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The Interactive Fly
Genes involved in tissue and organ development
Wing
Microarray profiling to discover genes expressed in the wing disc
Gene expression during Drosophila wing morphogenesis and differentiation
Tissue remodeling during maturation of the Drosophila wing
Model for the regulation of size in the wing imaginal disc of Drosophila
Cell competition, growth and size control in the Drosophila wing imaginal disc
The Drosophila wing hearts originate from pericardial cells and are essential for wing maturation
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 http://dev.biologists.org/supplemental/. Ninety-four transcripts show two-fold or greater enrichment in the body wall and 56 transcripts show two-fold or greater enrichment in the wing/hinge. Several of these genes were also found to be more highly expressed in wing discs than leg discs or eye-antennal discs, suggesting they may also have appendage-specific roles (Butler, 2003).
The rank order of transcripts correlates well with the spatial expression patterns of characterized genes. In the body wall, pannier (pan), twist (twi) and BarH1, which are enriched in the body-wall sample, are all known to be highly expressed in the presumptive body wall. In the wing, knot (kn), nubbin (nub) and Distal-less (Dll) are expressed at levels greater than 10-fold above those in the body wall. kn is expressed in the wing 3/4 intervein and hinge regions; nub is strongly expressed in the entire wing pouch and Dll is expressed along the dorsal-ventral (DV) margin exclusively in the wing pouch (Butler, 2003).
Other genes, known to have important roles in disc development, appear lower down the rank order. vestigial (vg), a key gene for development of the wing and hinge regions, shows only two-fold enrichment but this is consistent with the expression pattern of vg in the wing disc that extends into the body wall region. Transcripts with expression patterns restricted to the posterior compartment, [engrailed (en), invected (inv) and hedgehog (hh)], show approximately two-fold enrichment in the wing/hinge sample. The anterior-posterior compartment boundary splits the wing/hinge region into two equally sized compartments, but the position of the boundary in the body wall region produces a small posterior compartment representing approximately one-quarter of the total tissue. This is consistent with the approximately two-fold enrichment of posterior-specific transcripts found in the wing/hinge tissue sample. The E(spl)-Complex genes are expressed in developing sensory organs found in both the body wall and wing margin regions. Hence, these genes are not enriched in any one sample. The m6 gene is an exception (enriched in the body wall sample) and is known to be expressed only in the body wall region. In contrast, genes that show ubiquitous expression such as Ras or tubulin show no enrichment on the arrays (Butler, 2003).
Microarray analysis can therefore identify transcripts known to be differentially expressed in the wing/hinge and body wall regions of the disc. Few expression patterns of the identified genes have been described, so to verify the validity of the approach, and to discover more genes with potential roles in the development of these specific regions, in situ hybridizations were made for some of these uncharacterized genes (Butler, 2003).
Fifty transcripts that had strong enrichment (mostly three-fold or greater) were examined. For the body wall-enriched transcripts, the larger set (only transcripts for which clones are available in the Drosophila gene collections -- DGC1 and DGC2, Berkeley Drosophila Genome Project) were examined. For the wing/hinge region, transcripts were examined with three-fold or greater enrichment, systematically in rank order from the top, and PCR probes were generated when clones were not available. All transcripts tested showed expression patterns that were consistent with the microarray data, providing confirmation that the microarray analysis mirrors the spatial distribution of transcripts in vivo (Butler, 2003).
The wing disc comprises three cell layers: the squamous epithelium of the peripodial membrane; the columnar epithelium that becomes the adult epidermis, and the adepithelial layer that includes myoblast cells that give rise to adult thoracic muscles and tracheal cells that form air passages. The adepithelial layer extends from the proximal disc dorsally into the hinge region. The body wall fragment includes cells of all three layers, so the arrays also identified transcripts specific to muscle and tracheal cells (Butler, 2003).
pan and BarH1, which encode transcription factors, are expressed in the body wall epidermis and are involved in bristle patterning. Both transcripts were highly enriched on the arrays. Also highly enriched was tailup (tup), which encodes a LIM domain homeobox protein, and is expressed in the epithelium in a large region of the posterior body wall encompassing the presumptive postnotum, scutellum and scutum. No role for tup in patterning the mesothorax has been described. Another transcript with broad expression was thrombospondin/CG11326 (tsp), which is expressed in a similar region of the body wall to tup. tsp is also expressed in the ventral hinge and hence shows lower enrichment on the arrays. The other genes found to be specific to the epithelium showed highly localized expression: Obp56a/CG11797, CG10126, CG3244 and Glucose dehydrogenase. Obp56a/CG11797 encodes an odorant-binding protein and interestingly three other odorant-binding proteins showed enrichment on the arrays: Obp99a, CG9358 and Obp56d/CG1128. Idgf4, encoding an imaginal disc growth factor, is expressed in the peripodial membrane, primarily in dorsal cells. Presumably secretion of Idgf4 could influence development of the columnar epithelium (Butler, 2003).
Myoblast cells of the adepithelial layer develop into the direct and indirect flight muscles of the thorax, and genes involved in the development of these muscles have been shown to be expressed in the myoblasts during wing disc development. Several of these transcripts are enriched on the arrays: Mef2, twist (twi) and heartless (htl). Act57B is known to be regulated by Mef2 in the embryo, and Act57B is expressed in the myoblasts, suggesting this relationship also exists in these adult muscle precursors. Mef2 expression is activated by twi and may be inhibited by the transcriptional repressor, zinc finger homology 1 (zfh1). zfh1 is expressed in the myoblasts. stumps is also enriched on the arrays and expressed in the myoblasts. Together with htl, stumps has a role in the development of the tracheal cells. Viking (Vkg) encodes a component of collagen type IV and is known to be coexpressed with Cg25C, another collagen IV subunit in the embryo and in blood cells. Both transcripts are enriched on the arrays and show similar expression patterns in the adepithelial myoblasts and blood cells. Other genes showing specific expression in the myoblasts are BM-40/SPARC, a calcium-binding glycoprotein, which is expressed in the embryonic mesoderm, Elongation factor 1 alpha 100E (Ef1 alpha), CG8689, an alpha-amylase, and two transcripts encoding predicted proteins with unknown function CG11100 and CG15064 (Butler, 2003).
In the wing disc, cells of the larval and developing adult tracheal systems require activity of genes in the FGF pathway. Some of the key genes are expressed in the myoblasts (for example, htl and stumps), others in the epithelium (for example, branchless, bnl), and others in the tracheal cells themselves (for example, breathless, btl). htl and stumps showed enrichment on the arrays but bnl and btl were not detectable. For bnl this may be because expression is highly localized and apparently at very low levels. However, it is not clear why the arrays failed to detect btl expression because six genes were identified that are also expressed specifically in tracheal cells -- these are CG5397, an O-acyltransferase, CG4386, a serine-type endopeptidase, CG2663, an alpha-tocopherol transfer-like protein, and CG15353, CG6921 and CG9338 that have no known homologies. In particular, CG4386 is interesting since it is only expressed in the dorsal branch, and CG6921 is distinguished because it is very strongly expressed in the most proximal cells (Butler, 2003).
The wing/hinge fragment of the wing disc primarily contains cells of the peripodial membrane and the columnar epithelium, with only a few myoblasts that extend into the hinge region. Thus the genes detected by the arrays as enriched in this disc fragment are expressed in cells of one of the two epithelial layers (Butler, 2003).
Transcription factors comprise the largest category of genes (18/56) with elevated expression in the wing/hinge region. These are expected to have regulatory roles in patterning the region. Transcription factors with known expression domains and roles in wing development are present: kn, pox-n, nub, Dll, bifid/optomotor blind, rotund, ventral veins lacking, en, vg and in. pdm2, which is highly related to nub, also shows wing-enriched expression on the arrays and is expressed in a similar domain to nub. pdm2 apparently has no significant function in the wing. The roles of the remaining seven predicted transcription factors are unknown, although the expression pattern of zinc finger homology 2 (zfh2) and Sox 15 have been described and both are expressed specifically in the hinge region. defective proventriculous (dve), which encodes a homeodomain protein, and CG15000, which is similar to NGFI-A-binding protein 2, are broadly expressed in the wing pouch, although dve is downregulated at the DV compartment boundary. odd paired (opa), known for a role in embryonic segmentation, is discretely expressed in cells of the presumptive mesopleura and dorsal hinge. No role for opa in wing disc development has been reported. Dorsocross1 (Doc1) and Doc2/CG5187 are T-Box related factors that are expressed in what appears to be an identical domain in the wing disc. Both transcripts also accumulate in body wall cells and this probably lowers their position in the overall ranked list (Butler, 2003).
Eight transcripts encoding enzymes are enriched two-fold or greater in the wing/hinge region. This group includes the most highly enriched transcript detected in the analysis, a kazal-type serpin gene CG17278 (68-fold). CG17278 shows a strong and specific expression pattern in the wing encompassing most of the wing pouch. One of the potentially most interesting wing-enriched enzymes is a cytochrome P450 gene, Cyp310al. This gene is strongly expressed in the dorsal and ventral parts of the wing pouch but excluded from the DV and AP boundaries. Variable expression in anterior body wall cells is also observed that is consistent with the array data that indicate Cyp310al transcripts are also present in body wall RNA. Surprisingly, the ß-galactosidase gene (CG3132) was found to be enriched in the wing/hinge region. Weak expression was found in a cluster of cells in the hinge but the majority of expression is in blood cells, which adhere preferentially to the distal disc margin. Thus the ß-gal transcript probably appears as wing/hinge enriched primarily because it is expressed in blood cells. The expression patterns of two other enzymes were also determined: the metalloendopeptidase Nep1/CG5894 and UDP-glucosyl transferase (Ugt86Di) (Butler, 2003).
The alpha-integrin, inflated, which has a role in cell adhesion, is expressed in the ventral compartment and is thus enriched on the wing/hinge arrays. A novel gene, CG5758, is potentially involved in cell adhesion since it encodes a predicted protein with ß-Ig-H3/Fas domains and its expression is restricted to the dorsal hinge. CG8381 encodes a proline-rich protein with repeated 'PEVK' motifs also found in titin. This gene is strongly expressed in the wing pouch but repressed in cells of the future veins and cells at the DV margin. Despite intense expression in the wing pouch, CG8381 shows only modest enrichment on the arrays, probably reflecting the fact that the gene is also expressed in several groups of cells in the body wall region (Butler, 2003).
The expression of two receptors was determined. CG4861 encodes an ldl-receptor-like protein and is expressed at very low levels throughout the wing pouch. wengen /CG6531, which is a receptor of the TNFR family, is expressed strongly in the wing pouch and weakly in the body wall. On the arrays, its ligand, eiger, was undetectable in the wing/hinge region sample but enriched in the body wall sample (Butler, 2003).
Two structural proteins, CG6469, a larval cuticle protein, and CG14301, a chitin-binding protein, are the only genes identified as being expressed in the ventral peripodial membrane. CG6469 is expressed broadly in the peripodial membrane but at a higher level in the ventral region. CG14301 is expressed in cells of both epithelial layers, in the columnar epithelium at the anterior disc margin and in four patches of cells in the wing pouch and the overlying peripodial membrane (Butler, 2003).
In a group of genes with miscellaneous functions the expression of three genes was determined. anachronism (ana), a secreted glycoprotein, is expressed in five clusters of cells including one in the body wall region and in some individual neuroblasts. ana null mutants are viable and have no observable defects suggesting it is not required, or functions redundantly, in the wing. CG14534, which has a domain that has been recognized in several proteins but has an unknown function (DUF243), is expressed only in cells that will give rise to the posterior wing margin. CG8483, which has homology to a venom allergen, is expressed in a complex pattern suggestive of expression in peripheral sense organ precursors (Butler, 2003).
The expression pattern is described for five of eight genes for which the sequence reveals no homology to known protein domains. CG15489 and CG15488 are in a cluster of genes also including nub and pdm-2 that are expressed in similar domains and are adjacent in the genome. CG15001, consisting of only a single exon, is adjacent to another gene (CG15000), also discovered on the arrays, with a similar expression domain. BG:DS00797.2/CG9008 is expressed strongly in the wing pouch and also in the adepithelial cell layer. CG8780 is highly enriched on the arrays (31-fold), and expressed specifically in the hinge and ventral pleura (Butler, 2003).
The genes CG17278, Cyp310a1 and CG8381 all show very intense expression in the wing pouch but reduced expression at the DV margin. Wg is expressed at the DV margin forming a gradient that regulates the expression of target genes in a concentration-dependent manner. To determine whether Wg signaling represses the expression of CG17278, Cyp310a1 and CG8381, wg was ectopically expressed in the dorsal and ventral wing-pouch regions (71B-gal4; UAS-wg), or Wg function was inhibited at the DV margin by expressing a dominant-negative form of TCF (Pangolin), a transcription factor required for Wg-signal transduction (C96-GAL4; UAS-DN-dTCF). With higher levels of Wg activity in the wing pouch, expression of all three genes was inhibited. In contrast, inhibition of Wg signaling at the DV margin allowed ectopic expression of Cyp310a1 in all margin cells and increased the number of cells expressing CG17278 and CG8381. In the presumptive margin, cells continue to express wg in the absence of Wg activity; cell replication increases, and ectopic expression of dmyc appears in margin cells. Therefore, ectopic expression of the genes studied here is caused by loss of Wg-dependent repression rather than loss of the non-expressing cells from the presumptive margin. This does not imply that Wg-dependent repression must be direct. Without functional data on these potential target genes, their relationship to wg and their role in wing patterning remain unknown (Butler, 2003).
- Gene expression during Drosophila wing morphogenesis and differentiation
The simple cellular composition and array of distally pointing hairs has made the Drosophila wing a favored system for studying planar polarity and the coordination of cellular and tissue level morphogenesis. A gene expression screen was carried out to identify candidate genes that functioned in wing and wing hair morphogenesis. Pupal wing RNA was isolated from tissue prior to, during and after hair growth and used to probe Affymetrix Drosophila gene chips. 435 genes were identified whose expression changed at least 5 fold during this period and 1335 whose expression changed at least 2 fold. As a functional validation, 10 genes were chosen where genetic reagents existed but where there was little or no evidence for a wing phenotype. New phenotypes were found for 9 of these genes providing functional validation for the collection of identified genes. Among the phenotypes seen were a delay in hair initiation, defects in hair maturation, defects in cuticle formation and pigmentation and abnormal wing hair polarity. The collection of identified genes should be a valuable data set for future studies on hair and bristle morphogenesis, cuticle synthesis and planar polarity (Ren, 2005).
The primary goal in characterizing pupal wing gene expression was to identify genes that play an important role in pupal wing morphogenesis. ken and barbie (ken) encodes a DNA binding transcription factor that contains an N terminal BTB/POZ domain and 3 C2H2 zinc fingers. Its expression increased 6.8 fold from 32 to 40 hrs. Loss of function mutations in ken are semilethal. Escaper adults have been described as having unpigmented aristae and often lack external genitalia (hence the gene name). Wings were examined from ken mutant escapers and also in genetic mosaics. The triple row bristles on the wing margin were lightly pigmented reminiscent of the arista phenotype. This is most obvious in mosaics where the lightly pigmented bristles stand out from their wild type neighbors. No hair phenotype was seen, but a subtle hair pigmentation phenotype would be difficult to see (Ren, 2005).
The HMGS gene encodes the Drosophila HMG Coenzyme A synthase, a key enzyme in steroid and isoprenoid metabolism. Its expression increased 8.4 fold from 32 to 40 hrs. Individuals homozygous for a P insertion allele die as pharate adults or pupae. The pharate adults are notable for a melanotic liquid that accumulates principally near the ventral head. Mutations that result in weak cuticle often show such melanotic leakage, suggesting that HMGS may be required for normal cuticle elaboration. The reason for the phenotype being seen primarily in the ventral head is unclear. No evidence was seen for a specific wing phenotype (Ren, 2005).
The expression karst gene, which encodes betaHeavy-spectrin, increased 5.5 fold from 32 to 40 hrs. Spectrin typically contains 4 chains, 2 alpha and 2 beta; these chains are known to link the actin cytoskeleton to the plasma membrane. Somewhat surprisingly, kst mutants are viable (at reduced levels) and female sterile due to defects in the follicular epithelium. Adult kst mutants have rough eyes and their wings often are cupped downward. kst wings were examined and an additional mutant phenotype was found that is nicely correlated with its expression profile. kst wing cells produce normal looking hairs but the hairs are often found on a small pedestal. The wing cell surface (that is not hair) is rough and at times remnants of cell outlines are visible. This phenotype can also be seen in mosaic clones. The clones can be recognized under the stereo microscope because they are often associated with a dimpling of the wing surface (Ren, 2005).
The krotzkopf verkehrt (kkv) and knickkopf (knk) genes were both identified in a screen for having an unusual defect in embryonic cuticle, known as the blimp phenotype. Mutant embryo cuticles were seen to expand in cuticle preparations. The kkv gene encodes a chitin synthase implicating it in cuticle synthesis and its expression increased 4.9 fold from 32 to 40 hrs. The knk gene encodes a novel gene that is only well conserved in the ecdysozoa, suggesting a role in cuticle metabolism. The amino acid sequence shows homology to what is thought to be a dopamine binding domain suggesting Knk might be involved in cross linking of cuticle. The expression of knk increased 7 fold between 32 and 40 hrs. Mutations in both of these genes are embryonic lethals so mosaic clones of cells carrying mutations in either of these genes were examined. The phenotypes seen in the adult cuticle were quite similar to one another. Most notably wing mutant wing hairs displayed a lack of pigmentation and were thinner and flimsier than normal. This phenotype is dramatic and at low magnification it often appears as if hairs were not formed by mutant cells. The hairs appeared normal in size and shape when clones were examined in pupal wings arguing that the mutations affect a process after hair outgrowth (e.g., cuticle synthesis or maturation). Clones in other body regions such as the abdomen and thorax also showed a dramatic loss of pigmentation. In all of these cases the borders between pigmented and unpigmented were relatively sharp. Consistent with these mutations resulting in weak cuticle phenotypes, areas were often seen where internal tissues and hemolymph appeared to be erupting from the animal. This was usually seen on the dorsal abdomen, particularly in the region of the intersegmental membrane. The eruptions could be related to the blimp phenotype seen in embryos (Ren, 2005).
The expression of the brain tumor (brat) decreased 5.5 fold from 24 to 40 hrs. This gene has been studied primarily due to the neural tumor phenotype seen in loss of function mutants. The wings of bratts/Df brat flies raised at semi-permissive conditions were examined. No hair phenotype was seen but the occasional loss of sensory bristle shaft cells (principally distally along the anterior margin) was seen and occasional duplicated bristle cells (principally in the costa). These phenotypes are suggestive of a role for brat in specifying cell fate or in Notch mediated lateral inhibition (Ren, 2005).
The expression of dopa decarboxylase (Ddc) increased 6 fold from 24 to 32 hrs and then decreased 1.9 fold from 32 to 40 hrs. This well characterized gene is known to function in the epidermis for the cross linking of cuticle and in the formation of melanin. Loss of Ddc function results in fragile and pale cuticle with thin bristles. No detailed description of the wing phenotype had been reported previously. Ddc null alleles are recessive embryonic lethals adults that contained clones mutant for Ddc were examined. On the abdomen (and some other parts of the body) clones could be seen where there was lightly pigmented cuticle and bristles. No wing phenotype was seen other than apparent clones resulting in lighter triple row bristles. The abdominal clone boundaries were not sharp as seen for grh, knk or kkv, which also give rise to lightly pigmented cuticle suggesting that the Ddc cells might be rescued by the diffusion of dopamine from neighboring cells. Therefore adults homozygous for a temperature sensitive Ddc allele were examined. Animals raised at 25°C showed a much stronger phenotype in general than was in clones suggesting that Ddc acts nonautonously in the wing. The phenotype was even stronger in animals raised at 29°C. The wings of Ddc mutants were characterized by very thin wispy hairs, occasional multiple hair cells and an overall faint appearance. When Ddcts pupal wings were examined, the early hairs appeared normal in morphology. Thus, the wispy appearance of the adult wing hairs is presumably due to a late defect. It is suggested that Ddc dependent cross linking of the cuticle is essential for maintaining the structure of the hair and in the absence of this cross linking the hair collapses after the actin cytoskeleton is disassembled. Occasional multiple hair cells were seen in the Ddcts pupal wings; thus that defect is likely due to a different process also being affected in the mutant. The formation of multiple hair cells has previously been associated with planar polarity defects or due to disruptions of the cytoskeleton (Ren, 2005).
The HR46 gene (also known as DHR3) encodes a nuclear receptor and is an essential gene known to be important for the ecdysone cascade. Large clones of loss of function alleles result in wing (folded and curved) and notum defects (rough short bristles and pale pigmentation). The expression of this gene increased 250 fold from 24 to 32 hr and then decreased 4.3 fold from 32 to 40 hr. Moderate sized wing clones of cells lacking HR46 were examined, but no clear cut phenotype was seen. In pupal wing clones examined a couple of hours after hair formation mutant hairs appeared somewhat thicker but this alteration was transient (Ren, 2005).
The Eip78CD gene encodes a related nuclear receptor. The expression of this non-essential gene increased 3 fold from 24 to 32 hr followed by a three fold drop from 32 to 40 hr (but the differences were not significant) suggesting it might be functionally redundant with HR46. To test this hypothesis Eip78CD mutants, which also contained HR46 mutant clones, were examined. No mutant phenotypes were seen in the clones, suggesting either that there is an alternative redundant gene or that HR46 is not essential for hair morphogenesis. Since the level of HR46 expression fell dramatically between 32 and 40 hrs it seemed possible that declining HR46 expression could be important for hair development. To test this the overexpression of HR46 from a transgene containing a hs promoter was induced. This resulted in a dramatic loss of hair formation leading to wings with extensive bald regions. The strongest phenotype was seen when the transgene was induced by heat shocking 6-8 hrs prior to the time of hair initiation. The phenotype was dose sensitive and directly related to the number of transgenes and length and temperature of transgene induction (Ren, 2005).
The expression of the non-stop (not) gene decreased 3.9 fold from 24 to 40 hrs. Mutations in not result in photoreceptor neurons projecting through the lamina instead of terminating there. The mutations also result in approximately 20% of ommatidia being misoriented -- a planar polarity phenotype. Strong alleles of not die as prepupae so not clones were examined in both adult and pupal wings. Large numbers of clones were induced. Perhaps 25% of wing cells are found in clones. All adult wings of this genotype had regions where there were cells that failed to form hairs or that had very small hairs. These were found only in proximal medial regions on the ventral wing surface. All such wings also had subtle polarity abnormalities; small groups of hairs with slightly abnormal polarity in all regions of the wing. Consistently finding such defects leads to the conclusion that these were due to not clones. Of 47 such wings examined 27 also contained multiple hair cells and a further 10 contained regions with planar polarity defects reminiscent of genes such as fz and dsh. When marked not clones were examined in pupal wings most, but not all, showed cells where hair differentiation was delayed or absent. Such clones were seen in all wing regions. It is suggested that all not clones have delayed hair formation. When the clones are located in wing regions where hairs normally form first (distal or peripheral regions) the hairs form later than normal but still have enough time to reach a relatively normal length. In contrast, when clones are located in regions where hair formation is normally late (proximal and medial regions on the ventral wing surface) not enough time remains prior to cuticle deposition to produce a normal hair. The not gene encodes a ubiquitin carboxyterminal hydrolase likely to function in the removal of ubiquitin from proteins during protein degradation (Ren, 2005).
The Uch-L3 gene also encodes a ubiquitin carboxy hydrolase and its expression decreased 2.9 fold between 24 and 40 hrs. A P insertion mutation in this gene is semi-lethal and escapers have an abnormal eye. No homozygous Uch- L3J2b8 flies were found that eclosed but it was possible to examine animals that died as pharate adults. These animals displayed several morphological defects such as loss of tarsal leg joints, shorter and fatter leg segments, the loss of a discrete antennal segment 4 and a fatter arista that could be due to defects in cell shape or movement. Pupal wings from such animals were examined and wings were found that were wider and shorter than normal and regions were found with a loss of hairs. All of the phenotypes seen in Uch-L3 pupae and pharate adults showed variable expressivity (Ren, 2005).
- Tissue remodeling during maturation of the Drosophila wing
The final step in morphogenesis of the adult fly is wing maturation, a process not well understood at the cellular level due to the impermeable and refractive nature of cuticle synthesized some 30 h prior to eclosion from the pupal case. Advances in GFP technology now make it possible to visualize cells using fluorescence after cuticle synthesis is complete. Between eclosion and wing expansion, the epithelia within the folded wing begin to delaminate from the cuticle and that delamination is complete when the wing has fully expanded. After expansion, epithelial cells lose contact with each other, adherens junctions are disrupted, and nuclei become pycnotic. The cells then change shape, elongate, and migrate from the wing into the thorax. During wing maturation, the Timp gene product, tissue inhibitor of metalloproteinases, and probably other components of an extracellular matrix are expressed that bond the dorsal and ventral cuticular surfaces of the wing following migration of the cells. These steps are dissected using the batone and Timp genes and ectopic expression of αPS integrin, inhibitors of Armadillo/β-catenin nuclear activity and baculovirus caspase inhibitor p35. It is concluded that an epithelial-mesenchymal transition is responsible for epithelial delamination and dissolution (Kiger, 2007; full text of article).
The following outline is proposed of that program based upon cell behavior: delamination and severing contacts; changing cell shape; and migration and ECM synthesis.
Stage 1, delamination and severing contacts
A signaling role for integrins during the prepupal apposition has been proposed that prepares cells for integrin-based adhesion of the epithelia at the pupal apposition. The observation that wing epithelial cells persist in the blistered regions produced by ectopic αPS integrin expression suggests that the integrin interaction also prepares cells to respond to the later signal that induces epithelial delamination and dissolution. This signal is also blocked in the mutant batone, which prevents wing expansion. Some cells begin to delaminate from the cuticle before wing expansion has begun, and all have delaminated by the time expansion is complete. Delamination must involve severing of ECM contacts. The precision of the cellular array in a newly open wing must derive from cell–cell contacts between stretched cells that are maintained following delamination. Each cell then compacts and becomes round (as judged by the increase in fluorescence intensity). The round cells have evidently severed their junctions with adjacent cells because the precise array of cells begins to break up and Arm-GFP moves from the cell membrane to the cytoplasm (Kiger, 2007).
It would appear that disturbing the normal state of Arm/β-catenin signaling activity in epithelial cells blocks delamination. Delamination is blocked by ectopic expression of Pygo in the epithelial cells, which blocks expression of Arm target genes in a variety of tissues, and by ectopic expression of Shaggy, which blocks expression of Arm target genes by phosphorylating cytoplasmic Arm, promoting its degradation and depleting nuclear Arm. Ectopic expression of stabilized forms of Arm not subject to Shaggy phosphorylation evidently has a dominant-negative effect on Arm signaling activity in the maturing wing, blocking delamination of epithelial cells. This interpretation is supported by the following observations. First, no effect is produced by ectopic expression of wild-type Arm using the same Gal4-30A driver, consistent with other reports, very likely indicating the efficiency with which wild-type Arm is eliminated by phosphorylation and degradation through the proteasome. Second, a very low level of nuclear Arm is sufficient for target gene expression. The Arm-GFP fusion protein used here is fully active and completely covers homozygosity for a null arm allele, yet nuclear Arm-GFP cannot be detected in cells receiving a Wingless signal. Thus, it is reasonable that non-physiologically high levels of stable forms of Arm could have a dominant-negative effect, not unlike the inhibitory effect of over-expression of Pygo on Arm-directed transcription. (Kiger, 2007).
Arguing against an interpretation that the effects of ectopic gene expression might be non-specific, note that Gal4-30A-driven expression of p35 does not block delamination. Nor does Gal4-30A-driven expression of either αPS integrin or wild-type transcription factor Pangolin/dTCF/LEF-1, or a dominant-negative form of CREB have any effect on wing maturation (Kiger, 2007).
Stage 2, changing cell shape
The round cells then begin to change shape, extending thin cytoplasmic filaments, and elongate into spindles that associate with similarly shaped cells forming streams. The fact that p35 expression interrupts developmental progression at the round cell stage clearly separates Stage 1 from the changes in cell shape, cell migration, and ECM synthesis events that follow. In some cellular contexts caspase inhibition prevents cell migration independently of blocking apoptosis. It has been shown that the nuclei of wing cells cease to retain nuclear-targeted GFP and begin to fragment their DNA at what appears to be the round cell stage, consistent with the observation of pycnotic nuclei at this stage (Kiger, 2007).
Stage 3, migration and ECM synthesis
The cells migrate toward the hinge and into the body of the fly, leaving behind components, perhaps including tissue inhibitor of metalloproteinases, of an ECM that will bond dorsal and ventral cuticular surfaces. It is noteworthy that Timp deficiency does not interfere with cell migration. ECM assembly must be the final step in the developmental program. The nonautonomous action of Timp in bonding cuticle secreted by mutant Timp clones suggests that Timp is present in abundance and diffuses over large distances in the wing to participate in ECM formation (Kiger, 2007).
Precisely how ectopic expression of the various UAS transgenes studied in this paper produces wing blisters or collapsed wings is not wholly clear. It seems doubtful that cells that fail to delaminate during early phases of tissue remodeling would secrete ECM components normally. Yet a variable number of cells in these wings do delaminate and leave the wing, presumably because of variation in the level of Gal4-30A expression. These cells might be expected to secrete the necessary ECM components, although the level of critical component(s) may be insufficient for normal bonding to occur in some cases. Blister formation might also be caused by the presence of numbers of undelaminated cells physically preventing ECM from bonding the underlying cuticle. Note that when ectopic p35 expression is limited, a moderate number of round, delaminated cells can become bound in the wing without producing blisters (Kiger, 2007).
The presence of true hemocytes in the wing raises the question of whether these cells play a role in wing maturation. If Gal4-30A was to be expressed in these cells, as well as in epithelial cells, interpretation of ectopic expression studies would be complicated. No cells were detected expressing DsRedGFP fluorescence that did not express ywing-GFP fluorescence, suggesting that Gal4-30A is not expressed in true hemocytes. The observations that Hemese-Gal4-driven expression of Shaggy or of Pygo has no effect on wing maturation strongly suggest that the effects of Shaggy and Pygo on wing maturation are not mediated by true hemocytes exclusively, if at all. While the possibility that Timp and/or other ECM components are supplied by true hemocytes cannot be ruled out, the bulk of the evidence supports an active role for epithelial cells in bonding the wing surfaces. Precocious death of epithelial cells induced by Gal4-30A-driven expression of Ricin A in late pupal epithelial cells prevents bonding of dorsal and ventral cuticle after eclosion. Because the wing cuticle is fully formed, the induced cell death must have occurred after cuticle deposition but before eclosion. UV irradiation after eclosion blocks both epithelial cell delamination and bonding of the wing surfaces. In addition, it is clear that mitotic clones of defective epithelial cells affect bonding of the wing surfaces. Mitotic clones mutant for an integrin gene produce blisters in the wing cuticle as do mitotic clones ectopically expressing PKAc (Kiger, 2007).
These studies describe for the first time the developmental program that completes morphogenesis of the adult fly. The requirement for a normal state of Arm/β-catenin signaling activity suggests that an epithelial–mesenchymal transition (EMT) transforms epithelial cells into mobile fibroblasts in the wing (Kiger, 2007).
The best known example of an EMT in Drosophila is neuroblast delamination. In embryonic central nervous system formation, Wingless signaling has been shown to induce nonautonomously the delamination of specific neuroectoderm cells to form S2 neuroblasts. In peripheral nervous system formation, Wingless signaling is required for bristle formation at the wing margin, and ectopic expression of Wingless induces ectopic bristles in the wing blade. The ability of Wingless to induce neuroectoderm cells to form neuroblasts is tightly regulated by Notch in both the central and peripheral nervous systems. Evidence supports the idea that Notch modulates Wingless signaling by associating directly with Arm/β-catenin to regulate its transcriptional activity (Kiger, 2007).
Arm/β-catenin signaling appears to be characteristic of EMTs. Translocation of Arm/β-catenin into the nucleus precedes gastrulation in Drosophila, the sea urchin, and zebrafish. EMTs occur in the vertebrate neural crest when cells delaminate from the neural epithelium and migrate throughout the embryo. In the avian neural crest, dominant-negative forms of β-catenin and LEF/TCF inhibit delamination of cells from the epithelium, G1/S transition, and transcription of target genes. β-Catenin and LEF/TCF proteins are observed to translocate to the nuclei of avian neural crest cells only during delamination and to be absent during advanced stages of migration. EMTs are also a characteristic of cancer formation and can be initiated in some cancers by aberrant β-catenin activity (Kiger, 2007).
Multiple ways of activating Arm/β-catenin signaling exist. There are two independently regulated pathways that can target Arm/β-catenin to the proteasome, the Shaggy/Glycogen synthase kinase 3 degradation complex and the Seven in Absentia Homologue/ubiquitin ligase degradation complex. Multiple G-protein-coupled receptors target the Shaggy/Glycogen synthase kinase 3 degradation complex for inhibition. Further studies are necessary to identify the hormone(s), receptor(s) and signal transduction mechanisms acting in the wing maturation program and to relate this work to the extensive studies of the hormonal signals controlling wing expansion and cuticle tanning (Kiger, 2007).
- Model for the regulation of size in the wing imaginal disc of Drosophila
For animal development it is necessary that organs stop growing after they reach a certain size. However, it is still largely unknown how this termination of growth is regulated. The wing imaginal disc of Drosophila serves as a commonly used model system to study the regulation of growth. Paradoxically, it has been observed that growth occurs uniformly throughout the disc, even though Decapentaplegic (Dpp), a key inducer of growth, forms a gradient. This paper presents a model for the control of growth in the wing imaginal disc, which can account for the uniform occurrence and termination of growth. A central feature of the model is that net growth is not only regulated by growth factors, but by mechanical forces as well. According to the model, growth factors like Dpp induce growth in the center of the disc, which subsequently causes a tangential stretching of surrounding peripheral regions. Above a certain threshold, this stretching stimulates growth in these peripheral regions. Since the stretching is not completely compensated for by the induced growth, the peripheral regions will compress the center of the disc, leading to an inhibition of growth in the center. The larger the disc, the stronger this compression becomes and hence the stronger the inhibiting effect. Growth ceases when the growth factors can no longer overcome this inhibition. With numerical simulations it was shown that the model indeed yields uniform growth. Furthermore, the model can also account for other experimental data on growth in the wing disc (Aegerter-Wilmsen, 2007).
Since the wing imaginal disc serves as a model system to study the regulation of growth, a large amount of experimental data is already available. The model has been evaluated with experimental results from the literature. When clones with increased Dpp signaling are generated, they grow larger in the lateral regions than in the medial part. Furthermore, clones with decreased Dpp signaling survive better laterally than medially. A common explanation for these findings is that the medial cells are more competitive than the lateral cells because they receive higher levels of Dpp. Therefore, a clone with a fixed level of Dpp signaling is hindered more when growing in the medial part than when growing more laterally. The model may offer an additional, alternative explanation. A clone is stretched more and compressed less when growing laterally than when growing medially. Therefore, it grows faster laterally as long as its level of Dpp signaling is fixed. It is expected that both competition and differences in compression contribute to the difference of size among different clones (Aegerter-Wilmsen, 2007 and references therein).
Discs with homogeneous Dpp signaling are expanded along the dorsoventral boundary. According to the model, the total growth factor activity in these discs is highest along the dorsoventral boundary, thus accounting for the expansion along this boundary. Furthermore, it has been found that discs with homogeneous Dpp signaling do not show uniform growth. Instead the growth rate of cells in the lateral regions, close to the dorsoventral boundary, is higher than the growth rate of cells in the medial part of the disc. According to the model, the high growth factor activity along the dorsoventral boundary will promote additional growth along the whole boundary. This stretches the regions further away from the dorsoventral boundary. This stretching pulls the cells along the dorsoventral boundary toward the center of the disc. The cells in the center are thus being compressed. The closer the cells are located to the center, the more they are compressed and the more growth is inhibited, thus leading to the observed differences in growth rate (Aegerter-Wilmsen, 2007).
The Dpp pathway can be activated locally by expressing a constitutively active form of one of its receptors (tkvQ-D). Recently, it has been shown that activating the Dpp pathway in clones in this way can stimulate transient non-autonomous cell proliferation. When inhibiting the pathway, similar effects were seen. Clones with increased Dpp activity were modeled as a region with increased Dpp activity compared to its surrounding tissue with lower homogeneous Dpp activity. In that case, the cells with high Dpp signaling initially grow faster than the surrounding cells, thus stretching them. As in the wild-type situation at the start of growth, the stretching is highest in the cells closest to the region with high Dpp signaling and therefore growth is induced in these cells. This non-autonomous growth increases the stretching in the cells further away from the clone, which will increase their growth. Therefore, after some time, growth in the cells surrounding the clone will be homogeneous again, comparable with the situation in the wild type disc. Thus, the model accounts for the non-autonomous effect as well as for the observation that it only occurs transiently (Aegerter-Wilmsen, 2007).
Clones with decreased Dpp activity were modeled in a similar way. The cells surrounding the clone get stretched between the slow growing cells in the clone and the faster growing cells further away from the clone. Therefore growth is also induced non-autonomously in cells surrounding clones with decreased Dpp signaling, which is again in agreement with the data (Aegerter-Wilmsen, 2007).
Non-autonomous effects on cell proliferation were also assessed for clones in which growth is increased by overexpressing CyclinD and Cdk4 instead of by increased Dpp signaling. The non-autonomous proliferation was not observed in that case, even though this would in principle be expected based on the model. However, cell divisions are only slightly increased in these clones and apoptosis is increased, which is generally accompanied by basal extrusion. Therefore, it seems as if co-expression of CyclinD and Cdk4 causes only very little net overgrowth at the stage measured. For such clones the non-autonomous stimulation of proliferation is expected to be less pronounced and to occur at a relatively late point in time, which may explain why it has not been observed (Aegerter-Wilmsen, 2007).
Experimentally induced alterations in cell proliferation are often compensated for by changes in cell size, such that the final wing disc size is not changed. This suggests that wing disc size is not a function of cell numbers. In the model, the wing disc is considered as an elastic sheet with certain mechanical properties. As long as the mechanical properties of the tissue as a whole are not influenced by cell size, the final disc size is indeed not a function of cell numbers according to the model. Furthermore, according to the model, it would be expected that a reduction of growth in the center of the disc automatically leads to a reduction of growth in the peripheral regions. Indeed, when the size of the wing blade was decreased by down-regulating vestigial (vg) expression, non-autonomous reductions in surrounding WT territories were observed along all axes of growth. Lastly, the model predicts that stretching occurs in the peripheral regions. Therefore, it also predicts that, upon cutting the disc from the end toward the middle, tissue at both sides of the cut moves apart. In wound healing experiments, this was indeed observed. In contrast, the model predicts that the central region of the disc becomes compressed. The increased thickness of the (columnar layer of the) wing disc could be seen as an indication that compression indeed occurs (Aegerter-Wilmsen, 2007).
This paper has presented a model for the determination of final size in the wing imaginal disc. In the model, growth is negatively regulated by mechanical stresses, which are automatically generated as a result of growth rate differences in an elastic tissue. With the use of numerical simulations, it was demonstrated that the model naturally leads to uniform growth as was shown experimentally and that it leads to the observed final size of the wing disc. Furthermore, it was argued that the model can also account for other experimental data in literature (Aegerter-Wilmsen, 2007).
- Cell rearrangement and cell division during the tissue level morphogenesis of evaginating Drosophila imaginal discs
The evagination of Drosophila imaginal discs is a classic system for studying tissue level morphogenesis. Evagination involves a dramatic change in morphology and published data argue that this is mediated by cell shape changes. The evagination of both the leg and wing discs has been reexamined and it has been found that the process involves cell rearrangement and that cell divisions take place during the process. The number of cells across the width of the ptc domain in the wing and the omb domain in the leg decreases as the tissue extends during evagination and cell rearrangement was observed to be common during this period. In addition, almost half of the cells in the region of the leg examined divided between 4 and 8 h after white prepupae formation. Interestingly, these divisions were not typically oriented parallel to the axis of elongation. These observations show that disc evagination involves multiple cellular behaviors, as is the case for many other morphogenetic processes (Taylor, 2008).
This study established that cell rearrangement takes place during leg and wing evagination and contributes to the thinning and extension of the appendages. These observations are consistent with the pioneering results of Fristrom (1976) on evagination. The current data also established that cell rearrangement takes place throughout the appendage and is not restricted to a particular region along the proximal/distal axis. However, the observations are also consistent with cell rearrangement being non-uniform as some regions appeared to 'thin' more than others. For example, in the wing the width of the ptc domain at position M5 thinned more than at position M4 (refering to neuronal landmarks). The evaginating leg and wing cells retain their epithelial morphology with extensive apical junctional complexes. Rearrangement requires that cells change neighbors and hence must remove old junctions and generate new ones while maintaining tissue integrity. This problem is not restricted to evaginating discs but is a general one for epithelial tissues and is an issue that has concerned developmental/cell biologists for many years. Important insights into how this could be accomplished come from recent observations on germ band elongation in the Drosophila embryo. Several groups have provided evidence that junctional remodeling plays a key role in cell rearrangement in this epithelial tissue. This mechanism also appears to function in the repacking of pupal wing cells. It is suggested that it also plays a role in leg and wing evagination. No clear evidence is seen for the multicellular rosettes that have been implicated in germ band extension. Perhaps this is due to disc evagination being substantially slower than germ band extension (Taylor, 2008).
No evidence was seen of dramatic coordinated changes in cell shape. There was a small but significant increase in the length along the proximal/distal axis of evaginating omb domain tibia cells that should contribute to elongation. However, the change was not large enough to account for leg morphogenesis. No significant change was seen in cell shape in evaginating ptc domain wing cells although there was a hint of a possible small effect. It is worth noting that in these measurements cells from all positions along the relevant part of the proximal/distal axis were included. Casual observation suggested that there might be small regions with consistent changes but these would likely be counterbalanced by changes in shape elsewhere in the domain (Taylor, 2008).
It was not possible to image the earliest stages of leg disc evagination or the disc cells that form ventral thorax. Thus, these observations were not able to distinguish between the two proposed mechanisms of eversion (i.e., spreading vs. invasion hypotheses). Patterned cell death could in principle play an important role in disc evagination. Previous studies have not seen evidence for patterned cell death during wing blade evagination and the current observations support this conclusion. Cell death has been detected in evaginating legs but this is restricted to the regions of the tarsal segments where the leg joints form and hence is unlikely to contribute to the overall thinning of the omb domain of leg segments (Taylor, 2008).
Based on the literature, it was not expected that cell division takes place during evagination, but the current observations showed that it occurred. The most definitive experiments involved generating clones of cells marked by GFP expression and following these in vivo. These experiments provided compelling evidence for cell division. This was only done for the leg but other experiments provided strong evidence for cell division in evaginating wings. The size of wing clones was larger when they were induced at white prepupae than at the formation of the definitive pupae. Cell division was not rare in evaginating legs, and on average about 40% of the cells divided. Indeed, a majority of the cells divided in about 1/3 of clones examined. This amount of cell division is sufficient to account for the thickening of the omb domain that was observed from 6 to 8 h in developing legs. Observations on the size of wing clones suggested a similar fraction of wing cells divided during evagination. A limitation is that the in vivo imaging technique only allowed effective imaging of clones on the leg surface juxtaposed to the pupal case in the basitarsus and tibia (and occasionally tarsal) segments. Thus, data could not be obtained for much of the leg disc derivatives, and hence the overall proportion of evaginating leg cells that divide cannot be confidently estimated. The spindle in these dividing cells was not imaged but it was inferred that the spindle was not oriented parallel to the elongating axis, based on the position of the resulting daughter cells shortly after division. The two daughter cells usually filled up the area taken up by the parental cell prior to division, which helped in assigning a lineage. The leg epidermis is continuous without free 'space'. Hence, that daughter cells would occupy the space of the parental cell is not surprising. A parallel orientation for the spindle might be expected if the cell division plane was tightly linked to the mechanism of elongation. The inferred orientation of the cell divisions was most often between 46o and 60o. Thus, they would increase the number of cells both along the proximal/distal and anterior/posterior (and dorsal/ventral) axes. In the second day pupal leg, the width of the omb domain was narrower than it was in the evaginating leg. This could be a reflection of a later stage of convergent extension. However, legs were not followed throughout this period, other possibilities cannot be ruled out. It is interesting to note that cells in the pupal tibia and basitarsus have a spiral arrangement, and this appears to arise from 6 to 8 h after white prepupae. Thus, this arrangement could be at least in part a consequence of the orientation of the cell divisions (Taylor, 2008).
The fraction of dividing cells varied widely from one clone to another. This was not correlated with particular pupae or legs as both clones where a majority of the cells divided and clones where no cells divided were found in the same pupae and on the same leg. One possibility is that the variation is due to region specific differences. For example, cells in one region of the leg might never divide during evagination while a majority of cells in another region might always divide. No evidence is seen for this but the experiments were not compelling on this point. The observations on the omb domain did not examine a majority of leg cells and in the experiments where MARCM clones were followed, it could not be routinely said exactly where on the leg a clone was located. A second possibility is that the variation is due to the clustered distribution of S phase and mitotic cells in wing and leg discs. Any small clone could comprise a cluster (or not contain a cluster) and this could lead to a great deal of variation in observed cell division. The basis for the clustering is uncertain but could simply represent a pseudo-synchronization due to neighboring sister cells having been born at the same time (Taylor, 2008).
The observations suggest that several different factors play a role in evagination. At the start of evagination, the leg and wing discs are folded and some of the initial elongation is due to an unfolding of the tissue that presumably results from changes in the shape of cells along the apical/basal axis. During the period when leg discs evert and present the apical surface of their epithelial cells to the outside, elongation is also taking place and there is active pulsatile movement. This appears to be related to the movement of hemolymph in the prepupae and blood cells can often be seen to move in step with the pulses. This suggests that hydraulic pressure could be playing a role in eversion and elongation. The leg resembles a cylinder closed on one side (distal tip) and open to the body on the other (proximal). Thus, it is expected that hemolymph is pumped by the heart to produce a mechanical force that could help evert and/or elongate the leg. The pulsatile movement starts to decrease at about 4-4.5 h after white prepupae and largely ends by about 5 h. This is around the time of eversion, but the slowing clearly precedes eversion. It is suggested that the hydraulic pressure of the hemolymph helps drive the early stages of evagination, when the leg is short and unfolding of the tissue plays a major role. It is possible that after this time the increased leg length or increased leg stiffness limits the effectiveness of hemolymph hydraulic pressure. Alternatively, it is possible that there is a decline in the hydraulic pressure due to changes in heart pumping or other prepupal events. The lack of hydraulic pressure may be one reason for the less than optimal evagination of discs seen during in vitro culture (Taylor, 2008).
Mutations in many Drosophila genes result in changes in appendage morphology. It is expected that some of these produce their phenotype by interfering with the observed cell rearrangement. A particularly interesting candidate for such a gene is dachsous (ds), which encodes a large protein with many cadherin domains. Mutations in this gene result in shorter fatter wings and legs with an altered distribution of cells (e.g. an increase in the number of cells along the anterior posterior axis of the wing and a decrease in the number of cells along the proximal/distal axis). However, mutations in this gene are known to alter disc patterning and growth and this may be the cause of the altered shape (Taylor, 2008).
Another group of interesting candidate genes for altering cell rearrangement in evaginating legs is the cellular myosin encoded by zipper and the interacting Sqh (myosin regulatory light chain) and RhoA proteins. Mutations in these genes give rise to a crooked leg phenotype that has been interpreted as being due to the mutations altering cell shape. However, myosin has been implicated in the junctional remodeling associated with cell rearrangements in the extending germ band and it is possible that the leg phenotype is also due to an effect on junctional remodeling required for cell rearrangement. One of the interesting properties of extending germ band cells is the planar polarization of membranes so that the anterior/posterior edges of cells are distinct from the dorsal/ventral edges of cells in their content of proteins such as myosin. No evidence was seen for this in prepupal legs and wings but this point deserves further study as it is possible the experimental conditions were not favorable for seeing this (Taylor, 2008).
- Cell competition, growth and size control in the Drosophila wing imaginal disc
This paper reports experiments aimed at understanding the connections between cell competition and growth in the Drosophila wing disc. The principal assay has been to generate discs containing marked cells that proliferate at different rates and to study their interactions and their contribution to the final structure. It is known that single clones of fast-dividing cells within a compartment may occupy the larger part of the compartment without affecting its size. This has suggested the existence of interactions involving cell competition between fast- and slow-dividing cells directed to accommodate the contribution of each cell to the final compartment. This study shows that indeed fast-dividing cells can outcompete slow-dividing ones in their proximity. However, it is argued that this elimination is of little consequence because preventing apoptosis, and therefore cell competition, in those compartments does not affect the size of the clones or the size of the compartments. These experiments indicate that cells within a compartment proliferate autonomously at their own rate. The contribution of each cell to the compartment is exclusively determined by its division rate within the frame of a size control mechanism that stops growth once the compartment has reached the final arresting size. This is supported by a computer simulation of the contribution of individual fast clones growing within a population of slower dividing cells and without interacting with them. The values predicted by the simulation are very close to those obtained experimentally (Martín, 2009).
The main objective of this work was to study the role of cell competition in regulating growth and size in the wing disc of Drosophila. As the disc is composed of two (A and P) compartments, which behave as independent units of size control, these experiments refer to mechanisms operating within compartments (Martín, 2009).
To make some precise statements about the growth dynamics of the disc several developmental parameters were calculated, some of which had not been described in detail in previous publications. According to the data, the wing disc starts growth with about 55 cells, of which 34 would belong to the A and 21 to the P compartment. The final cell number is around 30,000 (about 19,000 A and 11,000 P). The total number of cell divisions is about 9.1. These estimates coincide well with previous ones, although the final number of 30,000 cells is somewhat lower than previous measurements (Martín, 2009).
This study has shown that the growth rate of the wild-type wing disc changes markedly through development: during the second larval period wild type (M+) cells divide at about 5.5-5.7 hours per cycle, then the length of the cell division cycle increases as development progresses, and in the second half of the third larval period it is as high as 30 hours. Thus most of the growth occurs during the second and early third larval period. A similar growth pattern is found in developmentally delayed M/+ discs, the cell doubling time (CDT) of which increases from 10-12 hours in the second larval period to 34-40 at the prepupal stage. It is not surprising that the major difference between wild-type and M/+ discs occurs during the early periods. Possibly the metabolic demand is greater in fast-proliferating cells and therefore the limitation in protein synthesis of M/+ cells is more noticeable (Martín, 2009).
Normally there is little apoptosis in the wing disc; therefore, cell competition cannot have a major role in normal circumstances. Nevertheless, it may be a safeguard mechanism to eliminate abnormal cells or to deal with unusual situations such as cells with different division rates, which may interfere with the growth of the disc. The significance was examined of these events of cell competition in the overgrowth of M+ clones, and in the control of compartment size (Martín, 2009).
The fact that M+ clones growing in M/+ discs can reach an average size more than ten times their normal size (when they grow in a M+ disc) while not altering the final size of the compartment, suggested that both clone overgrowth and size control may depend on cell competition. In this view the M+ clones would overgrow at the expense of the elimination of neighbouring M/+ cells. Moreover, the removal of the latter would ensure that they do not produce progeny that would give rise to an excess of cells in the compartment (Martín, 2009).
However, the experiments indicate that cell competition does not play a significant role in these processes; in the absence of cell competition, the M+ clones are able to overgrow as much as in the normal situation. Besides, the size of compartments is not affected by the presence of these clones, even though they can occupy the larger part of the compartment (Martín, 2009).
The authors believe that the key element to explain these results is the mechanism that controls the overall size of the compartment and that arrests growth once it has reached the final dimension. This mechanism would function without regard to the lineage of the cells or of their relative contribution to the final structure. It would also function autonomously in each compartment (Martín, 2009).
The authors ask us to consider a compartment containing M+ and M/+ cells from early in development. The cells proliferate at the rate dictated by their metabolic activity, according to their Minute genotype, and their division rate is not affected by interactions with neighbour cells. Because of their proliferation advantage the M+ cells occupy a large part of the compartment. In principle, if the M/+ cells were to proliferate at their normal rhythm for the whole duration of the M/+ larval period, the sum of the contribution of the M+ and M/+ cells would produce an excess of tissue. The reason why this is not the case is that the size control mechanism arrests growth as soon as the final size has been reached. In the presence of a large M+ clone the final arresting size of the compartment is reached earlier than in a one entirely made of M/+ cells. For this reason the M/+ cells contribute less than they would have in the absence of a M+ clone. That is, the developmental delay associated with the M/+ condition is partially abolished by the presence of the M+ clone. This is predicted by the computer simulation and is supported by the results. Using the expression of the vg and wg genes to monitor the developmental stage of the compartment, it was found that compartments with M+ clones are ahead and are therefore expected to reach the final arresting size earlier than those that are entirely M/+ (Martín, 2009).
The existence of a non-cell-autonomous mechanism governing the growth of the compartment is also suggested by observations about vg expression in compartments containing M+ clones. M+ clones can sometimes split the vg domain into M+ and M/+ territories. The significant finding is that both territories show the same pattern of expression. This very strongly suggests that the control of vg expression is determined by an overall mechanism probably measuring the size of the compartment in each moment and regardless of the individual lineages (Martín, 2009).
What, then, is the role of cell competition in regulating growth and size in the wing disc? Cell competition results from interactions between two types of viable cells and causes the elimination of one of them. That is, it is a mechanism to remove viable cells that are not developmentally adapted to the growing tissue. Unlike other situations that cause apoptosis, the 'loser' cells in the competition process are not necessarily damaged; they are poor competitors. In the cases reported here, it is the relatively slow proliferating cells that are eliminated, which may have a beneficial effect on the general fitness of the disc. Nevertheless, there may be other safeguard functions of greater biological significance. Cell competition may be instrumental in removing viable but developmentally abnormal cells, which, for instance, do not interpret developmental cues correctly. This would include tumour or transformed cells that may arise in development. The process would ensure that tumour cells would normally be outcompeted by non-tumour cells. In certain circumstances, however, the acquisition by the latter of some additional property may turn tumour cells into super-competitors, thus reversing the situation. It has been argued that cell competition may be a major factor in tumour progression in circumstances in which tumour cells are able to outcompete normal cells (Martín, 2009).
- The Drosophila wing hearts originate from pericardial cells and are essential for wing maturation
In addition to the heart proper, insects possess wing hearts in the thorax to ensure regular hemolymph flow through the narrow wings. In Drosophila, the wing hearts consist of two bilateral muscular pumps of unknown origin. This paper presents the first developmental study on these organs and reports that the wing hearts originate from eight embryonic progenitor cells arising in two pairs in parasegments 4 and 5. These progenitors represent a so far undescribed subset of the Even-skipped positive pericardial cells (EPC) and are characterized by the early loss of tinman expression in contrast to the continuously Tinman positive classical EPCs. Ectopic expression of Tinman in the wing heart progenitors omits organ formation, indicating a crucial role for Tinman during progenitor specification. The subsequent postembryonic development is a highly dynamic process, which includes proliferation and two relocation events. Adults lacking wing hearts display a severe wing phenotype and are unable to fly. The phenotype is caused by omitted clearance of the epidermal cells from the wings during maturation, which inhibits the formation of a flexible wing blade. This indicates that wing hearts are required for proper wing morphogenesis and functionality (Tögel, 2008).
Unlike in vertebrates, where an elaborate closed blood vessel system extends throughout the whole body, insects possess only one vessel, the tubular heart, in their otherwise open circulatory system. Once the hemolymph has left the heart, it moves freely between the internal organs and can not be directed into narrow body appendages such as antennae, legs or wings. To ensure sufficient hemolymph supply of these appendages additional circulatory organs evolved (Pass, 2000; Pass, 2006). In Drosophila, circulation in the wings is maintained by the so-called wing hearts (Krenn, 1995), a pair of autonomous muscular pumps located bilaterally in the scutellum, the dorsal elevation of the second thoracic segment. Due to this location, they are also referred to as scutellar pulsatile organs. Although known for many years, no developmental studies on the origin or morphogenesis of these organs have been performed. Probably, this was due to the lack of available methods to track their differentiation. However, studies on the origin of the thoracic somatic muscles in Drosophila and comparative anatomical investigations in insects suggested that the wing hearts originate from the cardiac mesoderm or from the heart itself (Tögel, 2008).
A previous study identified an enhancer region of the Drosophila hand gene that is able to drive reporter gene activity in the wing hearts (Sellin, 2006). In the present work, this reporter was used to identify the embryonic anlagen of the wing hearts and to elucidate the dynamics of their postembryonic development with in vivo time lapse imaging. It was found that the anlagen of the Drosophila wing hearts indeed derive from the cardiac mesoderm but, astonishingly, not from the muscular cardioblast lineage. Instead, they represent a so far undescribed subpopulation of the well-known Even-skipped (Eve) positive pericardial cells (EPCs) (Tögel, 2008).
In addition to their unknown origin, little is known about the contribution of wing hearts to wing morphogenesis and functionality. After eclosion, wings are unfolded by a sudden influx of hemolymph and subsequently undergo maturation. During this process, the epidermal cells that until then bonded the dorsal and ventral wing surfaces enter programmed cell death, delaminate from the cuticle, and disappear into the thorax (Kimura, 2004). Subsequently, the cuticles of the intervein regions become tightly bonded to form a flexible wing blade, while the cuticles of the vein regions form tubes, lined by living cells, through which hemolymph circulates in mature adult insects. Measurements of hemolymph flow in adult butterflies showed that wing hearts function as suction pumps that draw hemolymph out of the wings starting shortly after wing unfolding. Whether wing hearts might play a role in wing maturation was tested by generating flies lacking wing hearts. The findings demonstrate that the delaminated epidermal cells are removed from the wings by the hemolymph flow generated by the wing hearts. Loss of wing heart function leads to remains of epidermal cells resting between the unbonded dorsal and ventral wing surfaces which results in malformation of the wing blade and flightlessness. It is concluded that wing hearts are essential for wing maturation and, thus, for acquiring flight ability in Drosophila (Tögel, 2008).
A hand-C-GFP reporter was generated (Sellin, 2006) that reflects the described hand expression pattern and was found to be active in wing hearts. To confirm that the hand-C-GFP reporter is expressed in all cells of mature wing hearts, their morphology was examined based on the signal from the reporter in conjunction with histological sections. In the adult fly, wing hearts are located at the lateral angles of the scutellum, which are joined to the posterior wing veins by cuticular tubes. Each organ is curved in anterior–posterior direction as well as dorso-ventrally. It consists of about 7-8 horizontally arranged rows of prominent muscle cells, which are attached at their proximal side to a thin layer of cells that has a greater dorsal extension than the muscle cells. Both cell types are labeled by the reporter. The fine acellular strands that hold the wing hearts to the adjacent epidermal cells were not observed to be marked by the reporter. 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).
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Genes involved in organ development
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