BIOLOGICAL OVERVIEW

Antp is the last gene of the Antennapedia Complex (ANTP-C), that is, the most distal from the centromere, and the one expressed in the most posterior locale. Antp is distal to fushi tarazu and even more distant from Sex combs reduced The highest levels of transcription are found in the ventral portion of the second thoracic segment, where Antp is the initiator of a cascade of events that result in the development of an adult leg.

This begs an interesting question: why does a gene that regulates thoracic development get the name Antennapedia? Many of the earliest discovered mutations in Drosophila were dominant. This type of mutation is readily produced and easy to spot, since only one copy of the mutated gene is required in order to see a phenotypic effect. As early as 1949 dominant mutations had been found which converted antenna into the second leg [Image] (the mutant adult having no antenna, and two sets of second legs). The name Antennapedia (antenna-foot) was appropriately descriptive. Such homeotic transformations are exciting to contemplate, and serve as the basis of our understanding of gene function in Drosophila (Abbott, 1986).

It is important to understand that adult structures are derived from cells set aside during embryonic development. For example, imaginal discs store the potential for leg development throughout the larval stages. Discs begin to develop in the third instar larval stage, and the metamorphosis to adult takes place during the pupal stage. Therefore, a change in the regulation of the Antennapedia gene, resulting in its expression in the eye-antennal disc, is sufficient to cause an eye to leg transformation that will show up in the adult.

Later discovery of recessive alleles (null alleles) of Antennapedia were found to have an effect just opposite to the one first noted in 1949. The second or mesothoracic leg was transformed into an antenna. Antennapedia was thought to have a positive effect, that is, the promotion of leg fate. Another view posited that Antennapedia acted to repress genes whose expression in the thorax would result in head fate. Similarly, expression of Antp in the head could act to repress head fate, establishing ectopic leg in place of antenna. What these varying results have in common is the transformation of one organ into another, based on the existence of a unified cellular precursor for the whole organ, the imaginal disc. Careful observation reveals that such transformations are rarely complete. They depend on the period during development in which the ectopic expression is carried out and the level of gene expression (Scanga, 1995). Nevertheless, Antennapedia and other homeotic genes can modify the expression of the whole genetic hierarchy of events required for organogenesis.

Because of their widespread effect on cell fate, there is tremendous interest in identifying the downstream targets of homeotic genes. The search has been complicated because of the overlapping actions of the linked array of five segmentally acting genes of the Antennapedia complex. It isn't always clear which gene in the complex is having the noted effect.

One such downstream target for Antennapedia is the gene spalt. spalt is expressed in both anterior and posterior regions of the Drosophila embryo where it promotes head and tail patterns. sal has an added feature: it represses teashirt function. teashirt is known to promote trunk (thoracic) development. Ectopic expression of Antp in the head is likely to repress sal, and thus result in the expression of teashirt (otherwise repressed by sal), and promote trunk identity (Künlein, 1994).

Do Antp null alleles allow for the expression of sal in the leg disc? And if so, is this sufficient to promote a leg-into-antenna transformation? In the case of the spalt,gene, the effects of Antp are known to be recessive. It is not yet known exactly how homeotic genes transform the fate of a complete organ. One clue lies in the lack of specificity of the transformation. Recently it has been shown that ectopic expression of Ultrabithorax, abdominal-A and Abdominal-B cause similar transformations in some of the fruitfly appendages: antennal tissue into leg tissue and wing tissue into haltere tissue. Thus the homeotic requirement to form appendages is, in some cases, non-specific (Casares, 1996).

Antennapedia represses homothorax in leg discs. During the evolution of insects from a millipede-like ancestor, the Hox genes are thought to have promoted the diversification of originally identical body structures. In Drosophila, antennae and legs are homologous structures that differ from each other as a result of the Hox gene Antennapedia (Antp), which promotes leg identities by repressing unknown antennal-determining genes. Four lines of evidence are presented that identify extradenticle (exd) and homothorax (hth) as antennal-determining genes. (1) Removing the function of eitherexd or hth (which is required for the nuclear localization of Exd protein), transforms the antenna into leg; such transformations occur without activation of Antp. (2) In most antennal cells, hth is expressed and Exd is nuclear, whereas both are restricted to proximal cells of the leg. (3) Antp is a repressor of hth. (4) Ectopic expression of Meis1, a murine hth homolog, can trigger antennal development elsewhere in the fly. Taken together, these data indicate that hth is an antennal selector gene, and that Antp promotes leg development by repressing hth, consequently preventing the nuclear transport of Exd (Casares, 1998).

Now that it is clear that hth determines antennal fate, it is worthwhile reconsidering the transformation to leg that is produced by hth or exd mutant cells in the antenna. This is the same phenotype seen with dominant Antp mutants, but the leg develops without the activity of Antp, Scr or Ubx. It follows that a leg can be generated without Hox activity, suggesting that the leg pathway is the ground state for ventral appendages. Thus the ground pattern for both larvae and adults is thoracic. Nor does Antp "select" for a specific leg pathway -- it simply represses hth in the leg primordia, thereby blocking antennal development and allowing the development of legs by default. This supports the idea that Antp promotes a ground (mesothoracic) pattern by repressing cephalic genes. This basal pattern is modified by Scr toward prothoracic (first leg) or by Ubx toward metathoracic (third leg) in their respective primordia. The downregulation of hth by Antp explains the phenotype of the dominant Antp mutants is due to homothorax repression. It also explains the ability of other Hox genes such as Ubx, abdominal-A, and Abdominal-B to induce the transformation of antennae into legs. These genes prevent the nuclear translocation of Exd (most likely through hth repression), so the antennal to leg transformations are probable nonspecific and caused by a property that is common to Antp and other Hox proteins (Morata, 1998 and Casares, 1998).

During embryogenesis, in contrast with leg development, Antp selects for a specific developmental pathway. Loss-of-function mutations and experiments to induce ectopic expression show that Antp determines the larval mesothoracic pattern -- a function that is clearly distinct from the other Hox genes. Why legs should be different is not clear, but different Hox genes have similar effects on appendages, possibly because these appendages have no hth activity, without which the Hox genes lack specificity (Morata, 1998 and references).

Hox genes encoding homeodomain transcriptional regulators are known to specify the body plan of multicellular organisms and are able to induce body plan transformations when misexpressed. These findings led to the hypothesis that duplication events and misexpression of Hox genes during evolution have been necessary for generating the observed morphological diversity found in metazoans. It is known that overexpressing Antennapedia in the head induces antenna-to-leg as well as head-to-thorax transformation and eye reduction. At present, little is known about the exact molecular mechanism causing these phenotypes. The aim of this study was to understand the basis of inhibition of eye development. It has been demonstrated that Antp represses the activity of the eye regulatory cascade. By ectopic expression, it has been shown that Antp antagonizes the activity of the eye selector gene eyeless. Using both in vitro and in vivo experiments, it has been demonstrated that this inhibitory mechanism involves direct protein-protein interactions between the DNA-binding domains of Ey and Antp, resulting in mutual inhibition (Plaza, 2001).

If the Antp protein is able to block Ey activity, this mechanism should also function in other tissues. Therefore, ectopic eye formation should also be blocked by Antp. To test this prediction, ectopic eyes were induced on wing, antennae and legs using the UAS-GAL4 system. Results show that the ectopic eye formation induced by ey is completely blocked on co-expressing ey and Antp. Moreover, the Antp induced antenna-to-leg transformation is inhibited by ey. A series of similar tests employing hs-ey and hs-Antp transgenes, singly or in combination, leads to the same conclusions. Furthermore, these tests reveal a specific requirement for the Antp homeodomain (HD), since N-terminal deletions of the Antp protein do not affect its ability to inhibit Ey activity, whereas deletion of the HD results in a protein unable to inhibit ey function. Similarly, using the UAS-GAL4 system, the Antp HD-deleted protein is unable to repress ectopic eye formation. These results made it necessary to demonstrate that both proteins co-localize in the same cells of the discs. Upon examining protein accumulation by confocal microscopy, it was found that both proteins are efficiently co-expressed in these different tissues. Furthermore, immunostaining experiments performed using the ey antibody or the neuronal marker 22C10 confirm that, despite the presence of Ey in the disc, co-expression of Antp leads to inhibition of neuronal differentiation (Plaza, 2001).

In order to test whether the DNA-binding activity of Antp is not required for the inhibition of eye development, an Antp mutant was tested in which the DNA-binding specificity was changed (Q50K). Interestingly, this mutated protein is still able to repress eye development. In addition, mutagenesis experiments were performed to convert Q50 and N51, residues shown to be crucial for DNA contacts, into alanines. This mutant is unable to bind a DNA PS2 probe containing a Hox/Exd/Hth motif, even in the presence of EXD and HTH in the bandshift assay. This A50,A51 mutant protein is still capable of inhibiting eye development when expressed in the eye disc using a strong EyE-GAL4 line, although with a lower activity than the wild-type Antp protein (Plaza, 2001).

Based on these in vivo results it was asked whether Antp and Ey might interact directly and thereby inhibit each others activities. Potential in vitro interactions between Antp and Ey were examined using glutathione S-transferase (GST)-Antp fusion proteins immobilized on glutathione-Sepharose beads. These immobilized proteins were tested for their ability to retain in vitro synthesized ³⁵S-labeled Ey protein. Different portions of the Antp protein were produced and tested separately for their ability to interact with Ey. Only the C-terminal portion of Antp including the HD is able to interact with Ey (Plaza, 2001).

To define the regions within Ey and Antp that are required for the interaction of the two proteins, a set of deletion mutants of each protein was tested for the ability to interact in vitro. Structure-function studies of both proteins have delineated specific domains that contribute to their functions as transcription factors as well as their interactions with other proteins. The Antp HD that mediates DNA binding has also been shown to interact with other HD proteins such as Exd. The Ey protein contains two DNA-binding domains, a paired domain and an HD. The paired domain has been shown to interact with different transcription factors. These findings led to an investigation of whether Ey paired domains and HDs are involved in the interaction with Antp. Deleting either of these domains in the Ey protein results in a partial loss of the interaction with the Antp HD. Furthermore, either the Ey paired domain or the Ey HD alone is still able to interact with Antp. These experiments suggest that since each domain is able to interact with Antp, both domains might cooperate for efficient binding of Ey to Antp. Moreover, deletion of the C-terminal part of the Antp protein results in the loss of binding to both the paired domain and the HD of Ey, confirming that the Antp HD is essential for the interaction with Ey. Since the complexes were formed using Ey paired domain and Antp HD purified from bacteria, the two proteins appear to interact directly through their respective DNA-binding region. It was hypothesized that the DNA interface might be important to stabilize the interaction. Indeed, the interaction between the two proteins requires that one of them binds to DNA (Plaza, 2001).

The question of whether the ability of Antp to repress eye development and to interact with Ey can be extended to other homeotic genes. For this purpose, expression of Scr, Ubx, abdA and AbdB were targeted into the eye disc using dpp^blink-GAL4. Expression of these different genes also results in inhibition of eye development by inducing apoptosis. Interestingly, these different proteins are also able to interact with Ey in vitro. Deletion of the ABD-A HD region abrogates binding to Ey, suggesting that also for this protein, the HD is required for interaction with Ey (Plaza, 2001).

GENE STRUCTURE

Antp has two promoters, P1 and P2. The resulting ANTP mRNAs contain 1512-nucleotide (P1) and 1727-nucleotide (P2) 5'-noncoding regions, composed of exons A, B, D, and E (P1) or exons C, D, and E (P2), respectively. Multiple AUG (translational start) codons are present in exons A, B, and C. 252-nucleotide exon D, common to mRNAs from both transcription units and devoid of AUG codons, can mediate initiation of translation by internal ribosome binding in cultured cells. Many mRNAs in Drosophila contain long 5'-noncoding regions with apparently unused AUG codons, suggesting that internal ribosome binding may be a common mechanism of translational initiation, and possibly its regulation (Oh, 1992).

cDNA clone length - 5.0 kb and 3.5 kb depending on 3'UTR length

Bases in 5' UTR - P1 - 1512; P2 - 1727

Exons - P1 - seven; P2 - six

Bases in 3' UTR - 533. There are additional downstream termination signals.

PROTEIN STRUCTURE

Amino Acids - 378

Structural Domains

The six Drosophila proteins that belong to the antennapedia-type Homeobox subfamily are Antennapedia (ANTP), Abdominal-A (ABD-A), Deformed (DFD), Proboscipedia (PB), Sex combs reduced (SCR) and Ultrabithorax (UBX). The ExPASy World Wide Web (WWW) molecular biology server of the Geneva University Hospital and the University of Geneva provides extensive documentation for the 'Homeobox' antennapedia-type protein signature. The four paralogous Hox clusters of mammals contain eight ANTP homologs: HoxA6, HoxA7, HoxB6, HoxB7, HoxB8, HoxC6, HoxC8 and HoxD8.

The ANTP homeodomain is located near the C-terminal end. The protein is rich in prolines and glutamines. A glutamine rich region is found one third of the way into the protein (Stroeber, 1986 and Laughon, 1986).

See four paralogous Hox clusters of mammals for homologies of Antennapedia with mammalian hox proteins.

date revised: 20 March 2001 

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