Interactive Fly, Drosophila



Targets of Activity

The analysis of the expression of Scr in Antp mutant embryos reveals a case of tissue-specific regulation of Scr expression by Antp. In the epidermis, Antp has been shown to negatively regulate Scr, but it positively regulates Scr in the visceral mesoderm (Reuter, 1990).

Scr, Antp, Ubx and Abd-B repress Dfd both transcriptionally and at the phenotypic level, if their products are present in sufficient amounts (Gonzalez-Reyes, 1992).

teashirt is necessary for proper formation of anterior and central midgut structures. Antp activates tsh in anterior midgut mesoderm. In the central midgut mesoderm Ubx, abd-A, dpp, and wg are required for proper tsh expression. The control of tsh by Ubx and abd-A, and probably also by Antp, is mediated by secreted signaling molecules. By responding to signals as well as localized transcription regulators, the TSH transcription factor is produced in a spatial pattern distinct from any of the homeotic genes (Mathies, 1994).

Antennapedia activates teashirt mesodermal transcription at five sites within an upstream enhancer, 5 kb from transcriptional start. Ultrabithorax activates the teashirt enhancer in both epidermis and somatic mesoderm (McCormick, 1995). There are an additional three sites for Antennapedia binding at the proximal promoter and in the first intron of tsh. Far downstream of tsh, there is a enhancer that is active in epidermis and mesoderm. The downstream enhancer is regulated by Ultrabithorax, Abdominal-A and Abdominal-B (Mathies, 1994).

odd paired is positively regulated by Antennapedia and Abdominal-A at the location of the first and third midgut constructions respectively. Between these domains opa is negatively regulated by Ultrabithorax and Decapentaplegic (Cimbara, 1995).

Diversification of Drosophila segmental morphologies requires the function of Hox transcription factors. However, little information is available that describes pathways through which Hox activities effect the discrete cellular changes that diversify segmental architecture. Serrate is a Hox gene target. Serrate acts in many segments as a component of such pathways. In the embryonic epidermis, Serrate is required for morphogenesis of normal abdominal denticle belts and maxillary mouth hooks, both Hox-dependent structures. The Hox genes Ultrabithorax and abdominal-A are required to activate an early stripe of Serrate transcription in abdominal segments. In the abdominal epidermis, Serrate promotes denticle diversity by precisely localizing a single cell stripe of rhomboid expression, which generates a source of EGF signal that is not produced in thoracic epidermis. In the head, Deformed is required to activate Serrate transcription in the maxillary segment, a region where Serrate is required for normal mouth hook morphogenesis. However, Serrate does not require rhomboid function in the maxillary segment, suggesting that the Hox-Serrate pathway to segment-specific morphogenesis can be linked to more than one downstream function (Wiellette, 1999a).

Ser transcripts in the trunk are first detected at the extended germband stage in ventral patches in the middle of abdominal segments A2-A8 and in offset lateral patches. The ventral regions of thoracic segments do not exhibit Ser expression at this stage. As the germband retracts, the abdominal stripes intensify and develop sharp anterior borders. The first abdominal segment (A1) is unique: Ser expression begins later than in the other abdominal segments and forms a narrower stripe after germband retraction. After germband retraction, Ser transcripts can also be detected in the ventral regions of thoracic segments in broad, faint patches. Embryos mutant in all genes of the Bithorax Complex (BX-C), Ubx, abd-A and Abdominal-B (Abd-B), develop thoracic-type denticles throughout the trunk region. Consistent with this transformation, stage 11 and 12 BX-C mutant embryos have no Ser expression in ventral regions. Ventral Ser expression does begin in BX-C mutants after germband retraction, but the location and level of expression matches that of the thoracic segments. As expected, Ubx mutant embryos show a transformation of abdominal- to thoracic-type Ser expression only in A1; abd-A mutants show Ser transcript stripes in A2-A8 that are similar to the wild-type A1 pattern, and Abd-B mutants display no change in A1-A8 ventral Ser transcription. Thus Ubx function is sufficient to activate some Ser expression in the center of each segment, but abd-A function is required for the earlier, broader pattern of Ser transcription in A2-A8, a transcript pattern that correlates with complete diversification of denticle belts. Embryos lacking all trunk Hox functions express Ser at the margins of the anterior part of each trunk segment and at lower levels in the center of this region, a pattern almost the inverse of that seen in wild type. Transcription of Ser in the posterior-most region of each segment, probably corresponding to the posterior compartment, is completely suppressed. The delimitation of Ser expression to reiterated subsegmental stripes in the embryonic metameres suggests that segment polarity genes also regulate the Ser transcript pattern. ptc mutants lack ventral abdominal Ser transcripts, correlating with the loss of denticle diversity and number in ptc denticle belts. Ser transcription in wingless (wg) mutants appears in broad stripes, while hedgehog (hh) and engrailed (en) mutant embryos exhibit Ser transcription throughout almost the entire ventral epidermis of the abdominal segments. Broadened patterns of Ser transcription in these segment polarity mutants correspond to expanded fields of denticles that lack significant diversity of denticle type (Wiellette, 1999a).

A model is presented for the roles of Ser, rho and Hox genes in the generation of denticle belt patterns in the thorax and abdomen. Three Hox genes (Antp, Ubx, and abdA) serve to establish the segmentally specific levels of Ser expression in the third abdominal segment and in the first two thoracic segments respectively. Ser is activated at stage 11 in abdominal parasegments by Ubx and abd-A functions but not in thoracic parasegments where Antp is the principal Hox function. Ubx function is required for the A1-type abdominal expression pattern of Ser, which is narrower and fainter than the pattern in other abdominal segments. This pattern correlates with a narrower, less complex denticle pattern in A1 than in more posterior segments. abd-A function is required for the wider, more abundant Ser stripes in A2-A8. Expression of Ser in the embryonic epidermis results in context-dependent responses, including rho expression, denticle belt patterning and normal development of the mouth hooks. These embryonic roles of Ser are apparently different from its roles in wing margin determination and wing outgrowth. One similarity is the short range over which Ser function is exerted, either at the anterior border of its ventral A2-A8 expression pattern, or at the dorsal/ventral margin of its expression boundary in the wing pouch. The spitz-group gene rho can potentiate Egfr activation via the Spitz (Spi) ligand. Egfr activation is required from late stage 11 to early stage 13 for patterning of the denticle belts, and rho, unlike spi and Egfr, has a spatially and temporally regulated expression pattern. Abdomen-specific rho expression is required for patterning of abdominal denticle rows 1 through 4, probably by allowing secretion of Spitz protein from denticle row 2 and 3 cells, which activates Egfr in neighboring cells. Ser function is required for activation of the abdomen-specific posterior row of rho transcription, expression of which is also dependent on Ubx/abd-A. The evidence presented in this paper suggests that Ser provides a critical intermediate that translates broad Hox and segment polarity domains into narrow stripes of rho expression, which then specify diversification at the single cell level. Ser and rho mutants each show only a single row of denticles between rows 2 and 5 of A2-A8, indicating that Ser and rho are both required for normal development of rows 3 and 4. rho,Ser double mutants develop row 5-like denticle identities throughout the denticle belt. Thus, either gene alone provides some A/P denticle diversity, while the double mutant lacks any diversity. If the only role of Ser were regulation of rho in denticle row 3 cells, then rho,Ser mutants should develop the same phenotype as rho mutants. Since this is not observed, it is concluded that Ser has identity functions independent of rho regulation. Ser function is required in the cells immediately to its anterior expression boundary and within the most anterior row of Ser-expressing cells; the effect within its own domain of expression may be a result of signaling from cells within the same row, or from those to the posterior (Wiellette, 1999a and references).

An immunopurification method has been used to clone target genes of the Antennapedia protein (ANTP). Centrosomin (cnn) is expressed in the developing visceral mesoderm (VM) of the midgut and the central nervous system (CNS). In the VM, Antp and abdominal-A negatively regulate cnn, while Ultrabithorax shows positive regulation. In the CNS, cnn is regulated positively by Antp and negatively by Ubx and abd-A. Evidence suggests that the expression of the cnn gene in the VM correlates with the morphogenetic function of Ubx in that tissue, i.e., the formation of the second midgut construction (Heuer, 1995).

Connectin is a cell-surface molecule containing leucine-rich repeats. Connectin can mediate cell-cell adhesion, suggesting a direct link between homeotic gene function and processes of cell-cell recognition. A 4 kb restriction enzyme fragment of Connectin, encompassing the 100 base pair clone of the Connectin promoter immunopurified with anti Ultrabithorax antibody, gives a consistent pattern of expression corresponding to a subset of the total Connectin expression pattern. High levels of expression are detected in a small group of cells in the gnathal and thoracic segments and in a posterior segment. This fragment does not produce CNS expression. The reduced levels of expression of the 4 kb fragment suggest a down regulation by Ubx and the abdominal homeotic genes. In Ubx mutants, expression is derepressed in the abdominal segments A1 and A2. Derepression is more dramatic in a Ubx/abd-A double mutant indicating that both genes repress the 4 kb construct. Antp is required for the high levels of expression found in T2 and T3 of wild type embryos (Gould, 1992).

Notch signaling and the determination of appendage identity

The Notch signaling pathway defines an evolutionarily conserved cell-cell interaction mechanism that throughout development controls the ability of precursor cells to respond to developmental signals. Notch signaling regulates the expression of the master control genes eyeless, vestigial, and Distal-less, which in combination with homeotic genes induce the formation of eyes, wings, antennae, and legs. Therefore, Notch is involved in a common regulatory pathway for the determination of the various Drosophila appendages (Kurata, 2000).

Because Distal-less in combination with extradenticle (exd) and homothorax (hth) specifies the antennae, Dll expression was monitored in the eye discs that are capable of forming ectopic antennae. In wild-type larvae, Dll protein is expressed in the antennal but not in the eye disc. In all of the tested discs in ey-GAL4 UAS-Nactey2 animals that form ectopic antennae from the eye disc, significant Dll expression was detected ectopically. This indicates that Notch signaling directly or indirectly induces ectopic expression of Dll in the eye-antennal disc, leading to the ectopic induction of antennae (Kurata, 2000).

The observation that Nact can induce both ectopic eyes and, in a specific genetic background, antennae, led to a consideration of the possibility that Notch signaling also might induce the formation of other appendages in a different genetic context. To test this hypothesis, the activation of Notch signaling was combined with ectopic expression of Antennapedia (Antp). The latter is known to determine the identity of the second thoracic segment (T2), which on the dorsal side gives rise to a pair of wings and on the ventral side to a pair of second legs. For this purpose, transgenic flies of the constitution ey-GAL4 UAS-Nact UAS-Antp were generated. About 26 of the flies escaping pupal lethality were found to have ectopic wings on the head. Almost all ectopic wing structures consisted of dorsal and ventral wing blades bordered by bristles of the wing margin, but lacking wing veins. In contrast, in wing structures induced by the ectopic expression of vg, the wing margin is not formed, suggesting that Notch signaling and Antp are acting upstream of vg. Furthermore, about 17% of these flies show ectopic leg structures induced by secondary transformation of the ectopic antennal tissue into leg structures (e.g., arista into tarsus). Therefore, activation of Notch signaling when combined with the ectopic expression of Antp driven by ey-GAL4 is capable of inducing wing and leg structures on the head (Kurata, 2000).

In wild-type larvae, the vg gene is expressed in the wing but not in the eye disc. By contrast, in ey-GAL4 UAS-Nact UAS-Antp animals in which ectopic wing structures are induced in the eye disc all of the tested eye discs show significant ectopic expression of Vg protein. It therefore appears that activation of Notch signaling in the context of Antp expression induces vg expression in the eye discs and that there are synergistic effects between Notch signaling and Antp expression. Notch signaling pathway has been shown to be used to specifically activate the boundary enhancer of the vg gene necessary for dorso-ventral wing formation. The same enhancer also may be used for ectopic formation of the wing, a point that has to be investigated further. A dorso-ventral boundary also is established by Notch in the eye disc that controls growth and polarity in the Drosophila eye. In ey-GAL4 UAS-Nact UAS-Antp ectopic legs also are induced on the head; this is accompanied by Dll expression (Kurata, 2000).

In view of the above observations it is proposed that the effects of Notch signaling on the various appendages depend on the context provided by control genes such as ey and Antp. In the eye primordia, Notch signaling induces ey expression, which induces a cascade of downstream genes leading to eye morphogenesis. In conjunction with Antp, Notch signaling induces vg, leading to wing formation. At low levels of ey expression, Notch signaling induces Dll, leading to antenna morphogenesis. In the case of the leg, Notch also induces Dll expression that, in conjunction with Antp, leads to leg formation (Kurata, 2000).

Direct regulation of the muscle-identity gene apterous by a Hox protein in the somatic mesoderm

Hox genes control segment identity in the mesoderm as well as in other tissues. Most evidence indicates that Hox genes act cell-autonomously in muscle development, although this remains a controversial issue. apterous expression in the somatic mesoderm is under direct Hox control. A small enhancer element of apterous (apME680) has been identified that regulates reporter gene expression in the LT1-4 muscle progenitors. The product of the Hox gene Antennapedia is present in the somatic mesoderm of the second and third thoracic segments. Through complementary alterations in the Antennapedia protein and in its binding sites on apME680, it has been shown that Antennapedia positively regulates apterous in a direct manner, demonstrating unambiguously its cell-autonomous role in muscle development. LT1-4 muscles contain more nuclei in the thorax than in the abdomen and it is proposed that one of the segmental differences under Hox control is the number of myoblasts allocated to the formation of specific muscles in different segments (Capovilla, 2001).

A fragment of 680 bp, located in the second largest ap intron, is capable of directing lacZ reporter expression starting from stage 10 in clusters of cells very similar to those expressing ap at this stage. This fragment is called apME680 (for ap-muscle-enhancer-680) because it directs muscle-specific reporter gene expression. At stage 13, beta-galactosidase is detected in one continuous cluster in T2 and T3, while two smaller clusters, located at the dorsal and ventral limits of the thoracic clusters, are detected in segments A1-A7. In segment A8, a unique smaller cluster is detected. These beta-galactosidase-positive cells contribute to the formation of muscles LT1-4 in segments T2-A7 and to muscle LT1 in A8. These are a subset of the muscles originating from ap-expressing cells, since ap is expressed also in the progenitors of muscles VA2 and VA3. Thoracic muscles LT1-4 are differ slightly from the same abdominal muscles. In particular, muscle LT4 extends more dorsally and ventrally in the thorax than in the abdomen (Capovilla, 2001).

The question of the significance of the homeotic regulation of ap by Antp was addressed. The perdurance of beta-galactosidase allows the labeling of thoracic and abdominal LT1-4 mature muscles originating from the cells expressing ap starting from the early germ band extended stage. LT1-4 muscles present different characteristics in the thorax and in the abdomen. In the thorax, they contain more beta-galactosidase, they are more tightly packed and, at least in the case of muscle LT4, extend more dorsally and ventrally. These differences may be a consequence of more myoblasts contributing to the thoracic muscles than to the corresponding abdominal muscles. To investigate this hypothesis, double labeling experiments were performed using anti-beta-galactosidase to label muscles LT1- 4 and anti-MEF2 antibodies, which label all muscle nuclei. In wild-type embryos, LT1-4 thoracic muscles do contain more MEF2-positive nuclei than the same abdominal muscles. The number of nuclei was compared in the T2, T3 and A1 hemisegments of ten independent embryos. This quantitative analysis shows that, on average, T3 muscles contain a total of 28 nuclei, while A1 muscles contain 19 nuclei. This difference is statistically significant. No significant differences were observed between the number of nuclei in T2 and T3. Consistently, highly packed nuclei are present in the medial portion of T2 and T3 muscles, but are absent in the same region of abdominal muscles (Capovilla, 2001).

Homeotic Complex (Hox) gene regulation and homeosis in the mesoderm of the Drosophila melanogaster embryo: the roles of signal transduction and cell autonomous regulation

Homeosis and Homeotic Complex (Hox) regulatory hierarchies have been evaluated in the somatic and visceral mesoderm. Both Hox control of signal transduction and cell autonomous regulation are critical for establishing normal Hox expression patterns and the specification of segmental identity and morphology. Novel regulatory interactions have been identified associated with the segmental register shift in Hox expression domains between the epidermis/somatic mesoderm and visceral mesoderm. A proposed mechanism for the gap between the expression domains of Sex combs reduced (Scr) and Antennapedia (Antp) in the visceral mesoderm is provided. Previously, Hox gene interactions have been shown to occur on multiple levels: direct cross-regulation, competition for binding sites at downstream targets and through indirect feedback involving signal transduction. Extrinsic specification of cell fate by signaling can be overridden by Hox protein expression in mesodermal cells and the term autonomic dominance is proposed for this phenomenon. The endoderm was used to monitor target gene regulation by the Hox proteins (specifically wg, dpp and lab) through signal transduction (Miller, 2001).

There are two distinct processes involved in the development of the body wall musculature: founder cell pattern specification and myoblast recruitment. Muscle pattern specification by founder cells is dictated by genes such as nau and S59, while myoblast recruitment depends on genes such as myoblast city (mbc). The Hox genes Antp, Ubx and abd-A have been shown to specify a subset of the embryonic muscle patterns. The data suggest that nearly all the Hox proteins are capable of specifying/altering aspects of the ventral body wall musculature since ectopic mesodermal expression of the encoded proteins produces homeosis in this tissue (Miller, 2001).

The anterior ventral projection (AV) and posterior ventral projection (PV) pattern changes observed here likely reflect alterations in founder cell specification. However, some pattern transformations could also be indicative of alterations in apodeme attachments. The ventral muscles of the T2 segment are responsive to all of the Hox encoded proteins except Lab, while the metathorax (T3) shows less susceptibility and the A1 segment is only occasionally transformed. Otherwise, somatic mesoderm (sm) segmental identities in the abdomen are governed by the Hox regulatory hierarchies of posterior dominance and phenotypic suppression (i.e. posterior prevalence). The specification of the ventral T2 muscle pattern by Antp is influenced inductively from the adjacent epidermal layer due to an apparent absence of any Hox gene expression in this mesodermal tissue. In fact, Hox gene expression in the entire thorax is patchy and modulated such that only cell clusters exhibit Hox protein accumulation. Antp and Scr accumulation were examined in the T2 sm; there is little or no Hox expression in the ventral sm. This situation provides an opportunity to examine the hierarchial relationship between cell autonomous and inductive specification of segmental identity by the Hox genes. Since the ventral T2 sm is affected by nearly all the Hox genes it would appear that cell autonomous determination of segmental identity by these genes is dominant to inductive specification in the mesothorax (T2). This 'autonomic dominance' is distinct from other Hox regulatory hierarchies, such as posterior dominance and phenotypic suppression, since it establishes a hierarchial relationship between signal transduction and direct (cell autonomous) Hox gene specification of segmental identity. Moreover, since all of the Hox genes are normally expressed anterior to T2 (with the exception of lab) are capable of transforming the inductively specified ventral T2 sm, it would appear that the model of posterior prevalence does not fit this tissue in this segment (Miller, 2001).

The co-linear relationship to homeotic penetrance in the thoracic sm by ectopic Hox expression may be related to their relative homologies. For example, the frequency of transformation by these ectopic Hox proteins is representative of their chromosomal organization; namely (Lab<Pb<Dfd<Scr<Antp<Ubx). This ordering is similar to their respective homologies; this ordering is also a reflection of their dependence on Exd as a co-factor. Interestingly, this same order of penetrance is represented in reverse, when these Hox proteins are used to rescue lab mutant phenotypes in the embryonic CNS (i.e. Lab>Pb>Scr>Antp>Ubx>abdA>Abd-B). In summary, segmental identity was followed in ectopic Hox protein expression experiments by monitoring ventral muscle development. It was found that nearly all the homeotic proteins are capable of transforming the T2 segment in a pattern that is not predicted by previously described hierarchies such as posterior dominance and phenotypic suppression. Since the T2 segment musculature is at least in part inductively regulated by Hox gene expression in ectodermal tissue, 'autonomic dominance' is proposed as an additional component of the Hox regulatory hierarchy to explain this phenomena; namely, the ability of Hox encoded proteins to cell autonomously override an exogenous signal. A better understanding of signal transduction between germ layers is needed in order to determine the mechanism of autonomic dominance (Miller, 2001).

During an investigation of Hox cross-regulation in the midgut visceral mesoderm it was demonstrated that both Antp and Ubx are responsible for the proper maintenance of the posterior boundary of Scr expression in ps4. It is proposed that Ubx represses Scr at this position extrinsically from nearby tissues. The segmental register shift in Hox expression domains found between the epidermis/somatic mesoderm/CNS and visceral mesoderm juxtaposes Ubx expression (ps5) to a position where it can influence Scr expression in the visceral mesoderm (ps4). Since Ubx activates dpp, which represses Scr in the visceral mesoderm, it seems reasonable to conclude that the interaction seen involves the action of dpp. Hox cross-regulation studies demonstrate that ectodermal Gal4 drivers producing ectopic Ubx repress Scr in the visceral mesoderm while stimulating dpp-LacZ expression. Ubx expression in the somatic mesoderm, which is between the epidermis and visceral mesoderm, may be the tissue that actually contributes the signaling influence demonstrated in this interaction. However, the responder only contains the visceral mesoderm regulatory elements and does not demonstrate that dpp gene activation is the signaling source in these outer tissues. Interestingly, ectopic expression of Abd-A outside the visceral mesoderm also demonstrates a posterior expansion of Scr expression in the visceral mesoderm, presumably since it represses Ubx there. Similarly, Antp repression of Scr in ps5 of the visceral mesoderm appears to be through signaling. Scr and Antp expression does not entirely fill the gap when Ubx expression is removed. Additionally, in Antp null mutants, Scr accumulation is seen in cells that normally express Antp in the presence of normal Ubx expression. By counting Scr expressing cells in the visceral mesoderm, it was found that Antp represses Scr in this tissue, contrary to previous reports. Interestingly, ectopic Antp in ectodermal tissues has no effect on ectodermal Scr expression. Thus, both Ubx and Antp contribute to define the Scr domain at its posterior visceral mesoderm boundary apparently through signal transduction (Miller, 2001).

Ectopic Hox protein expression in the mesoderm can induce lab, lab-LacZ and dpp-LacZ expression in the midgut. Typically, the anterior ectopic endodermal lab expression parallels the observed expression pattern in the visceral mesoderm. The lack of ectopic lab expression posterior to ps7 is probably due to the unaltered high levels of wg expression, that repress lab. Normal lab induction in the endoderm requires wg, dpp and vein; however, dFos dependent (wg independent) lab transcription can be accomplished with high Dpp levels. Typically, lab induction by dpp, wg and vein is coordinated by sgg (GSK3) which may be responsible for the ps4 gap in lab, lab-LacZ, and expression patterns seen in experiments involving ectopic Antp visceral mesoderm expression. Moreover, the lack of expanded lab-LacZ expression (unlike native lab) by ectopic Antp indicates the existence of presently undefined cis-regulatory elements at the lab locus that are not contained in genomic fragments of the identified enhancers. Antp protein may be regulating other influential signaling pathways while the corresponding cis-acting elements are not located in the genomic lab enhancers tested. Antp expression is functionally linked to another TGF-beta agonist 60A (glass bottom boat), as well as the Wnt pathway agonist DWnt4 (Miller, 2001).

Signal transduction pathway cross-talk is probably involved in the process by which the Hox genes dictate segmental identity. For example, there is a difference in the response of dpp enhancers to different modes of ectopic Antp expression. One form of ectopic Antp expression regulates visceral mesoderm dpp-LacZ and subsequently, endodermal lab expression. Conversely, heat shock driven Antp expression has no effect on dpp expression. The stress response (heat shock) has been linked to signal transduction by pathway cross-talk from genes such as dorsal (NFkB) and cactus (IkB) as well as through kinases such as c-Jun N-terminal kinases, p38 mitogen-activated protein kinase, protein kinase B and casein kinase 2. The discrepancy between the two forms of ectopic Antp is perhaps due to pathway cross-talk. Despite the fact that no effect has been found on dpp regulation by prolonged or transient heat shock expression of Antp protein, the possibility that the accumulation of Antp by Gal4 activation produces significantly higher levels cannot be ruled out (Miller, 2001).

It is concluded that Hox gene interactions in the mesoderm are not always consistent with previous governing hierarchies: posterior dominance and phenotypic suppression. In the visceral mesoderm it is found that posterior dominance (Hox direct cross-regulation) seems legitimate but may be mediated by signal transduction. Phenotypic suppression is violated by morphological changes and target gene regulation. In the somatic mesoderm, more anterior Hox genes alter the identity of the ventral T2 segment, but this tissue is largely extrinsically regulated in the absence of direct Hox expression. In light of this result, the notion of autonomic dominance is proposed: Hox genes cell-autonomously dominate tissues regulated by signal transduction (Miller, 2001).

The predominant paradigm depends on whether cells are extrinsically or autonomously specified by Hox gene expression. It is argued that non-typical homeosis caused by ectopically expressed Hox proteins (i.e. not following the dictates of posterior prevalence) can be taken to indicate inductively specified tissues and hence, confer autonomic dominance. Interestingly, ectopic expression of the Hox proteins also exhibit non-typical homeosis in the chordotonal organs of the PNS and the thoracic cuticle, suggesting that inductive specification and autonomic dominance may not be restricted to the mesoderm. However, Hox regulatory hierarchies seem to be of limited value in other tissues as well. The mechanism responsible for autonomic dominance has not been determined in this study; only the correlation between autonomous Hox dominance over inductively specified tissue. Signal transduction pathway cross-talk could be the predominant cause of autonomic dominance phenotypes (homeosis) due to Hox regulation of signaling agonists. These agonists could then contribute to the signaling environment to alter the tissue, since these morphogens are potent factors in differentiation. Meanwhile, Hox genes cross-regulate each other cell autonomously and in nearby tissues through signal transduction. This occurs in a tissue specific manner that likely depends on both the signaling environment, transcriptional co-factors, and perhaps any of an estimated 100 target genes for a given Hox protein. The signaling environment of any given tissue is dictated primarily by Hox genes, which is critical for maintenance of Hox expression domains and subsequent differentiation, determination and morphogenesis. This complex set of intrinsic and extrinsic Hox controls are likely responsible for the means by which Hox genes were genetically identified for their abilities to dominate segmental identities as homeotic selector genes (Miller, 2001).

Multi-step control of muscle diversity by Hox proteins in the Drosophila embryo

Hox transcription factors control many aspects of animal morphogenetic diversity. The segmental pattern of Drosophila larval muscles shows stereotyped variations along the anteroposterior body axis. Each muscle is seeded by a founder cell and the properties specific to each muscle reflect the expression by each founder cell of a specific combination of 'identity' transcription factors. Founder cells originate from asymmetric division of progenitor cells specified at fixed positions. Using the dorsal DA3 muscle lineage as a paradigm, this study shows that Hox proteins play a decisive role in establishing the pattern of Drosophila muscles by controlling the expression of identity transcription factors, such as Nautilus and Collier (Col), at the progenitor stage. High-resolution analysis, using newly designed intron-containing reporter genes to detect primary transcripts, shows that the progenitor stage is the key step at which segment-specific information carried by Hox proteins is superimposed on intrasegmental positional information. Differential control of col transcription by the Antennapedia and Ultrabithorax/Abdominal-A paralogs is mediated by separate cis-regulatory modules (CRMs). Hox proteins also control the segment-specific number of myoblasts allocated to the DA3 muscle. It is concluded that Hox proteins both regulate and contribute to the combinatorial code of transcription factors that specify muscle identity and act at several steps during the muscle-specification process to generate muscle diversity (Enriquez, 2010).

Eve expression in the DA1 muscle lineage provided the first paradigm for studying the early steps of muscle specification. Detailed characterization of an eve muscle CRM showed that positional and tissue-specific information were directly integrated at the level of CRMs via the binding of multiple transcription factors, including dTCF, Mad, Pnt, Tin and Twi. Based on this transcription factor code and using the ModuleFinder computational approach, this study has identified a CRM, CRM276, that precisely reproduces the early phase of col transcription. This CRM also drove expression in cells of the lymph gland, another organ that is issued from the dorsal mesoderm where col is expressed. Parallel to this study, two col genomic fragments were selectively retrieved in chromatin immunoprecipitation (ChIP-on-chip) experiments designed to identify in vivo binding sites for Twi, Tin or Mef2 in early embryos. One fragment overlaps with CRM276. Based on this overlap and interspecies sequence conservation, a 1.4 kb subfragment of CRM276 that retained most of the transcription factor binding sites identified by ModuleFinder was tested, and it was found to specifically reproduced promuscular col expression. This in vivo validation shows that intersecting computational predictions and ChIP-on-chip data should provide a very efficient approach to identify functional CRMs on a genome-wide scale (Enriquez, 2010).

The eve and col early mesodermal CRMs are activated at distinct A/P and D/V positions. It is now possible to undertake a comparison of these two CRMs, in terms of the number and relative spacing of common activator and repressor sites and their expanded combinatorial code, in order to understand how different mesodermal cis elements perform a specific interpretation of positional information (Enriquez, 2010).

A progenitor is selected from the Col promuscular cluster in T2 and T3 but not T1. One cell issued from the Col-expressing promuscular cluster in T1 nevertheless shows transiently enhanced Col expression, suggesting that the generic process of progenitor selection is correctly initiated in T1. This process aborts, however, in the absence of a Hox input, as shown by the loss of progenitor Col expression and DA3 muscle in specific segments in Hox mutants. The similar changes in Nau and Col expression observed under Hox gain-of-function conditions leads to the conclusion that the expression of 'identity' transcription factor iTFs is regulated by Hox factors at the progenitor stage. The superimposition of Hox information onto the intrasegmental information thereby implements the iTF code in a segment-specific manner and establishes the final muscle pattern. Unlike DA3, a number of specific muscles are found in both T1 and T2-A7, such as the Eve-expressing DA1 muscle; other muscles form in either abdominal or thoracic segments, as illustrated by the pattern of Nau expression in stage 16 embryos. This diversity in segment-specific patterns indicates that Hox regulation of iTF expression is iTF and/or progenitor specific (Enriquez, 2010).

As early as 1994, Hox proteins were proposed to regulate the segment-specific expression of iTFs. Seven years later, an apterous mesodermal enhancer (apME680) active in the LT1-4 muscles was characterized and itwas proposed that regulation by Antp was direct. However, mutation of the predicted Antp binding sites present in apME680 abolished its activity also in A segments, suggesting that some of the same sites were bound by Ubx/AbdA. Evidence is now available that the regulation of col expression by Ubx/AbdA in muscle progenitors is direct and involves a single Hox binding site. However, regulation by Antp does require other cis elements. It remains to be seen whether regulation by Antp is also direct. Since Antp, Ubx and AbdA display indistinguishable DNA-binding preferences in vitro, the modular regulation of col expression by different Hox paralogs suggests that other cis elements and/or Hox collaborators contribute to Hox specificity. Direct regulation of col by Ubx has previously been documented in another cellular context, that of the larval imaginal haltere disc, via a wing-specific enhancer. In this case, Ubx directly represses col expression by binding to several sites, contrasting with col-positive regulation via a single site in muscle progenitors. This is the second example, in addition to CG13222 regulation in the haltere disc, of direct positive regulation by Ubx via a single binding site. Hox 'selector' proteins collaborate on some cis elements with 'effector' transcription factors that are downstream of cell-cell signaling pathways. In the DA3 lineage, it seems that Dpp, Wg and Ras signaling act on one col cis element and the Hox proteins on others. The regulation of col expression by Hox proteins in different tissues via different CRMs provides a new paradigm to decipher how different Hox paralogs cooperate and/or collaborate with tissue- and lineage-specific factors to specify cellular identity (Enriquez, 2010).

The DA3 muscle displays fewer nuclei in T2 and T3 than in A1-A7, an opposite situation to that described for an aggregate of the four LT1-4 muscles. It has been proposed that the variation in the number of LT1-4 nuclei was controlled by Hox proteins. These studies of the DA3 muscle extend this conclusion by showing that the variations due to Hox control are specific to each muscle and are exerted at the level of FCs. Since the number of nuclei is both muscle- and segment-specific, Hox proteins must cooperate and/or collaborate with various iTFs to differentially regulate the nucleus-counting process. As such, Hox proteins contribute to the combinatorial code of muscle identity. Identifying the nature of the cellular events and genes that act downstream of the iTF/Hox combinatorial code and that are involved in the nucleus-counting process represents a new challenge (Enriquez, 2010).

Homeotic effects

Mutations in the clustered homeotic genes (HOM-C genes) can cause specific homeotic transformation, suggesting that the HOM-C genes determine segmental identities by acting on different target genes. However, misexpression of several HOM-C genes in the antenna disc causes similar antenna-to-leg transformations. No HOM-C genes are normally expressed in the eye-antenna disc proper. It has been considered that Antp, when ectopically expressed in the eye-antenna disc, suppresses an antenna-determining gene. This study shows that Scr, Antp, Ubx, and abd-A HOM-C genes all exert their effects through a common mechanism: suppression of the transcription of the homothorax (hth) homeobox gene, thereby preventing the nuclear localization of the Extradenticle homeodomain protein. If hth is a key effector suppressed by these four HOM-C genes, addition of hth should reverse the antennal transformations. Coexpression of the hth and HOM-C genes completely or partially reverts the transformation phenotype. It is noted, however, that suppression of hth is probably not the only effect of HOM-C expression in the antenna disc, since Scr, Antp, and Ubx each induce the antenna to transform into leg, showing different segmental characters (i.e., thoracic 1, thoracic 2 and thoracic 3 legs, respectively). Ectopic hth expression can cause duplication of the proximodistal axis of the antenna, suggesting that it is involved in proximodistal development of the antenna (Yao, 1999).

A possible mechanism for the suppression of hth by different HOM-C proteins assumes that the HOM-C proteins compete with a factor required for hth transcription. One candidate protein that fits all of these criteria is Hth itself. The gene spineless exhibits a similar antenna-determining function. It is possible that hth and spineless represent separate pathways specifying antennal identity. Since hth and ss are expressed in the leg discs as well as in the antenna discs, it is not their simple presence that determines antennal identity. What then distinguishes the antenna vs. the leg? One possibility is that the detailed spatial and temporal expression pattern makes the difference. The broader expression pattern of hth in the antenna disc may distinguish the antenna from the leg. It is also possible that the level of spineless makes a difference: high levels of ss correlate with antennal identity and low levels of ss correlate with leg identity. The duplication in the antenna caused by ectopic hth could be explained by the creation of a new proximodistal interface in the distal portion region of the disc. In both antenna and leg discs, Distal-less is expressed in the distal regions and is required for distal development. The roughly complementary expression of hth/nuclear Exd vs. Dll, defines the proximal and distal domains of appendages, respectively. The combined action of Wg and Dpp signaling defines the two domains by activating Dll and repressing hth in the distal domain. Antennal duplication due to ectopic hth could be explained by the juxtaposition of distal (Dll expressing) and proximal (hth expressing) cells (Yao, 1999 and references)

In spite of its name Antennapedia is not normally expressed in the antenna imaginal disc, but in fact determines leg fate. The response of the antenna imaginal disc to ectopic Antennapedia gene expression was explored. The distal to proximal changes in morphological transformation in response to Antennapedia at different developmental stages were correlated with changing expression patterns of gene expression. At particular stages and doses of Antennapedia, cell differentiation of leg bristles was uncoupled from transformation of the third antennal segment to tarsus. The results suggest that determination for bristle type does not depend on a prior determination decision for organ type. The results also provide an avenue for exploring the nature of 'competence' at cellular and molecular levels (Scanga, 1995).

It is of interest to investigate the regulatory basis of classic Antp alleles that give rise to antenna to leg transformations. The spontaneous mutant allele of Antp, Nasobemia (AntpNs), consists of an internal 25-kb partial duplication of the Antp gene as well as a complex insertion of > 40 kb of new DNA. The duplication gives the mutant gene three Antp promoters, and transcripts from each of these are correctly processed to yield functional ANTP proteins. At least two of the promoters are ectopically active in the eye-antenna imaginal discs, leading to homeotic transformation of the adult head (Talbert, 1995).

A 60-aa peptide corresponding to the homeodomain of Antennapedia protein can translocate through the membrane of neurons in culture, accumulate in neuronal nuclei, and promote neurite growth. Three mutant versions of ANTP were constructed that differ in their ability to translocate through the membrane and to bind specifically the cognate sequence for homeodomains present in the promoter of HoxA5. Removing two hydrophobic residues of the third helix inhibits ANTP internalization and suppresses its neurotrophic activity. ANTP neurotrophic activity is lost when mutations are introduced in positions preserving its penetration and nuclear accumulation but abolishing its capacity to bind specifically the cognate DNA-binding motif for homeoproteins. These results suggest that Antennapedia protein neurotrophicity requires both its internalization and its specific binding to homeobox cognate sequences (Le Roux, 1993).

Control of the spineless antennal enhancer: direct repression of antennal target genes by Antennapedia

It is currently thought that antennal target genes are activated in Drosophila by the combined action of Distal-less, homothorax, and extradenticle, and that the Hox gene Antennapedia prevents activation of antennal genes in the leg by repressing homothorax. To test these ideas, a 62bp enhancer was isolated from the antennal gene spineless that is specific for the third antennal segment. This enhancer is activated by a tripartite complex of Distal-less, Homothorax, and Extradenticle. Surprisingly, Antennapedia represses the enhancer directly, at least in part by competing with Distal-less for binding. Antennapedia is required in the leg only within a proximal ring that coexpresses Distal-less, Homothorax and Extradenticle. It is concluded that the function of Antennapedia in the leg is not to repress homothorax, as has been suggested, but to directly repress spineless and other antennal genes that would otherwise be activated within this ring (Duncan, 2010).

This report examines the regulation of an enhancer from the antennal gene ss that drives expression specifically in the third antennal segment (A3). The work provides the first look at how the homeodomain proteins Dll, Hth, and Exd function in the antenna to activate antennal target genes. These proteins form a trimeric Dll/Hth/Exd complex on the enhancer, suggesting that Dll acts much like a Hox protein in antennal specification. This work also reveals how the Hox protein Antp functions in the leg to repress antennal development. The conventional view has been that the primary function of Antp is to repress hth in the distal leg, which then prevents the activation of all downstream antennal genes. However, this study found that Antp represses the ss A3 enhancer directly. This repression is essential within a proximal ring in the leg that coexpresses the antennal gene activators Dll, Hth, and Exd. Antp competes with Dll for binding to the enhancer, and this competition is part of a molecular switch that allows the ss A3 element to be activated in the antenna, but represses its activation in the leg. The results suggest that repression of antenna-specific genes in the proximal ring is the sole function of Antp in the leg imaginal disc (Duncan, 2010).

At 62 bp, the ss A3 enhancer (called D4) is one of the smallest enhancers to be identified in Drosophila, and yet it is quite strong; only a single copy is required to drive robust expression of lacZ reporters. The enhancer is also very specific, driving expression in A3 and nowhere else in imaginal discs. It has been proposed that antennal identity in Drosophila is determined by the combined action of Dll, Hth, and Exd. Consistent with this proposal, all three of these factors were found to be required for D4 expression. Although these activators are coexpressed in both A2 and A3, D4/lacZ expression is restricted to A3 by Cut, which represses the enhancer in A2. Like ss itself, D4/lacZ is also repressed by ectopically expressed Antp (Duncan, 2010).

A previous report (Emmons, 2007) showed that the antennal expression pattern of ss is reproduced by lacZ reporters containing a 522 bp fragment from the ss 5' region. This fragment contains five conserved (41%-90% identity) domains, each of which was deleted and tested for effect on expression in vivo. Expression in the arista and the third antennal segment (A3) prove to be under separate control; expression in the arista requires domains 1, 3 and 5, whereas expression in A3 is lost only when domain 4 is deleted. Moreover, reporters containing domain 4 alone show expression in A3 and nowhere else in imaginal discs. Thus, domain 4 is both necessary and sufficient for A3-specific expression. Domain 4 (D4) is 62 bp in length and is highly conserved, being invariant at 50/62 base pairs in the 12 Drosophila species sequenced (Duncan, 2010).

Surprisingly, Dll, Hth, Exd, Cut, and Antp all act directly upon D4. The activators Hth and Exd bind with strong cooperativity to directly adjacent sites. Their joint binding site matches the optimum site for in vitro binding of the mammalian homologs of Hth and Exd (Meis and Prep), consistent with the robust activity of the enhancer in vivo. Mutation of either of these sites abolishes activity of the enhancer. The coactivator Dll binds three sites in D4; one of these sites (Dlla) is required for almost all activity of the enhancer. Dll shows strong cooperativity with Hth and Exd for binding to D4, indicating that Dll interacts physically with these proteins. This interaction requires DNA binding, as Dll protein containing a missense change that blocks DNA binding (a change of asn51 to ala in the homeodomain) shows no ability to associate with D4-bound Hth and Exd. A curious feature of the cooperativity seen in the binding studies is that although Hth and Exd increase the affinity of Dll for D4, Dll appears to have little effect on the affinity of Hth and Exd for the enhancer. Since Hth and Exd already bind cooperatively with one another, it may be that additional cooperative interactions with Dll have little effect. Alternatively, it may be that Hth and Exd interact with Dll only after binding DNA. If so, Hth and Exd would be expected to increase Dll binding to D4, but Dll would have little effect on the binding of Hth and Exd, as observed. Interactions between Dll and Hth in the absence of DNA have been reported in immunoprecipitation experiments. However, this study was unable to repeat these observations. Moreover, the finding that the asn51 mutant of Dll fails to associate with D4-bound Hth and Exd argues strongly against such interactions (Duncan, 2010).

The repressor Cut also acts directly upon D4. Binding of Cut requires two sites, one overlapping Dlla and the other overlapping the joint Hth/Exd site. These binding sites suggest that D4 is controlled by Cut in much the same way that a structurally similar Abdominal-A (Abd-A) regulated enhancer from the rhomboid gene is controlled by the repressor Senseless (Sens). In the rhomboid enhancer, adjacent Hth and Exd sites are also present, and these create a binding site for Sens. Activity of the rhomboid enhancer is controlled by a competition between binding of the Sens repressor and binding of the activators Abd-A, Hth, and Exd. It seems likely that D4 is controlled similarly, with the repressor Cut competing for binding with the activators Dll, Hth, and Exd. It will be of interest to determine whether enhancers similar to D4 are used more widely to control Cut targets involved in its role as an external sense organ determinant (Duncan, 2010).

A key finding in this work is that Antp represses D4 by direct interaction. Antp binds a single site in D4, which overlaps or is identical to the Dlla binding site. Like Dll, Antp binds cooperatively with Hth and Exd. Using purified proteins, it was showm that binding of Dll and Antp to the Dlla site is mutually exclusive. This indicates that Antp represses the enhancer at least in part by competing with Dll for binding. Similar competition may occur at other enhancers; when Antp expression is driven artificially in the distal leg, variable deletions of the tarsal segments occur. These defects might arise because Antp competes with Dll for binding to its target genes in the distal leg. In most other contexts examined, Antp is an activator of transcription; why it fails to activate D4 is not clear. The similar behavior of Dll and Antp in binding to D4 supports the idea that Dll behaves like a Hox protein in activating D4 (Duncan, 2010).

Although the initial focus of this study was on the antenna, the finding that Antp interacts directly with D4 led to an examination of D4 regulation in the leg, where Antp is normally expressed. In second leg imaginal discs, Antp is required only in a proximal ring of cells that coexpresses Dll and Hth. This ring appears in the early third instar, and is of uncertain function. Large Antp clones in T2 leg discs that do not enter this ring appear to develop completely normally, regardless of whether they are located distal or proximal to the ring. However, clones that overlap the ring show activation of D4/lacZ within the ring cells. Importantly, such clones have no effect on the expression of Dll or Hth within the ring. By examining Antp clones of increasing age the following sequence of events is inferred. First, D4/lacZ is activated in cells of the ring that are included within Antp clones. Second, many such clones begin expressing the antennal markers Ss and Cut, indicating a transformation to antenna, and round up as if they have lost affinity for neighboring cells. Third, such clones appear to extend and move distally in the disc (Duncan, 2010).

The events described for Antp clones in the leg make sense of several previously enigmatic observations. It has been noted that many Antp clones in the leg do not transform to antenna and appear to develop normally. The finding that only clones that overlap the proximal ring undergo transformation accounts for this observation. Antp clones that do contain transformations usually show apparent nonautonomy in that not all cells in the clone are transformed to antenna. The current results account for this observation as well, since within an Antp leg clone only those cells located in the proximal ring undergo transformation to antenna; cells located elsewhere in the clone retain normal leg identity. Most importantly, these observations provide an explanation for why ss is controlled directly by Antp. Antp clones have no effect on hth or Dll expression in the proximal ring. Therefore, Antp must function in the ring at the target gene level to repress antennal genes that would otherwise be activated by combined Hth and Dll (and Exd). Since several such targets are known, it seems likely that several, perhaps many, antennal genes in addition to ss are repressed directly by Antp (Duncan, 2010).

Transformed Antp clones in the leg often show ectopic hth expression in distal locations. If hth is not directly controlled by Antp in the leg, as this study suggests, then why is hth ectopically expressed within such clones? A likely explanation is that downstream antennal genes that have become activated in such clones feed back to activate hth. This interpretation is strongly supported by the finding that ectopic expression of the antennal genes ss, dan, or danr in the distal leg causes ectopic activation of hth. Thus, the distal expression of hth seen in Antp leg clones is likely a consequence rather than a cause of the transformation to antenna. Whether repression of hth in the antenna by ectopic Antp is also indirect is not clear. Dll is also expressed ectopically in transformed Antp leg clones, suggesting that it is also subject to feedback activation by downstream antennal genes (Duncan, 2010).

The function of the proximal Dll- and Hth-expressing ring in the proximal leg is not well understood. The ring is highly conserved among the insects, and may serve as a boundary between the proximal and distal portions of the legs. In the context of this work, a striking feature of the ring is that it contains a microcosm of gene expression domains corresponding to the three major antennal segments. Thus, proceeding from proximal to distal through the ring, cells express hth alone, hth + Dll, and hth + Dll + strong dachshund. These expression combinations are characteristic of the A1, A2, and A3 antennal segments, respectively. Looked at in this way, the ring would appear to resemble a repressed antennal primordium within the leg (Duncan, 2010).

It has been known for almost thirty years that Antp is required in the leg to repress antennal identity. However, an understanding of how this repression occurs has been lacking. The current results indicate that Antp functions within the proximal ring to directly repress antennal genes that would otherwise be activated by combined expression of Dll, Hth, and Exd. This appears to be the only function of Antp in the leg, at least during the third instar larval stage. The results are entirely consistent with the idea that second leg is the 'ground state' ventral appendage (the limb type that develops in the absence of identity specification) and that the role of Antp in the leg is to preserve this ground state by repressing the activation of 'head-determining' genes (Duncan, 2010).

Segment-specific regulation of the Drosophila AP-2 gene during leg and antennal development

Segmentation involves subdivision of a developing body part into multiple repetitive units during embryogenesis. In Drosophila and other insects, embryonic segmentation is regulated by genes expressed in the same domain of every segment. Less is known about the molecular basis for segmentation of individual body parts occurring at later developmental stages. The Drosophila transcription factor AP-2 gene, dAP-2, is required for outgrowth of leg and antennal segments and is expressed in every segment boundary within the larval imaginal discs. To investigate the molecular mechanisms generating the segmentally repetitive pattern of dAP-2 expression, transgenic reporter analyses was performed and multiple cis-regulatory elements were isolated that can individually or cooperatively recapitulate endogenous dAP-2 expression in different segments of the appendages. An enhancer specific for the proximal femur region, which corresponds to the distal-most expression domain of homothorax (hth), was analyzed in the leg imaginal discs. Hth is known to be responsible for the nuclear localization and, hence, function of the Hox cofactor, Extradenticle (Exd). Both Hth and Exd were shown to be required for dAP-2 expression in the femur, and a conserved Exd/Hox binding site was found to be essential for enhancer activity. These loss- and gain-of-function studies further support direct regulation of dAP-2 by Hox proteins and suggest that Hox proteins function redundantly in dAP-2 regulation. This study reveals that discrete segment-specific enhancers underlie the seemingly simple repetitive expression of dAP-2 and provides evidence for direct regulation of leg segmentation by regional combinations of the proximodistal patterning genes (Ahn, 2011).

The segmentally repeated expression of dAP-2 in the developing leg and antennal discs may suggest that its expression in each segment is regulated in a similar manner by upstream segmentation genes. Alternatively, each domain (ring) of dAP-2 expression could result from the combinatorial activities of multiple transcription factors, which themselves are not expressed in a repeated pattern, but instead occupy distinct and broader domains along the PD axis of the appendages. Current data provide strong evidence that the latter strategy is utilized to establish dAP-2 expression in all but the tarsal segments. It seems that the tarsus has adopted a strategy different from that of other leg segments to regulate dAP-2 expression (Ahn, 2011).

In an effort to understand molecular mechanisms controlling dAP-2 expression during leg development, the regulatory potential of dAP-2 genomic fragments was tested using transgenic reporter analyses. Multiple enhancers were successfully isolated which can independently direct reporter expression in specific leg segments and together recapitulate, almost completely, the endogenous expression pattern. It is intriguing that the relative positions of these enhancers on the chromosome are well correlated with the position of their activity along the PD axis of the leg. Importantly, the presence of segment-specific enhancers suggests that dAP-2 expression is differentially regulated in each leg segment. It is likely that each domain of dAP-2 expression in the true joints is regulated by a combination of upstream regulators involved in PD patterning using segment-specific enhancers similar to the distinct enhancers used to regulate expression of the pair-rule gene, even-skipped, in every other parasegment during embryonic segmentation. Interestingly, dAP-2 expression in the coxa is differentially regulated along the DV axis and depends on two region-specific enhancers. In addition, the EB fragment displayed relatively weaker activity in the ventral region compared to the larger E6 fragment. These data raise the possibility that DV patterning genes are also involved in dAP-2 regulation in the proximal segments. It is possible that the use of multiple region-specific enhancers is a general mechanism establishing expression of segmentation genes during leg development (Ahn, 2011).

Current data indicate that dAP-2 expression in antennal discs also requires multiple region-specific enhancers. Some of the leg enhancers showed an antennal expression pattern similar to their leg patterns with respect to the PD axis. However, there are also enhancers specific for either antennal or leg discs implying that the genes required for normal identity of the two homologous appendages might be involved in regulation of dAP-2 expression in some segments. One of the features that distinguish antennae from legs is that in antennal discs, hth expression is expanded to the intermediate region where dac expression is missing. In contrast, the expression patterns of Dll, dac and hth are very similar in the proximal and distal regions of the two appendages. It is interesting to note that the dAP-2 enhancers for the most proximal and distal regions are shared between the two appendages while the intermediate region utilizes distinct enhancers. This implies that dAP-2 expression in the intermediate region is more likely to be regulated by antennal- or leg-specific regulatory pathways. For example, although the femur and the AIII are homologous structures, the Hox-dependent proximal femur enhancer is active in the leg, but not in the antenna. Likewise, the BE enhancer is active in the proximal AIII of the Hox-free antenna, but not in the leg (Ahn, 2011).

The Hox gene Antp has been considered to be a key factor in determining leg identity since Antp mutant clones in the T2 leg cause a leg-to-antenna transformation, mainly outside of the Hth domain. Previous studies suggested that Antp performs its selector function by acting as a repressor of hth and other antennal genes in the intermediate leg. In contrast, both Antp and hth are expressed in the proximal leg, and are required for growth and segmentation of this region. Therefore, it has been proposed that the role of Antp as a repressor of hth is limited to the intermediate leg, and that both Antp and Hth contribute to proper development of the proximal leg (Ahn, 2011).

The similar loss-of-function phenotypes of hth and exd suggest that Hth and Exd act on common target genes during development of the proximal leg. In certain developmental contexts, Hth can directly bind to DNA through its homeodomain in a ternary complex including Exd and Hox proteins to regulate expression of target genes. However, it has been shown that a Hth isoform lacking the homeodomain can execute the function of Hth in PD patterning of Drosophila leg discs indicating that direct DNA binding is not necessary for its function in proximal leg discs. Since no conserved, consensus Hth binding site were found in the proximal femur enhancer of dAP-2, Hth is likely functioning in dAP-2 expression through a mechanism independent of its direct binding to DNA through its homeodomain. Instead, Hth may regulate dAP-2 expression in the proximal femur by facilitating the nuclear localization of Exd or by interacting with other transcription factors which bind DNA (Ahn, 2011).

As a cofactor of Hox proteins, Exd, and its mammalian homolog Pbx, cooperatively bind DNA with Hox proteins and regulate expression of their target genes which are involved in a variety of developmental processes in both vertebrates and Drosophila. Although previous genetic analyses have revealed essential functions of Exd and Hox proteins in leg development, it has been unclear whether these factors act together on common target genes during this process. This study has identified a conserved Exd/Hox binding site which is required for activity of the proximal femur enhancer of dAP-2. Through clonal analyses, it was demonstrated that hth, exd and Antp are necessary for dAP-2 expression in the presumptive proximal femur of leg imaginal discs. This is the first example of a direct target gene of an Exd/Hox complex in Drosophila limb development. This study also provides insight into the molecular mechanism integrating the combinatorial actions of PD patterning genes in the regulation of region-specific expression of leg segmentation genes (Ahn, 2011).

Although Antp is expressed in all three pairs of legs, most of the prothoracic (T1) and metathoracic (T3) legs with Antp mutant clones appeared to be normal, except for a rare fusion between the femur and tibia. However, Scr/Antp double mutant clones in T1 legs and Antp/Ubx double mutant clones in T3 legs generated leg defects indistinguishable from those generated by Antp mutant clones in T2 legs. It was proposed that the low penetrance of the Antp mutant phenotypes in T1 and T3 legs is due to redundancy with Scr and Ubx, which are expressed in T1 and T3 leg discs, respectively. This idea is consistent with the previous observations that Scr and Ubx both can induce antenna-to-leg transformations when ectopically expressed in antennal discs. It is proposed that Antp, Scr and Ubx can redundantly activate dAP-2 expression in the proximal femur as Exd/Hox heterodimers based on the following observations. First, dAP-2 expression in T2 leg discs, but not in T1 and T3 leg discs, requires Antp. Secondly, EMSA results demonstrate that all three Hox proteins bind strongly to the binding site in the proximal femur enhancer as Exd/Hox heterodimers. Thirdly, all three Hox proteins can activate PrF enhancer function when ectopically expressed in antennal discs (Ahn, 2011).

Functional synthetic Antennapedia genes and the dual roles of YPWM motif and linker size in transcriptional activation and repression

Segmental identity along the anteroposterior axis of bilateral animals is specified by Hox genes. These genes encode transcription factors, harboring the conserved homeodomain and, generally, a YPWM motif, which binds Hox cofactors and increases Hox transcriptional specificity in vivo. In this study synthetic Drosophila Antennapedia genes were derived, consisting only of the YPWM motif and homeodomain, and their functional role throughout development was investigated. Synthetic peptides and full-length Antennapedia proteins cause head-to-thorax transformations in the embryo, as well as antenna-to-tarsus and eye-to-wing transformations in the adult, thus converting the entire head to a mesothorax. This conversion is achieved by repression of genes required for head and antennal development and ectopic activation of genes promoting thoracic and tarsal fates, respectively. Synthetic Antennapedia peptides bind DNA specifically and interact with Extradenticle and Bric-à-brac interacting protein 2 cofactors in vitro and ex vivo. Substitution of the YPWM motif by alanines abolishes Antennapedia homeotic function, whereas substitution of YPWM by the WRPW repressor motif, which binds the transcriptional corepressor Groucho, allows all proteins to act as repressors only. Finally, naturally occurring variations in the size of the linker between the homeodomain and YPWM motif enhance Antennapedia repressive or activating efficiency, emphasizing the importance of linker size, rather than sequence, for specificity. These results clearly show that synthetic Antennapedia genes are functional in vivo and therefore provide powerful tools for synthetic biology. Moreover, the YPWM motif is necessary -- whereas the entire N terminus of the protein is dispensable -- for Antennapedia homeotic function, indicating its dual role in transcriptional activation and repression by recruiting either coactivators or corepressors (Papadopoulos, 2011).

Earlier studies have shown that the HD plays an essential role in Hox gene function. However, ectopic expression of the HD alone results in little, if any, homeotic transformation. By combining the HD with the YPWM motif in synthetic gene constructs, the homeotic functions of two Hox genes, Scr and Antp were successfully reconstructed. Substitutions of the YPWM motif by WRPW retain the repressive activity of Antp, and C-terminal fusions of the WRPW motif to full-length proteins convert them into stronger repressors. On the other hand, the en repression domain - also able to interact with Gro in vivo - does not confer significant changes to Antp functioning as a repressor or activator. The role of the size of the linker between the YPWM motif and the HD was also evaluated, using naturally occurring splicing variants of Antp with an eight- or a four-amino acid linker. It was found that the long-linker variant renders the protein a stronger activator, and the short-linker makes the protein a stronger repressor of transcription in vivo. This result, combined with the finding that mutation of the linker sequence did not alter its specificity, suggests that Antp specificity depends partially on linker size but not on sequence. Finally, it was observed that synthetic peptides and full-length proteins can interact with Exd and Bip2, but all require an intact YPWM motif. The WRPW motif cannot substitute for YPWM in this case (Papadopoulos, 2011).

The YPWM motif is required for Antp homeotic function in the embryo, larva, and adult fly, but the flanking regions of YPWM have no effect on Antp function. The YPWM motif has a dual role in Antp function, because it is necessary and indispensable for activation and repression of all Antp target genes tested: repression of endogenous Scr, ectopic activation of Tsh, repression of Salm and dan, and ectopic activation of grn. These properties indicate the essential role of the YPWM motif in recruiting both corepressors and coactivators. The YPWM motif is also required for ectopic induction of mesothoracic leg structures and for interaction with Exd and Bip2 cofactors in gel shift and transactivation assays. However, substitution by WRPW retains to a large extent its repressor activity in both synthetic peptides and full-length proteins and results in partial to complete repression of reporter genes (Papadopoulos, 2011).

It has been shown previously that the YPWM motif acts as an activation domain in transactivation experiments. To examine whether the YPWM motif also can act as a repressor domain, it was fused to the C terminus of the Gal4 protein and its binding to UAS and its transactivation efficiency were tested. It was observed that the Gal4YPWM protein decreased transcription of the reporter by about 60%, whereas a quadruple-alanine fusion (Gal4AAAA) did not. Therefore, the YPWM motif, in addition to its role as an activation module, can act as a potent repressor module in vivo (Papadopoulos, 2011).

The differential requirements for a functional YPWM motif in Antp support the notion that different rules may apply in Hox-mediated transcriptional repression versus activation. Sequence comparisons among Hox binding preferences to direct targets show that Pbx-Hox sites usually are found in enhancers of genes activated by a Pbx-Hox complex, whereas regulatory elements bound by Hox factors alone (without any Pbx input) are distributed equally among the genes activated and repressed by Hox factors. This observation supports the idea that Hox-mediated activation depends largely on cofactors, whereas repression does not always require them (Papadopoulos, 2011).

Phenotypic observations in the embryo, in which both the YPWM,LLAntpWRPW and the AAAA,LLAntpWRPW proteins activated Tsh ectopically in the region of the embryonic head, but the synthetic or full-length AAAAAntp proteins did not, suggest that this target gene is activated indirectly in the embryo (possibly through repression of a repressor). However, the finding that the YPWM,LLAntpWRPW construct can ectopically activate grn, whereas the AAAA,LLAntpWRPW line cannot, indicates that Antp can act as a bona fide activator and that the activation of grn may be direct (Papadopoulos, 2011).

The substitution of the YPWM motif by alanines strongly decreases but does not entirely abolish Antp homeotic function in full-length and synthetic AAAAAntp constructs, because they still exhibit aristal transformations and display ectopic leg bristles on the A3 segment, suggesting weak transformations toward leg identity. Similar behavior of AAAA-substituted Antp has been reported in flies for the full-length AAAA,LLAntp, coinduced with a constitutively active Notch, or the beetle fushi tarazu gene encoding a protein with a YPWM-to-AAAA substitution (Papadopoulos, 2011).

Also, the phenotypes of AAAAAntp in the antenna are in line with the head-involution defects in the larval cuticle early in development. Although the AAAA-substituted synthetic peptides fail overall to exhibit significant phenotypes, they still infer mild malformations in the larval head, including defects in the formation of the mouth hooks and, in these cases, lethality. However, unlike their homeotic effect in the antenna, these lines retain T1 identity in the cuticle, as demonstrated by the presence of prothoracic beards (Papadopoulos, 2011).

In the embryo, the antennal disc, and in Drosophila S2 cells the long-linker Antp proteins behaved as potent activators, whereas the short-linker constructs acted as strong repressors. The long-linker variant is expressed predominantly in embryonic stages, and equal amounts of both variants are detected in larval, pupal, and adult stages. Thus, different Antp transcripts might play distinct, albeit similar, roles in development. Isoforms with different linker sizes also are found in Ultrabithorax (Ubx), and in Abdominal A mutations in the linker decrease its activation capacity on the wingless enhancer. Recently, the size of the linker between the YPWM and the HD of Ubx was found to play a regulatory role in Ubx function, with the long- and short-linker variants not being interchangeable in vivo. The short-linker variant was found to bind DNA more strongly than its long-linker relative, in the presence of Exd. In line with these arguments, the Antp short-linker variant also interacted more strongly with Exd and more weakly with Bip2 than did its long-linker counterpart (Papadopoulos, 2011).

One possible N-terminal domain to which potency could be attributed is the polyglutamine (poly-Q) stretch (present in the N terminus of Antp and absent in synthetic Antp peptides), which has been proposed to function as an activator domain in Bicoid and Abdominal B. Another conserved motif present in the N terminus of many homeoproteins and reported to participate in transcriptional potency in the fly Scr and the mouse Hoxa5 is the TSYF motif (SSYF in Scr). An alignment of various Antp orthologs showed that the N and C termini of all proteins are largely variable between insects and mammals, with the latter displaying shorter N termini devoid of poly-Q stretches. The residues flanking the YPWM motif are slightly conserved in arthropods but not between arthropods and mammals, an observation that is in line with the finding that their substitution by alanines does not affect the homeotic function of the protein. However, the linker between the HD and the YPWM motif, differentially spliced in the transcripts of at least some Hox transcripts (Antp, Ubx), could account for increased specificity of target selection in vivo. Recent evidence shows that the regions flanking the Ubx YPWM motif (alternatively spliced in Ubx isoforms) are responsible for Ubx specificity in vivo. Moreover, the YPWM motif and the linker region (termed 'the specificity module') of Deformed and Scr result in their unique regulatory behavior in vivo, and nonconserved domains N-terminal to this region allow subtle differences in Hox-DNA recognition properties (Papadopoulos, 2011).

These differences can have a considerable impact on the transcriptional output -- activation versus repression -- of target genes. Finally, decisions on whether certain Hox factors function as activators or repressors in a specific context can result from the abundance of their isoforms rather than from the regulatory sequences to which they bind. The finding that the same enhancer element upstream of the murine Six2 gene is activated by Hox11 and repressed by Hoxa2 substantiates this hypothesis (Papadopoulos, 2011).

This study has increased the understanding of synthetic genes. Hypothesizing that protein function is a sum of the functions of its protein domains, the latter being oriented in a precise 3D conformation that allows normal interactions to take place, attempting to engineer and possibly predict the functions of novel synthetic peptides on the basis of the structural architecture of their protein domains seems possible. Inversely, functions of proteins might be scaled down to fractions of their sequences, with each domain retaining only a subset of these functions. So far, to a great extent, the function of two Hox proteins, Antp and Scr, has been reconstructed successfully using their synthetic-peptide versions, and it has been demonstrated that they act predictably in vivo. If biomedically relevant proteins also fell in this category, the development and use of their corresponding synthetic peptides might present advantages over their full-length counterparts for application in vivo, because of their smaller size and more easily understood molecular properties. Novel proteins with predictable functions have been constructed successfully, e.g., with the neuronal Wiskott-Aldrich protein, N-WASP, and guanine nucleotide exchange factors, in which integrating artificial domains that respond to nonnative inputs led to the precise control of physiological responses, such as the formation of filopodia or lamellipodia (Papadopoulos, 2011).

Antennapedia: Biological Overview | Evolutionary Homologs | Transcriptional regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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