Department of Anatomy, University of Wisconsin, Madison, WI 53706, USA
*Author for correspondence (e-mail: gepangan{at}facstaff.wisc.edu)
Accepted 5 November 2001
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SUMMARY |
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Key words: Distal-less, homothorax, dachshund, bric a brac, spalt, spineless, Antenna, Leg, Wing, Appendage, Limb, Proximodistal
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INTRODUCTION |
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Although several genes are known to be expressed in segmentally reiterated patterns in the antenna and leg, most of these are unlikely to be regulated differently between these appendage types. For instance, Serrate, Delta and fringe, which function in the Notch pathway, are expressed and required for the formation of all joints and are not being limited to homologous subdomains of antenna and leg (Bishop et al., 1999; de Celis et al., 1998
; Mishra et al., 2001
; Parody and Muskavitch, 1993
; Rauskolb and Irvine, 1999
). Other genes, such as Bar and apterous are similarly expressed in narrow proximodistal (PD) subdomains that correspond to homologous segments of the antenna and leg (Kojima et al., 2000
; Larsen et al., 1996
; Pueyo et al., 2000
; Rincon-Limas et al., 1999
; Tsuji et al., 2000
). Thus although all of these genes contribute to limb segmentation, they probably do not contribute to the morphological differences between appendages.
In contrast, bric a brac (bab), which is required specifically for distal limb segmentation, is expressed in restricted PD subdomains and exhibits distinct expression patterns between antenna and leg. bab encodes a BTB/POZ domain protein necessary for normal development of most tarsal and distal antennal joints (Godt et al., 1993). bab mutations cause fusion of tarsal segments 2 through 5 (t2-t5) of the leg and of the fourth and fifth segments (a4, a5) and arista (ar) of the antenna (see Fig. 1A-D) (Godt et al., 1993
). The mature bab pattern consists of two concentric rings in the distal antenna and in four concentric rings in the distal leg (see Fig. 2C',F'). To understand how the antenna and leg develop different numbers of distal segments, we therefore have focussed on how bab becomes differentially expressed in the antenna and the leg.
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The observations that bab is differentially expressed between antenna and leg (Godt et al., 1993) (this work) and that its putative activators, Dll and Ss have distinct expression patterns and functions in the two limb types (Dong et al., 2000
; Dong et al., 2001
; Duncan et al., 1998
), led us to hypothesize that the regulation of bab was likely to vary between limbs. Here, we present evidence that supports this. In particular, we find that although Dll does serve as a bab activator in the antenna and also in the wing pouch, only part of this activation in the leg and none in the wing is mediated by Ss. Interestingly, the differences between antenna and leg expression of bab are not a consequence of differences in activators, but of differences in the expression of bab repressors. We conclude that bab is differentially regulated in different limbs by tissue-specific combinations of PD patterning genes. The unique gene networks that regulate bab in each limb type thereby contribute to morphological variation by regulating the number of distal segments.
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Materials and Methods |
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Fly strains
The following fly strains were employed: (1) dpp-GAL4 (A.3)/TM6B (Morimura et al., 1996); (2) act>CD2>GAL4 (=actin promoter-FRT-CD2-FRT-GAL4) (Pignoni and Zipursky, 1997
); (3) w; UAS-GFP-hth8/TM6B, Tb, Hu (Casares and Mann, 1998
); (4) w; FRT82B hthP2 (Pai et al., 1998
); (5) w; dac4 FRT40A, (Graeme Mardon); (6) w; FRT43D DllSA1 (Dong et al., 2000
); (7) y hs-FLPase; FRT43D 2piM (Dong et al., 2000
); (8) Dll1/CyO, wg-lacZ; (9) Dll3/CyO, wg-lacZ; (10) babARO7/TM6B, Tb Hu e ca (Frank Laski); (11) Df (3L) babAR07 th st cp FRT80B/TM3 (Artyom Kopp); (12) dac-lacZ (Graeme Mardon); (13) w; UAS-Dll/In (2LR) Gla, Gla Bc Elp (Konrad Basler); (14) Df(3R)ssD114.4/TM6 (Ian Duncan); (15) ssD114.7/TM6 (Ian Duncan); and (16) y hs-FLPase; FRT82B M piM (S. Blair). Stocks constructed by us for these experiments were: (1) y hs-FLPase; FRT82B 2piM; (2) y hs-FLPase; UAS-GFP-hth8/TM6B, Tb Hu; (3) y hs-FLPase; UAS-dac; (4) DllGAL4/CyO, wg-lacZ; (5) w; Df(2L)sal5 FRT40A; (6) y hs-FLPase; 2piM FRT40A; and (7) y hs-FLPase; UAS-sal.
Genetic manipulations
Dll hypomorphic larval imaginal discs were generated by crossing heterozygous Dll mutant animals in which each Dll mutant chromosome was balanced over CyO, wg-lacZ. Mutant animals were identified by the absence of X-gal staining in the larval tails. Ectopic expression of Dll, homothorax (hth) and dachshund (dac) was induced using the GAL4/UAS binary system (Brand and Perrimon, 1993). dpp-GAL4 was used to activate UAS-GFP-hth along the anterior-posterior compartment boundary of the developing imaginal discs. Clones of cells ectopically expressing Hth, Dac or Sal were generated using a modified GAL4/UAS system (Pignoni and Zipursky, 1997
) in which y hs-FLPase; UAS-GFP-hth/TM6B, Tb Hu (or y hs-FLPase; UAS-dac or y hs-FLPase; UAS-sal) flies were crossed to act>CD2>GAL4 flies. The resulting larvae were heatshocked at 37°C for 10 minutes at 72-96 hours after egg laying (AEL) to induce site-specific recombination between the FRT sites, which in turn results in constitutive GAL4 expression in the clones. Dll, hth, dac and sal null clones were generated using the FLP/FRT system (Xu and Rubin, 1993). Animals of the genotypes: (1) y hs-FLPase; FRT43D 2piM/FRT43D DllSA1; (2) y hs-FLPase; FRT82B piM/FRT82B hthP2; (3) y hs-FLPase; 2piM FRT40A/dac4 FRT40A; and (4) y hs-FLPase; 2piM FRT40A/Df(2L)sal5 FRT40A were heatshocked at 37°C for 1 hour at 48-72 hours AEL and examined in mid- to late-third instar. To make large hth null clones, the hth+ FRT chromosome carried a Minute (M) mutation. Animals of the genotype: y hs-FLPase; FRT82B M piM/FRT82B hthP2 were heatshocked at 37°C for 1 hour at 120-144 hours AEL and examined in mid- to late-third instar (because the heterozygous Minute animals develop slowly, this was
240-288 hours AEL). The ss null genotype examined was Df(3R)ssD114.4/ssD114.9. The bab null genotype examined was Df(3L)babARO7/Df(3L)babAR07 th st cp FRT80B/TM3.
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RESULTS |
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Dynamic expression of bab during antenna and leg development
In order to gain insight into possible positive and negative regulators of bab expression during appendage development, we first examined the dynamics of wild-type bab expression. In late second/early third instar antenna and leg discs, bab is expressed throughout most of the Dll domain, with the highest levels of expression in the centers of both antennal and leg discs (Fig. 2A,A',D,D'). By mid-third instar, bab expression is lost from the distalmost cells, and a ring of Bab is apparent in the antennal and leg discs (Fig. 2B,B',E,E'). Still later in the third instar, the bab expression pattern resolves into a set of concentric rings, two in the antenna and four in the leg (Fig. 2C,C',F,F'). Based on its wild-type expression, we postulated that bab is activated initially throughout the Dll domain, and that the mature bab expression patterns are achieved in part by subsequent repression distally and proximally. In addition, bab either is partially repressed or its expression is not maintained between concentric rings.
bab expression depends on Dll in the antenna, leg and wing
To examine the relationship between bab and Dll in the antenna, leg and wing, we investigated bab expression in Dll hypomorphic discs and in clones of cells lacking Dll. Bab is not detected in either Dll hypomorphic antenna (Fig. 3A) or leg (Campbell and Tomlinson, 1998) (not shown) discs, or in Dll null clones in either the antenna or leg (Fig. 3B,C,C'). Interestingly, although bab expression is lost cell-autonomously in Myc-marked Dll null clones in both antenna and leg (arrows in Fig. 3B,C,C' and not shown), bab expression is also lost non cell-autonomously under certain conditions. In the antenna, for instance, bab expression is lost in cells surrounding the Dll null clones when those clones are near the boundary of the normal dac and bab expression domains (arrowheads in Fig. 3B). Non cell-autonomous loss of the modulation between bab rings is also seen occasionally near these Dll null clones. These phenomena likely are due to the non cell-autonomous activation of dachshund (dac) around the Dll null clones (Dong et al., 2001
), since Dac is a bab repressor (see below and Discussion). In the leg, we also observe non cell-autonomous loss of bab expression around Dll null clones (e.g. arrowhead in Fig. 3C,C'). However, in this case Dll expression is also lost (J. C. and G. P., unpublished data). Thus the non cell-autonomous loss of bab expression in the leg is likely due to the loss of the bab activator Dll, and mechanistically different from the non cell-autonomous loss of bab expression in the antenna.
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bab expression partially depends on Ss in the antenna and leg
ss null mutations result in loss of part of t1 and of the t2, t3 and t4 segments of the leg, including their joints. Thus in ss null legs, there remains only a single distal joint positioned between t1 and t5 (Duncan et al., 1998). Consistent with the adult cuticular phenotype, in late third instar ss null animals, we observe a single ring of bab expression in the leg (Fig. 4A,A'). Double labeling of the ss mutant leg discs with both Bar antibodies that label the t4 and t5 segments (Kojima et al., 2000
) and Bab antibodies (not shown), indicates that the remaining Bab represents the distal-most ring. Thus while the proximal tarsal rings of bab depend on Ss, the distal ring expression of bab is Ss-independent. Similarly, in ss null antennal discs, there is a distal ring of bab expression (Fig. 4B,B'). This is consistent with the presence of a single distal joint, between the transformed a3 and the transformed arista of ss null animals (Duncan et al., 1998
). However, because the ss null antenna exhibit transformations toward leg, it is difficult to evaluate the requirements for ss in normal antennal joint formation. Thus, while we can conclude that Ss mediates only part of the Dll activation of bab in the leg, the relationship between ss and bab during normal antennal development remains unknown.
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Bab does not set the distal limit of dac expression in either the antenna or the leg
It has been proposed that mutually repressive interactions in the leg, e.g between Bar and aristaless (al) (Kojima et al., 2000) and between Dll and dac (Dong et al., 2001
), play critical roles in the subdivision of the PD axis. We therefore wished to test whether the antagonism of Dac for Bab is reciprocal. In bab null antenna and leg discs, Bab protein cannot be detected, but dac expression is normal (Fig. 5C and F). Consistent with this, dac is expressed in a medial ring instead of a circle that encompasses the bab domain prior to bab activation (not shown). Thus Dac represses bab, but Bab does not repress dac.
Restriction of bab expression by Spalt in the wing
We have also investigated bab regulation in the developing wing. In the wing pouch, bab expression resembles that of Dll, except that bab is not expressed in the middle of the wing pouch along the anterior-posterior compartment boundary. This absence of detectable bab expression coincides with the domain of spalt (sal) expression (Fig. 6A). sal encodes a zinc finger transcription factor (Kuhnlein et al., 1994) required to position the L2 wing vein (de Celis et al., 1996
; Sturtevant et al., 1997
). To test whether Sal represses bab, we made Df(2L)sal5 clones. We found that bab is derepressed in these sal null clones (arrowheads in Fig. 6B,B',B''). Therefore, bab expression is restricted by Sal in the wing. bab activation in the sal null clones is unlikely to be mediated via Dll, since Dll is not expressed or activated in many of these clones (arrowheads in Fig. 6B'''). bab is activated similarly in sal null clones in the haltere (Fig. 6C,D) and this is not mediated by Dll either (not shown). In fact, the wild-type expression of bab does not overlap with Dll in the haltere (not shown). Together, our results indicate that some, but not all, bab activation in the wing is mediated by Dll and that Sal is a potent repressor of bab in both wing and haltere.
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bab is derepressed weakly in sal null clones in a2 (Fig. 7F) and repressed in flipout clones ectopically expressing Sal (Fig. 7G). sal null clones do not exhibit derepression of bab outside of a2 (Fig. 7F and not shown). We conclude that Sal is a bab repressor in a2. bab often is derepressed in hth null clones in both a2 and a3 (Fig. 7I). We have shown previously that while sal is lost in hth null clones (Dong et al., 2000), dac is derepressed in hth null clones in both a2 and a3 (Dong et al., 2001
). Thus hth null clones in a2 and a3 often coexpress bab and its repressor dac. This supports the leg data described above indicating that Dac is insufficient for bab repression. We conclude that Dac probably requires Hth (or the product of a gene activated by Hth) as a corepressor in the antenna.
Interestingly, in large hth null clones that encompass much of the distal antenna, bab often (40/66 discs=61%) is derepressed uniformly throughout a2 and a3 (Fig. 8A), while at other times (26/66 discs=39%) the derepressed bab is modulated and appears in rings. Among the hth mutant antennal discs in which the derepressed bab is modulated, we frequently (11/26 discs=42%) see four rings (Fig. 8C). Thus large clones with four rings constitute 17% (11/66) of the total large hth null clones examined. This frequency resembles that (16%; 10/61) at which five distinct tarsal segments separated by four joints can be detected in adult antennae harboring large hth null clones (Fig. 8D). In these experiments, the remaining 51/61 antennae, while visibly transformed toward leg, possessed three or fewer distinct tarsal-like joints and four or fewer tarsal segments (Fig. 8B). Thus inter-ring modulation in hth mutant antennae can be correlated with joint formation, and bab modulation probably is essential for normal distal joint formation in both the antenna and the leg.
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Discussion |
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Activation of bab by Dll and Ss
Both ss and bab expression depend on Dll in the antenna and leg (Campbell and Tomlinson, 1998; Duncan et al., 1998
) and this work). We report here that only a subset of the Dll-dependent bab expression depends on Ss. In particular, the tarsal rings of bab expression in distal t1, distal t2 and distal t3 are both Dll and Ss dependent, whereas the ring of bab in distal t4 depends on Dll but not on Ss. Thus for the t1, t2 and t3 rings, Ss either mediates the Dll activation of bab or cooperates with Dll to activate bab, while the t4 ring is activated independent of Ss, possibly directly by Dll (Fig. 9). bab expression in the t4 ring may be regulated via a different enhancer than bab expression in the t1, t2 and t3 rings, but whether or not there are distinct bab enhancers for different rings is unclear. We think it likely that bab expression in each of the four tarsal rings represents differential activation in response graded Dpp and Wg signals in conjunction with the Dll and/or Ss selector(s).
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We find that only part of the bab expression depends on Dll in the wing, and that in a homologous structure, the haltere, bab is not Dll-dependent. It has been suggested that the insect wing may derive from a proximodorsal part of a branched limb of their aquatic ancestors (Averof and Cohen, 1997; Kukalova-Peck, 1978
). There is expression of bab proximal to the Dll domain in both antenna and leg discs. Thus if the wing has derived from the proximal leg, it could be that the regulation of bab expression in the wing shares features with that of the proximal leg.
Differential restriction of bab in different appendage types by Dac, Sal and Hth
Here we have demonstrated that repression by Dac establishes the proximal border of bab expression in the leg, that Sal restricts bab expression in the wing, and that Dac, Sal and Hth establish the proximal limit of bab expression in the antenna (Fig. 9). We have shown previously that coexpression of Dll and Hth/Exd determines antennal identity, and activates sal (Dong et al., 2000) and dac (Dong et al., 2001
). Here we demonstrate that differential expression of these genes leads to significant differences in bab expression. In particular, bab expression is restricted to a small domain containing two rings in the antenna in contrast to a larger domain of four rings in the leg. As bab is necessary for joint formation, we propose that these differences in bab expression contribute to the differences in the number of distal segments found in the antenna and the leg.
In both antenna and leg discs, bab expression is derepressed in dac null clones that are proximal to normal bab expression domain. However, the mechanisms of bab derepression probably differ between limb types. In the leg, derepression of bab is associated with derepression of Dll. Therefore, Dac likely restricts bab expression via bab activators. In the antenna, Dll and dac expression normally overlap, and Dll continues to be expressed in dac null clones. This suggests a more direct repression mechanism may exist in the antenna. There is substantial overlap between dac and bab expression in the leg at late third instar, indicating that the presence of Dac protein is not sufficient for bab repression and that the presence or absence other factors is required in order for Dac to repress bab. This is perhaps not surprising since Dac does not contain a known DNA binding motif and forms complexes with Eyes absent and Sine oculis to regulate transcription during eye development (Chen et al., 1997; Pignoni et al., 1997
). Based on existing genetic information, candidate Dac corepressors are Sal, Hth and n-Exd in the antenna, and Antennapedia (Antp) in the leg. Alternatively, because Dac overlaps with Dll in both the antenna and leg where Dac serves as a bab repressor, it also is possible that Dac functions by precluding the ability of Dll and/or Ss to activate bab.
Modulation between rings
At early third instar, bab is expressed throughout the Dll domain in the leg, with uniformly strong expression in the presumptive tarsal segments and weaker expression in presumptive femur and tibia. By late third instar, bab expression is modulated in the presumptive tarsus and is strongest at the distal part of tarsal segments 1-4, immediately proximal to each of the tarsal joints. Weaker expression of bab is observed between these concentric rings. This modulation persists at least through early pupal stages. Various mechanisms by which this modulation is achieved can be imagined, including both active repression and/or the failure to maintain bab in the inter-rings.
We have indirect evidence suggesting that active repression plays a role. Namely, in dac null antenna and leg discs, there is loss of bab inter-ring modulation as well as proximal derepression. This lack of inter-ring modulation correlates with joint loss and segment fusion in the dac null animals (Dong et al., 2001) (this work). In the leg, bab modulation by Dac occurs cell-autonomously. However in the antenna, there is loss of bab modulation even in cells that do not normally express detectable levels of Dac. Therefore in addition to repressing bab cell-autonomously, Dac also may non cell-autonomously regulate the production of bab inter-ring repressor(s) (Fig. 9). Since Dac is a nuclear protein, it could be that Dac directs the expression of a secreted molecule that in turn mediates inter-ring modulation.
Regulation of bab and limb evolution
We have presented evidence that bab is differentially regulated in different Drosophila appendage types and that this regulation contributes to variations in their morphology. It is necessary to determine whether bab is required for limb segmentation in other animals, and, if so, whether variations in bab regulation contribute to the interspecific variations of homologous appendages. Investigation of the genetic hierarchies that pattern the appendages of other arthropods and of closely related phyla are needed to address these issues and to provide further insights into the generation of morphological diversity.
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ACKNOWLEDGMENTS |
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