Howard Hughes Medical Institute, Department of Biology, Indiana University, Bloomington, IN 47405, USA
* These authors contributed equally to this work
Author for correspondence (e-mail: kaufman{at}bio.indiana.edu)
Accepted April 23, 2001
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SUMMARY |
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Key words: Drosophila, Proboscis, Labial disc, Homeotic, dachshund, extradenticle, Distal-less, spalt, proboscipedia, Sex combs reduced
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INTRODUCTION |
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Some of the more dramatic Drosophila homeotic mutations cause transformations of the proboscis into other types of appendages (Kaufman et al., 1990). A null mutation in the gene proboscipedia (pb), which is normally expressed in the labial discs, transforms the proboscis into a pair of tarsi; pb hypomorphic mutations, such as pb4, results in the transformation of the proboscis into a pair of aristae; and hypomorphic Scr mutations cause a transformation into what has been interpreted as a pair of maxillary palps (Kaufman et al., 1990) (A. M. Pattatucci, PhD thesis, Indiana University, 1991) (Aplin and Kaufman, 1997). Mutations in both pb and Scr transform the proboscis into a complete antenna (Percival-Smith et al., 1997). These transformations indicate that the proboscis is a highly derived appendage developmentally related to both antennae and legs.
In order to address the segmental organization and homology of the proboscis vis-a-vis legs and antennae, we have studied wild-type labial discs, as well as the individual and combined effects of pb and Scr mutations on the distribution of the known appendage and homeotic genes in the labial anlagen. Both hth and exd are required for proximal leg development and the formation of antennal structures (Casares and Mann, 1998). We found that wild-type labial discs expressed low levels of Hth and Exd, pb mutants displayed levels of Hth and Exd that were comparable with wild-type legs and antennae, and that ectopic expression of Pb in other imaginal discs suppressed Exd. Furthermore, we provide evidence that Pb is both required and sufficient to suppress dac in all appendages, thereby preventing formation of structures that correspond to the intermediate portions of legs or antennae. In addition, Pb suppresses sal expression; for example, ectopic expression of Pb in the eye-antennal disc suppresses sal and phenotypically transforms the antenna to maxillary palp. However, Teashirt (Tsh), another protein necessary for proximal leg development is not expressed either in wild-type labial discs or in those that are mutant for either Scr or pb. Dll, the most distally expressed appendage gene, is restricted to a small area located opposite the peripodial stalk in wild-type labial discs and is downregulated by both Scr and Pb. We conclude from these observations that the proboscis represents a highly modified appendage missing the regulatory program to properly specify the coxa (Cx), trochanter (Tr), femur (Fm) and, at least part of, the tibia (Tb). We argue that this modification is due in large part to the action of Pb and Scr. The distal portion of the appendage, homologous to the leg tarsus (Tar) and antennal arista (Ar), is present but is completely re-patterned, owing to the joint effect of Pb and Scr.
We have also investigated possible regulatory effects of the gnathal homeotic genes on each other. It has been shown that Dfd and Scr positively regulate pb and are required for its correct deployment during embryogenesis (Rusch and Kaufman, 2000). We confirm that Pb and Scr are co-expressed in most of the labial disc, while Dfd is expressed in a small proximal domain separate from the expression domains of Pb and Scr (Mahaffey and Kaufman, 1987; Randazzo et al., 1991). We report that Pb is necessary for Scr expression in the distal but not in the proximal half of the labial disc. We propose a model for the imaginal disc regulation of the gnathal Hox genes that is strikingly different from the one demonstrated for the same genes in the embryo (Rusch and Kaufman, 2000).
Dll is required for the development of the labellar structures on the distal part of the proboscis, such as pseudotracheae and border hairs (Cohen and Jürgens, 1989). The Dll expression domain for wild-type and mutant discs in the part of the labial disc opposite the peripodial stalk argues for a revision of the existing fate map (Kumar et al., 1979). We suggest that the proximodistal axis of the proboscis coincides with the main anatomical axis of the labial disc. This is supported by the expression domains of Pb and Scr, both of which are required for development of the labellar structures and are expressed chiefly in the distal two-thirds of the labial disc (Mahaffey and Kaufman, 1987; Randazzo et al., 1991; Pattatucci and Kaufman, 1991).
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MATERIALS AND METHODS |
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Immunohistochemistry
Imaginal discs were dissected and fixed as described in Pattatucci and Kaufman (Pattatucci and Kaufman, 1991) with the following modifications: inverted larvae were rocked for 20 minutes in 4% paraformaldehyde in phosphate-buffered saline (PBS) with 2.5% dimethylsulphoxide, then washed twice with absolute methanol. These were then stained as follows: after two quick washes in PBT (1xPBS with 0.1% Triton X-100) and four 10 minute washes in PBTB (PBT with 0.5% bovine serum albumin), larvae were blocked for 30 minutes with 30 µl normal goat serum in 270 µl of PBTB. Larvae were rocked overnight at room temperature after addition of the appropriate primary antibodies. After overnight incubation, larvae were washed four times for 10 minutes each with PBTB, reblocked with 30 µl normal goat serum in 470 µl of PBTB for 30 minutes, incubated with secondary antibody for 2 hours and then washed once with PBTB. After this wash, 100% glycerol was added, and the discs were dissected and mounted in Aqua Polymount. All fluorescent labeling was carried out with Jackson Labs fluorescently labeled secondary antibodies and were viewed using confocal microscopy.
Antibodies
The polyclonal rabbit ß-galactosidase antibody was produced and affinity purified in our laboratory by David Miller. The Dac monoclonal antibody, developed in the laboratory of Dr Rubin, was obtained from the Developmental Studies Hybridoma Bank. These and all other antibodies used and their dilutions were as follows:
Name Type Dilution Source or reference
Dfd Rabbit 1:200 Mahaffey et al., 1989
Scr Rabbit 1:300 Mahaffey and Kaufman, 1987
Scr Rat 1:500 Abzhanov and Kaufman, 1999
Pb E2 Rabbit 1:50 Kapoun and Kaufman, 1995
Pb E9 Rabbit 1:250 Cribbs et al., 1992
Dac Mouse 1:50 Mardon et al., 1994
Exd Rabbit 1:1000 Mann and Abu-Shaar, 1996
Exd Rat 1:200 González-Crespo and Morata, 1995
Hth Rabbit 1:100 Pai et al., 1998
Hth Chicken 1:500 Casares and Mann, 1998
Tsh Rabbit 1:3000 Wu and Cohen, 2001
Tsh Rat 1:500 Gallet et al., 1998
Dll Rabbit 1:200 Panganiban et al., 1994
Dll Mouse 1:500 Duncan et al., 1998
ß-Gal Rabbit 1:300 This work
ß-Gal Mouse 1:500 Promega
Sal Rabbit 1:30 Kuhnlein et al., 1994
Sal Rat 1:1000 Barrio et al. , 1999
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RESULTS |
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In summary, wild-type labial discs express no Dac, a reduced domain of Dll and low levels of Exd. pb5 null mutant labial discs express Dac, a larger domain of Dll and express Exd at levels comparable with those in leg discs, indicating that Pb is required for the negative regulation of dac, Dll and exd genes in labial discs.
Pb is sufficient for suppression of dac and exd
Using the GAL4 system (Brand and Perrimon, 1993) we examined whether Pb was sufficient for the negative regulation of dac, Dll and exd outside the normal expression domain of pb, namely the leg imaginal discs. We ectopically expressed Pb in a dpp pattern in the leg discs (dpp=>pb) and analyzed the expression of the appendage genes (Fig. 3A-H). Most dpp=>pb imaginal discs are mis-shapen because of ectopic Pb; the discs have abnormal folding and do not lay flat like wild-type discs. First, we determined that a stripe of ß-galactosidase, produced with the dpp: Gal4 driver (dpp=>lacZ), partly overlaps with the circular Dac domain in wild-type leg discs (Fig. 3A,B). However, in dpp=>pb flies, the Dac domain is disrupted by Pb-expressing cells (Fig. 3C,D). No nuclei accumulating both Pb and Dac are observed. A similar effect of Pb was observed on Exd Fig. 3E,F). In dpp=>lacZ animals, ß-galactosidase expression broadly overlaps the Dll domain (Fig. 3G). When Pb was expressed in the dpp pattern, although the pattern of Dll expression was altered, there was clear co-expression of these two proteins (Fig. 3H). So while Pb is sufficient for the repression of dac and exd, it is not sufficient for the repression of Dll.
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Molecular analysis of the labial discs from pb4 mutants
Unlike the transformation of the proboscis into a pair of tarsi in pb5 null mutants, the proboscis is transformed into a pair of aristae in pb4 hypomorphic mutants (Kaufman et al., 1990; Aplin and Kaufman, 1997). Recall that in wild-type labial discs, we observe low levels of Exd/Hth, no Dac and a small domain of Dll at the distal tip of the labial disc. We analyzed the molecular patterning of pb4 mutant labial discs and compared them with wild-type antennal disc patterning (Fig. 5A-G). In the wild-type antennal disc, the Hth domain overlaps the domain of Dll in a ring, which surrounds a Dll only region (Fig. 5B). Sal, an important antennal marker (Dong et al., 2000), is expressed in a circular domain at the junction of the Dll and Hth expression domains (Fig. 5A,C). In addition, the circular domain of Sal surrounds a crescent-shaped domain of Dac (Fig. 5G). No Sal was detected in the labial discs of wild-type animals (not shown). Scr is expressed at the base of the antennal disc but not in the disc proper, revealing little overlap with Sal accumulation in wild-type eye-antennal discs (Fig. 5G). Unlike wild-type labial discs, pb4 labial discs contain distinct domains of Dac, Dll, Hth and Sal expression (Fig 5D-F,H,I), similar to wild-type antennae but in diminished domains. We note that the Dac and Sal domains often form a spot or a short crescent-shaped stripe instead of the ring seen in wild-type eye-antennal discs (compare Fig. 5G with 5I).
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Distribution of Tsh in wild-type and mutant labial discs
We also studied the distribution of Tsh, a molecule required for development of the maxillae and antennae and the proximal segments of the leg (Bhojwani et al., 1997; Erkner et al., 1999). Using an enhancer trap, tsh expression has been shown to be absent from the proboscis in adults (Bhojwani et al., 1997). We confirmed the lack of Tsh accumulation in the labial discs with both rat and rabbit anti-Tsh antibodies (Table 1). This was in contrast to easily detectable protein levels in leg discs from the same animals (not shown). We also found that no ectopic Tsh expression is seen in pb5 mutant labial discs (not shown). This suggests that the leg-like structures of the pb5 homeotic mutants lack properly specified proximal podomeres.
Molecular analysis of Scr mutants
Hypomorphs of Scr that survive to adulthood exhibit a transformation of proboscis into what has been interpreted to be a maxillary palp identity (A. M. Pattatucci, PhD thesis, Indiana University, 1991). To understand the changes taking place at the molecular level, we analyzed the expression patterns of Pb and products of several appendage genes: dac, Dll, exd and sal in this class of Scr mutants.
Fig. 6A-C show the pattern of accumulation of Scr and Pb in a labial disc from an Scr4/Scr5 hypomorph. Levels of Scr accumulation in these mutants are variable and usually lower than in wild-type labial discs (Fig. 6B). However, there is easily detectable Pb protein in these discs (Fig. 6C). As these Scr lesions are leaky and because Scr has been shown to positively regulate pb in the embryo (Rusch and Kaufman, 2000), we thought it prudent to further investigate the regulatory relationship of the two homeotic genes in imaginal cells. Imaginal discs from dpp=>Scr animals were stained for Pb accumulation. No ectopic expression could be detected, despite readily detectable levels of the protein in the labial discs of these animals (not shown). Thus, in contrast to what is observed in the embryo (Rusch and Kaufman, 2000), Scr does not act as a positive regulator of pb in the imaginal discs.
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Molecular analysis of the labial discs from pb1Scr4/pb5Scr5 mutants
We also analyzed the labial discs of pb1Scr4/pb5Scr5 double mutants, where Scr function is impaired and Pb is absent. As Scr4/Scr5 is hypomorphic with variable but detectable levels of Scr accumulation, we observed several animals for their mutant proboscis phenotypes. These phenotypes varied from thick and shortened aristae with claws and pulvillae to apparently complete antennae with aristae, third and second antennal segments bearing characteristic bristles (Fig. 7A). This latter observation is consistent with the results of a clonal analysis in which Scrpb cells produced antennal structures (Percival-Smith et al., 1997). In accordance with the observed phenotypic variation, we found variable patterns of accumulation of Dac, Dll, Hth and Sal. Whereas no Dac (Fig. 2A), a small domain of Dll (Fig. 2B), low expression of Exd (Fig. 2C) and Hth, and no Sal (Fig. 7B) are expressed in wild-type labial discs, we observed expression patterns of these appendage genes that were very similar to those seen in either wild-type antennae (Dac not shown; compare Fig. 5A,G with Fig. 7C,D) or in wild-type legs (presumably owing to leaky expression of Scr). These antennal expression patterns (and importantly the presence of Sal) are consistent with the genetic make-up of the wild-type antennal disc, which does not exhibit any HOM-C gene expression in the antennal disc proper (Yao et al., 1999). As in pb4 mutant labial discs, which display a transformation to aristae, there is clear Sal expression where the domains of Hth and Dll overlap (Fig. 7D,E). However, unlike pb4 mutant discs, these pb Scr double mutant labial discs are more similar in shape and size to wild-type antennal discs, and the domain of Sal is more circular and not at the distal tip as in pb4 mutant labial discs (compare Fig. 7D,E with Fig. 5H).
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DISCUSSION |
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In addition to Dac, pb mutants display levels of Exd and Hth comparable with antennae and legs (Fig. 9); both are expressed at low levels in wild-type labial discs (Fig. 2; Fig. 7B). Exd is an important co-factor of many Hox genes and its absence can alter homeotic gene product function and specificity (Pinsonneault et al., 1997; Ryoo and Mann, 1999). The fact that pb suppresses the expression of Hth and Exd also implies that the three homeotic genes expressed in the labial disc (Dfd, Scr and pb) are functioning largely in the absence of the Exd co-factor and that this may contribute to the uniqueness of regulatory interactions in the labial appendage.
Dll, a protein needed for the development of most of the distal structures in both legs and antennae, is normally expressed at low levels (relative to antennal and leg discs) in a restricted distal domain in the labial disc (Fig. 9). Analysis of pb and Scr mutants demonstrates that Dll is expressed in the distal half of the labial disc at levels comparable with legs and antennae. Both pb and Scr are required for the development of the distiproboscis, a structure dependent at least in part on the function of Dll (Mahaffey and Kaufman, 1987; Cohen and Jürgens, 1989; Randazzo et al., 1991). As Pb is required for activation of Scr in the distal half of the labial disc and the expression domains of Scr and Dll are reciprocal in pb5 mutants, it is likely that Scr is the principal negative regulator of Dll expression in the labial disc.
We propose that the proboscis constitutes an appendage type that is distinct from both antenna and thoracic leg, owing at least in part to the repression in the labial discs by pb of several of the genes normally involved in patterning legs and antennae (Fig. 10). As a result, the fly proboscis appears to lack elements serially homologous to the proximal and intermediate leg segments. We support the hypothesis that the labellum, the distalmost part of the proboscis that bears the pseudotracheae, is homologous to the leg tarsus. However, our analysis demonstrates that the labellum is thoroughly modified and it is likely that other, as yet unidentified, genes are also specifically involved in the morphogenesis of this unique appendage and are controlled by both Scr and pb. Additionally, there may be loci not influenced by but similar to pb and Scr; for example, genes not normally expressed in the leg but involved in the suppression in the labial discs of limb segmental anlagen.
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Arista versus tarsus identity in pb mutant labial discs
One of the more interesting phenotypes observed is the aristal transformation in pb4 hypomorphs. What makes this interesting is that Scr, whose function normally is to specify tarsal identity, is present throughout the pb4 mutant labial discs except for the distal tip. Additionally, we observe that when Scr is ectopically expressed in the antennal disc using the Gal4 system, sal, a gene important for antennal/aristal development is repressed cell autonomously (Fig. 8). In addition, Scr can repress antennal identity and promote tarsal identity via repression of hth (Yao et al., 1999). In light of the fact that Scr is expressed in the proximal region of pb5 null labial discs, represses sal and specifies tarsus, one might predict that the more robust expression pattern of Scr in pb4 labial discs should be sufficient to repress Sal accumulation in these mutant discs as well. However, in pb4 mutant labial discs, at the point of decreased Scr expression, co-expression with Sal is observed. While it is possible that in the hypomorphic genotype, Scr levels fall below a threshold level in some cells (such that Scr cannot repress sal), we favor an alternative hypothesis. The products of several hypomorphic alleles of pb, including pb4, are partially functional, truncated peptides that still contain the DNA-binding homeodomain (Cribbs et al., 1992). A closer look at the pb4 adult phenotypes reveals that the proboscis of these mutants, in addition to having aristae, have fewer pseudotracheal rows compared with wild type, but do form these labial-specific structures (Cribbs et al., 1992). Moreover, antibody raised to epitopes encoded by the second exon of pb shows wild-type accumulation of the truncated pb4 encoded peptide (not shown). Therefore, the truncated Pb protein must still be sufficiently functional for specification of mouthpart components such as pseudotracheae, despite the concomitant expression of Scr. Additionally, while full-length Pb protein can suppress sal expression in the antennal disc (Fig. 8), it is evident that these truncated, partially functional proteins do not prevent Sal accumulation in the labial discs. We conclude therefore that the presence of the partially functional, truncated peptide is in some way interfering with the activity of Scr in the cells in which both proteins are accumulated. Determining the validity of this interference model will, of course, require further investigation. It should also be noted that the pb4 encoded peptide is deficient in its ability to repress dac expression in the labial discs, as easily detected levels of Dac are found in these mutant discs.
Finally, there are many appendage genes that are involved in antenna and leg patterning, but they have not been studied here. It is possible that yet another factor or co-factor is needed to activate tarsal identity, which the compromised Pb protein is still capable of repressing.
Distinct genetic environments in different imaginal discs
Pb suppresses dac gene expression cell autonomously in the labial discs and can suppress its expression in the antennal or leg anlagen; however, other regulatory relationships appear to be unique to the labial discs. An example of this is the regulation of Dll by either Pb or Scr. Mutants show clear de-repression of Dll to resemble Dll accumulation levels and pattern in leg and antennal discs, yet ectopic expression of either Pb or Scr shows that these proteins do not repress Dll in other imaginal discs. Thus, it would appear that other, as yet unidentified, factors are probably involved in the development and specification of the different imaginal discs. The fact that each disc type represents a different ontogenic environment is not an entirely surprising or unanticipated finding.
Fate map of the labial disc
The original fate map of the labial disc, based on surgical fragmentation and implantation experiments, indicated that the future distalmost part of the proboscis, which gives rise to the labellum and is surrounded by the border hairs, lay in the proximoventral region of the labial disc (Fig. 11; Kumar et al., 1979). Thus, the proximodistal axis of the adult proboscis on the published labial disc fate map is pointed towards the peripodial stalk (Fig. 11B). Analysis of Dll mutants has shown that, in part, the pseudotracheae of the labellum and the border hairs depend on the function of this gene (Cohen and Jürgens, 1989). We have shown in this analysis that Dll expression is localized to a well-defined domain opposite to and not near the peripodial stalk. This expression pattern suggests that the proximodistal axis of the proboscis points away from the stalk and coincides with the main anatomical axis of the balloon-shaped labial discs (Fig. 11B). Nevertheless, the map of the labial disc and the position of Dll accumulation become coincident, if (while the relative positions of the fate mapped structures are maintained) the map is simply rotated 180° (Fig. 11B). The expression pattern of Pb in wild-type labial discs supports this interpretation. Loss of Pb function is relative to the proximodistal axis of the proboscis, as mutants have a stronger effect on more distal structures (Boube et al., 1998). Notably, the highest expression levels of Pb are observed in the area of the labial disc opposite the peripodial stalk (these results).
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ACKNOWLEDGMENTS |
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