the spindle forms with the A/B axis. Based on the spindle orientation in wild-type embryos, all spindles with an angle >45° relative to the A/B axis during late mitosis are considered to be misoriented. The misoriented spindles in insc22 mutant embryos are limited; the great majority of NBs (90%) have their spindles oriented within 45° of the A/B axis, compared with 100% in wild-type NBs. In contrast to wild-type and insc NBs, in sna/esg/wor-deficient NBs, spindle orientation is completely randomized with almost equal distribution for each of the four quadrants. Moreover, a small number of NBs (10%) completely reverse their polarity, giving rise to a small apical GMC, which has never been reported in any known asymmetry mutant (Cai, 2001).

These observations indicate that removal of Insc alone has only a limited effect on NB asymmetric divisions in terms of basal protein localization and spindle orientation late in mitosis, suggesting that the Insc-dependent mechanism is not the only apparatus that controls the asymmetric divisions in NBs. It appears that an Insc-independent mechanism exists that functions in parallel to coordinate the asymmetry events at later stages (anaphase onwards) of mitosis. This Insc-independent asymmetry-controlling mechanism, which is responsible for the 'telophase rescue' phenomenon and for prevention of random spindle orientation in insc22 embryos, is destroyed upon removal of the three sna family genes. However, one might argue that the severe asymmetry defects seen in the absence of the sna family genes might be artifactual, caused by the combination of loss of insc expression and the absence of the mesoderm. This possibility is suggested because in insc/sna double mutant embryos, which lack both insc and the mesoderm, NBs exhibit phenotypes that are indistinguishable from those seen in the insc single mutant. It has therefore been concluded that in the absence of the sna family genes, both the Insc-dependent and -independent asymmetry-controlling mechanisms are destroyed, leading to asymmetry defects that are more severe than those seen in insc single mutants (Cai, 2001).

The existence of two distinct asymmetry-controlling mechanisms in wild-type NBs raises an interesting issue: how do these two mechanisms work in concert to mediate asymmetric divisions? Since embryos deficient for the sna family genes lack both mechanisms, it was reasoned that by restoring the Insc-dependent mechanism in these embryos the consequences of missing just the insc-independent mechanism could be assessed. Ectopic expression of full-length Insc protein with an early neural driver sca-gal4 in NBs of sna family gene mutant embryos shows complete rescue of the protein localization defects. The apical complex forms normally, as indicated by the formation of apical Insc as well as Pins and Baz crescents. The defects in basal protein localization are also completely rescued; Mir/Pros and Pon/Numb form tight basal crescents in mitotic NBs. These results suggest that, with respect to protein localization, Insc protein is the only component missing in the Insc-dependent asymmetry machinery, and replacement of Insc through ectopic expression is sufficient to restore wild-type localization of the apical and basal components. Furthermore, it indicates that the Insc-independent mechanism is cryptic with respect to protein localization since it is dispensable when the Insc-dependent mechanism is in place. Either mechanism alone is able to distribute basal proteins to the cortex of the future GMC 'budding site' with clear temporal and efficiency differences: the Insc-dependent mechanism localizes basal proteins starting in late prophase in the form of tight crescents, while the Insc-independent mechanism is only able to redistribute, sometimes partially, mislocalized basal proteins late in mitosis (telophase rescue) (Cai, 2001).

The spindle misorientation phenotype in sna family gene mutant embryos is also largely corrected by ectopic Insc expression. However, unlike protein localization, the rescue of mitotic spindle orientation is incomplete; the population of NBs with misoriented spindles drops from 45% to only 12%. These data suggest that both the Insc-dependent and -independent mechanisms are required for correct spindle orientation in wild-type embryos since ~10% of the mitotic spindles are misoriented in anaphase/telophase NBs defective for either mechanism. However, a complete randomization of spindle orientation is seen when both mechanisms are absent (Cai, 2001).

Thus, the underlying cause for the asymmetry defects associated with some deficiencies uncovering the 35B-D region of the genome, e.g. Df(2L)TE35BC-3, is the simultaneous loss of three members of the sna gene family: sna, esg and wor. All available lethal complementation groups uncovered by Df(2L)TE35BC-3, all deficiencies that remove only two out of the three sna family members and a sna/esg double mutant generated from recombination do not show any defects in any aspect of NB asymmetric division; only embryos double mutant for sna/esg, and further subjected to wor RNAi, reproduce the asymmetry defects seen in the deficiencies. These data indicate that the defects in sna/esg/wor-deficient embryos are caused by the simultaneous functional loss of all three sna family genes. The observation that the ectopic expression of sna, esg or wor reverses the asymmetry phenotypes in the segmented CNS of sna/esg/wor-deficient embryos further supports this conclusion. These conclusions are in agreement with an earlier study reporting that the sna family genes are required for CNS development (Cai, 2001).

It has been observed that in insc embryos, cell fate determinants such as Pros and Numb are mislocalized early during mitosis; however, in anaphase and telophase, the effect termed 'telophase rescue' causes the misplaced crescents to redistribute and overlie one spindle pole, enabling the basal cell fate determinants to segregate, exclusively or partially, to the GMCs. The insc loss-of-function alleles insc22, inscP49 and inscP72 all show telophase rescue. It has been found that essentially all NBs in insc embryos can redistribute Pros and Numb, at least partially, into GMCs. These observations suggest the existence of a second asymmetry-controlling mechanism that does not require insc functions, which operates late in mitosis to coordinate protein localization with spindle orientation. These observations explain why insc mutants have minimal effect on GMC cell fate. The Insc-independent mechanism corrects the earlier errors caused by absence of Insc during anaphase/telophase, thereby enabling cell fate determinants to be inherited by the GMC. This mechanism is apparently less efficient, as shown by the fact that in some insc NBs, normally basal components form a broad and loose crescent and are only partially sequestered into GMCs. Furthermore, the observation that mitotic spindle orientation is only mildly affected in insc NBs is also consistent with an Insc-independent compensatory mechanism (Cai, 2001).

Analysis of NB divisions in embryos deficient for the three sna family genes provides further support for the existence of an Insc-independent mechanism. In these embryos, the Insc-dependent mechanism is clearly abolished; both the transcription and the translation of insc are suppressed in the mutant NBs. In addition, telophase rescue no longer occurs; the normally basally localized components are misplaced in mitotic NBs and not redistributed to the future GMCs even at anaphase/telophase. Moreover, the spindle orientation in embryos deficient for the sna family genes becomes randomized; ~45% of NBs exhibit misoriented spindles with an angle >45° with respect to the A/B axis at anaphase/telophase, which is not seen in wild-type NBs and is at a much higher frequency than that seen in insc22 NBs. Thus, NBs deficient for the sna family genes show two defects that are not seen in insc NB: (1) the absence of telophase rescue, and (2) randomization of the spindle orientation late in mitosis. These observations indicate that both the Insc-dependent and -independent mechanisms require the sna family genes (Cai, 2001).

These two mechanisms can apparently function independently. In insc NBs, the Insc-independent mechanism functions in the absence of the Insc-dependent mechanism to correct the earlier (prophase to metaphase) asymmetry defects during anaphase/telophase. In sna/esg/wor-deficient NBs that have been forced to express Insc, the Insc-dependent mechanism can act in the absence of the Insc-independent mechanism to mediate the localization of the basal components from prophase to telophase, obviating the requirement for telophase rescue; however, although the Insc-dependent mechanism can reduce the extent of the mitotic spindle orientation defects seen in the sna/esg/wor NBs, it does not restore wild-type spindle orientation. Therefore, it appears that both mechanisms are required and act in concert to mediate mitotic spindle orientation. However, with respect to localization of the basal components, the effects of the Insc-independent mechanism are only visible when the Insc-dependent mechanism is absent (Cai, 2001).

For the Insc-dependent mechanism, three components have been identified: Baz, Insc and Pins are known to form an apically localized functional complex. The function of this complex requires the participation of all members. Insc appears to be the only component of the Insc-dependent mechanism missing in sna/esg/wor-deficient embryos since ectopic expression of Insc restores its function. Little information is available on the components of the Insc-independent mechanism. Other members of asymmetry machinery identified so far in NBs are the basal components such as Mir/Pros, Pon/Numb, Stau and pros RNA. These downstream components are controlled and coordinated by both Insc-dependent and -independent mechanisms (Cai, 2001).

In embryos deficient for the sna family genes, one of the major defects is the absence of Insc protein expression in the segmented CNS. RNA in situ hybridization indicates that the insc RNA transcripts are not detected in NBs of stage 9-10 embryos. Even in late stage 11 embryos when the insc RNA levels partially recover, Insc protein is never seen in the segmented CNS, indicating that the down-regulation of insc occurs at both the transcriptional and translational levels. In the procephalic region of these sna/esg/wor-deficient embryos, Insc expression is only partially affected. The 5'- and/or 3'-UTRs of the insc transcript appear to play an important role in the translational regulation of Insc expression. This is supported by two observations: (1) Insc protein can be detected in sna/esg/wor embryos following ectopic expression of a cDNA construct containing the complete insc coding region but with the 5'- and 3'-UTRs partially removed; (2) transcripts derived from lacZ driven by a 1.2 kb insc 5' CNS promoter sequence are not subjected to this translational repression in sna/esg/wor embryos, although their expression pattern is identical to that of Insc in the CNS. Given that the Sna family proteins are localized to nuclei, it is unlikely that they play a direct role in translational regulation. Other unknown intermediates must be involved (Cai, 2001).

Seizure suppression by gain-of-function escargot mutations

Suppressor mutations provide potentially powerful tools for examining mechanisms underlying neurological disorders and identifying novel targets for pharmacological intervention. Mutations are described that suppress seizures in a Drosophila model of human epilepsy. A screen utilizing the Drosophila easily shocked (eas) 'epilepsy' mutant identified dominant suppressors of seizure sensitivity. Among several mutations identified, neuronal escargot (esg) reduced eas seizures almost 90%. The esg gene encodes a member of the snail family of transcription factors. Whereas esg is normally expressed in a limited number of neurons during a defined period of nervous system development, the suppressor mutation caused normal esg to be expressed in all neurons and throughout development. This greatly ameliorates both the electrophysiological and the behavioral epilepsy phenotypes of eas. Neuronal esg appears to act as a general seizure suppressor in the Drosophila epilepsy model, since esg reduces the susceptibility of several seizure-prone mutants. esg must be ectopically expressed during nervous system development to reduce seizure susceptibility in adults. Furthermore, induction of esg in a small subset of neurons (interneurons) will reduce seizure susceptibility. A combination of microarray and computational analyses revealed 100 genes that represent possible targets of neuronal esg. It is anticipated that some of these genes may ultimately serve as targets for novel antiepileptic drugs (Hekmat-Scafe, 2005).

esg is defined as a seizure-suppressor gene on the basis of gain-of-function mutations that (1) revert the bang-sensitive behavioral phenotype associated with eas, sda, and bss/+ flies and (2) cause an increase in the seizure threshold of eas mutants. This conclusion is bolstered by the identification of five different esgEP alleles (all with independently derived P-element insertions) and one UAS-esg construct (located in a distinct cytological location) that all act as sda suppressors. On the basis of the lack of allele specificity of the esg-sda interaction, mutations of esg appear to be general seizure suppressors. This is expected since neither the esgEP alleles nor UAS-GAL4 would be expected to produce structurally altered gene products; suppression is presumably due to the ectopic expression of a structurally normal protein. It is expected that the five esgEP mutations identified in this study, as well as the UAS-esg insertion, show similar ectopic expression patterns under elav-GAL4 control and thereby produce seizure suppression in a similar gain-of-function manner (Hekmat-Scafe, 2005).

Neuronal induction of esgEP appears to reduce the fly's overall seizure susceptibility. This assertion is supported by the observation that wild-type, non-BS flies carrying elav-GAL4-activated esgEP display an increased seizure threshold. A general reduction in seizure sensitivity would also explain why neuronal esgEP suppresses a variety of BS mutations. Two of the BS mutations examined (eas and sda) encode very different products: eas is an ethanolamine kinase involved in synthesis of the phosphatidyl ethanolamine in neuronal membranes, and sda encodes an aminopeptidase. The third BS mutation is likely to encode yet another very different product. These three BS mutations may well reduce the fly's seizure threshold by different mechanisms. The elav-GAL4 activation of esgEP suppresses sda best of all; suppression of eas is intermediate and suppression of bss is the weakest. This is consistent with previous observations on general seizure suppressors that bss is the strongest of the three mutations (in terms of both its reduction of seizure threshold and the facility with which it can be suppressed by secondary mutations that reduce nervous system excitability) and sda is the weakest, with eas being intermediate (Hekmat-Scafe, 2005).

The gain-of-function esgEP mutations that act to suppress seizures cause no other obvious phenotypes whether in a wild-type or an eas background. Thus, esg mutant flies show no obvious nervous system excitability defects: they are not temperature-sensitive paralytics (hypoexcitability) and they do not shake their legs under ether anesthesia (hyperexcitability). Other behaviors also appear to be normal: flies groom, court, mate, jump, and fly. Flies that are eas+; esgEP2009; elav-GAL4 have a seizure threshold that is near the wild-type range (Hekmat-Scafe, 2005).

In the experiments presented in this study, the combined features of esgEP, elav-GAL4, and GeneSwitch (a conditional, RU486-dependent GAL4-progesterone fusion protein) begin to give a picture of seizure suppression. It is suggested that esgEP produces seizure suppression via its effect on immature postmitotic larval neurons that differentiate into interneurons of the adult CNS. It is further suggested that this could be due to cytoskeletal organization or reorganization that underlies the elaboration and strengthening of synaptic interconnections (Hekmat-Scafe, 2005).

Several other possible explanations for seizure suppression are not supported by the experiments presented here. For example, seizure suppression cannot occur by esgEP ameliorating some acute property of mature neurons in adults. Thus, esgEP suppression is probably not by manipulation of neurotransmitter metabolism, ion channel maintenance, or by other steady-state mechanisms used for maintaining or sustaining nervous system function or structure. This is because the bang-sensitive phenotype of eas is not suppressed by expression of esgEP in the adult nervous system as shown by the GeneSwitch experiment. Another alternative explanation is also not supported by the experiments presented in this study: esgEP-mediated seizure suppression must be unrelated to esg's normal role in facilitating neurogenesis or its role in polyploidization. This is because elav-GAL4 does not induce esgEP expression in either embryonic or larval neuroblasts. The GeneSwitch experiment also shows that embryonic expression of esgEP most likely does not account for its seizure suppression in adults. Furthermore, reducing the dosage of esg, which is normally expressed only in neuroblasts, has no effect on bang sensitivity (Hekmat-Scafe, 2005).

The GeneSwitch experiments show that seizure suppression in adult eas flies is apparently due primarily to esgEP induction in the larval stage. In larvae, there are four main classes of neurons, all of which should have esgEP expression driven by elav-GAL4:

  1. Some larval neurons undergo programmed cell death and make no contribution to the adult nervous system. These neurons would have no impact on seizure suppression in adults.
  2. Some larval neurons subserve similar functions in the adult and pass through metamorphosis with little or no restructuring. These neurons may be analogous to mature neurons of adults, suggesting that esgEP induction would have little effect on seizure suppression.
  3. Some larval neurons undergo considerable restructuring during metamorphosis because their adult function is much different from their larval role. The neurons in classes 2 and 3 appear to form the core of motor and neuromodulatory systems in the adult.
  4. Some adult-specific neurons have no function in the larva and differentiate after pupariation. Many of these adult-specific neurons appear to be interneurons in the sensory system.

Seizure suppression by esgEP could be due to its effects in several of these classes; however, the class of adult-specific neurons (class 4) is an especially attractive candidate. These interneurons are involved in integrating sensory signals such as those arising from mechanical 'bang' stimulation. Interneurons in the sensory system are numerous and are probably the neurons most greatly affected by electrical stimuli delivered to the brain by HFS. The observation that induction of esgEP in larval interneurons, but not motoneurons, produces adults with reduced seizure susceptibility is also consistent with the notion that esgEP is acting primarily in the class 4 neurons. Interneurons of class 4 undergo considerable development late in third instar lavae, but can also continue development after eclosion. This development may account for the continued progression of seizure suppression through days 2-3 of the adult stage (Hekmat-Scafe, 2005).

One possibility is that larval esg induction in developing interneurons affects their synaptic connections and thereby interferes with the spread of seizures. Class 3 larval neurons undergo both new outgrowth and pruning of their dendritic and axonal processes during metamorphosis, and arrested adult-specific neurons (class 4) begin to extend processes after pupariation. Since esgEP induced in larvae may well persist in pupae, it could affect the expression of genes whose products influence synapse formation or strengthening shortly after pupariation. Pupae do not take up the RU486, which might explain why the degree of suppression by esgEP is lower when activated by elav-GeneSwitch than when activated by elav-GAL4 (Hekmat-Scafe, 2005).

An intriguing possibility is that cytoskeletal elements are playing an important role in seizure sensitivity and resistance in Drosophila. Snail family transcription factors such as esg normally promote cell-cell separation during development, at least in part by inhibiting the expression of cadherin, a homophylic cell-cell adhesion molecule linked to components of the actin network. Filamin mutants are seizure sensitive in flies and humans with periventricular heterotopia; ß-integrin has been identified as a suppressor of eas; collagen type IV is a prominent target of aminopeptidase N (sda) in metastasis, and E-cadherin is downregulated by sna and potentially esg. Indeed, cytoskeletal reorganization is probably critical in morphological changes in dendritic spines associated with synaptic plasticit and epilepsy. Cytoskeletal organization and reorganization also plays a prominent role as scaffolding for proteins subserving membrane excitability. Thus, signaling efficiency, reliability, and stability appear to be greatly influenced by the subcellular colocalization of excitability proteins in the photoreceptor and the presynaptic terminal and at the postsynaptic membrane of the neuromuscular junction. Defects in this scaffolding function via abnormal cytoskeletal elements may contribute substantially to the kinds of excitability instability thought to underlie seizure disorders (Hekmat-Scafe, 2005).

The esg gene encodes a presumptive transcriptional repressor with restricted temporal and spatial expression in the nervous system. Presumably, ectopic expression of esg in larval interneurons mediates seizure suppression in adults via the activation or repression of one or more target genes. A combination of microarray and computational analyses revealed 100 genes that represent possible targets of neuronal esg. Although none of these genes encode cytoskeletal elements, a number of them encode serine protease inhibitors (induced by neuronal esg) or serine proteases (repressed by neuronal esg) that could act on cytoskeletal elements or other proteins that influence synaptic interconnections. Further experiments will be needed to determine which of the 100 genes actually mediates seizure suppression. Such a gene could ultimately serve as target for novel antiepileptic drugs (Hekmat-Scafe, 2005).

Isolation and examination of suppressor mutations is a potentially powerful approach to seizure disorders. It allows the identification of biological processes not previously associated with seizures through genes such as esg. In addition, esg has several properties that make it attractive as a candidate for new AED development: it reduces seizure susceptibility without apparent side effects. While it appears to be effective only during a window of time during neuronal development, this window may serve as an advantage in treating cases in which epilepsy develops early. Nevertheless, esg, or a seizure-suppressor gene with similar properties identified in this or future screens, may allow the development of powerful new treatments for the devastating effects of intractable epilepsy (Hekmat-Scafe, 2005).

Development of the male germline stem cell niche in Drosophila

Stem cells are found in specialized microenvironments, or 'niches', which regulate stem cell identity and behavior. The adult testis and ovary in Drosophila contain germline stem cells (GSCs) with well-defined niches, and are excellent models for studying niche development. This study investigates the formation of the testis GSC niche, or 'hub', during the late stages of embryogenesis. By morphological and molecular criteria, the development of an embryonic hub that forms from a subset of anterior somatic gonadal precursors (SGPs) were identified and followed in the male gonad. Embryonic hub cells form a discrete cluster apart from other SGPs, express several molecular markers in common with the adult hub and organize anterior-most germ cells in a rosette pattern characteristic of GSCs in the adult. The sex determination genes transformer and doublesex ensure that hub formation occurs only in males. Interestingly, hub formation occurs in both XX and XY gonads mutant for doublesex, indicating that doublesex is required to repress hub formation in females. This work establishes the Drosophila male GSC niche as a model for understanding the mechanisms controlling niche formation and initial stem cell recruitment, as well as the development of sexual dimorphism in the gonad (Le Bras, 2006).

The evidence indicates that an embryonic hub, which appears to give rise to the adult hub and create the male GSC niche, forms during the late stages of embryogenesis. A subset of anterior SGPs initiates expression of several molecular markers that are also expressed in the adult hub. These SGPs segregate into a tight cluster in a distinct region of the gonad, and a subset of germ cells organizes around these SGPs in a manner similar to the organization of GSCs around the adult hub. Since spermatogenesis begins by early larval stages, it is possible that the embryonic hub already forms a functional GSC niche. The formation of the hub, or indeed any stem cell niche, can be divided into the distinct issues of niche cell identity, niche morphogenesis, and stem cell recruitment (Le Bras, 2006).

The data indicate that the specification of hub cell identity occurs in two stages. During the first stage, some SGPs acquire an anterior identity that is sexually dimorphic, as indicated by the male-specific expression of esg and upd. Anterior SGP identity is positively regulated by abd-A, and is repressed by Abd-B, while sexual identity is regulated by tra and dsx. During the second stage of hub cell specification, a subset of these anterior SGPs acquires hub cell identity during stage 17 of embryogenesis. Only some anterior SGPs maintain esg expression, and the control of late gene expression in the hub appears to be distinct from early expression in anterior SGPs, since some esg and upd enhancer traps only exhibit gonad expression in the hub at this later stage. Furthermore, cells that maintain esg expression during stage 17 also express every other marker of adult hub identity tested, including Fasciclin 3, cdi, DN-cadherin and DE-cadherin. It is concluded that these cells are specified as hub cells at this time. The fate of the anterior SGPs that lose esg expression and do not form part of the hub is unknown. An intriguing possibility is that these cells could form another important somatic cell type: the cyst progenitor cells (somatic stem cells) that associate with the hub along with the GSCs (Le Bras, 2006).

Based on its expression pattern, the transcription factor esg would seem to be an excellent candidate for specifying hub cell identity. However, no changes were observed in the expression of other hub markers in esg null mutants; this includes expression of DE-cadherin, which is known to be regulated by esg in other tissues. It has been reported, however, that esg is required for hub maintenance, and that the hub is severely defective at later stages in esg mutants that survive embryogenesis. Thus, esg is critical for the male GSC niche, but is either not important for the initial formation of this structure, or acts redundantly with another factor (Le Bras, 2006).

It has been possible to follow the morphogenesis of the hub from the time of gonad formation until the embryonic hub is fully formed. At the time of gonad coalescence, anterior SGPs interact with other SGPs, and with the germ cells, in a manner that is indistinguishable from posterior SGPs. However, during stage 17, the hub cells undergo dramatic changes in their relationship to other SGPs and germ cells. Hub cells segregate away from other SGPs to one pole of the gonad, and coalesce tightly with one another. In addition, hub cells do not ensheath the germ cells at this stage. Instead, a defined interface between hub cells and germ cells forms which is labeled by DE- and DN-cadherin, but not Fasciclin 3. Thus, hub cells appear to maximize their interactions with one another, and minimize their interactions with other cells in the gonad, although they clearly still contact a subset of germ cells (Le Bras, 2006).

It is apparent that the changes in cell–cell contact and morphology that occur during hub formation require changes in cell adhesion. Indeed, characteristic changes have been found in expression of the homophilic adhesion molecules Fasciclin 3, DN-cadherin and DE-cadherin occur during hub formation; all three are significantly upregulated in the embryonic and adult hub. Increased homophilic adhesion among hub cells could account for their ability to maximize their contacts with one another, and sort away from other SGPs. However, no changes were observed in embryonic hub formation in mutants for these cell adhesion molecules. Thus, these proteins, and possibly others, may act redundantly in this process (Le Bras, 2006).

It is clear that a subset of germ cells organizes specifically with the developing hub as it forms. During the last stage of hub formation, germ cells become oriented in a rosette distribution around the developing hub in a manner characteristic of GSCs in the adult. These may represent the subset of germ cells that will become GSCs. The presence of DE- and DN-cadherin at sites of hub–germ cell contact suggests that cadherin-mediated adhesion may be important for niche–GSC interaction in the testis, as has been observed in the ovary. Interestingly, germ cells are not required for hub formation. Analysis of a number of hub identity markers indicates that these cell form normally from a subset of anterior SGPs in embryos that lack germ cells. The hub does not appear as well compacted in these embryos, consistent with observations of the adult hub, indicating that hub–germ cell contact (or hub–germ cell signaling) affects the final shape of the hub. Nevertheless, the GSC niche can form in the absence of one of its stem cell populations (somatic stem cells may still be present). It will be of great interest in the future to determine if the subset of germ cells organized around the male embryonic hub are, indeed, developing GSCs, and to study how their transition to stem cell identity might be regulated by the niche (Le Bras, 2006).

The formation of the male GSC niche is a sex-specific characteristic of anterior SGPs. Male-specific expression of esg and hub formation both require the sex determination genes tra and dsx. In some tissues, DSXM is required to promote male development and repress female development, while the opposite is true for DSXF. Interestingly, it was found that embryonic hub development is entirely masculinized in dsx null mutants; XX and XY individuals appear identical when mutant for dsx and both resemble wild type males. Thus, no role is seen for DSXM in promoting embryonic hub formation, while DSXF is required in females to repress hub formation. Since esg is expressed male-specifically, it is one candidate for being directly regulated by DSX (Le Bras, 2006).

We can compare the development of the anterior SGPs and hub with the development of another sexually dimorphic cell type, the msSGPs that join the posterior of the male gonad. First of all, these two cell types are distinct and do not depend on one another for their proper development. The hub still forms in Abd-B mutants that lack msSGPs, while msSGPs are still found in the gonad in Pc mutants, in which no anterior SGPs or hub cells form. Second, the mechanism for how sexual dimorphism is created differs between the two cell types. msSGPs are present only in males because they have undergone sex-specific apoptosis in females. In contrast, no apoptosis was observed in anterior SGPs. These cells appear to remain present in both sexes, but only form a hub in males. Thus, although the sex determination genes tra and dsx regulate sex-specific development of both cell types, the cellular mechanisms employed are different. Finally, as was observed for the hub, development of the msSGPs is completely masculinized in dsx mutant embryos. Thus, for both of these cell types, the male pattern of development in the embryonic gonad is the default state in the absence of dsx function, and it is the role of DSXF to repress male development in females. However, DSXM may well play a role in development of one or both of these gonad cell types at later stages, since proper testis development in males clearly requires dsx (Le Bras, 2006).

The sex determination pathway must also ensure that GSC niches form in females and are different from those in males. Recently, it has been shown that germ cells populating the anterior of the gonad in female embryos are predisposed to become GSCs in the adult ovary, while germ cells populating the posterior rarely become GSCs. This suggests that anterior SGPs in the female embryonic gonad may promote GSC identity, similar to what is proposed to happen in the male during hub formation. One possibility is that anterior SGPs give rise to GSC niches in both sexes, while genes such as tra and dsx control whether these niches will be male or female (Le Bras, 2006).

In conclusion, the development has been followed of the embryonic hub, which may represent the nascent GSC niche for the testis. This work provides a basis for further understanding the mechanisms controlling niche formation and GSC recruitment in Drosophila, and determining if these mechanisms are conserved in other stem cell systems, including the GSC niche of the mammalian testis (Le Bras, 2006).

Escargot restricts niche cell to stem cell conversion in the Drosophila testis

Stem cells reside within specialized microenvironments, or niches, that control many aspects of stem cell behavior. Somatic hub cells in the Drosophila testis regulate the behavior of cyst stem cells (CySCs) and germline stem cells (GSCs) and are a primary component of the testis stem cell niche. The shutoff (shof) mutation, characterized by premature loss of GSCs and CySCs, was mapped to a locus encoding the evolutionarily conserved transcription factor Escargot (Esg). Hub cells depleted of Esg acquire CySC characteristics and differentiate as cyst cells, resulting in complete loss of hub cells and eventually CySCs and GSCs, similar to the shof mutant phenotype. Esg-interacting proteins were identified, and an interaction was demonstrated between Esg and the corepressor C-terminal binding protein (CtBP), which is also required for maintenance of hub cell fate. These results indicate that niche cells can acquire stem cell properties upon removal of a single transcription factor in vivo (Voog, 2014).

Intrinsic dorsoventral patterning and extrinsic EGFR signaling genes control glial cell development in the Drosophila nervous system

Dorsoventral patterning and EGFR signaling genes are essential for determining neural identity and differentiation of the Drosophila nervous system. Their role in glial cell development in the Drosophila nervous system is not clearly established. This study demonstrates that the dorsoventral patterning genes, vnd, ind, and msh, are intrinsically essential for the proper expression of a master glial cell regulator, gcm, and a differentiation gene, repo, in the lateral glia. In addition, it was shown that esg is particularly required for their expression in the peripheral glia. These results indicate that the dorsoventral patterning and EGFR signaling genes are essential for identity determination and differentiation of the lateral glia by regulating proper expression of gcm and repo in the lateral glia from the early glial development. In contrast, overexpression of vnd, msh, spi, and Egfr genes repress the expression of Repo in the ventral neuroectoderm, indicating that maintenance of correct columnar identity along the dorsoventral axis by proper expression of these genes is essential for restrictive formation of glial precursor cells in the lateral neuroectoderm. Therefore, the dorsoventral patterning and EGFR signaling genes play essential roles in correct identity determination and differentiation of lateral glia in the Drosophila nervous system (Kim, 2015).

This study demonstrates that the DV patterning genes, ind, msh, and esg, are required for expression of the glial cell identity marker, gcm, and of the glial cell differentiation marker, Repo, in the proper region of the LTG in the Drosophila VNE. msh and esg acts locally in the formation and differentiation of the LG from the lateral column of the VNE, and esg strongly influences the formation and differentiation of the PG. ind is also locally involved in the initial formation and differentiation of the SG from the VNE. Considering that DV patterning genes, such as ind and msh, are required for the identity determination and formation of NBs in the intermediate and lateral columns along the DV axis, it is plausible that these two genes play essential roles in the proper development of the LTG in the corresponding columns. Interestingly, the zinc finger transcription factor, Esg, plays an important role in the formation and differentiation of the PG that originate from the lateral column, where esg is expressed. Although esg, together with snail and worniu, is required for the asymmetric division of NBs, the precise role of esg in embryonic CNS development has not been clearly determined. Thus, experimental results obtained in this study on esg's role in glial cell formation and differentiation is the first of its kind to analyze the role of esg in gliogenesis during embryonic CNS development (Kim, 2015).

Unexpectedly, vnd, which is essential for identity determination of the medial column NBs, showed the strongest influence on the proper formation and differentiation of all glia, including the LG, SG, and even PG in the VNE. Since the region of msh expression is ventrally expanded in the vnd mutant, disruption of the expression of gcm and Repo in the lateral column may have caused a decrease in the number of LG, LTG, and PG that originate from this region. In addition, the overexpression of vnd also repressed the expressions of Repo and MAPK in the Kr domain, presumably by promoting identity determination of the medial column in the intermediate and lateral columns. Original reports on the role of the vnd in formation and identity determination of the medial column NBs using the vnd target gene, NK6, showed that intermediate and lateral column identity markers are repressed by overexpression of vnd in the Kr-expression domain. One of the reasons for the wider influence of vnd in DV patterning than other DV patterning genes may be that vnd is expressed earliest among these genes, repressing expression of other DV patterning genes such as ind and msh in the medial column, in a process termed 'ventral dominance' (Kim, 2015).

The data revealed that the EGFR signaling receptor and ligand, Egfr and spi, play more global roles in glial cell development than do the DV patterning genes. Egfr and spi are required for initial glial cell formation as shown by reduced expression of gcm and Repo in the LGBs of the VNE. In addition, Repo expression in the differentiated glia was markedly reduced, especially in Egfr embryos, and in spi embryos, to a lesser degree. Interestingly, Repo expression is almost absent in the SG and remains only in the LGs of spi as well as of ind embryos. Since ind expression is activated by the EGFR signaling ligand, Spi, in the VNE to establish the identity of the intermediate column, it is plausible that glial phenotypes in spi and ind mutants are similar to each other. This result indicated that once the intermediate column identity is determined by ind-mediated repression of msh expression in the lateral column, EGFR signaling provides a consolidating extrinsic cue to make ind a repressor of some of the target genes in the intermediate column via MAPK-mediated phosphorylation. This interpretation is compatible with the results obtained by overexpression of Spi and Vn through Kr- and sca-Gal4 drivers, which show repressed Repo expression in the VNE due to the repressor activity of Ind, which in turn is activated by EGFR signaling. Thus, the results indicated that EGFR signaling globally activates many types of glial cell lineages in the VNE and delimits the area where glial cells originate by repressor activity that is chemically modified by EGFR signal transduction (Kim, 2015).

Establishment of proper identity along the DV axis by expression of the DV patterning and EGFR signaling genes is essential for correct formation and differentiation of glia from the VNE This study revealed that the DV patterning and EGFR signaling genes play important roles in the initial formation and differentiation of various types of glia in the Drosophila CNS. The DV patterning genes and EGFR signaling genes are locally and globally required, respectively, for glial cell formation and differentiation using loss-of function mutants of the genes. Unexpectedly, overexpression of the DV patterning and EGFR signaling genes also repressed the initial formation and differentiation of glia. Overexpression of vnd showed stronger repressor activity than msh on the Repo expression in most types of glial cells including the LG, whereas msh showed mild reduction in the Repo expression mainly in the SG, but not in the LG. The repressor activity of vnd started from the initial formation of the LGBs and continued until the glial cells differentiated into mature glia (Kim, 2015).

There are several possible explanations for the repressive effect in both loss-of-function and gain-of function mutants. First, vnd and EGFR signaling genes together play important roles in establishing identities of the medial and intermediate columns in DV patterning of the VNE. Therefore, overexpression of these genes also promote identities of the medial and intermediate in the lateral columns, where many glial cells, including the LG, PG, and some of the SG originate after neurons are formed. This identity change may block glial cell formation and differentiation from the lateral neuroectoderm. Second, overexpression of these genes may also promote neurogenesis over gliogenesis during developmental stages when overexpression was driven by Kr- and sca-Gal4. In addition, repressor activity appears to play a more dominant role than activator activity upon overexpression of vnd, considering that the DV patterning genes, vnd, int, and msh, act as successive repressors to establish and maintain their identity in the VNE. The results obtained using the loss-of-function and overexpression mutants demonstrate that the expression of a proper level of the DV patterning genes promote identity determination of neurons, while their overexpression represses formation of the glia in the VNE by default. In addition, repressor activity of the DV patterning genes appears to play a dominant role in the establishment of the three columnar divisions along the DV axis (Kim, 2015).

Similarly, overexpression of the EGFR signaling ligands, Spi and Vn, and the activated form of EGFR signaling receptor, EgfrAC, repressed Repo expression in all types of glial cells in the VNE. This may be due to the repressor activity of int, since activation of EGFR signaling induces phosphorylation of int and vnd to consolidate their repressor activity. In addition, since Egfr overexpression can cause expansion of vnd expression from the medial column to the lateral area, the intermediate and lateral columns may have acquired the medial identity, such that the LG and various types of other glia originating from the VNE are not generated after overexpression of Spi in the VNE (Kim, 2015).

These studies on the glial cell development in the Drosophila VNE revealed that the DV patterning and EGFR signaling genes play prominent roles in promoting neural identity, rather than glial identity during the early stages of CNS development, since their overexpression did not activate glial identity, but rather repressed it. Later, expression of the glial master gene, gcm, is required to promote glial cell identity in the VNE. It appears that the two-step mode of CNS development first ensures generation of a neural circuit and then provides supporting glial cells in the CNS. The results indicated that the DV patterning genes act locally to promote glial cell formation in their expression domains, but EGFR signaling genes act broadly throughout the VNE. Among the DV patterning genes, vnd, appears to influence glial cell formation and differentiation globally, since it represses int and msh to establish and maintain medial identity from the earliest developmental stage. It remains to be investigated how the DV patterning and EGFR signaling genes control the spatial and temporal regulation of glial cell formation and how they interact to promote glial identity in the CNS (Kim, 2015).


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escargot: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 25 October 2015

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