Sex combs reduced



scr is required during embryogenesis for labial and first thoracic segment development. Scr expression begins at gastrulation and is eventually apparent in three tissues: the labial and prothoracic (first thoracic) segments [Images] of the ectoderm, in parasegments two and three of the CNS, and in the visceral mesoderm of the anterior and posterior midgut. Scr is expressed in tissues that were not previously thought to accumulate SCR: a stripe of ectodermal cells in the parasegment 2 region of stage 5 embryos, the embryonic salivary glands, and the dorsal ridge (LeMotte, 1989 and Gorman, 1995).

The spatial accumulation of Proboscipedia overlaps partially with the distribution of the Deformed and Sex combs reduced proteins in the maxillary and labial segments, respectively. Sex combs reduced and Deformed, have different dorsal and ventral patterns of accumulation. Dorsally, these proteins are expressed in segmental domains; within the ventral region, a parasegmental register is observed. The boundary where this change in pattern occurs coincides with the junction between the ventral neurogenic region and the dorsal epidermis. After contraction of the germ band, when the nerve cord has completely separated from the epidermis, the parasegmental pattern is observed only within the ventral nerve cord while a segmental register is maintained throughout the epidermis (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).

Scr and Antp are expressed in the visceral mesoderm but not in the endoderm. The two genes are required for different aspects of the midgut morphogenesis. In Scr null mutant embryos the gastric caeca fail to form. Scr is expressed in the visceral mesoderm cells posterior to the primordia of the gastric caeca and appears to be indirectly required for the formation of the caeca (Reuter, 1990).

The expression of the ortholog of the Drosophila homeotic gene Sex combs reduced was examined in three divergent orders of insects: Hemiptera, Orthoptera and Thysanura. Whereas the anterior epidermal expression of Scr, in a small part of the posterior maxillary and all of the labial segment, is found in common among all four insect orders, the posterior (thoracic) expression domains vary. Unlike what is observed in flies, the Scr orthologs in other insects are not expressed broadly over the first thoracic segment, and instead are restricted to small patches. Scr is required for suppression of wings on the prothorax of Drosophila. Moreover, Scr expression at the dorsal base of the prothoracic limb in two other winged insects, crickets (Orthoptera) and milkweed bugs (Hemiptera), is consistent with Scr acting as a suppressor of prothoracic wings in these insects. Expression in this domain is a critical step in the evolution of modern winged insects and involved the repression of prothoracic wings. Scr is also expressed in a small patch of cells near the basitarsal-tibial junction of milkweed bugs, precisely where a leg comb develops, suggesting that SCR promotes comb formation, as it does in Drosophila. Surprisingly, the dorsal prothoracic expression of Scr is also present in the primitively wingless firebrat (Thysanura) and the leg patch is seen in crickets, which have no comb. The expression of Scr in the ventral and posterior epidermis of the prothorax is unique to Drosophila. This expression is required for the proper development of ventral structures, including specifying denticle types and the elaboration of the prothoracic beard. Mapping both gene expression patterns and morphological characters onto the insect phylogenetic tree demonstrates that in the cases of wing suppression and comb formation the appearance of Scr expression in the prothorax apparently precedes these specific functions. There is a striking conservation of the HOM/Hox anterior-to-posterior primary domain of expression throughout the metazoa. The order of expression (with respect to the anterior-most ectodermal expression domain) is generally co-linear with the known order of the genes within the homeotic complex of both chordates and insects. This provides further evidence that the HOM/Hox genes supply positional information and that this function is conserved (Rogers, 1997).

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

disco was initially identified in a screen for mutations affecting neural development. It was not until the discovery of disco-related (disco-r) that a patterning role was uncovered. The phenotype of terminal embryos lacking disco and disco-r is similar to those lacking the gnathal HOM-C genes Dfd and Scr; that is, structures from the gnathal segments (mandibular, maxillary and labial) are missing. This phenotype is due to reduced expression of Dfd and Scr target genes. Since HOM-C protein distribution is normal in disco, disco-r null embryos, and vice versa, these factors appear to act in parallel pathways (Robertson, 2004).

These studies have been extended and it is shown that: (1) Dfd can only direct maxillary developmental when Disco and/or Disco-R are present; (2) Tsh represses disco (and disco-r), helping to distinguish between trunk and gnathal segment types, and thereby establishing domains for appropriate HOM-C protein function, and (3) when ectopically expressed in the trunk, Disco represses trunk development and may transform these segments towards a gnathal segment type (Robertson, 2004).

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


Both Deformed and Sex combs reduced genes are transcribed during imaginal development: Dfd in a portion of the eye-antennal disc and Scr in the labial and prothoracic discs. SCR mRNA is also found in the adepithelial cells of all mesothoracic discs (Martinez-Arias, 1987).

The spatial pattern of Scr gene expression in imaginal tissues involved in the development of the adult thorax is governed in part by synapsis of homologous chromosomes in this region of the ANT-C. However, those imaginal discs that arise anterior to the prothorax do not appear to be sensitive to this form of gene regulation (Pattatucci, 1991b).

Neuroblast lineage identification and lineage-specific Hox gene action during postembryonic development of the subesophageal ganglion in the Drosophila central brain

The central brain of Drosophila consists of the supraesophageal ganglion (SPG) and the subesophageal ganglion (SEG), both of which are generated by neural stem cell-like neuroblasts during embryonic and postembryonic development. Considerable information has been obtained on postembryonic development of the neuroblasts and their lineages in the SPG. In contrast, very little is known about neuroblasts, neural lineages, or any other aspect of the postembryonic development in the SEG. This study characterized the neuroanatomy of the larval SEG in terms of tracts, commissures, and other landmark features as compared to a thoracic ganglion. Then clonal MARCM labeling was used to identify all adult-specific neuroblast lineages in the late larval SEG, and a surprisingly small number of neuroblast lineages, 13 paired and one unpaired, were found. The Hox genes iDfd, Scr, and Antp are expressed in a lineage-specific manner in these lineages during postembryonic development. Hox gene loss-of-function causes lineage-specific defects in axonal targeting and reduction in neural cell numbers. Moreover, it results in the formation of novel ectopic neuroblast lineages. Apoptosis block also results in ectopic lineages suggesting that Hox genes are required for lineage-specific termination of proliferation through programmed cell death. Taken together, these findings show that postembryonic development in the SEG is mediated by a surprisingly small set of identified lineages and requires lineage-specific Hox gene action to ensure the correct formation of adult-specific neurons in the Drosophila brain (Kuert, 2014).

A total of 14 identified postembryonic neuroblast lineages generate the adult-specific secondary neurons in the larval SEG. This is a surprisingly small number compared with the approximately 80 neuroblast lineages in the embryonic SEG. Cell counts indicate that only about one fourth of these ~80 neuroblasts are reactivated postembryonically. This is markedly different in the supraesophageal ganglion (SPG), where about 85 of the 100 embryonically active neuroblasts are reactivated and proliferate in larval stages. The experiments indicate that the fate of half of the embryonic SEG neuroblasts that are not present postembryonically is programmed cell death. This situation is comparable to that of embryonic neuroblasts in the abdominal ganglia where the majority of neuroblasts undergo apoptosis at the end of embryogenesis. The molecular cues that trigger cell death in these embryonic neuroblasts have not been studied. The fate of the other half of the embryonic SEG neuroblasts is unknown. They may terminate proliferation through other reaper/hid/grim-independent cell death mechanisms or through cell cycle exit at the end of embryogenesis. Further experiments will be necessary to elucidate this (Kuert, 2014).

The low number of postembryonic SEG lineages has interesting consequences for the relationship between primary neurons and secondary neurons in the mature SEG. Most neuroblasts generate 10-20 neural cells embryonically and 100-150 neural cells postembryonically. Thus, the ~80 embryonic SEG neuroblasts should generate 800-1600 primary neural cells per hemiganglion while the 14 postembryonic neuroblasts generate approximately 900 secondary neural cells (as estimated by cell counts) per hemiganglion. Assuming that most of the primary neurons survive metamorphosis, this suggests that a substantial fraction of the neurons in the adult SEG could be primary neurons that comprise the functional larval SEG before their integration into the adult brain (Kuert, 2014).

Previous work has shown that 75 neuroblast lineages generate the secondary neurons of the three thoracic neuromeres. This is in striking contrast to the 14 neuroblast lineages that generate secondary neurons in the three SEG neuromeres. This reduction is most evident in the SA region, where only one commissure (ISA) is present which is also formed by only one lineage, SA3. The labial neuromere is also reduced but not as dramatically. Moreover, it retains the two commissures (aI, pI) which are also characteristic of the thoracic neuromeres. This relatively small number of postembryonic neuroblast lineages in the SEG neuromeres is likely to reflect the marked reduction and fusion of segmental appendages in the three gnathal segments that are innervated by the SEG. From an evolutionary perspective, a loss/reduction of gnathal appendages in insects such as flies would eliminate or reduce the need for corresponding neural control circuitry at least in the adult. Interestingly, and in contrast to the VNC, no evidence was found for the presence of postembryonically generated motoneurons in the SEG, indicating that all secondary neurons in the SEG are interneurons. This notion is supported by the fact that none of the 14 SEG neuroblast lineages join the labial or pharyngeal nerves (which contain the motor axons from the proboscis), but instead they project their secondary axon tracts (SATs) to areas within the CNS (Kuert, 2014).

During embryonic and postembryonic brain development, the Hox genes Dfd, Scr, and Antp are regionally expressed in discrete and largely non-overlapping domains in the neuromeres of the SEG. In both cases Dfd is expressed in an anterior domain, Scr is expressed in a posteriorly adjacent domain, and Antp expression begins in a small labial domain adjacent to the prothoracic neuromere. Moreover, while the total number of neuroblast lineages that express a given Hox gene may be different embryonically and postembryonically, most of the postembryonic neuroblast lineages do express one of these genes suggesting that Hox gene expression is a stable developmental feature of SEG lineages. Indeed, most if not all of the Hox genes that are expressed in the embryonic CNS, are re-expressed in the neuroblast lineages of the postembryonic CNS (Kuert, 2014).

Hox genes are known to be expressed during CNS development in a number of bilaterian animal groups, including vertebrates, hemichordates, insects, and annelids, and in all of these animal groups the order of Hox gene expression domains in the developing CNS appears to be conserved. For example, the order of expression of orthologous Hox genes in the developing CNS of Drosophila, mouse, and human is virtually identical. Taken together, these findings suggest that a conserved pattern of Hox gene expression domains may be a common feature in the developing CNS of all bilaterians (Kuert, 2014).

This study reveals two types of lineage-specific requirement for Hox genes during postembryonic SEG development. The first is a requirement of the Hox genes Dfd, Scr and Antp for correct postembryonic development of a subset of those lineages that are normally present in the wildtype SEG. Hox genes are required for correct SAT projections in the lineages SA1 (Dfd), SA5 (Scr) and LB3 (Antp). Interestingly, in all three cases the lineage-specific loss-of-function of these Hox genes results in specific, reproducible SAT misprojections and not in randomized axonal misprojections. While this could, in principle, be the result of a homeotic transformation phenotype, no evidence was found for such a transformation, since in terms of their projection patterns mutant SATs of these three lineages do not resemble any of the wildtype SATs present in the larval SEG (Kuert, 2014).

Hox genes are also required for correct cell number in the lineages LB5 (Scr) and LB3 (Antp). While these Hox mutant lineages lose about half of their cells, which would suggest the involvement cell death in a hemilineage-dependent manner, no evidence was found for hemilineage-specific Hox gene expression in these lineages. Thus, further studies of Hox gene action in the lineages LB3 and LB5 are necessary to dissect the functional requirement of Scr and Antp in lineage-specific cell survival (Kuert, 2014).

The second type of lineage-specific requirement for Hox genes during postembryonic SEG development is the prevention of ectopic lineage formation. Thus, in addition to their requirement for correct development of normal wildtype lineages, the genes Dfd and Scr are also required for suppressing the appearance of aberrant ectopic lineages that are not normally present in the wildtype SEG. When Dfd or Scr mutant neuroblast clones are induced at early larval stages and recovered at late larval stages, five distinct types of ectopic neuroblast clones are found. Each of these is identifiable based on reproducible neuroanatomical features such as position, secondary axon tract projection and cell number. These ectopic lineages do not represent homeotic transformations of other wildtype neuroblast lineages, since all other SEG neuroblast lineages are present. Whether these ectopic lineages become functionally integrated into the adult brain of Drosophila is currently unknown. Evidence for an integration of ectopic neuron groups into a mature brain comes from mammalian studies, which show that Hoxa1 mutant hindbrain progenitors can establish supernumerary ectopic neural cell groups that escape apoptosis and give rise to a functional circuit in the postnatal brain (Kuert, 2014).

The molecular regulators through which the Hox genes Dfd, Scr and Antp exert their diverse roles in lineage-specific SEG development are currently not known. In terms of the Hox gene requirement for correct development of wildtype lineages, only 4 of the 14 SEG lineages (11 of which express Hox genes) show misprojection or cell number mutant phenotypes. However, in these 4 lineages, the Hox gene mutant phenotypes are highly penetrant and reproducible. The lineage-restricted nature of these mutant phenotypes suggests that Hox genes interact with other lineally acting control elements to determine the developmental features of the affected lineages. While the ensemble of these control elements is currently unknown, there is increasing evidence for the importance of transcription factor codes in controlling the expression of axonal guidance molecules. In terms of the Hox gene requirement for preventing the formation of ectopic lineages, the data suggest that this involves lineage-specific programmed cell death of the corresponding postembryonic neuroblasts. Indeed, all Hox genes studied to date have been implicated in some aspect of programmed cell death in postembryonic neuroblasts. The lab gene is required for the termination of specific tritocerebral neuroblasts, Dfd and Scr are required for lineage-specific neuroblast termination in the SEG, Antp und Ubx can trigger neuroblast death if misexpressed in thoracic lineages, and abd-A induces programmed cell death in neuroblasts of the central abdomen. It is therefore concluded that a general function of Hox genes in postembryonic neural development is in the regionalized termination of progenitor proliferation as a key mechanism for neuromere-specific differentiation and specialization of the adult brain (Kuert, 2014).

Effects of Mutation or Deletion

Both Proboscipedia (Pb) and Sex combs reduced (Scr) activities are required for determination of proboscis 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 in isolation, determine tarsal identity. In one model, expression of both 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 dominant negative 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 Bicoid class 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-Scr Q50K -containing complexes, would not only have diminished affinity 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 leg pathway, 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 leg identity, respectively (Percival-Smith, 1997 and references).

Scr gene function is required during mid and late embryogenesis for normal head involution to occur and for the proper formation of embryonic prothoracic and head structures, particularly those derived from the labial segment. Mutants show a labial to maxillary transformation (the maxillary segment is anterior to the labial segment (Pattatucci, 1991a).

The reported labial to maxillary transformation in embryos lacking Scr is surprising because DFD does not accumulate in the labial cells of an Scr mutant. Several lines of evidence indicate that Dfd is required for the production of maxillary structures. In Dfd mutant embryos, mouth hooks and cirri are not produced: however, the central portion of the maxillary sensory organ is still present. Further, when Dfd is ubiquitously expressed via a heat shock protein construct, ectopic mouth hooks and cirri are generated in the head and thoracic segments. After analyzing both the distribution of certain gene products in embryos lacking Scr and cuticular phenotypes of embryos with mutations that block head involution, it is suggested that a labial to maxillary transformation does not occur. It is proposed instead that a lack of Scr function causes a loss of labial identity (Pederson, 1996).

Gain-of-function mutations in Scr result in the presence of ectopic sex comb teeth on the first tarsal segment of mesothoracic and metathoracic legs of adult males. Heterozygous combinations of gain-of-function alleles with a wild-type Scr gene exhibit no evidence of ectopic protein localization in the second and third thoracic segments of embryos. However, mesothoracic and metathoracic leg imaginal discs can be shown to accumulate ectopically expressed SCR protein, implying a differential regulation of the Scr gene during these two periods of development (Pattatucci, 1991b).

The anterior-posterior extent of the salivary gland primordium, a placode of columnar epithelial cells derived from parasegment 2, is established by the positive action of Scr. Embryos mutant for Scr lack a detectable placode, while ectopic Scr expression leads to the formation of ectopic salivary glands. In contrast, the dorsal-ventral extent of the placode is regulated negatively (see Forkhead). Functions dependent on the decapentaplegic product place a dorsal limit on the placode, while dorsal-dependent genes act to limit the placode ventrally (Panzer, 1992).

The mutationally defined function of Scr is to specify the identity of the labial and prothoracic segments and to control the development of the gastric caeca (Gorman, 1995).

Scr and Antp are expressed in the visceral mesoderm but not in the endoderm. The two genes are required for different aspects of the midgut morphogenesis. In Scr null mutant embryos the gastric caeca fail to form. Scr is expressed in the visceral mesoderm cells posterior to the primordia of the gastric caeca and appears to be indirectly required for the formation of the caeca (Reuter, 1990).

Ectopic expression of homeotic genes, Dfd, Scr and Antp, results in the disruption of the developing PNS in the abdomen. Thus homeotic genes have specific roles in establishing the correct spatial patterns of sensory organs in their normal domains of expression (Heuer, 1992).

Sex combs reduced (Scr) activity is proposed to be required cell nonautonomously for determination of tarsus identity, and Extradenticle (Exd) activity is required cell autonomously for determination of arista identity. Using the ability of Proboscipedia to inhibit the Scr activity required for determination of tarsus identity, it was found that loss-of-Exd activity is epistatic to loss-of-Scr activity in tarsus vs. arista determination. That is, loss-of-Exd activity produces tarsus when there is no Scr activity, suggesting that Exd functions downstream of Scr. This suggests that in the sequence leading to arista determination, Scr activity is OFF while Exd activity is ON, and in the sequence leading to tarsus determination Scr activity is ON, which turns Exd activity OFF. Immunolocalization of Exd in early third-instar larval imaginal discs reveals that Exd is localized in the nuclei of antennal imaginal disc cells and localized in the cytoplasm of distal imaginal leg disc cells. It is propose that Exd localized to the nucleus suppresses tarsus determination and activates arista determination. It is further proposed that in the mesodermal adepithelial cells of the leg imaginal discs, Scr is required for the synthesis of a tarsus-inducer, which, when secreted, acts on the ectoderm cells inhibiting nuclear accumulation of Exd, such that tarsus determination is no longer suppressed and arista determination is no longer activated (Percival-Smith, 1998).

apontic is required for the formation of some but not all Deformed- and Sex combs reduced-dependent ventral gnathal structures. The lateral arms of the H-piece are missing and the lateralgraten are shortened in Dfd mutants; the hypostomal sclerites and dorsal pouch are missing in Scr mutants. Other Dfd- and Scr-dependent structures are intact in apt mutants, such as the ectostomal sclerites (Dfd-dependent), mouth hooks (Dfd-dependent) and cross bar of the H-piece (Scr-dependent). Thus, the apt phenotype suggests that apt might be contributing to the diversification of Dfd and Scr function in a specific cell population within each selector's domain. Such overlap in phenotypes could occur in principle by three different mechanisms: (1) apt could mediate Dfd and Scr functions by regulating their transcription in a particular region (i.e. apt acts upstream); (2) apt could be a target of Dfd and Scr in a discrete population of cells (apt acts downstream), or (3) apt could act in conjunction with Dfd and Scr (or with Dfd and Scr targets) to produce a distinct biological effect in a subpopulation of cells (apt acts in parallel). Whole mount in situ hybridizations of apt mutant embryos with DFD and SCR mRNA probes were performed their and patterns of transcription were found to be indistinguishable from wild type. Therefore, apt is not required to establish or maintain Dfd and Scr transcription. Conversely, apt transcription was examined in Dfd and Scr mutants by in situ hybridization and no changes were detected. Thus, neither Dfd nor Scr is required to establish or maintain apt transcription. It is concluded that apt acts in parallel with Dfd and Scr proteins to produce ventral gnathal structures (Gellon, 1997).

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).

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

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).

The Drosophila kismet gene is related to chromatin-remodeling factors and is required for both segmentation and segment identity

The Drosophila trithorax group gene kismet (kis) was identified in a screen for extragenic suppressors of Polycomb (Pc) and subsequently shown to play important roles in both segmentation and the determination of body segment identities. One of the two major proteins encoded by kis (Kis-L) is related to members of the SWI2/SNF2 and CHD families of ATP-dependent chromatin-remodeling factors. To clarify the role of Kis-L in gene expression, its distribution on larval salivary gland polytene chromosomes was examined. Kis-L is associated with virtually all sites of transcriptionally active chromatin in a pattern that largely overlaps that of RNA Polymerase II (Pol II). The levels of elongating Pol II and the elongation factors SPT6 and CHD1 are dramatically reduced on polytene chromosomes from kis mutant larvae. By contrast, the loss of Kis-L function does not affect the binding of PC to chromatin or the recruitment of Pol II to promoters. These data suggest that Kis-L facilitates an early step in transcriptional elongation by Pol II (Srinivasan, 2005).

The Drosophila kismet gene was identified in a screen for dominant suppressors of Polycomb, a repressor of homeotic genes. kismet mutations suppress the Polycomb mutant phenotype by blocking the ectopic transcription of homeotic genes. Loss of zygotic kismet function causes homeotic transformations similar to those associated with loss-of-function mutations in the homeotic genes Sex combs reduced and Abdominal-B. kismet is also required for proper larval body segmentation. Loss of maternal kismet function causes segmentation defects similar to those caused by mutations in the pair-rule gene even-skipped. The kismet gene encodes several large nuclear proteins that are ubiquitously expressed along the anteriorposterior axis. The Kismet proteins contain a domain conserved in the trithorax group protein Brahma and related chromatin-remodeling factors, providing further evidence that alterations in chromatin structure are required to maintain the spatially restricted patterns of homeotic gene transcription (Daubresse, 1999).

The genetic interactions between kis and Pc provided the first clue that kis plays an important role in the determination of body segment identity. kis mutations suppress the adult Pc phenotype by preventing the ectopic transcription of homeotic genes. Thus, kis is a member of the trithorax group of homeotic gene activators. Mosaic analyses reveal that loss of kis function causes homeotic transformations, including the transformation of first leg to second leg and the fifth abdominal segment to a more anterior identity. These phenotypes are identical to those associated with loss-of-function Scr and Abd-B mutations, respectively. Taken together, these findings suggest that kis acts antagonistically to Pc to activate the transcription of both Scr and Abd-B. It is intriguing that kis mutations alter the fate of only the fifth abdominal segment, since the identities of the fifth through ninth abdominal segments are determined by a single homeotic gene, Abd-B (Daubresse, 1999).

Variations in the levels of Abd-B protein result in the differences between these abdominal segments, with Abd-B expression being lowest in the fifth abdominal segment. Parasegment-specific cis-regulatory regions, termed infra-abdominal (iab) regions control Abd-B expression. Each iab region is named for the segment that it affects (iab-5 through iab-9). Mutations in both iab-5 and kis affect the identity of only the fifth abdominal segment, suggesting that the Kis protein may interact specifically with the iab-5 cis-regulatory element of Abd-B (Daubresse, 1999).

kis probably interacts not only with Scr and Abd-B, but with other homeotic genes as well. For example, the isolation of kis mutations as enhancers of loss-of-function Deformed (Dfd) mutations suggests that kis is probably also required to activate transcription of this ANTC homeotic gene. Furthermore, kis duplications strongly enhance the transformation of wing to haltere in Pc heterozygotes, a phenotype caused by the ectopic transcription of Ubx in the wing imaginal disc. However, kis mutations do not cause haltere-to-wing transformations due to decreased Ubx transcription. A possible explanation for the lack of homeotic transformations in kis clones in segments other than the prothoracic and fifth abdominal segment is that the mutations used in these studies are not null alleles. kis1 is a strong loss-of-function mutation. It has not been characterized at the molecular level, however, and may not completely eliminate kis function. It is also possible that sufficient levels of Kis protein persist in homozygous mutant tissue following mitotic recombination to support normal development. Further genetic studies, including the analysis of conditional kis alleles, will be necessary to distinguish between these possibilities (Daubresse, 1999).

Germline clonal analysis has revealed an unanticipated role for kis in segmentation. Embryos from mosaic kisS females exhibit a deletion or alteration of every other segment, while mutant embryos from mothers bearing germline clones of the stronger kis1 allele usually develop only half of the normal number of segments. This variation in phenotypic severity is closely correlated with the extent to which en expression is disrupted. The phenotypes associated with loss of maternal kis function resemble those caused by mutations in pair-rule segmentation genes that cause the deletion of the odd-numbered parasegments. kis thus appears to be necessary for the expression (or function) of one or more pair-rule genes. Recent genetic studies have suggested that kis may also be involved in the Notch signaling pathway. Thus it appears that kis plays roles in addition to the regulation of homeotic genes (Daubresse, 1999).

What pair-rule genes might require kis for their activity? Based on the kis mutant phenotype, perhaps the best candidates are eve and hairy (h), both of which are required for the formation of odd-numbered parasegments. Unlike eve, h and most other segmentation genes, kis is uniformly expressed in the early embryo. This raises the possibility that Kis functions as an essential cofactor or modifier of Eve or other pair-rule proteins. It is also possible that loss of kis function might result in pair-rule genes being transcribed outside of their normal expression domains. Additional work will be necessary to determine the molecular basis of the segmentation defects resulting from loss of maternal kis function (Daubresse, 1999).

Hox genes regulate the same character by different strategies in each segment

Hox genes control regional identity along the anterior-posterior axis in various animals. Each region contains morphological characteristics specific to that region as well as some that are shared by several different regions. The mechanism by which one Hox gene regulates region-specific characteristics has been extensively analyzed. However, little attention has been paid to the mechanism by which different Hox genes regulate the same characteristics in different regions. This study shows that two Hox genes in Drosophila, Sex combs reduced and Ultrabithorax, employ different mechanisms to achieve the same out-put, the absence of sternopleural bristles, in the prothorax and metathorax, respectively. Sternopleural bristles are characteristics of the mesothorax, and it was found that spineless is involved in their development. Analysis of the regulatory relationship between Hox genes and spineless indicated that ss expression is repressed by Sex combs reduced in the prothorax. Since sole misexpression of ss could induce ectopic sternopleural bristle formation in the prothorax irrespective of the expression of Sex combs reduced, spineless repression appears to be critical for inhibition of sternopleural bristles by Sex combs reduced. In contrast, spineless is expressed in the metathorax independently of Ultrabithorax activity, indicating that Ultrabithorax blocks sternopleural bristle formation through mechanisms other than spineless repression. This finding indicates that the same characteristics can be achieved in different segments by different Hox genes acting in different ways (Tsubota, 2008).

This study found that three genes, Antp, ss and al, are involved in sternopleural bristle formation. In the al mutant, no appreciable Ac expression in the T2 leg disc is detected and sternopleural bristles are not formed, indicating that the requirement of al is absolute. In contrast, Ac expression is detectable in the ss mutant T2 leg disc and in the Antp mutant clones, indicating that the requirement of both ss and Antp for ac expression is not absolute. However, sternopleural bristles were never found in the ss mutant, despite the fact that Antp expression was unaffected in the ss mutant clone in the T2 leg disc. In contrast, Antp mutant cells, in which ss is expressed normally, formed sternopleural bristles. In addition, sole misexpression of ss in the T1 segment produces sternopleural bristles ectopically, while that of Antp did not. Therefore, ss appears to be necessary and sufficient for sternopleural bristle formation, while Antp appears to be insufficient and not necessarily required. Moreover, Ac expression is ectopically induced in the T1 leg disc by misexpression of ss but not of Antp and in the ss mutant T2 leg disc is very weak, highly restricted, and only transient. This indicates that ss but not Antp appears to be one of the major activators of ac expression. Taken together, ss appears to be much more fundamental for sternopleural bristle formation than Antp (Tsubota, 2008).

The initiation of ac expression coincides with the initiation of ss expression. Since al and Antp are already expressed before ac induction in the early third instar stage, the timing of ac induction may be determined by the regulation of ss expression. Interestingly, the residual Ac expression seen in the ss mutant leg disc is first observed in the mid third instar as in the wild-type leg disc. This implies that at least one additional gene (referred to as X hereafter), whose expression or function is activated at the same stage as the initiation of ss expression, may be involved in ac induction. One possibility may be a gene functioning in hormonal regulation. Nonetheless, the ability of the sole misexpression of ss to induce ectopic ac expression and sternopleural bristle formation strongly indicates that ss is much more fundamental than X (Tsubota, 2008).

The restriction of ac expression to the overlap between the ss and al expression domains indicates the importance of determining the distal limit of ss expression and the proximal limit of al expression. Analysis of clones lacking ss activity or misexpressing ss indicates that ss has a repressive activity on al expression. How can al be expressed in the overlap domain? In the overlap domain, ss represses al expression when misexpressed at high levels but does not when misexpressed at approximately endogenous levels. The level of ectopic Al expression in the ss mutant clone located in a region proximal to the normal al expression domain is lower than that of endogenous Al expression. Moreover, Al expression in the wild-type leg disc gradually decays at its proximal edges. Considering all of these observations, the following hypothesis is suggested: al expression is activated according to the proximodistal information and the proximal limit of the al expression domain may be determined by a balance between activation according to the proximodistal information and repression by ss. The activation force may dominate the repressive activity of ss in the overlapping region but may gradually decay towards the proximal edges of the al expression domain. In contrast, ss expression does not appear to be regulated by al. As with the case of al activation, it may be possible that ss is repressed according to the proximodistal information (Tsubota, 2008).

The morphological identities of the T1 and T3 segments, including the absence of sternopleural bristles, are determined by Scr and Ubx, respectively. Analyses of the T1 leg disc with Scr mutant clones and the T2 leg disc with ectopic Scr activity indicate that both ss and Antp are repressed by Scr in the T1 leg disc. In addition, there is a possibility that the expression or function of gene X is repressed by Scr. Weak Ac expression is transiently observed in the ss mutant T2 leg disc, indicating that ac expression can be weakly activated without ss activity in the presence of gene X and Antp activity. In addition, Scr does not appear to repress ac expression directly, since ectopic induction of ac by ss misexpression in the T1 leg disc was not associated with an alteration in Scr expression. If gene X is active in the T1 leg disc, sole misexpression of Antp is expected to activate ac expression at least weakly and transiently. However, no ectopic Ac expression was found upon sole misexpression of Antp. Therefore, the activity of gene X is likely to be repressed in the T1 leg disc. For evaluating the significance of these three genes on Scr-dependent inhibition of sternopleural bristle formation, the ability of ss misexpression to induce ectopic ac expression and sternopleural bristle formation without affecting Scr expression is of crucial importance. At present, whether ac expression and sternopleural bristle formation can be induced solely by ss or only in a combination of ss and Antp and/or gene X is unclear. However, ss misexpression induced Antp expression and, thus, at least ss and Antp were coexpressed upon sole misexpression of ss. As for gene X, if it is not activated by ss misexpression, the results indicate that ac expression and sternopleural bristle formation can be induced without gene X activity at least in the presence of both ss and Antp expression. In contrast, if ac expression and sternopleural bristle formation require gene X activity, ss misexpression must activate gene X. After all, the results indicate that sole misexpression of ss can fulfill at least a minimum requirement for ac expression and sternopleural bristle formation. In other words, if Scr could not repress ss expression, ac expression would be activated and sternopleural bristles would be formed irrespective of the expression and function of Antp and gene X. Therefore, Scr must repress ss expression and this appears to be a key step to block sternopleural bristle formation in the T1 segment (Tsubota, 2008).

In contrast to the T1 leg disc, strong Ss expression was observed in the wild-type T3 leg disc and it is unaltered in Ubx mutant clones. Therefore, Ubx appears to act through a mechanism unrelated to ss expression. How does Ubx function? Simultaneous expression of both ss and Antp seemed insufficient for ac expression and sternopleural bristle formation in the T3 segment, since Antp misexpression failed to induce Ac expression in the T3 leg disc, in which ss is prominently expressed. It may be possible that Ubx represses ac expression directly. Alternatively, Ubx may compromise the function of the Ss protein directly or indirectly through regulation of its downstream gene products. Another possibility is that Ubx acts through repression of gene X activity. These possibilities are not mutually exclusive with each other (Tsubota, 2008).

The occurrence of ac expression and sternopleural bristle formation in the absence of Antp activity indicates that the absence of sternopleural bristles is not the ground state. However, the number of sternopleural bristles is variable in that condition, indicating that the complete formation of sternopleural bristles is not also the ground state. Since ss misexpression experiment suggests that sternopleural bristles can be formed as long as ss is expressed, one possible aspect of the ground state may be the expression of ss and the production of at least some kind of bristles. Antp may have acquired the ability to modify this state to produce the current-type of sternopleural bristles. On the other hand, Scr may have evolved the ability to block sternopleural bristle formation by acquiring the activity to repress ss expression and Ubx by acquiring another, yet unknown function. Taken together, the current state of sternopleural bristles in all three thoracic segments appears to be the derived state (Tsubota, 2008).

Analysis of the sequence and phenotype of Drosophila Sex combs reduced alleles reveals potential functions of conserved protein motifs of the Sex combs reduced protein

The Drosophila Hox gene, Sex combs reduced (Scr), is required for patterning the larval and adult, labial and prothoracic segments. Fifteen Scr alleles were sequenced and the phenotypes analyzed in detail. Six null alleles were nonsense mutations (Scr2, Scr4, Scr11, Scr13, Scr13A, and Scr16) and one was an intragenic deletion (Scr17). Five hypomorphic alleles were missense mutations (Scr1, Scr3, Scr5, Scr6, and Scr8) and one was a small protein deletion (Scr15). Protein sequence changes were found in four of the five highly conserved domains of SCR: the DYTQL motif (Scr15), YPWM motif (Scr3), Homeodomain (Scr1), and C-terminal domain (CTD) (Scr6), indicating importance for SCR function. Analysis of the pleiotropy of viable Scr alleles for the formation of pseudotracheae suggests that the DYTQL motif and the CTD mediate a genetic interaction with proboscipedia. One allele Scr14, a missense allele in the conserved octapeptide, was an antimorphic allele that exhibited three interesting genetic properties: (1) Scr14/Df had the same phenotype as Scr+/Df; (2) the ability of the Scr14 allele to interact intragenetically with Scr alleles mapped to the first 82 amino acids of SCR, which contains the octapeptide motif; (3) Scr6, which has two missense changes in the CTD, did not interact genetically with Scr14 (Sivanantharajah, 2009).

THE Homeotic selector (Hox) genes are required for patterning the anterior-posterior axis of all bilateral animals. In Drosophila, the Hox genes establish segmental identity in the embryo by controlling the spatial expression of target genes. Although much is known about the requirement of HOX activity in development, there is little known about internal domain structure of these proteins and how these transcription factors are regulated. In fact, the analysis of the functional domains of HOX proteins has proven difficult and often contradictory. For example, the insect specific QA motif of the HOX protein Ultrabithorax (UBX) is required for full Ubx repression of limb development in Drosophila when UBX is ectopically expressed. Noninsect UBX homologs lack a QA motif and lack the ability to suppress limb development when ectopically expressed in Drosophila; however, limb repression can be conferred to these noninsect UBX homologs by fusing the QA motif to the carboxyl termini. These ectopic expression experiments suggested that the QA motif was essential for UBX activity; therefore, it was surprising that a deletion of the QA motif within the Ubx locus produced only a subtle phenotype. The observation of differential pleiotropy in the Ubx and Antennapedia (Antp) loci offers a potential explanation for these difficulties: HOX proteins are made up of small independently acting peptide elements that alone make only a small contribution to HOX activity. Uniform pleiotropy is the same relative behavior of a set of alleles in a locus on two or more phenotypic characteristics, whereas, differential pleiotropy is a distinct relative behavior. Differential pleiotropy has been described in Ubx. Analysis of the Ubx-δQA allele revealed a differential requirement for the QA motif in the development of various UBX-dependent tissues. This preferential requirement for the QA motif in a subset of tissues is an example of differential pleiotropy. In addition, the YPWM motif of the HOX protein Antennapedia (ANTP), a motif that has been conserved across evolution in most HOX proteins, exhibits differential pleiotropy by being required for the formation of ectopic wing tissue but not the formation of ectopic leg tissue (Sivanantharajah, 2009 and references therein).

The sequence of Scr mutant alleles allowed the analysis of the requirement of highly conserved motifs of the HOX protein, Sex combs reduced (SCR). Like Ubx, Scr is haplo-insufficient, making it an excellent gene for identifying small changes in SCR activity because even subtle changes in levels of protein function are registered in SCR-dependent phenotypes. SCR function is essential for the development of labial derivatives, such as the adult proboscis and larval salivary glands, and for establishing the identity of the adult prothoracic legs. SCR activity is required with a second HOX protein, Proboscipedia (PB), for the formation of the proboscis but does not require PB for the formation of the sex comb bristles or the salivary gland. The SCR protein has five highly conserved regions. The octapeptide, YPWM motif and homeodomain (HD) are well conserved across evolution and are found in all SCR homologs. The MvDYTQLQPQRL sequence (DYTQL motif) and the carboxy-terminal domain (CTD) are insect and SCR specific. From analysis of an Scr antimorphic allele, it is suggested that the octapeptide of SCR participates in protein complex formation required for the formation of sex combs and pseudotrachea, and that the CTD inhibits protein complex formation by masking the octapeptide. In addition, analysis of the proboscis phenotype of viable Scr alleles in the presence of one or two copies of the pb locus suggests that the DYTQL motif and the CTD of SCR mediate a genetic interaction with pb (Sivanantharajah, 2009).

Scr14 is a Ser10-to-Leu change in the octapeptide motif of SCR. The octapeptide motif is found in all SCR homologs, and for SCR and murine HOXA5 the octapeptide is required for the formation of ectopic salivary glands in Drosophila. A submotif of the octapeptide, SSYF, is found in the Drosophila HOX proteins Labial, ANTP, Deformed, and UBX, which in the case of UBX is important for function. Therefore, it was surprising that the Scr14 change of a Ser10 to Leu of the most conserved residue of the octapeptide submotif had little affect on the number of sex comb bristles, pseudotracheal rows, and salivary gland nuclei when hemizygous over a Df. The only strong phenotype was a reduction of the number of sex comb bristles when heterozygous. This suggests that Scr14 is an antimorphic allele, and it is proposed that SCR14 forms inactive heterocomplexes with SCR+ resulting in a 50% reduction of total SCR activity. This model of inhibition of SCR at the protein level is favored over a mechanism of pairing-dependent repression because all null and hypomorphic alleles that interact with the Scr14 allele are DNA sequence changes that would result in an altered protein product. These alleles that interact with Scr14 encode proteins that result in a further reduction of total SCR activity. These alleles produce inactive or partially active SCR proteins that interact and inactivate SCR14. In the case of nonsense alleles, the only active complex left is that containing two SCR14 molecules (25%). This ability to interact with SCR14 maps to the first 82 amino acids of SCR, and the only conserved domain in this region of SCR is the octapeptide motif. However, two alleles Scr6 and Scr7 did not interact genetically with Scr14 (Sivanantharajah, 2009).

Scr7 mutants had reductions in the number of sex comb bristles, reductions in the number of rows of pseudotrachea, but no reductions in the number of salivary gland cells. Transcript and protein levels in this mutant do not differ significantly from wild type. The Scr7 phenotype may be caused by a subtle change in the pattern of SCR expression at the pupal stage. Therefore, it is possible that this allele may be a regulatory mutant. If Scr7 is a regulatory mutant, it is expected to show a weak genetic interaction with Scr14 because varying the ratio of SCR+ to SCR14 below 1 results in a theoretical maximum loss of 17.5% of total SCR activity. This weak effect is due to less inactive SCR14 SCR complexes forming as the expression of SCR decreases (Sivanantharajah, 2009).

The cold-sensitive Scr6 allele has two missense mutations in the conserved CTD of SCR, both these missense mutations result in amino acid changes of highly conserved amino acids of the CTD. The lack of an intragenic interaction between Scr6 and Scr14 suggests that SCR6 does not interact with SCR14 to form an inactive complex. The inability of SCR6 to interact with SCR14 is not due to a lack of CTD function because all proteins expressed from a nonsense allele lack the CTD but are still able to interact with SCR14. It is proposed that normally the CTD domain has a role in negative regulation of SCR activity by binding the octapeptide, and that the Scr6 missense mutations result in the expression of a protein that is hyperactive for a CTD function of binding and masking the octapeptide. This explains why SCR6 has less activity than SCR; the octapeptide is not available for complex formation. In addition, in SCR6 the octapepide is not available to interact with SCR14. The proposed intramolecular interaction of the octapeptide with the CTD in SCR6 would be temperature sensitive, rendering SCR6 activity cold sensitive (Sivanantharajah, 2009).

Many models can be proposed for the inactivity of the SCR14 SCR protein complex. But most of these models have difficulty explaining why the genetic interactions of null and hypomorphic alleles are specific to Scr14. One example is an incompatibility model where SCR14 and SCR form an inactive complex because the transcription machinery may not recognize the conformation that the heterotypic octapeptides adopt. In this model and others like it, it is assumed that SCR14 and SCR interact with the same affinity as that between two SCR or two SCR14 molecules. Therefore, complexes would form between the products of nonsense alleles and hypomorphic alleles, and because these complexes only have one HD they would be expected to be less active or inactive. This is not observed because the phenotype of a hypomorphic allele over a deletion is the same as that over a null nonsense allele (Sivanantharajah, 2009).

The favored model is a locked complex model, which to understand first requires presentation of a speculative model for SCR activity. It is conjectured that there is a dynamic equilibrium between four forms of SCR during adult sex comb and pseudotrachea formation. Three interactions of the octapeptide mediate the dynamic equilibrium: the octapepide interacting with a component(s) of the transcription machinery, the octapeptide interacting with another octapeptide motif to mediate complex formation of SCR, and the octapeptide interacting intramolecularly with the CTD. In two of the forms SCR is a monomer, and as a monomer SCR is in dynamic equilibrium with a form where the octapeptide is exposed for complex formation with another SCR molecule and a form where the octapeptide interacts with the CTD and is not available for complex formation. The two SCR protein complexes bound to DNA are in dynamic equilibrium between a complex held together by an interaction between two octapeptides, and a complex held together by an indirect interaction of the two octapeptides mediated by a component(s) of the transcriptional machinery. SCR is only active when it is interacting with the transcriptional machinery (Sivanantharajah, 2009).

In the locked complex model, the SCR14 SCR complex is inactive because the two octapeptides are unable to dissociate and interact with the transcriptional machinery and unable to dissociate to form monomers; dynamic equilibrium is lost. The locked complex model also explains why the genetic interactions are specific to Scr14. An interaction between nonsense null alleles and viable alleles is not observed, because all these alleles have a wild-type octapeptide sequence that is in dynamic equilibrium. The complexes that form between a truncated SCR protein and an SCR molecule encoded by a viable allele are transient, falling apart rapidly, and with the additional interaction between SCR protein complex and DNA, the formation of partially active complexes of the SCR proteins expressed from viable alleles is favored. This model, based on interpretation of genetic evidence, will require biochemical tests of the interaction of the octapeptide with itself and the CTD and of the formation of a locked complex between SCR and SCR14 (Sivanantharajah, 2009).

Scr3 encodes a protein in which Pro306 is changed to Leu, altering the sequence of the YPWM motif to YLWM. The effect of this change was the most severe of all the viable hypomorphic alleles on the number of pseudotracheal rows and salivary gland cells. The Scr3 allele had a weaker affect on the prothoracic leg identity. The YPWM motif is a highly conserved motif found in all HOX proteins, and is a binding site for two proteins: Extradenticle (EXD) and Bric-à-Brac Interacting Protein 2 (BIP2) (Joshi, 2007; Prince, 2008). The results are difficult to explain solely as a loss of EXD binding to SCR because although SCR and EXD are essential for salivary gland formation, EXD is not required for sex comb and pseudotrachea formation (Sivanantharajah, 2009).

The YPWM motif of SCR makes a protein- protein interaction with the hydrophobic pocket of the EXD HD (Joshi, 2007); therefore, a mutation in the YPWM motif may be expected to result in no salivary gland formation. Indeed, deleting the YPWM motif of the mammalian homolog, HOXA5, results in an inability to induce ectopic Forkhead (FKH) expression; however, this deletion of 16 amino acids includes a His residue important for minor groove interactions by SCR and EXD with the fhk enhancer element (Joshi, 2007). A potential explanation for the weak reduction of the salivary gland is that the Pro residue of the YPWM motif is not essential for binding to EXD. In fact, in the Apis mellifera SCR homolog, the YPWM motif is YSWM. Also, the YPWM motif is YKWM and HEWT in the Drosophila HOX proteins Labial and Abdominal-B, respectively. The structure of the vertebrate HOX-EXD (HoxB1, PBX1) homologous heterodimer was solved with a FDWM sequence. Therefore, the Pro306-to-Leu change may not completely inactivate the YPWM motif (Sivanantharajah, 2009).

An explanation for the observation that EXD is not required for pseudotracheae or sex comb development is that the YPWM of SCR interacts with a protein other than EXD. The YPWM motif of ANTP binds BIP2 (Prince, 2008). BIP2 is a TATA binding protein-associated factor, associated with the basal transcriptional machinery, that when coectopically expressed with ANTP promotes the formation of ectopic wing tissue in Drosophila. Since BIP2 is expressed widely throughout all of the imaginal discs of third instar larvae, there is a strong possibility that BIP2 may interact with the YPWM motifs of other HOX proteins such as SCR. If BIP2 binds to the SCR YPWM, the Pro306-to-Leu change observed in the YPWM motif of SCR3 could decrease the ability of these proteins to interact, explaining the proboscis toward maxillary palp transformation and reduction in sex comb bristle number in Scr3 mutants (Sivanantharajah, 2009).

Salivary gland formation requires both SCR and EXD for the expression of FKH, which is required for salivary gland formation. The evidence for SCR and EXD binding as a protein complex to a fkh enhancer is extensive at both functional and structural levels (Joshi, 2007). However, EXD is not required for the formation of sex comb bristles and pseudotrachea. In addition, the intragenic interaction between Scr14 and Scr alleles is observed for the formation of the sex combs and pseudotrachea, but not salivary gland nuclei. To resolve this inconsistency, it is suggested that SCR requires complex formation with itself for sex comb and pseudotracheae formation and complex formation with the HOX cofactor EXD for salivary gland formation. This phenomenon is similar to the observation that UBX does not require EXD for haltered development (Sivanantharajah, 2009).

The Glu365 residue of the SCR HD is well conserved through evolution and is found in all Drosophila HOX HDs; however, this residue does not mediate important contacts in the crystal structures of SCR, ANTP, and UBX or the NMR structure of ANTP. Glu365 is the first amino acid of the third α-helix of the HD, the helix that makes direct contacts with the major groove of DNA; therefore, the importance of this residue may lie in its position within the HD. A change from an acidic Glu residue to a bulkier, basic Lys residue may affect the structure of the third α-helix and subsequently the ability of the HD to bind DNA. Although the hypomorphic Scr1 allele may suggest that SCR has a HD independent activity like the pair rule protein Fushi tarazu, mutational studies have shown that the SCR HD is essential for SCR activity. Altering two amino acids in the N-terminal arm of the SCR HD to aspartic acids inactivated the SCR protein. Also changing Arg3 of the SCR HD resulted in an inability of the SCR protein to activate the fkh enhancer. The observation that changes in conserved amino acids of the HD result in a hypomorphic allele is not novel; three of four missense alleles with changes in highy conserved positions of the Proboscipedia HD were hypomorphic, only one was null (Sivanantharajah, 2009 and references therein).

Scr15 encodes a 35-amino-acid deletion of Thr83 through Pro117 encompassing the insect-specific DYTQL motif. Although, this change had a strong affect on all three phenotypes assessed, the DYTQL motif is not essential for SCR function, which is similar to the nonessential role of the insect-specific UBX QA motif. However, analysis of differential pleiotropy in flies with one or two copies of pb, suggests that the DYTQL motif and the CTD mediate an interaction with PB in pseudotrachea formation. It has been proposed that the CTD encoded by Scr6 is hyperactive; therefore, PB may have a role in overcoming the negative regulation of SCR activity mediated by the CTD. And since Scr15 is the loss of the DYTQL motif, it is proposed that the DYTQL motif may also have a role assisting in PB overcoming negative regulation of SCR activity possibly mediated by the CTD (Sivanantharajah, 2009).

How does Scr cause first legs to deviate from second legs?

All six legs of D. melanogaster have longitudinal rows of bristles, but only the first and third pairs have transverse rows. These 't-rows' serve as brushes for cleaning the eyes or wings during the grooming ritual, and the most distal t-row on the male foreleg basitarsus rotates during metamorphosis to form a sex comb that is used during the courtship ritual. The t-rows and sex comb pose a number of tantalizing riddles at the levels of development, genetics, behavior, and evolution (Held, 2010).

Evolution is thought to have inserted the t-rows and sex comb as modules into the more elementary midleg pattern. During development, the fore- and hindlegs are steered away from this midleg 'ground state' (toward their distinctive anatomies) by the Hox genes Sex combs reduced (Scr) and Ultrabithorax (Ubx). If either Scr or Ubx malfunctions, then the fore- or hindlegs (respectively) revert to midleg identity as a default (Held, 2010). How Ubx acts in hindlegs has been studied previously, but how Scr works in forelegs is less well understood (Held, 2010).

The advantage of using fly legs to probe Hox gene action in general is the high resolution of their rich cuticular detail. The sex comb in particular has become a popular tool for studying rapid evolution, evo-devo mechanisms, the dynamics of morphogenesis, and the genetics of dimorphisms. The universal roles of Hox genes in bilaterian phyla means that whatever is learned here may be widely applicable elsewhere (Held, 2010).

If the above evolutionary scenario is correct, then the foreleg is actually a quilt of distinct pattern territories, with the more recent (foreleg-specific) modules (t-rows and comb) under Scr control and the more ancient (midleg) background (longitudinal rows) independent of Scr. One way to tease apart these components is via the time dimension. Ever since 1970 one of the most incisive methods for temporal dissection has been the usage of temperature-sensitive mutations. In 2003, this technology was made even more powerful by a yeast mutation Gal80ts that allows any desired fly gene to be turned ON or OFF at any desired time (Held, 2010).

The sex comb (named for its presence in only one sex and its resemblance to a hair comb) is homologous to the most distal basitarsal t-row in females, but its bristles or 'teeth' are darker, thicker, blunter, more curved, and more numerous than trow bristles. Using a ts allele of the sex-transforming gene tra-2, it has been found that bristle number becomes fixed at the male (~10) or female (~7) level between ca -8 h BPF and +8 h APF at 25°C. In the present study the RNAi-mediated transformation was not from male to female, but rather from T1 to T2, so it is not surprising that the maximal effect is a reduction to zero teeth (T2 state) at -12 h BPF. Nor is it surprising that recovery to a T1 state occurs over the same span (-8 to +8 h) as the tra-2ts1 temperature sensitive period (TSP). What is surprising is that Scr-LOF affects tooth number as early as two days BPF. As the number decreases to zero, the missing teeth are usually not replaced by t-row bristles. This rule implies that in wild-type flies Scr makes teeth directly from ordinary epidermal cells, rather than first inducing t-row bristles and then modifying them into teeth. Disabling doublesex via UAS-dsx-RNAi partly transforms teeth into t-row bristles, so Scr is probably not acting via dsx. The sinusoidal shape of this curve suggests a gradual (analog) process like tissue growth (leading to more bristles) or movement (leading to sex comb rotation), as opposed to a spike, which implies a binary (digital) switch. If so, then Scr would be driving each process along its entire course, instead of just launching it. If evolution did indeed 'shoehorn' t-rows and sex combs into a midleg background, then intercalary growth (and movement) may have been required to make room for the new modules within the old pattern (Held, 2010).

Another salient difference between the sex comb and its female t-row counterpart is that the former rotates to a longitudinal orientation, whereas the latter remains transverse. This 90° rotation occurs at 16-28 h APF. Disabling Scr via RNAi inhibits rotation with a time course that parallels the effects of Scr-LOF on the number of teeth, though the maximal effect is difficult to gauge when fewer than 3 teeth are present. This parallelism is perplexing because rotation occurs a day after the number of sex comb teeth is fixed. Why should Scr be needed so much earlier than the overt process it controls? Another surprise is a 'bent-comb' anomaly seen in 40% of legs from pulses at 0-12 or 6-18 h APF (or 6-h pulses at 0-6 or 3-9 h APF) and in 10% of legs from flanking 12-h periods. In most cases the bend is midway along the comb, with the proximal and distal halves joined at a right angle. Such combs seem to be 'caught in the act,' with Scr having been already used by the distal half to license its rotation but not yet used by the proximal half, which has been prevented from doing so by the pulse. The remaining combs from the affected pulse periods tend to be curved rather than bent, and their arc is likewise concave distally. A similar arc is seen in proximal portions of wild-type combs during the normal rotation process, so the arc anomaly might represent a natural phase that has been frozen in time due to deprivation of Scr action (Held, 2010).

The t-row area is operationally defined as the area subtended by the most proximal and distal rows containing at least three adjacent bristles with osculating sockets. All bristles in the area thus defined were counted, regardless of whether they were organized in rows. The t-row area normally is rectangular on the basitarsus but triangular on the tibia. On both segments t-row bristles decline at about the same rate (midpoints -42 to -24 h) as the number of sex comb teeth, but they both recover from their minima more slowly, with the tibia recovering even more slowly than the basitarsus. Distal-to-proximal gradients of this sort (e.g., basitarsus preceding tibia) have been uncovered along the leg for bristles and bracts. When the basitarsus lacks recognizable t-rows, the last t-row on the tibia is still present, which explains why the tibial curve does not fall to zero like the basitarsal one. Both curves return to normal shortly after the bristle precursor cells undergo their differentiative mitoses at ~9-17 h APF (Held, 2010).

Bristles of the basitarsal t-rows are normally yellower than the surrounding (brown) bristles, and tibial t-rows are even lighter still. Removal of Scr function at any time from +6 to +30 h APF turns t-row bristles as brown as their neighbors, if not browner. The yellow color only returns with pulses at +36 h APF. Despite how late this stage may seem relative to TSPs discussed above, it is still two days before overt melanization, which only commences in the tarsus at +77 h APF (Held, 2010).

In addition to being yellower, the tibial t-row bristles in wild-type flies are noticeably thinner than nearby bristles. The pulse period +18 to +30 h (midpoint +24) makes t-row bristles thicker than nearby bristles. Indeed, these fatter t-row bristles vaguely resemble sex comb teeth in both shape and color. Similar effects have been reported previously for flies carrying inserts of a male-specific doublesex (dsx) gene linked to the hsp70 heat-shock promoter, regardless of whether heat shocks were administered. In wild-type forelegs dsx is expressed mainly in the sex comb area, so this partial conversion of t-row bristles into teeth implies a possible expansion of dsx activity into the t-row area (where Scr is normally expressed). Why disabling Scr (via RNAi) should enhance dsx (rather than suppress it) is unclear since (1) dsx is thought to regulate Scr rather than the other way around, and the interaction is thought to be positive rather than negative (Held, 2010).

At about the same time as t-row bristles thicken, t-row alignment is disrupted (midpoints +18 and +24). Anomalies include Y-shaped intersections of adjacent t-rows. More commonly, there are jumbled clumps of bristles. Both types of defects are also seen when the EGFR pathway is disabled, and their respective TSPs overlap. These flaws are intriguing because they offer clues to how bristle cells 'self assemble' into rows. Scr could be acting via the EGFR pathway (Held, 2010).

Tibial t-rows differ from basitarsal ones insofar as they lack bracts, except at the edges. Bracts are tiny pigmented cuticular protrusions (of no known function) associated with mechanosensory bristles on distal leg segments. They are normally absent from chemosensory bristles and tibial t-row bristles. Bracts arise from ordinary epidermal cells by induction from adjacent bristle cells via an EGFR signal emitted around +17 to +35 h APF. In the present investigation, tibial t-row bristles display bracts with pulses from -36 h BPF to +24 h APF. This TSP is remarkable for its length (2.5 days), as well as for its ~100% penetrance and expressivity. Tibial t-rows begin to recover their bractless state at +30 h APF, when bract induction is thought to occur (Held, 2010).

Midlegs have two large bristles (macrochaetes) on the distal tibia. The pre-Apical bristle also exists on forelegs, so it is not a useful marker for homeosis, but the Apical bristle is distinctively large, dark, and bractless, so it can serve that function. The precursor cell of the Apical bristle is first detectable at puparium formation and undergoes two mitoses over the next ~8 h. The TSP for T1-->T2 homeosis to an Apical bristle lasts a day (-36 to -12 h BPF) and reaches its peak about one day (27 h) before the precursor cell arises (Held, 2010).

Just proximal to the midleg's Apical bristle are ~5 peg-shaped 'spur' bristles arranged in a transverse row that differs from a foreleg t-row insofar as the sockets of its bristles are not always in contact. Its precursor cells presumably arise along with other tibial microchaetes at ~9 h APF and undergo differentiative mitoses over the next ~8 h. The peak of the TSP for T1-->T2 homeosis to spur bristles occurs at -12 h BPF, which is about a day (21 h) before the precursor cells arise. This hiatus is comparable to the analogous period for Apical bristle homeosis described above. Scr is, therefore, apparently needed to suppress midleg-specific bristle development at a fixed time interval before precursor initiation, regardless of the size or location of the bristles (Held, 2010).

Acquisition of a leucine zipper motif as a mechanism of antimorphy for an allele of the Drosophila Hox gene Sex combs reduced

In 1932, Muller first used the term 'antimorphic' to describe mutant alleles that have an effect that is antagonistic to that of the wild-type allele from which they were derived. In a previous characterization of mutant alleles of the Drosophila melanogaster Hox gene, Sex combs reduced (Scr), the missense, antimorphic allele Scr14 was identified, that is a Ser10 to Leu change in the N-terminally located, Bilateran-specific octapeptide motif. It is proposed that the cause of Scr14 antimorphy is the acquisition of a leucine zipper oligomerization motif spanning the octapeptide motif and adjacently located, Protostome-specific LASCY motif. Analysis of the primary and predicted secondary structures of the Scr N-terminus, suggests that while the SCR+ encodes a short, alpha-helical region containing one putative heptad repeat, the same region in SCR14 encodes a longer, alpha-helical region containing two putative heptad repeats. In addition, in vitro crosslinking assays demonstrated strong oligomerization of SCR14 but not SCR+. For in vivo sex comb formation, reciprocal inhibition of endogenous SCR+ and SCR14 activity was observed by ectopic expression of truncated SCR14 and SCR+ peptides, respectively. The acquisition of an oligomerization domain in SCR14 presents a novel mechanism of antimorphy relative to the dominant negative mechanism, which maintains oligomerization between the wild type and mutant protein subunits (Sivanantharajah, 2014).

Common origin of insect trachea and endocrine organs from a segmentally repeated precursor

Segmented organisms have serially repeated structures that become specialized in some segments. The Drosophila corpora allata, prothoracic glands, and trachea are shown to have a homologous origin and can convert into each other. The tracheal epithelial tubes develop from ten trunk placodes, and homologous ectodermal cells in the maxilla and labium form the corpora allata and the prothoracic glands. The early endocrine and trachea gene networks are similar, with STAT and Hox genes inducing their activation. The initial invagination of the trachea and the endocrine primordia is identical, but activation of Snail in the glands induces an epithelial-mesenchymal transition (EMT), after which the corpora allata and prothoracic gland primordia coalesce and migrate dorsally, joining the corpora cardiaca to form the ring gland. It is proposed that the arthropod ectodermal endocrine glands and respiratory organs arose through an extreme process of divergent evolution from a metameric repeated structure (Sanchez-Higueras, 2013).

The endocrine control of molting and metamorphosis in insects is regulated by the corpora allata (ca) and the prothoracic glands (pg), which secrete juvenile hormone and ecdysone, respectively. In Diptera, these glands and the corpora cardiaca (cc) fuse during development to form a tripartite endocrine organ called the ring gland. While the corpora cardiaca is known to originate from the migration of anterior mesodermal cells, the origin of the other two ring gland components is unclear (Sanchez-Higueras, 2013).

The tracheae have a completely different structure consisting of a tubular network of polarized cells. The tracheae are specified in the second thoracic to the eighth abdominal segments (T2-A8) by the activation of trachealess (trh) and ventral veinless (vvl) (Sanchez-Higueras, 2013).

The enhancers controlling trh and vvl in the tracheal primordia have been isolated and shown to be activated by JAK/ STAT signaling. While the trh enhancers are restricted to the tracheal primordia in the T2-A8 segments, the vvl1+2 enhancer is also expressed in cells at homologous positions in the maxilla (Mx), labium (Lb), T1, and A9 segments in a pattern reproducing the early transcription of vvl. The fate of these nontracheal vvl-expressing cells was unknown, but it was shown that ectopic trh expression transforms these cells into tracheae. To identify their fate, vvl1+2-EGFP and mCherry constructs were made (Sanchez-Higueras, 2013).

Although the vvl1+2 enhancer drives expression transiently, the stability of the EGFP and mCherry proteins labels these cells during development. It was observed that while the T1 and A9 patches remained in the surface and integrated with the embryonic epidermis, the patches in the Mx and Lb invaginated just as the tracheal primordia did. Next, the Mx and Lb patches fused, and a group of them underwent an epithelial-mesenchymal transition (EMT) initiating a dorsal migration toward the anterior of the aorta, where they integrate into the ring gland. To find out what controls the EMT, the expression of the snail (sna) gene, a key EMT regulator, was studied. Besides its expression in the mesoderm primordium, it was found that sna is also transcribed in two patches of cells that become the migrating primordium. Using sna bacterial artificial chromosomes (BACs) with different cis-regulatory regions, the enhancer activating sna in the ring gland primordium (sna-rg). A sna-rg-GFP construct labels the subset of Mx and Lb vvl1+2-expressing cells that experience EMT and migrate to form the ring gland. Staining with seven-up (svp) and spalt (sal) (also known as salm) markers, which label the ca and the pg, respectively, showed that the sna-rg-GFP cells form these two endocrine glands. The sna-rg-GFP-expressing cells in the Mx activated svp, and those in the Lb activated sal before they coalesced, indicating that the ca and pg are specified in different segments before they migrate (Sanchez-Higueras, 2013).

To test whether Hox genes, the major regulators of anteroposterior segment differentiation, participate in gland morphogenesis, vvl1+2-GFP embryos were stained, and it was found that the Mx vvl1+2 primordium expressed Deformed (Dfd) and the Lb primordium Sex combs reduced (Scr), while the T1 primordium expressed very low levels of Scr. Dfd mutant embryos lacked the ca, while Scr mutant embryos lacked the pg. Dfd and Scr expression in the gland primordia was transient, suggesting that they control their specification. Consistently, in Dfd, Scr double-mutant embryos, vvl1+2 was not activated in the Mx and Lb patches, and the same was true for vvl transcription. In these mutants, the sna-rg-GFP expression was almost absent, and the ca and pg did not form. In each case, Dfd controlled the expression of the Mx patch and Scr of the Lb patch (Sanchez-Higueras, 2013).

The capacity of different Hox genes to rescue the ring gland defects of Scr, Dfd double mutants was tested. Induction of Dfd with the sal-Gal4 line in these mutants restored the expression of vvl1+2 and sna-rg-GFP in the Mx and the Lb. However, in contrast to the wild-type, both segments formed a ca as all cells express Svp. Similarly, induction of Scr also restored the vvl1+2 and sna-rg-GFP expression, but both primordia formed a pg as they activate Sal and Phantom, an enzyme required for ecdysone synthesis. The capacity of both Dfd and Scr to restore vvl expression, regardless of the segment, led to a test of whether other Hox proteins could have the same function. Induction of Antennapaedia (Antp), Ultrabithorax (Ubx), abdominal-A (abd-A), or Abdominal-B (Abd-B) restored vvl1+2 expression in the Mx and Lb, but these cells formed tubes instead of migratory gland primordia. These cephalic tubes are trachea, as they do not activate sna-rg, they express Trh, and their nuclei accumulate Tango (Tgo), a maternal protein that is only translocated to the nucleus in salivary glands and tracheal cells, indicating that the trunk Hox proteins can restore vvl expression in the Mx and Lb but induce their transformation to trachea (Sanchez-Higueras, 2013).

To investigate whether vvl and trh expression is normally under Hox control in the trunk, focus was placed on Antp, which is expressed at high levels in the tracheal pits. In double-mutant Dfd, Antp embryos, vvl1+2 was maintained in the Lb where Scr was present, while the Mx, T1, and T2 patches were missing. In T3-A8, vvl1+2 expression, although reduced, was present, probably due to the expression of Ubx, Abd-A, and Abd-B in the posterior thorax and abdomen. Thus, Antp regulates vvl expression in the tracheal T2 primordium. Surprisingly, in Dfd, Antp double mutants, Trh and Tgo were maintained in the T2 tracheal pit, indicating that although Hox genes can activate ectopic trh expression, in the tracheal primordia they may be acting redundantly with some other unidentified factor, explaining why the capacity of Hox proteins to specify trachea had not been reported previously (Sanchez-Higueras, 2013).

sna null mutants were studied to determine sna's requirement for ring gland development, but their aberrant gastrulation precluded analyzing specific ring gland defects. To investigate sna function in the gland primordia, the sna mutants were rescued with the sna-squish BAC, which drives normal Sna expression except in the ring gland. These embryos have a normal gastrulation and activate the sna-rg- GFP; however, the gland primordia degenerate and disappear. To block apoptosis, these embryos were made homozygous for the H99 deficiency, which removes three apoptotic inducers. In this situation, the ca and pg primordia invaginated and survived, but they did not undergo EMT. As a result, the gland primordia maintain epithelial polarity, do not migrate, and form small pouches that remain attached to the epidermis. Vvl is required for tracheal migration. In vvl mutant embryos, sna-rg-GFP expression was activated, but the cells degenerated. In vvl mutant embryos also mutant for H99, the primordia underwent EMT and migrated up to the primordia coalescence; however, the later dorsal migration did not progress (Sanchez-Higueras, 2013).

This study has shown that the ca and pg develop from vvl-expressing cephalic cells at positions where other segments form trachea, suggesting that they could be part of a segmentally repeated structure that is modified in each segment by the activity of different Hox proteins. As the cephalic primordia are transformed into trachea by ectopic expression of trunk Hox, tests were performed to see whether the trachea primordia could form gland cells. Ectopic expression of Dfd with arm- Gal4 resulted in the activation of sna-rg-GFP on the ventral side of the tracheal pits. These sna-rg-GFP0-expressing cells also expressed vvl1+2 and Trh and had nuclear Tgo, showing that they conserve tracheal characteristics. These sna-rg-GFP-positive cells did not show EMT and remained associated to the ventral anterior tracheal branch. The strength of ectopic sna-rg-GFP expression increased when ectopic Dfd was induced in trh mutant embryos. However, migratory behaviors in the sna-rg-GFP cells were only observed if Dfd was coexpressed with Sal. Thus, sal is expressed several times in the gland primordia, first at st9-10 repressing trunk Hox expression in the cephalic segments and second from st11 in the prothoracic gland. It is uncertain whether the sal requirement for migration is linked to the first function or whether it represents an additional role (Sanchez-Higueras, 2013).

These results show that the endocrine ectodermal glands and the respiratory trachea develop as serially homologous organs in Drosophila. The identical regulation of vvl in the primordia of trachea and gland by the combined action of the JAK/STAT pathway and Hox proteins could represent the vestiges of an ancestral regulatory network retained to specify these serially repeated structures, while the activation of Sna for gland development and Trh and Tgo for trachea formation could represent network modifications recruited later by specific Hox proteins during the functional specialization of each primordium. This hypothesis or alternative possibilities should be confirmed by analyzing the expression of these gene networks in various arthropod species. The diversification of glands and respiratory organs must have occurred before the split of insects and crustaceans, as there is a correspondence between the endocrine glands in both classes, with the corpora cardiaca corresponding to the pericardial organ, the corpora allata to the mandibular organ, and the prothoracic gland to the Y gland. Despite their divergent morphology, a correspondence between the insect trachea and the crustacean gills can also be made, as both respiratory organs coexpress vvl and trh during their organogenesis. Divergence between endocrine glands and respiratory organs may have occurred when the evolution of the arthropod exoskeleton required solving two simultaneous problems: the need to molt to allow growth, and the need for specialized organs for gas exchange (Sanchez-Higueras, 2013).

Sex combs reduced: Biological Overview | Evolutionary Homologs | Regulation | Targets of Activity, Homeotic Effects, Post-Transcriptional Regulation and Protein Interactions | Developmental Biology | References

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