proboscipedia

Promoter Structure

A conserved region located in the second intron of proboscipediais essential for proper formation of the adult mouthparts. This region directs pb expression of embryonic labial, maxillary and mandibular head segments, sub-esophageal ganglia, and supraesophageal ganglia, and in larvae, the labial discs, sub- and supra-esophageal ganglia (Randazzo, 1991). A 0.5 kb fragment from this region has beenshown to direct expression in both embryos and third instar labial discs whencombined with a 600 bp pb basal promoter sequence. A 32 bp element contained within the 0.5kb region functions as a labial disc enhancers for pb. Surprisingly, the conserved second intron pbenhancers do not function properly with a heterologous hsp70 promoter, suggesting thatpromoter-specific interactions occur at the pb locus. The pb transcription unit does not require sequences upstream of -98 bp to provide pb functionin the labial discs. Rather, pb's upstream DNA appears to contain negative regulatory DNArequired for silencing PB accumulation in inappropriate domains of third instar imaginal discs (Kapoun, 1995).

Transcriptional Regulation

The gene proboscipedia (pb) is a member of the Antennapedia complex in Drosophila and is required for the proper specification of the adult mouthparts. In the embryo, pb expression serves no known function despite having an accumulation pattern in the mouthpart anlagen that is conserved across several insect orders. Several of the genes necessary to generate this embryonic pattern of expression have been identified. These genes can be roughly split into three categories based on their time of action during development. (1) Prior to the expression of pb, the gap genes are required to specify the domains where pb may be expressed. (2) The initial expression pattern of pb is controlled by the combined action of the genes Deformed (Dfd), Sex combs reduced (Scr), cap'n'collar (cnc), and teashirt (tsh). cnc and tsh act as as negative regulators of pb expression in the mandible and first thoracic segments, respectively. (3) Maintenance of this expression pattern later in development is dependent on the action of a subset of the Polycomb group genes. These interactions are mediated in part through a 500-bp regulatory element in the second intron of pb. Dfd protein binds in vitro to sequences found in this fragment. This is the first clear demonstration of autonomous positive cross-regulation of one Hox gene by another in Drosophila and the binding of Dfd to a cis-acting regulatory element indicates that this control might be direct (Rusch, 2000).

Many of the genes that are members of either the gap, pair rule, or segment polarity genes have some effect on the pattern of pb accumulation. For the most part, mutations in genes of these classes reduce the number of cells expressing pb but do not eliminate Pb entirely from the affected segments. In no case do they cause pb to accumulate ectopically. The most striking results were caused by zygotic mutations in the genes buttonhead (btd), giant (gt), and hunchback (hb). btd is a head gap gene required for formation of the mandibular segment. In btd mutants, no mandibular structures are seen and no pb accumulation occurs anterior of the maxillary segment. pb accumulation is normal in the other gnathal segments. Mutations in both gt and hb disrupt the formation of the labial lobe and result in concomitant loss of pb expression therein. When the pb reporter #7 was tested in a hb mutant background no lacZ expression in the presumptive labial segment was found. In the case of gt, pb expression is not entirely extinguished. Weak pb accumulation can sometimes be seen in the most dorsal and posterior cells of the presumptive labial segment, overlapping with the few remaining cells of the engrailed stripe in the labial segment. For both gt and hb, this reduction or loss of pb expression in the labial lobe cannot be attributed to alterations in the Scr pattern because Scr accumulates in the cells posterior to the maxillary segment (Rusch, 2000).

Nearly all the pair rule and segment polarity genes affect morphology of the gnathal segments and to varying degrees perturb pb accumulation. In general, mutations in the pair rule genes eliminate either the maxillary or labial lobe, as well as reduce the width of the respective segment. Despite these effects on morphology, pb expression can often be seen in the affected segments. Mutations in the segment polarity genes affect the morphology of both the maxillary and labial lobes. The overall effect is a reduction in the size of these lobes, resulting in a correspondingly reduced number of pb-expressing cells. Of the segment polarity genes tested, wingless has the strongest effect on pb expression. At early stages in wg^CX4 mutants, no pb expression is apparent in the presumptive labial lobe, though later, as head involution commences, some of these cells do begin to express pb (Rusch, 2000).

On the basis of these results and previous work, a working model for the regulation of pb has been developed. This model accounts for both temporal and spatial aspects of pb expression. In effect, the regulation of pb can be broken down into early, middle, and late phases. The early phase represents the period prior to the onset of pb expression, during which the gap genes define the domain of pb expression through a presumably indirect mechanism. During the middle phase, the genes cnc, Dfd, Scr, and tsh act to establish the initial expression pattern of pb along the A/P axis. During the late phase it is proposed that the PcG and trxG genes assume responsibility for maintaining the pattern of pb expression through the later stages of embryogenesis (Rusch, 2000).

The early phase reflects a requirement for gap gene function for normal expression of pb to occur during later stages. Specifically, btd, gt, and hb have been identified as being required for proper gnathal expression of pb. The function of the head gap gene btd has been shown to be required only during early stages of embryogenesis. The expression patterns of gt and hb are such that they are no longer expressed in the labial segment at the time when pb expression begins. This is taken as a strong indication that the gap genes influence pb indirectly. Consistent with this hypothesis, no gt or hb binding sites could be detected in the regulatory elements of the pb reporter. In the case of hb, the role that various trans-acting factors might play in mediating loss of pb expression in the labial segment was investigated. Expression of Scr, the positive regulator of pb in the labial segment, is not eliminated. Further, repression of pb is not attributable to expansion of tsh expression. One possibility is that another negative regulator is being expressed such that Scr can no longer activate pb. Given the negative regulatory interactions that occur between the gap genes, it is possible that one of the other gap genes might be misexpressed and downregulate pb. However, it may be misexpression of cnc or some other gene that has yet to be identified. Alternatively, the 'hit-and-run' hypothesis, proposed to explain the long-term repression of Ultrabithorax (Ubx) by hb, may describe how transient expression of the gap genes is required very early in development to permit later expression of pb. In this hypothesis, heritable changes in chromatin structure, mediated by the PcG genes, are invoked to explain how hb regulates Ubx long after hb expression has ceased. In the case of pb regulation, one or more of these gap genes may be required to alter chromatin structure in and around the pb locus, thereby allowing the various trans-acting factors access to the pb cis-acting regulatory elements (Rusch, 2000).

During the middle phase, the initial expression pattern of pb is set by a variety of trans-acting factors. Focus was placed on the identification of those factors that determine the ectodermal pattern of pb expression along the A/P axis of the embryo. The Hox genes Dfd and Scr act as positive regulators of pb and Dfd can bind to pb regulatory elements in vitro. It is thought likely that Scr also regulates pb directly based on the similarity with which mutations in Dfd and Scr affect expression of pb and the pb reporter. In addition to the Hox genes, the region-specific homeotics cnc and tsh have been identified as negative regulators of pb and serve to restrict pb expression to the gnathos. It is not clear whether these genes regulate pb directly, though in the case of tsh the sequence TGGAAAAGT has been identified in the 500-bp regulatory fragment used in the pb reporter; this sequence is very similar to the identified tsh binding site. While this regulatory paradigm does not completely describe the regulation of the endogenous gene, based on the presence of pb residual expression, it is sufficient to explain the behavior of the 500-bp pb reporter. This mechanism of regulation places pb downstream of the Hox genes and is the first instance in Drosophila where one Hox gene is positively and directly regulated by another, a distinction previously accorded only to vertebrate Hox genes. Studies by others have suggested that wg may be mediating the nonautonomous residual expression of pb that is uncovered by mutations in Dfd and/or Scr. With the exception that wg has the strongest effect on pb expression of the segment polarity genes tested, the results shed little light on the mechanism that underlies this phenomenon. However, non-cell autonomous signaling has been implicated to explain regulation of ectodermal pb function by mesodermal expression of Scr; perhaps the residual expression in the embryo is an example of this pathway. Further experiments, including identification of an enhancer that mediates this residual expression, are needed (Rusch, 2000).

Finally, during the late phase, two PcG genes, Psc and ph, have been identified that are involved in maintaining repression of pb outside its normal domain of expression. This result supersedes a previous report that the PcG genes do not regulate pb. No trxG genes have been identified that are required for the maintenance of pb expression. To function, the PcG genes are thought to assemble on DNA in large multimeric complexes. Unlike the genes of the BX-C, which are regulated, to a greater and lesser extent, by all the PcG genes that have been tested, pb is not regulated by the majority of known PcG genes. Assuming that the PcG genes function similarly at the pb locus, the implication is that not all multimeric complexes can be equal. However, it is not clear how these differences are established. One possibility is that complexes composed of different combinations of PcG genes are formed at different times during development, thereby regulating different loci. Interestingly, a vertebrate homolog of Psc has been shown to bind a specific DNA sequence. This exact sequence is also found in the regulatory elements of the pb reporter construct, indicating that Psc may bind directly, though this remains to be shown (Rusch, 2000).

In addition to forming quantitatively different complexes, the timing of complex formation may be crucial to the proper expression of pb. Normally, the PcG genes are required after the expression pattern of pb has been established to prevent ectopic expression. Presumably, this ectopic expression would result from newly expressed transcription factors acting inappropriately at the pb locus. This hypothesis may offer an explanation for the differences seen between the expression pattern of endogenous pb and the pb reporter in tsh mutant backgrounds. In this scenario, Scr expression in T1 begins concurrently with the initiation of PcG-mediated repression at the pb locus. In the absence of tsh, competition between activation by Scr and repression by the PcG genes results in the weak and variable pb expression. Because the PcG genes do not regulate ß-galactosidase expression from the pb reporter, pb is free to respond strongly to Scr accumulation in T1. Further experiments are required to support this hypothesis (Rusch, 2000).

In Drosophila, pb plays a role in specifying limbs to become specialized for feeding. In other insects where it has been examined, pb is expressed in and required for the formation of the larval mouthparts. Overall, the expression patterns of Dfd and Scr have also been conserved in other insects. Given these results, it is interesting to speculate about whether the regulation of pb may be conserved in other insects. Preliminary results from studies in Tribolium suggest that at least some aspects of pb regulation may be conserved. A deficiency that deletes many of the Tribolium Hox genes except for the lab, pb, and Abd-B homologs has been isolated. Embryos homozygous for this deficiency display a mutant phenotype in which every segment of the Tribolium larva is transformed toward an antennal segment and bears a pair of antennae. These larvae have no mouthparts or walking legs. Further, gain-of-function mutations in Tribolium pb, which result in ectopic expression of Tribolium pb in the antennal segment, are known to transform the antennae into generic feeding palps. Given these two results, it can be inferred that pb is not being expressed in embryos containing this deficiency because the antennae are not transformed into feeding palps. If pb is not being expressed, this implies that the positive regulators of pb have been removed by this deficiency. The simplest interpretation that is consistent with these results and the results presented here is that the Dfd and Scr homologs in Tribolium may be required to positively regulate the Tribolium pb much like their counterparts do in Drosophila. Actual proof of this possibility requires in situ data on the expression pattern of the pb homolog in various Tribolium Hox mutants (Rusch, 2000).

Cross-regulation of Homeotic Complex (Hox) genes by ectopic Hox proteins during the embryonic development of Drosophila was examined using Gal4 directed transcriptional regulation. The expression patterns of the endogenous Hox genes were analyzed to identify cross-regulation while ectopic expression patterns and timing were altered using different Gal4 drivers. Evidence is provided fortissue specific interactions between various Hox genes and the induction of endodermal labial (lab) by ectopically expressedUltrabithorax outside the visceral mesoderm (VMS). Similarly, activation and repression of Hox genes in the VMS from outside tissuesseems to be mediated by decapentaplegic gene activation. Additionally, it has been found that proboscipedia (pb) is activated in the epidermis byectopically driven Sex combs reduced (Scr) and Deformed (Dfd); however, mesodermal pb expression is repressed by ectopic Scr in thistissue. Mutant analyses demonstrate that Scr and Dfd regulate pb in their normal domains of expression during embryogenesis. EctopicUltrabithorax and Abdominal-A repress only lab and Scr in the central nervous system (CNS) in a timing dependent manner; otherwise,overlapping expression in the CNS in tolerated. A summary of Hox gene cross-regulation by ectopically driven Hox proteins is tabulated for embryogenesis (Miller, 2001).

The pb gene is normally expressed during embryogenesisbut mutants have no apparent embryonic phenotype. However, ectopic Pb protein in the embryo does produce homeotic transformations in embryos. These observations suggest that the regulation of pb expression during embryogenesis may be importantfor proper development. Both Scr and Dfd are necessary for establishing the proper expression patterns of pb during embryogenesis. Althoughectopic Scr and Dfd function equivalently to activate pb inthe antennal segment epidermis, they have an oppositeeffect on native pb expression in the mandibular mesoderm(Mn). Ectopic Dfd accumulation has no significanteffect on pb expression in the Mn, which is part of the Dfdexpression domain. However, ectopic expression of Scr by the prd and 69B drivers represses pb expression in the Mn, demonstrating the opposite tissue specific regulation of pb by Scr (Miller, 2001).

Genetic analyses demonstrate native regulatory interactions between pb, Dfd and Scr during embryogenesis. The reduction of pb expression in Dfd and Scr mutant backgrounds shows that normal pb expression is dependent on these genes. Dfd is required in the mandibular mesoderm (Mn) and anterior maxillary (Mx) segments (Miller, 2001).

Similarly, Scr is necessary in the posterior Mx and Labial(Lb) segments. The functional significance of these regulatory interactions is debatable due to the lack of mutant embryonic phenotypes in pb nulls; however, the evolutionary implications are perhaps more interesting. Positive cross-regulation between Hox genes has not been previously demonstrated in Drosophila exceptthrough signal transduction. Nevertheless, vertebrate enhancers that are responsible for direct positive cross-regulation exhibit similar activity when tested inDrosophila suggesting that these differences are probably due to evolutionary changes at cis-regulatory elements. Since there is no clear mutant pb embryonicphenotype, these cis-regulatory elements apparently direct positive Hox regulatoryinteractions between Scr, Dfd and pb may be atavistic andnon-functional in derived insects such as Drosophila. However, these cis-regulatory elements may also be necessary for proper Hox gene expression later in development (Miller, 2001).

Epidermal Hox interactions seem to fit the posterior dominance model best.That is, the observed effects on resident Hox gene expression caused by ectopic Hox protein accumulation usually exhibit repression of the more anteriorly expressed gene. Antp represents the predominant exception to this hierarchy. Ectopic Antpprotein represses lab expression in epidermal cells but hasno significant effect on pb, Dfd or Scr expression in thistissue. However, Antp normally restricts theposterior domain of Scr in the VMS in a manner that appearsto be mediated through short-range signaling. Since there is such a clear effect on the most anteriorly expressed Hox gene lab, while the three Hox genes(expressed more posterior to lab yet anterior to Antp'sdomain) appear to be refractory to Antp's negative control,it would appear these indifferent Hox genes or some other factor is negating Antp's influence. However, a resolution of the underlying cause of this observation awaitsfurther experimentation (Miller, 2001).

In summary, cross-regulation of the Hox genes cannot bedescribed effectively by generalized models such as posterior prevalence. Among the Hox genes, different tissues exhibit unique characteristics and timing dependent interactions. Moreover, signal transduction pathways complicateinterpretations of cross-regulation between Hox genes inthese ectopic expression experiments since these pathwaysare not always subject to current regulatory paradigms. Observations that Scr and Dfd positively regulate pb in a tissue specific manner suggests that some interactions may be atavistic or perhaps, only significant in other developmentalstages. Posterior prevalence occurs frequently but specific interactions demonstrate extensive variability; however, much of this variability is likely due to indirect signaling cascades set up by the Hox genes themselves (Miller, 2001).

During animal development, the HOM-C/HOX proteins direct axial patterning by regulating region-specific expression of downstream target genes. Though much is known about these pathways, significant questions remain regarding the mechanisms of specific target gene recognition and regulation, and the role of co-factors. From studies of the gnathal and trunk-specification proteins Disconnected (Disco) and Teashirt (Tsh), respectively, evidence is presented for a network of zinc-finger transcription factors that regionalize the Drosophila embryo. Not only do these proteins establish specific regions within the embryo, but their distribution also establishes where specific HOM-C proteins can function. In this manner, these factors function in parallel to the HOM-C proteins during axial specification. In tsh mutants, disco is expressed in the trunk segments, probably explaining the partial trunk to head transformation reported in these mutants, but more importantly demonstrating interactions between members of this regionalization network. It is concluded that a combination of regionalizing factors, in concert with the HOM-C proteins, promotes the specification of individual segment identity (Robertson, 2004).

Though HOM-C genes have a clear role in establishing segmentidentities, ectopic expression often has only a limited effect. The dataindicate that, for Dfd, this restriction arises because of the limiteddistribution of Disco in the trunk segments. There are two importantconclusions from these observations: (1) the spatial distribution of Disco establishes where cells can respond to Dfd, and this is probably true for Scr as well. Cells expressing disco develop a maxillary identity when provided with Dfd, even though this may not have been their original HOM-C-specified fate. This highlights (2) -- the combination of Disco and Dfd overrides normal trunk patterning, without altering expression of tsh and trunk HOM-C genes. As with the maxillary segment, identity is lost in the mandibular and labial segments when embryos lack disco and disco-r. This indicates that Disco and Disco-R may have similar roles in all gnathal segments. That co-expression of Disco and Scr in the trunk activates the Scr gnathal target gene pb strengthens this conclusion. Therefore, it is proposed that Disco defines the gnathal region, and establishes where the gnathal HOM-C proteins Dfd and Scr can function (Robertson, 2004).

Homeotic effects

Two independent pb minigene P-element insertion lines completely rescuethe labial palp-to-first leg homeotic transformation caused by pb null mutations; thus, a homeoticgene of the ANTP-C can properly carry out its homeotic function outside of the complex. Despitethe complete rescue of the null, the minigene expresses PB protein in only a subset of pb's normaldomains of expression (Randazzo, 1991).

The cut locus codes for a homeodomain protein and controls the identity of a subset of cells in the peripheral nervous system in Drosophila. During a screen to identify cut-interacting genes, it was observed that flies containing a hypomorphic cut mutation and a heterozygous deletion of the Antennapedia complex exhibit a transformation of mouthparts into leg and antennal structures similar to that seen in homozygous proboscipedia (pb) mutants. The same phenotype is produced with all heterozygous pb alleles tested and is fully penetrant in two different cut mutant backgrounds. This phenotype is accompanied by pronounced changes in the expression patterns of both cut and pb in labial discs. These experiments implicate cut in the regulation of expression and/or function of two homeotic genes (Johnston, 1998).

The adult mouthparts are produced from the labial imaginal discs. Proboscipedia is expressed in nuclei of labial disc cells in third instar larvae; Cut is expressed in a pattern that substantially overlaps that of Pb and is also nuclear. In wild-type and ctL188 discs, Cut and Pb are expressed throughout the entire disc, however in ctL188; pb5/+ double mutant discs both the level and the pattern of expression for both proteins is altered. The level of expression for both proteins is significantly decreased overall and entirely lost in some of the cells. Where present, Cut expression appears more punctate in comparison to wild-type discs. The mutant discs are morphologically abnormal. Staining of ctL188; pb5/+ labial discs with acridine orange shows no consistent increase in apoptotic cell death relative to that in control discs at this stage. Pb expression is undetectable in pb1/pb5 mutant labial discs and the pattern of Cut expression is altered to resemble that of a leg imaginal disc, in which Cut is expressed in two rows of cells in the position of the future claw organ in the most distal segment (Johnston, 1998).

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