Sex combs reduced

DEVELOPMENTAL BIOLOGY

Embryonic

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).

Larval

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).

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 (Scr^ScxP and hsp70-pb [HSPB] mutations). This effect of the Scr^ScxP 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 Scr^ScxP 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 kis^S 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).

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