Just as gnathal buds are beginning to form, (5.5 to 6 hours) PB protein appears in the presumptive mesoderm, just behind the stomodeum. Cells expressing pb migrate away from the midline as the maxillary and labial lobes take shape, separating into two groups, bilateraly. After only seven hours, approximately six to eight cells with strong staining are left, internal to each mandibular lobe. As the germ band retracts, PB protein becomes more concentrated in ectodermal nuclei of the labial and maxillary lobes. At the end of germ band retraction, staining is found in the sternal region. As labial lobes fuse at the ventral midline and involute into the stomodeum (10 to 12 hours), PB staining reaches a maximum. During head involution staining is present first in the superesophageal ganglion, then the subesophageal ganglion, and ultimately in the central nervous system (the ventral cord) in a subset of neural precursors (Pultz, 1988).

The spatial accumulation partially overlaps with the distribution of the Deformed and Sex combs reduced proteins in the maxillary and labial segments, respectively (Mahaffey, 1989).

To gain further insights into homeotic gene action during CNS development, the role of the homeotic genes was characterized in embryonic brain development of Drosophila. Neuroanatomical techniques were used to map the entire anteroposterior order of homeotic gene expression in the Drosophila CNS. This order is virtually identical in the CNS of Drosophila and mammals. All five genes of the Antennapedia Complex are expressed in specific domains of the developing brain. The labial gene has the smallest spatial expression domain; it is only expressed in the posterior part of the tritocerebral anlage. This contrasts with previous reports that lab is expressed throughout the tritocerebral (intercalary) neuromere. The proboscipedia gene has the largest anteroposterior extent of expression, however, in contrast to other homeotic genes, pb is only found in small segmentally repeated groups of 15-20 cells per neuromere. These groups of pb-expressing cells range from the posterior deutocerebrum toward the end of the VNC. Since pb-expressing cells are found anterior to the lab-expressing cells in the brain, this is an exception to the spatial colinearity rule. (Spatial colinearity is conserved in the epidermis, where pb expression is posterior to lab expression). The Deformed gene is expressed in the mandibular neuromere and the anterior half of the maxillary neuromere and the Sex combs reduced gene is expressed in the posterior half of the maxillary neuromere and the anterior half of the labial neuromere. The Antennapedia gene is expressed in a broad domain from the posterior half of the labial neuromere toward the end of the VNC. The three genes of the Bithorax Complex are expressed in the VNC. Ultrabithorax gene expression extends in a broad domain from the posterior half of the T2 neuromere to the anterior half of the A7 neuromere, with highest expression levels in the posterior T3/anterior A1 neuromeres. The abdominal-A gene is expressed from the posterior half of the A1 neuromere to the posterior half of the A7 neuromere. For the above mentioned genes, the anterior border of CNS expression remains stable from stage 11/12 until the end of embryogenesis. In contrast, the anterior border of CNS expression for the Abdominal-B gene shifts at stage 14. Before this stage Abd-B expression extends from the posterior half of neuromere A7 to the end of the VNC; afterwards, it extends from the posterior half of neuromere A5 to the end of the VNC with the most intense expression localized to the terminal neuromeres. With the exception of the Dfd gene, the anterior limit of homeotic gene expression in the CNS is always parasegmental (Hirth, 1998).

Specification of individual adult motor neuron morphologies by combinatorial transcription factor code

How the highly stereotyped morphologies of individual neurons are genetically specified is not well understood. This study identified six transcription factors (TFs; Ems, Zfh1, Pb, Zfh2, Pros and Toy) expressed in a combinatorial manner in seven post-mitotic adult leg motor neurons (MNs) that are derived from a single neuroblast in Drosophila. Unlike TFs expressed in mitotically active neuroblasts, these TFs do not regulate each other's expression. Removing the activity of a single TF resulted in specific morphological defects, including muscle targeting and dendritic arborization, and in a highly specific walking defect in adult flies. In contrast, when the expression of multiple TFs was modified, nearly complete transformations in MN morphologies were generated. These results show that the morphological characteristics of a single neuron are dictated by a combinatorial code of morphology TFs (mTFs). mTFs function at a previously unidentified regulatory tier downstream of factors acting in the NB but independently of factors that act in terminally differentiated neurons (Enriquez, 2015).

Neurons are the most morphologically diverse cell types in the animal kingdom, providing animals with the means to sense their environment and move in response. In Drosophila, neurons are generated by neuroblasts (NBs), specialized stem cells dedicated to the generation of neurons and glia. As they divide, NBs express a temporal sequence of transcription factors (TFs) that contribute to the generation of neuronal diversity. For example, in the embryonic ventral nerve cord (VNC), most NBs express a sequence of five TFs (Hunchback, Krüppel, Pdm1/Pdm2, Castor, and Grainyhead), while in medulla NBs and intermediate neural progenitors of the Drosophila larval brain a different series of TFs have been described. In vertebrates, analogous strategies are probably used by neural stem cells, e.g., in the cerebral cortex and retina, suggesting that this regulatory logic is evolutionarily conserved. Nevertheless, although temporally expressed NB TFs play an important role in generating diversity, this strategy cannot be sufficient to explain the vast array of morphologically distinct neurons present in nervous systems. For example, in the Drosophila optic lobe there is estimated to be ~40,000 neurons, classified into ~70 morphologically distinct types, each making unique connections within the fly's visual circuitry neurons (Enriquez, 2015).

A second class of TFs has been proposed to specify subtypes of neurons. For example, in the vertebrate spinal cord, all motor neurons (MNs) express a common set of TFs at the progenitor stage (Olig2, Nkx6.1/6.2, and Pax6) and a different set of TFs after they become post-mitotic (Hb9, Islet1/2, and Lhx3). Hox6 at brachial and Hox10 at lumbar levels further distinguish MNs that target muscles in the limbs instead of body wall muscles. Subsequently, limb-targeting MNs are further refined into pools, where all MNs in a single pool target the same muscle. Each pool is molecularly defined by the expression of pool-specific TFs, including a unique combination of Hox TFs. In Drosophila embryos, subclasses of MNs are also specified by unique combinations of TFs: evenskipped (eve) and grain are expressed in six MNs that target dorsal body wall, and Hb9, Nkx6, Islet, Lim3, and Olig2 are required for ventral-targeting MNs. However, each neuronal subtype defined by these TFs includes multiple morphologically distinct neurons, leaving open the question of how individual neuronal morphologies are specified neurons (Enriquez, 2015).

A third class of TFs suggested to be important for neuronal identity is encoded by terminal selector genes. Initially defined in C. elegans, these factors maintain a neuron's terminally differentiated characteristics by, for example, regulating genes required for the production of a particular neurotransmitter or neuropeptide. Consequently, these TFs must be expressed throughout the lifetime of a terminally differentiated neuron. Notably, as with neurons that are from the same subtype, neurons that share terminal characteristics, and are therefore likely to share the same terminal selector TFs, can have distinct morphological identities. For example, in C. elegans two terminal selector TFs, Mec-3 and Unc-86, function together to maintain the expression of genes required for a mechanosensory fate in six morphologically distinct touch sensitive neurons neurons (Enriquez, 2015).

In contrast to the logic revealed by these three classes of TFs, very little is known about how individual neurons, each with their own stereotyped dendritic arbors and synaptic targets, obtain their specific morphological characteristics. This paper addresses this question by focusing on how individual MNs that target the adult legs of Drosophila obtain their morphological identities. The adult leg MNs of Drosophila offer several advantages for understanding the genetic specification of neuronal morphology. For one, all 11 NB lineages that generate the ~50 leg-targeting MNs in each hemisegment have been defined. More than two-thirds of these MNs are derived from only two lineages, Lin A (also called Lin 15) and Lin B (also called Lin 24), which produce 28 and 7 MNs, respectively, during the second and third larval stages. Second, each leg-targeting MN has been morphologically characterized-both dendrites and axons-at the single-cell level. In the adult VNC, the leg MN cell bodies in each thoracic hemisegment (T1, T2, and T3) are clustered together. Each MN extends a highly stereotyped array of dendrites into a dense neuropil within the VNC and a single axon into the ipsilateral leg, where it forms synapses onto one of 14 muscles in one of four leg segments: coxa (Co), trochanter (Tr), femur (Fe), and tibia (Ti). Not only does each MN target a specific region of a muscle, the pattern of dendritic arbors of each MN is also stereotyped and correlates with axon targeting. The tight correlation between axon targeting and dendritic morphology has been referred to as a myotopic map. The stereotyped morphology exhibited by each MN suggests that it is under precise genetic control that is essential to its function neurons (Enriquez, 2015).

This study demonstrates that individual post-mitotic MNs express a unique combination of TFs that endows them with their specific morphological properties. Focus was placed on Lin B, which generates seven MNs, and six TFs were identified that can account for most of the morphological diversity within this lineage. Interestingly, these TFs do not cross-regulate each other and are not required for other attributes of MN identity, such as their choice of neurotransmitter (glutamine) or whether their axons target muscles in the periphery, i.e., they remain terminally differentiated leg motor neurons. Consistent with the existence of a combinatorial code, when two or three, but not individual, TFs were simultaneously manipulated nearly complete transformations in morphology were observed. However, removing the function of a single TF, which is expressed in only three Lin B MNs, resulted in a highly specific walking defect that suggests a dedicated role for these neurons in fast walking. Together, these findings reveal the existence of a regulatory step downstream of temporal NB factors in which combinations of morphology TFs (mTFs) control individual neuron morphologies, while leaving other terminal characteristics of neuronal identity unaffected neurons (Enriquez, 2015).

Inherent in the concept of a combinatorial TF code is the idea that removing or ectopically expressing a single TF will only generate a transformation of fate when a different wild-type code is generated. Consistent with this notion, only when the expression of two or three mTFs were simultaneously manipulated was it possible to partially mimic a distinct mTF code and, as a result, transform the identity of one Lin B MN into another. In contrast, manipulating single TFs typically resulted in aberrant or neo-codes that are not observed in wild-type flies. For example, removing pb function from Lin B resulted in two MNs with a code (Ems+Zfh1) and MN morphology that are not observed in wild-type Lin A and Lin B lineages. Analogously, ectopic Pb expression in Lin A, which normally does not express this TF, generated aberrant codes and MN morphologies. This latter experiment was particularly informative because although Pb redirected a subset of Lin A dendrites to grow in an anterior region of the neuropil, it did not alter the ability of these dendrites to cross the midline. Thus, the dendrites of these MNs had characteristics of both Pb-expressing Lin B MNs (occupying an antero-ventral region) and Pb-non-expressing Lin A MNs (competence to cross the midline). Axon targeting of these MNs was also aberrant: although they still targeted leg muscles, Pb-expressing Lin A MNs frequently terminated in the coxa, which is not a normal characteristic of Pb-expressing Lin B MNs or of any Lin A MN. These observations suggest that the final morphological identity of a neuron is a consequence of multiple TFs executing functions that comprise a complete morphological signature. Some functions, such as the ability to occupy the antero-ventral region of the neuropil, can be directed by a single TF (e.g., Pb), while other functions, such as the ability to accurately target the distal femur, require multiple TFs (e.g., Pb+Ems). Further, because it was possible to generate MNs that have both Lin B and Lin A morphological characteristics, hte results argue against the idea that there are lineage-specific mTFs shared by all progeny derived from the same lineage. Instead, the data are more consistent with the idea that the final morphological identity of an MN depends on its mTF code neurons (Enriquez, 2015).

Drosophila NBs, and perhaps vertebrate neural stem cells, express a series of TFs that change over time and have therefore been referred to as temporal TFs. For Lin B, the sequence of these factors is unknown, in part because the Lin B NB is not easily identified in the second-instar larval VNC, the time at which it is generating MNs. Nevertheless, each MN derived from Lin B and Lin A has a stereotyped birth order, consistent with the idea that temporal TFs play an important role in directing the identities of MNs derived from these lineages and, therefore, the mTFs they express. For Lin B, this birth order is Co1->Tr1->Fe1->Tr2->Co2->Co3->Co4. Interestingly, according to the mTF code proposed in this study, each of these MNs differs by at most two mTFs in any successive step. For example, Tr1 has the code [Zfh1, Ems, Pb, Zfh2] while Fe1, the next MN to be born, has the code [Zfh1, Ems, Pb]. Thus, it is posited that the sequence of temporal TFs acting in the NB is responsible for directing each successive change in mTF expression in postmitotic MNs (e.g., in the Tr1->Fe1 step, repression of zfh2). Although a link between temporal TFs and TFs expressed in postmitotic neurons has been proposed in Drosophila, the role of these TFs in conferring neuron morphologies is not known. Further, there may be additional diversity-generating mechanisms in lineages that produce many more neurons than the seven MNs generated by Lin B. One additional source of diversity may come from NB identity TFs, which distinguish lineages based on their position. Such spatial information could in principle allow the same temporal TFs to regulate different sets of mTFs in different NB lineages. It is also likely that differences in the levels of some mTFs may contribute to neuronal identities. Consistent with this idea, the levels of Zfh2 and Pros differ in the Lin B MNs expressing these TFs, differences that are consistent in all three thoracic segments and between animals. Further, Zfh1 levels vary between Lin B MNs and its levels control the amount of terminal axon branching. Previous studies also demonstrated that TF levels are important for neuron morphology, including Antp in adult leg MNs derived from Lin A and Cut in the control of dendritic arborization complexity in multidendritic neurons. If the levels of mTFs are important, it may provide a partial explanation for why the transformations of morphological identity generated in this study with the MARCM technique, which cannot control levels, are typically only partially penetrant neurons (Enriquez, 2015).

Another distinction between temporal TFs and mTFs is that no evidence has been observed of cross-regulation between mTFs. In situations when mTFs were either removed (e.g., pb-/-; emsRNAi) or ectopically expressed (e.g., UAS-pb + UAS-ems) in postmitotic Lin B MARCM clones, the expression of the remaining mTFs was unchanged. In contrast, when an NB lineage is mutant for a temporal TF, the prior TF in the series typically continues to be expressed. These observations suggest that the choice of mTF expression is made in the NB and that once the postmitotic code is established, it is not further influenced by coexpressed mTFs neurons (Enriquez, 2015).

The data further suggest that mTFs are distinct from terminal selector TFs. In mutants for the mTFs studied here, the resulting neurons remain glutamatergic leg motor neurons: they continue to express VGlut, which encodes a vesicular glutamate transporter, expressed by all Drosophila MNs, and they still exit the VNC to target and synapse onto muscles in the adult legs. Thus, whereas terminal selector TFs maintain the terminal characteristics of fully differentiated neurons, mTFs are required transiently to execute functions required for each neuron's specific morphological characteristics. Together, it is suggested that the combined activities of terminal selector TFs and mTFs specify and maintain the complete identity of each post-mitotic neuron neurons (Enriquez, 2015).

Although the mTFs defined in this study, e.g., Ems, Pb, and Toy, do not fit the criteria for a terminal selector TF, it is plausible that some TFs function both as mTFs and terminal selector TFs. One example may be Apterous, a TF that is expressed in six interneurons in the thoracic embryonic segments and that functions with other TFs to control the terminal differentiation state of these neuropeptide-expressing neurons. In addition to the loss of neuropeptide expression, these neurons display axon pathfinding defects in the absence of apterous. Despite the potential for overlapping functions, it is conceptually valuable to consider the specification of neuronal morphologies as distinct from other terminal characteristics, as some mTFs regulate morphology without impacting these other attributes. It is also plausible that some of the TFs that have been previously designated as determinants of subtype identity may also be part of mTF codes. For example, eve is required for the identity of dorsally directed MNs inDrosophila embryogenesis, but the TFs required for distinguishing the individual morphologies of these neurons are not known. It may be that Eve is one component of the mTF code and that it functions together with other mTFs to dictate the specific morphologies of these neurons neurons (Enriquez, 2015).

Flies containing a single pb mutant Lin B clone exhibited a highly specific walking defect: when walking at high speed, these flies were significantly more unsteady compared to control flies. The restriction of this defect to high speeds suggests that the Pb-dependent characteristics of these MNs may be specifically required when the walking cycle is maximally engaged, raising the possibility that Tr1, Tr2, and Fe1 are analogous to so-called fast MNs described in other systems. Further, these data support the idea that the highly stereotyped morphology of these MNs is critical to the wild-type function of the motor circuit used for walking. In particular, the precise dendritic arborization pattern exhibited by these MNs, which is disrupted in the pb mutant, is likely to be essential for their function. Although it cannot be excluded that other pb-dependent functions contribute to this walking defect, these observations provide strong evidence that the myotopic map, in which MNs that target similar muscle types have similar dendritic arborization patterns, is important for the fly to execute specific adult behaviors neurons (Enriquez, 2015).

Effects of Mutation or Deletion

In pb null mutants labial palps are transformed to prothoracic legs [Image], whereas maxillary palps are small and malformed (Pultz, 1988 and Cribbs, 1992b).

The Drosophila homeotic gene proboscipedia specifies labial identity and directs formation of the adult distiproboscis from the labial imaginal discs. pb null alleles result in the homeotic transformation of the distiproboscis into prothoracic (T1) legs. Homology with other transcription factors, localization to the nucleus, and restricted embryonic and imaginal expression implicate the PB protein as a transcription factor. In order to examine the possible roles that PB may play in the specification of adult mouthparts, PB was expressed in cells of wing, leg and eye-antennal imaginal discs, and the effects on the development of adult structures were observed. The ectopic expression of PB in the imaginal discs under the control of the inducible GAL4 system under control of a dpp imaginal disc enhancer alters the developmental program of adult legs into maxillary or labial palps. Labial-like structures observed include pseudotrachea, shot hairs resembling basiconica, and patches of smooth cuticle usually associated with the labellar bolster at the distal-most end of the labial palps. Leg patterning defects resulting from ectopic PB expression do not include a replacement of the entire leg by labial palps. Instead, an appendage of mixed identity is produced, containing both leg- and mouth-specific structures. These homeotic transformations have an equal effect on all three sets of legs, indicating an activity that is not solely dependent upon the unique combinations of other homeotic genes present in each of the leg discs. Wings expressing PB do not exhibit a homeotic transformation, but are smaller in size than wild type, are missing veins, have ectopic socketed bristles growing from the wing blade surface, and display a generalized crumpled appearance. Segment polarity genes required for establishing the AP compartment boundary are found to be undisturbed by ectopic PB. Furthermore, normal patterns of apoptosis are observed in animals expressing ectopic PB, indicating that PB does not alter or affect cell death. The normal domain of activity of pb is in the labial imaginal discs, tissues that are derived from the embryonic labial segments. The fact that pb can alter the segmental identity of the thoracic imaginal discs, derived from segments more posteriorly located than the labial segment, indicates that pb does not follow the general rule of "posterior dominance" of the HOM-C genes. These results suggest that molecular events occurring downstream of the establishment of the compartment boundary are affected by ectopic PB expression in imaginal discs and point to a general role in "palp" formation, in addition to the specification of labial identity (Alpin, 1997).

Mutations of the Drosophila homeotic proboscipedia gene (pb, the Hox-A2/B2 homolog) provoke dose-sensitive defects. These effects were used to search for dose-sensitive dominant modifiers of pb function. Two identified interacting genes are the proto-oncogene Ras1 and its functional antagonist Gap1, prominent intermediaries in known signal transduction pathways. Ras1+ is a positive modifier of pb activity both in normal and ectopic cell contexts, while Gap1, the Ras1-antagonist, has an opposite effect. Ras1-modulated changes were observed in homeotic effects on cell identity (bristle to distal sex combs, wing trichomes to veins, veins to trichomes or veins to bristles). Only a small number of cell identities in precise contexts are changed by HSPB activity. This suggests that most cells are aware of their positions and their correctly associated fates, perhaps as a consequence of cell-cell communication. Ras1-dependent modifications of segmental identity are also observed. These occur in a concerted fashion on groups of adjacent cells, again suggesting cell communication. A general role for Ras1 in homeotic function is likely, since Ras1+ activity also modulates functions of the homeotic loci Sex combs reduced and Ultrabithorax. These data suggest that the modulation occurs by an independent mechanism for the transcriptional control of the homeotic loci themselves, or of the Ras1/Gap1 genes. Taken together the data support a role for Ras1-mediated cell signaling in the homeotic control of segmental differentiation (Boube, 1997).

The Drosophila homeotic gene proboscipedia (a HoxA2/B2 homolog) is required for the development of adult mouthparts. Ectopic Pb protein expression from a transgenic heat shock promoter (HSPB) results in transformation of adult antennae to maxillary palps. In contrast, most tissues appear refractory to Pb-induced effects. To study the basis of homeotic tissue specificity, mutations that modify dominant HSPB-induced phenotypes have been characterised. One HSPB point mutation (Arg5 of the homeodomain mutated to His) removes homeotic activity in the mouthparts and antennae, but provokes a dose-sensitive eye loss. Eye loss can be induced by Pbproteins that no longer effectively bind to DNA. The dose-sensitive eye loss thus appears to be mediated by specific, context-dependent protein-protein interactions. Dominant eye loss may reflect the titration of limiting proteins factor(s) through specific interactions with the altered heat shock induced protein (Benassayag, 1997a).

A transgenic Hsp70-proboscipedia (HSPB) element that rescues pb mutations also induces the dominant transformation of antennae to maxillary palps. To identify sequences essential to PB protein function, EMS-induced HSPB mutations were sought that lead to phenotypic reversion of the HSPB transformation. Ten revertants harbor identified point mutations in HSPB coding sequences. The point mutations that remove all detectable phenotypes in vivo reside either within the homeodomain or, more unexpectedly, in evolutionarily nonconserved regions outside the homeodomain. Two independent homeodomain mutations that change the highly conserved Arginine-5 in the N-terminal hinge show effects on adult eye development, suggesting a previously unsuspected role for Arg5 in functional specificity. Three additional revertant mutations outside the homeodomain reduce but do not abolish PB+ activity, identifying protein elements that contribute quantitatively to pb function. One of the three is in the N-terminus of the protein, a second is 25 residues downstream of the homeodomain, and a third mutations deletes the C-terminal 123 amino acids. This in vivo analysis shows that apart from the conserved motifs of PB, other elements throughout the protein make important contributions to homeotic function (Benassayag, 1997b).

Effects of Mutation: Mediator complex subunits act as genetic modifiers of Pb

A new Drosophila gene, poils aux pattes (pap; cytological locus 78A1-3), has been identified in a P-element screen for dominant genetic modifiers of cell identity functions of the homeotic loci Scr (Hox-A5/B5) and proboscipedia (pb; Hox-A2/B2). The Scr selector gene confers prothoracic identity, while pb alone induces maxillary identity. Together, the Scr and pb selectors show a combinatorial behavior leading to specification of the adult labial palps (mouthparts). Low-level ectopic expression of PB protein from an hsp70-pb mini-gene, the HSPB element, induces several dose-sensitive cell-identity phenotypes that have been used to screen for second-site dominant modifier mutations. Of 5000 new autosomal P insertions tested, only one, in the pap locus, shows dose-sensitive enhancement of the distal sex comb induced by the HSPB element. Starting from the P-element molecular tag, a 50-kb pair interval encompassing the pap gene was cloned. Analysis of genomic and complementary DNA (cDNA) sequences indicates a transcription unit spanning at least 22 kb and generating a ~10-kb mRNA. The exonic P insertion resides upstream of the first in-frame ATG of an open reading frame (ORF) of 2618 amino acids. Full reversion of lethality by mobilizing the P element, and the rescue of lethality by a ubiquitin-cDNA construct, confirms that this ORF corresponds to pap. The ORF encodes the unique Drosophila counterpart of TRAP240/ARC250, recently identified as a subunit of the human thyroid hormone receptor-associated protein (TRAP) or activator-recruited cofactor (ARC) protein complexes. The Drosophila PAP protein shows 27% overall identity (40% similarity) with human TRAP240 and 27% identity (39% similarity) with its C. elegans counterpart. This conservation extends across the proteins but is highest in the N- and C-terminal regions. pap is therefore considered as the presumptive fly homolog of TRAP240. A second Drosophila TRAP, dTRAP80, is described that is necessary for cell viability (Boube, 2000).

TRAPs are components of the mediator complex (MED). Work of the last 10 years has brought to light a new class of transcription factor complex, the mediator. The first known mediator components, encoded by the yeast SRB/MED genes, were identified by dominant mutations suppressing the conditional lethality caused by a C-terminal domain (CTD) mutation of the Pol II large subunit. The biochemically purified Srb proteins interact physically with core RNA Pol II in the form of large protein complexes. Mediator complexes have likewise been identified in mammalian cells where, as in budding yeast, they associate with the core Pol II to form a giant holoenzyme. The mammalian MED complexes capable of stimulating basal transcription initiation in vitro contain ~20 subunits, including at least five proteins homologous to yeast Srb/MED proteins. Several related complex forms that mediate transcription in vitro have been isolated through their physical contact with a spectrum of mammalian transcription factors. These include nuclear targets for different transcription factors, including thyroid hormone (TRAP complex), VP16, the p65 subunit of NF-kappaB, SREBP-1a, Sp1 (CRSP), E1A, and p53. Alternative protocols have yielded related mammalian complexes (human or mouse mediator; SMCC) or subcomplexes (negative regulator of activated transcription; NAT), associated with human Srb10/Cdk8). These related complexes are viewed as versatile interfaces that link specific transcription factors and the general Pol II machinery in a complex equilibrium. The MED complexes are most often considered transcriptional coactivators, and this property has been used as the basis of their biochemical purification. Importantly, however, these complexes are not dedicated activators, and some forms have also been described as corepressors (Boube, 2000 and references therein).

MED complexes appear to integrate regulatory information from multiple transcription factors and relay that information to the core Pol II. In support of this view, recent work demonstrates that human ARC complex can interact with two transcription factors and parlay this input into a synergistic transcriptional response. In metazoans, the dynamic developmental process requires fine control of the gene expression program, presumably involving their MED complexes. However, for the moment little is yet known of their in vivo functions in development. The first known mutation of a metazoan MED subunit, in the sur2 locus of the nematode C. elegans, was isolated as a suppressor of activated Ras in vulval development. A MED complex has been identified in C. elegans and suggested to participate in regulation of developmental target genes. Recently, gene inactivations have been described for two mouse subunits, Srb7 and TRAP220. Murine Srb7 corresponds to a core MED subunit required for yeast cell viability and is apparently required for cell viability in the mouse embryo as well. The inactivation of TRAP220, a subunit implicated in ligand-dependent binding to thyroid hormone receptor, reveals diverse developmental defects in a variety of tissues (Boube, 2000 and references therein).

The recent availability of the Drosophila genomic sequence and the collection of corresponding cDNAs through the Berkeley Drosophila Genome Project has facilitated the identification a single putative Drosophila homolog for each of the 23 known human TRAP/ARC subunits. For simplicity, these proteins and their genes will be referred to as TRAPs when more than one name exists for the same entity. While the existence of a biochemical entity remains to be demonstrated, the observed structural conservation of such a large number of MED genes provides clear circumstantial evidence for the existence of a fly mediator complex (dMED) similar to the purified complexes from worms and mammals (Boube, 2000).

Among the new Drosophila MED genes identified is dTRAP80 at cytological position 90F1-2 on chromosome 3R. It encodes a predicted dTRAP80 protein of 642 amino acids exhibiting 40% identity (59% similarity) to its human counterpart. The majority of putative Drosophila MED genes, including pap, appear to lack a homolog in the complete Saccharomyces cerevisiae genome sequence. S. cerevisiae SRB4 encodes a core component of the Srb mediator complex required for the expression of virtually all yeast genes. The gene was identified by dominant mutations that directly suppress a Pol II CTD mutation. For the dTRAP80 protein, low but potentially significant overall structural conservation (16% identity, 48% similarity) was detected with Srb4 proteins from the MED complexes of the yeast S. cerevisiae and Schizosaccharomyces pombe. The overall identity between these two yeast Srb4 proteins is only 25% (60% similarity), with conservation most pronounced in a region with predicted alpha-helical character between amino acids 214-313 of S. cerevisiae Srb4 (40% identity, 72% similarity). Both primary sequence and predicted helical character are conserved within this interval in metazoan TRAP80 moieties, attaining 34% identity between human TRAP80 and S. pombe SRB4. The corresponding sequences appear unique in the budding yeast and Drosophila genomes, arguing against a novel reiterated domain. Thus, despite the low level of overall identity, these observations are good evidence for homology of the metazoan TRAP80 genes with yeast SRB4 (Boube, 2000).

One lethal P-insertion mutation from the BDGP collection is situated within the dTRAP80 coding sequence. This insertion, dTRAP801, is located downstream of the apparent initiator ATG within the same exon. The cloning and the identification of mutations in these two putative Drosophila MED genes allowed for an initiation of an in vivo assessment of their physiological roles in normal development. dTRAP801 mutants die as second-instar larvae with no obvious cuticular defect. The initial pap1 P-element insertion and most derived imprecise excisions (including the molecular null allele pap53) are recessive embryonic lethals. pap- embryos appear normal apart from discrete cuticular defects of the embryonic mouthparts. Thus, both functions are essential for viability, and pap is detectably required for normal embryonic development. Ubiquitous accumulation of pap and dTRAP80 mRNA is observed by in situ hybridization in embryos of all stages and in larval imaginal discs. The presence of mRNA in early embryos further suggests that a maternal contribution partially compensates for the absence of zygotic expression for both pap and dTRAP80. In overexpression experiments, strong anti-PAP staining is limited to posterior cells, whereas in clones of pap53 cells, the signal was no longer detected. Immunostaining experiments with this specific anti-PAP serum show that pap mRNA is translated throughout the imaginal tissues, as predicted by the mRNA distributions. PAP protein accumulation is predominantly nuclear, in agreement with a role in the general transcription machinery (Boube, 2000).

The prototypical Srb4 protein is required for transcription of nearly all Pol II-dependent promoters in yeast. If dTRAP80 encodes the functional homolog of Srb4, it is predicted to participate in all aspects of mediator function, and the dTRAP80- condition should be cell lethal. The survival of dTRAP80- embryos to second-instar larvae (above) suggests that maternally contributed dTRAP80 mRNA suffices for embryonic survival. To test the consequences of removing dTRAP80 while minimizing the complication of maternal contribution, mitotic recombination was employed. From heterozygous mother cells, twin clones of daughter cells homozygous for each of the two chromosome arms were induced, one carrying dTRAP801 (or dTRAP80+) and the other its wild-type homolog (plus the associated cuticular markers Stubble [Sb] and ebony [e]). The dTRAP80+/+ or dTRAP80-/- cells of interest were identified by their bristle shape (Sb+). Where dTRAP80+ yielded 150 clones, none were observed with dTRAP80-/- for an equivalent sample size. It has been concluded that dTRAP80 is required for cell viability in the adult epidermis. This result provides independent support for a general cellular role of dTRAP80 consistent with the sequence-based interpretation that it encodes a fly Srb4 (Boube, 2000).

To examine functional requirements for the essential pap gene in adult development, mitotic clones of mutant cells were generated. In marked contrast to dTRAP80, clones were obtained showing that normal pap function is not required for cell viability. Clones induced during larval development lead to distinct consequences in different tissues. (1) Adult Drosophila melanogaster males normally align a single row of specialized bristles, the sex comb teeth, on the first tarsal segment of the prothoracic (T1) leg. These sex comb teeth are not found elsewhere. In contrast, clones of pap mutant cells situated in the distal second tarsal segment differentiate as ectopic sex comb teeth. Normal pap function thus opposes sex comb cell fate in this position. This role appears cell autonomous, since all observed ectopic sex comb teeth were mutant for pap. In contrast, clones within the normal sex comb or bordering it do not affect the number of cells adopting this fate. (2) Clones localized elsewhere in the T1 leg, or at any position in the T2 and T3 legs, are without effect. (3) Clones in the maxillary palps are associated with malformations. (4) Large clones in the wing blade, the notum, or the antennae lead to apparently normal pattern. Therefore in contrast with ubiquitous accumulation of PAP in epidermal cells, pap function is required for normal development in only a subset of those cells. Taken together, these data strongly suggest that developmental pap activity may be regulated according to the tissue and cell, being required for some identities but dispensable for others (Boube, 2000).

The ectopic distal sex comb induced by pap clones is a readily visible cell identity marker that reflects normal pap function. Ectopic distal sex comb teeth are induced in appropriately positioned cells lacking any pap function. This phenotype is also observed at low frequency with certain Scr and pb gain-of-function alleles (ScrScxP and hsp70-pb [HSPB] mutations). This effect of the ScrScxP allele is enhanced in pap heterozygotes. Functions of the homeodomain transcription factor Scr specify prothoracic identity, including sex comb cell fate. The induction of distal sex combs by HSPB also depends on Scr activity, since it is no longer detected in Scr heterozygotes. Ectopic sex comb differentiation is enhanced in HSPB/pap53 heterozygotes, but this effect is abolished in Scr heterozygotes. These observations of dose-sensitive interactions indicate a synergistic functional link between Scr and pap in this cell identity specification. pap and dTRAP80 both encode the sole detected fly homologs to human proteins identified by their presence in the MED complex. If the enhancement of the distal sex comb phenotype in pap heterozygotes is caused by limiting mediator function, double heterozygotes with dTRAP80 should aggravate this condition. Therefore, whether dTRAP80 acts together with pap in this cell identity decision was tested. The loss-of-function allele dTRAP801 is fully recessive; pap53 is likewise fully recessive. In contrast, 10% of pap53 +/+ dTRAP801 males possess an ectopic distal sex comb tooth, revealing a cooperative function in these cells. Synergistic enhancement of the ectopic sex comb caused by ScrScxP is likewise observed in double heterozygotes. These functional data indicate a shared function of PAP and dTRAP80, again suggesting the existence of a Drosophila mediator complex. They further suggest that at least one common function of PAP and dTRAP80 acts to antagonize Scr activity in distal sex comb differentiation (Boube, 2000).

Apart from the prothorax, normal Scr activity is also required for development of the adult labial palps, where it acts in a combinatorial fashion with the homeotic pb gene. Thus the effects of pap and dTRAP80 mutations on adult mouthparts formation were examined. The wild-type labium is typified by the presence of pseudotracheal rows used for drinking and the absence of a segmental appendage. The hypomorphic pb4/pb5 genotype leads to a transformation of distal labium to antennal arista, with a concomitant reduction of the pseudotracheae. In this sensitized context, changes in relative Scr activity can be readily detected. Reduced pap or dTRAP80 activity in heterozygotes enhances the labial-to-leg transformation, as seen by the appearance of leg-specific cell types: sex comb teeth in males, bracted bristles, and terminal claws. In pap dTRAP80, double heterozygotes leg structures often entirely replace labial pseudotracheae. As in the leg, this effect of pap and dTRAP80 mutants on labial development is synergistic. These data provide further support for a shared role of PAP and dTRAP80 proteins opposed, in this case, to the leg-forming activity of Scr in the labial tissue (Boube, 2000).

The observed effects of pap and dTRAP80 mutations in the T1 legs and labium may most simply be rationalized as consequences of increased Scr activity. This could result from augmented regulatory activity of Scr protein toward Scr target genes. Alternatively, it might reflect higher Scr gene expression with greater quantities of Scr protein. To distinguish between these two possibilities, Scr homeoprotein accumulation was examined by indirect immunofluorescence in pb4/pb5 labial imaginal discs that give rise to mixed labial/antennal or pb4/pap1 pb5 dTRAP801, yielding T1 leg identities. Nuclear SCR protein does not detectably increase with the transition to T1 leg identity. These data indicate that PAP and dTRAP80, acting in parallel or downstream of Scr, negatively modulate Scr protein function in labial tissue (Boube, 2000).

The above experiments were performed in heterozygotes for pap and dTRAP80. In the sensitized genetic context employed, slight but functionally important changes in Scr accumulation could potentially pass undetected in this test. The molecular epistatic relations between pap and Scr were therefore in homozygous mutant cells to determine whether Scr gene expression depends on normal pap function and vice versa. Both Scr and pap are normally expressed throughout the labial and T1 leg imaginal discs, and both confer detectable phenotypes there as described above. Mitotic clones of homozygous pap-/- or Scr-/- cells were induced in first- and second-instar larvae and identified in mature third-instar imaginal discs by the cell autonomous GFP marker and by the accumulation of Scr or PAP proteins examined in these cells of known genotype. No change in Scr accumulation is detected in pap-/- cells compared with neighboring wild-type cells in either tissue. These results obtained in homozygous pap- cells confirm that Scr gene expression, as measured by accumulation of the nuclear homeodomain protein, is not detectably affected by altered pap function in these tissues. Conversely, PAP protein accumulation is unchanged in Scr-/- cells, indicating that pap transcription is likewise independent of Scr function in these tissues. These reciprocal experiments, coupled with the results in heterozygotes described above, provide molecular evidence that the pap and dTRAP80 loci act in parallel with homeotic Scr function in distal sex comb and labial identity specification (Boube, 2000).

Proboscipedia (PB) is a HOX protein required for adult maxillary palp and proboscis formation. To identify domains of Pb important for function, 21 pb point mutant alleles were sequenced. Twelve pb alleles had DNA sequence changes that encode an altered Pb protein product. The DNA sequence changes of these 12 alleles fell into 2 categories: missense alleles that effect the Pb homeodomain (HD), and nonsense or frameshift alleles that result in C-terminal truncations of the Pb protein. The phenotypic analysis of the pb homeobox missense alleles suggests that the Pb HD is required for maxillary palp and proboscis development and pb-Sex combs reduced (Scr) genetic interaction. The phenotypic analysis of the pb nonsense or frameshift alleles suggests that the C-terminus is an important region required for maxillary palp and proboscis development and pb-Scr genetic interaction. Pb and Scr do not interact directly with one another in a co-immunoprecipitation assay and in a yeast two-hybrid analysis, which suggests the pb-Scr genetic interaction is not mediated by a direct interaction between Pb and Scr (2004).

A direct functional antagonism of proboscipedia and eyeless in Drosophila head development

Diversification of Drosophila segmental and cellular identities requires the combinatorial function of homeodomain-containing transcription factors. Ectopic expression of the mouthparts selector proboscipedia (pb) directs a homeotic antenna-to-maxillary palp transformation. It also induces a dosage-sensitive eye loss that was used to screen for dominant Enhancer mutations. Four such Enhancer mutations were alleles of the eyeless (ey) gene that encode truncated Ey proteins. Apart from eye loss, these new eyeless alleles led to defects in the adult olfactory appendages -- the maxillary palps and antennae. In support of these observations, both ey and pb were seen to be expressed in cell subsets of the prepupal maxillary primordium of the antennal imaginal disc, beginning early in pupal development. Transient co-expression is detected early after this onset, but is apparently resolved to yield exclusive groups of cells expressing either Pb or Ey proteins. A combination of in vivo and in vitro approaches indicates that Pb suppresses Ey transactivation activity via protein-protein contacts of the Pb homeodomain and Ey Paired domain. The direct functional antagonism between Pb and Ey proteins suggests a novel crosstalk mechanism integrating known selector functions in Drosophila head morphogenesis (Benassayag, 2003).

To better understand the relationship between Pb and Ey in normal development, the phenotypic effects of ey mutations were studied in the sensitized HSPbsy genetic context (ectopic expression of pb). Two copies of the HSPbsy transgene (the sensitizing condition) showed no marked effect. In contrast, pharate adult females with 2x HSPbsy and homozygous for eyJD showed strong maxillary palp and antennal defects. In some cases, the maxillary palp, whose identity is indicated by the distinctive distal bristles, remains adjoined to the antennal appendage. However, differentiation of the proboscis (which likewise depends on pb function) is not affected. Thus in the sensitized context, ey mutations can provoke strong defects of the maxillary and antennal appendages. The phenotype suggests that ey+ may participate in partitioning imaginal disc cells into antenna and maxillary palp during morphogenesis (Benassayag, 2003).

To confirm a role of eyeless in this process, HSPbsy was removed from the genetic background to examine the effects of the new eyeless mutations alone. All four ey alleles appear recessive in a non-sensitized background as shown for eyJD, and can be interpreted as loss-of-function mutations in accord with their molecular lesions. All give homozygous escapers with visible defects, allowing for the composition of an allelic series, from weakest to strongest: ey11>eyD1Da>eyEH>eyJD. Analysis of the phenotypes of hemizygotes with Df(4)BA led to the same conclusion (Benassayag, 2003).

eyJD homozygotes display eye reduction or loss, low viability and strong brain defects associated with abnormal behavior. Furthermore, a minority of surviving eyJD homozygotes (10%-20%, after outcrossing) show alterations in the size and/or shape of maxillary palps and antennae. The altered maxillary palps of eyJD homozygotes still harbor the two characteristic sensilla trichodea, suggesting that maxillary identity per se is not affected. Reduced, malformed maxillary palps are often accompanied by enlarged, misshapen antennae. Similar although weaker defects are likewise detected for eyEH homozygotes, as well as for certain trans-heterozygous combinations with other Enhancer alleles in the sensitized background. The reciprocal effect of eyJD on appendages that derive from the same antennal imaginal disc constitutes evidence of a potential role for ey in apportioning the maxillary portion of this disc. The defects observed in eyJD homozygotes appeared stronger than in the hemizygous combination, eyJD/Df(4)BA. Thus, the truncated protein may have a limited antimorphic character not detected in the presence of wild-type protein (Benassayag, 2003).

Although mutant phenotypes implicated both pb and now ey in maxillary development, no gene expression had been detected in the maxillary portion of the antennal disc in the third instar larvae. Pb and Ey expression were examined later, during the prepupal stage when maxillary and antennal structures evaginate from the eye-antenna imaginal disc. Using a rabbit anti-Pb serum directed against the C-terminal region (anti-E9), Pb accumulation was detected in the central part of the maxillary primordium beginning approximately eight hours after puparium formation, during evagination of the antennal and maxillary appendages from the composite disc. Ey protein as visualized by a rabbit anti-Ey serum accumulates in the same primordium and, within discrimination, at the same time. This expression appears to be limited to the borders of the primordium, rather than the center as for Pb. Results of tests for co-expression of the two proteins were mitigated: in situ hybridization or immunostaining experiments were inconclusive, whereas available antibodies that gave acceptable signals in this tissue were both rabbit polyclonal antisera. To address whether endogenous pb and ey patterns in the maxillary palps may overlap, a pb-GAL4 mini-gene was used based on descriptions of the pb-promoter region. Using a pb-GAL4 driver insertion to direct ß-galactosidase expression (pb-GAL4>UAS-lacZ), the patterns of pb>lacZ and ey expression were examined by double-immunofluorescence labeling and confocal microscopy. Early Pb expression is limited to a small number of cells in the distal maxillary primordium. At this stage, Ey expression can likewise be detected in a small group of cells partially overlapping those expressing Pb. Co-expression appears to be very limited in the progression of a dynamic pattern. In later prepupae, the expression patterns of pb>lacZ and ey in the maxillary primordium are adjacent but exclusive. Taken together, these data show a previously undisclosed co-temporal expression of both pb and ey in the maxillary primordium of prepupae, and support an ephemeral co-expression of these genes in a small number of cells. This is in agreement with the known function of pb in maxillary determination, and with the newly established function of ey in this tissue (Benassayag, 2003).

Thus, ctopically expressed homeotic Pb protein, even a form bereft of DNA-binding capacity, can suppress eye development in a dose-sensitive manner. Genetic and molecular results indicate a central role for direct contacts between conserved domains of the Hox selector protein Pb and the eye selector Ey. One physiological situation where the interaction between pb and ey is likely to be relevant was identified, based on their genetic interaction, mutant phenotypes and expression patterns in forming the adult antennae and maxillary palps (Benassayag, 2003).

This work has identified a previously unrecognized role for ey in the development of the maxillary palps and antennae. The mutation employed for most of these experiments, eyJD, behaves as a strong allele affecting viability, formation of the adult eyes and brain mushroom bodies, but also of antennal and maxillary differentiation. Consistent with a late requirement for ey in the antennal disc, ey expression in the maxillary primordium appears in early stages of metamorphosis when both eye-antennal discs have fused, and the antennal and maxillary appendages start to evaginate. After evagination, the maxillary primordium migrates to join the labial disc in forming the adult mouthparts, whereas the antennal primordium remains near the eye. When a contiguous epidermal cell layer has been completed, the head sac is abruptly evaginated under the internal pressure. Maxillary ey expression in early stages of prepupal metamorphosis is limited to the boundary between the maxillary primordium and the antenna, and ey mutant phenotypes often involved simultaneously reduced palps and enlarged antennae. These reciprocal effects are consistent with communicating cell populations, suggesting that ey may contribute to a partitioning of the antennal disc permitting the establishment of two separate appendages. Further analysis of this process will require new maxillary-specific markers permitting the fates of these cells to be followed (Benassayag, 2003).

Starting from a dose-sensitive eye loss provoked by ectopic Pb, new ey alleles isolated as eye loss Enhancers were identified. These mutations reveal a role for ey, and a potential biological relevance for this Hox-PAX6 interaction, in the development of the antennal and maxillary sensory palps. ey loss-of-function defects in the sensory palps are exacerbated by Pbsy. The most direct interpretation of the enhanced ey loss-of-function phenotype with HSPbsy is that the newly isolated alleles retain a partial function that can be negated by adequate Pb levels. The molecular characterization of these alleles is consistent with this hypothesis, because all four alleles should encode truncated proteins that contain most or all of the interacting PD (Benassayag, 2003).

To better understand the in vivo relationship between these two selector genes, attempts were made to examine the effects of double mutants for pb and ey. Although homozygotes for pb- or for the new ey mutants showed viabilities of up to 50% compared with heterozygotes, a double mutant adult was never obtained for any of the four ey alleles. This result, although suggesting that the double mutant is synthetic lethal, does not offer insight into the tissue(s) implicated in this lethality (Benassayag, 2003).

One tissue in which an interaction is clearly indicated from this analysis is the maxillary palp primordium, where a dynamic expression was detected of Ey and of Pb (directly or via the pbGAL4 driver) during pre-pupal development. Transient early co-expression of Pb and Ey in pre-pupae is limited to a small number of cells, whereas later expression appears exclusive. This result can be rationalized in two ways: first, co-expressing cells might be rapidly eliminated by apoptosis, through a coordinate gene-activation process triggered by a Hox-Pax dimer; second, co-expression of the Ey and Pb transcription factors could induce a developmental pathway interference resulting in a G1 cell-cycle arrest. These possibilities are not fully exclusive. Indeed, one or both mechanisms could serve to refine the boundaries between antennal and maxillary cell populations within the antennal disc (Benassayag, 2003).

In vertebrates, Pax6 has multiple known or inferred roles in eye, brain and nasal development. Apart from the fly eye, several groups have identified an eyeless function required for development of the mushroom bodies, neural structures important for olfactory perception and learning. This study describes a specific role for Ey in concert with Pb in the maxillary and antennal appendages; both of these are derived from the antennal disc and constitute the adult olfactory system. An analysis of mutations producing headless flies has revealed a role for Drosophila Pax6 in head morphogenesis and thereby suggests a requirement of ey for the development of all structures derived from eye-antennal discs. These studies involve mutations truncating the Ey protein, which induces head defects. Interestingly, because these truncated Ey proteins still contain the PD, the fact that the phenotypes obtained reflect an allele-specific antimorphic effect of the PD cannot be excluded. Taken together, these results strongly suggest that eyeless, apart from its known role in eye morphogenesis, may also play multiple other roles in head formation (notably for brain and olfactory sensory systems) (Benassayag, 2003).

The development of olfactory and visual systems has several common features in Drosophila. Both systems are derived from the composite eye-antennal imaginal disc. Moreover, both have similar signal transduction pathways and appear to share regulatory networks. However, when the expression of ey, so, eya and dac was examined in the pre-pupal maxillary primordium, only ey expression was detected. This observation suggests that ey acts there via a distinct combinatorial code of regulatory genes compared with eye development. One possibility proposed in this study is that ey activity is modulated by other co-factors or transcription factors whose activity is likely to be sensitive to Pb. In this light, it is worth noting that other Enhancer mutations isolated also similarly affect maxillary palps, either singly or in combination with ey. It will be of fundamental interest to better understand the molecular basis for how a single protein might function in multiple, distinct networks (Benassayag, 2003).

The ey mutants studied here were identified as dominant enhancers of pb-induced eye reduction. Consistent with the antagonism observed in vivo, Ey and Pb proteins interact directly in vitro, via the Ey Paired domain and the Pb homeodomain. This interaction with Pb that renders Ey unable to activate its downstream target genes can be extended to other homeotic genes because Antp, Scr, Ubx, abdA and AbdB repress eye development while increasing apoptosis in the eye disc, and their protein products likewise interact in vitro with Ey protein. This suggests a combinatorial interaction of homeodomain-containing proteins (Hox and Pax) to specify a given body segment. An inhibition through physical association has been proposed between Pax6 and En-1 during eye development in quail, and between Pax3 and Msx1 for muscle development in chicken. Moreover, a similar inhibitory mechanism involving a Hox protein HD has been reported in vertebrates; in contrast, physical interaction with Hox-B1 protein leads to increased Pax6 activity in Hela cells, raising the possibility that additional context-dependent partners modulate the action of Hox-Pax combinations to generate functional diversity. Based upon genetic and molecular data, it is proposed that variations on a PD-HD interface can serve to mediate combinatorial or hierarchical functional relationships among Hox and Pax genes in normal development (Benassayag, 2003).

The results presented here appear to favor a specific role for discrete protein-protein interactions rather than an indirect interference mechanism. Indeed, (1) by analysing the residues of Pb protein involved in its homeotic function, a Pbsy protein was identified with diminished DNA binding but still able to inhibit eye development; (2) using this mutant in a genetic screen to isolate Pb functional partners, four independent eyeless mutations were isolated, all of them leading to a shortened Ey protein; (3) genetic interaction tests showed that Pbsy-induced eye loss is highly sensitive to levels of ey function but independent of several other eye-determining genes including eyg, eya or so, and (4) ectopically expressed Pb interferes with ey activity in the eye imaginal disc by inhibiting so and eya activation without affecting ey transcription or Ey accumulation (Benassayag, 2003).

In conclusion, these results suggest that a specific Hox/Pax interaction between Pb and Ey is involved in a normal developmental process defining the boundary between the antenna and maxillary palp. More generally, the formation of such protein couples could afford a sensitive and delicate measure of the balance of Pax6 level, permitting a finely tuned integration to generate distinct transcriptional outputs during development (Benassayag, 2003).


Aplin, A. C. and Kaufman, T. C. (1997). Homeotic transformation of legs to mouthparts by proboscipedia expression in Drosophila imaginal discs. Mech. Dev. 62 (1): 51-60.

Barrow, J. R. and Capecchi, M. R. (1996). Targeted disruption of the Hoxb-2 locus in mice interferes with expression of Hoxb-1 and Hoxb-4. Development 122: 3817-3828

Barrow, J. R. and Capecchi, M. R. (1999). Compensatory defects associated with mutations in Hoxa1 restore normal palatogenesis to Hoxa2 mutants. Development 126: 5011-5026.

Barrow, J. R., Stadler, H. S. and Capecchi, M. R. (2000). Roles of Hoxa1 and Hoxa2 in patterning the early hindbrain of the mouse. Development 127: 933-944.

Benassayag, C., et al. (1997a). A homeodomain point mutation of the Drosophila proboscipedia protein provokes eye loss independently of homeotic function. Mech. Dev. 63(2): 187-198.

Benassayag, C., et al. (1997b). Point mutations within and outside the homeodomain identify sequences required for proboscipedia homeotic function in Drosophila. Genetics 146(3): 939-949.

Benassayag, C., et al. (2003). Evidence for a direct functional antagonism of the selector genes proboscipedia and eyeless in Drosophila head development. Development 130: 575-586. 12490563

Boube, M., et al. (1997). Ras1-mediated modulation of Drosophila homeotic function in cell and segment identity. Genetics 146(2): 619-628.

Boudreau, N., et al. (1997). Induction of the angiogenic phenotype by Hox D3. J. Cell Biol. 139: 257-264

Brooke, N. M., Garcia-Fernandez, J. and Holland, P. W. (1998). The ParaHox gene cluster is an evolutionary sister of the Hox gene cluster. Nature 392(6679): 920-922.

Brown, S., et al. (1999). Characterization of the Tribolium Deformed ortholog and its ability to directly regulate Deformed target genes in the rescue of a Drosophila Deformed null mutant. Dev. Genes Evol. 209: 389-398.

Bobola, N., et al. (2003). Mesenchymal patterning by Hoxa2 requires blocking Fgf-dependent activation of Ptx1. Development 130: 3403-3414. 12810588

Boube, M., et al. (2000). Drosophila homologs of transcriptional mediator complex subunits are required for adult cell and segment identity specification. Genes Dev. 14: 2906-2917. 11090137

Core, N., et al. (1997). Altered cellular proliferation and mesoderm patterning in Polycomb-M33-deficient mice. Development 124, 721-729

Creuzet, S., et al. (2002). Negative effect of Hox gene expression on the development of the neural crest-derived facial skeleton. Development 129: 4301-4313. 12183382

Cribbs, D. L., et al. (1992a). Ectopic expression of the Drosophila homeotic gene proboscipedia under Antennapedia P1 control causes dominant thoracic defects. Genetics 132: 699-711

Cribbs, D. L., et al. (1992b). Structural complexity and evolutionary conservation of the Drosophila homeotic gene proboscipedia. EMBO J 11: 1437-49

Cribbs, D. L., et al. (1995). Levels of homeotic protein function can determine developmental identity: evidence from low-level expression of the Drosophila homeotic gene proboscipedia under Hsp70 control. EMBO J 14: 767-778

Davenne, M., et al. (1999). Hoxa2 and Hoxb2 control dorsoventral patterns of neuronal development in the rostral hindbrain. Neuron 22(4): 677-91.

DeCamillis, M. A., et al. (2001). Interactions of the Tribolium Sex combs reduced and proboscipedia orthologs in embryonic labial development. Genetics 159: 1643-1648. 11779803

DeCamillis, M. and ffrench-Constant, R. (2003). Proboscipedia represses distal signaling in the embryonic gnathal limb fields of Tribolium castaneum. Dev. Genes Evol. 213(2): 55-64. 12632174

Enriquez, J., Venkatasubramanian, L., Baek, M., Peterson, M., Aghayeva, U. and Mann, R. S. (2015). Specification of individual adult motor neuron morphologies by combinatorial transcription factor codes. Neuron 86(4):955-70. PubMed ID: 25959734

Ferretti, E., et al. (2000). Segmental expression of Hoxb2 in r4 requires two separate sites that integrate cooperative interactions between Prep1, Pbx and Hox protein. Development 127: 155-166.

Frasch, M., Chen, K. and Lufkin, T. (1995). Evolutionary-conserved enhancers direct region-specific expression of the murine Hoxa-1 and Hoxa-2 loci in both mice and Drosophila. Development 121: 957-974

Maconochie, M. K., et al. (2001). Differences in Krox20-dependent regulation of Hoxa2 and Hoxb2 during hindbrain development. Dev. Bio. 233: 468-481. 11336508

Gaufo, G. O., Flodby, P. and Capecchi, M. R. (2000). Hoxb1 controls effectors of sonic hedgehog and Mash1 signaling pathways. Development 127: 5343-5354.

Gaufo, G. O., Wu, S. and Capecchi, M. R. (2004). Contribution of Hox genes to the diversity of the hindbrain sensory system. Development 131: 1259-1266. 14960494

Gavalas, A., et al. (1997). Role of Hoxa-2 in axon pathfinding and rostral hindbrain patterning. Development 124: 3693-3702.

Grammatopoulos, G. A., et al. (2000). Homeotic transformation of branchial arch identity after Hoxa2 overexpression. Development 127: 5355-5365.

Grapin-Botton, A., Bonnin, M.-A. and Le Douarin, N. M. (1997). Hox gene induction in the neural tube depends on three parameters: competence, signal supply and paralogue group. Development 124: 849-859

Grapin-Botton, A., et al (1998). Defined concentrations of a posteriorizing signal are critical for MafB/Kreisler segmental expression in the hindbrain. Development 125(7): 1173-1181.

Guazzi, S., et al. (1998). Regulatory interactions between the human HOXB1, HOXB2, and HOXB3 proteins and the upstream sequence of the Otx2 gene in embryonal carcinoma cells. J. Biol. Chem. 273(18): 11092-9.

Guidato, S., Prin, F. and Guthrie, S. (2003). Somatic motoneurone specification in the hindbrain: the influence of somite-derived signals, retinoic acid and Hoxa3. Development 130: 2981-2996. 12756180

Harrison, K. A., et al. (1994). A novel human homeobox gene distantly related to proboscipedia is expressed in lymphoid and pancreatic tissues. J Biol Chem 269: 19968-19975

Hirth, F., Hartmann, B. and Reichert, H. (1998). Homeotic gene action in embryonic brain development of Drosophila. Development 125: 1579-1589.

Hughes, C. L. and Kaufman, T. C. (2000). RNAi analysis of Deformed, proboscipedia and Sex combs reduced in the milkweed bug Oncopeltus fasciatus: novel roles for Hox genes in the Hemipteran head. Development 127: 3683-3694

Hughes, C. L. and Kaufman, T. C. (2002). Exploring the myriapod body plan: expression patterns of the ten Hox genes in a centipede. Development 129: 1225-1238. 11874918

Hunt, P., et al. (1998). Stability and plasticity of neural crest patterning and branchial arch Hox code after extensive cephalic crest rotation. Dev. Biol. 198(1): 82-104.

Hunter, M. P. and Prince, V. E. (2002). Zebrafish Hox paralogue group 2 genes function redundantly as selector genes to pattern the second pharyngeal arch. Dev. Biol. 247: 367-389. 12086473

Irvine, S. Q. and Martindale, M. Q. (2000). Expression patterns of anterior Hox genes in the polychaete Chaetopterus: Correlation with morphological boundaries. Dev. Biol. 217: 333-351.

Jacobs, Y., Schnabel, C. A. and Cleary, M. L. (1999). Trimeric association of Hox and TALE homeodomain proteins mediates Hoxb2 hindbrain enhancer activity. Mol. Cell. Biol. 19: 5134-5142.

Johnston, L. A., et al. (1998). The homeobox gene cut interacts genetically with the homeotic genes proboscipedia and Antennapedia. Genetics 149(1): 131-142.

Joulia, L., Bourbon, H. M. and Cribbs, D. L. (2005). Homeotic proboscipedia function modulates hedgehog-mediated organizer activity to pattern adult Drosophila mouthparts. Dev. Biol. 278(2): 496-510. 15680366

Jungbluth, S., Bell, E. and Lumsden, A. (1999). Specification of distinct motor neuron identities by the singular activities of individual Hox genes. Development 126(12): 2751-2758.

Kanzler, B., et al. (1998). Hoxa-2 restricts the chondrogenic domain and inhibits bone formation during development of the branchial area. Development 125(14): 2587-2597.

Kapoun, A. M. and Kaufman, T. C. (1995). A functional analysis of 5', intronic and promoter regions of the homeotic gene proboscipedia in Drosophila melanogaster. Development 121: 2127-2141

Karmakar, K., Narita, Y., Fadok, J., Ducret, S., Loche, A., Kitazawa, T., Genoud, C., Di Meglio, T., Thierry, R., Bacelo, J., Luthi, A. and Rijli, F. M. (2017). Hox2 genes are required for tonotopic map precision and sound discrimination in the mouse auditory brainstem. Cell Rep 18(1): 185-197. PubMed ID: 28052248

Kutejova, E., Engist, B., Mallo, M., Kanzler, B. and Bobola, N. (2005). Hoxa2 downregulates Six2 in the neural crest-derived mesenchyme. Development 132(3): 469-78. 15634706

Maconochie, M. K., et al. (1997). Cross-regulation in the mouse HoxB complex: the expression of Hoxb2 in rhombomere 4 is regulated by Hoxb1. Genes Dev. 11(14): 1885-1895.

Maconochie, M., et al. (1999). Regulation of Hoxa2 in cranial neural crest cells involves members of the AP-2 family. Development 126(7): 1483-1494.

Mahaffey, J. W., Diederich, R. J. and Kaufman, T. C. (1989). Novel patterns of homeotic protein accumulation in the head of the Drosophila embryo. Development 105: 167-74

Manley, N. R., et al. (2001). Hoxb2 and Hoxb4 act together to specify ventral body wall formation. Dev. Bio. 237: 130-144. 11518511

Metscher, B. D. et al. (1997). Homeobox genes in axolotl lateral line placodes and neuromasts. Dev. Genes Evol. 207: 287-295

Miller, C. T., Maves, L. and Kimmel, C. B. (2004). moz regulates Hox expression and pharyngeal segmental identity in zebrafish. Development 131: 2443-2461. 15128673

Miller, D. F. B., et al. (2001). Cross-regulation of Hox genes in the Drosophila melanogaster embryo. Mech. Dev. 102: 3-16. 11287177

Nonchev, S., et al. (1996). Segmental expression of Hoxa-2 in the hindbrain is directly regulated by Krox-20. Development 122: 543-554.

Ohnemus, S., et al. (2001). Different levels of Hoxa2 are required for particular developmental processes. Mech. Dev. 108: 135-147. 11578867

Pasqualetti, M., et al. (2000). Ectopic Hoxa2 induction after neural crest migration results in homeosis of jaw elements in Xenopus. Development 127: 5367-5378.

Percival-Smith, A., et al. (1997). Genetic characterization of the role of the two HOX proteins, Proboscipedia and Sex Combs Reduced, in determination of adult antennal, tarsal, maxillary palp and proboscis identities in Drosophila melanogaster. Development 124(24): 5049-5062.

Prince, V. E., et al. (1998). Zebrafish hox genes: expression in the hindbrain region of wild-type and mutants of the segmentation gene, valentino. Development 125(3): 393-406.

Pultz, M.A., et al. (1988). The proboscipedia locus of the Antennapedia Complex: a molecular and genetic analysis. Genes Dev. 2: 901-20

Qiu, M., et al. (1997). Role of the Dlx homeobox genes in proximodistal patterning of the branchial arches: mutations of Dlx-1, Dlx-2, and Dlx-1 and -2 alter morphogenesis of proximal skeletal and soft tissue structures derived from the first and second arches. Dev. Biol. 185(2): 165-184

Randazzo, F. M., Cribbs, D. L. and Kaufman, T. C. (1991). Rescue and regulation of proboscipedia: a homeotic gene of the Antennapedia Complex. Development 113: 257-71

Robertson, L. K., et al. (2004). An interactive network of zinc-finger proteins contributes to regionalization of the Drosophila embryo and establishes the domains of HOM-C protein function. Development 131: 2781-2789. 15142974

Rozowski, M. and Akam, M. (2002). Hox gene control of segment-specific bristle patterns in Drosophila. Genes Dev. 16: 1150-1162. 12000797

Rusch, D. B. and Kaufman, T. C. (2000). Regulation of proboscipedia in Drosophila by homeotic selector genes. Genetics 156: 183-194.

Samad, O. A., et al. (2004). Integration of anteroposterior and dorsoventral regulation of Phox2b transcription in cranial motoneuron progenitors by homeodomain proteins. Development 131: 4071-4083. 15289435

Santagati, F., Minoux, M., Ren, S. Y. and Rijli, F. M. (2005). Temporal requirement of Hoxa2 in cranial neural crest skeletal morphogenesis. Developmen 132(22): 4927-36. 16221728

Schilling, T. F., Prince, V. and Ingham, P. W. (2001). Plasticity in zebrafish hox expression in the hindbrain and cranial neural crest. Dev. Bio. 231: 201-216. 11180963

Searcy, R. D. and Yutzey, K. E. (1998). Analysis of Hox gene expression during early avian heart development. Dev. Dyn. 213(1): 82-91.

Smith, R. C., et al. (1997). p21CIP1-mediated inhibition of cell proliferation by overexpression of the gax homeodomain gene. Genes Dev. 11:1674-1689.

Takihara, Y., et al. (1997). Targeted disruption of the mouse homologue of the Drosophila polyhomeotic gene leads to altered anteroposterior patterning and neural crest defects. Development 124: 3673-3682.

Tayyab, I., Hallahan, H. M. and Percival-Smith, A. (2004). Analysis of Drosophila proboscipedia mutant alleles. Genome 47(3): 600-9. 15190377

Vesque, C., et al. (1996). Hoxb-2 transcriptional activation in rhombomeres 3 and 5 requires an evolutionarily conserved cis-acting element in addition to the Krox-20 binding site. EMBO J 15(19): 5383-5396.

Vigano, M. A., et al., (1998). Definition of the transcriptional activation domains of three human HOX proteins depends on the DNA-binding context. Mol. Cell. Biol. 18(11): 6201-12.

Wassef, M. A., et al. (2008). Rostral hindbrain patterning involves the direct activation of a Krox20 transcriptional enhancer by Hox/Pbx and Meis factors. Development 135: 3369-3378. PubMed Citation: 18787068

proboscipedia: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 15 July 2015 

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