BIOLOGICAL OVERVIEW

The dorsal/ventral boundary of the developing wing imaginal disc structures the growth of the entire wing. Interacting here are apterous, expressed in the dorsal margin, and the genes scalloped and vestigial (vg), the latter coding for a novel nuclear protein. apterous expression structures the dorsal and ventral expression of the adhesive integrins. Interactions between dorsal and ventral cells in the growing imaginal disc induce vestigial gene expression through a discrete, extraordinarily conserved imaginal disc-specific enhancer. The link between dorsal/ventral compartmentalization and wing formation distinguishes the development of this sheet-like appendage from that of legs and antennae (Williams, 1994).

scalloped is also expressed in the central and peripheral nervous systems of the developing larva, where it is required for the differentiation of sensory organs. The role of scalloped and vestigial in wing and sensory organ morphogenesis recalls the role of hairy in structuring sensory organs in the wing and leg. scalloped interacts with cut, a proneural gene involved at the wing margin (Jack, 1992). Thus scalloped and vestigial are termed patterning genes, because of their effects on wing sensory organ distribution. However, these two genes are responsible as well for a more global pattern, with ramifications for all of wing development.

The scalloped gene is a downstream effector molecule in the wingless pathway of wing imaginal discs. sd is required for early expression of vestigial. In one-good-turn-deserves-another fashion, vg is later required to maintain sd expression. Early expression of vg, and presumably of sd as well, is widespread and unaltered in wingless mutants, but the phenotypic effect of mutations in both genes is the same: a disrupted wing margin.

Both apterous and wingless are required to retain vestigial. Accompanying vestigial is the presumed scalloped expression, flanking the dorso-ventral margin of the wing imaginal disc. Since early scalloped and vestigial expression do not require wingless, sd and vg are the earliest tissue specific pro-wing genes whose appearance reflects the initial specification of the wing. In this model ap and wg are subsequently required for dorso-ventral compartmentalization (Williams, 1993).

The questions continue: what is the role of the homeodomain proteins distal-less and aristaless in regulation of the early expression of scalloped in wing specification? Is scalloped dependent or independent of these organizers of the distal tip of imaginal discs?

Scalloped is required for Vg function: altering Sd and Vg cellular levels relative to one another inhibits wing formation. Whereas Vg expression is normally restricted to the wing and haltere imaginal discs, a subset of cells within almost all imaginal discs normally express sd. Thus, when Vg is ectopically produced a supply of Sd is already present in those tissues. Since Sd is required for formation of the normal wing, a test was performed to see if there is a similar requirement for Sd in the formation of Vg-induced ectopic wings. The induction of wing tissue overgrowths by ectopic Vg is partially suppressed in animals heterozygous for a strong viable allele of sd and is completely suppressed in hemizygotes for the same viable allele. These observations demonstrate that Vg requires Sd to transform cells to wing fates. This requirement does not appear to reflect a role for Sd as a downstream effector of Vg function, because the expression of Sd alone, whether under the control of dpp or other promoters, does not induce the formation of ectopic wing tissue. Instead, these observations suggest that Sd and Vg could act in parallel to induce wing cell fates (Simmonds, 1998).

In vitro, Vg binds directly to both Sd and its human homolog, Transcription Enhancer Factor-1. The interaction domains map to a small region of Vg that is essential for Vg-mediated gene activation and to the carboxy-terminal half of Sd. To map Vg-Sd and Vg-TEF-1 interaction domains, a Far Western blotting assay was used to screen 15 deleted proteins that remove terminal or internal regions of Vg. Only Vg proteins that contain amino acids 279-335 have any significant affinity for Sd. The Vg-Sd interaction appears to be limited to this 56-amino-acid domain, since Sd does not bind to a deleted Vg protein missing only these amino acids, and a construct encoding only this portion of the protein will still bind to Sd. Significantly, a duplicate panel of Vg deletion proteins probed with TEF-1 shows that TEF-1 interacts with Vg via the same protein domain. Affinity columns containing this protein fragment of Vg bind Sd and TEF-1 protein as well as does full-length Vg. This Sd/TEF-1-binding domain of Vg is serine rich and includes putative phosphorylation sites. Phosphorylation of Vg at these sites may potentially modify the Vg-Sd interaction. This region is highly conserved in Vg proteins from Drosophila virilis and Aedes aegypti. Similar sequences also occur in mammalian genomic and expressed sequence tag databases. The amino- and carboxy-terminal portions of Sd were also tested to map which region of Sd interacts with Vg. Previous studies with TEF-1 have demonstrated that regions mediating interaction with cell-specific TIFs are separable from the DNA-binding TEA/ATTS domain. The Vg-binding region of Sd maps to the carboxy-terminal half of the protein, separable from the TEA/ATTS domain in the amino-terminal half. The carboxy-terminal portion of Sd is also highly similar to TEF-1, which is consistent with the observation that TEF-1 binds to Vg with the same affinity as does Sd. To confirm the direct protein-protein interaction between Vg and Sd in a cellular environment, a yeast two-hybrid assay was used. In yeast, Vg and Sd proteins show a specific and reciprocal interaction when fused to either Gal4-binding domain (pGBDU) or Gal4 activation domain (pACT) fusion constructs. Activation of target genes sd and cut by Vg requires the Sd-Vg interaction domain identified in vitro, implying that this activation is Sd dependent. Moreover, a UAS-vg construct with the Sd-binding domain deleted is unable to induce the formation of ectopic wing tissue, consistent with the observation that this induction is sd dependent (Simmonds, 1998).

A wide variety of studies have suggested that TEA/ATTS domain proteins require tissue-specific transcriptional intermediary factors (TIFs), although relatively little progress has been made toward identifying and characterizing these TIFs . According to the definitions established by the analysis of TEF-1, a TIF for Sd would be expected to bind directly to Sd, to show a restricted pattern of expression, and to be required for Sd function in vivo. These observations presented here, together with the analysis of the coordinate regulation of downstream target genes by Sd and Vg, argue that Vg functions as a tissue-specific TIF for Sd. Although it is possible that Vg interacts with proteins other than Sd, genetic studies argue against this, because all vg mutant phenotypes are shared by sd. In contrast, sd is required for the development of other tissues in which vg is not required. Thus, it is likely that there are other trans-acting factors in Drosphila that interact with Sd. Although the DNA target sequence of Sd is as yet uncharacterized, one target of a yeast TEA/ATTS domain protein (TEC-1) is an element in the TEC-1 promoter. Likewise, in flies, one target of Sd-Vg is likely to be the sd promoter itself, since activation of sd during early development is dependent on Vg, and ectopic Vg induces elevated expression of sd. This suggests a model whereby low levels of Sd expression within wing imaginal discs are elevated in the presence of Vg by positive autoregulation. The dependence on sd of elevated levels of Vg in the wing disc suggests that vg is also a target of positive autoregulation, and direct evidence for this now been obtained. The TEA/ATTS domain protein family is involved in developmental processes as diverse as mammalian neuronal and cardiac muscle development to conidial formation in Aspergillus and pseudohyphal growth of Saccharomyces cerevisiae. Although Vg homologs have not yet been identified in these organisms, genes containing sequences related to the Sd/TEF-1 interaction domain of Vg are conserved in mammals; these genes are thus candidate TIFs for mammalian TEF-1-related proteins. One of these candidate TIFs is expressed in fetal heart tissue, which is intriguing, given that gene-targeted mutations in TEF-1 result in cardiac defects. Future challenges will be to determine whether genes encoding these putative Sd-interacting domains actually function as TIFs for TEF-1 or related proteins, and whether distinct regulatory TIFs have evolved that adapt the transcriptional activities of conserved Sd/TEF-1 homologs to specific functions in different tissues in their respective organisms (Simmonds, 1998).

GENE STRUCTURE

Bases in 5' UTR - 596

Exons - 12

Bases in 3' UTR - 1021

PROTEIN STRUCTURE

Structural Domains

The TEA domain, homologous to that of human TEF-1, is a DNA binding domain. (Campbell, 1992). There is an N terminal serine-rich region.

date revised: 10 February 98

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