Achaete regulation in imaginal discs: Role of wingless
armadillo, dishevelled, and shaggy encode elements of a unique 'wingless signalling pathway' that is used several times throughout development. cut and achaete are targets of Shaggy signalling in the wing margin region, reflecting the activity of wingless and probably mediating its function. achaete is negatively regulated by the wingless pathway. The functional relationship between these genes and wg is the same as that which exists during the patterning of the larval epidermis (Couso, 1994).
In Drosophila wing imaginal discs, the Wingless (Wg) protein acts as a morphogen, emanating from the dorsal/ventral (D/V) boundary of the disc to directly define cell identities along the D/V axis at short and long range. High levels of a Wg receptor, Drosophila frizzled 2 (Dfz2), stabilize Wg, allowing it to reach cells far from its site of synthesis. Wg signaling represses Dfz2 expression, creating a gradient of decreasing Wg stability moving toward the D/V boundary. This repression of Dfz2 is crucial for the normal shape of Wg morphogen gradient as well as the response of cells to the Wg signal. In contrast to other ligand-receptor relationships where the receptor limits diffusion of the ligand, Dfz2 broadens the range of Wg action by protecting it from degradation. Slender and chemosensory bristle cell fates are determined during the third larval instar by proneural genes such as ac, whose expression is wg-dependent. ac is initially expressed at mid-third instar in the anterior compartment in a stripe on each side of the D/V boundary. As was found for ac, the expression domain of another short-range target, Delta (Dl), normally expressed in a narrow stripe on either side of the wg stripe, is much broader in Dfz2 mutant wing discs. Thus, Dfz2-mediated stabilization of Wg can, in large part, explain the biphasic nature of the Wg morphogen gradient. This work raises additional questions regarding Wg function (Cadigan, 1998).
In developing organs, the regulation of cell proliferation and patterning of cell fates is coordinated. How this coordination is achieved, however, is unknown. In the developing Drosophila wing, both cell proliferation and patterning require the secreted morphogen Wingless at the dorsoventral compartment boundary. Late in wing development, Wg also induces a zone of non-proliferating cells at the dorsoventral boundary. This zone gives rise to sensory bristles of the adult wing margin. How Wg coordinates the cell cycle with patterning has been investigated by studying the regulation of this growth arrest. Wg, in conjunction with Notch, induces arrest in both the G1 and G2 phases of the cell cycle in separate subdomains of the zone of non-proliferating cells (ZNC). The ZNC is composed of three subdomains, each about four cells wide. Cells in the central domain express wg. This domain is flanked by dorsal and ventral domains, which, in the anterior compartment, express Achaete and Scute. Cells in the ZNC stop proliferating 30 h before most of the other cells in the disc but re-enter the cell cycle for two or three divisions after pupariation. This arrest is seen by an absence of cells in the S phase of mitosis. The domain architecture of the ZNC is suggested by the expression of string and the G2 cyclins A and B. In the anterior compartment, cells in the dorsal and ventral domains do not express STG messenger RNA but accumulate high leves of G2 cyclins in the cytoplasm. Since Stg is required for mitosis and Stg and the G2 cyclins are degraded at cell division, these patterns are indicative of arrest in G2. In contrast, in the central domain CycA and CycB proteins are undetectable, but STG mRNA is expressed. This indicates that these cells may be arrested in G1. G1 arrest may be due to inactivation of dE2F, a factor required to activate the transcription of genes needed for DNA replication (Johnston, 1998).
Loss of wingless function during disc development abolishes both the G1 and G2 arrests and allows string expression in the anterior dorsal and ventral domains. Four observations suggest that the proneural genes achaete and scute regulate the G2 arrest of the ZNC:
Together, these results indicate that Wg induces G2 arrest in two subdomains by inducing the proneural genes achaete and scute, which downregulate the mitosis-inducing phosphatase String (Cdc25). Notch activity creates a third domain by preventing arrest at G2 in wg-expressing cells, resulting in their arrest in G1. To test whether Notch directly regulates the G1 arrest, discs were constructed lacking Wg activity, but expressing activated Notch in the ZNC. These discs do not form a ZNC at all. Thus, in the absence of Wg activity, Notch is not sufficient to induce a G1 arrest. It is noted that the string promoter contains putative Ac/Sc-binding sites, indicating that these basic helix-loop-helix proteins can repress string expression directly (Johnston, 1998).
wingless is required for the expression of the proneural ac-sc complex genes and the subsequent formation of the sensory bristles along the margin. In the developing wing margin of Drosophila, wingless is normally expressed in a narrow stripe of cells adjacent to the proneural cells that form the sensory bristles of the margin. Loss of scute expression and of sensory precursors was observed if clones substantially included the normal region of wingless expression. These 'anti-proneural' phenotypes were associated with the loss of wingless expression, a loss that may be partially or wholly responsible for the anti-proneural phenotype. The role of Notch in the regulation of wingless expression precedes the requirement for lateral inhibition in proneural cells. Furthermore, overexpression of wingless with a heat shock-wingless construct rescued the loss of sensory precursors associated with the early loss of Notch (Rulifson, 1995).
Demonstrating an opposite effect of Wingless in the eye than in the wing, ectopic expression of wingless results in a reduced expression of achaete, and blockage of sensory organ precursor formation. Interommatidial bristles are mechanosensory organs composed of four cells that are derived from a single sensory organ precursor. The process involves achaete and scute. achaete expression is greatly reduced when wingless is expressed in the eye disc. The effect of Wingless is non-cell autonomous, and wingless signaling in the eye involves the same components as in the wing: porcupine, dishevelled, shaggy and armadillo. As in the wing, Wingless can still regulate achaete expression in homozygous clones for a Notch null allele. Thus, a direct role for Notch (as a receptor, for example) in wingless signaling is unlikely. It is unknown whether the effects of Wingless on achaete are direct, or whether wingless signaling modifies negative inputs from other bHLH proteins such as Extramachrocheate and Hairy (Cadigan, 1996).
Short-range interaction between dorsal and ventral (D and V) cells establishes an organizing center at the DV compartment boundary that controls growth and specifies cell fate along the dorsal-ventral axis of the Drosophila wing. The secreted signaling molecule Wingless (WG) is expressed by cells at the DV compartment boundary and has been implicated in mediating its long-range patterning activities. Does WG acts directly at a long-range to specify cell fates in the wing? To investigate this question, mutant clones of two components of the WG transduction pathway, dishevelled and armadillo were examined. Cells mutant for dsh show reduced levels of Dll and vg expression. Cells mutant for a temperature sensitive hypomorphic allele of arm, likewise show loss of expression of Dll and vg when larvae are shifted to non-permissive temperatures. Reducing WG levels at the margin reduces both the maximum level of Dll expression near the DV boundary and the distance from the DV boundary at which Dll can be activated. An intermediate level of WG activity is not sufficient to support the specification of wing margin bristles, suggesting that WG has fallen below a critical threshold for activation of AS-C gene expression, while remaining above the respective thresholds of activation for both Dll and vg. Thus, WG acts directly, at long range, to define the expression domains of its target genes, Distal-less and vestigial. Expression of the Achaete-scute genes, Distal-less and vestigial at different distances from the DV boundary is controlled by WG in a concentration-dependent manner, with AS-C requiring the highest levels of WG. Dll, expressed in a wider range, requires the next highest level, and vg, which is expressed across the entire wing pouch, requires the lowest levels. It is proposed that WG acts as a morphogen in patterning the D/V axis of the wing (Neumann, 1997).
Unlike the leg, Dll null clones can be recovered anywhere in the wing even when they are generated early in development. The primary effect of these clones is on the differentiation of the wing margin. The characteristic hairs or bristles found at the margin are deleted or rudimentary in Dll null clones located at the margin. The effect of these clones is found to be autonomous so that, for example, a clone situated only on the ventral side of the margin will not affect the adjacent margin bristles or hairs in the dorsal region. In the mature wing disc, Dll is expressed in the wing pouch in a graded fashion centered on the wing margin and appears to be downstream of wg in this appendage. Its function in the wing is quite distinct from that in the leg. It is not required for growth and axis formation in the wing, because a wing disc in which Dll has been almost completely removed by clones is morphologically normal. Additionally, it does not appear to affect cell adhesion in the wing because Dll clones generated early in development can be recovered anywhere in the adult wing. However, these clones do have distinct phenotypes, the most striking being an autonomous deletion of the bristles and hairs normally found at the wing margin. The margin is characterized by the expression of a number of genes including wg, cut and achaete: wg and cut are expressed normally in Dll clones in the wing; this is not surprising because wg appears to be upstream of Dll and the phenotype of wg and cut mutations in the wing is more severe than Dll. However, ac expression at the margin is absent in cells lacking Dll, showing that Dll is required for the normal differentiation of the wing margin (Campbell, 1998).
Two types of sensory organs, large bristles (macrochaetes) and small bristles (microchaetes), develop in fixed numbers at constant positions on the dorsal part of the mesothorax (also called the notum) of Drosophila. The accurate positioning of the macrochaetes is established within the epithelial sheets of the notum region of the wing imaginal discs during the third larval to early pupal stage. For convenience, this region of the wing disc will be referred to as the 'thoracic disc' to distinguish it from the wing pouch region. Initially, in the thoracic disc, group of cells (termed proneural clusters) are formed and characterized by the expression of the proneural genes achaete (ac) and scute (sc). These proneural clusters form around the positions where macrochaetes will form. Next, one or a few sensory mother cells (SMCs) are singled out from the proneural cluster, and each SMC subsequently undergoes two rounds of cell division to form four progeny cells that differentiate into the components of a sensory bristle. Thus, precise positioning of the macrochaete on the notum depends on the complex expression pattern of the ac and sc genes in the thoracic disc. ac and sc expression patterns are controlled through the action of enhancer-like cis-regulatory elements. These elements are presumed to respond to a prepattern established by local specific combinations of factors. The identity of these pattern producing factors is largely unknown (Tomoyasu, 1998).
Two large bristles, an anterior-dorsocentral bristle (aDC) and a posterior-dorsocentral bristle (pDC) are formed along the anterior/posterior (A/P) axis on the notum. It has been shown that wg activity is necessary for the formation of both aDC and pDC. Wingless is expressed in an anterior-dorsal (medial) to posterior-ventral (lateral) stripe in the thoracic discs. However, the SMCs are not induced all along the wg expression domain, but induced only adjacent to the dorsal posterior side, behind the wg expression domain in the anterior compartment of the thoracic disc. This suggests that Wg signaling alone is insufficient to induce SMCs in aDCs and pDCs, and that another factor(s), which resides on the dorsal posterior side of the thoracic disc, is also required for inducing these SMCs. One candidate factor is Dpp. In the thoracic disc, Dpp is induced in a stripe of cells located posterior to the dorsocentral SMCs. This expression pattern and the property of Dpp as a morphogen suggests that Dpp signaling may also participate in prepattern formation of the macrochaetes on the notum (Tomoyasu, 1998).
The role of Dpp signaling in dorsocentral bristle formation has been examined by either ectopically activating or conditionally reducing Dpp signaling. Ubiquitous activation of Dpp signaling in the notum region of the wing imaginal disc induces additional dorsocentral proneural clusters all along the dorsal side of the wg expression domain, and alters wg expression. Conditional loss-of-function of Dpp signaling during disc development results in the inhibition of dorsocentral proneural cluster formation and expansion of the wg expression domain. These results suggest that Dpp signaling has two indispensable roles in dorsocentral bristle formation: induction of the dorsocentral proneural cluster in cooperation with Wg signaling and restriction of the wg expression domain in the notum region of the wing imaginal disc (Tomoyasu, 1998).
There is a substantial distance between dorsocentral SMCs and the dpp expression domain in wild-type discs. One explanation for the existence of this gap is that the highest level of Dpp signaling inhibits the formation of proneural clusters. A down shift of the Dpp activity slope would release the area in which proneural induction is inhibited by the highest levels of Dpp signaling. The mechanism by which the highest levels of Dpp signaling inhibits proneural induction is unclear and should be studied at the molecular level. It is worth noting that the effective range of wg from its source for proneural cluster induction seem to be different from that of dpp. The dorsocentral proneural cluster is formed within approximately five cell diameters from the wg expression domain, whereas it can be formed more than ten cell diameters from the dpp source. This difference must contribute to the oval shape of the proneural cluster, which is longest along the A/P axis. wg expression is not uniform in the notal stripe: it is lower at the A/P compartment border. It is possible that the difference in wg expression levels along the A/P axis also affects the precise positioning of the dorsocentral proneural cluster (Tomoyasu, 1998).
Wingless and Decapentaplegic cell signaling pathways act synergistically in their contribution to macrochaete (sense organ) patterning on the notum of Drosophila. The analysis of the origin of sense organ precursor prepatterning has focused on the specification and positioning of the anterior and posterior dorsocentral macrochaetes (aDC, pDC) two large mechanosensory organs located in precise positions relative to surrounding rows of microchaetes. The aDC and pDC SOPs form sequentially on the proximal edge of a single DC proneural cluster where Achaete and Scute expression depends on a cis-activating enhancer sequence, the DC enhancer. Ac expression in the DC proneural cluster requires the activity of wingless. The DC SOPs form adjacent to the stripe of cells expressing wg in the presumptive notum during the third larval instar. To probe the nature of gene interaction required for macrochaetae formation, the Wingless-signaling pathway was ectopically activated by removing Shaggy activity (the homolog of vertebrate glycogen synthase kinase 3) in mosaics. Proneural activity is asymmetric within the Shaggy-deficient clone of cells and shows a fixed polarity with respect to body axis, independent of the precise location of the clone. This asymmetric response indicates the existence in the epithelium of a second signal, possibly Decapentaplegic. Ectopic expression of Decapentaplegic induces extra macrochaetes only in cells that also receive the Wingless signal. Outside the Wg-activated domain, in the medial scutum and prescutum, clones that ectopically express Dpp make only microchaetes. In the Wg-activated domain, within and lateral to the DC meridian, clones of cells ectopically expressing dpp are associated with many extra macrochaetes, which are formed both within and around the Dpp-expressing clones. It is concluded that in areas of the notum where the WG transduction pathway is inactive, Dpp alone is insufficient for macrochaete formation. Activation of Hedgehog signaling generates a long-range signal (Dpp) that can promote macrochaete formation in the Wingless activity domain. This signal depends on decapentaplegic function. Autonomous activation of the Wingless signal response in cells causes them to attenuate or sequester this signal. Extramacrochaetae (a proneural antagonist) is required to limit the anterior/posterior extent of this cluster. If the level of emc is reduced, extra macrochaetes form primarily anterior but also posterior to the normal DCs along the proximal edge of the wg stripe. Further reduction of emc results in additional extra macrochaetes along the dorsal edge of the stripe. These results suggest a novel patterning mechanism that determines sense organ positioning in Drosophila (Phillips, 1999).
On each half of the dorsal mesothorax (heminotum), 11 large bristles (macrochaetae) occupy precisely constant positions. The location of each macrochaeta is specified during the third instar larval and early pupal stages by the emergence of its precursor cell (sensory mother cell: SMC) at a precise position in the imaginal wing discs, the precursors of the epidermis of most of the mesothorax and wings. The accurate positioning of SMCs is thought to be the culmination of a multistep process in which positional information is gradually refined. The GATA family transcription factor Pannier and the Wnt secreted protein Wingless are known to be important for the patterning of the notum. Thus, both proteins are necessary for the development of the dorsocentral mechanosensory bristles. Pannier has been shown to directly activate the proneural genes achaete and scute by binding to the enhancer responsible for the expression of these genes in the dorsocentral proneural cluster. Moreover, the boundary of the expression domain of Pannier appears to delimit the proneural cluster laterally, while antagonism of Pannier function by U-shaped, a Zn-finger protein, sets its limit dorsally. Therefore, Pannier and U-shaped provide positional information for the patterning of the dorsocentral cluster. In contrast and contrary to previous suggestions, Wingless does not play a similar role, since the levels and vectorial orientation of its concentration gradient in the dorsocentral area can be greatly modified without affecting the position of the dorsocentral cluster. Thus, Wingless has only a permissive role on dorsocentral achaete-scute expression. Evidence is provided indicating that Pannier and U-shaped are main effectors of the regulation of wingless expression in the presumptive notum (Garcia-Garcia, 1999).
An enhancer that directs expression specifically at the DC proneural cluster is present within a 5.7 kb fragment of AS-C DNA. Different subfragments were assayed for enhancer activity in vivo. A 1.4 kb subfragment (AS1.4DC) directs lacZ transcription from a minimal hsp70 promoter in the DC proneural cluster: beta-galactosidase and Scute endogenous accumulations precisely colocalized at this cluster. This fragment and the corresponding region of the AS-C from D. virilis were sequenced. Stretches of conserved DNA were present throughout the fragment, although they appeared to cluster within three regions. Subfragments containing each one of these regions were assayed for DC enhancer activity. Only the most 3' subfragment (PB0.5DC) shows such an activity, but to a much lesser extent than AS1.4DC. Interestingly, the activity is usually limited to only one cell, which is the posterior DC SMC. However, when assayed with the sc promoter, the PB0.5DC fragment directs lacZ activity in most cells of the DC cluster. Consequently, the sequences essential for specifying transcription in the DC cluster are contained within the PB0.5DC subfragment, although additional sequences that reinforce this expression are present in the larger AS1.4DC fragment. The AS1.4DC fragment was used to study DC enhancer activity (Garcia-Garcia, 1999).
The Pnr protein, which is a GATA-1 transcription factor, is known to regulate ac-sc expression at the DC cluster by acting directly or indirectly through the DC enhancer. The sequence of AS1.4DC was examined: within it, seven putative GATA-1 factor binding sites were found. Three of them fit the vertebrate consensus sequence (WGATAR: sites 1, 2 and 4); three comply with the consensus obtained in a random oligonucleotide selection experiment performed with Pnr protein (GATAAG: sites 3, 5 and 6), and one fits both consensus sequences (site 7). Interestingly, sites 5, 6 and 7 are within the PBO.5DC subfragment and two of them are conserved in D. virilis. Site-directed mutagenesis of site 7 strongly decreases enhancer activity of the AS1.4DC-lacZ construct (abbreviated DC-lacZ). Additional mutagenesis of other sites displaying the vertebrate consensus does not further reduce the residual activity. However, mutagenesis of all seven sites completely abolishes activity. These data suggest that Pnr interacts with some of these sites and that this interaction is essential for DC-lacZ activity. The capacity of Pnr to activate transcription of an AdhCAT reporter gene linked to either the complete AS1.4DC enhancer fragment or to each of the three subfragments was tested in transfection assays performed in chicken embryonic fibroblast (CEF) cells. Pnr stimulates transcription to similar levels from the complete enhancer and from subfragment PB0.5DC. In contrast, no stimulation was detected with the other subfragments. Notably, PB0.5DC displays DC enhancer activity in flies and contains three putative Pnr-binding GATA sites. Mutagenesis of only one of these (site 7) does not affect AdhCAT activity. But simultaneous removal of two sites (either sites 5 and 7, or 6 and 7) strongly impairs activity and mutagenesis of all three sites essentially abolished it. This suggests that a minimum of two GATA sites are necessary for transcriptional activation. Further evidence for a direct interaction of Pnr with the GATA sites of the enhancer was obtained in electrophoretic mobility-shift assays (EMSA) conducted with two different GST-Pnr fusion proteins that included the DNA binding domain of Pnr. Additonally, it has been shown that although relatively high levels of Wg protein are necessary for full DC-lacZ activity, the precise levels of this protein and the orientation of its gradient do not convey information for the position and the shape of the DC cluster (Garcia-Garcia, 1999).
A model is provided for the dorsal-lateral patterning of the DC area by Pnr and Ush. In the third instar wing disc and in the dorsalmost part of the prospective notum, Ush is present at high concentrations and the Pnr/Ush heterodimers are relatively abundant. These heterodimers would act as repressors and prevent activation of downstream genes. In the DC area, defined along the dorso-lateral axis by lower concentrations of Ush and the presence of Pnr, there is sufficient free Pnr to activate genes like ac-sc, DC-lacZ and wg. ac-sc is transcribed in the more dorsal part of the area because its activation requires relatively high concentrations of Pnr. wg is only transcribed at the edge of the Pnr domain because its expression is very sensitive to both Pnr and Pnr/Ush, and consequently low concentrations of the former are sufficient for activation and low concentrations of the latter, even in the presence of high concentrations of free Pnr, impose repression. The inability of extra doses of the activator Pnr to revert the repression by Pnr/Ush in the dorsalmost region of the notum suggests that activator and repressor do not compete for overlapping sites at the DC as-sc and notal wg enhancers. The presence of Pnr/Ush at their site(s) would block the activating effect of bound Pnr. Additional inputs, notably decapentaplegic, are known to act on the DC enhancer (Garcia-Garcia, 1999).
Achaete regulation in imaginal discs: Role of genes other than wingless
Continued: Achaete Transcriptional regulation part 3/3 | back to part 1/3
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