Effects of Mutation or Deletion

Since Dll mutations are lethal, it is impossible to observe the effects on adult animals. Larvae however, have rudimentary limbs. In the absence of Dll, these vestigial limbs are deleted. Keilin organs, a distal sensory apparatus of larval appendages, are associated with the developing leg imaginal disc primordia. Thus these sense organs are the rudimentary legs of Drosophila embryos. In Dll mutants, the sensory hairs of Keilin's organs are deleted (S.M. Cohen, 1989).

Dll protein can be detected in a central domain in leg discs throughout most of larval development; in mature discs this domain corresponds to the distal-most regions of the leg: the tarsus and the distal tibia. Clonal analysis reveals that late in development these are the only regions in which Dll function is required. Dll3 is the strongest hypomorph in which all of the tarsus is deleted and the tibia and femur are reduced in size. The expression of two genes required for the patterning of the tarsus, al and bric à brac (bab) was examined in Dll3 leg discs. In wild-type discs, al is expressed in the center of the disc and bab in the rest of the presumptive tarsus. In Dll3 leg discs no al or bab expression can be detected in the center of the discs. Clonal analysis was performed with a Dll null allele. Clones were generated at various times during development and the resulting adult legs were compared to legs containing wild-type clones generated at the same time. Dll clones generated early in development fail to be recovered in the region more distal than the coxa, while later in development phenotypically wild-type Dll clones (but lacking bracts) could be recovered in the proximal tibia and femur but not in more distal regions, where they segregate out as cuticular vesicles. The requirement for Dll in the femur and most of the tibia is lost by about the early third instar. Additional observations reveal that there is a clear difference in the time at which normally patterned Dll null clones can be recovered in the dorsal femur, as compared to the ventral femur (here 'ventral' corresponds only to the ventral third): Dll null clones can be recovered in the dorsal femur when they are generated at any stage in development, although early in development their frequency is reduced when compared to wild-type. In the trochanter, almost no wild-type Dll clones are recovered at any stage in development; there is a proximal ring of Dll expression in the third instar leg disc that probably corresponds to the trochanter. When a leg is composed almost entirely of Dll null mutant tissue then the region more distal to the coxa is represented only by a small stump of tissue. A marked reduction in the P/D axis can be identified in leg discs consisting almost entirely of Dll null tissue, showing that the leg truncations produced by loss of Dll are not caused simply by cell death late in development but may be caused by disruption of normal patterning and growth or cell survival during development. In discs containing larger regions of wild-type tissue, this tissue is generally found in the center of the disc surrounded by Dll null tissue, in contrast to wild-type clones that form irregular patterns contributing to any region of the leg. Legs derived from these types of discs develop normal distal regions, but the leg between this region and the coxa is aberrant: there is a marked reduction in growth, the division into segments is disrupted and the size and density of bristles is reduced (Campbell, 1998).

Proximodistal axis formation in the Drosophila leg: subdivision into proximal and distal domains by Homothorax and Distal-less

The developing legs of Drosophila are subdivided into proximal and distal domains by the activity of the homeodomain proteins Homothorax (Hth) and Distal-less (Dll). The expression domains of Dll and Hth are initially reciprocal. In the mature third instar disc, Dll is expressed in a large central domain that corresponds to the presumptive tarsus and distal tibia. Dll is also expressed in a secondary ring. X-gal staining of adult legs carrying a Dll-lacZ reporter gene shows that this ring is located at the proximal edge of the femur, possibly extending slightly into the distal trochanter. The central domain of Dll expression is controlled by Wg and Dpp. The proximal ring arises in third instar and does not depend on Wg or Dpp activity. The leg disc is a continuous single-layered epithelial sheet that forms a series of folds as it grows. The peripheral region of the disc forms the proximal segments. This region is folded back over the central region where Dll is expressed. The domain of Hth expression extends from the peripodial membrane at the top, through the coxa and trochanter segment primordia. The distal-most portion of the Hth domain overlaps the proximal part of the dac-lacZ domain within the proximal ring of Dll expression in the femur. Dll is expressed alone in the central folds of the disc (which correspond to tarsal segment primordia). In proximal tarsus and tibia, Dll and Dac overlap. Dac is expressed alone in the presumptive femur. Because the disc is highly folded, horizontal optical sections make proximal and distal regions of the disc appear to be closely apposed, although they are actually far apart along the PD axis in the plane of the disc epithelium. Hth is expressed in the upper layer and around the lateral sides of the epithelial sac. Dll is expressed in the center of the lower layer. The two expression domains abut, but do not overlap. dac-lacZ is not detectably expressed at this stage, but can be reliably detected in slightly older discs at the transition from second to third instar. These observations suggest that the primary subdivision of the disc is into two domains: a central Dll-expressing domain and a proximal Hth-expressing domain. Wg and Dpp act together to induce Dll and Dac in the center of the leg disc. Wg and Dpp repress Hth and Teashirt, but not through activation of Dll (Wu, 1999).

The expression patterns of Dll and Hth/Exd reflect an early subdivision of the disc into proximal and distal domains. At early stages of disc development, Dll and Hth/Exd are expressed in reciprocal domains that account for all cells of the disc. At this stage, Dac is not yet expressed. What is the relationship between Dll and Hth/Exd expression in the early disc? The Dll domain is defined by Wg and Dpp signaling. The same signals repress nuclear localization of Exd and Hth expression. The reciprocity of Dll and Hth expression suggests a model in which Wg and Dpp act through Dll to repress Hth in the early disc. However, the analysis of marked Dll mutant clones reported here shows that this is not the case. Clones of Dll mutant cells located in the distal region of the leg do not express Hth. This contrasts with recent reports by González-Crespo (1998) and Abu-Shaar (1998) in which evidence is presented for ectopic expression of Exd and Hth in Dll mutant clones. How can the difference in the results between these reports be reconciled? In both studies, the clones were induced in second instar larvae using the same allele of Dll. In the experiments reported here, clones were marked by the absence of Dll protein and by the absence of a neutral beta-gal marker, which permits definitive genotyping of the cells independent of Dll expression. In the other reports, clones were marked only by the absence of Dll. The disc epithelium is highly folded and the proximal Hth-expressing epithelium is very close to the distal Dll-expressing epithelium. Unless cells in the clone are definitively genotyped, it is difficult to distinguish a genuine clone from a patch of the overlying Hth-expressing proximal epithelium that has been pushed downward into the plane of the optical section. Serial optical sections of wild-type discs show that this type of distortion of the disc epithelium can occur in damaged discs as well as in discs that are not obviously damaged. How is Hth repressed by Wg and Dpp? Dac is induced by Wg and Dpp toward the end of second instar. Hth expands distally, to some extent, in Dac mutant discs. These observations suggest that Dac contributes to Hth repression. However, Hth is repressed prior to the onset of Dac expression indicating that Dac cannot be the primary repressor. Whether Wg and Dpp act directly to repress Hth expression or act via another as yet unidentified repressor remains to be determined (Wu, 1999).

In conclusion, Hth and Dll expression appear to define alternative fates in the second instar disc. Under normal circumstances, there does not appear to be a cell lineage restriction between these populations (i.e. no compartment boundary). These results suggest that cells can cross between these territories if they are able to switch between Hth and Dll expression. This situation appears to be analogous to the DV subdivision of the leg disc (as opposed to the proximal distal subdivision reported here). DV subdivision is stable at the level of gene expression in a cell population, but is not a clonal lineage restriction boundary. Similarly, the separation of proximal and distal cell populations requires Hth function. These results suggest that cells at the interface between these two territories are specialized to allow integration of otherwise immiscible populations of cells (Wu, 1999 and references).

Drosophila terminalia as an appendage-like structure

This study reports the expression pattern of Dll in the genital disc, the requirement of Dll activity for the development of the terminalia and the activation of Dll by the combined action of the morphogenetic signals Wingless (Wg) and Decapentaplegic (Dpp). In Drosophila, the terminalia comprise the entire set of internal and external genitalia (with the exception of the gonads), and includes the hindgut and the anal structures. They arise from a single imaginal disc of ventral origin that has a complex organization and shows bilateral symmetry. The genital disc shows extreme sexual dimorphism. Early in development, the anlage of the genital disc of both sexes consists of three primordia: the female genital primordium (FGP); the male genital primordium (MGP), and the anal primordium (AP). In both sexes, only two of the three primordia develop: the corresponding genital primordium and the anal primordium. These in turn develop, according to the genetic sex, into female or male analia. The undeveloped genital primordium is the repressed primordium (either RFP or RMP, for the respective female and male genital primordia) (Gorfinkiel, 1999).

During the development of the two components of the anal primordium -- the hindgut and the analia -- only the latter is dependent on Dll and hedgehog (hh) function. The hindgut is defined by the expression of the homeobox gene even-skipped. The lack of Dll function in the anal primordia transforms the anal tissue into hindgut by the extension of the eve domain. Meanwhile targeted ectopic Dll represses eve expression and hindgut formation. The Dll requirement for the development of both anal plates in males and only for the dorsal anal plate in females, provides further evidence for the previously held idea that the analia arise from two primordia. In addition, evaluation was made of the requirement for the optomotor-blind (omb) gene which, as in the leg and antenna, is located downstream of Dpp. These results suggest that the terminalia show similar behavior as the leg disc or the antennal part of the eye-antennal disc, consistent with both the proposed ventral origin of the genital disc and the evolutive consideration of the terminalia as an ancestral appendage (Gorfinkiel, 1999).

The expression pattern of Dll in the genital disc was analyzed. Dll is neither expressed in the embryonic terminalia nor in the embryonic precursor cells of the genital disc. In the female third larval instar genital disc, Dll shows a localized distribution; it is strongly expressed in a large spot in the central part of the anal primordium and in a faint band of cells in the genital primordium. It is not detected in the RMP. Similarly, in the male genital disc, Dll is expressed in a large spot both in the anal primordium and in the male genital primordium but not in the RFP. Several GAL4 insertions in the Dll locus were used and these permitted the identifcation of the adult regions where Dll is expressed according to the observed X-Gal staining. In females, Dll is expressed in the vaginal plates and in the anal plates. In the dorsal anal plate, Dll is expressed in a generalized manner, while the ventral plate shows fainter Dll expression, which is stronger in the distal part of the plate. In males, Dll is expressed in the claspers and anal plates. The expression, both in male and female external terminalia, is as predicted by the prospective fate map of the genital disc. The internal structures are not well defned in term of Dll expression (Gorfinkiel, 1999).

To investigate whether there is a functional requirement for Dll in the terminalia, the phenotype of different viable Dll mutant combinations was analyzed. These Dll hypomorphic mutant combinations were initially described by their phenotype in the leg and antenna. In the allelic series homozygous Dll3, DllIB/DllMP and Dll3/DllMP, the female dorsal anal plate is reduced whereas the ventral anal plate is normal. The vaginal plates are disorganized. In males, the anal plates are strongly reduced. The external genital structures are, however, unaffected. These phenotypes show that the Dll expression domains do not fully correspond to its requirement. This led to a search for alterations in the internal genital structures. In both males and females, the internal genitalia appear normal. Surprisingly, under the strongest hypomorphic conditions (Dll3/DllMP), the hindgut is enlarged in both females and males. The few anal structures that remain are surrounded by hindgut tissue. This result suggests an expansion of hindgut territory at the expense of the anal plates. To further investigate the requirement for Dll, Dll SAI clones (marked with yellow) were induced during the larval stages. Dll2 clones do not develop anal plates (males) or dorsal anal plates (females). These clones were recognized since some of them still differentiate yellow (y) bristles. Dll2 clones do not show detectable alterations in the male external genitalia. Since only a few structures of the genitalia can be analyzed with the y marker, it is possible that minor phenotypic alterations may go undetected. Dll 2clones do not affect the development of the female ventral anal plates. Thus, although Dll is also expressed in the ventral anal plate in females and the claspers in males, it seems that it is not required for the formation of these structures. These results are in agreement with those observed using the viable Dll mutant combinations (Gorfinkiel, 1999).

Both the female and male anal primordia give rise to two different adult structures: the hindgut and the anal plates. These territories are well defined by the complementary expression of the homeotic genes Dll and even-skipped (eve). Adult regions that express Dll and eve were defined by X-Gal staining of Dll-GAL4/UAS-LacZ and eve-lacZ- flies, respectively. These two genes show a complementary expression pattern. Dll is expressed in the anal plates of both females and males but not in the hindgut. In contrast, eve is expressed in the hindgut of both females and males. Some residual Dll expression is detected in the rectal papillae, but these structures are not derived from the genital disc. Thus, the adult analia and hindgut are defined by Dll and eve expression patterns, respectively. Also in the genital disc, eve labels the prospective hindgut that occupies the central part of the anal primordium while Dll marks the primordia of the anal plates located at both ends of the primordia in both females and males. This eve expression both in discs and adults suggests that eve is required for hindgut development (Gorfinkiel, 1999).

In the Dll hypomorphic combinations the hindgut is enlarged and the anal plates are reduced. This phenotype correlates with gene expression, since in Dll2clones, eve expression extends into the anal territory both in females and males. In some Dll2 clones eve is only activated in the part of the clone nearest to the normal eve-expression domain. This indicates that there is a region capable of activating eve that is then transformed to hindgut. This region could correspond to the prospective dorsal analia in females where Dll is specifically required (Gorfinkiel, 1999).

To test if a mutually repressive interaction between the homeotic genes Dll and eve in the anal primordium can lead to their complementary expression domains, either Dll or eve were ectopically expressed in the presumptive cells of both the hindgut and analia using the cad-GAL4 line. In UAS-Dll/cad-GAL4 discs, eve is not expressed and there is a reduction of the whole primordium. The Dll domain is also reduced in all ventral discs upon Dll ectopic expression because an excess of Dll represses its own expression. The adult flies do not show hindgut structure and the anal plates are also reduced. In UAS-eve/cad-Gal4 discs there is a reduction of the Dll domain along with an enlargement of the hindgut primordium, but there are still cells that co-express Dll and eve. Therefore, it is concluded that the complementary expression domains of Dll and eve in the anal primordium is due to eve repression by Dll (Gorfinkiel, 1999).

Hh signal is required to form the genital and anal structures but not the hindgut. In the leg and antennal discs, the expression of Dll depends on the Hh signaling pathway. Using the hh ts2 allele, it was observed that in the genital disc, Hh is also required for Dll activation: after 4 days at the restrictive temperature, the genital discs are very small and show no Dll expression. In the same hh ts2 larvae, residual Dll expression can be detected in the trochanter region of the leg disc. However, eve expression in the anal primordia is maintained and occupies most of the reduced genital disc. This result indicates that Dll, but not eve expression, depends on Hh and that all the terminalia with the exception of the hindgut require Hh function. To further analyze this hh requirement for Dll activation, the effect of smoothened (smo) lack of function was examined. In smo2 cells, Hh reception is impeded because smo is a component of the Hh receptor complex. In the genital disc, Dll expression only disappears in smo2 clones when the clone is large enough to cover most of the Dll expression domain. Accordingly, eve expression is also ectopically activated in smo2 mutant cells; although in Dll2 cells eve cannot be activated in certain regions of the clones. These results indicate once again that Dll is dependent on Hh function while eve is not (Gorfinkiel, 1999).

Large smo2 clones close to the A/P compartment transform some structures of the external genitalia and analia. In the female genitalia, smo2 clones duplicate the long bristle of the vaginal plates and clones in T8 to produce tissue overgrowth with y2 bristles. Large smo2 clones reduce the female dorsal anal plate, whereas the female ventral anal plate is rarely affected. Some clones produce segregated tissue in the female analia labelled with y bristles in the perianal region. However, small clones or clones located outside the A/P compartment border have no effect. In the male genitalia, smo2 clones duplicate the genital arc, part of the claspers and the hypandrium bristle. All these structures are located close to the A/P compartment border. As in Dll2 clones, large smo2 clones delete the anal plate in males. In both males and females, only when the clone is large enough can Dll expression not be activated in the disc primordia, giving rise to the Dll2 phenotype. This result suggests that only in large smo2 clones both wg and dpp are not activated and therefore are unable to induce Dll expression (Gorfinkiel, 1999).

The hh requirement for the analia but not for the hindgut has also been confirmed by the ectopic expression of Cubitus interruptus (Ci). ci encodes a transcription factor that acts as an activator of the target genes of the Hh pathway. The overexpression of Ci in the anal primordia of cad-GAL4/UAS-ci flies, leads to the enlargement and fusion of the anal plates. Accordingly, the Dll expression domain in the genital disc is expanded to cover most of the primordia and the eve domain is reduced. This again demonstrates the complementary and exclusive nature of the eve and Dll domains in the anal primordia (Gorfinkiel, 1999).

The requirement for the Hh signal in Dll activation might be mediated by Wg and Dpp signals. This occurs in other ventral discs. Dll expression arises at the juxtaposition of Wg and Dpp expressing cells as revealed by double staining for Dll and Dpp, and Dll and Wg. In both genital and anal primordia, Dll expressing cells overlap those that express wg and dpp. It has been previously reported that the ectopic expression of both Wg and Dpp produces several phenotypic alterations in both female and male terminalia. Similar types of transformations are also induced by the lack of function of either patched (ptc) or Protein kinase A (Pka). In these mutants, the Hh pathway is constitutively active giving rise to the derepression of Wg and Dpp. The lack of Pka function in the genital disc induces ectopic Dll. This Dll induction requires both Wg and Dpp signals in the same cells since Dll is not activated in Pka2;dpp2 and in Pka2;wg2 double mutant clones, as occurs in other discs of ventral origin (Gorfinkiel, 1999).

In the male repressed primordium (RMP) of the female genital disc, wg is expressed but not dpp. Consequently, Dll is not expressed because Dll is only activated in cells that express both dpp and wg. Ectopic Dpp expression in the wg expression domain driven by the MS248-GAL4 line induces Dll 'de novo' in the RMP, which shows an increase in size. However, these changes do not allow the development of adult structures from this primordium since there is no activation of the male specifc cyto-differentiation genes because the genetic sex has not changed. Dll is not activated in the repressed female primordium (RFP) of the male genital disc despite the fact that, in this primordium, both wg and dpp are normally expressed. This activation does not occur even if the levels of Dpp are increased. These results suggest that specific genes expressed in the RFP can exert a negative control of Dll expression (Gorfinkiel, 1999).

In order to find other genes involved in the development of the terminal structures, the expression pattern and the functional requirement for optomotor-blind (omb) were examined. This gene encodes a protein with a DNA-binding domain (T domain) and behaves as a downstream gene of the Hh pathway in other imaginal discs. In the genital disc, Omb is detected in the dpp expression domains, abutting the wg expressing cells. This behaviour of omb expression is similar to that found in the leg and antennal discs. In the genital disc, omb is also regulated by the Hh signaling pathway since Pka2 clones also ectopically express omb. The phenotypes produced due to omb lack of function using the allele omb282 were examined; homozygous females for this allele could not be obtained but some male pharates were analyz

ed. In males, the dorsal bristles of the claspers and the hypandrium bristles are absent. Also, the hypandrium is devoid of hairs and the hypandrium fragma is reduced. Surprisingly, the anal plates are mostly somewhat enlarged in the ventral region and reduced in the dorsal areas. The structures affected in omb2 are duplicated when omb is overexpressed in the dpp domain using the dpp-GAL4/UAS-omb combination. In males, the dorsal bristles of the clasper and the hypandrium bristles are duplicated. These phenotypes are similar to the ones obtained as a result of ectopic Dpp (Gorfinkiel, 1999).

The present findings provide further evidence that the terminalia can be considered a ventral appendage. In addition, this is the first evidence for a similar genetic pathway operating in both the ventral and genital/anal appendages. From an evolutionary point of view, this lends support to the idea that the terminalia arose as the result of the modification of a primitive appendage in an ancestor common to all arthropods. The Dll requirement for some of the external genital structures broadens the definition of the evolutionarily conserved Dll function to cover a more fundamental role than that of the proximo/distal selector gene in the appendages (Gorfinkiel, 1999).

The Dll requirement for the formation of only the dorsal anal plate in females and for both anal plates in males lends further support to the idea that the anal plates form from two primordia. It is proposed that the anal primordia develop as follows: at the blastoderm stage, the anal primordium divides into two cell populations, one of which will form the dorsal analia in a female or the complete analia in a male (homologous anal primordium), while the other set of cells will form the ventral analia in a female and give rise to no structure in a male (non-homologous primordium). Previous reports using the transformed2ts (tra2ts) mutation are also consistent with this organization. In the switch from male to female development, using the tra2ts mutant, the ventral anal plate requires more time to reach a normal female phenotype than the dorsal anal plate. In the shift from the female-to-male determining temperature, the homologous anal primordium switches into the male program while the non-homologous primordium is brought into the repressed state. The functional requirement for Dll only in the homologous anal primordium supports the idea that the anal plates originate from two primordia. It also suggests the existence of other gene(s) responsible for the formation of the ventral anal plate in females (Gorfinkiel, 1999).

Coexpression of the homeobox genes Distal-less and homothorax determines Drosophila antennal identity

The Distal-less gene is known for its role in proximodistal patterning of Drosophila limbs. However, Distal-less has a second critical function during Drosophila limb development, that of distinguishing the antenna from the leg. The antenna-specifying activity of Distal-less is genetically separable from the proximodistal (PD) patterning function because certain Distal-less allelic combinations exhibit antenna-to-leg transformations without proximodistal truncations. Distal-less has been shown to act in parallel with homothorax (a previously identified antennal selector gene) to induce antennal differentiation. While mutations in either Distal-less or homothorax cause antenna-to-leg transformations, neither gene is required for the others expression, and both genes are required for antennal expression of spalt. Coexpression of Distal-less and homothorax activates ectopic spalt expression and can induce the formation of ectopic antennae at novel locations in the body, including the head, the legs, the wings and the genital disc derivatives. Ectopic expression of homothorax alone is insufficient to induce antennal differentiation from most limb fields, including those of the wing. Distal-less therefore is required for more than induction of a proximodistal axis upon which homothorax superimposes antennal identity. hth encodes a TALE-class homeodomain protein required for the nuclear localization of a PBC-class homeodomain protein encoded by extradenticle. Based on their genetic and biochemical properties, it is proposed that Homothorax and Extradenticle may serve as antenna-specific cofactors for Distal-less (Dong, 2000).

Animals heterozygous for Dll null alleles exhibit partial antenna-to-leg transformations, indicating that Dll levels may be important for antennal determination. Weak hypomorphic combinations of Dll alleles also lead to partial transformation of the third antennal segment (a3) and the arista into leg-like structures. Intermediate hypomorphic combinations of Dll alleles transform the medial antenna toward leg and exhibit distal truncations. Strong combinations of Dll alleles exhibit more severe antennal truncations. These same allelic combinations result in progressively more severe truncations of the distal leg. Notably, the antenna-to-leg transformations are not a property of a specific subset of Dll alleles, but are observed with all Dll alleles when assayed in appropriate combinations. For the transformation phenotype to be apparent, Dll PD function must be largely intact. This is likely due to the fact that the PD axis must be manifest in order for either antennal or leg identity to occur. The fact that transformation is observed without limb truncation indicates that the antennal selector function is more sensitive to Dll dosage than its PD function. Together, the results of Dll phenotypic analysis indicate that Dll is required for antennal identity, as well as for limb outgrowth (Dong, 2000).

Antp represses hth, thereby restricting hth expression to the proximal region of the leg. Antp also represses sal in the leg. Because antennal sal expression is dependent upon both Dll and hth, it was hypothesized that Antp repression of sal might be mediated via Antp repression of hth, which in turn prevents the overlap of Dll and Hth in the distal leg. Consistent with this possibility, Sal is expressed in Antp null clones in the Dll domain where hth is derepressed. It was therefore thought likely that Antp may be repressing sal expression indirectly by preventing hth from being expressed in the Dll domain of the leg. Since both Dll and Hth are required for antennal differentiation, by preventing the coexpression of Dll and Hth, Antp can preclude antennal development. The explanation for this favored by the authors is that when Hth is ectopically expressed using the GAL4/UAS system, Dll expression is downregulated in the cells producing Hth. These cells would then have Hth, but insufficient Dll. If both are required for antennal differentiation, antennal differentiation would not be possible. Consistent with this idea, a decrease in Dll expression in leg cells ectopically expressing Hth is seen (Dong, 2000).

Could Dll form a functional complex with Hth and Exd in the antenna? Given that Dll and Hth cooperate to regulate antennal differentiation, it is of interest to elucidate the molecular basis of this synergy. Exd and its vertebrate counterpart, Pbx, are known cofactors for a variety of homeodomain proteins, including Labial, Engrailed and Ultrabithorax. Hth is required for retention of Exd in the nucleus and may form part of the functional Exd/Hox complex. Vertebrate homologs of Hth, the Meis and Prep proteins, have been shown to form trimeric complexes with Hox and Pbx proteins. Several lines of evidence now support the idea that Exd and Hth are cofactors for the Dll homeodomain protein in the developing Drosophila antenna. These include: (1) the similar antenna-to-leg transformation phenotypes of Dll, hth and exd mutants; (2) the known physical interactions of Exd and Hth with other homeodomain proteins; (3) the fact that Dll and hth function in parallel to regulate antennal development, and (4) the fact that ectopically expressing Hth can mimic loss of Dll function in the antenna. Testing whether Dll, Hth and Exd interact physically and whether such a complex activates antennal enhancers will be important steps toward understanding limb development and tissue-specific gene regulation (Dong, 2000).

Proximodistal domain specification and interactions in developing Drosophila appendages

The morphological diversification of appendages represents a crucial aspect of animal body plan evolution. The arthropod antenna and leg are homologous appendages, thought to have arisen via duplication and divergence of an ancestral structure. To gain insight into how variations between the antenna and the leg may have arisen, the epistatic relationships among three major proximodistal patterning genes, Distal-less, dachshund and homothorax, have been compared in the antenna and leg of Drosophila. Drosophila appendages are subdivided into different proximodistal domains specified by specific genes, and limb-specific interactions between genes and the functions of these genes are crucial for antenna-leg differences. In particular, in the leg, but not in the antenna, mutually antagonistic interactions exist between the proximal and medial domains, as well as between medial and distal domains. The lack of such antagonism in the antenna leads to extensive coexpression of Distal-less and homothorax, which in turn is essential for differentiation of antennal morphology. Furthermore, a fundamental difference between the two appendages is the presence in the leg and absence in the antenna of a functional medial domain specified by dachshund. These results lead to a proposal that the acquisition of particular proximodistal subdomains and the evolution of their interactions has been essential for the diversification of limb morphology (Dong, 2001).

Each segment in the Drosophila leg is considered to be homologous to part or all of a segment in the antenna. The correspondences are based on reproducible homeotic transformations that can occur between parts of the two limbs. Such correlation enables a comparison of the expression domains of Dll, dac and hth between the antenna and the leg. The relative wild-type expression of these three important PD patterning genes of the leg differs in the antenna, indicating that their PD axes are differentially subdivided (Dong, 2001).

For example, at late third instar, Dll expression extends more proximally in the antenna into regions homologous to the leg trochanter. In addition, dac is expressed at lower levels and is expressed in fewer segments in the antenna than in the leg. The dac expression domain in the antenna lies completely within the Dll expression domain. In contrast, the dac and Dll domains in the leg are exclusive when dac expression is activated and remain largely non-overlapping at late third instar. hth is expressed only proximally in the leg, but is expressed throughout the antenna disc until early larval stages when it is lost from distal cells. Because Dpp and Wg, which regulate Dll, dac and hth in the leg, are similarly expressed in the antenna, it is thought unlikely that the differences in Dll, dac and hth expression could be accounted for by variations in Dpp and Wg expression. Instead, it is hypothesized that the differences are due to limb type-specific interactions between Dll, dac and hth. The results of experiments described here confirm that this is the case (Dong, 2001).

Gradients of the morphogens, Wg and Dpp, initiate the PD organization of the Drosophila leg by activating Dll and repressing dac distally and by repressing hth in the distal and medial leg. This creates three domains, distal, medial and proximal, that are specified by the expression Dll, dac and hth, respectively. The expression of dac is derepressed in clones of Dll-null cells in the presumptive distal region of the leg disc. The reciprocal is observed in dac null clones, where Dll expression expands into the medial domain. Mutually repressive interactions between the distal and medial domains therefore are required to keep these domains distinct from one another (Dong, 2001).

If the antenna is homologous to the leg, one might expect to find many genetic parallels, particularly with respect to the three major PD patterning genes of the leg, Dll, dac and hth. As in the leg, Dll and hth are required to specify the distal and proximal domains of the antenna. However, dac has a different function in the antenna. No deletions of antennal segments are observed in dac-null flies. In addition, the genetic relationships between Dll, dac and hth are different in the developing antenna. Specifically, the extensive overlap in expression of these three genes in the antenna indicates that domains are not kept separated as they are in the leg. The normal expression domain of dac in the antenna lies completely within an area of hth and Dll coexpression, making it unlikely that dac represses either gene. Nonetheless, because Dll and hth appear to have slightly lower levels of expression where dac is normally expressed, a test was performed to see whether either Hth or Dll levels would be elevated if dac were removed. No detectable changes in the levels of either Dll or Hth were observed in clones of cells that lack Dac. Therefore unlike the situation in the leg, Dac is insufficient to antagonize the expression of either Dll or hth in the antenna. Taken together, these data indicate that mutual antagonism is not a universal feature of appendage development (Dong, 2001).

The antennal regulation of dac by Dll also differs from that of the leg. The regulation of dac by Dll in the antenna varies depending on the proximodistal location. Dll can be a dac repressor or activator, or exert no effect on dac. Dac expression is not activated in Dll-null clones in the presumptive arista, whereas Dll-null clones in the presumptive base of the arista (segments a4 and a5) exhibit non-cell-autonomous dac activation, and Dll-null clones in the presumptive third antennal segment (a3), where dac is normally expressed, result in loss of dac. These data indicate that the regulation of dac by Dll in the antenna is different from that in the leg. They also indicate that the normal antennal expression of dac both requires Dll and has PD regional specificities. Because both Dll and Hth are required for antennal identity and are coexpressed with dac, Hth may also be required for the antennal expression of dac. Consistent with this view, ectopic expression of either Dll in antennal cells expressing Hth or of Hth in antennal cells expressing Dll can activate dac, as can ectopic coexpression of Dll and Hth in the wing disc. Furthermore, antennal dac expression, is not efficiently repressed by ectopic Hth (Dong, 2001).

Unlike Dll-null clones, both Dll hypomorphs and hth-null clones exhibit antenna-to-leg transformations. Examination of Dll hypomorphs and hth-null clones therefore reveals their homeotic functions. One such function may be the repression of leg dac. Leg expression of dac encompasses more segments and occurs at higher levels compared with the antenna. As in Dll hypomorphic leg discs, in Dll hypomorphic antenna discs, dac expression expands distally. hth-null clones exhibit derepression of dac in a1, a2 and a4 and elevation of Dac levels in a3. It is therefore proposed that the derepression of dac in Dll hypomorphs and in hth-null clones may represent leg-specific dac expression. Conclusive evidence for this awaits identification of dac enhancer elements and analysis of their regulatory inputs. Nonetheless, taken together, these data support the view that the regulation of leg and antennal dac expression occurs via distinct mechanisms and that the homeotic functions of Dll and hth are mediated not only through activation of antenna-specific genes such as spalt, but also through the active repression of leg development (Dong, 2001).

Appendages are subdivided by mutually antagonistic domains. Gradients of the morphogens Dpp and Wg initiate the PD organization of the Drosophila leg by activating Dll and repressing dac and hth distally, and by allowing the activation of dac while repressing hth medially. This creates three domains, distal, medial and proximal, that are specified respectively by expression of Dll, dac and hth. Further refinement and maintenance of the borders between domains requires mutually antagonistic interactions between proximal and medial domains as well as between medial and distal domains. Specifically, Dll and dac are mutually repressive. Also, mutually repressive interactions between the proximal and medial domains do exist via Tsh repression of dac and Dac repression of hth. Thus, pattern formation in the leg requires mutually antagonistic interactions among all three domains in order to refine and maintain borders that initially were set up by morphogens (Dong, 2001).

In contrast to the situation in the Drosophila leg, Dll, dac and hth are expressed in largely overlapping patterns in the antenna. This suggests that there is not mutual antagonism between Dll and hth in the antenna. Furthermore, that the entire antennal expression domain of dac lies within an area of Dll and hth coexpression indicates that Dac was unlikely to repress the antennal expression of either Dll or hth. Analysis of dac mutants confirms that Dac does not antagonize either proximal or distal development in the antenna but it does so in the leg. Therefore mutual antagonism is not a universal feature of appendage development (Dong, 2001).

Interestingly, in more basal insects like the cricket, Acheta domesticus, Dll and n-Exd expression are exclusive in the antenna. Since n-Exd is normally coincident with hth expression, it is inferred that Dll and Hth expression are exclusive in the cricket antenna. If exclusion reflects mutual antagonism, this in turn could indicate that mutual antagonism between proximal and distal domains is lost in the antenna within the insect lineage during the course of dipteran evolution (Dong, 2001).

It is noted that the absence of antagonism of any single PD domain towards another leads to overlap of otherwise exclusively expressed transcription factors. This, in turn, may permit the coexpressed factors to execute additional functions. Indeed, while Hth is required for proximal patterning of both antenna and leg, and Dll is required for distal patterning of both antenna and leg, their coexpression leads to the differentiation of antenna-specific cell fates. Thus, expression of distinct combinations of transcription factors such as Dll, Dac and n-Exd/Hth both in specific domains along the PD axis and between appendage types is likely to activate and repress particular suites of target genes, thereby contributing to differences in appendage morphologies (Dong, 2001).

The ability of Dll, Dac and n-Exd/Hth to repress the expression of one another undoubtedly is context-dependent. However, the only known factor involved in context specification is the Hox protein Antp. In the presence of Antp in the antenna, Dll and Hth are no longer coexpressed. Conversely, when Antp is removed from the leg, hth is derepressed in cells expressing Dll. Thus Antp appears to play a role in some aspects of domain antagonism. It remains unclear whether Antp directly modulates interactions among Dll, Dac and n-Exd/Hth or whether there are other molecules that intervene (Dong, 2001).

n-Exd/hth and Dll, and their homologs are expressed respectively in the proximal and distal domains in the appendages of animals as diverse as arthropods and vertebrates, and are required for the proximal and distal development in many Drosophila appendages. It is therefore suggested that the existence of both proximal and distal domains in appendages pre-dates the evolution of the arthropods. However, with the available information, it cannot be said whether these domains in the ancestral appendage were distinct, as they are in the modern Drosophila leg, or overlapping, as they are in the Drosophila antenna. It is speculated that n-Exd and hth, and their vertebrate homologs, the Pbx and Meis genes, were ancestrally expressed in the body wall because they are in modern animals and that as limbs evolved, they were originally expressed throughout the entire outgrowth. Subsequent antagonism by distal factors such as Dll could have allowed for the evolution of additional domains within different appendages (Dong, 2001).

This comparison of the Drosophila antenna and leg leads to the conclusion that a fundamental difference between these homologous appendages is the presence of a functional medial domain in the leg, specified by dac. The antenna has fewer segments, with dac expressed at relatively low levels and in only one of the segments, whereas dac is expressed in at least four leg segments. Loss of dac results in medial deletions in the leg but not in the antenna. Repression of proximal and distal genes by dac is not observed in the antenna, as it is in the leg. Consequently, the antennal expression of n-Exd/hth and Dll are not separated in the antenna by a medial domain that expresses dac. For these reasons, it is proposed that the acquisition of a medial domain, possibly through the use of dac, may have been a distinct step in appendage evolution. Consistent with this, increasing the territory and levels of dac expression in the antenna leads to repression of hth and Dll and to the differentiation of medial leg structures (Dong, 2001).

Two scenarios by which the existing Drosophila domain organizations may have arisen can be envisioned, given primitive appendages that had only proximal and distal domains. One possibility is that the medial domains were initially acquired by both the antenna and leg, but lost from the antenna sometime prior to the evolution of Drosophila. A second possibility is that the medial domain is an innovation of only the leg and may never have existed in the antenna. The expression of dac in the legs and its absence in the antennae of other arthropods may provide support for the latter scenario. Comparison of the relative domains of expression and the functions of Dll, dac and hth in other organisms will undoubtedly lead to further insights into how distinct PD domains were acquired and became patterned during the course of appendage evolution (Dong, 2001).

Ectopic expression of DREF induces DNA synthesis, apoptosis, and unusual morphogenesis in the Drosophila eye imaginal disc: possible interaction with Polycomb and trithorax group proteins

The promoters of Drosophila genes encoding DNA replication-related proteins contain transcription regulatory element DRE (5'-TATCGATA) in addition to E2F recognition sites. A specific DRE-binding factor, DNA replication-related element factor or DREF, positively regulates DRE-containing genes. In addition, it has been reported that DREF can bind to a sequence in the hsp70 scs' chromatin boundary element that is also recognized by boundary element-associated factor, and thus DREF may participate in regulating insulator activity. To examine DREF function in vivo, transgenic flies were established in which ectopic expression of DREF was targeted to the eye imaginal discs. Adult flies expressing DREF exhibited a severe rough eye phenotype. Expression of DREF induces ectopic DNA synthesis in the cells behind the morphogenetic furrow that are normally postmitotic, and abolishes photoreceptor specifications of R1, R6, and R7. Furthermore, DREF expression caused apoptosis in the imaginal disc cells in the region where commitment to R1/R6 cells takes place, suggesting that failure of differentiation of R1/R6 photoreceptor cells might cause apoptosis. The DREF-induced rough eye phenotype is suppressed by a half-dose reduction of the E2F gene, one of the genes regulated by DREF, indicating that the DREF overexpression phenotype is useful to screen for modifiers of DREF activity. Among Polycomb/trithorax group genes, it was found that a half-dose reduction of some of the trithorax group genes involved in determining chromatin structure or chromatin remodeling (brahma, moira, and osa) significantly suppresses and that reduction of Distal-less enhances the DREF-induced rough eye phenotype. The results suggest a possibility that DREF activity might be regulated by protein complexes that play a role in modulating chromatin structure. Genetic crosses of transgenic flies expressing DREF to a collection of Drosophila deficiency stocks allowed identification of several genomic regions, deletions of which caused enhancement or suppression of the DREF-induced rough eye phenotype. These deletions should be useful to identify novel targets of DREF and its positive or negative regulators (Hirose, 2001).

Distal-less: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | References

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