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
  • Proteome-wide association studies identify biochemical modules associated with a wing-size phenotype in Drosophila melanogaster
  • 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
  • Localized JNK signaling regulates organ size during development
  • A novel role for the 3'-5' exoribonuclease Dis3L2 in controlling cell proliferation and tissue growth
  • 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
  • Patterned anchorage to the apical extracellular matrix defines tissue shape in the developing appendages of Drosophila
  • Enhanced flight performance by genetic manipulation of wing shape 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
  • Control of lysosomal biogenesis and Notch-dependent tissue patterning by components of the TFEB-V-ATPase axis in Drosophila melanogaster
  • 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
  • Cell mixing induced by myc is required for competitive tissue invasion and destruction
  • Critical role for Fat/Hippo and IIS/Akt pathways downstream of Ultrabithorax during haltere specification in Drosophila
  • The Gyc76C receptor Guanylyl cyclase and the Foraging cGMP-dependent kinase regulate extracellular matrix organization and BMP signaling in the developing wing of Drosophila melanogaster
  • A local difference in Hedgehog signal transduction increases mechanical cell bond tension and biases cell intercalations along the Drosophila anteroposterior compartment boundary
  • Establishment of a developmental compartment requires interactions between three synergistic cis-regulatory modules
  • Curly encodes Dual Oxidase, which acts with Heme Peroxidase Curly Su to shape the adult Drosophila wing
  • Decanalization of wing development accompanied the evolution of large wings in high-altitude Drosophila
  • The gene expression program for the formation of wing cuticle in Drosophila
  • Tissue nonautonomous effects of fat body methionine metabolism on imaginal disc repair in Drosophila
  • Proprioceptive feedback determines visuomotor gain in Drosophila
  • Intra-organ growth coordination in Drosophila is mediated by systemic ecdysone signaling
  • Asymmetric distribution of Spalt in Drosophila wing squamous and columnar epithelia ensures correct cell morphogenesis
  • Ecdysone signaling induces two phases of cell cycle exit in Drosophila cells

    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).

    Proteome-wide association studies identify biochemical modules associated with a wing-size phenotype in Drosophila melanogaster

    The manner by which genetic diversity within a population generates individual phenotypes is a fundamental question of biology. To advance the understanding of the genotype-phenotype relationships towards the level of biochemical processes, a proteome-wide association study (PWAS) was performed of a complex quantitative phenotype. The variation of wing imaginal disc proteomes was quantified in Drosophila genetic reference panel (DGRP) lines using SWATH mass spectrometry. In spite of the very large genetic variation (1/36 bp) between the lines, proteome variability is surprisingly small, indicating strong molecular resilience of protein expression patterns. Proteins associated with adult wing size form tight co-variation clusters that are enriched in fundamental biochemical processes. Wing size correlates with some basic metabolic functions, positively with glucose metabolism but negatively with mitochondrial respiration and not with ribosome biogenesis. This study highlights the power of PWAS to filter functional variants from the large genetic variability in natural populations (Okada, 2016).

    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).

    Localized JNK signaling regulates organ size during development

    A fundamental question of biology is what determines organ size. Despite demonstrations that factors within organs determine their sizes, intrinsic size control mechanisms remain elusive. This study shows that Drosophila wing size is regulated by JNK signaling during development. JNK is active in a stripe along the center of developing wings, and modulating JNK signaling within this stripe changes organ size. This JNK stripe influences proliferation in a non-canonical, Jun-independent manner by inhibiting the Hippo pathway. Localized JNK activity is established by Hedgehog signaling, where Ci elevates dTRAF1 expression. As the dTRAF1 homolog, TRAF4, is amplified in numerous cancers, these findings provide a new mechanism for how the Hedgehog pathway could contribute to tumorigenesis, and, more importantly, provides a new strategy for cancer therapies. Finally, modulation of JNK signaling centers in developing antennae and legs changes their sizes, suggesting a more generalizable role for JNK signaling in developmental organ size control (Willsey, 2016).

    Two independently generated antibodies that recognize the phosphorylated, active form of JNK (pJNK) specifically label a stripe in the pouch of developing wildtype third instar wing discs. Importantly, localized pJNK staining is not detected in hemizygous JNKK mutant discs, in clones of JNKK mutant cells within the stripe, following over-expression of the JNK phosphatase puckered (puc), or following RNAi-mediated knockdown of bsk using two independent, functionally validated RNAi lines (Willsey, 2016).

    The stripe of localized pJNK staining appeared to be adjacent to the anterior-posterior (A/P) compartment boundary, a location known to play a key role in organizing wing growth, and a site of active Hedgehog (Hh) signaling. Indeed, pJNK co-localizes with the Hh target gene patched (ptc). Expression of the JNK phosphatase puc in these cells specifically abrogated pJNK staining, as did RNAi-mediated knockdown of bsk. Together, these data indicate that the detected pJNK signal reflects endogenous JNK signaling activity in the ptc domain, a region of great importance to growth control. Indeed, while at earlier developmental stages pJNK staining is detected in all wing pouch cells, the presence of a localized stripe of pJNK correlates with the time when the majority of wing disc growth occurs (1000 cells/disc at mid-L3 stage to 50,000 cells/disc at 20 hr after pupation, so it is hypothesized that localized pJNK plays a role in regulating growth (Willsey, 2016).

    Inhibition of JNK signaling in the posterior compartment previously led to the conclusion that JNK does not play a role in wing development. The discovery of an anterior stripe of JNK activity spurred a reexamination of the issue. Since bsk null mutant animals are embryonic lethal, JNK signaling was conditionally inhibited in three independent ways in the developing wing disc. JNK inhibition was achieved by RNAi-mediated knockdown of bsk (bskRNAi#1or2), by expression of JNK phosphatase (puc), or by expression of a dominant negative bsk (bskDN). These lines have been independently validated as JNK inhibitors. Inhibition of JNK in all wing blade cells (rotund-Gal4, rn-Gal4) or specifically in ptc-expressing cells (ptc-Gal4) resulted in smaller adult wings in all cases, up to 40% reduced in the strongest cases. Importantly, expression of a control transgene (UAS-GFP) did not affect wing size. This contribution of JNK signaling to size control is likely an underestimate, as the embryonic lethality of bsk mutations necessitates conditional, hypomorphic analysis. Nevertheless, hypomorphic hepr75/Y animals, while pupal lethal, also have smaller wing discs, as do animals with reduced JNK signaling due to bskDN expression. Importantly, total body size is not affected by inhibiting JNK in the wing. Even for the smallest wings generated (rn-Gal4, UAS-bskDN), total animal body size is not altered (Willsey, 2016).

    To test whether elevation of this signal can increase organ size, eiger (egr), a potent JNK activator, was expressed within the ptc domain (ptc-Gal4, UAS-egr). Despite induction of cell death as previously reporte and late larval lethality, a dramatic increase was observed in wing disc size without apparent duplications or changes in the shape of the disc. While changes in organ size could be due to changing developmental time, wing discs with elevated JNK signaling were already larger than controls assayed at the same time point. Similarly, inhibition of JNK did not shorten developmental time. Thus, changes in organ size by modulating JNK activity do not directly result from altering developmental time. Finally, the observed increase in organ size is not due to induction of apoptosis, as expression of the pro-apoptotic gene hid does not increase organ size. In contrast, it causes a decrease in wing size. Furthermore, co-expression of diap1 or p35 did not significantly affect the growth effect of egr expression, while the effect was dependent on Bsk activity (Willsey, 2016).

    In stark contrast to known developmental morphogens, no obvious defects were observed in wing venation pattern following JNK inhibition, suggesting that localized pJNK may control growth in a pattern formation-independent manner. Indeed, even a slight reduction in Dpp signaling results in dramatic wing vein patterning defects. Second, inhibiting Dpp signaling causes a reduction in wing size along the A-P axis, while JNK inhibition causes a global reduction. Furthermore, ectopic Dpp expression increases growth in the form of duplicated structures, while increased JNK signaling results in a global increase in size. Molecularly, it was confirmed that reducing Dpp signaling abolishes pSMAD staining, while quantitative data shows that inhibiting JNK signaling does not. Furthermore, it was also found that Dpp is not upstream of pJNK, as reduction in Dpp signaling does not affect pJNK. Together, the molecular data are consistent with the phenotypic results indicating that pJNK and Dpp are separate programs in regulating growth. Consistent with these findings it has been suggested that Dpp does not play a primary role in later larval wing growth control (Akiyama, 2015). Finally, it was found that inhibition of JNK does not affect EGFR signaling (pERK) and that inhibition of EGFR does not affect the establishment of pJNK (Willsey, 2016).

    A difference in size could be due to changes in cell size and/or number. Wings with reduced size due to JNK inhibition were examined and no changes in cell size or apoptosis were found, suggesting that pJNK controls organ size by regulating cell number. Consistently, the cell death inhibitor p35 was unable to rescue the decreased size following JNK inhibition. Indeed, inhibition of JNK signaling resulted in a decrease in proliferation, while elevation of JNK signaling in the ptc domain resulted in an increase in cell proliferation in the enlarged wing disc. Importantly, this increased proliferation is not restricted to the ptc domain, consistent with previous reports that JNK can promote proliferation non-autonomously (Willsey, 2016).

    To determine the mechanism by which pJNK controls organ size, canonical JNK signaling through its target Jun was considered. Interestingly, RNAi-mediated knockdown of jun in ptc cells does not change wing size, consistent with previous analysis of jun mutant clones in the wing disc. Furthermore, in agreement with this, a reporter of canonical JNK signaling downstream of jun (puc-lacZ) is not expressed in the pJNK stripe. Finally, knockdown of fos (kayak, kay) alone or with junRNAi did not affect wing size. Together, these data indicate that canonical JNK signaling through jun does not function in the pJNK stripe to regulate wing size (Willsey, 2016).

    In search of such a non-canonical mechanism of JNK-mediated size control, the Hippo pathway was considered. JNK signaling regulates the Hippo pathway to induce autonomous and non-autonomous proliferation during tumorigenesis and regeneration via activation of the transcriptional regulator Yorkie (Yki). Recently it has been shown that JNK activates Yki via direct phosphorylation of Jub. To test whether this link between JNK and Jub could account for the role of localized pJNK in organ size control during development, RNAi-mediated knockdown of jub was performed in the ptc stripe, and adults with smaller wings were observed. Indeed, the effect of JNK loss on wing size can be partially suppressed in a heterozygous lats mutant background and increasing downstream yki expression in all wing cells or just within the ptc domain can rescue wing size following JNK inhibition. These results suggest that pJNK controls Yki activity autonomously within the ptc stripe, leading to a global change in cell proliferation. This hypothesis predicts that the Yki activity level within the ptc stripe influences overall wing size. Consistently, inhibition of JNK in the ptc stripe translates to homogeneous changes in anterior and posterior wing growth. Similarly, overexpression or inhibition of Yki signaling in the ptc stripe also results in a global change in wing size (Willsey, 2016).

    It is important to note that the yki expression line used is wild-type Yki, which is still affected by JNK signaling. For this reason, the epistasis experiment was also performed with activated Yki, which is independent of JNK signaling. Expression of this activated Yki in the ptc stripe caused very large tumors and lethality. Importantly, inhibiting JNK in this context did not affect the formation of these tumors or the lethality. Furthermore, inhibiting both JNK and Yki together does not enhance the phenotype of Yki inhibition alone, further supporting the idea that Yki is epistatic to JNK, instead of acting in parallel processes (Willsey, 2016).

    Mutants of the Yki downstream target four-jointed (fj) have small wings with normal patterning, and fj is known to propagate Hippo signaling and affect proliferation non-autonomously. Although RNAi-mediated knockdown of fj in ptc cells does not cause an obvious change in wing size, it is sufficient to block the Yki-induced effect on increasing wing size . However, overexpression of fj also reduces wing size, which makes it not possible to test for a simple epistatic relationship. Overall, these data are consistent with the notion that localized pJNK regulates wing size not by Jun-dependent canonical JNK signaling, but rather by Jun-independent non-canonical JNK signaling involving the Hippo pathway (Willsey, 2016).

    While morphogens direct both patterning and growth of developing organs, a link between patterning molecules and growth control pathways has not been established. pJNK staining is coincident with ptc expression, suggesting it could be established by Hh signaling. During development, posterior Hh protein travels across the A/P boundary, leading to activation of the transcription factor Cubitus interruptus (Ci) in the stripe of anterior cells. To test whether localized activation of JNK is a consequence of Hh signaling through Ci, RNAi-mediated knockdown of ci was performed, and it was found that the pJNK stripe is eliminated. Consistently, adult wing size is globally reduced. In contrast, no change was observed in pJNK stripe staining following RNAi-mediated knockdown of dpp or EGFR. Expression of non-processable Ci leads to increased Hh signaling. Expression of this active Ci in ptc cells leads to an increase in pJNK signal and larger, well-patterned adult wings. The modest size increase shown for ptc>CiACT is likely due to the fact that higher expression of this transgene (at 25 ° C) leads to such large wings that pupae cannot emerge from their cases. For measuring wing size, this experiment was performed at a lower temperature so that the animals were still viable. Furthermore, inhibition of JNK in wings expressing active Ci blocks Ci's effects, and resulting wings are similar in size to JNK inhibition alone . Together, these data indicate that Hh signaling through Ci is responsible for establishing the pJNK stripe (Willsey, 2016).

    To determine the mechanism by which Ci activates the JNK pathway, transcriptional profiles of posterior and ptc domain cells isolated by FACS from third instar wing discs were compared. Of the total 12,676 unique genes represented on the microarray, 50.4% (6,397) are expressed in ptc domain cells, posterior cells, or both. Hh pathway genes known to be differentially expressed were identified. It was next asked whether any JNK pathway genes are differentially expressed, and and it was found that dTRAF1 expression is more than five-fold increased in ptc cells, while other JNK pathway members are not differentially expressed (Willsey, 2016).

    dTRAF1 is expressed along the A/P boundary and ectopic expression of dTRAF1 activates JNK signaling. Thus, positive regulation of dTRAF1 expression by Ci could establish a stripe of pJNK that regulates wing size. Indeed, Ci binding motifs were identified in the dTRAF1 gene, and a previous large-scale ChIP study confirms a Ci binding site within the dTRAF1 gene. Consistently, a reduction in Ci led to a 29% reduction in dTRAF1 expression in wing discs. Given that the reduction of dTRAF1 expression in the ptc stripe is buffered by Hh-independent dTRAF1 expression elsewhere in the disc, this 29% reduction is significant. Furthermore, inhibition of dTRAF1 by RNAi knockdown abolished pJNK staining. Finally, these animals have smaller wings without obvious pattern defects. Conversely, overexpression of dTRAF1 causes embryonic lethality, making it not possible to attempt to rescue a dTRAF1 overexpression wing phenotype by knockdown of bsk. Nevertheless, it has been shown that dTRAF1 function in the eye is Bsk-dependent. Finally, inhibition of dTRAF1 modulates the phenotype of activated Ci signaling. Together, these data reveal that the pJNK stripe in the developing wing is established by Hh signaling through Ci-mediated induction of dTRAF1 expression (Willsey, 2016).

    Finally, localized centers of pJNK activity were detected during the development of other imaginal discs including the eye/antenna and leg. Inhibition of localized JNK signaling during development caused a decrease in adult antenna size and leg size. Conversely, increasing JNK signaling during development resulted in pupal lethality; nevertheless, overall sizes of antenna and leg discs were increased. Together, these data indicate that localized JNK signaling regulates size in other organs in addition to the wing, suggesting a more universal effect of JNK on size control (Willsey, 2016).

    Intrinsic mechanisms of organ size control have long been proposed and sought after. This study reveals that in developing Drosophila tissues, localized, organ-specific centers of JNK signaling contribute to organ size in an activity level-dependent manner. Such a size control mechanism is qualitatively distinct from developmental morphogen mechanisms, which affect both patterning and growth. Aptly, this mechanism is still integrated in the overall framework of developmental regulation, as it is established in the wing by the Hh pathway. These data indicate that localized JNK signaling is activated by Ci-mediated induction of dTRAF1 expression. Furthermore,it is not canonical Jun-dependent JNK signaling, but rather non-canonical JNK signaling that regulates size, possibly through Jub-dependent regulation of Yki signaling, as described for regeneration. As the human dTRAF1 homolog, TRAF4, and Hippo components are amplified in numerous cancers, these findings provide a new mechanism for how the Hh pathway could contribute to tumorigenesis. More importantly, these findings offer a new strategy for potential cancer therapies, as reactivating Jun in Hh-driven tumors could lead tumor cells towards an apoptotic fate (Willsey, 2016).

    A novel role for the 3'-5' exoribonuclease Dis3L2 in controlling cell proliferation and tissue growth

    This study has shown that the exoribonuclease Dis3L2 is required for regulation of proliferation in the wing imaginal discs in Drosophila. Dis3L2 is a member of a highly conserved family of exoribonucleases that degrade RNA in a 3'-5' direction. Knockdown of Dis3L2 results in substantial wing overgrowth due to increased cellular proliferation rather than an increase in cell size. Imaginal discs are specified in the embryo before proliferating and differentiating to form the adult structures of the fly. Using RNA-seq, a small set of mRNAs was identified that are sensitive to Dis3L2 activity. Of the mRNAs which increase in levels and are therefore potential targets of Dis3L2, two were identified that change at the post-transcriptional level but not at the transcriptional level, namely CG2678 (a transcription factor) and pyrexia (a TRP cation channel). A compensatory effect between Dis3L2 and the 5'-3' exoribonuclease Pacman was identified, demonstrating that these two exoribonucleases function to regulate opposing pathways within the developing tissue. This work provides the first description of the molecular and developmental consequences of Dis3L2 inactivation in a non-human animal model. The work is directly relevant to the understanding of human overgrowth syndromes such as Perlman syndrome (Towler, 2016).

    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).

    Patterned anchorage to the apical extracellular matrix defines tissue shape in the developing appendages of Drosophila

    How tissues acquire their characteristic shape is a fundamental unresolved question in biology. While genes have been characterized that control local mechanical forces to elongate epithelial tissues, genes controlling global forces in epithelia have yet to be identified. This study describes a genetic pathway that shapes appendages in Drosophila by defining the pattern of global tensile forces in the tissue. In the appendages, shape arises from tension generated by cell constriction and localized anchorage of the epithelium to the cuticle via the apical extracellular-matrix protein Dumpy (Dp). Altering Dp expression in the developing wing results in predictable changes in wing shape that can be simulated by a computational model that incorporates only tissue contraction and localized anchorage. Three other wing shape genes, narrow, tapered, and lanceolate, encode components of a pathway that modulates Dp distribution in the wing to refine the global force pattern and thus wing shape (Ray, 2015).

    Enhanced flight performance by genetic manipulation of wing shape in Drosophila

    Insect wing shapes are remarkably diverse and the combination of shape and kinematics determines both aerial capabilities and power requirements. However, the contribution of any specific morphological feature to performance is not known. Using targeted RNA interference to modify wing shape far beyond the natural variation found within the population of a single species, this study shows a direct effect on flight performance that can be explained by physical modelling of the novel wing geometry. Data show that altering the expression of a single gene (narrow) can significantly enhance aerial agility and that the Drosophila wing shape is not, therefore, optimized for certain flight performance characteristics that are known to be important. This technique points in a new direction for experiments on the evolution of performance specialities in animals (Ray, 2016).

    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).

    Control of lysosomal biogenesis and Notch-dependent tissue patterning by components of the TFEB-V-ATPase axis in Drosophila melanogaster

    In vertebrates, TFEB (transcription factor EB) and MITF (microphthalmia-associated transcription factor) family of basic Helix-Loop-Helix (bHLH) transcription factors regulate both lysosomal function and organ development. However, it is not clear whether these 2 processes are interconnected. This study shows that Mitf, the single TFEB and MITF ortholog in Drosophila, controls expression of vacuolar-type H+-ATPase pump (V-ATPase) subunits. Remarkably, it was also found that expression of Vha16-1 and Vha13, encoding 2 key components of V-ATPase, is patterned in the wing imaginal disc. In particular, Vha16-1 expression follows differentiation of proneural regions of the disc. These regions, that will form sensory organs in the adult, appear to possess a distinctive endo-lysosomal compartment and Notch (N) localization. Modulation of Mitf activity in the disc in vivo alters endo-lysosomal function and disrupts proneural patterning. Similar to these findings in Drosophila, in human breast epithelial cells, it was observed that the impairment of the Vha16-1 human ortholog ATP6V0C changes the size and function of the endo-lysosomal compartment and depletion of TFEB reduces ligand-independent N signaling activity. These data suggest that lysosomal-associated functions regulated by the TFEB-V-ATPase axis might play a conserved role in shaping cell fate (Tognon, 2016).

    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).

    Cell mixing induced by myc is required for competitive tissue invasion and destruction

    Cell-cell intercalation is used in several developmental processes to shape the normal body plan. There is no clear evidence that intercalation is involved in pathologies. This study used the proto-oncogene myc to study a process analogous to early phase of tumour expansion: myc-induced cell competition. Cell competition is a conserved mechanism driving the elimination of slow-proliferating cells (so-called 'losers') by faster-proliferating neighbours (so-called 'winners') through apoptosis and is important in preventing developmental malformations and maintain tissue fitness. Using long-term live imaging of myc-driven competition in the Drosophila pupal notum and in the wing imaginal disc, this study showed that the probability of elimination of loser cells correlates with the surface of contact shared with winners. As such, modifying loser-winner interface morphology can modulate the strength of competition. Elimination of loser clones requires winner-loser cell mixing through cell-cell intercalation. Cell mixing is driven by differential growth and the high tension at winner-winner interfaces relative to winner-loser and loser-loser interfaces, which leads to a preferential stabilization of winner-loser contacts and reduction of clone compactness over time. Differences in tension are generated by a relative difference in F-actin levels between loser and winner junctions, induced by differential levels of the membrane lipid phosphatidylinositol (3,4,5)-trisphosphate. These results establish the first link between cell-cell intercalation induced by a proto-oncogene and how it promotes invasiveness and destruction of healthy tissues (Levayer, 2015).

    To analyse quantitatively loser cell elimination, long-term live imaging was performed of clones showing a relative decrease of the proto-oncogene myc in the Drosophila pupal notum, a condition known to induce cell competition in the wing disc. Every loser cell delamination was counted over 10 h, and the probability of cell elimination was calculated for a given surface of contact shared with winner cells. A significant increase was observed of the proportion of delamination with winner-loser shared contact, whereas this proportion remained constant for control clones. The same correlation was observed in ex vivo culture of larval wing disc. Cell delamination in the notum was apoptosis dependent and expression of flowerlose (fwelose), a competition-specific marker for loser fate, was necessary and sufficient to drive contact-dependent delamination. Moreover it was confirmed that contact-dependent death is based on the computation of relative differences of fwelose between loser cells and their neighbours. Thus, cell delamination in the notum recapitulates features of cell competition (Levayer, 2015).

    This suggests that winner-loser interface morphology could modulate the probability of eliminating loser clones. Using the wing imaginal disc, winner-loser contact was reduced by inducing adhesion- or tension-dependent cell sorting and observed a significant reduction of loser clone elimination. This rescue was not driven by a cell-autonomous effect of E-cadherin (E-cad) or active myosin II regulatory light chain (MRLC) on growth, death or cell fitness but rather by a general diminution of winner-loser contact. Competition is ineffective across the antero-posterior compartment boundary, a frontier that prevents cell mixing through high line tension. Accordingly, there was no increase in death at the antero-posterior boundary in wing discs overexpressing fweloseA in the anterior compartment. However, reducing tension by reducing levels of myosin II heavy chains was sufficient to increase the shared surface of contact between cells of the anterior and posterior compartments, and induced fwelose death at the boundary. Altogether, it is concluded that the reduction in surface contact between winners and losers is sufficient to block competition, which explains how compartment boundaries prevent competition (Levayer, 2015).

    Loser clones have been reported to fragment more often than controls, whereas winner clones show convoluted morphology, suggesting that winner-y actin levels, and F-actin levels were higher in WT clones than M-/+ cells. This prompted a test of the role of actin organization in winner-loser mixing. Downregulating the formin Diaphanous (Dia, a filamentous actin polymerization factor) by RNA interference (RNAi) or by using a hypomorphic mutant was sufficient to obtain a high proportion of fragmen-cell intercalation independent of death. To assess the contribution of each phenomenon, the proportion of clones fragmented 48 h after clone induction (ACI) was systematically counted. A twofold increase was observed in the frequency of split clones in losers (wild type (WT) in tub-dmyc) versus WT in WT controls. Overexpressing E-cad or active myosin II was sufficient to prevent loser clone splitting, whereas blocking apoptosis or blocking loser fate by silencing fwelose did not reduce splitting. Finally, the proportion of split clones was also increased for winner clones either during myc-driven competition (UAS-myc, UAS-p35) or during Minute-dependent competition (WT clones in M-/+ background). Altogether, this suggested that winner-loser mixing is increased independently of loser cell death or clone size by a factor upstream of fwe, and could be driven by cell-cell intercalation. Accordingly, junction remodelling events leading to disappearance of a loser-loser junction were three times more frequent at loser clone boundaries than control clone boundaries in the pupal notum. The rate of junction remodelling was higher in loser-loser junctions and in winner-winner junctions than in winner-loser junctions. The preferential stabilization of winner-loser interfaces should increase the surface of contact between winner and loser cells over time. Accordingly, loser clone compactness in the notum decreased over time whereas it remains constant on average for WT clones in WT background. Similarly, the compactness of clones in the notum also decreased over time for conditions showing high frequency of clone splitting in the wing disc, whereas clone compactness remained constant for conditions rescuing clone splitting. Altogether, it is concluded that both Minute- and myc-dependent competition increase loser-winner mixing through cell-cell intercalation (Levayer, 2015).

    It was then asked what could modulate the rate of junction remodelling during competition. The rate of junction remodelling can be cell-autonomously increased by myc. Interestingly, downregulation of the tumour suppressor PTEN is also sufficient to increase the rate of junction remodelling through the upregulation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3). It was reasoned that differences in PIP3 levels could also modulate junction remodelling during competition. Using a live reporter of PIP3 that could detect modulations of PIP3 in the notum, a significant increase of PIP3 was observed in the apico-lateral membrane of tub-dmyc-tub-dmyc interfaces compared with WT-WT and WT-tub-dmyc interfaces. Moreover, increasing/reducing Myc levels in a full compartment of the wing disc was sufficient to increase/decrease the levels of phospho-Akt (a downstream target of PIP3, whereas fweloseA overexpression had no effect. Similarly, levels of phospho-Akt were relatively higher in WT clones than in the surrounding M-/+ cells. Thus differences in PIP3 levels might be responsible for winner-loser mixing (Levayer, 2015).

    Accordingly, reducing PIP3 levels by overexpressing a PI3 kinase dominant negative (PI3K-DN) or increasing PIP3 levels by knocking down PTEN (UAS-pten RNAi) were both sufficient to induce a high proportion of fragmented clones and to reduce clone compactness over time in the notum, whereas increasing PIP3 in loser clones was sufficient to prevent cell mixing. Moreover, abolishing winner-loser PIP3 differences through larval starvation prevented loser clone fragmentation, the reduction of clone compactness over time in the notum and could rescue WT clone elimination in tub-dmyc background. It is therefore concluded that differences in PIP3 levels are necessary and sufficient for loser-winner mixing and required for loser cell elimination (Levayer, 2015).

    It was then asked which downstream effectors of PIP3 could affect junction stability. A relative growth decrease can generate mechanical stress that can be released by cell-cell intercalation. Accordingly, growth reduction through Akt downregulation is sufficient to increase clone splitting and could contribute to loser clone splitting. However, Akt is not sufficient to explain winner-loser mixing because, unlike PIP3, increasing Akt had no effect on clone splitting. PIP3 could also modulate junction remodelling through its effect on cytoskeleton and the modulation of intercellular adhesion or tension. No obvious modifications of E-cad, MRLC or Dachs (another regulator of tension) was detected in loser cells. However, a significant reduction of F-actin levels and a reduction of actin turnover/polymerization rate were observed in loser-loser and loser-winner junctions in the notum. Similarly, modifying Myc levels in a full wing disc compartment was sufficient to modify actin levels, and F-actin levels were higher in WT clones than M-/+ cells. This prompted a test of the role of actin organization in winner-loser mixing. Downregulating the formin Diaphanous (Dia, a filamentous actin polymerization factor) by RNA interference (RNAi) or by using a hypomorphic mutant was sufficient to obtain a high proportion of fragmented clones and to reduce clone compactness over time, whereas overexpressing Dia in loser clones prevented clone splitting (UAS-dia::GFP) and compactness reduction. This effect was specific to Dia as modulating Arp2/3 complex (a regulator of dendritic actin network) had no effect on clone splitting. Thus, impaired filamentous actin organization was necessary and sufficient to drive loser-winner mixing. These actin defects were driven by the differences in PIP3 levels between losers and winners. Thus Dia could be an important regulator of competition through its effect on cell mixing. Overexpression of Dia was indeed sufficient to reduce loser clone elimination significantly (Levayer, 2015).

    Filamentous actin has been associated with tension regulation. It was therefore asked whether junction tension was modified in winner and loser junctions. The maximum speed of relaxation of junction after laser nanoablation (which is proportional to tension) was significantly reduced in loser-loser and winner-loser junctions compared with winner-winner junctions. This distribution of tension has been proposed to promote cell mixing. Accordingly, decreasing PIP3 in clones reduced tension both in low-PIP3-low-PIP3 and low-PIP3-normal-PIP3 junctions, whereas overexpressing Dia in loser clones or starvation were both sufficient to abolish differences in tension, in agreement with their effect on winner-loser mixing and the distribution of F-actin. Thus the lower tension at winner-loser and loser-loser junctions is responsible for winner-loser mixing. Altogether, it is concluded that the relative PIP3 decrease in losers increases winner-loser mixing through Akt-dependent differential growth and the modulation of tension through F-actin downregulation in winner-loser and loser-loser junctions (Levayer, 2015).

    Several modes of tissue invasion by cancer cells have been described, most of them relying on the departure of the tumour cells from the epithelial layer. This study suggests that some oncogenes may also drive tissue destruction and invasion by inducing ectopic cell intercalation between cancerous and healthy cells, and subsequent healthy cell elimination. myc-dependent invasion could be enhanced by other mutations further promoting intercalation (such as PTEN). Stiffness is increased in many tumours, suggesting that healthy cell-cancer cell mixing by intercalation might be a general process (Levayer, 2015).

    Critical role for Fat/Hippo and IIS/Akt pathways downstream of Ultrabithorax during haltere specification in Drosophila

    In Drosophila, differential development of wing and haltere, which differ in cell size, number and morphology, is dependent on the function of Hox gene Ultrabithorax (Ubx). This paper reports studies on Ubx-mediated regulation of the Fat/Hippo and IIS/dAkt pathways, which control cell number and cell size during development. Over-expression of Yki or down regulation of negative components of the Fat/Hippo pathway, such as expanded, caused considerable increase in haltere size, mainly due to increase in cell number. These phenotypes were also associated with the activation of Akt pathways in developing haltere. Although activation of Akt alone did not affect the cell size or the organ size, dramatic increase was observed in haltere size when Akt was activated in the background where expanded is down regulated. This was associated with the increase in both cell size and cell number. The organ appeared flatter than wildtype haltere and the trichome morphology and spacing resembled that of wing suggesting homeotic transformations. Thus, these results suggest a link between cellular growth and pattern formation and the final differentiated state of the organ (Singh, 2015).

    Wing and haltere are the dorsal appendages of second and third thoracic segments, respectively, of adult Drosophila. They are homologous structures, although differ greatly in their morphology. The homeotic gene Ultrabithorax (Ubx), which is required and sufficient to confer haltere fate to epithelial cells, is known to regulate many wing patterning genes to specify haltere, but the mechanism is still poorly understood (Singh, 2015).

    There are a number of differences between wing and haltere at the cellular and organ levels. Wing is a large, flat and thin structure, while haltere is a small globular structure, although both are made up of 2-layered sheet of epithelial cells. Space between the two layers of cells in haltere is filled with haemocytes. Cuticle area of each wing cell is 8 fold more than a haltere cell. Haltere has smaller and fewer cells than the wing. Trichomes of wing cells are long and thin, while haltere trichomes are short and stout in morphology. The ratio of anterior to posterior compartment size in the haltere (~2.5:1) is much different from that in the wing (~1.2:1). Haltere also lacks wing-type vein and sensory bristles. Haltere cells are more cuboidal compared to flatter wing cells (Roch, 2000). Thus, cell number, size and shape all add to the differences in the size and shape of the two organs (Singh, 2015).

    However, cells of the third instar larval wing and haltere discs are similar in size and shape (Makhijani, 2007). The difference between cell size and shape becomes evident at late pupal stages (Roch, 2000). Wing cells become much larger, compared to haltere cells. At pupal stages, they also exhibit differences in the organization of actin cytoskeleton elements viz. F-actin levels are much higher in haltere cells compared to wing cells (Roch, 2000) (Singh, 2015).

    In the context of final shape of wings and halteres, one needs to understand the mechanism by which Ubx influences cell size, shape and arrangement. It is possible that Ubx regulates overall shape of the haltere by regulating either cell size and/or shape. The current understanding of mechanisms by which wing and haltere differ at cellular, tissue and organ level is ambiguous (Sanchez-Herrero, 2013). For example, while removal of Ubx from the entire haltere, or at least from one entire compartment, leads to haltere to wing transformation with increased growth of Ubx minus tissues, mitotic clones of Ubx (using the null allele Ubx6.28) show similar sized twin spot in small clones (Crickmore, 2006, De Navas, 2006; Makhijani, 2007). Only when very large clones of Ubx6.28Ubx6.28 are generated, one can see increased growth compared to their twin spots (Crickmore, 2006). This suggests that unless a certain threshold level of growth factors is de-repressed, the haltere does not show any overgrowth phenotype (Singh, 2015).

    There have been several efforts to identify functional and molecular mechanisms by which Ubx regulates genes/pathways to provide haltere its distinct morphology. Various approaches have been used to identify targets of Ubx that are expected to differentially express between wing and haltere, e.g., loss-of-function genetics, deficiency screens, enhancer-trap screening and genome wide approaches such as microarray analysis and chromatin immunoprecipitation (ChIP). Targets include genes involved in diverse cellular functions like components of the cuticle and extracellular matrix, genes involved in cell specification, cell proliferation, cell survival, cell adhesion, or cell differentiation, structural components of actin and microtubule filaments, and accessory proteins controlling filament dynamics (reviewed in Sanchez-Herrero, 2013; Singh, 2015).

    Decapentaplegic (Dpp), Wingless (Wg), and Epidermal growth factor receptor (EGFR) are some of the major growth and pattern regulating pathways that are repressed by Ubx in the haltere (Weatherbee, 1998, Shashidhara, 1999; Prasad, 2003; Mohit, 2006; Crickmore, 2006, Pallavi, 2006; De Navas, 2006; Makhijani, 2007). However, over-expression of Dpp, Wg, Vestigial (Vg) or Vein (Vn) provides only marginal growth advantage to haltere compared to the wildtype. In this context, additional growth regulating pathways amongst the targets of Ubx were examined. Genome wide studies have identified many components of Fat/Hippo and Insulin-insulin like/dAkt signalling (IIS/dAkt) pathways as potential targets of Ubx. The Fat/Hippo pathway is a crucial determinant of organ size in both Drosophila and mammals. It regulates cell proliferation, cell death, and cell fate decisions and coordinates these events to specify organ size. In contrast, the IIS/dAkt pathway is known to regulate cell size (Singh, 2015).

    Recent studies have revealed that the Fat/Hippo pathway networks with other signalling pathways. For example, during wing development, Fat/Hippo pathway activities are dependent on Four-jointed (Fj) and Dachous (Ds) gradients, which are influenced by Dpp, Notch, Wg and Vg. Glypicans, which play a prominent role in morphogen signalling, are regulated by Fat/Hippo signalling (Baena-Lopez, 2008). EGFR activates Yorkie (Yki; effector of Fat/Hippo pathway) through its EGFR-RAS-MAPK signalling by promoting the phosphorylation of Ajuba family protein WTIP (Reddy, 2013). However, EGFR negatively regulates events downstream of Yki (Herranz, 2012). The Fat/Hippo pathway is also known to inhibit EGFR signalling, which makes the interaction between the two pathways very complex and context-dependent. IIS/dAkt pathway is also known to activate Yki signalling and vice-versa. Thus, Fat/Hippo pathway may specify organ size by regulating both cell number (directly) and cell size (via regulating IIS/dAkt pathway) (Singh, 2015).

    This study reports studies on the functional implication of regulation of Fat/Hippo and IIS/dAkt pathways by Ubx in specifying haltere development. Over-expression of Yki or down regulation of negative components of the Fat/Hippo pathway, such as expanded (ex), induced considerable increase in haltere size, mainly due to increase in cell number. Although activation of dAkt alone did not affect the organ size or the cell size, activation of Yki or down regulation of ex in the background of over-expressed dAkt caused dramatic increase in haltere size, much severe than Yki or ex alone. In this background, increase was observed in both cell size and cell number. The resulted haltere appeared flatter than wildtype haltere and the morphology of trichomes and their spacing resembled that of wing suggesting homeotic transformations. Thus, these results suggest a link between cellular growth and pattern formation and the final differentiated state of the organ (Singh, 2015).

    The findings suggest that, downstream of Ubx, the Fat/Hippo pathway is critical for haltere specification. It is required for Ubx-mediated specification of organ size, sensory bristle repression, trichome morphology and arrangement. The Fat/Hippo pathway cooperates with the IIS/dAkt pathway, which is also a target of Ubx, in specifying cell size and compartment size in developing haltere. The fact that over-expression of Yki or downregulation of ex show haltere-to-wing transformations at the levels of organ size and shape, and trichome morphology and arrangement, suggest that regulation of the Fat/Hippo pathway by Ubx is central to the modification of wing identity to that of the haltere (Singh, 2015).

    The observations made in this study pose new questions and suggest various interesting possibilities to study the Fat/Hippo pathway with a new perspective.

    (1) It was observed that while Yki is nuclear in haltere discs, it appears to be non-functional. Yki is a transcriptional co-activator protein, which requires other DNA-binding partners for its activity. In this context, understanding the precise relationship between Yki and Ubx may provide an insight into mechanism of haltere specification (Singh, 2015).

    (2) The Fat/Hippo pathway (along with the IIS/dAkt pathway) may be involved in the specification of cell size, trichome morphology and their arrangement, all of which are important parameters in determining organ morphology. Recent studies indicate that the Fat/Hippo pathway regulates cellular architecture and the mechanical properties of cells in response to the environment. It would be interesting to study the role of the Fat/Hippo pathway in regulating the cytoskeleton of epithelial cells during development. Haltere cells at pupal stages exhibit higher levels of F-actin than wing cells. One possible mechanism that is currently being investigated is lowering of F-actin levels in transformed haltere cells due to over-expression of Yki or down regulation of ex. This may cause flattening of cells during morphogenesis leading to larger organ size (Singh, 2015).

    (3) Reversing cell size and number was sufficient to induce homeotic transformations at the level of haltere morphology. This suggests the importance of negative regulation of genetic mechanisms that determine cell size and number, in specifying an organ size and shape. As a corollary, Ubx-mediated regulation of Fat/Hippo and IIS/dAkt pathways provides an opportunity to study cooperative repression of cell number and cell size during organ specification (Singh, 2015).

    (4) Certain genetic backgrounds investigated in this study showed severe effect on cell proliferation in haltere discs than in wing discs. This could be due to the fact that, the wing disc has already attained a specific size by the third instar larval stage (the developmental stage examined in this study), which is controlled by several pathways. Any change to this size may need more drastic alteration to the controlling mechanisms. As Ubx specifies haltere by modulating various wing-patterning events, there may still exist a certain degree of plasticity in mechanisms that determine the size of the haltere. However, in absolute terms, the haltere is also resistant to changes in growth control due to regulation by Ubx at multiple levels. Thus, differential development of wing and haltere provides a very good assay system to study not only growth control, but also to dissect out function of important growth regulators (tumour suppressor pathways) such as the Fat/Hippo pathway using various genome-wide approaches (Singh, 2015).

    The Gyc76C receptor Guanylyl cyclase and the Foraging cGMP-dependent kinase regulate extracellular matrix organization and BMP signaling in the developing wing of Drosophila melanogaster

    The developing crossveins of the wing of Drosophila melanogaster are specified by long-range BMP signaling and are especially sensitive to loss of extracellular modulators of BMP signaling such as the Chordin homolog Short gastrulation (Sog). However, the role of the extracellular matrix in BMP signaling and Sog activity in the crossveins has been poorly explored. Using a genetic mosaic screen for mutations that disrupt BMP signaling and posterior crossvein development, this study has identified Gyc76C, a member of the receptor guanylyl cyclase family that includes mammalian natriuretic peptide receptors. Gyc76C and the soluble cGMP-dependent kinase Foraging, likely linked by cGMP, are necessary for normal refinement and maintenance of long-range BMP signaling in the posterior crossvein. This does not occur through cell-autonomous crosstalk between cGMP and BMP signal transduction, but likely through altered extracellular activity of Sog. This study identified a novel pathway leading from Gyc76C to the organization of the wing extracellular matrix by matrix metalloproteinases and shows that both the extracellular matrix and BMP signaling effects are largely mediated by changes in the activity of matrix metalloproteinases. Parallels and differences between this pathway and other examples of cGMP activity in both Drosophila melanogaster and mammalian cells and tissues are discussed (Schleede, 2015).

    The vein cells that develop from the ectodermal epithelia of the Drosophila melanogaster wing are positioned, elaborated and maintained by a series of well-characterized intercellular signaling pathways. The wing is easily visualized, and specific mutant vein phenotypes have been linked to changes in specific signals, making the wing an ideal tissue for examining signaling mechanisms, for identifying intracellular and extracellular crosstalk between different pathways, and for isolating new pathway components (Schleede, 2015).

    The venation defect, the loss of the posterior crossvein (PCV), is used to identify and characterize participants in Bone Morphogenetic Protein (BMP) signaling. The PCV is formed during the end of the first day of pupal wing development, well after the formation of the longitudinal veins (LVs, numbered L1-L6), and requires localized BMP signaling in the PCV region between L4 and L5. Many of the homozygous viable crossveinless mutants identified in early genetic screens have now been shown to disrupt direct regulators of BMP signaling, especially those that bind BMPs and regulate BMP movement and activity in the extracellular space. The PCV is especially sensitive to loss of these regulators because of the long range over which signaling must take place, and the role many of these BMP regulators play in the assembly or disassembly of a BMP-carrying 'shuttle' (Schleede, 2015).

    The BMP Decapentaplegic (Dpp) is secreted by the pupal LVs, possibly as a heterodimer with the BMP Glass bottom boat (Gbb). This stimulates autocrine and short-range BMP signaling in the LVs that is relatively insensitive to extracellular BMP regulators. However, Dpp and Gbb also signal over a long range by moving into the intervein tissues where the PCV forms. In order for this to occur, the secreted BMPs must bind the D. melanogaster Chordin homolog Short gastrulation (Sog) and the Twisted gastrulation family member Crossveinless (Cv, termed here Cv-Tsg2 to avoid confusion with other 'Cv' gene names). The Sog/Cv-Tsg2 complex facilitates the movement of BMPs from the LVs through the extracellular space, likely by protecting BMPs from binding to cell bound molecules such as their receptors. In order to stimulate signaling in the PCV, BMPs must also be freed from the complex. The Tolloid-related protease (Tlr, also known as Tolkin) cleaves Sog, lowering its affinity for BMPs, and Tsg family proteins help stimulate this cleavage. Signaling is further aided in the PCV region by a positive feedback loop, as BMP signaling increases localized expression of the BMP-binding protein Crossveinless 2 (Cv-2, recently renamed BMPER in vertebrates). Cv-2 also binds Sog, cell surface glypicans and the BMP receptor complex, and likely acts as a co-receptor and a transfer protein that frees BMPs from Sog. The lipoprotein Crossveinless-d (Cv-d) also binds BMPs and glypicans and helps signaling by an unknown mechanism (Schleede, 2015).

    PCV development takes place in a complex and changing extracellular environment, but while there is some evidence that PCV-specific BMP signaling can be influenced by changes in tissue morphology or loss of the cell-bound glypican heparan sulfate proteoglycans, other aspects of the environment have not been greatly investigated. During the initial stages of BMP signaling in the PCV, at 15-18 hours after pupariation (AP), the dorsal and ventral wing epithelia form a sack that retains only a few dorsal to ventral connections from earlier stages; the inner, basal side of the sack is filled with extracellular matrix (ECM) proteins, both diffusely and in laminar aggregates. As BMP signaling in the PCV is maintained and refined, from 18-30 hours AP, increasing numbers of dorsal and ventral epithelial cells adhere, basal to basal, flattening the sack. The veins form as ECM-filled channels between the two epithelia, while in intervein regions scattered pockets of ECM are retained basolaterally between the cells within each epithelium; a small amount of ECM is also retained at the sites of basal-to-basal contact. This changing ECM environment could potentially alter BMP movement, assembly of BMP-containing complexes, and signal reception, as has been demonstrated in other developmental contexts in Drosophila (Schleede, 2015).

    This study demonstrates the strong influence of the pupal ECM on PCV-specific long-range BMP signaling, through the identification of a previously unknown ECM-regulating pathway in the wing. In a screen conducted for novel crossveinless mutations on the third chromosome, a mutation was found in the guanylyl cyclase at 76C (gyc76C) locus, which encodes one of five transmembrane, receptor class guanylyl cyclases in D. melanogaster. Gyc76C has been previously characterized for its role in Semaphorin-mediated axon guidance; Malpighian tubule physiology, and the development of embryonic muscles and salivary glands. Like the similar mammalian natriuretic peptide receptors NPR1 and NPR2, the guanylyl cyclase activity of Gyc76C is likely regulated by secreted peptides, and can act via a variety of downstream cGMP sensors (Schleede, 2015).

    The evidence suggests that Gyc76C influences BMP signaling in the pupal wing by changing the activity of the cGMP-dependent kinase Foraging (For; also known as Dg2 or Pkg24A), also a novel role for this kinase. But rather than controlling BMP signal transduction in a cell-autonomous manner, evidence is provided that Gyc76C and Foraging regulate BMP signaling non-autonomously by dramatically altering the wing ECM during the period of BMP signaling in the PCV. This effect is largely mediated by changing the levels and activity of matrix metalloproteinases (Mmps), especially Drosophila Mmp2. Genetic interactions suggest that the ECM alterations affect the extracellular mobility and activity of the BMP-binding protein Sog (Schleede, 2015).

    This provides the first demonstration of Gyc76C and For activity in the developing wing, and the first evidence these proteins can act by affecting Mmp activity. Moreover, the demonstration of in vivo link from a guanylyl cyclase to Mmps and the ECM, and from there to long-range BMP signaling, may have parallels with findings in mammalian cells and tissues. NPR and NO-mediated changes in cGMP activity can on the one hand change matrix metalloproteinase expression secretion and activity, and on the other change in BMP and TGFβ signaling (Schleede, 2015).

    Mutation 3L043, uncovered by a genetic screen to identify homozygous lethal mutations required for PCV development, is a novel allele of gyc76C, a transmembrane peptide receptor that, like vertebrate NPRs, acts as a guanylyl cyclase. gyc76C is likely linked by cGMP production to the activity of the cGMP-dependent kinase For, and that Gyc76C and For define a new pathway for the regulation of wing ECM. This pathway appears to act largely through changes in the activity of ECM-remodeling Mmp enzymes. Loss of gyc76C or For alter both the organization of the wing ECM and the levels of the two D. melanogaster Mmps, and the gyc76C knockdown phenotype can be largely reversed by knockdown of Mmp2. This is the first indication of a role for cGMP, Gyc76C and For function in the developing wing, and their effects on the ECM provides a novel molecular output for each (Schleede, 2015).

    Gyc76C and For are necessary for the normal refinement and maintenance of long-range BMP signaling in the posterior crossvein region of the pupal wing; in fact, crossvein loss is the most prominent aspect of the adult gyc76C knockdown phenotype. The evidence suggests that this effect is also mediated by changes in Mmp activity, and most likely the Mmp-dependent reorganization of the ECM. In fact, analysis using genetic mosaics finds no evidence for a reliable, cell autonomous effect of cGMP activity on BMP signal transduction in the wing. Thus, this apparent crosstalk between receptor guanylyl cyclase activity and BMP signaling in the wing is mediated by extracellular effects (Schleede, 2015).

    It is noteworthy that the cGMP activity mediated by NPR or nitric oxide signaling can change also Mmp gene expression, secretion or activation in many different mammalian cells and tissues. Both positive and negative effects have been noted, depending on the cells, the context, and the specific Mmp. Given the strong role of the ECM in cell-cell signaling, the contribution of cGMP-mediated changes in Mmp activity to extracellular signaling may be significant. There is also precedent for cGMP activity specifically affecting BMP and TGFβ signaling in mammals. cGMP-dependent kinase activity increases BMP signaling in C2C12 cells, and this effect has been suggested to underlie some of the effects of nitric oxide-induced cGMP on BMP-dependent pulmonary arterial hypertension. Conversely, atrial natriuretic peptide stimulates the guanylyl cyclase activities of NPR1 and NPR2 and can inhibit TGFβ activity in myofibroblasts; this inhibition has been suggested to underlie the opposing roles of atrial natriuretic peptide and TGFβ during hypoxia-induced remodeling of the pulmonary vasculature. However, unlike the pathway observed in the fly wing, these mammalian effects are thought to be mediated by the intracellular modulation of signal transduction, with cGMP-dependent kinases altering BMP receptor activity or the phosphorylation and nuclear accumulation of receptor-activated Smads. Nonetheless, it remains possible that there are additional layers of regulation mediated through extracellular effects, underscoring the importance of testing cell autonomy (Schleede, 2015).

    Aside from its role in adult Malpighian tubule physiology, Gyc76C was previously shown to have three developmental effects: in the embryo it regulates the repulsive axon guidance mediated by Semaphorin 1A and Plexin A, the proper formation and arrangement of somatic muscles, and lumen formation in the salivary gland. All these may have links to the ECM. Loss of gyc76C from embryonic muscles affects the distribution and vesicular accumulation of the βintegrin Mys, and reduces laminins and the integrin regulator Talin in the salivary gland. The axon defects likely involve a physical interaction between Gyc76C and semaphorin receptors that affects cGMP levels; nonetheless, gyc76C mutant axon defects are very similar to those caused by loss of the perlecan Trol (Schleede, 2015).

    The parallels between the different contexts of Gyc76C action are not exact, however. First, only the wing phenotype has been linked to a change in Mmp activity. Second, unlike the muscle phenotype, the wing phenotype is not accompanied by any obvious changes in integrin levels or distribution, beyond those caused by altered venation. Finally, most gyc76C mutant phenotypes are reproduced by loss of the Pkg21D (Dg1) cytoplasmic cGMP-dependent kinase, instead of For (Dg2, Pkg24A) as found in the wing, and thus may be mediated by different kinase targets (Schleede, 2015).

    For has been largely analyzed for behavioral mutant phenotypes, and the overlap between Pkg21D and For targets is unknown. While many targets have been identified for the two mammalian cGMP-dependent kinases, PRKG1 (which exists in alpha and beta isoforms) and PRKG2, it is not clear if either of these is functionally equivalent to For. One of the protein isoforms generated by the for locus has a putative protein interaction/dimerization motif with slight similarity to the N-terminal binding/dimerization domains of alpha and beta PRKG1, but all three For isoforms have long N-terminal regions that are lacking from PRKG1 and PRKG2. In fact, a recent study suggested that For is instead functionally equivalent to PRKG2: Like PRKG2, For can stimulate phosphorylation of FOXO, and is localized to cell membranes in vitro. But For apparently lacks the canonical myristoylation site that is thought to account for the membrane localization and thus much of the target specificity of PRKG2. FOXO remains the only identified For target, and foxo null mutants are viable with normal wings (Schleede, 2015).

    The loss of long range BMP signaling in the PCV region caused by knockdown of gyc76C can, like the ECM, be largely rescued by knockdown of Mmp2. Two results suggest that it is the alteration to the ECM that affects long-range BMP signaling, rather than some independent effect of Mmp2. First, the BMP signaling defects caused by gyc76C knockdown were rescued by directly manipulating the ECM through the overexpression of the perlecan Trol. Second, when Mmp activity is inhibited by overexpression of the diffusible Mmp inhibitor TIMP, this not only rescued the PCV BMP signaling defects caused by gyc76C knockdown, but also led to ectopic BMP signaling, not throughout the region of TIMP expression, but only in those regions with abnormal accumulation of ECM (Schleede, 2015).

    The Mmp2-mediated changes in the ECM likely affect long-range BMP signaling by altering the activity of extracellular BMP-binding proteins, particularly Sog. The BMPs Dpp and Gbb produced in the LVs bind Sog and Cv-Tsg2, shuttle into the PCV region, and are released there by Tlr-mediated cleavage of Sog and transfer to Cv-2 and the receptors. Genetic interaction experiments suggest that knockdown of gyc76C both increases Sog's affinity for BMPs and reduces the movement of the Sog/Cv-Tsg2/BMP complex into the crossvein region (Schleede, 2015).

    Collagen IV provides the best-studied example for how the ECM might affect Sog activity. The two D. melanogaster collagen IV chains regulate BMP signaling in other contexts, and they bind both Sog and the BMP Dpp. Results suggest that collagen IV helps assemble and release a Dpp/Sog/Tsg shuttling complex, and also recruits the Tld protease that cleaves Sog cleavage and releases Dpp for signaling. D. melanogaster Mmp1 can cleave vertebrate Collagen IV. Since reduced Gyc76C and For activity increases abnormal Collagen IV aggregates throughout the wing and diffuse Collagen IV in the veins, it ishypothesized that these Collagen IV changes both foster the assembly or stability of Sog/Cv-Tsg2/BMP complexes and tether them to the ECM, favoring the sequestration of BMPs in the complex and reducing thelong-range movement of the complex into the region of the PCV (Schleede, 2015).

    While few other D. melanogaster Mmp targets have been identified, it is likely that Mmp1 and Mmp2 share the broad specificity of their mammalian counterparts, so other ECM components, known or unknown, might be involved. For instance, vertebrate Perlecan and can be cleaved by Mmps. Trol regulates BMP signaling in other D. melanogaster contexts, and Trol overexpression rescue gyc76C knockdown's effects on BMP signaling. But while null trol alleles are lethal before pupal stages, normal PCVs were formed in viable and even adult lethal alleles like trolG0023, and actin-Gal 4-driven expression of trol-RNAi using any of four different trol-RNAi lines did not alter adult wing venation. Loss of the D. melanogaster laminin B chain shared by all laminin trimers strongly disrupts wing venation, and a zebrafish laminin mutation can reduce BMP signaling (Schleede, 2015).

    Finally, it was recently shown that Dlp, one of the two D. melanogaster glypicans, can be removed from the cell surface by Mmp2. While gyc76C knockdown did not detectably alter anti-Dlp staining in the pupal wing, it is noteworthy that Dlp and the second glypican Dally are required non-autonomously for BMP signaling in the PCV and that they bind BMPs and other BMP-binding proteins.

    A local difference in Hedgehog signal transduction increases mechanical cell bond tension and biases cell intercalations along the Drosophila anteroposterior compartment boundary

    Tissue organization requires the interplay between biochemical signaling and cellular force generation. The formation of straight boundaries separating cells with different fates into compartments is important for growth and patterning during tissue development. In the developing Drosophila wing disc, maintenance of the straight anteroposterior (AP) compartment boundary involves a local increase in mechanical tension at cell bonds along the boundary. The biochemical signals that regulate mechanical tension along the AP boundary, however, remain unknown. This study shows that a local difference in Hedgehog signal transduction activity between anterior and posterior cells is necessary and sufficient to increase mechanical tension along the AP boundary. This difference in Hedgehog signal transduction is also required to bias cell rearrangements during cell intercalations to keep the characteristic straight shape of the AP boundary. Moreover, severing cell bonds along the AP boundary does not reduce tension at neighboring bonds, implying that active mechanical tension is upregulated, cell bond by cell bond. Finally, differences in the expression of the homeodomain-containing protein Engrailed also contribute to the straight shape of the AP boundary, independently of Hedgehog signal transduction and without modulating cell bond tension. The data reveal a novel link between local differences in Hedgehog signal transduction and a local increase in active mechanical tension of cell bonds that biases junctional rearrangements. The large-scale shape of the AP boundary thus emerges from biochemical signals inducing patterns of active tension on cell bonds (Rudolf, 2015).

    This study has analyzed the links between the determination of cell fate and the physical and mechanical mechanisms shaping the AP boundary of larval Drosophila wing discs. Previous work has shown a role for the transcription factors Engrailed and Invected and the Hedgehog signal transduction pathway in organizing the segregation of anterior and posterior cells of the wing disc. This study now shows that a difference in Hedgehog signal transduction between anterior and posterior cells significantly contributes to the straight shape of the AP boundary by autonomously and locally increasing mechanical cell bond tension that in turn biases the asymmetry of cell rearrangements during cell intercalations. Furthermore, Engrailed and Invected also contribute to maintaining the characteristic straight shape of the AP boundary by mechanisms that are independent of Hedgehog signal transduction and do not appear to modulate cell bond tension (Rudolf, 2015).

    In the wild-type wing disc, anterior cells transducing the Hedgehog signal are juxtaposed to posterior cells that do not transduce the Hedgehog signal. Three cases were genereated to test whether this difference in Hedgehog signal transduction is important for the straight shape of the AP boundary, the morphological and molecular signature of cells along the AP boundary, and the local increase in cell bond tension. In case I, Hedgehog signal transduction was low (or absent) in both A and P cells. In case II, Hedgehog signal transduction was high in both A and P cells. And in case III, Hedgehog signal transduction was high in P cells, but low in A cells, reversing the normal situation. In cases I and II the AP boundary was no longer as straight as in the wild-type situation. Moreover, the increased apical cross-section area of cells along the AP boundary that is characteristic for the wild type was no longer seen. Finally, the levels of F-actin and cell bond tension were no longer increased along the AP boundary. In case III, it was found that the difference in Hedgehog signal transduction is sufficient to maintain the characteristic straight shape of the AP boundary, to induce the morphological signatures of cells along the AP boundary and to increase F-actin and mechanical tension. Taken together, these experiments establish that the difference in Hedgehog signal transduction between anterior and posterior cells plays a key role in increasing cell bond tension along the AP boundary, in maintaining the characteristic shape of the AP boundary, and in defining the molecular and morphological signatures of cells along the AP boundary. These findings account for the observation that while Hedgehog signal transduction is active within the strip of anterior cells, the increase in mechanical tension is confined to cell bonds along the AP boundary, where cells with highly different Hedgehog signal transduction activities are apposed. The small differences in Hedgehog signal transduction activity that might exist between neighboring rows of anterior cells in the vicinity of the AP boundary appear to be insufficient to increase cell bond tension. Importantly, Hedgehog signal transduction per se does not increase cell bond tension along the AP boundary. The role of Hedgehog signal transduction along the AP boundary thus differs from its roles during other morphogenetic processes in which all cells that transduce the Hedgehog signal, for example, respond by accumulation of F-actin and a change in shape. It will be interesting to elucidate the molecular mechanisms by which cells perceive a difference in Hedgehog signal transduction, and how such a difference in Hedgehog signal transduction results in increased cell bond tension (Rudolf, 2015).

    F-actin and Myosin II are enriched along the AP boundary. Based on similar observations, the existence of actomyosin cables has been proposed for several compartment boundaries, including the AP boundary in the Drosophila embryonic epidermis, the DV boundary of Drosophila wing discs and the rhombomeric boundaries in zebrafish embryos. Actomyosin cables have been proposed to maintain the straight shape of compartment boundaries by acting as barriers of cell mixing between cells of the adjacent compartments. Actomyosin cables are also characteristic of additional processes, e.g. dorsal closure and germband extension in the Drosophila embryo, tracheal tube invagination and neural plate bending and elongation. During Drosophila germ band extension, it has been shown that mechanical tension is higher at cell bonds that are part of an actomyosin cable compared with isolated cell bonds, indicating that cell bond tension is influenced by higher-order cellular organization during this process. The results, based on laser ablation experiments, show that the increased cell bond tension along the AP boundary can be induced by single cells and does not depend on the integrity of the actomyosin cable. Thus, these data instead indicate that increased cell bond tension is autonomously generated cell bond by cell bond along the AP boundary. This suggests that differences in Hedgehog signal transduction activity regulate the structure and mechanical properties of cell junctions between adjacent cells and in particular upregulate an active mechanical tension, mediated by actomyosin contractility (Rudolf, 2015).

    The cell cortex is a thin layer of active material that is under mechanical tension. In addition to viscous and elastic stresses, active stresses generated by actomyosin contractility are an important contribution. Adherens junctions are adhesive structures that include elements of the cell cortices of the adhering cells. Locally generated active tension, therefore, can largely determine the cell bond tension as long as cell bonds do not change length or rearrange. As a consequence, locally generated active tension also sets the cell bond tension at the actomyosin cable along the AP boundary. This view is consistent with experiments in which cell bond tension remains high even if the integrity of the actomyosin cable is lost. These mechanical properties of cell junctions along the AP boundary are thus different from those of a conventional string or cable in which elastic stresses are associated with stretching deformations. Such elastic stresses relax and largely disappear when the cable is severed. Thus, this work suggests that the mechanical properties of the actomyosin cable along the AP boundary are very different from those of a conventional cable, but fit well in the concepts of active tension studied in the cell cortex, e.g., in Caenorhabditis elegans. This active tension is a local property that can be set by local signals irrespective of the local force balances. Force balances rather determine movements and rearrangements, e.g. upon laser ablation (Rudolf, 2015).

    How does a local increase in actively generated cell bond tension contribute to the straight shape of the AP boundary? Previous work showed that cell intercalations promote irregularities in the shape of compartment boundaries. The local increase in active cell bond tension enters the force balances during cell rearrangements. During cell intercalation, differences in active cell bond tension between junctions along the AP boundary and neighboring junctions are balanced by frictional forces associated with vertex movements. As a result, vertex movements are biased such that the AP boundary remains straight and cell mixing between neighboring compartments is suppressed. The observation that a local difference in Hedgehog signal transduction upregulates active cell bond tension leads to the prediction that cell rearrangements along the AP boundary should not be biased if there is no difference in Hedgehog signal transduction. This is indeed what was found in case II (Rudolf, 2015).

    It has been previously suggested that the engrailed and invected selector genes play a role in maintaining the separation of anterior and posterior cells that is independent of Hedgehog signal transduction. Quantitative analysis of clone shapes in this study supports this notion. It is speculated that this Hedgehog-independent pathway contributes to the remarkably straight shape of the AP boundary in cases I and II, in which Hedgehog signal transduction activities between anterior and posterior cells have been nearly equalized. Two lines of evidence indicate that the Hedgehog-independent pathway shapes the AP boundary without modulating cell bond tension. First,several cases have been generated in which neighboring cell populations differed in the expression of Engrailed and Invected, but not in Hedgehog signal transduction activity. In none of these cases was an increase in cell bond tension detected along the interface of these two cell populations. Second, in cases in which a difference was created in Hedgehog signal transduction between two cell populations in the absence of differences in Engrailed and Invected expression, the same increase was detected in cell bond tension between these cell populations compared with the wild-type compartment boundary (Rudolf, 2015).

    Previously studies have described several physical mechanisms that shape the DV boundary of wing discs. In addition to a local increase in mechanical tension along the DV boundary, evidence was provided that oriented cell division and cell elongation created by anisotropic stress contribute to the characteristic shape of the DV boundary. It is therefore conceivable that the Hedgehog-independent pathway influences the shape of the AP boundary by one or more of these mechanisms (Rudolf, 2015).

    It is proposed that the AP boundary is shaped by mechano-biochemical processes that integrate signaling pathways with patterns of cell mechanical properties. In tjos model, Engrailed and Invected shape the AP boundary with the help of two different mechanisms. (1) Engrailed and Invected result in a difference in Hedgehog signal transduction between anterior and posterior cells. This difference leads to a cell-autonomous increase in F-actin and active cell bond tension along the AP boundary. The local increase in active cell bond tension then biases the asymmetry of cell rearrangements during cell intercalations and thereby contributes to maintaining the straight shape of the AP boundary. (2) Engrailed and Invected contribute independently of Hedgehog signal transduction to the straight shape of the AP boundary by an as yet unknown mechanism not involving the modulation of cell bond tension. The first mechanism uses biochemical signals to create mechanical patterns that subsequently guide junctional dynamics to organize a straight compartment boundary. It is speculated that the second mechanism also involves a mechano-chemical process, even though the nature of this process is currently unknown. The current work suggests that the large-scale shape of the AP boundary thus emerges from the collective behavior of many cells that locally exchange biochemical signals and regulate active mechanical tension (Rudolf, 2015).

    Establishment of a developmental compartment requires interactions between three synergistic cis-regulatory modules

    The subdivision of cell populations in compartments is a key event during animal development. In Drosophila, the gene apterous (ap) divides the wing imaginal disc in dorsal vs ventral cell lineages and is required for wing formation. ap function as a dorsal selector gene has been extensively studied. However, the regulation of its expression during wing development is poorly understood. This study analyzed ap transcriptional regulation at the endogenous locus and identified three cis-regulatory modules (CRMs) essential for wing development. Only when the three CRMs are combined, robust ap expression is obtained. In addition, the trans-factors that regulate these CRMs were genetically and molecularly analyzed. The results propose a three-step mechanism for the cell lineage compartment expression of ap that includes initial activation, positive autoregulation and Trithorax-mediated maintenance through separable CRMs (Bieli, 2015).

    Genetic and cis-regulatory analysis has provided information about the logic of ap expression during wing development. It is proposed that ap expression is controlled by at least three CRMs that act in combination. The first element, apE is the earliest to be activated in proximal wing disc cells via the EGFR pathway; its expression subsequently weakens in the wing pouch. Deletion of this early enhancer (e.g., apDG12 or apC1345) completely abolishes wing formation. The asymmetry of ap expression to the proximal domain of the wing disc is probably due to the localized activation of the EGFR pathway by its ligand Vn and a distal repression by Wg signaling. The initial activation of the apE by the EGFR pathway was genetically and molecularly confirmed; however, other inputs are required for the continuous activation of this CRM in later wing discs (Bieli, 2015).

    A few hours after apE activation, a second CRM, apDV, is activated in a subset of apE positive cells. In contrast to apE, apDV is restricted to the dorsal-distal domain of the wing pouch by direct positive inputs from Ap and Vg/Sd. The direct Ap autoregulatory input defines the time window when the apDV element is activated; apDV can only be active after the induction of Ap by the early enhancer (apE). It has been shown that Ap induces vg expression by triggering Notch signaling at the D/V boundary. Thus, the (direct) input of Vg/Sd on apDV can be regarded as an indirect positive autoregulation, which delimits the spatial domain where apDV can be actived. Consequently, the interface of Ap and Vg expression defines the region of apDV activity via positive autoregulation (Bieli, 2015).

    The third ap CRM is the ap PRE/TRE region (apP), that, when deleted, leads to a strong hypomorphic wing phenotype (apc1.2b). The apP requires Trx input and maintains ap expression when placed in cis with the apDV and apE CRMs. Only the combination of the three CRMs faithfully reproduces ap expression in the wing disc. Moreover, the regulatory in locus deletion and in situ rescue analysis provide strong functional relevance for these CRMs (Bieli, 2015).

    Ultimately, this cascade of ap CRMs provides a mechanism to initiate, refine and maintain ap expression during wing imaginal disc development, in which the later CRMs depend on the activity of the early ones. A similar mechanism has been described for Distal-less (Dll) regulation in the leg primordia where separate CRMs trigger and maintain Dll expression in part by an autoregulatory mechanism (Bieli, 2015).

    It has been proposed that positive autoregulation may help to maintain the epigenetic memory of differentiation. In the case of ap, this study demonstrates that autoregulation works in conjunction with a PRE/TRE system; this might make the system very robust and refractory to perturbations (Bieli, 2015).

    ChIP experiments have shown that many developmentally important genes are associated with a promoter proximal PRE as found at ap. The role of such a PRE has been studied at the engrailed (en) locus. It has been demonstrated that in imaginal discs, the promoter as well as the promoter proximal PRE are important for the long-range action of en enhancers. It has been proposed that this PRE brings chromatin together, allowing both positive and negative regulatory interactions between distantly located DNA fragments (Bieli, 2015).

    The current results indicate that sequences around the transcription start of ap (apP) may serve a similar function. First, this element, when placed in cis with the ap CRMs (apE and apDV), maintains the ap expression pattern and keeps reporter gene expression off in cells where low or no activity of apDV and apE has been observed. Second, in the absence of trx, the expression of ap and apDV+E+P-lacZ is strongly reduced. All these data suggest that sequences within the apP integrate Trx input, thereby maintaining ap expression in a highly proliferative tissue such as the wing disc. Interestingly, trx mutant clones were not round and did not show ectopic wg activation, which is a hallmark of ap loss-of-function clones. This suggests that in trx mutant clones enough Ap protein is still present to maintain wg expression off. However, derepression of the ventral-specific integrin αPS2 was found in trx mutant clones in the wing pouch as previously described for ap mutant clones (Bieli, 2015).

    It has been suggested that TrxG proteins could act passively antagonizing PcG silencing, rather than playing an active role as co-activators of gene transcription. For example, Ubx expression in the leg and haltere does not require Trx in the absence of Polycomb repression. These possibilities were tested and trx mutant clones were generated that were also mutant for the PcG member Sex combs on midlegs (Scm). Dorsally-located Scm- trx- double mutant clones still downregulate ap-lacZ expression while ventral-induced ones are unable to derepress ap-lacZ as was observed for Scm- single mutant clones. Therefore, the results suggest that TrxG maintains ap expression in dorsal cells, while ap expression is repressed in the ventral compartment by PcG proteins. Moreover, it has been shown that the sequences around the ap transcription start, including the PRE, are occupied by PcG complexes PRC1 and PRC2, as well as Trx (Bieli, 2015).

    Enhancers-promoter interactions initiate transcription but their dynamics during development have remained poorly understood. A Chromosome conformation capture (3C) experiment provides evidence for the direct interaction between the ap CRMs apE and apDV with the maintenance element encoded by the apP. Beyond this, it was also found that these elements cooperate continuously during wing development. Flip-out experiments, in which the apDV and apE CRMs were removed at different time points, suggest that these elements need to be present continuously to ensure correct ap expression. Additionally, flies carrying apE only on one chromosome and apDV only on the homologue were unable to fully rescue wing development suggesting that these CRMs need to be in cis. It is conceivable that in cis configuration of the three ap CRMs facilitates and stabilizes enhancer-promoter looping. It could also help to rapidly establish relevant chromatin contacts after each cell division. These results are in accordance with previous observations, in which constant interactions between ap enhancers and promoter during embryogenesis have been described. The current results extend these observations to the wing disc, a highly proliferative tissue, where the expression of the trans-factors that regulate the activity of the apE and apDV is very dynamic. This raises the question on how this contact is re-assembled over many cell generations. It is possible that some epigenetic modifications are laid down in the activated apE and apDV CRMs, which are then inherited during cell divisions to ensure contact with apP. Studies of the chromatin status of these elements will be required to fully understand this process (Bieli, 2015).

    A key question in developmental biology is how transcriptional regulation is coupled to tissue growth to precisely regulate gene expression in a spatio-temporal manner. For example, during Drosophila leg development, initial activation of the ventral appendage gene Dll by high levels of Wg and Dpp initiates a cascade of cross-regulation between Dll and Dachshund (Dac) and positive feedback loops that patterns the proximo-distal axis. Other mechanisms to expand gene expression patterns depend on memory modules such as PREs, as it is the case for the Hox genes or other developmental genes like hh. To direct wing formation, expression of ap in the highly proliferative tissue of the wing disc must be precisely induced to generate and maintain the D/V border. These in-depth analyses at the ap locus provide a functional and molecular explanation of how expression of this dorsal selector gene is initiated, refined at the D/V border, and maintained during wing disc development. It is proposed that this three-step mechanism may be common for developmental patterning genes to make the developmental program robust to perturbations (Bieli, 2015).

    Curly encodes Dual Oxidase, which acts with Heme Peroxidase Curly Su to shape the adult Drosophila wing

    Curly, described almost a century ago, is one of the most frequently used markers in Drosophila genetics. Despite this the molecular identity of Curly has remained obscure. This study shows that Curly mutations arise in the gene dual oxidase (duox), which encodes a reactive oxygen species (ROS) generating NADPH oxidase. Using Curly mutations and RNA interference (RNAi), this study demonstrated that Duox autonomously stabilizes the wing on the last day of pupal development. Through genetic suppression studies, this study identified a novel heme peroxidase, Curly Su (Cysu; CG5873) that acts with Duox to form the wing. Ultrastructural analysis suggests that Duox and Cysu are required in the wing to bond and adhere the dorsal and ventral cuticle surfaces during its maturation. In Drosophila, Duox is best known for its role in the killing of pathogens by generating bactericidal ROS. This work adds to a growing number of studies suggesting that Duox's primary function is more structural, helping to form extracellular and cuticle structures in conjunction with peroxidases (Hurd, 2015).

    Decanalization of wing development accompanied the evolution of large wings in high-altitude Drosophila

    In higher organisms, the phenotypic impacts of potentially harmful or beneficial mutations are often modulated by complex developmental networks. Stabilizing selection may favor the evolution of developmental canalization-that is, robustness despite perturbation-to insulate development against environmental and genetic variability. In contrast, directional selection acts to alter the developmental process, possibly undermining the molecular mechanisms that buffer a trait's development, but this scenario has not been shown in nature. This study examined the developmental consequences of size increase in highland Ethiopian Drosophila melanogaster. Ethiopian inbred strains exhibited much higher frequencies of wing abnormalities than lowland populations, consistent with an elevated susceptibility to the genetic perturbation of inbreeding. Mutagenesis was then used to test whether Ethiopian wing development is, indeed, decanalized. Ethiopian strains were far more susceptible to this genetic disruption of development, yielding 26 times more novel wing abnormalities than lowland strains in F2 males. Wing size and developmental perturbability cosegregated in the offspring of between-population crosses, suggesting that genes conferring size differences had undermined developmental buffering mechanisms. These findings represent the first observation of morphological evolution associated with decanalization in the same tissue, underscoring the sensitivity of development to adaptive change (Lack, 2016).

    The gene expression program for the formation of wing cuticle in Drosophila

    The cuticular exoskeleton of insects and other arthropods is a remarkably versatile material with a complex multilayer structure. This study isolated cuticle synthesizing cells in relatively pure form by dissecting pupal wings and used RNAseq to identify genes expressed during the formation of the adult wing cuticle. Dramatic changes in gene expression during cuticle deposition were observed, and combined with transmission electron microscopy, candidate genes for the deposition of the different cuticular layers were identified. Among genes of interest that dramatically change their expression during the cuticle deposition program are ones that encode cuticle proteins, ZP domain proteins, cuticle modifying proteins and transcription factors, as well as genes of unknown function. A striking finding is that mutations in a number of genes that are expressed almost exclusively during the deposition of the envelope (the thin outermost layer that is deposited first) result in gross defects in the procuticle (the thick chitinous layer that is deposited last). An attractive hypothesis to explain this is that the deposition of the different cuticle layers is not independent with the envelope instructing the formation of later layers. Alternatively, some of the genes expressed during the deposition of the envelope could form a platform that is essential for the deposition of all cuticle layers (Sobala, 2016).

    Tissue nonautonomous effects of fat body methionine metabolism on imaginal disc repair in Drosophila

    Regulatory mechanisms for tissue repair and regeneration within damaged tissue have been extensively studied. However, the systemic regulation of tissue repair remains poorly understood. To elucidate tissue nonautonomous control of repair process, it is essential to induce local damage, independent of genetic manipulations in uninjured parts of the body. This study developed a system in Drosophila for spatiotemporal tissue injury using a temperature-sensitive form of diphtheria toxin A domain driven by the Q system to study factors contributing to imaginal disc repair. Using this technique, it was demonstrated that methionine metabolism in the fat body, a counterpart of mammalian liver and adipose tissue, supports the repair processes of wing discs. Local injury to wing discs decreases methionine and S-adenosylmethionine, whereas it increases S-adenosylhomocysteine in the fat body. Fat body-specific genetic manipulation of methionine metabolism results in defective disc repair but does not affect normal wing development. The data indicate the contribution of tissue interactions to tissue repair in Drosophila, as local damage to wing discs influences fat body metabolism, and proper control of methionine metabolism in the fat body, in turn, affects wing regeneration (Kashio, 2016).

    Proprioceptive feedback determines visuomotor gain in Drosophila

    Multisensory integration is a prerequisite for effective locomotor control. In aerial performance of flies, continuous visual signalling from the compound eyes is fused with phasic proprioceptive feedback to ensure precise neural activation of wing steering muscles (WSM) within narrow temporal phase bands of the stroke cycle. This phase-locked activation relies on mechanoreceptors distributed over wings and gyroscopic halteres. This study investigated visual steering performance of tethered flying fruit flies with reduced haltere and wing feedback signalling. Using a flight simulator, visual object fixation behaviour, optomotor altitude control and saccadic escape reflexes were evaluated. The behavioural assays show an antagonistic effect of wing and haltere signalling on visuomotor gain during flight. Compared with controls, suppression of haltere feedback attenuates while suppression of wing feedback enhances the animal's wing steering range. The results suggest that the generation of motor commands owing to visual perception is dynamically controlled by proprioception. Collectively, the findings contribute to a general understanding how moving animals integrate sensory information with dynamically changing temporal structure (Bartussek, 2016).

    Functional gustatory role of chemoreceptors in Drosophila wings

    Neuroanatomical evidence argues for the presence of taste sensilla in Drosophila wings; however, the taste physiology of insect wings remains hypothetical, and a comprehensive link to mechanical functions, such as flight, wing flapping, and grooming, is lacking. This study shows that the sensilla of the Drosophila anterior wing margin respond to both sweet and bitter molecules through an increase in cytosolic Ca2+ levels. Conversely, genetically modified flies presenting a wing-specific reduction in chemosensory cells show severe defects in both wing taste signaling and the exploratory guidance associated with chemodetection. In Drosophila, the chemodetection machinery includes mechanical grooming, which facilitates the contact between tastants and wing chemoreceptors, and the vibrations of flapping wings that nebulize volatile molecules as carboxylic acids. Together, these data demonstrate that the Drosophila wing chemosensory sensilla are a functional taste organ and that they may have a role in the exploration of ecological niches (Raad, 2016).

    Intra-organ growth coordination in Drosophila is mediated by systemic ecdysone signaling

    In developing Drosophila, perturbing the growth of one imaginal disc - the parts of a holometabolous larva that become the external adult organs - has been shown to retard growth of other discs and delays development, resulting in tight inter-organ growth coordination and the generation of a correctly proportioned adult. This study used the wing imaginal disc in Drosophila to study and identify mechanisms of intra-organ growth coordination. Larvae were generated in which the two compartments of the wing imaginal disc have ostensibly different growth rates (wild-type or growth-perturbed). It was found that there is tightly coordinated growth between the wild-type and growth-perturbed compartments, where growth of the wild-type compartment is retarded to match that of the growth-perturbed compartment. Crucially, this coordination is disrupted by application of exogenous 20-hydroxyecdysone (20E), which accelerates growth of the wild-type compartment. The role of 20E signaling in growth coordination was further elucidate by showing that in wild-type discs, compartment-autonomous up-regulation of 20E signaling accelerates compartment growth and disrupts coordination. Interestingly, growth acceleration through exogenous application of 20E is inhibited with suppression of the Insulin/Insulin-like Growth Factor Signaling (IIS) pathway. This suggests that an active IIS pathway is necessary for ecdysone to accelerate compartment growth. Collectively, these data indicate that discs utilize systemic mechanisms, specifically ecdysone signaling, to coordinate intra-organ growth (Gokhale, 2016).

    The results reveal that growth among developmental compartments in an organ is tightly coordinated, such that even if the growth of one compartment is perturbed, both compartments grow at more-or-less the same relative rate as observed in wild-type flies. This growth coordination between compartments is disrupted by exogenously feeding 20E to growth-perturbed larvae, resulting in acceleration in the growth rate of the unperturbed compartment. This growth acceleration upon feeding 20E is dependent on IIS in the unperturbed compartment. Collectively these data support a model of imaginal disc growth regulation whereby growth perturbation in one compartment causes a systemic reduction in circulating ecdysteroids, which results in reduction in growth rate of the adjacent compartment (Gokhale, 2016).

    These data are surprising in light of previous studies that suggest that imaginal discs and individual compartments within imaginal discs can autonomously grow to their target size. A previous study cultured WT imaginal discs in the abdomen of adults hosts and found that these discs grow autonomously to their normal size. Another study generated 'fast' discs and compartments in M-/+ larvae and demonstrated that these compartments have higher growth rates relative to the body as a whole and to adjacent compartments. It was further demonstrated that the 'fast' compartments and discs are developmentally advanced as compared to M-/+controls. Collectively, these data support the hypothesis that imaginal disc possesses an autonomous mechanism for arresting growth once they reach a target size, and that this mechanism operates at the level of developmental compartments. Whilst compartments may possess a target size, the current data suggest that they do not grow independently to this size, at least in vivo. Rather growth between developmental compartments is coordinated even when one compartment is growth perturbed, and this growth coordination appears to be regulated by systemic rather than disc-autonomous mechanisms, at least in part (Gokhale, 2016).

    The conclusions are supported by data from Mesquita (2010), who also looked at inter-compartmental growth in the Drosophila wing imaginal disc. They observed that slowing the growth of one compartment non-autonomously slowed the growth of the adjacent compartment. They further demonstrate that the signal from the growth-perturbed compartment is dependent on Drosophila p53. However, they do not elucidate what the signal is. The current results suggest that the signal involves ecdysone. This is surprising given the current understanding of wing imaginal disc growth. Recent models of disc growth suggest that growth of the wing imaginal disc is driven mainly by morphogen gradients formed by the patterning genes Wg, Dpp, and Vg, which drive cellular proliferation within the disc. Recent studies further implicate disc-autonomous mechanisms in regulating the relative size of different compartments within the wing (Ferreira, 2015). The current data show that systemic signaling, mediated by ecdysone, is also critical for regulating growth rates among different parts of the disc (Gokhale, 2016).

    The involvement of ecdysone in intra-organ growth coordination echoes its known role in inter-organ growth coordination. As noted above, growth among organs is tightly coordinated when one organ is growth perturbed-a consequence of the growth-perturbed organ suppressing ecdysone synthesis. Addition of ecdysone to these growth-perturbed larvae is able to rescue the growth rate of undamaged imaginal discs. Ecydsone is however not able to rescue the growth rate of the growth perturbed tissues, most likely because the inherent growth perturbation of these tissues prevents them from responding to ecdysone. Similar to these studies on inter-organ growth coordination, the current data suggest ecdysone is able to rescue the growth rate of wild-type compartments in M-/+larvae, and this is mediated by compartment-autonomous ecdysone signaling (Gokhale, 2016).

    While the current data indicate that ecdysone is an important growth-coordinating signal among developmental compartments, it is unclear precisely which tissue is influencing ecdysone synthesis. It is possible that in larvae with antfast:postslow discs the limitation on ecdysone synthesis might be an autonomous effect of the Minute mutation on the prothoracic gland, since the whole of the rest of the larvae is Minute. However, the data demonstrate that knock-down of RpS3 using engrailed-GAL4, which is not expressed in the prothoracic gland, still retards disc growth. This suggests that the growth coordination mechanism is regulated by a signal from the compartments themselves. As discussed above, in studies where systemic growth is retarded through localized tissue damage, including knock-down of ribosomal proteins, it is the damaged/growth-perturbed tissue itself that inhibits ecdysone synthesis by signaling via dILP8. Therefore, in larvae with antfast:postslow discs, ecdysteroidgenesis could be limited via a dILP8-dependent mechanism. Which compartment is generating a putative dILP8 signal is, however, unclear. dILP8 levels are highest at the L2-L3 transition and decline during L3, before increasing somewhat before pupariation. It is possible, therefore, that in larvae with antfast:postslow discs, it is the immature slow-growing posterior compartment that is secreting dILP8. Conversely, the residual generation and death of M-/- cells in the anterior compartment through mitotic recombination early in L3 may also drive dILP8 synthesis. Further experiments exploring the role of dILP8 in intra-organ growth coordination are clearly necessary (Gokhale, 2016).

    A key feature of growth coordination is that ecdysone acts as a promoter of growth for imaginal discs. This appears contrary to previous findings that show that ecdysone inhibits larval body growth by inhibiting IIS or Myc in the fat body. However, evidence from other insect species suggests that ecdysone can function as either a growth promoter or inhibitor, depending on its concentration. Specifically, in vitro evidence from Manduca shows that low concentrations of ecdysone can promote growth of imaginal tissues, while higher concentrations stimulate differentiation, and stop cell proliferation. Further evidence from Manduca suggests that ecdysone promotes mitosis by regulating the cell cycle, and thus acts as a mitogen. These data echo data from Drosophila that suggests that ecdysone regulates cell cycle progression and promotes imaginal disc growth via the ecdysone inducible gene crooked legs. Collectively, it is apparent, therefore, that ecdysone is a central regulator of larval and imaginal tissue growth, although the tissue-specific effects and molecular mechanisms involved have not yet been completely elucidated. Research from this and other labs supports the hypothesis that imaginal discs reduce their growth rates in response to low levels of ecdysone. At the same time, low levels of ecdysone increase body growth rate and final adult body size. Together these data suggest that ecdysone suppresses the growth of larval tissue (which comprises the majority of the larva) but promotes growth of imaginal tissues. This hypothesis has intuitive appeal in that a key function of ecdysone is to 'prepare' the larva for pupariation and metamorphosis, a process that involves breakdown and autophagy of the larval tissues to provide nutrients for final growth and differentiation of the imaginal discs (Gokhale, 2016).

    Research over the past decade has elucidated mechanisms by which ecdysone functions as a suppressor of larval growth. These studies demonstrate a role for IIS in ecdysone-mediated suppression of larval growth. Specifically, ecdysone signaling in the fat body suppresses IIS, which in turn inhibits systemic IIS and larval growth through repression of dILP2 release from the brain and promotes fat body autophagy. How ecdysone promotes imaginal disc growth is less clear, however. A recent paper by Herboso (2015), indicated that ecdysone promotes growth by suppressing Thor signaling in the imaginal discs. Discs from larvae with reduced ecdysone synthesis have elevated levels of Thor, a repressor of growth that is a target of the IIS pathway. The hypothesis that ecdysone regulates and coordinates growth via IIS/TOR signaling is further supported by the observation that down-regulation of Inr activity prevents the wild-type compartment of antfast:postslow discs from increasing its relative growth rate in response to ecdysone (Gokhale, 2016).

    However, additional data suggest a more nuanced role for IIS in coordinating growth among developmental compartments. In particular, changes in Inr activity in the anterior compartment do not affect relative compartment growth rate in larvae that are otherwise wholly wild-type. Rather, changes in Inr activity increase or decrease relative compartment size, presumably due to changes in compartment growth earlier in development. This is surprising, given that mutations of Inr reduce the growth and proliferation of clones in the wing imaginal disc during L3. In antfast:postslow discs, however, changes in Inr activity does alter growth coordination during L3, but in a counterintuitive way: reduced Inr activity increases relative growth rate, whilst increased Inr activity decreases relative growth rate. This is the opposite of what would be predicted if ecdysone promotes growth by directly upregulating IIS. One interpretation of these data is that the anterior compartments of the antfast:postslow disc adjust their relative growth rate to rescue the final anterior:posterior size ratio, presumably using a mechanism independent of ecdysone. Why this rescue is not evident in wild-type larvae is unclear, but suggest that the rescue mechanism is able to override the ecdysone-regulated mechanism that coordinates growth rates between compartments with different potential growth rates (Gokhale, 2016).

    From the current study and those of others, it seems unlikely that ecdysone promotes imaginal disc growth only through its effects on IIS. In particular, the role of ecdysone in the regulation of differentiation and patterning genes such as broad, senseless, and cut has been well elucidated. Patterning genes are known to regulate cell proliferation. It is therefore possible that ecdysone also regulates imaginal disc growth by regulating the expression of patterning genes in the imaginal disc. One of the challenges in elucidating the role of ecdysone signaling in imaginal disc development is that manipulating ecdysone-signaling organ-autonomously in imaginal discs is technically difficult. This study likely only subtly up-regulated ecdysone signaling by knocking down EcR compartment-autonomously and found that this mild knockdown accelerated compartment growth. It is seems likely that this effect is related to the degree of the knockdown, however, for two reasons. First, complete knockdown of EcR will ultimately block ecdysone signaling, even if it de-represses the expression of certain genes. Second, ecdysone levels can both promote and inhibit insect growth and development depending on its level. As discussed above, moderate level of ecdysone are sufficient to stimulate imaginal disc growth in vitro, while high levels suppress cell proliferation. More precise methods of manipulating ecdysone signaling at a cellular and tissue level are therefore needed (Gokhale, 2016).

    In summary, this study provides evidence for an ecdysone-dependent mechanism that coordinates growth between compartments in the wing imaginal disc of Drosophila. The data suggest that the control of cell proliferation across the imaginal disc is not an entirely autonomous process, but is coordinated through humoral signaling. This research also highlights the crosstalk between different systemic signaling mechanisms - insulin/IGF- and ecdysone-signaling - in the generation of correctly proportioned organs. The developmental mechanisms regulating organ size, while best studied in Drosophila, are conserved across all animals. There is considerable evidence that localized growth perturbation causes systemic growth retardation in humans. For example, children suffering from chronic inflammatory diseases such as Crohn's disease have systemic growth hormone insensitivity and experience severe growth retardation as a complication of the disease. The utilization of systemic signaling mechanisms to coordinate growth within and between organs may thus be a conserved mechanism across all animals (Gokhale, 2016).

    Asymmetric distribution of Spalt in Drosophila wing squamous and columnar epithelia ensures correct cell morphogenesis

    The Drosophila wing imaginal disc is a sac-like structure that is composed of two opposing cell layers: peripodial epithelium (PE, also known as squamous epithelia) and disc proper (DP, also known as pseudostratified columnar epithelia). The molecular mechanism of cell morphogenesis has been well studied in the DP but not in the PE. Although proper Dpp signalling activity is required for proper PE formation, the detailed regulation mechanism is poorly understood. This study found that the Dpp target gene spalt (sal) is only expressed in DP cells, not in PE cells, although pMad is present in the PE. Increasing Dpp signalling activity cannot activate Sal in PE cells. The absence of Sal in the PE is essential for PE formation. The ectopic expression of sal in PE cells is sufficient to increase the PE cell height. Down-regulation of sal in the DP reduced DP cell height. It was further demonstrated that the known PE cell height regulator Lines, which can convert PE into a DP cell fate, is mediated by sal mis-activation in PE because sal-RNAi and lines co-expression largely restores PE cell morphology. By revealing the microtubule distribution, it was demonstrated that Lines- and Sal-heightened PE cells are morphologically similar to the intermediate cell with cuboidal morphology (Tang, 2016).

    The wing disc is a sac-like structure composed of PE, DP, and intermediate cells linking the PE and DP. To investigate the potential role of Dpp signalling in PE morphogenesis, the distribution of Dpp signalling activity in late 3rd instar (L3) wing imaginal discs was revealed in both the x-y and x-z views. Using an antibody of phospho-Mothers against dpp (pMad) to reveal Dpp signal transduction activity, it was found that Dpp signal transduction was ubiquitously present in both the PE and DP. The pMad level was relatively reduced in the central PE compared with the central DP. Dpp target gene expression patterns were detected in the PE. The main Dpp target genes are brinker (brk), omb, and sal in the L3 wing discs. brk was transcribed in both the PE and DP, with a relatively weaker level in the PE, as indicated by a brk-lacZ reporter. However, both omb and sal were transcribed only in the DP, not in the PE. These data indicate that the Dpp target genes omb and sal are asymmetrically expressed in the PE and DP. Brk is also a repressor of other Dpp target genes, including sal and omb, and thereby restricts their expression domains to the medial DP region. The presence of Brk in the PE might be a direct cause of the absence of Omb and Sal in the PE. To assess this possibility, brk-RNAi was expressed in the PE. Sal expression was not detectable in the central PE. The efficiency of brk-RNAi was demonstrated by the elevation of Brk targets Omb and Sal in lateral wing discs of C765>brk-RNAi. To further confirm that Dpp signalling cannot induce sal expression in the PE, a constitutive active form of the Dpp receptor tkvQD was expressed in the PE. Sal was not induced in central PE. When tkvQD clones were generated, Sal was induced only in clones within the DP and not in clones located in the PE). Similarly, Omb was not induced in clones located in the PE. Ubiquitous expression of tkvQD failed to induce Omb in the PE. These data demonstrate that Dpp signalling cannot activate omb and sal in PE (Tang, 2016).

    The expression patterns of Dpp target genes are well studied in wing DP. Dpp controls target genes (sal and omb) expression indirectly through repression of the transcriptional repressor Brk. Dpp target gene expression patterns have not been studied in wing PE to date. The results revealed that pMad is ubiquitously present in both DP and PE. However, brk-lacZ was still present in PE. Either suppressing brk or elevating Dpp signalling by expressing tkvQD cannot induce Sal and Omb in the PE. Except for Lin, other factors, such as Bowl, Wg, and EGFR, cannot induce Sal in the PE. The factors that suppress sal in the PE require further investigation (Tang, 2016).

    As omb and sal are expressed only in the DP, not the PE, it was therefore asked whether this asymmetric transcription of omb and sal is essential for correct PE formation. To test this possibility, sal was mis-expressed in the PE using the Gal4-UAS system. dpp-Gal4 line is expressed in narrow stripes in the lateral PE and in the middle DP. When sal was ectopically expressed in the dpp-Gal4 domain, the height of lateral PE was notably elongated to a height similar to that of intermediate cells. Then, sal was expressed driven by C765-Gal4, which is ubiquitously expressed in both DP and PE. A similar elongation phenotype was observed in the PE. The cell height of the central PE was elongated to a height similar to that of cuboidal cells. The extent of elongation as a result of dpp-Gal4 was stronger than that of C765-Gal4. This difference might be due to the differences in Gal4 activity because dpp-Gal4 is stronger than C765-Gal4. The quantification of cell height, using the ratio between PE and DP cells within one wing disc, revealed a significant increase in sal mis-expression discs. Consistently, the PE height ratio between sal mis-expression and control also revealed a significant increase. To confirm this result, sal over-expression clones were generated in PE. From the x-z cross view, the clonal height was apparently elongated to a height similar to that for cuboidal cells. Therefore, it is concluded that sal is sufficient to elongate PE height (Tang, 2016).

    Unlike the effect of sal mis-expression, the elongation of PE height in case of omb ectopic expression was not apparent, however, the differences in the PE/DP ratio and normalized PE height for C765>omb wing discs are statistically significant. Strong overexpression of omb induces severe extrusion and basal delamination, and cell motility can thicken the wing disc. Although a relatively weaker UAS-omb line was used, the side effect from cell movement may remain, thus leading to the statistic difference in the PE measurement. A previous report demonstrates that if Dpp signalling is suppressed in the PE by Ubx-Gal4 driven dad, a portion of the PE cells are elongated to a cuboidal shape. Therefore, suppressing Dpp signalling in the PE and expressing sal in the PE exhibit similar effects. When carefully assessing the Ubx-Gal4 expression domain, a portion of the expressing cells were, surprisingly, located in the DP. Thus, a non-autonomous effect from a loss of Dpp signalling in the DP in the Ubx>dad wing disc is reasonable. Because when dad-expressing clones were generated within PE, the height of PE did not increase. When Omb-RNAi was driven by hh-Gal4 which is expressed in PE and the posterior compartment of DP, the posterior DP height was reduced. Interestingly, the height of opposite PE was increased. Thus, it is possible that there is a connection between DP and PE during cell morphogenesis. To directly confirm the non-autonomous effect on PE elongation, the DP height was shortened by expression of either dad or brk within the DP specific nub-Gal4 domain. Consistently, the PE height was apparently increased (Tang, 2016).

    Previous studies have revealed that mis-expressing lin induces ectopic sal expression in the PE. Thus, sal may mediate lin’s role in PE elongation. First, the experiment of lin mis-expression in the PE was repeated and the elongation phenotype was consistently observed in the PE. Then, the transcription state of sal was revealed using a sal-lacZ reporter. sal was apparently transcribed in the PE. The sal gene complex is composed of two functionally redundant genes: spalt major (salm) and spalt-related (salr). A rescue experiment was performed by co-expressing lin and salm-RNAi. The morphology of the wing imaginal discs was rescued to an approximate normal state, and PE height was no longer elongated. The cell heights of the corresponding genotypes were also measured. sal down-regulation largely rescued the abnormal cell height induced by lin mis-expression. These results indicate that sal mediates the role of lin in promoting PE elongation. However, Lin elongated PE to a greater extent than Sal did, according to the statistic measurements. Other mediators may be involved downstream of Lin. Since that Ubx-Gal4 line is also expressed in part of the DP cells, potential non-autonomous effects between DP and PE can not be ruled out (Tang, 2016).

    Given that the mis-expression of sal in the PE elongates cell height, whether down-regulating Dpp-Sal signalling in DP is sufficient to shorten the DP was assessed. nub-Gal4 is only expressed in the wing pouch region in the DP. When Dpp signalling was mildly inhibited by expressing a dominant negative form of the Dpp receptor, tkvDN, in the nub-Gal4 domain, DP cell height was reduced. Unlike the strong inhibition of Dpp signalling by expressing dad, the non-autonomous effect on PE height was not apparent in nub>tkvDN. The Dpp signalling activities in discs of nub>tkvDNand nub>dad were revealed by anti-Sal staining. The DP height was slightly reduced when sal was down-regulated either by salm-RNAi or salr-RNAi; however, the extent of this reduction was weaker than that of tkvDN. Then, sal mutant clones were generated, marked by the loss of GFP in the DP. The intensity of F-actin labelled by Phalloidin was much stronger in the clone regions. The x-z cross-section showed that the apical side of sal mutant clones in the DP was retracted toward the basal side. These data suggested that Dpp-Sal signalling is required to maintain DP elongation. Interestingly, a similar retraction phenotype was also observed in sal-overexpressing clones. Therefore, both sal loss- and gain-of-function clones induce an apical retraction phenotype in the DP. This phenotype is observed in both omb loss- and gain-of-function clones in the DP. Omb exhibits a graded distribution in the DP along the A/P axis and specifies unknown apically distributed adhesion molecules. A continuous Omb level is essential for maintaining the epithelial integrity of the wing disc. Therefore, sharp discontinuity in either Omb or Sal levels in the DP induces apical retraction of cells. To confirm this conclusion, a sharp discontinuity of Sal was generated in the DP using dpp-Gal4 driven UAS-sal. Sal continuity was disrupted at the A/P boundary and where deep apical folds were formed. The expression domain of sal in the DP is narrower than that of omb and vg. Beyond the sal domain, omb and vg can ensure the correct cell morphogenesis in the DP. Clones lacking Vg function are also extruded from the DP layer (Tang, 2016).

    Microtubule cytoskeleton is polarized during cell morphogenesis in the wing imaginal disc. To reveal microtubule-based cytoskeleton changes induced by either lin or sal mis-expression and the microtubule dynamics during normal development, the microtubule level was monitored via antibody staining. In the 2nd instar, all cells were cuboidal shape, and microtubules were uniformly distributed. During the early 3rd instar, the cell shape begins to differentiate. PE cells were largely shortened, whereas DP cells were remarkably elongated. Correlating with DP elongation, the microtubule network was asymmetrically enriched to the apical side of the DP. When lin was mis-expressed in the PE by Ubx-Gal4, sal was activated in the PE. Both direct and indirect sal expression induced PE height elongation with an even microtubule distribution. The microtubule levels of PE were increased compared with the wild type PE. The microtubule distribution in Sal-elongated PE was similar to that in very lateral PE and intermediate cells in the L3 stage or undifferentiated cells in earlier larval stages. In the rescue experiment, lin and salm-RNAi were co-expressed. PE height was restored were similar to that in wild type PE. Therefore, based on the cell height and microtubule distribution, Sal mis-expression converts the PE into a cuboidal cell shape (Tang, 2016).

    During tissue morphogenesis, cell-shape changes always accompany microtubule cytoskeleton rearrangement. Dpp signalling activity has been proposed to play a basic function in microtubule organization. Dpp signalling is graded in the DP along the A/P axis, with higher levels in the medial DP region, which is enriched in apical microtubules. Thus, a correlation is noted between Dpp signalling activity and microtubule levels in the DP. Clones with loss-of-function of Dpp receptors in the DP appear extruded (cell height is severely shortened) and exhibit reduced apical microtubule levels. Consistently, clones with both loss- and gain-of-function sal and omb in the DP consistently exhibit severe apical retraction with shortened cell height and loss of apical microtubule enrichment. Therefore, the data support the hypothesis that Dpp-Omb/Sal signalling activity plays a more general function in microtubule-based cell morphogenesis. Other transcription factors that induce reductions in DP cell height also correlate with the loss of apical microtubule enrichment. The Tbx6 subfamily gene cluster Dorsocross (Doc) initiates wing hinge/blade fold formation. In the Doc expression domain in the DP, cells are shortened from the apical side with severe loss of apical microtubules (Tang, 2016).

    Ecdysone signaling induces two phases of cell cycle exit in Drosophila cells

    During development cell proliferation and differentiation must be tightly coordinated to ensure proper tissue morphogenesis. Because steroid hormones are central regulators of developmental timing, understanding the links between steroid hormone signaling and cell proliferation is crucial to understanding the molecular basis of morphogenesis. This study examined the mechanism by which the steroid hormone ecdysone regulates the cell cycle in Drosophila. A cell cycle arrest induced by ecdysone in Drosophila cell culture is analogous to a G2 cell cycle arrest observed in the early pupa. In the wing, ecdysone signaling at the larva to puparium transition induces Broad which in turn represses the cdc25c phosphatase String. The repression of String generates a temporary G2 arrest that synchronizes the cell cycle in the wing epithelium during early pupa wing elongation and flattening. As ecdysone levels decline after the larva to puparium pulse during early metamorphosis, Broad expression plummets allowing String to become re-activated, which promotes rapid G2/M progression and a subsequent synchronized final cell cycle in the wing. In this manner, pulses of ecdysone can both synchronize the final cell cycle and promote the coordinated acquisition of terminal differentiation characteristics in the wing (Guo, 2016).

    This study presents a model for how the pulse of ecdysone at the larval to pupal transition impacts the cell cycle dynamics in the wing during metamorphosis. Ecdysone signaling at the larva to puparium transition induces Broad, which in turn represses Stg to generate a temporary G2 arrest, which synchronizes the cell cycle in the wing epithelium. As ecdysone levels decline, Broad expression plummets, allowing Stg to be re-activated resulting in a pulse of cdc2 activity that promotes a rapid G2/M progression during the final cell cycle in the wing. This ultimately culminates in the relatively synchronized cell cycle exit at 24h APF, coinciding with the second large pulse of ecdysone. This second pulse in the pupa activates a different set of transcription factors (not Broad), promoting the acquisition of terminal differentiation characteristics in the wing. In this way, two pulses of ecdysone signaling can both synchronize the final cell cycle by a temporary G2 arrest and coordinate permanent cell cycle exit with the acquisition of terminal differentiation characteristics in the wing (Guo, 2016).

    Over 30 years ago it was shown that 20-HE exposure in Drosophila tissue culture cells induces a cell cycle arrest in G2-phase. This response appears to be shared among 3 different cell lines, Cl-8, Kc and S2. This study shows that in Kc cells, pulsed 20-HE exposure also leads to a G2 arrest followed by rapid cell cycle re-entry after 20-HE removal and a subsequent prolonged G1. This cell cycle response to a pulse of 20-HE is reminiscent of the cell cycle changes that occur during early metamorphosis in the pupal wings and legs (Guo, 2016).

    It is worth considering why Kc and S2 cells, which are thought to be derived from embryonic hemocytes would exhibit a similar cell cycle response to 20-HE to the imaginal discs. Relatively little is known about how ecdysone signaling impacts embryonic hemocytes, although recent work suggests that ecdysone signaling induces embryonic hemocyte cell death under sensitized conditions. More is known about larval hemocytes, which differentiate into phagocytic macrophages and disperse into the hemolymph during the first 8h of metamorphosis. Ecdysone is involved in this maturation process, as lymph glands of ecdysoneless (ecd) mutants fail to disperse mature hemocytes and become hypertrophic in the developmentally arrested mutants. This suggests that the high levels of systemic ecdysone signaling at the larval-puparium transition mediates a switch from proliferation to cell cycle arrest and terminal differentiation for lymph gland hemocytes during metamorphosis. Without ecdysone signaling, hemocytes may continue to proliferate and fail to undergo terminal differentiation leading to the hypertrophic lymph gland phenotype observed. Interestingly, while the loss of broad also prevents proper differentiation of hemocytes similar to loss of ecd, loss of broad does not lead to the hypertrophy observed in ecd mutants. Further studies will be needed to examine whether the ecdysone induced cell cycle arrest in larval hemocytes occurs in the G2 phase, or whether their cell cycle arrest proceeds via a similar pathway to that shown in this study for the wing (Guo, 2016).

    Multiple lines of evidence suggest that the ecdysone receptor complex in the larval wing acts as a repressor for certain early pupa targets and that the binding of ecdysone to the receptor relieves this repression. For example loss of EcR by RNAi or loss of the EcR dimerization partner USP, de-represses ecdysone target genes that are high in the early pupal wing such as Broad-Z1 and βFtz-F1. The EcR/USP heterodimer also cooperates with the SMRTR co-repressor in the wing to prevent precocious expression of ecdysone target genes such as Broad-Z1. Consistent with the hypothesis that a repressive EcR/USP complex prevents precocious expression of Broad-Z1 and thereby a precocious G2 arrest, inhibition of SMRTR can also cause a G2 arrest. Thus, in the context of the early pupal wing, it is proposed that the significant pulse of ecdysone at the larval to puparium transition relieves the inhibition of a repressive receptor complex, leading to Broad-Z1 activation. Consistent with this model, high levels of Broad-Z1 in the larval wing lead to precocious neural differentiation at the margin and precocious inhibition of stg expression in the wing pouch. Interestingly, a switch in Broad isoform expression also occurs during the final cell cycle in the larval eye, such that Broad-Z1 becomes high in cells undergoing their final cell cycle and entering into terminal differentiation. However in this case, Broad-Z1 expression is not associated with a G2 arrest and occurs in an area of high Stg expression, suggesting the downstream Broad-Z1 targets in the eye may be distinct or regulated differently from those in the wing (Guo, 2016).

    The ecdysone receptor has also been shown to down regulate Wingless expression via the transcription factor Crol at the wing margin, to indirectly promote CycB expression. While a loss of EcR at the margin decreased CycB protein levels, the effects of EcR loss on CycB levels in the wing blade outside of the margin area were not obvious. It is suggested that in the wing, the role for EcR outside of the margin acts on the cell cycle via a different mechanism through stg. Consistent with a distinct mechanism acting in the wing blade, over-expression of Cyclin B in the early prepupal wing could not promote increased G2 progression or bypass the prepupal G2 arrest. Instead the results on the prepupal G2 arrest are consistent with previous findings that Stg is the rate-limiting component for G2-M cell cycle progression in the fly wing pouch and blade (Guo, 2016).

    In order to identify the gene expression changes in the wing that occur in response to the major peaks of ecdysone during metamorphosis, RNAseq was performed on a timecourse of pupal wings. Major changes were observed in gene expression in this tissue during metamorphosis. In addition, known ecdysone targets were identified that are affected differently in the wing during the first larval-to-pupal ecdysone pulse and the second, larger pulse at 24h APF. Ecdysone signaling induces different direct targets with distinct kinetics. Furthermore specific targets, for example Ftz-F1 can modulate the expression of other ecdysone targets, to shape the response to the hormone. Thus, it is expected that a pulse of ecdysone signaling leads to sustained effects on gene expression and the cell cycle, even after the ecdysone titer returns to its initial state. These factors together with the differences in the magnitude of the ecdysone pulse may contribute to the differences in the response to the early vs. later pulses in the wing (Guo, 2016).

    Ecdysone signaling can also affect the cell cycle and cell cycle exit via indirect mechanisms such as altering cellular metabolism. This is used to promote cell cycle exit and terminal differentiation in neuroblasts, where a switch toward oxidative phosphorylation leads to progressive reductive divisions, (divisions in the absence of growth) leading to reduced neuroblast cell size and eventually terminal differentiation. Although reductive divisions do occur in the final cell cycle of the pupa wing, this type of mechanism does not provide a temporary arrest to synchronize the final cell cycle in neuroblasts as is see in wings. Importantly, a striking reduction is seen in the expression of genes involved in protein synthesis and ribosome biogenesis in the wing during metamorphosis, consistent with the lack of cellular growth. Instead the increased surface area of the pupal wing comes from a flattening, elongation and apical expansion of the cells due to interactions with the extracellular matrix creating tension and influencing cell shape changes. This is also consistent with the findings that a significant number of genes associated with protein targeting to the membrane are increased as the wing begins elongation in the early pupa. Further studies will be needed to determine whether the changes in expression of genes involved in ribosome biogenesis and protein targeting to the membrane are controlled by ecdysone signaling, or some other downstream event during early wing metamorphosis (Guo, 2016).

    Perhaps the most interesting and least understood aspect of steroid hormone signaling is how a diversity of cell-type and tissue-specific responses are generated to an individual hormone. Cell cycle responses to ecdysone signaling are highly cell type specific. For example abdominal histoblasts, the progenitors of the adult abdominal epidermis, become specified during embryogenesis and remain quiescent in G2 phase during larval stages. During pupal development, the abdominal histoblasts must be triggered to proliferate rapidly by a pulse of ecdysone to quickly replace the dying larval abdominal epidermis. This is in contrast to the behavior of the wing imaginal disc, where epithelial cells undergo asynchronous rapid proliferation during larval stages, but during metamorphosis the cell cycle dynamics become restructured to include a G2 arrest followed by a final cell cycle and entry into a permanently postmitotic state, in a manner coordinated with tissue morphogenesis and terminal differentiation (Guo, 2016).

    How does the same system-wide pulse of ecdysone at the larval to puparium transition lead to such divergent effects on the cell cycle in adult progenitors? Surprisingly it seems to be through divergent effects on tissue specific pathways that act on the same cell cycle targets. In the abdominal histoblasts the larval to puparium pulse of ecdysone triggers cell cycle re-entry and proliferation via indirect activation of Stg, by modulating the expression of a microRNA miR-965 that targets Stg. This addition of the microRNA essentially allows ecdysone signaling to act oppositely on the same cell cycle regulatory target as Broad-Z1 does in the wing. Thus, tissue specific programs of gene regulatory networks can create divergent outcomes from the same system- wide hormonal signal, even when they ultimately act on the same target (Guo, 2016).

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    Genes involved in organ development

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