ABSTRACT
Patterning and proliferation are coordinately controlled in the development of Drosophila imaginal discs. Localized expression of decapentaplegic (dpp) at the anterior-posterior and wingless (wg) at the dorsal-ventral compartment boundaries controls growth of the wing with respect to the A/P and D/V axes. The growth-promoting effects of these organizers are thought to be indirect, since growth is dispersed throughout the disc, and is not localized near the sources of wg or dpp. wg has also been implicated in proximal-distal patterning of the wing hinge. In this report, we present evidence that wg is principally required for local cell proliferation in the hinge. Loss of wg expression leads to a local reduction in cell division, resulting in the deletion of a distinct set of wing hinge structures. Ectopic activation of the wg pathway in cells of the wing hinge leads to over-proliferation without repatterning, indicating that wg acts as a mitogen in this part of the disc. By contrast, overexpression of wg in the wing blade leads to repatterning and only secondarily to proliferation. These results suggest that the Wg signal elicits very different responses in different regions of the wing imaginal disc.
INTRODUCTION
Among the more intriguing problems in pattern formation is how positional information is used to control cell proliferation. It is necessary to know how cell proliferation is integrated with patterning events and how final cell numbers are specified. Recent work has established the basic principles by which pattern is generated in the appendages of Drosophila (reviewed in Blair, 1995; Brook et al., 1996), making this developmental process a good starting point for examining the coordination of proliferation with patterning.
Drosophila appendages are subdivided into compartments (Garcia-Bellido, 1975). The nascent wing imaginal disc is divided into anterior-posterior compartments in the embryonic ectoderm (reviewed in Cohen, 1993) and becomes further sub-divided into dorsal-ventral and proximal-distal compartments during the second larval instar (Garcia-Bellido et al., 1973). Crick and Lawrence (1975) suggested that compartment boundaries may serve as organizing centers responsible for generating spatial pattern in developing appendages, and recently a lot of evidence in favor of this hypothesis has accumulated (reviewed in Blair, 1995; Brook et al., 1996).
Interactions between the compartments have been shown to induce specialized cells near the compartment boundaries that control patterning and proliferation in the disc. Thus engrailed activates the secreted signaling molecule Hedgehog in posterior cells, which in turn induces the expression of decapentaplegic in nearby anterior cells (Basler and Struhl, 1994). Decapentaplegic is also a secreted signaling protein, and has been shown to be the mediator of the anterior-posterior organizer (Capdevila and Guererro, 1994; Diaz-Benjumea et al., 1994; Zecca et al., 1995). Ectopic expression of decapentaplegic in either compartment leads to a duplication of the anterior-posterior axis. Similarly, apterous specifies dorsal cell fate (Diaz-Benjumea and Cohen, 1993; Blair, 1993). fringe and Serrate signal from dorsal to ventral cells and induce expression of Wingless and Vestigial at the dorsal-ventral boundary (Irvine and Wieschaus, 1994; Kim et al., 1995; Diaz-Benjumea and Cohen, 1995; Couso et al., 1995). The D-V boundary corresponds to the wing margin in the adult wing and wingless function is required for the formation of wing margin structures (Couso et al., 1994; Phillips and Whittle, 1993). wingless (wg) function in the wing margin is also required for cell proliferation and/or cell survival in the wing blade, and ectopic expression of wg in the wing pouch not only locally respecifies cells to assume wing margin fate, but also stimu-lates overproliferation of surrounding cells (Diaz-Benjumea and Cohen, 1995). Thus wg mediates the organizing effects of the D-V boundary both on patterning and on proliferation. Analysis of the mutation nubbin has suggested the presence of a proximal-distal organizing center in the wing hinge which is responsible for growth of the wing (Ng et al., 1995). In the third instar wing imaginal disc, wg is expressed in two rings surrounding the wing pouch, one of which is missing in a nubbin mutant background. This observation has suggested that this domain of wg expression might be implicated in formation of the wing hinge and in growth of the wing (Ng et al., 1995).
While the molecular mechanisms by which organizers are established are starting to be understood, and key mediators of organizer function have been identified, downstream events are not well understood. Thus it is not at all clear how organizers regulate cell proliferation. Here we present evidence that the ring of wingless expression surrounding the wing pouch is required for local cell proliferation in the wing hinge, but not for long-range patterning. We also show that ectopic activation of the wg pathway can induce overproliferation without causing repatterning, indicating that wg is acting as a mitogen in this part of the wing disc. The mitogenic effect of wg in the hinge contrasts with its effects in the wing blade, where it directly specifies cell fates and apparently indirectly promotes proliferation (Diaz-Benjumea and Cohen, 1995). Region-specific differences in the mitogenic and fate specification responses to Wnt-1, the vertebrate orthologue of wg, have also been observed in the mouse central nervous system (Dickinson et al., 1994), raising the possibility that the pathways by which cells distinguish between growth and cell fate specification in response to Wnt signals may be conserved.
MATERIALS AND METHODS
Drosophila stocks
wgCX4 is described in Baker (1987) and van den Heuvel et al. (1993). spdfg is described in Lindsley and Zimm (1992). wgr0727, Df(2L)spdj2 and Df(2L)spdhL2 are described below. neuralized-lacZ is described in Ghysen and O’Kane (1989). UAS-Wg is described in Lawrence et al. (1995). The GAL4 driver MS1096 is decribed in Capdevila and Guerrero (1994).
P-element-generated alleles
wgr0727 is a pZ P-element inserted into the wg locus (a gift of Ulrike Gaul). It is embryonic lethal, although the segment polarity phenotype of wgr0727 homozygous embryos is weaker than that of wgCX4 homozygous embryos, arguing that it is a strong wg hypomorph (unpublished observation). By performing plasmid rescue, we mapped the insertion site of wgr0727 to about 100 bp 5′ of the wg tran-scription start site (see Fig. 4). We used this P-element to generate both Df(2L)spdj2 and Df(2L)spdhL2. Df(2L)spdj2 was generated by imprecise excision of wgr0727 (scoring for loss of the rosy+ marker). Hybridization of genomic DNA from flies heterozygous for Df(2L)spdj2 with a probe spanning the insertion site of wgr0727 showed that the left flank is gone, while the right flank is still intact, thus leaving the wg transcript untouched (data not shown). Cytological analysis showed that the imprecise P-element excision has generated a large deficiency covering the 27C1-28A1 interval (Fig. 4). Df(2L)spdhL2 was also generated by imprecise excision of the wgr0727 P-element. Southern blot analysis indicated that at least 30 kb are deleted in both the 5′ and the 3′ regions of wg. Df(2L)spdhL2 is not visible cytologically and complements the lethal complementation groups (‘G’ and ‘J’) that Tiong and Nash (1990) identified on either side of the wg locus.
Histochemical methods
Whole-mount in situ hybridization was performed as described by Tautz and Pfeifle (1989) using a wg RNA probe. X-gal staining of pharate adults carrying wg-lacZ was done as in Hama et al. (1990). Anti-Dll staining was as in Diaz-Benjumea and Cohen (1995). Acridine orange staining was performed as in Masucci et al. (1990). Bromodeoxyuridine (BrdU) labeling was done as in Usui and Kimura (1992). BrdU incorporation was for 30 minutes for the experiment in Fig. 6 and for 10 minutes in Fig. 8.
Molecular methods
Southern blot analysis was performed following standard procedures (Ausubel et al. 1994). Enhancer activity of the 1.2 kb EcoRI fragment (indicated as probe c in Fig. 4D) was tested in the Casper-hs43-AUG-βGal vector (Thummel and Pirrotta, 1991). Several inde-pendent transformants inserted at different chromosomal locations produced the same expression pattern. UAS-Dsh was prepared by cloning a full-length dsh cDNA (Klingensmith et al., 1994) as an EcoRI fragment into pUAST (Brand and Perrimon, 1993).
RESULTS
The mutation nubbin identifies a class of genes required for patterning the hinge region of the Drosophila wing (Ng et al., 1995). In adult viable nubbin mutants, almost the whole wing is lost. However, removing nubbin function in clones of cells indicates that wild-type activity is not required in cells giving rise to most of the wing blade itself, but only in cells located in the hinge region. This indicates that there may be a pat-terning center in the hinge region which has both local and long-range influences on wing development. To learn more about this patterning center, we sought to identify other mutations with a similar phenotype to nubbin. One such mutation is spadeflag (spdfg). Here we show that spdfg is a reg-ulatory mutation of wg that specifically abolishes wg function in the hinge.
The spadeflag mutant phenotype results from the loss of wg expression in the wing hinge
spdfg was originally identified as a viable spontaneous mutation that causes an overall reduction of wing size, as well as a reduction of the alula and a variable loss of posterior wing margin structures (Lindsley and Zimm, 1992). A detailed characterization of the spdfg phenotype indicates that the most penetrant aspect of this phenotype is the loss of specific hinge structures (Fig. 1). The hinge structures lost in spdfg include the medial costa, the humeral cross-vein, septum 2 and the part of the dorsal radius which lies between the medial costa and the alula (Fig. 1B,D). The alula is sometimes completely absent and sometimes vestigial. The Sc1 campaniform sensillum is always absent, while the Sc12 group of campaniform sensillae is reduced, with more proximally located sensillae sometimes remaining. More proximal hinge structures, such as the proximal costa and the axillary cord are not affected by spdfg.
The structures lost in the hinge of spdfg mutants are centered on a domain of wg expression that runs through the hinge (Fig. 1B,D). In the third instar wing imaginal disc, wg is expressed in several domains including two rings surrounding the wing pouch (Fig. 2B). Examination of wg-lacZ expression in the adult wing shows that both of these rings of wg expression run through the hinge structures of the adult. The inner of the two rings in the imaginal disc corresponds to the more distal stripe running through the adult hinge, and we refer to this as the ‘inner ring’. The inner ring runs from the medial costa through the humeral cross-vein and the dorsal radius and then curves around to the base of the alula. The outer ring runs from the base of the costa to the base of the axillary cord (see Phillips and Whittle, 1993; Fig. 1B). The inner ring of wg expression lies in the hinge domain, which it is deleted in spdfg mutants.
The inner ring of wg expression is also absent in nubbin mutant wing discs which correlates well with the nubbin hinge phenotype (Ng et al., 1995). These observations suggest that spdfg may reduce wg function associated with the inner ring of wg expression in the wing imaginal disc.
To test this hypothesis directly, we examined wg expression in spdfg mutant imaginal discs by whole-mount in situ hybridization. The inner ring of wg expression is not detectable in the spdfg mutant disc (Fig. 2). Careful examination also suggests that the intensity of staining in the wing margin is reduced relative to a wild-type control (compare Fig. 2A and B). The reduced levels of wg RNA in the wing margin of spdfg mutants correlates well with the observation that spdfg homozygotes show some loss of margin bristles, as noted by Couso et al. (1994).
spadeflag is allelic to wg
The observation that spdfg removes wg expression in the inner ring of the wing hinge and reduces it in the wing margin is consistent with the proposal that spd may be a regulatory allele of wg (Couso et al., 1994; Tiong and Nash, 1990). However, spd shows an ambiguous complementation behavior towards different wg alleles (Tiong and Nash, 1990). We sought to clarify this issue by crossing spdfg to three well-characterized wg alleles: a wg null point mutant, a deficiency removing only the 5′ region of wg, and a deficiency removing both the 5′ and the 3′ regions of wg.
wgCX4 is a null point mutant caused by a small deletion removing the wg promoter (van den Heuvel et al., 1993). Flies of the genotype spdfg/wgCX4 show a milder version of the hinge phenotype seen in spdfg homozygotes (Fig. 3A,B) and there are no reductions of margin structures. If spd and wg are different loci that show a dominant genetic interaction, it is expected that a deficiency removing both would show the same phenotype as the spdfg/wgCX4 heterozygous combination. However, deficiencies that uncover the whole region do not show any phenotype, suggesting that spdfg is allelic to wg. This raises the question why the heteroallelic combination gives a weaker phenotype than either homozygote. This effect is probably due to transvection, i.e.. because the relevant enhancer on the wgCX4 chromosome is driving the expression of the wg gene on the spdfg chromosome. There is further genetic evidence that the wg locus is subject to transvection (Neumann and Cohen, 1996). For example, the mutation wg1, which has been shown to be a small deletion in the 3′ regulatory region of wg (Baker, 1987; van den Heuvel et al., 1993), is largely complemented by wgCX4.
We also crossed spdfg to two new alleles of wg generated by P-element mobilization. Df(2L)spdj2 is a large deficiency that breaks about 100 bp 5′ of the wg transcription start site and removes the entire 5′ regulatory region of wg (Fig. 4). Df(2L)spdj2 behaves as an embryonic lethal allele of wg in trans to wgCX4 (Neumann and Cohen, 1996). When heterozygous with spdfg, Df(2L)spdj2 produces a hinge phenotype indistinguishable from that of spdfg homozygotes (Fig. 3C,D). This shows that removing the 5′ sequences of wg abolishes the ability to complement spdfg. As the 5′ regulatory sequences of wg are present on the wgCX4 chromosome and wgCX4 can partially complement spdfg, we conclude that the defect on the spdfg chromo-some must lie in the 5′ regulatory region of wg.
Df(2L)spdhL2 is a small deficiency that removes the wg gene and flanking DNA extending at least 30 kb in both directions. Df(2L)spdhL2 does not include the other known lethal complementation groups flanking the wg locus (Tiong and Nash, 1990). Flies of the genotype spdfg/Df(2L)spdhL2 have a hinge phenotype that is only slightly stronger than that of spdfg homozygotes (Fig. 3E,F). The proximal costa is present, as is the proximal part of the distal costa, but the alula is absent. In addition, the anterior and posterior wing margins are absent, and adjacent wing blade tissue is scalloped. This wing margin phenotype is much stronger than that of spdfg homozygotes and of flies of the genotype spdfg/Df(2L)spdj2.
Taken together with the effects of spdfg on wg expression, these observations suggest that spdfg is a regulatory mutation that removes wg function in the hinge while only reducing it in the wing margin. Consistent with this suggestion, removal of wg function during third instar using the wgts mutation leads to a number of similar defects in wing development (Phillips and Whittle, 1993; Couso et al., 1994). Although not described in those reports, wgts produces a hinge defect indistinguishable from that of spdfg homozygotes (see Fig. 5D in Couso et al., 1994). The hinge phenotype is partially masked by transvection when spdfg is heterozygous over a point mutant of wg, but is uncovered by a deficiency that removes the 5′ regulatory region of wg The full margin phenotype is only uncovered by a deficiency that removes both the 5′ and the 3′ regulatory regions of wg.
spadeflag deletes an enhancer located 5′ of wg that drives wg expression in the hinge and in the wing margin
The observation that Df(2L)spdj2 uncovers the spdfg mutant phenotype suggested that spdfg must affect sequences located 5′ of wg. We tested DNA in this region from spdfg mutants for chromosome rearrangements and found a restriction fragment length polymorphism indicative of a small deletion located about 9 kb 5′ of the wg promoter (Fig. 4B-D). Comparison of the restriction enzyme digestion pattern of the mutant DNA with wild-type DNA shows that there is a deletion of about 1 kb that removes most of a 1.2 kb EcoRI fragment in the wild-type DNA (probe c in Fig. 4D). The 1.2 kb EcoRI fragment contains an enhancer element sufficient to direct reporter gene expression in a ring around the wing pouch and in a stripe along the wing margin (Fig. 5). lacZ expression driven by this DNA fragment is activated in second instar in a diffuse pattern (Fig. 5A) which resolves into a wing margin stripe and a ring around the wing pouch during early-mid third instar (Fig. 5B,C). The dynamics of this expression are similar to those of the endogenous wg gene in the margin and the inner ring.
Staining of adult flies shows that the ring of lacZ expression corresponds to the more distal ring of wg expression in the hinge (not shown). These results suggest that the spdfg mutant phenotype is due to the deletion of an enhancer element that drives wg expression in the inner ring of the wing hinge and in the wing margin. Although the enhancer drives strong expression in the wing margin, removal of this fragment in spdfg mutants does not cause a severe loss of wing margin structures, indicating that this aspect of wg regulation is partially redundant.
The spadeflag phenotype is due to decreased proliferation in the hinge region
To further investigate the spdfg phenotype, we examined the expression pattern of the neuronal marker neuralised/A101 (Ghysen and O’Kane, 1989; Huang et al., 1991) in the spdfg mutant background (Fig. 6A-D). Specific groups of sense-organ precursors, including all or part of the Sc12 group of campaniform sensillae, are missing in late third instar wing imaginal discs of spdfg mutants (Fig. 6C,D). These cells are inside, or close to the inner ring of wg expression (compare Figs 2B and 5D with 6A). Examination of the spdfg mutant also shows that there is tissue missing in this area, bringing the Sc25 group of campaniform sensillae closer to the sensillae located on vein 3 (compare Fig. 6B with D). This indicates that the structures that are deleted in the adult hinge are already absent in late third instar wing imaginal discs of spdfg mutants.
To determine whether the loss of distal hinge structures in spdfg is due to cell death, we stained wing imaginal discs of spdfg mutants with acridine orange, which labels apoptotic cells (Masucci et al., 1990). No abnormal cell death was detected in any part of the disc throughout third instar (data not shown). To further address this question, we crossed the spdfg enhancer-lacZ reporter gene into the spdfg mutant background. If the absence of wg activity in the inner ring leads to localized cell death, it is expected that, after correct initial activation, the expression of this reporter would fade away, as the cells in which it is expressed would die. However, this is not what we observe. During late third instar, when the loss of tissue in the hinge of spdfg individuals is already apparent, the enhancer is still strongly expressed (Fig. 6E,F). Furthermore, we labeled spdfg mutant discs as well as wild-type discs carrying the spdfg enhancer-lacZ gene for lacZ expression and simultaneously for BrdU incorporation, which marks dividing cells. In wild-type discs, many cells expressing the lacZ reporter in the hinge are dividing (Fig. 6G). In the spdfg mutant background, however, only very few cells expressing the lacZ reporter in the hinge can be seen to divide (Fig. 6H). Taken together, these results indicate that the absence of distal hinge structures in spdfg mutants is not due to cell death. Instead, they suggest that wg activity in the inner ring is required to promote local cell pro-liferation in the wing hinge.
Ectopic activation of the wingless pathway causes overproliferation in the hinge region of the wing
It has been shown that wg is able to induce wing margin cell fates in the wing pouch (Diaz-Benjumea and Cohen, 1995). To determine whether wg is also sufficient to specify pattern elements in the hinge region, we misexpressed wg using the GAL4:UAS system (Brand and Perrimon, 1993). As a GAL4 driver we used MS1096, which has been shown to express GAL4 in the dorsal wing pouch (Capdevila an Guerrero, 1994). However, we found that it is also expressed in the ventral wing pouch, as well as in the dorsal hinge, although at lower levels (Fig. 7A). When crossed to this driver, a UAS-wg line utilizing the wild-type wg cDNA (Lawrence et al., 1995) is able to activate Distal-less (Dll) expression throughout the wing pouch (Fig. 7C). Dll has been shown to be a target gene of wg in the wing pouch (Diaz-Benjumea and Cohen, 1995). Pharate adults recovered from this cross also have a high density of wing margin bristles on both wing surfaces, although sometimes with a lower density ventrally (Fig. 7D). We also observed that there is an increase in the size of the dorsal hinge in this com-bination (visualized by the area that expresses UAS-lacZ under the control of MS1096 in the dorsal hinge, Fig. 7C). This cor-relates with a broadening of the hinge region of the pharates (compare Fig. 7B with 7D), suggesting that there may be extra growth in the hinge induced by ectopic wg activity.
dishevelled (dsh) is required to transduce the wg signal (reviewed in Klingensmith and Nusse, 1994). Dsh is a phos-phoprotein that becomes hyper-phosphorylated in response to the wg signal, and it has been shown that overexpression of Dsh is sufficient to cause its hyper-phosphorylation and activation of the wg pathway, as assayed by the accumulation of Armadillo protein in cultured cells (Yanagawa et al., 1995).
Sokol et al. (1995) have shown that injection of Xdsh mRNA into Xenopus oocytes can mimic the effect of injection of Wnt mRNA, even though Xdsh mRNA is present ubiquitously in the oocyte. These results suggested that overexpression of dsh in Drosophila could have a similar effect. Indeed, expression of UAS-dsh by several GAL4 drivers can phenocopy ectopic expression of wg in the wing and leg imaginal discs (Fig. 7E,F, and data not shown).
UAS-dsh crossed to MS1096 induces Dll expression throughout the wing pouch, as observed with UAS-wg (Fig. 7C,E). The dorsal hinge is greatly overgrown, as indicated by the abnormal folds of cells expressing UAS-lacZ and UAS-dsh (Fig. 7E). The pharates resulting from this cross also have densely packed wing margin bristles all over the wing blade surfaces, although in some cases the ventral surface is more sparsely covered (Figs 7F, 8B). The region occupied by the proximal dorsal hinge is greatly expanded. This effect of UAS-dsh is much stronger than that of UAS-wg.
To determine whether the expansion of the dorsal hinge region in MS1096:UAS-dsh individuals is due to increased proliferation, we pulse labeled imaginal discs with BrdU. When incubated with BrdU for 10 minutes, only few cells are labeled in a wild-type wing disc (not shown). By contrast, both the frequency and intensity of labeling is increased in the dorsal hinge of MS1096:UAS-dsh wing discs (compare the region between the arrows in Fig. 8A with the regions of the disc on either side, which resemble control discs). This result indicates that overexpression of dsh in the hinge region strongly stimulates cell division.
Examination of the extra tissue in the proximal hinge of MS1096:UAS-dsh pharates indicates that it is not patterned. There does not appear to be a duplication of distal hinge structures more proximally. To further address this point, we crossed the neuronal marker A101 into this background (Fig. 8B). It is possible to locate most of the sensillar precursors in the hinge region in the correct location in these discs and in the correct numbers. This is consistent with the observation that sensory mother cells are selected from mitotically quiescent clusters of cells (Usui and Kimura, 1992). However, the spacing of cells within clusters of sensillae is abnormal. Thus the Sc25 group of sensillae appears as a compact cluster in wild-type discs, but is stretched into a long line in discs of the genotype MS1096:UAS-dsh (compare Fig. 6B with Fig. 8B). These results indicate that ectopic activation of the wg pathway in the hinge region of the wing does not respecify pattern, but instead stimulates cell division. However, the greatly increased cell numbers disrupt the organization of the hinge.
DISCUSSION
Distinct mitogenic and fate specification responses to the Wg signal
We have shown here that wingless function is required to promote proliferation of cells in the wing hinge. Reduction of wg gene activity in the wing hinge leads to underproliferation of this region and loss of distal hinge structures. Conversely, overexpression of wg or activation of the wg signal transduction pathway through dsh expression leads to local overgrowth of the proximal hinge. The effects of Wg in the proximal hinge suggest a rather direct mitogenic effect in this region of the disc. This contrasts with the effects of Wg in the wing pouch, where the primary effect of increasing Wg activity is to respecify cell fate toward wing margin. The difference in response to the Wg signal in the hinge and the wing blade is reminiscent of the effects observed when Wnt-1 is overex-pressed in the mouse CNS using a HOXb-4 Region A enhancer (Dickinson et al., 1994). Although Wnt-1 is required for spec-ification of fates at the mid-brain hind-brain junction, ectopic expression in the ventral CNS causes overproliferation without cell fate respecification. Skaer and Martinez-Arias (1992) have shown that wg stimulates cell division in the anlage of the Malpighian tubules, and several Wnt genes, including Wnt-1, the vertebrate orthologue of wg, have been found to have an oncogenic effect on mammary epithelial cells (reviewed in Nusse and Varmus, 1992).
Although wg is required for proliferation in the distal hinge region of the wild-type wing imaginal disc, this does not result in a zone of proliferation that stands out above that in the sur-rounding area. This suggests that other factors are stimulating proliferation in the rest of the disc at a similar rate. In this context it is noteworthy that, while removal of the inner ring of wg expression does not lead to a reduction of proliferation in the proximal hinge region, ectopic activation of the wg pathway strongly induces overproliferation in this region. This suggests that wg can synergize with the factor(s) that are reg-ulating cell division in the proximal hinge.
wg locally mediates the effect of the P/D organizer
The reduction of the total size of the wing in spdfg homozygotes is mostly due to the absence of hinge structures. This indicates that the wg expression domain in the inner ring surrounding the hinge does not have any long distance effects on wing development, unlike the wg expression domain in the wing margin (Diaz-Benjumea and Cohen, 1995). Therefore, wg cannot be the sole mediator of the putative hinge-organizing center identified by nubbin and only part of the nubbin phenotype can be due to removal of the inner ring of wg expression (Ng et al., 1995). However, the spdfg phenotype suggests that wg locally mediates the stimulatory effect of the putative proximal-distal organizer on growth of the wing and suggests that one mechanism whereby organizers exert their influence on cell division may be to establish subdomains of cells in which proliferation is regulated independently from the rest of the disc.