Antennapedia
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).
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).
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).
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).
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).
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.