BIOLOGICAL OVERVIEW

How are boundaries formed between various tissues? One solution to this problem is illustrated by the action of the selector gene apterous. Selector genes establish the autonomy and direct the development of compartments with respect to other adjoining compartments. In the selector affinity model (Garcia-Bellido, 1975) it is proposed that the presence or absence of selector gene expression controls the affinity of cells for each other. Simply put, selector-expressing cells prefer to associate with other selector expressing cells, and to not associate with non-selector-expressing cells. A boundary is formed between cell groups to minimize contact between them (Garcia-Bellido, 1975 and Blair, 1995).

Two groups of cells separated by their adhesive properties are called compartments. The cells in different compartments are lineage restricted, that is, cells of one lineage do not cross into another compartment made up of cells from another lineage, because of their higher self affinity.

Such a boundary process occurs in wing morphogenesis. apterous expressing cells in the dorsal domain of the wing produce one type of integrin (alpha PS1 beta PS), and cells in the ventral half produce another type of integrin (alpha PS2 beta PS). myospheroid codes for beta PS integrin; multiple edematous wings codes for alpha PS1 integrin, and inflated codes for alpha PS2 integrin.

Integrins are cell surface adhesion molecules that act like specific glue, attaching the cells bearing them to specific protein molecules on the surface of other cells or on extracellular matrix molecules in their vicinity. Integrins can communicate information about the extracellular environment to the inside of the cell, and thus alter cell fate. apterous mutation or ectopic expression results in mis-expression of the adhesive integrin molecules, resulting in a breakdown in dorsal/ventral separation of wing cells (Blair, 1994).

The way Apterous works appears to be complex, having at least two distinct outputs. First, Apterous is responsible for making the dorsal cell distinct from ventral, a property that may be due to its activating the gene Dorsal wing (Tiong, 1995). Second, Apterous regulates the expression of boundary determining proteins such as Fringe and Serrate. Serrate is thought to act as a ligand of Notch (Kim, 1995), an adhesive protein that can communicate to the inside of the cell, information about the extracellular milieu. Serrate appears to signal from dorsal to ventral cells to elicit the production of a long range morphogen, perhaps Wingless (Lawrence, 1996). Fringe is remarkable because a boundary forms wherever fringe-expressing and nonexpressing cells meet, a boundary that can organize long-range pattern (Irvine, 1994). Fringe is actually responsible for the induction of Serrate, by an apterous independent mechanism (Kim, 1995). These kinds of proteins insure the separation of cellular compartments and the ongoing developmental pathways of the separated cell lineages.

Serrate, known to be regulated by Apterous, is not required for the initiation of wing development but rather for the expansion and early patterning of the wing primordium. apterous is expressed in dorsal cells of the wing disc; in the absence of ap, the wing blade does not develop, an effect thought to be due to the loss of Ser expression. Consistent with this, ectopic expression of Ser, or of fringe (fng), which leads to the expression of Ser, rescues the loss of the wing in ap mutants. However, the Ser and ap mutant phenotypes are not identical: while in the absence of ap there is no trace of the wing blade, Ser mutants do bear a small marginless wing blade. To explain this difference, the expression of wingless (wg) and vestigial (vg) were compared during wing development in these mutants. The expression of vg decays at the beginning of the third instar in Ser mutants, but is never activated in the wing region of ap mutants. In Ser mutants, the expression of wg never spreads along the wing disc DV interface and resolves into two rings, which fate map the small wing characteristic of Ser mutants. However, in ap mutants, wg expression comes to outline a single circle of expression, which defines proximal hinge structures and the absence of a wing blade, a deficit that is characteristic of these mutants. This is a phenotype very similar to that of vg null alleles. These results suggest that the phenotype of ap mutants cannot be accounted for simply by the absence of Ser. In ap mutants, the development of the wing blade is never initiated; in the absence of Ser, this process is initiated normally, but is aborted early on. Furthermore, since the expansion of wg expression in the AP direction is required for the establishment of the proper size of the primordium, its failure to occur in Ser mutants indicates that, in addition to its role in the establishment of the wing margin, Ser is required to define the proper size of the wing primordium (Klein, 1998).

apterous is also also required for the expression of a second Notch ligand, Delta. The initial stages of the development of the wing blade require Notch signaling and lead to the activation of the vestigial boundary enhancer (vgBE) at the interface between dorsal and ventral cells. Ser is not involved in this event and therefore there ought to be another Notch ligand, under the control of ap, that is responsible for the activation of the vgBE. The product of the Delta gene is a good candidate for this function. During the second instar, Dl is expressed throughout the wing disc, but it is slightly upregulated over the ventral region and shortly afterwards, its pattern of expression is identical to that of vgBE, i.e. a 2- to 3- cell-wide stripe that straddles the DV interface. Furthermore, the expression of Delta is similar to that of the vgBE in Ser and ap mutant discs: in ap mutant wing discs, expression of Dl is lost at the time when the wing primordium is induced, whereas in Ser mutants expression is detected until early third instar. This suggests that Delta might be the activating ligand for the Notch-dependent expression of the vgBE, which operates in the absence of Ser. Consistent with this possibility, ectopic expression of Dl can rescue the loss of wing blade tissue and of wing margin characteristic of ap and Ser mutants (Klein, 1998).

Serrate, in turn, along with Delta, refines Notch function at the DV interface. After the establishment and expansion of the wing primordium, there is a new requirement for Notch signalling in the growth and patterning of the wing blade. In this process, both Serrate and Delta act as ligands for Notch and, as in earlier stages, have different patterns of gene expression, which suggests that they might have different functions. However, ectopic expression of either Dl or Ser will rescue the loss of wing tissue and of wing margin characteristic of ap mutants, and this raises the question of why are there two different ligands to achieve the activation of Notch in these early stages of wing development. It is believed that coexpression of both ligands might result in a different degree of Notch activation than would be achieved individually -- this might allow for a finer degree of regulation for Notch activity. In the presence of Serrate, Delta is able to signal although with a reduced activity level, which perhaps reflects a competition between Serrate and Delta for Notch (Klein, 1998).

A feedback mechanism exists in which Fringe functions to inhibit Serrate by targeting Notch. In contrast to Delta, the effects of ectopic expression of Ser on wild-type discs are restricted to ventral cells. This has led to the suggestion that there is an inhibitor of Serrate activity in dorsal cells and that this inhibitor is under the control of ap. Consistent with this proposal, ectopic expression of Ser in ap mutants is found to be able to induce the expression of downstream targets of Notch in 'dorsal' cells. A variety of arguments have led to the proposal that the dorsal inhibitor of Serrate function is encoded by the fng gene. For example, ectopic expression of Ser with ptcGAL4 results in the activation of Notch targets in two parallel stripes in ventral cells of the developing wing blade, and this can be observed as early as the beginning of the third instar. When Ser is coexpressed with fng, the anterior stripe, but not the posterior one, is lost completely in late third instar discs. Correspondingly the ectopically induced margin structures are reduced to a posterior stripe with characteristics of the posterior compartment. While Fringe suppresses the function of Serrate cell autonomously, it enhances its signaling ability in a nonautonomous manner. Fringe is thought to dampen Serrate signaling by affecting its interaction with Notch, but no evidence has been presented to support this suggestion. Increasing the concentration of Notch appears to be able to titrate the effects of fng. Furthermore, the effects of ectopic expression of fng are partially suppressed by the expression of Notch with fng and are exaggerated by expressing dominant negative Notch molecules with fng. Altogether, these results strongly suggest that a target of Fringe activity is the Notch molecule itself (Klein, 1998).

Fringe functions to inhibit Serrate signaling via Notch. The activity of Fringe can inhibit Serrate signaling by enhancing the intrinsic dominant negative activity of Serrate over Notch. Expression of Ser throughout the late wing disc leads to a strong broadening of the wing veins and a moderate increase in the number of bristles in the notum. Both of these neurogenic phenotypes can be suppressed by coexpressing Notch with Ser, indicating that they are due to a dominant negative effect of Serrate. Ectopic expression of fng alone in the same pattern results in nicked wings with normal veins and a reduction of bristles in the notum, which is associated with the loss of sensory organ precursors. Coexpression of fng with Ser suppresses the extra vein phenotype caused by misexpression of Ser and, therefore, supports the notion that Fringe reduces the ability of Serrate to bind Notch. Fringe is shown to impinge on Notch signaling by the observation that the action of Fringe requires the activity of Su(H). Fringe is not able to rescue the defects caused by Su(H) mutants (Klein, 1998).

GENE STRUCTURE

Bases in 5' UTR - 941

Exons - six

Bases in 3' UTR - 1759; within the 3'UTR are several copies of ATTTA thought to result in transient instability.

PROTEIN STRUCTURE

Amino Acids - 469

Structural Domains

In addition to two Lim domains, Apterous contains a C-terminal homeodomain (Bourgouin, 1992).

The predicted Islet protein contains two LIM domains and a C-terminal homeodomain, with extensive homology to the vertebrate Islet-1 and Islet-2 proteins. The homology is highest in the homeodomain (95% identity to Islet 1 and 2) and somewhat lower in the LIM domains (85%). Overall the Drosophila and vertebrate proteins show 57% identity. A highly conserved 16 amino acids stretch located C-terminal to the homeodomain denotes the Islet-specific domain, and is found in all members of the Islet subfamily, but not in other LIM-HD proteins. The Drosophila Islet homolog shows greater similarity to the vertebrate Islet proteins than to other Drosophila LIM-HD proteins. For example, within the homeodomain, Drosophila Islet shows only 38% amino acid identity to Apterous, which is 92% identical to its vertebrate homolog LH-2. Mutations affecting the isl locus completely abolish immunoreactivity with antibodies that recognize both vertebrate Islet-1 and Islet-2, suggesting that only a single islet homolog exists in Drosophila (Thor, 1997).

The LIM domain is a cysteine-rich domain composed of 2 special zinc fingers joined by a 2-amino acid spacer. Some proteins are made up of only LIM domains, while others contain a variety of different functional domains. LIM proteins form a diverse group that includes transcription factors and cytoskeletal proteins. The primary role of LIM domains appears to be in protein-protein interaction, through the formation of dimers with identical or different LIM domains or by binding distinct proteins. In LIM homeodomain proteins, LIM domains seem to function as negative regulatory domains. LIM homeodomain proteins are involved in the control of cell lineage determination and the regulation of differentiation, and LIM-only proteins may have similar roles. LIM-only proteins are also implicated in the control of cell proliferation since several genes encoding such proteins are associated with oncogenic chromosome translocations. In analyzing sequence relationships among various LIM domains it is suggested that they may be arranged into 5 groups that appear to correlate with the structural and functional properties of the proteins containing these domains. All N-terminal LIM domains (LIM1) are segregated into cluster A, whereas all LIM2 domains of the same proteins constitute cluster B. This relationship suggests that the putative duplication leading to the LIM A and B domains is ancient, preceding their association with different structural motifs (e.g., homeodomains, kinases). Furthermore, the sequence relationships between the LIM domains (LIM1 and LIM2) in the same protein may be conserved by functional constraints based on cooperation between LIMA and B domains. In contrast, the two type C LIM (another LIM domain cluster) domains of some of the LIM-only proteins like CRP are more similar to one another, implying the possibility of a more recent duplication. Cluster D is a rather divergent set of LIM domains that includes the cytoskeletal proteins Zyxin and Paxillin. The closest homologs of Apterous, for both LIM1 and LIM2 domains, are human and rat LH2. The Islet LIM1 and LIM2 domains define Islet as a cohesive subfamily of LIM proteins (Dawid, 1995).

The presence of the LIM domain of mammalian Isl-1 (Drosophila homolog: Islet) inhibits binding of the homeodomain to its DNA target. This in vitro inhibition can be released either by denaturation/renaturation of the protein or by truncation of the LIM domains. A similar inhibition is observed in vivo using reporter constructs. LIM domains in a chimeric protein can inhibit binding of the Ultrabithorax homeodomain to its target. The ability of LIM domains to inhibit DNA binding by the homeodomain provides a possible basis for negative regulation of LIM-homeodomain proteins in vivo (Sanchez-Garcia, 1993).

date revised: 5 Jan 97 

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