Notch wing phenotype

The Interactive Fly

Genes involved in tissue and organ development


Illustration:[select image for enlarged view] Notch wings, [left to right] average, extreme

condition, nearly normal, T.H.Morgan, THE PHYSICAL BASIS OF HEREDITY, 1919.

  • Development of the wing
  • Microarray profiling to discover genes expressed in the wing disc
  • Gene expression during Drosophila wing morphogenesis and differentiation
  • Tissue remodeling during maturation of the Drosophila wing
  • Homeodomain interacting protein kinase (HIPK) is required for collective death of the wing epithelium
  • Model for the regulation of size in the wing imaginal disc of Drosophila
  • Cell rearrangement and cell division during the tissue level morphogenesis of evaginating Drosophila imaginal discs
  • Cell competition, growth and size control in the Drosophila wing imaginal disc
  • The Drosophila wing hearts originate from pericardial cells and are essential for wing maturation
  • Identification of genes affecting wing patterning through a loss-of-function mutagenesis screen and characterization of med15 function during wing development
  • Differential division rates and size control in the wing disc
  • Integrating force-sensing and signaling pathways in a model for the regulation of wing imaginal disc size
  • Wg and Wnt4 provide long-range directional input to planar cell polarity orientation in Drosophila
  • Cytonemes are required for the establishment of a normal Hedgehog morphogen gradient in Drosophila epithelia
  • The Elongin complex antagonizes the chromatin factor Corto for Vein versus intervein cell identity in Drosophila wings
  • Mechanisms of regulating tissue elongation in Drosophila wing: impact of oriented cell divisions, oriented mechanical forces, and reduced cell size
  • A common set of DNA regulatory elements shapes Drosophila appendages
  • Gain of cis-regulatory activities underlies novel domains of wingless gene expression in Drosophila
  • Proteasome, but not autophagy, disruption results in severe eye and wing dysmorphia: a subunit- and regulator-dependent process in Drosophila
  • Elimination of unfit cells maintains tissue health and prolongs lifespan

    Genes involved in wing morphogenesis

    Development of the wing

    The wing is derived from the wing imaginal disc, formed from the embryonic ectoderm by an invagination at the intersection of a dorsal/ventral stripe of Wingless with an anterior-posterior stripe of Decapentaplegic. These cells come from the posterior compartment of the second thoracic parasegment and the anterior compartment of the third (Cohen, 1993). One or two cells expressing aristaless, a homeobox protein, invaginate along with the presumptive imaginal disc. The aristaless expressing cells are fated to become the distal most cells of the wing (Campbell, 1993).

    The nuclear proteins, Distal-less and Vestigial are the earliest known markers for the leg and wing imaginal discs, and are required for pattern formation along the proximal-distal axis in the adult. However, their involvement in imaginal disc formation is not clear since imaginal discs are formed in the absence of Dll and vg. Ventral leg and dorsal wing primordia appear to originate from a common imaginal primordium. Cell lineage tracing study has shown that in stage 12, the wing disc cells expressing vg segregate and move dorsally away from Dll expressing cells. Of major importance is the role of escargot and snail in the initial specification of the wing disc. Although Snail is known best for its role in mesoderm formation, it is expressed later in the ectodermal wing primordium. In an esg sna double mutant, the apical constriction of the wing primordial cells is not observed. This supports the idea that in the absence of esg and sna the wing primordium is transformed into epidermis. A two step model for wing disc formation is proposed. In the first step, an extrinsic signal, such as the combined activity of Dpp and Wg, induces vg, esg and sna expression. In a second step esg and sna initiate a program of auto- and crossactivation to stabilize their own expression. This second step is likely to be responsible for irreversible and autonomous fate commitment of the wing primordium (Fuse, 1996).

    The disc is structured into three axes. The anterior/posterior axis is structured by the segment polarity genes engrailed, hedgehog and dishevelled on either side of a stripe expressing decapentaplegic. The proximal/distal axis is strucured by the genes distal-less and aristaless. The dorso-ventral axis is structured by vestigial. wingless serves to structure the sensory hairs at the edges of the wing.

    For a discussion of the hierarchy of genes involved in wing vein formation (Sturtevant, 1995), see the biological overview of rhomboid. Essential information is also found at the vein site.

    Microarray profiling to discover genes expressed in the wing disc

    The Drosophila wing disc is divided along the proximaldistal axis into regions giving rise to the body wall (proximal), wing hinge (central) and wing blade (distal). DNA microarray analysis has been applied to discover genes with potential roles in the development of these regions. A set of 94 transcripts, enriched two fold or greater, were identified in the body wall and 56 enriched transcripts in the wing/hinge region. Transcripts that are known to have highly restricted expression patterns, such as pannier, twist and Bar-H1 (body wall) and knot, nubbin and Distal-less (wing/hinge), show strong differential expression on the arrays. In situ hybridization for 50 previously uncharacterized genes similarly revealed that transcript enrichment identified by the array analysis is consistent with the observed spatial expression. There was a broad spectrum of patterns, in some cases suggesting that the genes could be targets of known signaling pathways. Three of these genes respond to wingless signaling. Genes likely to play specific roles in tracheal and myoblast cell types were also discovered, since these cells are part of the body wall fragment. In summary, the identification of genes with restricted expression patterns using whole genome profiling suggests that many genes with potential roles in wing disc development remain to be characterized (Butler, 2003).

    To identify genes with expression patterns enriched in the presumptive wing/hinge or body wall regions, wing imaginal discs were cut into two fragments at the boundary between the body wall and the wing hinge. Folds associated with the hinge provide morphological features to allow precise cutting. RNA expression profiles of these samples were determined using oligonucleotide microarrays representing approximately 13,500 known and predicted genes in the Drosophila genome (Genechip Drosophila Genome Array 1, Affymetrix). Information for all genes is available at Ninety-four transcripts show two-fold or greater enrichment in the body wall and 56 transcripts show two-fold or greater enrichment in the wing/hinge. Several of these genes were also found to be more highly expressed in wing discs than leg discs or eye-antennal discs, suggesting they may also have appendage-specific roles (Butler, 2003).

    The rank order of transcripts correlates well with the spatial expression patterns of characterized genes. In the body wall, pannier (pan), twist (twi) and BarH1, which are enriched in the body-wall sample, are all known to be highly expressed in the presumptive body wall. In the wing, knot (kn), nubbin (nub) and Distal-less (Dll) are expressed at levels greater than 10-fold above those in the body wall. kn is expressed in the wing 3/4 intervein and hinge regions; nub is strongly expressed in the entire wing pouch and Dll is expressed along the dorsal-ventral (DV) margin exclusively in the wing pouch (Butler, 2003).

    Other genes, known to have important roles in disc development, appear lower down the rank order. vestigial (vg), a key gene for development of the wing and hinge regions, shows only two-fold enrichment but this is consistent with the expression pattern of vg in the wing disc that extends into the body wall region. Transcripts with expression patterns restricted to the posterior compartment, [engrailed (en), invected (inv) and hedgehog (hh)], show approximately two-fold enrichment in the wing/hinge sample. The anterior-posterior compartment boundary splits the wing/hinge region into two equally sized compartments, but the position of the boundary in the body wall region produces a small posterior compartment representing approximately one-quarter of the total tissue. This is consistent with the approximately two-fold enrichment of posterior-specific transcripts found in the wing/hinge tissue sample. The E(spl)-Complex genes are expressed in developing sensory organs found in both the body wall and wing margin regions. Hence, these genes are not enriched in any one sample. The m6 gene is an exception (enriched in the body wall sample) and is known to be expressed only in the body wall region. In contrast, genes that show ubiquitous expression such as Ras or tubulin show no enrichment on the arrays (Butler, 2003).

    Microarray analysis can therefore identify transcripts known to be differentially expressed in the wing/hinge and body wall regions of the disc. Few expression patterns of the identified genes have been described, so to verify the validity of the approach, and to discover more genes with potential roles in the development of these specific regions, in situ hybridizations were made for some of these uncharacterized genes (Butler, 2003).

    Fifty transcripts that had strong enrichment (mostly three-fold or greater) were examined. For the body wall-enriched transcripts, the larger set (only transcripts for which clones are available in the Drosophila gene collections -- DGC1 and DGC2, Berkeley Drosophila Genome Project) were examined. For the wing/hinge region, transcripts were examined with three-fold or greater enrichment, systematically in rank order from the top, and PCR probes were generated when clones were not available. All transcripts tested showed expression patterns that were consistent with the microarray data, providing confirmation that the microarray analysis mirrors the spatial distribution of transcripts in vivo (Butler, 2003).

    The wing disc comprises three cell layers: the squamous epithelium of the peripodial membrane; the columnar epithelium that becomes the adult epidermis, and the adepithelial layer that includes myoblast cells that give rise to adult thoracic muscles and tracheal cells that form air passages. The adepithelial layer extends from the proximal disc dorsally into the hinge region. The body wall fragment includes cells of all three layers, so the arrays also identified transcripts specific to muscle and tracheal cells (Butler, 2003).

    pan and BarH1, which encode transcription factors, are expressed in the body wall epidermis and are involved in bristle patterning. Both transcripts were highly enriched on the arrays. Also highly enriched was tailup (tup), which encodes a LIM domain homeobox protein, and is expressed in the epithelium in a large region of the posterior body wall encompassing the presumptive postnotum, scutellum and scutum. No role for tup in patterning the mesothorax has been described. Another transcript with broad expression was thrombospondin/CG11326 (tsp), which is expressed in a similar region of the body wall to tup. tsp is also expressed in the ventral hinge and hence shows lower enrichment on the arrays. The other genes found to be specific to the epithelium showed highly localized expression: Obp56a/CG11797, CG10126, CG3244 and Glucose dehydrogenase. Obp56a/CG11797 encodes an odorant-binding protein and interestingly three other odorant-binding proteins showed enrichment on the arrays: Obp99a, CG9358 and Obp56d/CG1128. Idgf4, encoding an imaginal disc growth factor, is expressed in the peripodial membrane, primarily in dorsal cells. Presumably secretion of Idgf4 could influence development of the columnar epithelium (Butler, 2003).

    Myoblast cells of the adepithelial layer develop into the direct and indirect flight muscles of the thorax, and genes involved in the development of these muscles have been shown to be expressed in the myoblasts during wing disc development. Several of these transcripts are enriched on the arrays: Mef2, twist (twi) and heartless (htl). Act57B is known to be regulated by Mef2 in the embryo, and Act57B is expressed in the myoblasts, suggesting this relationship also exists in these adult muscle precursors. Mef2 expression is activated by twi and may be inhibited by the transcriptional repressor, zinc finger homology 1 (zfh1). zfh1 is expressed in the myoblasts. stumps is also enriched on the arrays and expressed in the myoblasts. Together with htl, stumps has a role in the development of the tracheal cells. Viking (Vkg) encodes a component of collagen type IV and is known to be coexpressed with Cg25C, another collagen IV subunit in the embryo and in blood cells. Both transcripts are enriched on the arrays and show similar expression patterns in the adepithelial myoblasts and blood cells. Other genes showing specific expression in the myoblasts are BM-40/SPARC, a calcium-binding glycoprotein, which is expressed in the embryonic mesoderm, Elongation factor 1 alpha 100E (Ef1 alpha), CG8689, an alpha-amylase, and two transcripts encoding predicted proteins with unknown function CG11100 and CG15064 (Butler, 2003).

    In the wing disc, cells of the larval and developing adult tracheal systems require activity of genes in the FGF pathway. Some of the key genes are expressed in the myoblasts (for example, htl and stumps), others in the epithelium (for example, branchless, bnl), and others in the tracheal cells themselves (for example, breathless, btl). htl and stumps showed enrichment on the arrays but bnl and btl were not detectable. For bnl this may be because expression is highly localized and apparently at very low levels. However, it is not clear why the arrays failed to detect btl expression because six genes were identified that are also expressed specifically in tracheal cells -- these are CG5397, an O-acyltransferase, CG4386, a serine-type endopeptidase, CG2663, an alpha-tocopherol transfer-like protein, and CG15353, CG6921 and CG9338 that have no known homologies. In particular, CG4386 is interesting since it is only expressed in the dorsal branch, and CG6921 is distinguished because it is very strongly expressed in the most proximal cells (Butler, 2003).

    The wing/hinge fragment of the wing disc primarily contains cells of the peripodial membrane and the columnar epithelium, with only a few myoblasts that extend into the hinge region. Thus the genes detected by the arrays as enriched in this disc fragment are expressed in cells of one of the two epithelial layers (Butler, 2003).

    Transcription factors comprise the largest category of genes (18/56) with elevated expression in the wing/hinge region. These are expected to have regulatory roles in patterning the region. Transcription factors with known expression domains and roles in wing development are present: kn, pox-n, nub, Dll, bifid/optomotor blind, rotund, ventral veins lacking, en, vg and in. pdm2, which is highly related to nub, also shows wing-enriched expression on the arrays and is expressed in a similar domain to nub. pdm2 apparently has no significant function in the wing. The roles of the remaining seven predicted transcription factors are unknown, although the expression pattern of zinc finger homology 2 (zfh2) and Sox 15 have been described and both are expressed specifically in the hinge region. defective proventriculous (dve), which encodes a homeodomain protein, and CG15000, which is similar to NGFI-A-binding protein 2, are broadly expressed in the wing pouch, although dve is downregulated at the DV compartment boundary. odd paired (opa), known for a role in embryonic segmentation, is discretely expressed in cells of the presumptive mesopleura and dorsal hinge. No role for opa in wing disc development has been reported. Dorsocross1 (Doc1) and Doc2/CG5187 are T-Box related factors that are expressed in what appears to be an identical domain in the wing disc. Both transcripts also accumulate in body wall cells and this probably lowers their position in the overall ranked list (Butler, 2003).

    Eight transcripts encoding enzymes are enriched two-fold or greater in the wing/hinge region. This group includes the most highly enriched transcript detected in the analysis, a kazal-type serpin gene CG17278 (68-fold). CG17278 shows a strong and specific expression pattern in the wing encompassing most of the wing pouch. One of the potentially most interesting wing-enriched enzymes is a cytochrome P450 gene, Cyp310al. This gene is strongly expressed in the dorsal and ventral parts of the wing pouch but excluded from the DV and AP boundaries. Variable expression in anterior body wall cells is also observed that is consistent with the array data that indicate Cyp310al transcripts are also present in body wall RNA. Surprisingly, the ß-galactosidase gene (CG3132) was found to be enriched in the wing/hinge region. Weak expression was found in a cluster of cells in the hinge but the majority of expression is in blood cells, which adhere preferentially to the distal disc margin. Thus the ß-gal transcript probably appears as wing/hinge enriched primarily because it is expressed in blood cells. The expression patterns of two other enzymes were also determined: the metalloendopeptidase Nep1/CG5894 and UDP-glucosyl transferase (Ugt86Di) (Butler, 2003).

    The alpha-integrin, inflated, which has a role in cell adhesion, is expressed in the ventral compartment and is thus enriched on the wing/hinge arrays. A novel gene, CG5758, is potentially involved in cell adhesion since it encodes a predicted protein with ß-Ig-H3/Fas domains and its expression is restricted to the dorsal hinge. CG8381 encodes a proline-rich protein with repeated 'PEVK' motifs also found in titin. This gene is strongly expressed in the wing pouch but repressed in cells of the future veins and cells at the DV margin. Despite intense expression in the wing pouch, CG8381 shows only modest enrichment on the arrays, probably reflecting the fact that the gene is also expressed in several groups of cells in the body wall region (Butler, 2003).

    The expression of two receptors was determined. CG4861 encodes an ldl-receptor-like protein and is expressed at very low levels throughout the wing pouch. wengen /CG6531, which is a receptor of the TNFR family, is expressed strongly in the wing pouch and weakly in the body wall. On the arrays, its ligand, eiger, was undetectable in the wing/hinge region sample but enriched in the body wall sample (Butler, 2003).

    Two structural proteins, CG6469, a larval cuticle protein, and CG14301, a chitin-binding protein, are the only genes identified as being expressed in the ventral peripodial membrane. CG6469 is expressed broadly in the peripodial membrane but at a higher level in the ventral region. CG14301 is expressed in cells of both epithelial layers, in the columnar epithelium at the anterior disc margin and in four patches of cells in the wing pouch and the overlying peripodial membrane (Butler, 2003).

    In a group of genes with miscellaneous functions the expression of three genes was determined. anachronism (ana), a secreted glycoprotein, is expressed in five clusters of cells including one in the body wall region and in some individual neuroblasts. ana null mutants are viable and have no observable defects suggesting it is not required, or functions redundantly, in the wing. CG14534, which has a domain that has been recognized in several proteins but has an unknown function (DUF243), is expressed only in cells that will give rise to the posterior wing margin. CG8483, which has homology to a venom allergen, is expressed in a complex pattern suggestive of expression in peripheral sense organ precursors (Butler, 2003).

    The expression pattern is described for five of eight genes for which the sequence reveals no homology to known protein domains. CG15489 and CG15488 are in a cluster of genes also including nub and pdm-2 that are expressed in similar domains and are adjacent in the genome. CG15001, consisting of only a single exon, is adjacent to another gene (CG15000), also discovered on the arrays, with a similar expression domain. BG:DS00797.2/CG9008 is expressed strongly in the wing pouch and also in the adepithelial cell layer. CG8780 is highly enriched on the arrays (31-fold), and expressed specifically in the hinge and ventral pleura (Butler, 2003).

    The genes CG17278, Cyp310a1 and CG8381 all show very intense expression in the wing pouch but reduced expression at the DV margin. Wg is expressed at the DV margin forming a gradient that regulates the expression of target genes in a concentration-dependent manner. To determine whether Wg signaling represses the expression of CG17278, Cyp310a1 and CG8381, wg was ectopically expressed in the dorsal and ventral wing-pouch regions (71B-gal4; UAS-wg), or Wg function was inhibited at the DV margin by expressing a dominant-negative form of TCF (Pangolin), a transcription factor required for Wg-signal transduction (C96-GAL4; UAS-DN-dTCF). With higher levels of Wg activity in the wing pouch, expression of all three genes was inhibited. In contrast, inhibition of Wg signaling at the DV margin allowed ectopic expression of Cyp310a1 in all margin cells and increased the number of cells expressing CG17278 and CG8381. In the presumptive margin, cells continue to express wg in the absence of Wg activity; cell replication increases, and ectopic expression of dmyc appears in margin cells. Therefore, ectopic expression of the genes studied here is caused by loss of Wg-dependent repression rather than loss of the non-expressing cells from the presumptive margin. This does not imply that Wg-dependent repression must be direct. Without functional data on these potential target genes, their relationship to wg and their role in wing patterning remain unknown (Butler, 2003).

    Gene expression during Drosophila wing morphogenesis and differentiation

    The simple cellular composition and array of distally pointing hairs has made the Drosophila wing a favored system for studying planar polarity and the coordination of cellular and tissue level morphogenesis. A gene expression screen was carried out to identify candidate genes that functioned in wing and wing hair morphogenesis. Pupal wing RNA was isolated from tissue prior to, during and after hair growth and used to probe Affymetrix Drosophila gene chips. 435 genes were identified whose expression changed at least 5 fold during this period and 1335 whose expression changed at least 2 fold. As a functional validation, 10 genes were chosen where genetic reagents existed but where there was little or no evidence for a wing phenotype. New phenotypes were found for 9 of these genes providing functional validation for the collection of identified genes. Among the phenotypes seen were a delay in hair initiation, defects in hair maturation, defects in cuticle formation and pigmentation and abnormal wing hair polarity. The collection of identified genes should be a valuable data set for future studies on hair and bristle morphogenesis, cuticle synthesis and planar polarity (Ren, 2005).

    The primary goal in characterizing pupal wing gene expression was to identify genes that play an important role in pupal wing morphogenesis. ken and barbie (ken) encodes a DNA binding transcription factor that contains an N terminal BTB/POZ domain and 3 C2H2 zinc fingers. Its expression increased 6.8 fold from 32 to 40 hrs. Loss of function mutations in ken are semilethal. Escaper adults have been described as having unpigmented aristae and often lack external genitalia (hence the gene name). Wings were examined from ken mutant escapers and also in genetic mosaics. The triple row bristles on the wing margin were lightly pigmented reminiscent of the arista phenotype. This is most obvious in mosaics where the lightly pigmented bristles stand out from their wild type neighbors. No hair phenotype was seen, but a subtle hair pigmentation phenotype would be difficult to see (Ren, 2005).

    The HMGS gene encodes the Drosophila HMG Coenzyme A synthase, a key enzyme in steroid and isoprenoid metabolism. Its expression increased 8.4 fold from 32 to 40 hrs. Individuals homozygous for a P insertion allele die as pharate adults or pupae. The pharate adults are notable for a melanotic liquid that accumulates principally near the ventral head. Mutations that result in weak cuticle often show such melanotic leakage, suggesting that HMGS may be required for normal cuticle elaboration. The reason for the phenotype being seen primarily in the ventral head is unclear. No evidence was seen for a specific wing phenotype (Ren, 2005).

    The expression karst gene, which encodes betaHeavy-spectrin, increased 5.5 fold from 32 to 40 hrs. Spectrin typically contains 4 chains, 2 alpha and 2 beta; these chains are known to link the actin cytoskeleton to the plasma membrane. Somewhat surprisingly, kst mutants are viable (at reduced levels) and female sterile due to defects in the follicular epithelium. Adult kst mutants have rough eyes and their wings often are cupped downward. kst wings were examined and an additional mutant phenotype was found that is nicely correlated with its expression profile. kst wing cells produce normal looking hairs but the hairs are often found on a small pedestal. The wing cell surface (that is not hair) is rough and at times remnants of cell outlines are visible. This phenotype can also be seen in mosaic clones. The clones can be recognized under the stereo microscope because they are often associated with a dimpling of the wing surface (Ren, 2005).

    The krotzkopf verkehrt (kkv) and knickkopf (knk) genes were both identified in a screen for having an unusual defect in embryonic cuticle, known as the blimp phenotype. Mutant embryo cuticles were seen to expand in cuticle preparations. The kkv gene encodes a chitin synthase implicating it in cuticle synthesis and its expression increased 4.9 fold from 32 to 40 hrs. The knk gene encodes a novel gene that is only well conserved in the ecdysozoa, suggesting a role in cuticle metabolism. The amino acid sequence shows homology to what is thought to be a dopamine binding domain suggesting Knk might be involved in cross linking of cuticle. The expression of knk increased 7 fold between 32 and 40 hrs. Mutations in both of these genes are embryonic lethals so mosaic clones of cells carrying mutations in either of these genes were examined. The phenotypes seen in the adult cuticle were quite similar to one another. Most notably wing mutant wing hairs displayed a lack of pigmentation and were thinner and flimsier than normal. This phenotype is dramatic and at low magnification it often appears as if hairs were not formed by mutant cells. The hairs appeared normal in size and shape when clones were examined in pupal wings arguing that the mutations affect a process after hair outgrowth (e.g., cuticle synthesis or maturation). Clones in other body regions such as the abdomen and thorax also showed a dramatic loss of pigmentation. In all of these cases the borders between pigmented and unpigmented were relatively sharp. Consistent with these mutations resulting in weak cuticle phenotypes, areas were often seen where internal tissues and hemolymph appeared to be erupting from the animal. This was usually seen on the dorsal abdomen, particularly in the region of the intersegmental membrane. The eruptions could be related to the blimp phenotype seen in embryos (Ren, 2005).

    The expression of the brain tumor (brat) decreased 5.5 fold from 24 to 40 hrs. This gene has been studied primarily due to the neural tumor phenotype seen in loss of function mutants. The wings of bratts/Df brat flies raised at semi-permissive conditions were examined. No hair phenotype was seen but the occasional loss of sensory bristle shaft cells (principally distally along the anterior margin) was seen and occasional duplicated bristle cells (principally in the costa). These phenotypes are suggestive of a role for brat in specifying cell fate or in Notch mediated lateral inhibition (Ren, 2005).

    The expression of dopa decarboxylase (Ddc) increased 6 fold from 24 to 32 hrs and then decreased 1.9 fold from 32 to 40 hrs. This well characterized gene is known to function in the epidermis for the cross linking of cuticle and in the formation of melanin. Loss of Ddc function results in fragile and pale cuticle with thin bristles. No detailed description of the wing phenotype had been reported previously. Ddc null alleles are recessive embryonic lethals adults that contained clones mutant for Ddc were examined. On the abdomen (and some other parts of the body) clones could be seen where there was lightly pigmented cuticle and bristles. No wing phenotype was seen other than apparent clones resulting in lighter triple row bristles. The abdominal clone boundaries were not sharp as seen for grh, knk or kkv, which also give rise to lightly pigmented cuticle suggesting that the Ddc cells might be rescued by the diffusion of dopamine from neighboring cells. Therefore adults homozygous for a temperature sensitive Ddc allele were examined. Animals raised at 25°C showed a much stronger phenotype in general than was in clones suggesting that Ddc acts nonautonously in the wing. The phenotype was even stronger in animals raised at 29°C. The wings of Ddc mutants were characterized by very thin wispy hairs, occasional multiple hair cells and an overall faint appearance. When Ddcts pupal wings were examined, the early hairs appeared normal in morphology. Thus, the wispy appearance of the adult wing hairs is presumably due to a late defect. It is suggested that Ddc dependent cross linking of the cuticle is essential for maintaining the structure of the hair and in the absence of this cross linking the hair collapses after the actin cytoskeleton is disassembled. Occasional multiple hair cells were seen in the Ddcts pupal wings; thus that defect is likely due to a different process also being affected in the mutant. The formation of multiple hair cells has previously been associated with planar polarity defects or due to disruptions of the cytoskeleton (Ren, 2005).

    The HR46 gene (also known as DHR3) encodes a nuclear receptor and is an essential gene known to be important for the ecdysone cascade. Large clones of loss of function alleles result in wing (folded and curved) and notum defects (rough short bristles and pale pigmentation). The expression of this gene increased 250 fold from 24 to 32 hr and then decreased 4.3 fold from 32 to 40 hr. Moderate sized wing clones of cells lacking HR46 were examined, but no clear cut phenotype was seen. In pupal wing clones examined a couple of hours after hair formation mutant hairs appeared somewhat thicker but this alteration was transient (Ren, 2005).

    The Eip78CD gene encodes a related nuclear receptor. The expression of this non-essential gene increased 3 fold from 24 to 32 hr followed by a three fold drop from 32 to 40 hr (but the differences were not significant) suggesting it might be functionally redundant with HR46. To test this hypothesis Eip78CD mutants, which also contained HR46 mutant clones, were examined. No mutant phenotypes were seen in the clones, suggesting either that there is an alternative redundant gene or that HR46 is not essential for hair morphogenesis. Since the level of HR46 expression fell dramatically between 32 and 40 hrs it seemed possible that declining HR46 expression could be important for hair development. To test this the overexpression of HR46 from a transgene containing a hs promoter was induced. This resulted in a dramatic loss of hair formation leading to wings with extensive bald regions. The strongest phenotype was seen when the transgene was induced by heat shocking 6-8 hrs prior to the time of hair initiation. The phenotype was dose sensitive and directly related to the number of transgenes and length and temperature of transgene induction (Ren, 2005).

    The expression of the non-stop (not) gene decreased 3.9 fold from 24 to 40 hrs. Mutations in not result in photoreceptor neurons projecting through the lamina instead of terminating there. The mutations also result in approximately 20% of ommatidia being misoriented -- a planar polarity phenotype. Strong alleles of not die as prepupae so not clones were examined in both adult and pupal wings. Large numbers of clones were induced. Perhaps 25% of wing cells are found in clones. All adult wings of this genotype had regions where there were cells that failed to form hairs or that had very small hairs. These were found only in proximal medial regions on the ventral wing surface. All such wings also had subtle polarity abnormalities; small groups of hairs with slightly abnormal polarity in all regions of the wing. Consistently finding such defects leads to the conclusion that these were due to not clones. Of 47 such wings examined 27 also contained multiple hair cells and a further 10 contained regions with planar polarity defects reminiscent of genes such as fz and dsh. When marked not clones were examined in pupal wings most, but not all, showed cells where hair differentiation was delayed or absent. Such clones were seen in all wing regions. It is suggested that all not clones have delayed hair formation. When the clones are located in wing regions where hairs normally form first (distal or peripheral regions) the hairs form later than normal but still have enough time to reach a relatively normal length. In contrast, when clones are located in regions where hair formation is normally late (proximal and medial regions on the ventral wing surface) not enough time remains prior to cuticle deposition to produce a normal hair. The not gene encodes a ubiquitin carboxyterminal hydrolase likely to function in the removal of ubiquitin from proteins during protein degradation (Ren, 2005).

    The Uch-L3 gene also encodes a ubiquitin carboxy hydrolase and its expression decreased 2.9 fold between 24 and 40 hrs. A P insertion mutation in this gene is semi-lethal and escapers have an abnormal eye. No homozygous Uch- L3J2b8 flies were found that eclosed but it was possible to examine animals that died as pharate adults. These animals displayed several morphological defects such as loss of tarsal leg joints, shorter and fatter leg segments, the loss of a discrete antennal segment 4 and a fatter arista that could be due to defects in cell shape or movement. Pupal wings from such animals were examined and wings were found that were wider and shorter than normal and regions were found with a loss of hairs. All of the phenotypes seen in Uch-L3 pupae and pharate adults showed variable expressivity (Ren, 2005).

    Tissue remodeling during maturation of the Drosophila wing

    The final step in morphogenesis of the adult fly is wing maturation, a process not well understood at the cellular level due to the impermeable and refractive nature of cuticle synthesized some 30 h prior to eclosion from the pupal case. Advances in GFP technology now make it possible to visualize cells using fluorescence after cuticle synthesis is complete. Between eclosion and wing expansion, the epithelia within the folded wing begin to delaminate from the cuticle and that delamination is complete when the wing has fully expanded. After expansion, epithelial cells lose contact with each other, adherens junctions are disrupted, and nuclei become pycnotic. The cells then change shape, elongate, and migrate from the wing into the thorax. During wing maturation, the Timp gene product, tissue inhibitor of metalloproteinases, and probably other components of an extracellular matrix are expressed that bond the dorsal and ventral cuticular surfaces of the wing following migration of the cells. These steps are dissected using the batone and Timp genes and ectopic expression of αPS integrin, inhibitors of Armadillo/β-catenin nuclear activity and baculovirus caspase inhibitor p35. It is concluded that an epithelial-mesenchymal transition is responsible for epithelial delamination and dissolution (Kiger, 2007; full text of article).

    The following outline is proposed of that program based upon cell behavior: delamination and severing contacts; changing cell shape; and migration and ECM synthesis.

    Stage 1, delamination and severing contacts

    A signaling role for integrins during the prepupal apposition has been proposed that prepares cells for integrin-based adhesion of the epithelia at the pupal apposition. The observation that wing epithelial cells persist in the blistered regions produced by ectopic αPS integrin expression suggests that the integrin interaction also prepares cells to respond to the later signal that induces epithelial delamination and dissolution. This signal is also blocked in the mutant batone, which prevents wing expansion. Some cells begin to delaminate from the cuticle before wing expansion has begun, and all have delaminated by the time expansion is complete. Delamination must involve severing of ECM contacts. The precision of the cellular array in a newly open wing must derive from cell–cell contacts between stretched cells that are maintained following delamination. Each cell then compacts and becomes round (as judged by the increase in fluorescence intensity). The round cells have evidently severed their junctions with adjacent cells because the precise array of cells begins to break up and Arm-GFP moves from the cell membrane to the cytoplasm (Kiger, 2007).

    It would appear that disturbing the normal state of Arm/β-catenin signaling activity in epithelial cells blocks delamination. Delamination is blocked by ectopic expression of Pygo in the epithelial cells, which blocks expression of Arm target genes in a variety of tissues, and by ectopic expression of Shaggy, which blocks expression of Arm target genes by phosphorylating cytoplasmic Arm, promoting its degradation and depleting nuclear Arm. Ectopic expression of stabilized forms of Arm not subject to Shaggy phosphorylation evidently has a dominant-negative effect on Arm signaling activity in the maturing wing, blocking delamination of epithelial cells. This interpretation is supported by the following observations. First, no effect is produced by ectopic expression of wild-type Arm using the same Gal4-30A driver, consistent with other reports, very likely indicating the efficiency with which wild-type Arm is eliminated by phosphorylation and degradation through the proteasome. Second, a very low level of nuclear Arm is sufficient for target gene expression. The Arm-GFP fusion protein used here is fully active and completely covers homozygosity for a null arm allele, yet nuclear Arm-GFP cannot be detected in cells receiving a Wingless signal. Thus, it is reasonable that non-physiologically high levels of stable forms of Arm could have a dominant-negative effect, not unlike the inhibitory effect of over-expression of Pygo on Arm-directed transcription. (Kiger, 2007).

    Arguing against an interpretation that the effects of ectopic gene expression might be non-specific, note that Gal4-30A-driven expression of p35 does not block delamination. Nor does Gal4-30A-driven expression of either αPS integrin or wild-type transcription factor Pangolin/dTCF/LEF-1, or a dominant-negative form of CREB have any effect on wing maturation (Kiger, 2007).

    Stage 2, changing cell shape

    The round cells then begin to change shape, extending thin cytoplasmic filaments, and elongate into spindles that associate with similarly shaped cells forming streams. The fact that p35 expression interrupts developmental progression at the round cell stage clearly separates Stage 1 from the changes in cell shape, cell migration, and ECM synthesis events that follow. In some cellular contexts caspase inhibition prevents cell migration independently of blocking apoptosis. It has been shown that the nuclei of wing cells cease to retain nuclear-targeted GFP and begin to fragment their DNA at what appears to be the round cell stage, consistent with the observation of pycnotic nuclei at this stage (Kiger, 2007).

    Stage 3, migration and ECM synthesis

    The cells migrate toward the hinge and into the body of the fly, leaving behind components, perhaps including tissue inhibitor of metalloproteinases, of an ECM that will bond dorsal and ventral cuticular surfaces. It is noteworthy that Timp deficiency does not interfere with cell migration. ECM assembly must be the final step in the developmental program. The nonautonomous action of Timp in bonding cuticle secreted by mutant Timp clones suggests that Timp is present in abundance and diffuses over large distances in the wing to participate in ECM formation (Kiger, 2007).

    Precisely how ectopic expression of the various UAS transgenes studied in this paper produces wing blisters or collapsed wings is not wholly clear. It seems doubtful that cells that fail to delaminate during early phases of tissue remodeling would secrete ECM components normally. Yet a variable number of cells in these wings do delaminate and leave the wing, presumably because of variation in the level of Gal4-30A expression. These cells might be expected to secrete the necessary ECM components, although the level of critical component(s) may be insufficient for normal bonding to occur in some cases. Blister formation might also be caused by the presence of numbers of undelaminated cells physically preventing ECM from bonding the underlying cuticle. Note that when ectopic p35 expression is limited, a moderate number of round, delaminated cells can become bound in the wing without producing blisters (Kiger, 2007).

    The presence of true hemocytes in the wing raises the question of whether these cells play a role in wing maturation. If Gal4-30A was to be expressed in these cells, as well as in epithelial cells, interpretation of ectopic expression studies would be complicated. No cells were detected expressing DsRedGFP fluorescence that did not express ywing-GFP fluorescence, suggesting that Gal4-30A is not expressed in true hemocytes. The observations that Hemese-Gal4-driven expression of Shaggy or of Pygo has no effect on wing maturation strongly suggest that the effects of Shaggy and Pygo on wing maturation are not mediated by true hemocytes exclusively, if at all. While the possibility that Timp and/or other ECM components are supplied by true hemocytes cannot be ruled out, the bulk of the evidence supports an active role for epithelial cells in bonding the wing surfaces. Precocious death of epithelial cells induced by Gal4-30A-driven expression of Ricin A in late pupal epithelial cells prevents bonding of dorsal and ventral cuticle after eclosion. Because the wing cuticle is fully formed, the induced cell death must have occurred after cuticle deposition but before eclosion. UV irradiation after eclosion blocks both epithelial cell delamination and bonding of the wing surfaces. In addition, it is clear that mitotic clones of defective epithelial cells affect bonding of the wing surfaces. Mitotic clones mutant for an integrin gene produce blisters in the wing cuticle as do mitotic clones ectopically expressing PKAc (Kiger, 2007).

    These studies describe for the first time the developmental program that completes morphogenesis of the adult fly. The requirement for a normal state of Arm/β-catenin signaling activity suggests that an epithelial–mesenchymal transition (EMT) transforms epithelial cells into mobile fibroblasts in the wing (Kiger, 2007).

    The best known example of an EMT in Drosophila is neuroblast delamination. In embryonic central nervous system formation, Wingless signaling has been shown to induce nonautonomously the delamination of specific neuroectoderm cells to form S2 neuroblasts. In peripheral nervous system formation, Wingless signaling is required for bristle formation at the wing margin, and ectopic expression of Wingless induces ectopic bristles in the wing blade. The ability of Wingless to induce neuroectoderm cells to form neuroblasts is tightly regulated by Notch in both the central and peripheral nervous systems. Evidence supports the idea that Notch modulates Wingless signaling by associating directly with Arm/β-catenin to regulate its transcriptional activity (Kiger, 2007).

    Arm/β-catenin signaling appears to be characteristic of EMTs. Translocation of Arm/β-catenin into the nucleus precedes gastrulation in Drosophila, the sea urchin, and zebrafish. EMTs occur in the vertebrate neural crest when cells delaminate from the neural epithelium and migrate throughout the embryo. In the avian neural crest, dominant-negative forms of β-catenin and LEF/TCF inhibit delamination of cells from the epithelium, G1/S transition, and transcription of target genes. β-Catenin and LEF/TCF proteins are observed to translocate to the nuclei of avian neural crest cells only during delamination and to be absent during advanced stages of migration. EMTs are also a characteristic of cancer formation and can be initiated in some cancers by aberrant β-catenin activity (Kiger, 2007).

    Multiple ways of activating Arm/β-catenin signaling exist. There are two independently regulated pathways that can target Arm/β-catenin to the proteasome, the Shaggy/Glycogen synthase kinase 3 degradation complex and the Seven in Absentia Homologue/ubiquitin ligase degradation complex. Multiple G-protein-coupled receptors target the Shaggy/Glycogen synthase kinase 3 degradation complex for inhibition. Further studies are necessary to identify the hormone(s), receptor(s) and signal transduction mechanisms acting in the wing maturation program and to relate this work to the extensive studies of the hormonal signals controlling wing expansion and cuticle tanning (Kiger, 2007).

    Homeodomain interacting protein kinase (HIPK) is required for collective death of the wing epithelium

    Post-eclosion elimination of the Drosophila wing epithelium was studied in vivo where collective 'suicide waves' promote sudden, coordinated death of epithelial sheets without a final engulfment step (see Collective Cell Death and Canonical Pathways). Like apoptosis in earlier developmental stages, this unique communal form of cell death is controlled through the apoptosome proteins, Dronc and Dark, together with the IAP antagonists, Reaper, Grim, and Hid. Genetic lesions in these pathways caused intervein epithelial cells to persist, prompting a characteristic late-onset blemishing phenotype throughout the wing blade. This phenotype was leveraged in mosaic animals to discover relevant genes. This study establish that homeodomain interacting protein kinase (HIPK) is required for collective death of the wing epithelium. Extra cells also persisted in other tissues, establishing a more generalized requirement for HIPK in the regulation of cell death and cell numbers (Link, 2007).

    Elimination of the wing epithelium in newly eclosed adults is predictable, easily visualized, and experimentally tractable. The major histomorphologic events involve cell death, delamination, and clearance of corpses and cell remnants. Recent studies established that post-eclosion PCD is under hormonal control and involves the cAMP/PKA pathway (Kimura, 2004). While dying cells in the adult wing present apoptotic features (e.g., sensitivity to p35 and TUNEL positive), elimination of the epithelium is distinct from classical apoptosis in several important respects. First, unlike most in vivo models, overt engulfment of cell corpses does not occur at the site of death. Instead, dead or dying cells and their remnants are washed into the thoracic cavity via streaming of material along and through wing veins. Second, extensive vacuolization is seen in ultrastructural analyses, which could indicate elevated autophagic activity. Third, widespread and near synchronous death that occurs in this context defines an abrupt group behavior. The process affects dramatic change at the tissue level, causing wholesale loss of intervein cells and coordinated elimination of the entire layer of epithelium. Rather than die independently, these cells die communally, as if responding to coordinated signals propagated throughout the entire epithelium, perhaps involving intercellular gap junctions. This group behavior contrasts with canonical in vivo models where a single cell, surrounded by viable neighbors, sporadically initiates apoptosis (Link, 2007).

    One study proposed that an epithelial-to-mesenchymal transition (EMT) accounts for the removal of epithelial cells after eclosion (Kiger, 2007). Although the results do not exclude EMT associated changes in the newly eclosed wing epithelium, compelling lines of evidence establish that post-eclosion loss of the wing epithelium occurs by PCD in situ—before cells are removed from the wing (Kimura, 2004 and this study). First, before elimination, wing epithelial cells label prominently with TUNEL. Second, every mutation in canonical PCD genes so far tested failed to effectively eliminate the wing epithelium, and at least two of these were recovered in the current screen. Third, elimination of the wing epithelium was reversed by induction of p35, a broad-spectrum caspase inhibitor (Kimura, 2004). Fourth, using time-lapse microscopy, condensing or pycnotic nuclei were clearly detected, followed by the rapid removal of all cell debris in time frames (minutes) not consistent with active migration. Instead, removal of cell remnants occurred by a passive streaming process, involving perhaps hydrostatic flow of the hemolymph (Link, 2007).

    This study sampled over one fifth of all lethal genes and nearly 10% of all genes in the fly genome for the progressive blemish phenotype, a reliable indicator of PCD failure in the wing epithelium. Nearly half of the mutants that produced melanized wing blemishing also displayed a cell death–defective phenotype when examined with the vg:DsRed reporter. The precise link between these defects is unclear, but a likely explanation suggests that as the surrounding cuticle fuses, persisting cells, now deprived for nutrients and oxygen, become necrotic and may initiate melanization. Mutants could arrest at upstream steps, involving the specification or execution of PCD, or they might affect proper clearance of cell corpses from the epithelium. New alleles were recovered of dark (l(2)SH0173) and a likely hypermorph of thread (l(3)S048915), which provides reassuring validation of this prediction (Link, 2007).

    By leveraging this distinct phenotype, novel cell death genes, were captured including the Drosophila orthologue of HIPK. Though first identified as an NK homeodomain binding partner, this gene was found to be an essential regulator of PCD and cell numbers in diverse tissue contexts. Of the four mammalian HIPK genes, HIPK2, the predicted orthologue of Drosophila HIPK, has been placed in the p53 stress-response apoptotic pathway, but whether the Drosophila counterpart similarly impacts this network is not yet known (Link, 2007).

    Model for the regulation of size in the wing imaginal disc of Drosophila

    For animal development it is necessary that organs stop growing after they reach a certain size. However, it is still largely unknown how this termination of growth is regulated. The wing imaginal disc of Drosophila serves as a commonly used model system to study the regulation of growth. Paradoxically, it has been observed that growth occurs uniformly throughout the disc, even though Decapentaplegic (Dpp), a key inducer of growth, forms a gradient. This paper presents a model for the control of growth in the wing imaginal disc, which can account for the uniform occurrence and termination of growth. A central feature of the model is that net growth is not only regulated by growth factors, but by mechanical forces as well. According to the model, growth factors like Dpp induce growth in the center of the disc, which subsequently causes a tangential stretching of surrounding peripheral regions. Above a certain threshold, this stretching stimulates growth in these peripheral regions. Since the stretching is not completely compensated for by the induced growth, the peripheral regions will compress the center of the disc, leading to an inhibition of growth in the center. The larger the disc, the stronger this compression becomes and hence the stronger the inhibiting effect. Growth ceases when the growth factors can no longer overcome this inhibition. With numerical simulations it was shown that the model indeed yields uniform growth. Furthermore, the model can also account for other experimental data on growth in the wing disc (Aegerter-Wilmsen, 2007).

    Since the wing imaginal disc serves as a model system to study the regulation of growth, a large amount of experimental data is already available. The model has been evaluated with experimental results from the literature. When clones with increased Dpp signaling are generated, they grow larger in the lateral regions than in the medial part. Furthermore, clones with decreased Dpp signaling survive better laterally than medially. A common explanation for these findings is that the medial cells are more competitive than the lateral cells because they receive higher levels of Dpp. Therefore, a clone with a fixed level of Dpp signaling is hindered more when growing in the medial part than when growing more laterally. The model may offer an additional, alternative explanation. A clone is stretched more and compressed less when growing laterally than when growing medially. Therefore, it grows faster laterally as long as its level of Dpp signaling is fixed. It is expected that both competition and differences in compression contribute to the difference of size among different clones (Aegerter-Wilmsen, 2007 and references therein).

    Discs with homogeneous Dpp signaling are expanded along the dorsoventral boundary. According to the model, the total growth factor activity in these discs is highest along the dorsoventral boundary, thus accounting for the expansion along this boundary. Furthermore, it has been found that discs with homogeneous Dpp signaling do not show uniform growth. Instead the growth rate of cells in the lateral regions, close to the dorsoventral boundary, is higher than the growth rate of cells in the medial part of the disc. According to the model, the high growth factor activity along the dorsoventral boundary will promote additional growth along the whole boundary. This stretches the regions further away from the dorsoventral boundary. This stretching pulls the cells along the dorsoventral boundary toward the center of the disc. The cells in the center are thus being compressed. The closer the cells are located to the center, the more they are compressed and the more growth is inhibited, thus leading to the observed differences in growth rate (Aegerter-Wilmsen, 2007).

    The Dpp pathway can be activated locally by expressing a constitutively active form of one of its receptors (tkvQ-D). Recently, it has been shown that activating the Dpp pathway in clones in this way can stimulate transient non-autonomous cell proliferation. When inhibiting the pathway, similar effects were seen. Clones with increased Dpp activity were modeled as a region with increased Dpp activity compared to its surrounding tissue with lower homogeneous Dpp activity. In that case, the cells with high Dpp signaling initially grow faster than the surrounding cells, thus stretching them. As in the wild-type situation at the start of growth, the stretching is highest in the cells closest to the region with high Dpp signaling and therefore growth is induced in these cells. This non-autonomous growth increases the stretching in the cells further away from the clone, which will increase their growth. Therefore, after some time, growth in the cells surrounding the clone will be homogeneous again, comparable with the situation in the wild type disc. Thus, the model accounts for the non-autonomous effect as well as for the observation that it only occurs transiently (Aegerter-Wilmsen, 2007).

    Clones with decreased Dpp activity were modeled in a similar way. The cells surrounding the clone get stretched between the slow growing cells in the clone and the faster growing cells further away from the clone. Therefore growth is also induced non-autonomously in cells surrounding clones with decreased Dpp signaling, which is again in agreement with the data (Aegerter-Wilmsen, 2007).

    Non-autonomous effects on cell proliferation were also assessed for clones in which growth is increased by overexpressing CyclinD and Cdk4 instead of by increased Dpp signaling. The non-autonomous proliferation was not observed in that case, even though this would in principle be expected based on the model. However, cell divisions are only slightly increased in these clones and apoptosis is increased, which is generally accompanied by basal extrusion. Therefore, it seems as if co-expression of CyclinD and Cdk4 causes only very little net overgrowth at the stage measured. For such clones the non-autonomous stimulation of proliferation is expected to be less pronounced and to occur at a relatively late point in time, which may explain why it has not been observed (Aegerter-Wilmsen, 2007).

    Experimentally induced alterations in cell proliferation are often compensated for by changes in cell size, such that the final wing disc size is not changed. This suggests that wing disc size is not a function of cell numbers. In the model, the wing disc is considered as an elastic sheet with certain mechanical properties. As long as the mechanical properties of the tissue as a whole are not influenced by cell size, the final disc size is indeed not a function of cell numbers according to the model. Furthermore, according to the model, it would be expected that a reduction of growth in the center of the disc automatically leads to a reduction of growth in the peripheral regions. Indeed, when the size of the wing blade was decreased by down-regulating vestigial (vg) expression, non-autonomous reductions in surrounding WT territories were observed along all axes of growth. Lastly, the model predicts that stretching occurs in the peripheral regions. Therefore, it also predicts that, upon cutting the disc from the end toward the middle, tissue at both sides of the cut moves apart. In wound healing experiments, this was indeed observed. In contrast, the model predicts that the central region of the disc becomes compressed. The increased thickness of the (columnar layer of the) wing disc could be seen as an indication that compression indeed occurs (Aegerter-Wilmsen, 2007).

    This paper has presented a model for the determination of final size in the wing imaginal disc. In the model, growth is negatively regulated by mechanical stresses, which are automatically generated as a result of growth rate differences in an elastic tissue. With the use of numerical simulations, it was demonstrated that the model naturally leads to uniform growth as was shown experimentally and that it leads to the observed final size of the wing disc. Furthermore, it was argued that the model can also account for other experimental data in literature (Aegerter-Wilmsen, 2007).

    Cell rearrangement and cell division during the tissue level morphogenesis of evaginating Drosophila imaginal discs

    The evagination of Drosophila imaginal discs is a classic system for studying tissue level morphogenesis. Evagination involves a dramatic change in morphology and published data argue that this is mediated by cell shape changes. The evagination of both the leg and wing discs has been reexamined and it has been found that the process involves cell rearrangement and that cell divisions take place during the process. The number of cells across the width of the ptc domain in the wing and the omb domain in the leg decreases as the tissue extends during evagination and cell rearrangement was observed to be common during this period. In addition, almost half of the cells in the region of the leg examined divided between 4 and 8 h after white prepupae formation. Interestingly, these divisions were not typically oriented parallel to the axis of elongation. These observations show that disc evagination involves multiple cellular behaviors, as is the case for many other morphogenetic processes (Taylor, 2008).

    This study established that cell rearrangement takes place during leg and wing evagination and contributes to the thinning and extension of the appendages. These observations are consistent with the pioneering results of Fristrom (1976) on evagination. The current data also established that cell rearrangement takes place throughout the appendage and is not restricted to a particular region along the proximal/distal axis. However, the observations are also consistent with cell rearrangement being non-uniform as some regions appeared to 'thin' more than others. For example, in the wing the width of the ptc domain at position M5 thinned more than at position M4 (refering to neuronal landmarks). The evaginating leg and wing cells retain their epithelial morphology with extensive apical junctional complexes. Rearrangement requires that cells change neighbors and hence must remove old junctions and generate new ones while maintaining tissue integrity. This problem is not restricted to evaginating discs but is a general one for epithelial tissues and is an issue that has concerned developmental/cell biologists for many years. Important insights into how this could be accomplished come from recent observations on germ band elongation in the Drosophila embryo. Several groups have provided evidence that junctional remodeling plays a key role in cell rearrangement in this epithelial tissue. This mechanism also appears to function in the repacking of pupal wing cells. It is suggested that it also plays a role in leg and wing evagination. No clear evidence is seen for the multicellular rosettes that have been implicated in germ band extension. Perhaps this is due to disc evagination being substantially slower than germ band extension (Taylor, 2008).

    No evidence was seen of dramatic coordinated changes in cell shape. There was a small but significant increase in the length along the proximal/distal axis of evaginating omb domain tibia cells that should contribute to elongation. However, the change was not large enough to account for leg morphogenesis. No significant change was seen in cell shape in evaginating ptc domain wing cells although there was a hint of a possible small effect. It is worth noting that in these measurements cells from all positions along the relevant part of the proximal/distal axis were included. Casual observation suggested that there might be small regions with consistent changes but these would likely be counterbalanced by changes in shape elsewhere in the domain (Taylor, 2008).

    It was not possible to image the earliest stages of leg disc evagination or the disc cells that form ventral thorax. Thus, these observations were not able to distinguish between the two proposed mechanisms of eversion (i.e., spreading vs. invasion hypotheses). Patterned cell death could in principle play an important role in disc evagination. Previous studies have not seen evidence for patterned cell death during wing blade evagination and the current observations support this conclusion. Cell death has been detected in evaginating legs but this is restricted to the regions of the tarsal segments where the leg joints form and hence is unlikely to contribute to the overall thinning of the omb domain of leg segments (Taylor, 2008).

    Based on the literature, it was not expected that cell division takes place during evagination, but the current observations showed that it occurred. The most definitive experiments involved generating clones of cells marked by GFP expression and following these in vivo. These experiments provided compelling evidence for cell division. This was only done for the leg but other experiments provided strong evidence for cell division in evaginating wings. The size of wing clones was larger when they were induced at white prepupae than at the formation of the definitive pupae. Cell division was not rare in evaginating legs, and on average about 40% of the cells divided. Indeed, a majority of the cells divided in about 1/3 of clones examined. This amount of cell division is sufficient to account for the thickening of the omb domain that was observed from 6 to 8 h in developing legs. Observations on the size of wing clones suggested a similar fraction of wing cells divided during evagination. A limitation is that the in vivo imaging technique only allowed effective imaging of clones on the leg surface juxtaposed to the pupal case in the basitarsus and tibia (and occasionally tarsal) segments. Thus, data could not be obtained for much of the leg disc derivatives, and hence the overall proportion of evaginating leg cells that divide cannot be confidently estimated. The spindle in these dividing cells was not imaged but it was inferred that the spindle was not oriented parallel to the elongating axis, based on the position of the resulting daughter cells shortly after division. The two daughter cells usually filled up the area taken up by the parental cell prior to division, which helped in assigning a lineage. The leg epidermis is continuous without free 'space'. Hence, that daughter cells would occupy the space of the parental cell is not surprising. A parallel orientation for the spindle might be expected if the cell division plane was tightly linked to the mechanism of elongation. The inferred orientation of the cell divisions was most often between 46o and 60o. Thus, they would increase the number of cells both along the proximal/distal and anterior/posterior (and dorsal/ventral) axes. In the second day pupal leg, the width of the omb domain was narrower than it was in the evaginating leg. This could be a reflection of a later stage of convergent extension. However, legs were not followed throughout this period, other possibilities cannot be ruled out. It is interesting to note that cells in the pupal tibia and basitarsus have a spiral arrangement, and this appears to arise from 6 to 8 h after white prepupae. Thus, this arrangement could be at least in part a consequence of the orientation of the cell divisions (Taylor, 2008).

    The fraction of dividing cells varied widely from one clone to another. This was not correlated with particular pupae or legs as both clones where a majority of the cells divided and clones where no cells divided were found in the same pupae and on the same leg. One possibility is that the variation is due to region specific differences. For example, cells in one region of the leg might never divide during evagination while a majority of cells in another region might always divide. No evidence is seen for this but the experiments were not compelling on this point. The observations on the omb domain did not examine a majority of leg cells and in the experiments where MARCM clones were followed, it could not be routinely said exactly where on the leg a clone was located. A second possibility is that the variation is due to the clustered distribution of S phase and mitotic cells in wing and leg discs. Any small clone could comprise a cluster (or not contain a cluster) and this could lead to a great deal of variation in observed cell division. The basis for the clustering is uncertain but could simply represent a pseudo-synchronization due to neighboring sister cells having been born at the same time (Taylor, 2008).

    The observations suggest that several different factors play a role in evagination. At the start of evagination, the leg and wing discs are folded and some of the initial elongation is due to an unfolding of the tissue that presumably results from changes in the shape of cells along the apical/basal axis. During the period when leg discs evert and present the apical surface of their epithelial cells to the outside, elongation is also taking place and there is active pulsatile movement. This appears to be related to the movement of hemolymph in the prepupae and blood cells can often be seen to move in step with the pulses. This suggests that hydraulic pressure could be playing a role in eversion and elongation. The leg resembles a cylinder closed on one side (distal tip) and open to the body on the other (proximal). Thus, it is expected that hemolymph is pumped by the heart to produce a mechanical force that could help evert and/or elongate the leg. The pulsatile movement starts to decrease at about 4-4.5 h after white prepupae and largely ends by about 5 h. This is around the time of eversion, but the slowing clearly precedes eversion. It is suggested that the hydraulic pressure of the hemolymph helps drive the early stages of evagination, when the leg is short and unfolding of the tissue plays a major role. It is possible that after this time the increased leg length or increased leg stiffness limits the effectiveness of hemolymph hydraulic pressure. Alternatively, it is possible that there is a decline in the hydraulic pressure due to changes in heart pumping or other prepupal events. The lack of hydraulic pressure may be one reason for the less than optimal evagination of discs seen during in vitro culture (Taylor, 2008).

    Mutations in many Drosophila genes result in changes in appendage morphology. It is expected that some of these produce their phenotype by interfering with the observed cell rearrangement. A particularly interesting candidate for such a gene is dachsous (ds), which encodes a large protein with many cadherin domains. Mutations in this gene result in shorter fatter wings and legs with an altered distribution of cells (e.g. an increase in the number of cells along the anterior posterior axis of the wing and a decrease in the number of cells along the proximal/distal axis). However, mutations in this gene are known to alter disc patterning and growth and this may be the cause of the altered shape (Taylor, 2008).

    Another group of interesting candidate genes for altering cell rearrangement in evaginating legs is the cellular myosin encoded by zipper and the interacting Sqh (myosin regulatory light chain) and RhoA proteins. Mutations in these genes give rise to a crooked leg phenotype that has been interpreted as being due to the mutations altering cell shape. However, myosin has been implicated in the junctional remodeling associated with cell rearrangements in the extending germ band and it is possible that the leg phenotype is also due to an effect on junctional remodeling required for cell rearrangement. One of the interesting properties of extending germ band cells is the planar polarization of membranes so that the anterior/posterior edges of cells are distinct from the dorsal/ventral edges of cells in their content of proteins such as myosin. No evidence was seen for this in prepupal legs and wings but this point deserves further study as it is possible the experimental conditions were not favorable for seeing this (Taylor, 2008).

    Cell competition, growth and size control in the Drosophila wing imaginal disc

    This paper reports experiments aimed at understanding the connections between cell competition and growth in the Drosophila wing disc. The principal assay has been to generate discs containing marked cells that proliferate at different rates and to study their interactions and their contribution to the final structure. It is known that single clones of fast-dividing cells within a compartment may occupy the larger part of the compartment without affecting its size. This has suggested the existence of interactions involving cell competition between fast- and slow-dividing cells directed to accommodate the contribution of each cell to the final compartment. This study shows that indeed fast-dividing cells can outcompete slow-dividing ones in their proximity. However, it is argued that this elimination is of little consequence because preventing apoptosis, and therefore cell competition, in those compartments does not affect the size of the clones or the size of the compartments. These experiments indicate that cells within a compartment proliferate autonomously at their own rate. The contribution of each cell to the compartment is exclusively determined by its division rate within the frame of a size control mechanism that stops growth once the compartment has reached the final arresting size. This is supported by a computer simulation of the contribution of individual fast clones growing within a population of slower dividing cells and without interacting with them. The values predicted by the simulation are very close to those obtained experimentally (Martín, 2009).

    The main objective of this work was to study the role of cell competition in regulating growth and size in the wing disc of Drosophila. As the disc is composed of two (A and P) compartments, which behave as independent units of size control, these experiments refer to mechanisms operating within compartments (Martín, 2009).

    To make some precise statements about the growth dynamics of the disc several developmental parameters were calculated, some of which had not been described in detail in previous publications. According to the data, the wing disc starts growth with about 55 cells, of which 34 would belong to the A and 21 to the P compartment. The final cell number is around 30,000 (about 19,000 A and 11,000 P). The total number of cell divisions is about 9.1. These estimates coincide well with previous ones, although the final number of 30,000 cells is somewhat lower than previous measurements (Martín, 2009).

    This study has shown that the growth rate of the wild-type wing disc changes markedly through development: during the second larval period wild type (M+) cells divide at about 5.5-5.7 hours per cycle, then the length of the cell division cycle increases as development progresses, and in the second half of the third larval period it is as high as 30 hours. Thus most of the growth occurs during the second and early third larval period. A similar growth pattern is found in developmentally delayed M/+ discs, the cell doubling time (CDT) of which increases from 10-12 hours in the second larval period to 34-40 at the prepupal stage. It is not surprising that the major difference between wild-type and M/+ discs occurs during the early periods. Possibly the metabolic demand is greater in fast-proliferating cells and therefore the limitation in protein synthesis of M/+ cells is more noticeable (Martín, 2009).

    Normally there is little apoptosis in the wing disc; therefore, cell competition cannot have a major role in normal circumstances. Nevertheless, it may be a safeguard mechanism to eliminate abnormal cells or to deal with unusual situations such as cells with different division rates, which may interfere with the growth of the disc. The significance was examined of these events of cell competition in the overgrowth of M+ clones, and in the control of compartment size (Martín, 2009).

    The fact that M+ clones growing in M/+ discs can reach an average size more than ten times their normal size (when they grow in a M+ disc) while not altering the final size of the compartment, suggested that both clone overgrowth and size control may depend on cell competition. In this view the M+ clones would overgrow at the expense of the elimination of neighbouring M/+ cells. Moreover, the removal of the latter would ensure that they do not produce progeny that would give rise to an excess of cells in the compartment (Martín, 2009).

    However, the experiments indicate that cell competition does not play a significant role in these processes; in the absence of cell competition, the M+ clones are able to overgrow as much as in the normal situation. Besides, the size of compartments is not affected by the presence of these clones, even though they can occupy the larger part of the compartment (Martín, 2009).

    The authors believe that the key element to explain these results is the mechanism that controls the overall size of the compartment and that arrests growth once it has reached the final dimension. This mechanism would function without regard to the lineage of the cells or of their relative contribution to the final structure. It would also function autonomously in each compartment (Martín, 2009).

    The authors ask us to consider a compartment containing M+ and M/+ cells from early in development. The cells proliferate at the rate dictated by their metabolic activity, according to their Minute genotype, and their division rate is not affected by interactions with neighbour cells. Because of their proliferation advantage the M+ cells occupy a large part of the compartment. In principle, if the M/+ cells were to proliferate at their normal rhythm for the whole duration of the M/+ larval period, the sum of the contribution of the M+ and M/+ cells would produce an excess of tissue. The reason why this is not the case is that the size control mechanism arrests growth as soon as the final size has been reached. In the presence of a large M+ clone the final arresting size of the compartment is reached earlier than in a one entirely made of M/+ cells. For this reason the M/+ cells contribute less than they would have in the absence of a M+ clone. That is, the developmental delay associated with the M/+ condition is partially abolished by the presence of the M+ clone. This is predicted by the computer simulation and is supported by the results. Using the expression of the vg and wg genes to monitor the developmental stage of the compartment, it was found that compartments with M+ clones are ahead and are therefore expected to reach the final arresting size earlier than those that are entirely M/+ (Martín, 2009).

    The existence of a non-cell-autonomous mechanism governing the growth of the compartment is also suggested by observations about vg expression in compartments containing M+ clones. M+ clones can sometimes split the vg domain into M+ and M/+ territories. The significant finding is that both territories show the same pattern of expression. This very strongly suggests that the control of vg expression is determined by an overall mechanism probably measuring the size of the compartment in each moment and regardless of the individual lineages (Martín, 2009).

    What, then, is the role of cell competition in regulating growth and size in the wing disc? Cell competition results from interactions between two types of viable cells and causes the elimination of one of them. That is, it is a mechanism to remove viable cells that are not developmentally adapted to the growing tissue. Unlike other situations that cause apoptosis, the 'loser' cells in the competition process are not necessarily damaged; they are poor competitors. In the cases reported here, it is the relatively slow proliferating cells that are eliminated, which may have a beneficial effect on the general fitness of the disc. Nevertheless, there may be other safeguard functions of greater biological significance. Cell competition may be instrumental in removing viable but developmentally abnormal cells, which, for instance, do not interpret developmental cues correctly. This would include tumour or transformed cells that may arise in development. The process would ensure that tumour cells would normally be outcompeted by non-tumour cells. In certain circumstances, however, the acquisition by the latter of some additional property may turn tumour cells into super-competitors, thus reversing the situation. It has been argued that cell competition may be a major factor in tumour progression in circumstances in which tumour cells are able to outcompete normal cells (Martín, 2009).

    The Drosophila wing hearts originate from pericardial cells and are essential for wing maturation

    In addition to the heart proper, insects possess wing hearts in the thorax to ensure regular hemolymph flow through the narrow wings. In Drosophila, the wing hearts consist of two bilateral muscular pumps of unknown origin. This paper presents the first developmental study on these organs and reports that the wing hearts originate from eight embryonic progenitor cells arising in two pairs in parasegments 4 and 5. These progenitors represent a so far undescribed subset of the Even-skipped positive pericardial cells (EPC) and are characterized by the early loss of tinman expression in contrast to the continuously Tinman positive classical EPCs. Ectopic expression of Tinman in the wing heart progenitors omits organ formation, indicating a crucial role for Tinman during progenitor specification. The subsequent postembryonic development is a highly dynamic process, which includes proliferation and two relocation events. Adults lacking wing hearts display a severe wing phenotype and are unable to fly. The phenotype is caused by omitted clearance of the epidermal cells from the wings during maturation, which inhibits the formation of a flexible wing blade. This indicates that wing hearts are required for proper wing morphogenesis and functionality (Tögel, 2008).

    Unlike in vertebrates, where an elaborate closed blood vessel system extends throughout the whole body, insects possess only one vessel, the tubular heart, in their otherwise open circulatory system. Once the hemolymph has left the heart, it moves freely between the internal organs and can not be directed into narrow body appendages such as antennae, legs or wings. To ensure sufficient hemolymph supply of these appendages additional circulatory organs evolved (Pass, 2000; Pass, 2006). In Drosophila, circulation in the wings is maintained by the so-called wing hearts (Krenn, 1995), a pair of autonomous muscular pumps located bilaterally in the scutellum, the dorsal elevation of the second thoracic segment. Due to this location, they are also referred to as scutellar pulsatile organs. Although known for many years, no developmental studies on the origin or morphogenesis of these organs have been performed. Probably, this was due to the lack of available methods to track their differentiation. However, studies on the origin of the thoracic somatic muscles in Drosophila and comparative anatomical investigations in insects suggested that the wing hearts originate from the cardiac mesoderm or from the heart itself (Tögel, 2008).

    A previous study identified an enhancer region of the Drosophila hand gene that is able to drive reporter gene activity in the wing hearts (Sellin, 2006). In the present work, this reporter was used to identify the embryonic anlagen of the wing hearts and to elucidate the dynamics of their postembryonic development with in vivo time lapse imaging. It was found that the anlagen of the Drosophila wing hearts indeed derive from the cardiac mesoderm but, astonishingly, not from the muscular cardioblast lineage. Instead, they represent a so far undescribed subpopulation of the well-known Even-skipped (Eve) positive pericardial cells (EPCs) (Tögel, 2008).

    In addition to their unknown origin, little is known about the contribution of wing hearts to wing morphogenesis and functionality. After eclosion, wings are unfolded by a sudden influx of hemolymph and subsequently undergo maturation. During this process, the epidermal cells that until then bonded the dorsal and ventral wing surfaces enter programmed cell death, delaminate from the cuticle, and disappear into the thorax (Kimura, 2004). Subsequently, the cuticles of the intervein regions become tightly bonded to form a flexible wing blade, while the cuticles of the vein regions form tubes, lined by living cells, through which hemolymph circulates in mature adult insects. Measurements of hemolymph flow in adult butterflies showed that wing hearts function as suction pumps that draw hemolymph out of the wings starting shortly after wing unfolding. Whether wing hearts might play a role in wing maturation was tested by generating flies lacking wing hearts. The findings demonstrate that the delaminated epidermal cells are removed from the wings by the hemolymph flow generated by the wing hearts. Loss of wing heart function leads to remains of epidermal cells resting between the unbonded dorsal and ventral wing surfaces which results in malformation of the wing blade and flightlessness. It is concluded that wing hearts are essential for wing maturation and, thus, for acquiring flight ability in Drosophila (Tögel, 2008).

    A hand-C-GFP reporter was generated (Sellin, 2006) that reflects the described hand expression pattern and was found to be active in wing hearts. To confirm that the hand-C-GFP reporter is expressed in all cells of mature wing hearts, their morphology was examined based on the signal from the reporter in conjunction with histological sections. In the adult fly, wing hearts are located at the lateral angles of the scutellum, which are joined to the posterior wing veins by cuticular tubes. Each organ is curved in anterior–posterior direction as well as dorso-ventrally. It consists of about 7-8 horizontally arranged rows of prominent muscle cells, which are attached at their proximal side to a thin layer of cells that has a greater dorsal extension than the muscle cells. Both cell types are labeled by the reporter. The fine acellular strands that hold the wing hearts to the adjacent epidermal cells were not observed to be marked by the reporter. PubMed ID: Movies are provided to demonstrate the location and the beating of wing hearts (Tögel, 2008).

    The hand-C-GFP reporter was tested for expression in earlier stages of wing heart development and it was found to be active throughout the entire organogenesis. This enabled identification of the embryonic anlagen of the wing hearts, which consist of eight progenitor cells located dorsally and anterior to the heart, in two pairs in the second and third thoracic segment from stage 16/17 onward. The progenitors exhibit a flattened triangular shape and are interconnected by thin cytoplasmic extensions. In addition, the second and the fourth pair of the progenitors are closely associated with the dorsal tracheal branches at their interconnection in the second and third thoracic segments. The characteristic pairwise arrangement and the connection to the tracheae are retained during the subsequent three larval stages. Proliferation starts at about the transition from the second to the third larval instar, leading to eight clusters of cells that remain arranged in four pairs in the anterior region until 1h after puparium formation (APF). Between 1 and 10h APF, the cell number increases significantly and the anterior three pairs of cell clusters are retracted to join the last pair of clusters, eventually forming one large median cluster. Between 13 and 50h APF, the single large cluster splits along its anterior-posterior axis into two groups of cells that migrate laterally in the forming scutellum, thereby adopting the characteristic arched appearance of the adult wing hearts. During this process some of the cells on either side form the underlying thin layer while the remaining cells arrange in horizontal rows along that layer. First contractions of the mature organs were observed at about 45-50h APF (Tögel, 2008).

    The expression of the bHLH transcription factor Hand in the wing heart progenitors, which serves as a general marker for all classes of heart cells in Drosophila, prompted a to screen for the expression of genes known to be active in cardiac lineages. Analysis of Even-skipped (Eve) expression revealed that the embryonic wing heart progenitors arise through the same lineage as the well described Eve expressing pericardial cells (EPCs). At stage 10 in embryogenesis, 12 Eve clusters are present on either side of the embryo, located in parasegments (PS) 2 to 12. Each cluster gives rise to a pair of EPCs, except for the most posterior cluster in PS 14, which generates only one EPC. During subsequent development, the first and the second pair of EPCs, located in parasegment 2 and 3, turn toward the midline of the embryo to accompany the tip of the heart, which later bends ventrally into the embryo. The third and the fourth pair of EPCs in PS 4 and 5 are shifted anteriorly in relation to the heart. This step is not based on migration but on the remodeling of the embryo during head involution, since the cells remain in their PS close to the likewise Eve positive anlagen of the DA1 muscle. The EPCs in PS 4 and 5 subsequently differentiate into the later wing heart progenitors, while all others become the classical EPCs and accompany the heart in a loosely associated fashion. At least from PS 4 to 12, all pairs of Eve positive cells (wing heart progenitors and classical EPCs) are interconnected by cytoplasmic extensions forming a rope ladder-like strand above the heart after dorsal closure at stage 16/17. This mode of contact between the cells persists in the wing heart progenitors in postembryonic stages and might be essential for proper relocation in the prepupae (Tögel, 2008).

    Although the Drosophila wing hearts have been known for many years, their origin and development have remained unknown. This study provides the first developmental approach on these organs using in vivo time lapse imaging as well as genetic and immunohistochemical methods. It was found that the wing hearts develop from embryonic anlagen that consist of eight progenitor cells located anterior to the heart. Analysis of gene expression in these progenitors confirmed the hypothesis that the wing hearts originate from the cardiac mesoderm, but not from the contractile cardioblast lineage, as has been suggested based on anatomical data. Surprisingly, the embryonic anlagen derive from a particular subset of the well-known EPCs. EPCs arise in pairs in PS 2 to 12 from the dorsal progenitor P2, which divides asymmetrically into the founder of the dorsal oblique muscle 2 and the founder of the EPCs in a numb-dependent lineage decision. Additionally, a single EPC arises in PS 14. The subsequent differentiation of the founders into EPCs requires the activity of the transcription factors Zfh1 and Eve. This study shows that the EPCs located in PS 4 and 5 are relocated in relation to the heart during head involution at stage 14/15 of embryogenesis and subsequently differentiate into the wing heart progenitors. Until this step, no difference to the EPCs in the anterior and posterior PS could be detected. Like the classical EPCs, which remain close to the heart, the EPCs that give rise to the wing heart progenitors depend on factors involved in asymmetric cell division, e.g. Insc or Numb, and fail to differentiate in embryos mutant for zfh1 as well as in animals lacking mesodermal Eve. Loss of tinman expression is the only event that could be identified that discriminates between a classical EPC fate and the specification of wing heart progenitors. Consistently, ectopic expression of Tinman in the wing heart progenitors effectively represses their specification, probably by committing them to a classical EPC fate, indicating that Tinman plays a crucial role in the involved regulatory pathway (Tögel, 2008).

    So far, the biological role of pericardial cells (PCs), and EPCs in particular, is not well understood. In the embryo, three populations of PCs arise in each segment, which are characterized by the expression of different combinations of genes (Odd positive PCs, Eve positive PCs, and Tinman positive PCs). During postembryonic stages, the number of PCs decreases, raising the question which population contributes to the final set of PCs in the adult and whether all PCs have the same function throughout development. Recent studies have shown that postembryonic PCs express Odd and Eve, a combination which is not observed in the embryo, and are dispensable for cardiac function. Genetic ablation of all larval PCs had no effect on heart rate, but increased sensitivity to toxic stress. In contrast, the specification of the correct number of embryonic PCs is crucial for normal heart function. Loss of mesodermal Eve during embryogenesis results in fewer larval pericardial cells, which causes a reduction in heart rate and lifespan. Conversely, hyperplasia of embryonic PCs has no effect on heart rate but causes decreased cardiac output. This was explained by an excess of Pericardin secreted by the PCs into the extracellular matrix enveloping the heart (Johnson, 2007). Taken together, embryonic PCs seem to influence cardiac development by e.g., secreting substances whereas postembryonic PCs function as nephrocytes. However, in this study, functional data is provided on a subset of embryonic EPCs, which differentiate into adult progenitors giving rise to a myogenic lineage. This represents a completely new function of PCs, raising the question whether EPCs might in general have myogenic potential and rather represent a population of adult progenitors, than PCs in a functional sense (Tögel, 2008).

    The organogenesis of the wing hearts is a highly dynamic process, which includes distinct cellular interactions. At first, adjacent EPCs (including the wing heart progenitors) on either side of the embryo establish contact via cytoplasmic extensions. After dorsal closure of the embryo, interconnections are also formed between opposing EPCs resulting in a rope ladder-like strand above the heart. These interconnections are assumed to be needed to retain contact between the wing heart progenitors during the subsequent development. During larval stages, some of the wing heart progenitors establish a second contact to specific tracheal branches and proliferation starts. In the prepupa, a relocation event joins all wing heart progenitors in one large cluster. During this step, the progenitors are probably passively relocated in conjunction with the tracheal branches to which they are connected. Finally, the wing heart progenitors initiate active migration and form the mature wing hearts in the pupa. Considering the complexity of their development, it is proposed that wing hearts provide an ideal model for studying organogenesis on several different levels such as signaling, cell polarity, or path finding (Tögel, 2008).

    Elimination of the embryonic progenitors by ectopic expression of tinman or by laser ablation causes the loss of wing hearts, which results in a specific wing phenotype in conjunction with flightlessness. In the identified phenotype, the delaminated epidermal cells are not cleared from the wings during wing maturation and bonding of the dorsal and ventral wing surfaces is omitted. Recently, it was reported that the epidermal cells transform into mobile fibroblasts and actively migrate out of the wings. However, in in vivo time-lapse studies migration of epidermal cells could not be observed during wing clearance. Conversely, their movements correlated with the periods of wing heart beating, indicating that they are passively transported by the hemolymph flow. One-sided ablation of mature wing hearts in pupae, confirms that wing hearts play a crucial physiological role in wing maturation, since the wing phenotype occurs only on the treated side, but in the same genetic background. In contrast, mutations in genes coding for proteins involved in cell adhesion, e.g. integrins, or in adhesion to the extra cellular matrix, cause a blistered wing phenotype. In the latter phenotype, the epidermal cells of the immature wings are not attached to their opposing cells or to the cuticle and the wing surfaces are separated during unfolding by the sudden influx of hemolymph. In contrast, in animals lacking wing hearts the wings resemble those of the wild-type shortly after unfolding. The epidermal cells also delaminate later from the cuticle, as indicated by their disarrayed pattern, but are not removed from the wings due to the missing hemolymph circulation and probably impede spatially the bonding of the dorsal and the ventral cuticle. Thus, the wings remain in their immature state and do not acquire aerodynamic properties, which accounts for the flightlessness. It is concluded that wing hearts are crucial for establishing proper wing morphology and functionality in Drosophila (Tögel, 2008).

    Wing hearts occur in all winged insects, but differ considerably in their morphology. However, their function is highly conserved, since they all function as suction pumps that draw hemolymph from the wings. In the basal condition, the heart itself is directly connected to the scutellum and constitutes the pump. This connection was lost several times during evolution and other muscles, e.g. the separate wing hearts in Drosophila, were recruited to retain the function indicating a high selection pressure on wing circulation. It is suggested that this is due to the crucial role of wing hearts during wing maturation. Since proper wing morphogenesis is essential for flight ability, insect flight might not have been possible before the evolution of wing hearts (Tögel, 2008).

    Identification of genes affecting wing patterning through a loss-of-function mutagenesis screen and characterization of med15 function during wing development

    The development of the Drosophila wing depends on the correct regulation of cell survival, growth, proliferation, differentiation, and pattern formation. These processes, and the genes controlling then, are common to the development of epithelia in many different organisms. To identify additional genes contributing to wing development a genetic screen was performed in mosaic wings carrying clones of homozygous mutant cells. Twelve complementation groups were obtained corresponding to genes with a proven role in wing formation such as smoothened, thick veins, mothers against dpp, expanded, and fat and 71 new complementation groups were obtained affecting the pattern of veins and the size of wing. One of these groups mapped to the mediator15 gene (med15), a component of the Mediator complex. Med15 and other members of the Mediator complex were shown to be required, among other processes, for the transcription of decapentaplegic target genes (Terriente-Félix, 2010).

    The complementation group formed by the 77A and 133A1 mutants was analyzed in some detail. These mutations are alleles of med15, a gene encoding one component of the Mediator complex. Thus, they fail to complement other med15 alleles, and med15133A1 is associated with a stop codon that could truncate the protein in the N-terminal region after the KIX domain. The Mediator multiprotein complex promotes the transcription of inducible genes, acting as a link between the RNApolII holoenzyme and several sequence-specific transcription factors. The human homolog of Med15, MED105, is included in all Mediator complexes identified so far and forms part of a module named the tail that is the main target for the transcriptional activators. Thus, Med15 homologs can bid to different transcription factors such as Gcn4 and Gal4 in Saccharomyces cerevisiae, and, more interesting from the perspective of these data, to Smad2/3 and Smad4 in Xenopus. Other members of the Mediator complex that were previously analyzed are kohtalo and skuld (Med12 and Med13, respectively), which form part of the conserved Cdk8 module. Interestingly, mouse Cdk8 and Cdk9 phosphorylate Smad proteins, regulating their transcriptional activity and turnover (Alarcón). However, kohtalo and skuld are required for sensory organ development, for some aspects of Notch and Hedgehog signaling, and for the transcription of Wingless downstream genes (Terriente-Félix, 2010).

    Med15 mutations result in smaller than normal wings and loss of mainly the L2 vein. They also affect the fusion between the left and the right hemithorax and leg morphogenesis. The reduction in the level of expression of other components of the Mediator complex, most notably med20, med27, and med30, also results in smaller than normal wings and failures in vein differentiation, in addition to causing some levels of cell death. Although these phenotypes were similar, they are not identical, which might indicate specific requirements of these subunits or, alternatively, a different degree in the effectiveness of each interference RNA used. Mutant med15 cells display specific defects in gene expression, suggesting a requirement limited to particular enhancer-promotor interactions. In particular, the expression of spalt, a direct target of Dpp signaling, is compromised in med15 mutant cells. There are no known transcriptional targets of TGFβ signaling in the wing, and consequently it could not be determined directly whether the activity of this pathway is diminished in med15 mutants. A direct requirement of Med15 for the transcription of TGFβ target genes is nonetheless suggested by the similar phenotypes of wing size reduction observed in med15 mutants and in baboon mutations (Terriente-Félix, 2010).

    Differential division rates and size control in the wing disc

    Wing disc compartments were generated that contain marked fast growing M+ clones surrounded by slow dividing M/+ cells. Under these conditions the interactions between fast and slow dividing cells at the clone borders frequently lead to cell competition. However, an assay suppressing apoptosis indicates that cell competition plays no major role in size control. It is argued that cells within a compartment proliferate according to their genotype independently of each other and that their contribution to the final structure will depend solely on their proliferation rate. This model is supported by a computer simulation that predicts values similar to those found experimentally. The results on the growth of M+ clones within compartments and on the expression of developmental genes like vestigial and wingless suggest the existence of a non-cell autonomous mechanism that functions at the level of the entire cell population. It measures the population size in each moment, determines the corresponding expression levels of developmental genes and establishes the time to arrest growth (Morata, 2010).

    Integrating force-sensing and signaling pathways in a model for the regulation of wing imaginal disc size

    The regulation of organ size constitutes a major unsolved question in developmental biology. The wing imaginal disc of Drosophila serves as a widely used model system to study this question. Several mechanisms have been proposed to have an impact on final size, but they are either contradicted by experimental data or they cannot explain a number of key experimental observations and may thus be missing crucial elements. This study has modeled a regulatory network that integrates the experimentally confirmed molecular interactions underlying other available models. Furthermore, the network includes hypothetical interactions between mechanical forces and specific growth regulators, leading to a size regulation mechanism that conceptually combines elements of existing models, and can be understood in terms of a compression gradient model. According to this model, compression increases in the center of the disc during growth. Growth stops once compression levels in the disc center reach a certain threshold and the compression gradient drops below a certain level in the rest of the disc. This model can account for growth termination as well as for the paradoxical observation that growth occurs uniformly in the presence of a growth factor gradient and non-uniformly in the presence of a uniform growth factor distribution. Furthermore, it can account for other experimental observations that argue either in favor or against other models. The model also makes specific predictions about the distribution of cell shape and size in the developing disc, which were confirmed experimentally (Aegerter-Wilmsen, 2012).

    This paper presents a new model for the regulation of wing disc size. The model contains a rather complex regulatory network, which consists of a considerable number of interactions, receives nonuniform input of protein activities, and interacts with a mechanical stress pattern that emerges over time and space. It is assumed that the regulatory network represents protein activities and interactions that regulate these activities. The model does not distinguish between interactions at the transcriptional and protein activity level, but considers effects on net activities. All protein activities emerge from the network, except for those of Dpp, Wg and N, which are implemented in the model. In the regulatory network, differences in Ds and Fj concentrations between neighboring cells lead to activation of Dichate (D) by changing its intracellular localization. In addition, it is assumed that a weighted average of the area of a cell and its neighbors is a good readout for mechanical stress, that cells do not rearrange when exposed to mechanical tension, and that the planar polarization of D imposes a bias on the direction of the division plane. The interactions are hypothetical and form the main untested assumptions underlying the model. The regulation of ds by mechanical compression is not essential for the principle behind size regulation in the model, but improves the fit of simulation results with experimental data (Aegerter-Wilmsen, 2012).

    A qualitative understanding can be gained by considering it in terms of a compression gradient model. During growth, compression increases in the center of the disc. Growth ceases when compression in the center reaches a certain threshold and the gradient of the compression gradient drops below a certain threshold in the rest of the disc. Read-out of the compression gradient is accomplished by a mechanism that involves Vg and the Hippo pathway. Numerical simulations were used to show that the model can account for growth termination and that it reproduces a large range of additional data on growth regulation, including some emergent properties of the system. Based upon the principle underlying the model, predictions can be made with respect to cell shape patterns. In order to take into account the curved surface of the wing pouch, an open source image analysis program was developed. The results showed that the general dynamics of the formation of cell shape patterns is indeed similar to the one predicted by the model. This analysis is, however, based on images from different discs and, especially during the early stages, there is variation among discs. It would therefore be interesting to assess whether the predicted dynamics is also present in the temporal evolution of single discs. However, this first requires the development of experimental methods with which single discs can be followed over time (Aegerter-Wilmsen, 2012).

    Even though the development of cell shape patterns constitutes a fundamental prediction of the model, it would be an interesting future experimental challenge to test the model's basic assumptions directly, i.e., the regulation of Yki, Arm and ds by mechanical forces. The regulation of Yki by mechanical compression is most relevant for the model's behavior and appears necessary to obtain growth termination in combination with roughly uniform growth. The regulation of Arm by compression seems to be involved in stabilizing the Vg gradient, which could be relatively unstable if it would be regulated by Vg autoregulation alone. In addition, this interaction smoothens the compression gradient, which might have implications for the 3D structure of the wing disc. Last, the regulation of ds by mechanical forces is not essential for the principle behind size regulation, but improves the modeling results and also contributes to smoothening of the compression gradient. While developing the model, focus was placed on its ability to reproduce specific features of growth dynamics, as well as a number of key experiments that are used to argue in favor and against current models. One of the latter results, the decrease of medial growth upon induction of uniform Dpp signaling, could not be reproduced. In the simulations, these discs grow very fast. It is conceivable that such growth rates cannot be sustained in vivo because of a limited availability of nutrients and oxygen. When imposing a maximum total growth rate on disc growth, it is indeed possible to obtain growth rates in the medial part that are lower than those in wildtype discs, whereas lateral growth rates are higher, in agreement with experiments. Thus, with this additional assumption, the model can reproduce the results it was aimed to reproduce (Aegerter-Wilmsen, 2012).

    There are currently no experimental data available on the parameters underlying the model and therefore they were fitted manually. As has become clear from the parameter analysis, there are only a few parameter combinations that can reproduce all results. However, it is not known whether this set is reproduced robustly in vivo and there is no natural selection on reproducing experimental manipulations robustly. Nevertheless, it is entirely possible that a larger set of parameter values should reproduce the results. In addition, even though the model can reproduce the selected set of experimentally observed features, there are related observations it cannot reproduce. For example, the final size reached in the model is too small, the experimentally observed nonautonomous growth induction by clones overexpressing brk is nearly absent in the model, and growth induction along the boundary of ds overexpressing clones extends further inside the clone than measured experimentally. It would be interesting to study whether there are factors missing in the model, which would make the parameter space less strict. For example, the parameter space was strongly restricted by the stipulation to reproduce the absence of Vg-BE activity in ap0 mutants upon ectopic wg expression. If it could be assumed that smaller discs have a different geometry in vivo than larger ones, the number of possible parameter combinations would increase. It will be interesting to assess the geometrical properties of discs in young larvae and evaluate whether the model should be adjusted in this respect (Aegerter-Wilmsen, 2012).

    Very recently, another model has been formulated for growth regulation that assumes that growth is regulated by increases of Dpp signaling levels over time. However, growth is increased in wing discs in which Brk and Dpp signaling are removed. This either contradicts this model or the current understanding of Dpp signaling needs to be revised. The current model reproduces increased growth in such mutants, including its non-uniformity (Aegerter-Wilmsen, 2012).

    The adult wing is covered by bristles, which point towards the distal part of the wing. This orientation is regulated by planar polarity genes. Regulation of planar polarity seems to be related to growth regulation. For example, Ds and Fj are not only important for growth regulation, but are also required for the development of a proximodistal polarity pattern. It is currently not clear whether Ds and Fj are directly involved in regulating planar polarity. If this were the case, then the model would suggest that planar polarity may, at least in part, arise from an interplay between morphogens and mechanical forces. The model presented in this study was developed for the wing imaginal disc of Drosophila. It would be interesting to see whether a similar model could also reproduce size regulation and additional experimental results in other systems. For other imaginal discs, it has been shown that their centers are also compressed at the end of growth. The precise regulatory networks involved in growth and size regulation are different for the different discs, but it would be interesting to see whether certain principles are conserved. In mammals, mechanical forces regulate growth in many tissues. However, the situation is often very different from that in the wing disc in that most mammalian tissues reach their final size while they perform a biological function. Thus, it would be interesting to study whether principles similar to those described here apply for mammalian organs early during development (Aegerter-Wilmsen, 2012).

    Wg and Wnt4 provide long-range directional input to planar cell polarity orientation in Drosophila

    Planar cell polarity (PCP) is cellular polarity within the plane of an epithelial tissue or organ. PCP is established through interactions of the core Frizzled (Fz)/PCP factors and, although their molecular interactions are beginning to be understood, the upstream input providing the directional bias and polarity axis remains unknown. Among core PCP genes, Fz is unique as it regulates PCP both cell-autonomously and non-autonomously, with its extracellular domain acting as a ligand for Van Gogh (Vang). This study demonstrates in Drosophila wings that Wg (Wingless) and dWnt4 (Drosophila Wnt homologue) provide instructive regulatory input for PCP axis determination, establishing polarity axes along their graded distribution and perpendicular to their expression domain borders. Loss-of-function studies reveal that Wg and dWnt4 act redundantly in PCP determination. They affect PCP by modulating the intercellular interaction between Fz and Vang, which is thought to be a key step in setting up initial polarity, thus providing directionality to the PCP process (Wu, 2013).

    The data indicate that Wg/dWnt4 regulate the establishment of Fz–PCP axes by modulating the Fz–Vang intercellular interactions in a graded, dosage dependent manner. Consequently they might generate different levels of Fz–Vang interactions across a Wg/dWnt4 gradient experienced by cells. This process is reiterated across the tissue, and the directionality of Fz–Vang binding is subsequently reinforced by intracellular core PCP factor interactions. The data are consistent with a model in which Wg/dWnt4 generate a Fz ‘activity'), suggesting that both of these light sensors are necessary for light avoidance behavior.' gradient models. Accordingly, PCP axes are orientated towards the Wg/dWnt4 source, which is evident in (at least) the wing and eye. The early wing PCP axis (late larval to early pupal stages) correlates well with Wg/dWnt4 margin expression and, similarly, in the eye polarity is oriented in the dorsoventral axis towards the poles where Wg/Wnt4 are expressed. This model, relying on a Fz–Vang interaction, is also compatible with the addition of Fmi to this scenario, with intercellular (homophilic) Fmi–Fmi interactions also being required for PCP specification. As Fmi forms complexes with both Fz and Vang, the full complement of intercellular interactions includes Fz/Fmi–Fmi/Vang complexes, and these interactions would also be modulated by Wnt binding to Fz, either directly as proposed in this model or possibly by modulating the Fmi–Fmi interactions by Fmi being associated with Fz that is bound to different levels of Wg/Wnt4. In vivo, Fmi helps to enrich both Fz and Vang to the subapical junctional region, and Fmi–Fmi interactions bring Fz and Vang to close molecular proximity (Wu, 2013).

    Intercellular Fmi–Fmi interactions are strong, as Fmi-expressing S2 cells form cell aggregates through homophilic Fmi interactions. The interaction between Fz and Vang is weaker, and cell–cell contacts between the two cell groups are infrequent. It was suggested that PCP signal sensing complexes include both Fmi and Fz on one cell interacting with Fmi/Vang at the surface of a neighbouring cell. Within these complexes, Fz is required for sending a polarity signal, whereas Fmi and Vang are involved in its reception, consistent with the data and model. Although it has been suggested that Fmi is capable of sensing Fz/Fmi signals in the absence of Vang, the 'Fz-sensing' capability of cells with Fmi alone (lacking Vang) is much weaker than that of cells with Vang. It will be interesting to determine if there are other PCP regulators directly involved in modifying Fmi–Fmi interactions (Wu, 2013).

    How do these data relate to previous models and why was the Wg/Wnt4 requirement not observed before? Previous work attempted to address the role for the wing margin in PCP by examining either mutants affecting wing margin cells without eliminating wg/Wnt expression or in clones. Although cellular hairs near the site of wing margin loss point towards remaining wing margin areas, the effect Is considered weak. Potential effects were examined of Wnt LOF clones of Df(2L)NL, lacking wnt4, wg, wnt6 and wnt10. In contrast to the global reduction of Wg/Wnt4 through the temperature sensitive wg allele, such clones cause only mild PCP perturbations. There are several reasons why clonal loss of Wnt expression in the margin only mildly affects PCP orientation: cells can respond to Wnts from several sources/cells from remaining Wnt-expressing wing margin regions; polarization strengths (measured by nematic order) in the first few rows of cells near the margin are much weaker than those in cells further away (at 14-17 h APF) and weak PCP reorientation in cells neighbouring wing margin clones could thus reflect the initial weak polarization in these cells; and PCP orientation changes from its initial radial polarity towards the proximodistal polarity during hinge contraction morphogenesis and associated cell flow, probably leading to significant corrections of subtle defects near the margin. Similarly, PCP orientation in cells near the margin is only very weak early (at 14-16 h APF), probably because cells close to the Wnt-producing cells are exposed to saturated Wnt levels (and not a Wnt gradient), or because the presence of other organizers (directing polarity parallel to the margin) weakens the effect of Wnts. PCP in these cells is established/corrected through more local interactions during the feedback loops among neighbouring cells (Wu, 2013).

    To determine the direct role for Wg/Wnts on Fz–PCP signalling, it was examined at pupal stages, as the patterning role for canonical Wg signalling is much reduced then and PCP still correlates well with Wg/Wnt4 expression. Importantly, Wnt4 does not affect expression of patterning genes through canonical signalling at larval or pupal stages, yet Wnt4 alters PCP orientation, consistent with the model that Wnt4/Wg act directly on Fz-PCP interactions. The observation that Wnt4 requires Fz to affect neighbouring cells further supports this model. It is likely that, as well as the Wg/Wnt4 input and mechanism identified in this study, both early and late PCP axes depend on further cues, provided for instance by the parallel Ft/Ds-PCP system or other morphogenetic organizers. Strikingly, such a scenario would suggest that Wg regulates PCP directionality through both PCP systems, affecting Fz-PCP interactions directly and through canonical Wg signalling transcriptionally regulating graded fj and ds expression in eyes and wings. In summary, these data provide insight into Wnt-mediated mechanisms to directly regulate long-range Fz–PCP orientation by modulating Fz–Vang/PCP interactions during tissue morphogenesis (Wu, 2013).

    Cytonemes are required for the establishment of a normal Hedgehog morphogen gradient in Drosophila epithelia

    Hedgehog (Hh) signalling is important in development, stem cell biology and disease. In a variety of tissues, Hh acts as a morphogen to regulate growth and cell fate specification. Several hypotheses have been proposed to explain morphogen movement, one of which is transport along filopodia-like protrusions called cytonemes. This study analysed the mechanism underlying Hh movement in the wing disc and the abdominal epidermis of Drosophila melanogaster. In both epithelia, cells were shown to generate cytonemes in regions of Hh signalling. These protrusions are actin-based and span several cell diameters. Various Hh signalling components localize to cytonemes, as well as to punctate structures that move along cytonemes and are probably exovesicles. In vivo imaging was used show that cytonemes are dynamic structures and that Hh gradient establishment correlates with cytoneme formation in space and time. Indeed, mutant conditions that affect cytoneme formation reduce both cytoneme length and Hh gradient length. The results suggest that cytoneme-mediated Hh transport is the mechanistic basis for Hh gradient formation (Bischoff, 2013).

    The localization of several Hh signalling components at cytonemes has suggested a role for these structures in Hh signalling. This study characterize cytonemes in two Drosophila paradigms, the wing disc and the abdominal epidermis, and investigate their role in Hh gradient formation. Evidence is presented that cytonemes play an active role in gradient formation: the establishment of the Hh signalling gradient correlates dynamically in space and time with cytoneme formation in vivo; experimental shortening and lengthening of cytonemes affects the gradient accordingly; the analysis of ttv−/−,botv−/− mutant clones implicates cytonemes in Hh transport. Overall, the results support a model in which cytonemes of signal-producing cells are involved in long-range Hh transport (Bischoff, 2013).

    In wing discs, however, both sending and receiving cells generate cytonemes raising the question of which role the cytonemes of receiving cells play. Expression of Ihog in A compartment cells leads to a depletion of Hh from the P compartment cells close to the A/P border, which suggests that A compartment cytonemes might actively engage in Hh reception. Hence, cytonemes of both sending and receiving cells might contribute to Hh transport. Interestingly, A compartment cytonemes are rare in histoblasts, suggesting that cytonemes of receiving cells play a minor role in the abdomen (Bischoff, 2013).

    Ihog–RFP puncta were observed that associated with and moved along cytonemes. Frequently, such puncta were released from cytonemes. Puncta were also observed when labelling cytonemes with CD4–Tomato. This suggests that cytonemes might transport exovesicles that act as a vehicle for Hh or are the structure where exovesicles are being released. Accordingly, the knockdown of genes involved in exovesicle production/release has a significant effect on Hh gradient length, and Ihog can be detected in baso-lateral exovesicles at the ultrastructural level. However, the characterization of these exovesicles as well as their implication in Hh gradient formation requires further analysis. A role of exosomes in morphogen gradient formation has recently been suggested. Active Wnt proteins are secreted in exosomes in cultured cells and in the wing disc. In addition, vesicular release of SonicHh has been implicated in the determination of left–right asymmetry in vertebrates. Very recently, particles containing SonicHh and CDO (the vertebrate homologue of Ihog) that travel along filopodia-like extensions have been described in the chicken limb bud (Bischoff, 2013).

    The mechanisms by which cytonemes could transport morphogens to their targets must ensure specificity and accuracy. One possibility is that cytonemes established contact between sending and receiving cells. Alternatively, cytonemes could act as a structure of morphogen release and uptake without cell–cell contacts involved. In vivo imaging showed that cytonemes are dynamic structures. Cytonemes might grow towards a receiving cell and then retract after a signalling event has taken place, or their dynamics could be determined intrinsically by the stability of their cytoskeleton. Moreover, not just cytoneme length but also their number could shape the gradient, as its brightest section coincides with the dense array of shorter cytonemes. This cytoneme-based model challenges the previous diffusion-based models (Bischoff, 2013).

    Cytonemes have been described in a variety of signalling pathways. The Dpp receptor Thickveins is present in punctate structures moving along cytonemes. Air sac precursors extend cytonemes towards FGF-expressing cells. Tracheal cells were reported to have at least two types of cytoneme; one type that carries an FGF receptor, and another type that carries the Dpp receptor. This suggests that cytonemes are ligand specific. In the context of Notch signalling, filopodia mediate lateral inhibition between non-neighbouring cells of the pupal notum. Interestingly, the dynamic behaviour of these processes is crucial for signalling. Spitz/EGF is delivered through polarized actin protrusions to spatially bias the specification of a particular cell of the Drosophila leg. In another example, short cytonemes mediate the delivery of a juxtacrine Hh signal to maintain germline stem cells in the Drosophila ovary. This study has shown that cytonemes also play a pivotal role in long-range Hh signalling in wing disc cells, histoblasts and LECs. Therefore, it is believed that cytonemes are a general feature of signalling events of all epithelial cells (Bischoff, 2013).

    The Elongin complex antagonizes the chromatin factor Corto for Vein versus intervein cell identity in Drosophila wings

    Drosophila wings mainly consist of two cell types, vein and intervein cells. Acquisition of either fate depends on specific expression of genes that are controlled by several signaling pathways. The nuclear mechanisms that translate signaling into regulation of gene expression are not completely understood, but they involve chromatin factors from the Trithorax (TrxG) and Enhancers of Trithorax and Polycomb (ETP) families. One of these is the ETP Corto that participates in intervein fate through interaction with the Drosophila EGF Receptor -- MAP kinase ERK pathway. Precise mechanisms and molecular targets of Corto in this process are not known. This study shows that Corto interacts with the Elongin transcription elongation complex. This complex, that consists of three subunits (Elongin A, B, C), increases RNA polymerase II elongation rate in vitro by suppressing transient pausing. Analysis of phenotypes induced by EloA, B, or C deregulation as well as genetic interactions suggest that the Elongin complex might participate in vein vs intervein specification, and antagonizes corto as well as several TrxG genes in this process. Chromatin immunoprecipitation experiments indicate that Elongin C and Corto bind the vein-promoting gene rhomboid in wing imaginal discs. It is proposed that Corto and the Elongin complex participate together in vein vs intervein fate, possibly through tissue-specific transcriptional regulation of rhomboid (Rougeot, 2013).

    In Drosophila as in mammals, the three Elongin proteins Elo A, B, and C are mainly nuclear and interact two by two. EloC/B and EloC/A interactions may be direct, as they were observed without cross-linking treatment. By contrast, EloA/B interaction is more labile and may thus be indirect. It is possible that Drosophila EloC mediates the interaction between EloA and EloB, as previously shown in mammals. This study also showed that the ETP Corto interacts with all three Elo proteins, suggesting that Corto interacts with the Elongin complex. Hence, Corto and the Elongin Complex could share transcriptional targets. Several studies have shown that EloC binds its partners through a degenerate BC box motif, defined as (L,M)XXX(C,S)XXX(Í). Two putative BC boxes (aa 357-365 and aa 542-550) are present in the C-terminal part of Corto. However, deletion of these sequences did not impair co-immunoprecipitation between Corto and EloC, suggesting that these two proteins interact through another unidentified sequence (Rougeot, 2013).

    This study presents the first characterization of lines allowing deregulation of EloB or EloC expression. EloB or EloC loss-of-function mutations induce early lethality (before the third larval instar), demonstrating that EloB and EloC, like EloA (Gerber, 2004), are essential proteins. Clonal and tissue-specific analyses of EloC mutant cells reveal that EloC is critically required all through wing development. By contrast, RNAi-mediated EloA down-regulation only induced lethality during the pupal stage (Gerber, 2004), indicating either a less efficient reduction of EloA mRNA or a longer perdurance of maternal EloA. Alternatively, requirement of EloB and EloC in other complexes, such as an E3 ubiquitin ligase complex, might explain this difference (Rougeot, 2013).

    EloB/C loss-of-function as well as EloA over-expression induced wing phenotypes, mostly vein phenotypes. Interestingly, these loss-of-function and over-expression phenotypes are opposite (i.e truncated L5 vein for loss-of-function, ectopic veins for over-expression). Furthermore, whereas EloA over-expression induced ectopic veins, no phenotype was observed when over-expressing EloB and EloC. This result suggests that the amount of catalytic subunit EloA might be critical for Elongin complex function. In mammals, EloA is indeed the limiting component of the Elongin complex, EloB and EloC being in large excess (100 to 1000-fold more abundant than EloA). Curiously, a previous study reported that mitotic clones for a deficiency that uncovers EloA, produced ectopic wing veins. As this deletion uncovers more than 10 genes that may influence vein formation, the hypothesis is favored, in agreement with all data presented above, that EloA loss- of-function leads to loss of vein tissue. Alternatively, EloB and EloC, which also belong to ubiquitin ligase complexes, might modulate vein vs intervein cell fate in this context (Rougeot, 2013).

    Altogether, the observations suggest that the Elongin A, B, C subunits promote vein cell identity. On the opposite, Corto maintains intervein cell identity, possibly via interaction with TrxG complexes. As Corto and EloC co-localize at a few sites on polytene chromosomes, they might have common transcriptional targets. A balance between Corto and the Elongin complex might fine-tune transcription of such genes (Rougeot, 2013).

    In corto mutants, previous study has shown that ectopic veins perfectly match with ectopic expression of rho, the first vein-promoting gene to be expressed (Mouchel-Vielh, 2011). As Elo gene mutations counteract corto mutations during formation of ectopic veins, it is proposed that rho could be a common target of Corto and the Elongin complex in intervein cells. In agreement with this hypothesis, immunoprecipitation using chromatin from late third instar wing imaginal discs, that can be assimilated to chromatin of intervein cells, revealed the presence of both Corto and EloC on rho. Two independent genome-wide studies on whole embryos and embryonic S2 cells have shown that poised RNA-PolII binds the rho promoter, suggesting that rho expression is controlled by 'pause and release' of the transcriptional machinery. Interestingly, this studu found that Corto is slightly enriched just after the rho TSS, a position usually occupied by paused RNA-PolII. Corto shares many sites on polytene chromosomes with paused RNA-PolII-S5p, suggesting that it is involved in transcriptional pausing. On the other hand, this study found that EloC co-localizes with H3K36me3, that characterizes transcriptional elongation, and the Elongin complex was shown to suppress transient RNA-PolII pausing. Hence, in future intervein cells, Corto and the Elongin complex could apply opposite forces on the transcriptional machinery at the rho promoter. Corto would block rho transcription whereas the Elongin complex would be ready to accompany rho elongation if release should occur. In future vein cells on the other hand, the Elongin complex could actively participate in rho transcriptional elongation, since loss of function mutants for EloB and EloC exhibit loss of vein tissue. In these cells, rho expression would be independent of Corto, since corto mutants never present truncated veins (Rougeot, 2013).

    The results suggest that the Elongin complex might participate in determination of vein and intervein cell identity during wing development. It is proposed that this complex might interact with the ETP Corto at certain target genes and fine-tune their transcription in a cell-type specific manner. One of these targets could be the vein-promoting gene rho. In intervein cells, binding of Corto to the Elongin complex could prevent transcription of rho. Corto could also recruit other chromatin factors, such as the BAP chromatin-remodeling complex that was previously shown to inhibit rho expression in intervein cells. By contrast, in vein cells, the Elongin complex could participate in rho transcriptional elongation independently of Corto (Rougeot, 2013).

    Mechanisms of regulating tissue elongation in Drosophila wing: impact of oriented cell divisions, oriented mechanical forces, and reduced cell size

    Regulation of cell growth and cell division plays fundamental roles in tissue morphogenesis. However, the mechanisms of regulating tissue elongation through cell growth and cell division are still not well understood. The wing imaginal disc of Drosophila provides a model system that has been widely used to study tissue morphogenesis. This study used a recently developed two-dimensional cellular model to study the mechanisms of regulating tissue elongation in Drosophila wing. The effects of directional cues on tissue elongation were simulated. Also the role of reduced cell size was computationally analyzed. The simulation results indicate that oriented cell divisions, oriented mechanical forces, and reduced cell size can all mediate tissue elongation, but they function differently. Oriented cell divisions and oriented mechanical forces were shown to act as directional cues during tissue elongation. Between these two directional cues, oriented mechanical forces have a stronger influence than oriented cell divisions. In addition, the novel hypothesis is raised that reduced cell size may significantly promote tissue elongation. It was found that reduced cell size alone cannot drive tissue elongation. However, when combined with directional cues, such as oriented cell divisions or oriented mechanical forces, reduced cell size can significantly enhance tissue elongation in Drosophila wing. Furthermore, the simulation results suggest that reduced cell size has a short-term effect on cell topology by decreasing the frequency of hexagonal cells, which is consistent with experimental observations. Thse simulation results suggest that cell divisions without cell growth play essential roles in tissue elongation (Li, 2014).

    A common set of DNA regulatory elements shapes Drosophila appendages

    Animals have body parts made of similar cell types located at different axial positions, such as limbs. The identity and distinct morphology of each structure is often specified by the activity of different 'master regulator' transcription factors. Although similarities in gene expression have been observed between body parts made of similar cell types, how regulatory information in the genome is differentially utilized to create morphologically diverse structures in development is not known. This study used genome-wide open chromatin profiling to show that among the Drosophila appendages, the same DNA regulatory modules are accessible throughout the genome at a given stage of development, except at the loci encoding the master regulators themselves. In addition, open chromatin profiles change over developmental time, and these changes are coordinated between different appendages. It is proposed that master regulators create morphologically distinct structures by differentially influencing the function of the same set of DNA regulatory modules (McKay, 2013).

    This paper addresses a long-standing question in developmental biology: how does a single genome give rise to a diversity of structures? The results indicate that the combination of transcription factors expressed in each thoracic appendage acts upon a shared set of enhancers to create different morphological outputs, rather than operating on a set of enhancers that is specific to each tissue. This conclusion is based upon the surprising observation that the open chromatin profiles of the developing appendages are nearly identical at a given developmental stage. Therefore, rather than each master regulator operating on a set of enhancers that is specific to each tissue, the master regulators instead have access to the same set of enhancers in different tissues, which they differentially regulate. It was also found that tissues composed of similar combinations of cell types have very similar open chromatin profiles, suggesting that a limited number of distinct open chromatin profiles may exist at a given stage of development, dependent on cell-type identity (McKay, 2013).

    Different tissues were dissected from developing flies to compare their open chromatin profiles. These tissues are composed of different cell types, each with its own gene expression profile. Formaldehyde-assisted isolation of regulatory elements (FAIRE) data thus represent the average signal across all cells present in a sample. However, data from embryos and imaginal discs indicate that FAIRE is a very sensitive detector of functional DNA regulatory elements. For example, the Dll01 enhancer is active in 2–4 neurons of the leg imaginal disc; yet, the FAIRE signal at Dll01 is as strong as the Dll04 enhancer, which is active in hundreds of cells of the wing pouch. Thus, FAIRE may detect nearly all of the DNA regulatory elements that are in use among the cells of an imaginal disc. This study does not rule out the existence of DNA regulatory elements that are not marked by open chromatin or are otherwise not detected by FAIRE (McKay, 2013).

    Despite this sensitivity, the approach of this study does not identify which cells within the tissue have a particular open chromatin profile. For a given locus, it is possible that all cells in the tissue share a single open chromatin profile or that the FAIRE signal originates from only a subset of cells in which a given enhancer is active. Comparisons between eye-antennal discs, larval CNS, and thoracic discs suggest that the latter scenario is most likely, with open chromatin profiles among cells within a tissue shared by cells with similar identities at a given developmental stage (McKay, 2013).

    The observation that halteres and wings share open chromatin profiles demonstrates that Hox proteins like Ubx can differentially interpret the DNA sequence within the same subset of enhancers to modify one structure into another. This is consistent with the idea that morphological differences are largely dependent on the precise location, duration, and magnitude of expression of similar genes, and it is further supported by the similarity in gene expression profiles observed between Drosophila appendages and observed between vertebrate limbs. However, that such dramatic differences in morphology could be achieved by using the same subset of DNA regulatory modules in different tissues genome-wide was not known. The current findings provide a molecular framework to support the hypothesis that Hox factors function as 'versatile generalists,' rather than stable binary switches. The similarity in open chromatin profiles between wings and legs suggests that this framework also extends to other classes of master regulators beyond the Hox genes. It is also noted that, like the Drosophila appendages, vertebrate limbs are composed of similar combinations of cell types that differ in their pattern of organization. Moreover, the Drosophila appendage master regulators share a common evolutionary origin with the master regulators of vertebrate limb development, suggesting that the concept of shared open chromatin profiles may also apply to human development (McKay, 2013).

    The data suggest that open chromatin profiles vary both over time for a given lineage and between cell types at a given stage of development. Given the dramatic differences in the FAIRE landscape observed during embryogenesis and between the CNS and the appendage imaginal discs during larval stages, it appears as though the alteration of the chromatin landscape is especially important for specifying different cell types from a single genome. After cell-type specification, open chromatin profiles in the appendages continued to change as they proceeded toward terminal differentiation, suggesting that stage-specific functions require significant opening of new sites or the closing of existing sites. These findings contrast with those investigating hormone-induced changes in chromatin accessibility, in which the majority of open chromatin sites did not change after hormone treatment, including sites of de novo hormone-receptor binding. Thus, it may be that genome-wide remodeling of chromatin accessibility is reserved for the longer timescales and eventual permanence of developmental processes rather than the shorter timescales and transience of environmental responses (McKay, 2013).

    Different combinations of 'master regulator' transcription factors, often termed selector genes, are expressed in the developing appendages. Selectors are thought to specify the identity of distinct regions of developing animals by regulating the expression of transcription factors, signaling pathway components, and other genes that act as effectors of identity. One property attributed to selectors to explain their unique power to specify identity during development is the ability to act as pioneer transcription factors. In such models, selectors are the first factors to bind target genes; once bound, selectors then create a permissive chromatin environment for other transcription factors to bind. The finding that the same set of enhancers are accessible for use in all three appendages, with the exception of the enhancers that control expression of the selector genes themselves and other primary determinants of appendage identity, suggests that the selectors expressed in each appendage do not absolutely control the chromatin accessibility profile; otherwise, the haltere chromatin profile (for example) would differ from that of the wing because of the expression of Ubx (McKay, 2013).

    What then determines the appendage open chromatin profiles? Because open chromatin is likely a consequence of transcription factor binding, two nonexclusive models are possible. First, different combinations of transcription factors could specify the same open chromatin profiles. In this scenario, each appendage's selectors would bind to the same enhancers across the genome. For example, the wing selector Vg, with its DNA binding partner Sd, would bind the same enhancers in the wing as Dll and Sp1 bind in the leg. In the second model, transcription factors other than the selectors could specify the appendage open chromatin profiles. Selector genes are a small fraction of the total number of transcription factors expressed in the appendages. Many of the non-selector transcription factors are expressed at similar levels in each appendage, and thermodynamic models would predict them to bind the same enhancers. This model could also help to explain how the appendage open chromatin profiles coordinately change over developmental time despite the steady expression of the appendage selector genes during this same period. It is possible that stage-specific transcription factors determine which enhancers are accessible at a given stage of development. This would help to explain the temporal specificity of target genes observed for selectors such as Ubx. Recent work supports the role of hormone-dependent transcription factors in specifying the temporal identity of target genes in the developing appendages (Mou, 2012). Further experiments, including ChIP of the selectors from each of the appendages, will be required to determine the extent to which either of these models is correct (McKay, 2013).

    Binding of Ubx results in differential activity of enhancers in the haltere imaginal disc relative to the wing, despite equivalent accessibility of the enhancers in both discs, indicating that master regulators control morphogenesis by differentially regulating the activity of the same set of enhancers. It is likely that functional specificity of enhancers is achieved through multiple mechanisms. These include differential recruitment of coactivators and corepressors, modulation of binding specificity through interactions with cofactors, differential utilization of binding sites within a single enhancer, or regulation of binding dynamics through an altered chromatin context. This last mechanism would allow for epigenetic modifications early in development to affect subsequent gene regulatory events. For example, the activity of Ubx enhancers in the early embryo may control recruitment of Trithorax or Polycomb complexes to the PREs within the Ubx locus, which then maintain Ubx in the ON or OFF state at subsequent stages of development. Consistent with this model, Ubx enhancers active in the early embryo are only accessible in the 2-4 hr time point, whereas the accessibility of Ubx PREs varies little across developmental time or between tissues at a given developmental stage (McKay, 2013).

    The current results also have implications for the evolution of morphological diversity. Halteres and wings are considered to have a common evolutionary origin, but the relationship between insect wings and legs is unresolved. The observation that wings and legs share open chromatin profiles supports the hypothesis that wings and legs also share a common evolutionary origin in flies. Because legs appear in the fossil record before wings, the similarity in their open chromatin profiles suggests that the existing leg cis-regulatory network was co-opted for use in creation of dorsal appendages during insect evolution (McKay, 2013).

    Gain of cis-regulatory activities underlies novel domains of wingless gene expression in Drosophila

    Changes in gene expression during animal development are largely responsible for the evolution of morphological diversity. However, the genetic and molecular mechanisms responsible for the origins of new gene-expression domains have been difficult to elucidate. This study sought to identify molecular events underlying the origins of three novel features of wingless (wg) gene expression that are associated with distinct pigmentation patterns in Drosophila guttifera. The activity of cis-regulatory sequences (enhancers) across the wg locus in D. guttifera and Drosophila melanogaster were compared, and strong functional conservation was found among the enhancers that control similar patterns of wg expression in larval imaginal discs that are essential for appendage development. For pupal tissues, however, three novel wg enhancer activities were found in D. guttifera associated with novel domains of wg expression, including two enhancers located surprisingly far away in an intron of the distant Wnt10 gene. Detailed analysis of one enhancer (the vein-tip enhancer) revealed that it overlapped with a region controlling wg expression in wing crossveins (crossvein enhancer) in D. guttifera and other species. These results indicate that one novel domain of wg expression in D. guttifera wings evolved by co-opting pre-existing regulatory sequences governing gene activity in the developing wing. It is suggested that the modification of existing enhancers is a common path to the evolution of new gene-expression domains and enhancers (Koshikawa, 2015).

    A large body of comparative studies has shown that changes in the spatiotemporal expression of toolkit genes and the target genes they regulate correlate with the evolution of morphological traits. In a considerable number of instances, these spatiotemporal changes in gene expression have been demonstrated to involve the modification of enhancers. However, there are relatively few cases in which the origins of new enhancers have been elucidated, and none involving regulatory genes themselves (Koshikawa, 2015).

    This study has shown that three novel domains of wg expression in D. guttifera are governed by three novel enhancers, respectively. The evolution of wg cis-regulatory sequences within the D. guttifera lineage played a role in the gain of each enhancer activity, and the evolution of trans-acting regulatory factors was also necessary for the activity of two elements (gutCS and gutTS). Detailed analysis of the D. guttifera vein-tip enhancer revealed that it evolved within another conserved enhancer, whereas two other enhancers (the campaniform sensilla and thoracic stripe enhancers) arose within in an intron of the distant Wnt10 locus. These results bear on the understanding of the mechanisms underlying the evolution of new enhancers and domains of gene expression (Koshikawa, 2015).

    The D. guttifera vein-tip enhancer activity was localized within a 756-bp DNA segment that was also active in the developing pupal crossveins. This DNA segment is orthologous to segments of DNA in D. melanogaster and D. deflecta that were only active in the crossveins. The segments are all collinear, and contain numerous blocks of identical sequence, which suggests that the vein-tip enhancer activity evolved within the pre-existing crossvein enhancer (Koshikawa, 2015).

    One explanation for the presence of two activities in this one fragment is that they share functional sites: that is, binding sites for common transcription factors. Because both activities appear in the pupal wing, it is likely that they use common tissue-specific (wing) and temporal (pupal) inputs. The evolution of a new activity in the vein tips could have arisen through the addition of DNA-binding sites for transcription factors that were already present active in cells at vein tips. In this scenario, the novel enhancer activity would have resulted from the evolutionary co-option of an existing enhancer (Koshikawa, 2015).

    There is precedent for multifunctional enhancers and for this mechanism of co-option. For example, one enhancer of the D. melanogaster even-skipped gene governs two domains of gene expression that are controlled by shared inputs. In addition, it has been demonstrated that a novel optic lobe enhancer of the Drosophila santomea Neprilysin-1 gene arose via co-option of an existing enhancer. Moreover, it was shown that co-option had occurred in just a few mutational steps. The co-option of existing elements is an attractive explanation for the evolution of novel enhancers because it requires a relatively short mutational path (Koshikawa, 2015).

    One surprising property of enhancers is their ability to control gene transcription at promoters located at considerable linear distances away in the genome. For example, the enhancer that drives Sonic hedgehog (Shh) expression in the developing amniote limb bud is located in the intron of another gene ~1 Mb from the Shh locus. A growing body of evidence indicates that long segments of DNA are looped out in accommodating long-range enhancer-promoter interactions. The ability of enhancers to act over such long ranges suggests that new enhancers could evolve at considerable distances from the promoters that they regulate (Koshikawa, 2015).

    This study identified two enhancers in an intron of the D. guttifera Wnt10 gene that control transcription of the wg gene from a distance of ~70 kb, and separated by the Wnt6 locus. The data suggest that the gutTS enhancer preferentially regulates wg transcription and not Wnt10 or Wnt6 transcription, although the authors cannot explain this preference. The origins of the gutCS and gutTS enhancers are not as clear as the vein-tip enhancer. No pupal enhancer activity was detected in the orthologous DNA segments of D. melanogaster, there was no evidence of enhancer co-option. Nor were any obvious insertions found in these DNA segments such as a transposon. Nevertheless, the discovery of these novel, distant elements reflects the functional flexibility of cis-regulatory elements and their contribution to the evolution of gene regulation and morphological diversity (Koshikawa, 2015).

    Proteasome, but not autophagy, disruption results in severe eye and wing dysmorphia: a subunit- and regulator-dependent process in Drosophila

    Proteasome-dependent and autophagy-mediated degradation of eukaryotic cellular proteins represent the two major proteostatic mechanisms that are critically implicated in a number of signaling pathways and cellular processes. Deregulation of functions engaged in protein elimination frequently leads to development of morbid states and diseases. In this context, and through the utilization of GAL4/UAS genetic tool, this study examined the in vivo contribution of proteasome and autophagy systems in Drosophila eye and wing morphogenesis. By exploiting the ability of GAL4-ninaE. GMR and P{GawB}Bx(MS1096) genetic drivers to be strongly and preferentially expressed in the eye and wing discs, respectively, this study proved that proteasomal integrity and ubiquitination proficiency essentially control fly's eye and wing development. Indeed, subunit- and regulator-specific patterns of severe organ dysmorphia were obtained after the RNAi-induced downregulation of critical proteasome components (Rpn1, Rpn2, alpha5, beta5 and beta6) or distinct protein-ubiquitin conjugators (UbcD6, but not UbcD1 and UbcD4). Proteasome deficient eyes presented with either rough phenotypes or strongly dysmorphic shapes, while transgenic mutant wings were severely folded and carried blistered structures together with loss of vein differentiation. Moreover, transgenic fly eyes overexpressing the UBP2-yeast deubiquitinase enzyme were characterized by an eyeless-like phenotype. Therefore, the proteasome/ubiquitin proteolytic activities are undoubtedly required for the normal course of eye and wing development. In contrast, the RNAi-mediated downregulation of critical Atg (1, 4, 7, 9 and 18) autophagic proteins revealed their non-essential, or redundant, functional roles in Drosophila eye and wing formation under physiological growth conditions, since their reduced expression levels could only marginally disturb wing's, but not eye's, morphogenetic organization and architecture. However, Atg9 proved indispensable for the maintenance of structural integrity of adult wings in aged flies. In all, these findings clearly demonstrate the gene-specific fundamental contribution of proteasome, but not autophagy, in invertebrate eye and wing organ development (Velentzas, 2013).

    Elimination of unfit cells maintains tissue health and prolongs lifespan

    Viable yet damaged cells can accumulate during development and aging. Although eliminating those cells may benefit organ function, identification of this less fit cell population remains challenging. Previously, a molecular mechanism, based on 'fitness fingerprints' displayed on cell membranes, was identifed that allows direct fitness comparison among cells in Drosophila. This study reports the physiological consequences of efficient cell selection for the whole organism. The study found that fitness-based cell culling is naturally used to maintain tissue health, delay aging, and extend lifespan in Drosophila. A gene, ahuizotl (azot), was identified that ensures the elimination of less fit cells. Lack of azot increases morphological malformations and susceptibility to random mutations and accelerates tissue degeneration. On the contrary, improving the efficiency of cell selection is beneficial for tissue health and extends lifespan (Merino, 2015).

    Individual cells can suffer insults that affect their normal functioning, a situation often aggravated by exposure to external damaging agents. A fraction of damaged cells will critically lose their ability to live, but a different subset of cells may be more difficult to identify and eliminate: viable but suboptimal cells that, if unnoticed, may adversely affect the whole organism (Merino, 2015).

    What is the evidence that viable but damaged cells accumulate within tissues? The somatic mutation theory of aging proposes that over time cells suffer insults that affect their fitness, for example, diminishing their proliferation and growth rates, or forming deficient structures and connections. This creates increasingly heterogeneous and dysfunctional cell populations disturbing tissue and organ function. Once organ function falls below a critical threshold, the individual dies. The theory is supported by the experimental finding that clonal mosaicism occurs at unexpectedly high frequency in human tissues as a function of time, not only in adults an embryos (Merino, 2015).

    Does the high prevalence of mosaicism in our tissues mean that it is impossible to recognize and eliminate cells with subtle mutations and that suboptimal cells are bound to accumulate within organs? Or, on the contrary, can animal bodies identify and get rid of unfit viable cells (Merino, 2015)?

    One indirect mode through which suboptimal cells could be eliminated is proposed by the 'trophic theory,' which suggested that Darwinian-like competition among cells for limiting amounts of surv ead to removal of less fit cells. However, it is apparent from recent work that trophic theories are not sufficient to explain fitness-based cell selection, because there are direct mechanisms that allow cells to exchange 'cell-fitness' information at the local multicellular level (Merino, 2015).

    In Drosophila, cells can compare their fitness using different isoforms of the transmembrane protein Flower. The 'fitness fingerprints' are therefore defined as combinations of Flower isoforms present at the cell membrane that reveal optimal or reduced fitness. The isoforms that indicate reduced fitness have been called FlowerLose isoforms, because they are expressed in cells marked to be eliminated by apoptosis called 'Loser cells.' However, the presence of FlowerLose isoforms at the cell membrane of a particular cell does not imply that the cell will be culled, because at least two other parameters are taken into account: (1) the levels of FlowerLose isoforms in neighboring cells: if neighboring cells have similar levels of Lose isoforms, no cell will be killed; (2) the levels of a secreted protein called Sparc, the homolog of the Sparc/Osteonectin protein family, which counteracts the action of the Lose isoforms (Merino, 2015 and references therein).

    Remarkably, the levels of Flower isoforms and Sparc can be altered by various insults in several cell types, including: (1) the appearance of slowly proliferating cells due to partial loss of ribosomal proteins, a phenomenon known as cell competition; (2) the interaction between cells with slightly higher levels of d-Myc and normal cells, a process termed supercompetition; (3) mutations in signal transduction pathways like Dpp signaling; or (4) viable neurons forming part of incomplete ommatidia. Intriguingly, the role of Flower isoforms is cell type specific, because certain isoforms acting as Lose marks in epithelial cells are part of the fitness fingerprint of healthy neurons. Therefore, an exciting picture starts to appear, in which varying levels of Sparc and different isoforms of Flower are produced by many cell types, acting as direct molecular determinants of cell fitness. This study aimed to clarify how cells integrate fitness information in order to identify and eliminate suboptimal cells. Subsequently, the physiological consequences were analyzed of efficient cell selection for the whole organism (Merino, 2015).

    In order to discover the molecular mechanisms underlying cell selection in Drosophila, this study analyzed genes transcriptionally induced using an assay where WT cells (tub>Gal4) are outcompeted by dMyc-overexpressing supercompetitors (tub>dmyc) due to the increased fitness of these dMyc-overexpressing cells. The expression of CG11165 was strongly induced 24 hr after the peak of flower and sparc expression. In situ hybridization revealed that CG11165 mRNA was specifically detected in Loser cells that were going to be eliminated from wing imaginal discs due to cell competition. The gene, which was named ahuizotl (azot) after a multihanded Aztec creature selectively targeting fishing boats to protect lakes, consists of one exon. azot's single exon encodes for a four EF-hand-containing cytoplasmic protein of the canonical family that is conserved, but uncharacterized, in multicellular animals (Merino, 2015).

    To monitor Azot expression, a translational reporter was designed resulting in the expression of Azot::dsRed under the control of the endogenous azot promoter in transgenic flies. Azot expression was not detectable in most wing imaginal discs under physiological conditions in the absence of competition. Mosaic tissue was generated of two clonal populations, which are known to trigger competitive interactions resulting in elimination of otherwise viable cells. Cells with lower fitness were created by confronting WT cells with dMyc-overexpressing cells, by downregulating Dpp signaling, by overexpressing FlowerLose isoforms, in cells with reduced Wg signaling, by suppressing Jak-Stat signaling or by generating Minute clones. Azot expression was not detectable in nonmosaic tissue of identical genotype, nor in control clones overexpressing UASlacZ. On the contrary, Azot was specifically activated in all tested scenarios of cell competition, specifically in the cells undergoing negative selection. Azot expression was not repressed by the caspase inhibitor protein P35 (Merino, 2015).

    Because Flower proteins are conserved in mammals, tests were made to see if they are also able to regulate azot. Mouse Flower isoform 3 (mFlower3) has been shown to act as a 'classical' Lose isoform, driving cell elimination when expressed in scattered groups of cells, a situation where azot was induced in Loser cells but is not inducing cell selection when expressed ubiquitously a scenario where azot was not expressed. This shows that the mouse FlowerLose isoforms function in Drosophila similarly to their fly homologs (Merino, 2015).

    Interestingly, azot is not a general apoptosis-activated gene because its expression is not induced upon eiger, hid, or bax activation, which trigger cell death. Azot was also not expressed during elimination of cells with defects in apicobasal polarity or undergoing epithelial exclusion-mediated apoptosis (dCsk) (Merino, 2015).

    azot expression was analyzed during the elimination of peripheral photoreceptors in the pupal retina, a process mediated by Flower-encoded fitness fingerprints. Thirty-six to 38hr after pupal formation (APF), when FlowerLose-B expression begins in peripheral neurons, no Azot expression was detected in the peripheral edge. At later time points (40 and 44hr APF), Azot expression is visible and restricted to the peripheral edge where photoreceptor neurons are eliminated. This expression was confirmed with another reporter line, azot{KO; gfp}, where gfp was directly inserted at the azot locus using genomic engineering techniques (Merino, 2015).

    From these results, it is concluded that Azot expression is activated in several contexts where suboptimal and viable cells are normally recognized and eliminated (Merino, 2015).

    To understand Azot function in cell elimination, azot knockout (KO) flies were generated by deleting the entire azot gene. Next, Azot function was analyzed using dmyc-induced competition. In the absence of Azot function, loser cells were no longer eliminated, showing a dramatic 100-fold increase in the number of surviving clones. Loser cells occupied more than 20% of the tissue 72hr after clone induction (ACI). Moreover, using azot{KO; gfp} homozygous flies (that express GFP under the azot promoter but lack Azot protein), it was found that loser cells survived and showed accumulation of GFP. From these results, it is concluded that azot is expressed by loser cells and is essential for their elimination.

    In addition, clone removal was delayed in an azot heterozygous background (50-fold increase, 15%), compared to control flies with normal levels of Azot. Cell elimination capacity was fully restored by crossing two copies of Azot::dsRed into the azot-/- background demonstrating the functionality of the fusion protein. Silencing azot with two different RNAis was similarly able to halt selection during dmyc-induced competition. Next, in order to determine the role of Azot's EF hands, a mutated isoform of Azot (Pm4Q12) was generated and overexpressed, that carryed, in each EF hand, a point mutation known to abolish Ca2+ binding. Although overexpression of wild-type azot in negatively selected cells did not rescue the elimination, overexpression of the mutant AzotPm4Q12 reduced cell selection, functioning as a dominant-negative mutant. This shows that Ca2+ binding is important for Azot function. Finally, staining for apoptotic cells corroborated that the lack of Azot prevents cell elimination, because cell death was reduced 8-fold in mosaic epithelia containing loser cells (Merino, 2015).

    The role of azot in elimination of peripheral photoreceptor neurons in the pupal retina was examined using homozygous azot KO flies. Pupal retinas undergoing photoreceptor culling (44hr APF) of azot+/+ and azot-/- flies were stained for the cell death marker and the proapoptotic factor. Consistent with the expression pattern of Azot, the number of Hid and TUNEL-positive cells was dramatically decreased in azot-/- retinas compared to azot+/+ retinas (Merino, 2015).

    Those results show that Azot is required to induce cell death and Hid expression during neuronal culling. Therefore, tests were performed to see that was also the case in the wing epithelia during dmyc-induced competition. Hid was found to be expressed in loser cells and the expression was found to be strongly reduced in the absence of Azot function (Merino, 2015).

    Finally, forced overexpression of FlowerLose isoforms from Drosophila were unable to mediate WT cell elimination when Azot function was impaired by mutation or silenced by RNAi (Merino, 2015).

    These results suggested that azot function is dose sensitive, because heterozygous azot mutant flies display delayed elimination of loser cells when compared with azot WT flies. Therefore advantage was taken of the functional reporter Azot::dsRed to test whether cell elimination could be enhanced by increasing the number of genomic copies of azot. Tissues with three functional copies of azot were more efficient eliminating loser cells during dmyc-induced competition and most of the clones were culled 48hr ACI. From these results, it is concluded that azot expression is required for the elimination of Loser cells and unwanted neurons (Merino, 2015).

    Next, it was asked what could be the consequences of decreased cell selection at the tissue and organismal level. To this end, advantage was taken of the viability of homozygous azot KO flies. An increase of several developmental aberrations was observed. Focus was placed on the wings, where cell competition is best studied and, because aberrations, including melanotic areas, blisters, and wing margin nicks, were quantified. Wing defects of azot mutant flies could be rescued by introducing two copies of azot::dsRed, showing that the phenotypes are specifically caused by loss of Azot function (Merino, 2015).

    Next, it was reasoned that mild tissue stress should increase the need for fitness-based cell selection after damage. First, in order to generate multicellular tissues scattered with suboptimal cells, larvae were exposed to UV light and Azot expression was monitored in wing discs of UV-irradiated WT larvae that were stained for cleaved caspase-3, 24hr after treatment. Under such conditions, Azot was found to be expressed in cleaved caspase-3-positive cells. All Azot-positive cells showed caspase activation and 17% of cleaved caspase-positive cells expressed Azot. This suggested that Azot-expressing cells are culled from the tissue. To confirm this, later time points (3 days after irradiation) were examined; the increase in Azot-positive cells was no longer detectable. The elimination of azot-expressing cells after UV irradiation required azot function, because cells revealed by reporter azot{KO; gfp}, that express GFP instead of Azot, persisted in wing imaginal discs from azot-null larvae. Tests were performeed to see if lack of azot leads to a faster accumulation of tissue defects during organ development upon external damage. azot-/- pupae 0 stage were irradiated, and the number of morphological defects in adult wings was compared to those in nonirradiated azot KO flies. It was found that aberrations increased more than 2-fold when compared to nonirradiated azot-/- flies (Merino, 2015).

    In order to functionally discriminate whether azot belongs to genes regulating apoptosis in general or is dedicated to fitness-based cell selection, whether azot silencing prevents Eiger/TNF-induced cell death was exanubed. Inhibiting apoptosis (UASp35) or eiger (UASRNAieiger) rescued eye ablation, whereas azot silencing and overexpression of AzotPm4Q12 did not. Furthermore, azot silencing did not impair apoptosis during genitalia rotation or cell death of epithelial precursors in the retina. These results highlight the consequences of nonfunctional cell-quality control within developing tissues (Merino, 2015).

    The next part of the analysis demonstrated that the azot promoter computes relative FlowerLose and Sparc Levels. Epistasis analyses were performed to understand at which level azot is transcriptionally regulated. For this purpose, the assay where WT cells are outcompeted by dMyc-overexpressing supercompetitors was used. It was previously observed that azot induction is triggered upstream of caspase-3 activation and accumulates in outcompeted cells unable to die. Then, upstream events of cell selection were genetically modified. Silencing fweLose transcripts by RNAi or overexpressing Sparc both blocked the induction of Azot::dsRed in WT loser cells. In contrast, when outcompeted WT cells were additionally 'weakened' by Sparc downregulation using RNAi, Azot is detected in almost all loser cells compared to its more limited induction in the presence of endogenous Sparc. Inhibiting JNK signaling with UASpuc did not suppress Azot expression (Merino, 2015).

    The activation of Azot upon irradiation was examined. Strikingly, it was found that all Azot expression after irradiation was eliminated when Flower Lose was silenced and also when relative differences of Flower Lose where diminished by overexpressing high levels of Lose isoforms ubiquitously. On the contrary, Azot was not suppressed after irradiation by expressing the prosurvival factor Bcl-2 or a p53 dominant negative. These results show that Azot expression during competition and upon irradiation requires differences in Flower Lose relative levels (Merino, 2015).

    Finally, the regulation of Azot expression in neurons was analyzed. Silencing fwe transcripts by RNAi blocked the induction of Azot::dsRed in peripheral photoreceptors. Because Wingless signaling induces FlowerLose-B expression in peripheral photoreceptors, tests were performed to see if overexpression of Daxin, a negative regulator of the pathway, affected Azot levels. Axin overespression completely inhibited Azot expression. Similarly, overexpression of the cell competition inhibitor Sparc also fully blocked Azot endogenous expression in the retina. Finally, ectopic overexpression of FlowerLose-B in scattered cells of the retina was sufficient to trigger ectopic Azot activation. These results show that photoreceptor cells also can monitor the levels of Sparc and the relative levels of FlowerLose-B before triggering Azot expression (Merino, 2015).

    These results suggest that the azot promoter integrates fitness information from neighboring cells, acting as a relative 'cell-fitness checkpoint.'

    To test if fitness-based cell selection is a mechanism active not only during development, but also during adult stages, WT adult flies were exposed to UV light and monitor Azot and Flower expression were monitored in adult tissues. UV irradiation of adult flies triggered cytoplasmic Azot expression in several adult tissues including the gut and the adult brain. Likewise, UV irradiation of adult flies triggered Flower Lose expression in the gut and in the brain. Irradiation-induced Azot expression was unaffected by Bcl-2 but was eliminated when Flower Lose was silenced or when relative differences of Flower Lose where diminished in the gut. This suggests that the process of cell selection is active throughout the life history of the animal. Further confirming this conclusion, Azot function was essential for survival after irradiation, because more than 99% of azot mutant adults died 6 days after irradiation, whereas only 62.4% of WT flies died after the same treatment. The percentage of survival correlated with the dose of azot because adults with three functional copies of azot had higher median survival and maximum lifespan than WT flies, or null mutant flies rescued with two functional azot transgenes (Merino, 2015).

    The next part of the study addressed the role of cell selection during aging. Lack of cell selection could affect the whole organism by two nonexclusive mechanisms. First, the failure to detect precancerous cells, which could lead to cancer formation and death of the individual. Second, the time-dependent accumulation of unfit but viable cells could lead to accelerated tissue and organ decay. We therefore tested both hypotheses (Merino, 2015).

    It has been previously shown that cells with reduced levels for cell polarity genes like scrib or dlg are eliminated but can give rise to tumors when surviving. Therefore this study checked if azot functions as a tumor suppressing mechanism in those cells. Elimination of dlg and scrib mutant cells was not affected by RNAi against azot or when Azot function was impaired by mutation, in agreement with the absence of azot induction in these mutant cells. However, azot RNAi or the same azot mutant background efficiently rescued the elimination of clones with reduced Wg signaling (Merino, 2015).

    Moreover, the high number of suboptimal cells produced by UV treatment did not lead to tumoral growth in azot-null background. Thus, tumor suppression mechanisms are not impaired in azot mutant backgrounds, and tumors are not more likely to arise in azot-null mutants (Merino, 2015).

    Also tests were performed to see whether the absence of azot accelerates tissue fitness decay in adult tissues. Focused was placed on the adult brain, where neurodegenerative vacuoles develop over time and can be used as a marker of aging. The number was compared of vacuoles appearing in the brain of flies lacking azot (azot-/-), WT flies (azot+/+), flies with one extra genomic copy of the gene (azot+/+; azot+), and mutant flies rescued with two genomic copies of azot (azot-/-;azot+/+). For all the genotypes analyzed, a progressive increase was observed in the number and size of vacuoles in the brain over time. Interestingly, azot-/- brains showed higher number of vacuoles compared to control flies (azot+/+ and azot-/-;azot+/+) and a higher rate of vacuole accumulation developing over time. In the case of flies with three genomic copies of the gene (azot+/+; azot+), vacuole number tended to be the lowest (Merino, 2015).

    The cumulative expression of azot was analyzed during aging of the adult brain. Positive cells were detected as revealed by reporter azot{KO; gfp}, in homozygosis, that express GFP instead of Azot. A time-dependent accumulation of azot-positive cells was observed (Merino, 2015).

    From this, it is concluded that azot is required to prevent tissue degeneration in the adult brain and lack of azot showed signs of accelerated aging. This suggested that azot could affect the longevity of adult flies. Flies lacking azot (azot-/-) had a shortened lifespan with a median survival of 7.8 days, which represented a 52% decrease when compared to WT flies (azot+/+), and a maximum lifespan of 18 days, 25% less than WT flies (azot+/+). This effect on lifespan was azot dependent because it was completely rescued by introducing two functional copies of azot. On the contrary, flies with three functional copies of the gene (azot+/+; azot+) showed an increase in median survival and maximum lifespan of 54% and 17%, respectively (Merino, 2015).

    In conclusion, azot is necessary and sufficient to slow down aging, and active selection of viable cells is critical for a long lifespan in multicellular animals (Merino, 2015).

    The next part of the study demonstrates that death of unfit cells is sufficient and required for multicellular fitness maintenance. The results cited above show the genetic mechanism through which cell selection mediates elimination of suboptimal but viable cells. However, using flip-out clones and MARCM, this study found that Azot overexpression was not sufficient to induce cell death in wing imaginal discs. Because Hid is downstream of Azot, it was wondered whether expressing Hid under the control of the azot regulatory regions could substitute for Azot function (Merino, 2015).

    In order to test this hypothesis, the whole endogenous azot protein-coding sequence was replaced by the cDNA of the proapoptotic gene hid (azot{KO; hid}) flies. In a second strategy, the whole endogenous azot protein-coding sequence was replaced by the cDNA of transcription factor Gal4, so that the azot promoter can activate any UAS driven transgene (azot{KO; Gal4} flies. The number of morphological aberrations was compared in the adult wings of six genotypes: first, homozygous azot{KO; Gal4} flies that lacked Azot; second, azot{KO; hid} homozygous flies that express Hid with the azot pattern in complete absence of Azot; third, azot+/+ WT flies as a control; and finally three genotypes where the azot{KO; Gal4} flies were crossed with UAShid, UASsickle, another proapoptotic gene, or UASp35, an apoptosis inhibitor. In the case of UASsickle flies, a second azot mutation was introduced to eliminate azot function. Interestingly, the number of morphological aberrations was brought back to WT levels in all the situations where the azot promoter was driving proapoptotic genes (azot{KO; hid}, azot{KO; Gal4} × UAShid, azot{KO; Gal4} × UASsickle with or without irradiation. On the contrary, expressing p35 with the azot promoter was sufficient to produce morphological aberrations despite the presence of one functional copy of azot. Likewise, p35-expressing flies (azot{KO; Gal4}/azot+; UASp35) did not survive UV treatments, whereas a percentage of the flies expressing hid (26%) or sickle (28%) in azot-positive cells were able to survive (Merino, 2015).

    From this, it is concluded that specifically killing those cells selected by the azot promoter is sufficient and required to prevent morphological malformations and provide resistance to UV irradiation (Merino, 2015).

    The next part of the study demonstrated that death of unfit cells extends lifespan It was asked whether the shortened longevity observed in azot-/- flies could be also rescued by killing azot-expressing cells with hid in the absence of Azot protein. It was found that azot{KO; hid} homozygous flies had dramatically improved lifespan with a median survival of 27 days at 29°C, which represented a 125% increase when compared to azot-/- flies, and a maximum lifespan of 34 days, 41% more than mutant flies (Merino, 2015).

    Similar results were obtained at 25°C. It was found that flies lacking azot (azot-/-) had a shortened lifespan with a median survival of 25days, which represented a 24% decrease when compared to WT flies (azot+/+), and a maximum lifespan of 40 days, 31% less than WT flies (azot+/+). On the contrary, flies with three functional copies of the gene (azot+/+; azot+) or flies where azot is replaced by hid (azot{KO; hid} homozygous flies) showed an increase in median survival of 54% and 63% and maximum lifespan of 12% and 24%, respectively (Merino, 2015).

    Finally, the effects of dietary restriction on longevity of those flies was tested. It was found that dietary restriction could extend both the median survival and the maximum lifespan of all genotypes. Interestingly, dietary restricted flies with three copies of the gene azot showed a further increase in maximum lifespan of 35%. This shows that dietary restriction and elimination of unfit cells can be combined to maximize lifespan (Merino, 2015).

    In conclusion, eliminating unfit cells is sufficient to increase longevity, showing that cell selection is critical for a long lifespan in Drosophila (Merino, 2015).

    This study has shown that active elimination of unfit cells is required to maintain tissue health during development and adulthood. The gene (azot), whose expression is confined to suboptimal or misspecified but morphologically normal and viable cells. When tissues become scattered with suboptimal cells, lack of azot increases morphological malformations and susceptibility to random mutations and accelerates age-dependent tissue degeneration. On the contrary, experimental stimulation of azot function is beneficial for tissue health and extends lifespan. Therefore, elimination of less fit cells fulfils the criteria for a hallmark of aging (Merino, 2015).

    Although cancer and aging can both be considered consequences of cellular damage, no evidence was found for fitness-based cell selection having a role as a tumor suppressor in Drosophila. The results rather support that accumulation of unfit cells affect organ integrity and that, once organ function falls below a critical threshold, the individual dies (Merino, 2015).

    Azot expression in a wide range of 'less fit' cells, such as WT cells challenged by the presence of 'supercompetitors,' slow proliferating cells confronted with normal proliferating cells, cells with mutations in several signaling pathways (i.e., Wingless, JAK/STAT, Dpp), or photoreceptor neurons forming incomplete ommatidia. In order to be expressed specifically in 'less fit' cells, the transcriptional regulation of azot integrates fitness information from at least three levels: (1) the cell's own levels of FlowerLose isoforms, (2) the levels of Sparc, and (3) the levels of Lose isoforms in neighboring cells. Therefore, Azot ON/OFF regulation acts as a cell-fitness checkpoint deciding which viable cells are eliminated. It is proposed that by implementing a cell-fitness checkpoint, multicellular communities became more robust and less sensitive to several mutations that create viable but potentially harmful cells. Moreover, azot is not involved in other types of apoptosis, suggesting a dedicated function, and - given the evolutionary conservation of Azot - pointing to the existence of central cell selection pathways in multicellular animals (Merino, 2015).

    genes involved in wing morphogenesis

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    Genes involved in organ development

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