BIOLOGICAL OVERVIEW

The Drosophila wing imaginal disc gives rise to three main regions along the proximodistal axis of the dorsal mesothoracic segment: the notum, proximal wing, and wing blade. Development of the wing blade requires the Notch and Wingless signalling pathways to activate vestigial at the dorsoventral boundary. However, in the proximal wing, Wingless activates a different subset of genes, e.g., homothorax. This raises the question of how the downstream response to Wingless signalling differentiates between proximal and distal fate specification. A temporally dynamic response to Wingless signalling is shown to sequentially elaborate the proximodistal axis. In the second instar, Wingless activates genes involved in proximal wing development; later in the third instar, Wingless acts to direct the differentiation of the distal wing blade. The expression of a novel marker for proximal wing fate, Zn finger homeodomain 2 (zfh-2), is initially activated by Wingless throughout the 'wing primordium', but later is repressed by the activity of Vestigial and Nubbin, which together define a more distal domain. Thus, activation of a distal developmental program is antagonistic to previously established proximal fate. In addition, Wingless is required early to establish proximal fate, but later when Wingless activates distal differentiation, development of proximal fate becomes independent of Wingless signalling. Since P-element insertions in the zfh-2 gene result in a revertable proximal wing deletion phenotype, it appears that zfh-2 activity is required for correct proximal wing development. These data are consistent with a model in which Wingless first establishes a proximal appendage fate over notum, then the downstream response changes to direct the differentiation of a more distal fate over proximal. Thus, the proximodistal domains are patterned in sequence and show a distal dominance (Whitworth, 2003).

The Drosophila wing imaginal disc gives rise to the structures of the dorsal mesothoracic segment. This is subdivided into three main regions: the notum, the wing blade, and the proximal wing and hinge. The wing is attached to the thorax via a complex joint comprising a small portion of the appendage, the hinge, which consists of several interlocking sclerites and plates. The wing blade tapers toward the body, forming a short, narrow region that is attached at the hinge. This region shall be referred to as the proximal wing since it is morphologically and mechanically distinct from the hinge itself. Fate mapping of the late third instar imaginal disc has determined that the central portion, the wing pouch, develops as wing blade, a ring surrounding the wing pouch develops as proximal wing and hinge, and the large dorsal territory and a narrow ventral domain form the notum and ventral pleura (Whitworth, 2003).

Previous studies have attempted to follow the development of the proximal part of the wing by analysis of genes that have some expression in the proximal region of the wing disc, e.g., wg or nub, or by the exclusion of markers for notum and wing fates, e.g., teashirt (tsh) and vg, respectively. The identification and analysis is described of a novel marker for proximal wing fate that specifically demarcates the whole of the developing proximal wing tissue, the zinc-finger homeodomain gene zfh-2 (Fortini, 1991). In third larval instar (L3) wing discs, Wg is expressed in a stripe along the D/V boundary, forming the wing margin, and in two concentric rings around the wing pouch. In the adult wing, expression of a wg-lacZ reporter indicates that the two rings of wg delimit the proximal wing. The inner (distal) ring runs from the medial costa, through the humeral crossvein to the alula, and the outer (proximal) ring runs from the proximal end of the proximal costa to the axillary cord (Whitworth, 2003).

In both L3 wing discs and adult wings, Zfh-2 is expressed in a domain that completely overlaps the rings of Wg expression. In L3 wing discs, Zfh-2 does not extend either proximally into the notum or distally into the wing pouch. These observations indicate that, in late stages, Zfh-2 is specifically expressed throughout the developing proximal wing and therefore may be used as a useful marker for proximal wing fate. To determine the extent of coexpression of Zfh-2 and Wg, early L2 discs were examined with anti- Zfh-2 and anti-Wg antisera. Zfh-2 is expressed at this stage in a pattern that directly overlaps with Wg. As development proceeds, Zfh-2 quickly expands to cover the whole of the ventral portion of the wing disc, accompanying the expansion of the Wg domain (Whitworth, 2003).

Taken together, these observations suggest that, at the beginning of L2, the wing imaginal disc is divided into the presumptive notum and the appendage or 'wing' primordium, and that the wing primordium is undifferentiated with respect to the proximodistal axis. This is supported by reports that tsh is also expressed throughout L2 wing discs, but is later restricted to the presumptive notum and hinge regions, and also that vgQE is not yet activated to differentiate the wing pouch. From the dynamic expression pattern of the proximal wing marker zfh-2, it appears that the elaboration of distal elements within the disc, marked by the disappearance of Zfh-2 and concomitant activation of the vgQE, is initiated at the start of L3 at the center of the wing disc where the A/P and D/V boundaries intersect. This suggests that the proximal wing and wing pouch differentiate sequentially, the distal wing pouch being induced later than the already established proximal wing. The early expression of zfh-2 indicates that it is a specific marker for proximal fate (Whitworth, 2003).

At the beginning of the second larval instar, the wing imaginal disc expresses markers of proximal fate, hth and tsh, in the entire anlage. During early L2, the expression of wg and zfh-2 is initiated in an anterior-ventral wedge pattern. The data indicate that Wg function is required to activate zfh-2 expression at this stage, since early removal of Wg function leads to a simultaneous loss of zfh-2 expression. As development proceeds, wg and zfh-2 expression rapidly expands filling the whole of the ventral portion of the wing disc by the end of the second instar. Concomitant with the expansion of wg and zfh-2, both hth and tsh become repressed in the ventral portion of the disc. This transition appears to mark the first P-D differentiation of the wing disc into appendage and notum. However, since zfh-2 is expressed in the entire wing anlage at this time, it is believed that the appendage has not differentiated proximal wing and blade. Around the L2-L3 transition, the wing blade markers nub and vgQE are activated by the combined activity of the Wg and N signalling pathways. Nub and Vg, acting together or independently, repress zfh-2 expression in the center of the disc. This marks the second phase of P-D elaboration where the appendage anlage is split into proximal wing and blade. It is noted that, at this time, hth and tsh remain coexpressed in the notum, where zfh-2 is not expressed. The pattern of zfh-2 expression at this stage suggests that it is still influenced by Wg signalling since it remains restricted to areas of high Wg expression. During L3, the division of the wing disc into three distinct domains is maintained and refined as the individual domains undergo their characteristic patterning. At this time, Hth and Wg are upregulated in the proximal wing anlage, where their activities are interdependent, while zfh-2 expression persists but becomes independent of Wg activity (Whitworth, 2003).

These data further support a qualitative difference in the activity of Wg in the proximal wing compared with wing blade. In addition to the activation of different effectors, previous investigations have shown that ectopic Wg expression in the proximal wing causes large overgrowth of proximal tissue, but similar overexpression in the wing blade produces no overgrowth. This indicates that a different mitogenic response to Wg signalling is activated in the wing pouch compared with proximal wing (Whitworth, 2003).

Therefore, these observations suggest a model in which the wing disc is sequentially partitioned in a proximal to distal direction: notum, proximal wing, and finally wing blade. This view of temporal specification of PD identities is supported by transplantation experiments where L2 wing disc fragments can only differentiate proximal wing structures, whereas L3 disc fragments can produce wing blade elements. In support of the more general applicability of these findings, a study of PD patterning in the Drosophila leg has shown that Wg and Dpp act early to establish the PD axis, but later are not required. These data appear strikingly similar to the results presented in this study and suggest an important common mechanism for PD axis elaboration that has previously been unappreciated. This investigation also serves to emphasize the importance of considering the development of the imaginal disc as an extremely dynamic field, with respect to rapid changes in both size and patterning (Whitworth, 2003).

GENE STRUCTURE

cDNA clone length - 10233 bp

Bases in 5' UTR - 31

Exons - 11

Bases in 3' UTR - 1164

PROTEIN STRUCTURE

Amino Acids - 3005

Structural Domains

Drosophila ZFH-1 has one homeodomain and nine C2-H2 zinc fingers in contrast to the Drosophila protein ZFH-2 with three homeodomains and sixteen C2-H2 zinc fingers. The two proteins can be considered divergent homologs: their homeodomains are both more similar to ATBF1 homeodomains (ATBF1 is a human zinc finger homeodomain protein) than than they are to each other (Hashimoto, 1992). Comparison of each individual homeodomain sequence of Drosophila ZFH-1 and ZFH-2 to other homeodomain sequences indicates that the closest match is to mec-3, a LIM homeodomain expressed in mechanosensory neurons in C. elegans (See Drosophila Islet). Drosophila ZFH-1 contains two isolated fingers, a cluster of four fingers about a third of the way through the protein and a C-terminal cluster of three fingers. Alignment of the ZFH-1 zinc-finger sequences reveals that in their central portions, fingers 3-5 resemble fingers 7-9. This similarity, together with the tandem arrangement of each set of three fingers, raises the possibility that the different zinc-finger regions of ZFH-1 protein were generated by a gene duplication event. In comparison to Drosophila ZFH-1, the fingers of Drosophila ZFH-2 are less clustered (Fortini, 1991). All zinc fingers in ATBF1 and Drosophila Zfh proteins belong to the C2-H2 type except for two each in ATBF1 and Drosophila ZFH-1, which are of the C2HC class, and one in ZFH-2, which has a SCH2 arrangement (Hashimoto, 1992).

The third homeodomain of Drosophila ZFH-2 most closely resembles the one homeodomain of Drosophila ZFH-1. The first homeodomain of ZFH-1 may be a nonfunctional 'pseudohomeodomain', since two unorthodox amino acid residues in the homeodomain may prevent binding to DNA. Both proteins possess regions rich in certain amino acid residues, notable alanine, glutamic acid, serine, proline and glutamine. A particularly long run of glutamines in ZFH-1 is encoded by an opa repeat, a motif of uncertain function associated with Drosophila homeobox genes (Fortini, 1991).

The homeodomains of ATBF1 show 32% or lower sequence identity with the Antennapedia-class homeobox sequences, indicating the divergent nature of these homeodomains. In contrast, the third homeodomain of ATBF1 is 51% identical with the Drosophila ZFH-1 homeodomain, indicating a closer affinity of the homeodomains of the zinc-finger homeodomain to each other than to the Antennapedia-class homeodomain. Higher levels of sequence conservation are observed between ATBF1 and Drosophila ZFH-2 homoeodomains. The first three homeodomains of ATBF1 share 77%, 69% and 61% identity with the corresponding homeodomains of AFH-2. The more C-terminal fourth homeodomain of ATBF1 shows a 46% identity with ZFH-2 third homeodomain. ZFH-1 and ZFH-2 homeodomains homologies are less than 39%, indicating that they are more similar to human ATBF1 homeodomains than to one another (Hashimoto, 1992).

The entire coding region of ZFH-1 is present in some of the larger cDNA clones analyzed. RNA blot analysis of embryos detects a single AFH-1 transcript of 7.5 kb and three ZFH-2 transcripts of 10.5, 11 and 13 kb. Nevertheless, these data cannot rule out the possibliity that the open reading frames encode large precursor proteins that are processed into polypeptides bearing single DNA-binding domains. Different anti-ZFH-1 sera all recognize a common polypeptide species that migrates on denaturing gels with an apparent molecular weight of 145 kDa (which is only slightly larger than the 117 kDa expected for the zfh-1 long open reading frame sequence). These results strongly suggest that the mature zfh-1 gene product contains both the homeodomain and the zinc fingers predicted by its DNA sequence. As sera representing five nonoverlapping regions of the ZFH-2 protein display identical staining patterns in whole-mount embryos, it is thought that the zfh-2 long open reading frame is translated into a single large protein (Fortini, 1991).

date revised: 5 May 2003

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