BIOLOGICAL OVERVIEW

aristaless is involved in both embryonic development and pattern formation in appendages. Embryonically expressed al is involved in the ontogeny of specific head segments and the initiation of appendage development. al expression in larval imaginal discs has a direct involvement in axis specification of appendages. It has also been suggested that wingless, decapentaplegic and aristaless, when expressed in the tips of appendages, serve as organizers for the proximodistal axis.

Expression patterns form the basis of the most persuasive arguement that aristaless determines the presumptive distal tip of imaginal discs. al is expressed in the center of leg discs, the area of the presumptive leg tip. In a partially everted leg disc, al expression does in fact mark the tip. The same holds true for the antennal and wing discs. In the wing, Distal-less is expressed close to the intersection of the wingless expressing dorso/ventral boundary and the dpp expressing anterior/posterior boundary. In a partially everted wing disc, aristaless is expressed in a broad anterior region and at the tip (Campbell, 1993).

aristaless expression in the developing embryo, like that of Distal-less , is found at the intersection of wingless and dpp expressing cells (Schneitz, 1993). Since Distal-less is known to be downstream of wg, hh and dpp, one may ask whether al is also downstream or instead, is a target of Dll (Diaz-Benjumea, 1994). In the absence of available aristaless null mutants, overproduction of Aristaless has been studied to provide some information about its function. Wing duplications are apparent, but duplications of appendages derived from ventral discs (legs) are much less apparent (Campbell, 1993).

The roles played by al and Dll in patterning the legs and wings have been investigated through loss of function studies. In the developing leg, al is expressed at the presumptive tip; a molecularly defined null allele of al reveals that its only function in patterning the leg appears to be to direct the growth and differentiation of the structures at the tip. Null al homozygotes die as embryos with no obviously mutant phenotype. To characterize al function in the development of the leg and wing, large homozygous clones of al null cells were generated early in larval development. In the wing the only clear phenotype associated with these clones is a deletion of part of vein II. In the leg the only region affected by the clones is the tip of the leg where the claw organ is completely deleted. To delete both claws, clones have to be present in both anterior and posterior compartments. Phenotypes like this are identical to those produced by homozygotes of a strong partially functional allele, but are more severe than those of another molecularly defined allele, in which an inversion breaks in the 3' end of the gene. Other than in their appendages, al null clones, adults show extreme al phenotypes in the sternopleurum and scutellum (Campbell, 1988).

In contrast, Dll has been shown to be required for the development of all of the leg more distal than the coxa. Dll protein can be detected in a central domain in leg discs throughout most of larval development, and in mature discs this domain corresponds to the distal-most region of the leg, the tarsus and the distal tibia. Clonal analysis reveals that late in development these are the only regions in which Dll function is required. However, earlier in development Dll is required in more proximal regions of the leg, suggesting it is expressed at high levels in these cells early in development but not later. This reveals a correlation between a temporal requirement for Dll and position along the proximodistal axis; there is discussion of how this may relate to the generation of the P/D axis. Dll is required in the distal regions of the leg for the expression of tarsal-specific genes, including al and bric-a-brac. Dll mutant cells in the leg sort out from wild-type cells suggesting one function of Dll here is to control adhesive properties of cells. Dll is also required for the normal development of the wing, primarily for the differentiation of the wing margin (Campbell, 1998).

The combination of the two secreted signaling molecules Wg and Dpp induces the formation of the P/D axis in the leg of Drosophila. It was originally suggested that the Wg/Dpp combination may establish an organizer at the distal tip that controls patterning along the P/D axis and that this organizer is characterized by expression of the aristaless homeobox gene. Even if such an organizer does exist then al is not absolutely required for its activity because removing al at the tip using a null allele does not prevent formation of the P/D axis, although it does prevent the formation of the structures normally found at the tip of the leg. Ectopic al can induce outgrowths in the wing and these are associated with ectopic Wg expression, but there is no clear explanation for this phenomenon; it is possible that al may have some redundant function in maintaining Wg expression at the tip (Campbell, 1998).

Interaction between Clawless/C15 and Aristaless

The aristaless/clawless cooperation found in Bar repression in the pretarsus may possibly stem from the interactions between Al and Cll. GST pull-down assay was first conducted in vitro to confirm this possibility. Cll was tagged with GST, and a possible binding of Cll to Al was monitored by Western blotting of the eluents from a GST column with anti-Al antibody. GST-Cll was prepared using E. coli cells, and Al was synthesized using reticulocyte lysates. Al signals were detected only when a mixture of GST-Cll and Al was applied to and then eluted from the GST column, indicating that Al and Cll are capable of binding to each other in the absence of DNA (Kojima, 2005).

A polymerase chain reaction-based approach, the systematic evolution of ligands by exponential enrichment (SELEX), was undertaken to determine a possible consensus DNA sequence for the binding of the Al/Cll complex. The nucleotide sequence alignment of 48 fragments obtained after five rounds of enrichment revealed a consensus sequence of 5′-(T/C)TAATTAA(T/A)(T/A)G-3′, which differs from the consensus sequences for the vertebrate homologs of Al (TAATNNNATTA; Alx and Cart proteins) and those for Cll homologs (CGGTAA(T/G)(T/C)(G/C)G; Hox11/tlx proteins (Kojima, 2005 and references therein).

Protein-DNA interactions were examined using the electrophoretic mobility shift assay (EMSA). A double-stranded oligonucleotide containing the SELEX consensus sequence was used as a probe. No or weak retardation bands were detected for Cll or All alone. In contrast, a very strong retardation signal was observed for a combination of Al and Cll. A few base substitutions in the consensus sequences results in a significant reduction in or the abolishment of retardation signals. Thus, the Al/Cll complex is significantly different in target-sequence specificity from Al and Cll, and only the Al/Cll complex can strongly bind to the SELEX-determined consensus sequence (Kojima, 2005).

It may thus be concluded that, in the pretarsus, Al and Cll form a complex capable of binding to specific sequences, which cannot be well recognized solely by Al or Cll, and that the resultant complex plays a central role in al/cll-dependent gene regulation in the future pretarsus. However, it should be noted that the possibility cannot be formally excluded that Al and Cll separately bind to their own consensus sequences and function cooperatively in the pretarsus (Kojima, 2005).

Thus al and cll seem to act cooperatively through the formation of the complex between their protein products. To determine whether vertebrate Al and Cll homologs possess similar properties, possible interactions between the Al/Cll consensus sequence and either one of vertebrate Al homolog, Cart1, or a Cll homolog, Hox11L1 (also called as Tlx2), were assessed. Cart1 is capable of binding to the Al/Cll consensus binding site to some extent, but Hox11L1 can not at all. A considerably strong signal is detected when a mixture of Cart1 and Hox11L1 is subjected to gel retardation. Moreover, strong retardation signals are detected for a mixture of Al and Hox11L1 and that of Cart1 and Cll. Thus, the formation of an Al/Cll-type complex may be an evolutionally conserved feature of Al-type and Hox11/tlx-type homeodomain protein family members (Kojima, 2005).

GENE STRUCTURE

Bases in 5' UTR - 305

Exons - five

Bases in 3' UTR - 237

PROTEIN STRUCTURE

Structural Domains

aristaless has a paired type homeo domain as well as a proline/glycine rich domain, a proline/glutamine rich domain, and a Thr/Ser-rich domain. There is no paired domain (Schneitz, 1993).

date revised: 30 July 98

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