BIOLOGICAL OVERVIEW

Proboscipedia is a homeotic protein required for the formation of labial and maxillary palps. It is a member of the Antennapedia Complex (ANTP-C), a linked array of homeodomain proteins. Both Proboscipedia (Pb) and Sex combs reduced (Scr) activities are required for determination ofproboscis identity, while Scr determines tarsus identity. Simultaneous removal of Pb and Scr activity results in a proboscis-to-antenna transformation. Previous genetic observations suggest that Pb and Scr activity may interact. Five pieces of evidence support an interaction between Pb and Scr: (1) the proboscis of a null pb mutant is transformed into a pair of tarsi (the terminal segments of the leg), and (2) these alleles also result in reduced maxillary palps, which some investigators have interpreted as a transformation of the maxillary palps into antennae. (3) Ectopic expression of Pb from a heat-shock promoter/pb fusion gene, or in a small clone of cells from a Tubulin a1 (Tub a1) promoter/ pb fusion gene result in the transformation of the antennae into maxillary palps. (4) Ectopic expression of Scr from a heat-shock promoter/Scr fusion gene results in the transformation of the aristae into tarsi. (5) The proboscis of semilethal loss-of-function Scr alleles, and clones of Scr null mutant cells in the proboscis adopt maxillary palp identity (Percival-Smith, 1997 and references).

That both Pb and Scr activities are required for determination of proboscis identity, and that individual expression of Pb and Scr activities determines maxillary palp and tarsus identities, respectively, suggests a simple model for determination of four developmental identities. It is proposed that the expression patterns of Pb and Scr determine antenna, maxillary palp, tarsus and proboscis identities. Specifically, the absence of Pb and Scr expression, the default state, leads to antennal identity, expression of only Pb activity leads to maxillary palp identity, expression of only Scr activity leads to tarsus identity, and expression of both Pb and Scr activities leads to proboscis identity. A prediction of this simple model is that a proboscis primordial cell that is unable to express either Pb or Scr will adopt antennal identity (Percival-Smith, 1997).

Two mechanisms for the role of Pb and Scr in proboscis determination may be proposed. In both models,Pb regulates a set of Pb-regulated genes which, when expressed in isolation, determine maxillary palp identity. Similarly, Scr regulates a set of Scr-regulated genes that, when expressed inisolation, determine tarsal identity. In one model, expression ofboth sets of Pb-regulated genes and Scr-regulated genes in the same cell determines proboscis identity. In a second model, expression of Pb and Scr proteins in the same cell leads to formation of a Pb-Scr-containing, heteromeric, protein complex that regulates a novel set of genes that determines proboscis identity, the Pb-Scr-regulated genes. If the second model is correct, it should be possible to design dominantnegative Pb and Scr molecules that will inhibit one another's activity (Percival-Smith, 1997).

In choosing the mutations used for the designed dominant negative Pb and Scr molecules, the properties of previously described change of DNA-binding specificity mutants made them ideal candidates. Both Pb and Scr have a glutamine at position 50 of the homeodomain (HD): pb and Scr genes have been created where this glutamine has been substituted for a lysine. This change is expected to change the DNA-binding specificity of Pb and Scr from Antennapedia class DNA-binding sites to Bicoidclass DNA-binding sites, as has been extensively documented for other HDs. The result of this change would be that the Pb Q50K and Scr Q50K molecules, as well the Pb Q50K Scr and Pb-ScrQ50K -containing complexes, would not only have diminishedaffinity for their normal interaction site, but would also have an increased affinity for another set of sites, dragging away from their normal site of interaction the Pb Q50K and Scr Q50K molecules, as well as the Pb Q50K Scr and Pb-Scr Q50K -containing complexes (Percival-Smith, 1997).

Dominant negative Pb molecules inhibit the activity of Scr indicating that Pb and Scr interact in a multimeric protein complex in determination of proboscis identity. These data suggest that the expression pattern of Pb and Scr and the ability of Pb and Scr to interact in a multimeric complex control the determination of four adult structures (see above: antenna, maxillary palp, tarsus and proboscis). However, the Pb-Scr interaction is not detectable in vitro and is not detectable genetically in the head region during embryogenesis, indicating the Pb-Scr interaction may be regulated and indirect (for example, an additional factor binding to both proteins). This regulation may also explain why ectopic expression of Scr(Q50K) and Scr does not result in the expected transformation of the maxillary palp to an antennae and proboscis, respectively. Previous analysis of the requirements of Scr activity for adult pattern formation has shown that ectopic expression of Scr results in an antenna-to-tarsus transformation, but removal of Scr activity in a clone of cells does not result in a tarsus-to-arista transformation. In five independent assays the reason for this apparent contradictory requirement of Scr activity in tarsus determination is shown. Scr activity is required cell nonautonomously for tarsus determination. Specifically, it is proposed that Scr activity is required in the mesodermal adepithelial cells of all leg imaginal discs at late second/early third instar larval stage for the synthesis of a mesoderm-specific, tarsus-inducing, signaling factor, which after secretion from the adepithelial cells acts on the overlaying ectodermal cells determining tarsus identity (Percival-Smith, 1997).

It is suggested that the Drosophila leg is made up of two developmental fields: the tarsus and the proximal leg. These two developmental fields may correlate with the nuclear (proximal) versus cytoplasmic (distal) intracellular localization of Extradenticle, and the distal expression of Distalless. It is also proposed that there are four genetic pathways working in leg determination. The first pathway is the cell nonautonomous Scr-dependent, tarsus-inducing, signal pathway, and this lays down the plan for the basic unmodified tarsus. The second pathway is the relatively cell autonomous proximal legpathway, which can be activated by the expression of Scr, Antp or Ubx and which lays out the basic plan for the proximal leg. The third and fourth pathways are cell autonomous pathways that Scr and Ubx control. A basic leg plan results in second leg identity, but expression of Scr or Ubx in both the proximal and distal portions of this basic plan brings about modifications resulting in first or third legidentity, respectively (Percival-Smith, 1997 and references).

When pb is mutated, labial palps are transformed to prothoracic legs and maxillary palps are small and malformed. pb has proven an ideal gene to study the role of homeotic proteins in regional determination and regulation of other homeotic genes. Two examples are given below.

In the first example, a particular deletion mutation in the ANTP-C results in diminished function of proboscipedia, and consequently in a defective head phenotype. This mutant also has a dominant thoracic defect related to diminished expression of the homeotic Antennapedia (Antp) gene. The reduced expression of Antp is a consequence of ectopic pb expression in the thorax. In the mutant the pb gene has come under the control of a second promoter, Antp P1, inaddition to pb's own promoter. Ectopic PB protein expression occurs under Antp P1 control and results in diminished head transcription of pb and diminished accumulation of ANTP protein in the imaginal disc cells where Antp P1 is normally expressed. Thus PBprotein is capable of participating in the negative regulation of a more posteriorly expressedhomeotic gene (Antp), as well as serving a homeotic "selector" function in the head (Cribbs, 1992a).

In a second example of homeotic gene function, a mini-gene for pb was combined with a heat shock promoter. Expression of this Heat shock:proboscipedia element (HSPB) directs a dominant transformation of antennae toward maxillary palps. This kind of transformation is called a homeotic transformation and is characteristic of the fate determining roles of the homeotic genes. The extent of the transformation depends on the level of PB protein. HSPB function overrides the dominant antennal transformations (from antenna to ectopic leg) caused by Antennapedia mutations in a dose-sensitive manner, directing a switch of the antennal disc-derived leg to ectopic maxillary palp. The switch from ectopic leg to maxillary palp is caused by expression of pb and its function in inhibiting Antp. This switch correlates with strikingly reduced ANTP protein accumulation when PB concentrations exceeded a genetically defined threshold level. Thus PB is able to quantitatively determine segmental identity and can actually compete with other homeotic proteins to alter fate determination (Cribbs, 1995).

Homeotic proboscipedia function modulates hedgehog-mediated organizer activity to pattern adult Drosophila mouthparts

Drosophila proboscipedia (HoxA2/B2 homolog) mutants develop distal legs in place of their adult labial mouthparts. How pb homeotic function distinguishes the developmental programs of labium and leg has been examined. The labial-to-leg transformation in pb mutants occurs progressively over a 2-day period in mid-development, as viewed with identity markers such as dachshund (dac). This transformation requires hedgehog activity, and involves a morphogenetic reorganization of the labial imaginal disc. These results implicate pb function in modulating global axial organization. Pb protein acts in at least two ways. (1) Pb cell autonomously regulates the expression of target genes such as dac; (2) Pb acts in opposition to the organizing action of hedgehog. This latter action is cell-autonomous, but has a nonautonomous effect on labial structure, via the negative regulation of wingless and decapentaplegic. This opposition of Pb to hedgehog target expression appears to occur at the level of the conserved transcription factor cubitus interruptus/Gli that mediates hedgehog signaling activity. These results extend selector function to primary steps of tissue patterning, and leads to the notion of a homeotic organizer (Joulia, 2005).

The labial palps, the drinking and taste apparatus of the adult fly head, are highly refined ventral appendages homologous to legs and antennae. As for most adult structures, these mouthparts are derived from larval imaginal discs, the labial discs. Wild-type pb selector function acts together with a second Hox locus, Scr, to direct the development of the labial discs giving rise to the adult proboscis. In the absence of pb activity, the adult labium is transformed to distal prothoracic (T1) legs, reflecting the ongoing expression and function of Scr in the same disc. Though the pb locus shows prominent segmental embryonic expression, as for the other Drosophila homeotic genes of the Bithorax and Antennapedia complexes, it is unique in that it has no detected embryonic function and null pb mutants eclose as adults that are unable to feed. Thus, normal pb selector function is required relatively late, in the labial imaginal discs that proliferate and differentiate during larval/pupal development to yield the adult labial palps. Though the genetic pathway guiding development of the ventral labial imaginal discs to adult mouthparts remains relatively unexplored both in flies and elsewhere, study of P-D patterning has identified several genes subject to pb regulation in the labial discs (notably Dll, dac, and hth) and a distinct organization of normal labial discs has been indicated compared to other imaginal discs (Joulia, 2005).

This study pursued an investigation of how pb homeotic function distinguishes between labial and leg developmental programs. The results implicate pb function at the level of global axial organization. Employing identity markers such as dachshund (dac), a 2-day period late in larval development has been identified when normal pb function is required for labial development. The labial-to-leg transformation occurs during the third larval instar stage, involves a progressive morphogenetic reorganization of the labial imaginal disc, and is hedgehog-dependent. This analysis of the transformation indicates that normal pb action is required at least at two distinct levels. One is in the cell-autonomous regulation of target genes such as dac likely to be implicated in cell identity. A second level involves an autonomous action with a nonautonomous effect on labial structure, through the negative regulation of wingless and decapentaplegic downstream of hh signaling. This opposition to hh targets is likely to occur at the level of the transcription factor cubitus interruptus/Gli, a crucial and conserved mediator of hh signaling activity. These results led to a proposal that homeotic function may exist in intimate functional contact with the hedgehog organizer signaling system: the 'homeotic organizer' (Joulia, 2005).

Segmental organization in the imaginal discs involves the reiterated deployment of segment polarity genes that organize the fundamental segmental form. This involves a cascade proceeding from posteriorly expressed Engrailed protein through a short-range Hh morphogen gradient in anterior cells favoring the activator form of Ci transcription factor, which in turn activates wg and dpp to establish two concurrent, instructive concentration gradients that structure gene expression along the proximo-distal axis. In contrast with this elaborate choreography of the segment polarity genes, the homeodomain proteins encoded by Hox genes are expressed in a segmental register, which obscures how they can direct the differentiation of distinct cell types within the segment. The present investigation of homeotic proboscipedia function during labial palp formation indicates a multipronged action for pb in the labial disc. Pb acts cell-autonomously in the negative regulation of target genes including dac, which is normally extinguished in Pb-expressing cells of labial or leg imaginal discs but is activated in labial discs in the absence of pb activity. This activation of dac in mutant labial cells is hh-dependent and is likely a response to wg and dpp morphogen signals as for leg discs. The data further indicate that pb acts cell autonomously to regulate the level of both wg and dpp expression in response to hh. Thus, pb appears to negatively regulate dac expression directly, but also by withholding positive instructions from Wg and Dpp morphogens. The interweaving of homeotic selector proteins with strategic target genes including morphogens (wg, dpp) and targets of signaling activity (dac, Dll) may influence segment patterning from global size and shape to specific local pattern and cell identity. This positioning offers a powerful yet economical mode of selector function that helps to better understand how a single selector gene can integrate global patterning with cellular identity (Joulia, 2005).

This view invoking multiple and overlapping modes of regulation by a homeotic selector protein supports and extends the vision from analyses seeking to explain how Ultrabithorax (Ubx) selector function differentiates between the serially homologous wing and haltere appendages. This analysis supports a role for Ubx in fruit flies transforming a dorsal default state (wing) to haltere, by repressing the accumulation of Wg in the posterior part of the haltere, and by regulating a subset of Dpp or Wg activated targets such as vestigial and spalt related. Additionally, clear evidence has been presented for a nonautonomous action of Ubx via its activity in cells of the D-V organizer where wg is expressed. Ubx thus acts to down-regulate wg in the haltere, but also intervenes to modulate the expression of targets of both dpp and wingless signaling pathways. An analysis of mutants for maxillopedia (mxp), the Tribolium pb homolog, revealed augmented transcription of flour beetle wg within the transformed labial segment. This observation, in full accord with the above results for Ubx, and the current results for Drosophila pb, supports a conserved role for homeotic regulation of nonautonomous signaling input in appendage development. At the same time, mxp mutants show a precocious maxilla-to-leg transformation in larvae, demonstrating a prior, embryonic requirement for mxp. This result is of particular interest since it highlights a temporal aspect of pb action in the fly labial disc: the absence of pb function early has no apparent effect on the labial discs in early L3 larvae, which appear normal. It is only subsequently that these diverge toward leg structure. Thus, the globally conserved activity of mxp/pb in equivalent beetle or fly organs is nonetheless employed in temporally different ways among species. Though it is not clear whether this reflects the existence of species-specific co-factors or rather of the effects of expression dosage and timing, such modifications might offer important possibilities for changing form. Variations on all these themes can probably contribute to the diversification of organism form, within and among species (Joulia, 2005)

The roles of diffusible Wg and Dpp morphogens induced by Hh at the A-P boundary, and the transcriptional programs they induce according to their concentrations within a gradient, are considered central to organizing the group of cells constituting a segment. The present work indicates that pb normally acts downstream of Hh within the organizer, where it maintains Wg and Dpp at low levels in labial imaginal tissue. Overexpressing Wg or Dpp in the labial discs results in malformed, overgrown or transformed 'labial' tissue. These observations support the viewpoint that limiting morphogen accumulation is essential to ensuring that the labial program is correctly applied. This study underlines the potential importance of the absolute levels of wg and dpp-encoded signaling molecules deployed for tissue organization. While a gradient may in principle be formed from any source, part of the spectrum of threshold levels necessary for stimulating specific gene responses is likely removed from the repertoire in the labial environment. The absolute level of activation or inhibition of diverse signaling pathways thus may be in itself a tissue-specific property, allowing gradients of related form but with different instructive capacities that can be a distinctive element in guiding tissue formation and specifying ultimate identity. This integration of diverse sorts of information -- the hh organizer linked to the Hox selector -- may confer order to tissue organization and identity (Joulia, 2005).

The fine-tuning of morphogen signals by Hox selectors coupled with the concomitant regulation of downstream targets thus appears to offer a strategic control point for achieving reliable developmental control coupled with evolutionary flexibility. The modulation of different cell signaling pathways by pb activity implies it can regulate both the tissue “context” generated by the signaling pathways activated in a tissue, and the cellular response to this context. This capacity to meld large-scale patterning with cellular identities merits emphasis (Joulia, 2005).

While the logic described above appears to be conserved, its application leads to widely different results according to the species and the tissue. Quite recently, an analysis of vertebrate Hox function has led to the identification of an intimate developmental link between Hox selector function and hedgehog signaling. This analysis reveals a direct physical interaction between the mouse Ci homolog Gli and Hox homeodomain transcription factors. It thus provides a compelling complement to the present work, since the molecular framework of a direct link between Gli and Hox proteins goes far to rationalise the dose-sensitive interplay between Ci and Pb that was observed in Drosophila. If Hox proteins indeed compete for available nuclear Gli/Ci, this molecular mechanism may also help to understand other phenomena including phenotypic suppression in flies or posterior prevalence in mice. Correspondingly, the current data place Pb in antagonism to Ci within the hedgehog organizer, where it modulates output from the wg and dpp genes and the instructive morphogens they encode. These complementary observations from insect and vertebrate models suggest the existence of an evolutionarily conserved machinery with enormous potential for generating morphological diversity. It will be exciting to know more about how the homeotic selector function is integrated in known cascades that make use of conserved molecules both to ensure the fidelity of normal form, as well as to generate new form (Joulia, 2005).

GENE STRUCTURE

pb is found between labial, the most proximal gene of the ANTP-C, and the rogue homeobox gene zerknült (Pultz, 1988).

Using alternatuive splicing, coding sequences across a 34 kb interval yield four identified mRNA formsthat differ immediately upstream of the homeobox. As a consequence, the homeodomain isexpected to reside in four different contexts in the predicted protein isoforms (Cribbs, 1992b).

PROTEIN STRUCTURE

Amino Acids - 634

Structural Domains

A collectionof pb mutant alleles were examined using antisera directed against either the N-terminal region, the center or theC-terminal region of the protein. Surprisingly, several partial loss-of-function pb allelesappear to generate partially functional proteins truncated at their C-termini. This suggests that asignificant portion of the protein contributes quantitatively to PB function, but is partiallydispensable (Cribbs, 1992b).

See four paralogous Hox clusters of mammals for homologies of Probosipedia with mammalian Hox cluster proteins.

date revised: 2 February 98  

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