eagle (eg) is expressed in neuroblasts and is involved in the fate determination of serotonergic neurons. Serotonin is an evolutionarily conserved neurotransmitter, found in both invertebrates and vertebrates, and involved in locomotor and behavioral roles. Serotonin is produced in descendents of neuroblast NB 7-3. NB7-3 expresses several genes including engrailed, huckebein, seven-up, pdm1 and eagle. Although eg is expressed in both lateral and medial NB 7-3 derived serotonin cells, the eg loss-of-function mutants often affect the development of only one serotonin cell from each pair. The two cells can be distinguished from one another by differential expression of zinc finger homeobox 2 (zfh-2). This dual domain transcription factor has been shown to bind to and activate the DDC gene (Lundell, 1992 and 1994). The differential expression of zfh-2 and of another gene, pdm-1, can be used to determine that the remaining serotonin positive single cell in eg mutants expresses markers characteristic of the more lateral serotonin cell. In a wild-type CNS, both zfh-2 and pdm1 are selectively expressed in the more lateral serotonin cell but not in the more medial cell. engrailed and eagle are expressed in both these serotonin cells. In eg mutants only the medial cell consistently fails to become a serotonin cell. Therefore, even though eg is normally expressed in both serotonin cells, the absence of Eg protein has a more dramatic effect on the fate of the more medial neuron. This important observation suggests that the lateral serotonin cell can maintain its fate in the absence of Eg (Lundell, 1998).
Analysis of gene expression in eg mutants shows that expression of zfh-2 and en is dependent on eg function but expression of pdm1 is independent of eg function. Loss of eg function appears to have no affect on the expression of pdm1. Clearly, the serotonin cell phenotype in eg mutants is not directly related to the expression of pdm1. Loss of eg function affects the expression of zfh-2 in the lateral serotonin neuron. Loss of eg function affects the expression of en in both serotonin neurons. Thus, eagle is necessary for the maintenance of both engrailed and zfh-2 expression in the serotonin neurons (Lundell, 1998).
The simplest explanation for the difference between the medial and lateral serotonin neurons is that the lateral cell contains a redundant mechanism that allows continued synthesis of serotonin in the absence of Eg protein. This redundant mechanism is not 100% efficient, since not all segments in an eg mutant CNS contain serotonin cells. Since zfh-2 but not pdm1, expression is affected in eg mutants, it is suggested that zfh-1 is a potential factor for this redundant pathway, which establishes eg-independent serotonin synthesis. In an eg-loss-of-function mutant, the loss of Ddc expression is always accompanied by the loss of en expression, but can occur independently in the two serotonin cells. In a hemisegment where both cells fail to express Ddc, neither cell shows en expression. In a hemisegment where only the lateral serotonin cells continues to express Ddc, this lateral cell shows en expression but the medial cell does not. It is concluded that the two serotonin cells have distinctive regulatory networks. In the medial cell, eagle is required for the serotonin fate, while in the lateral cell, engrailed and zhf-1 are required but eagle is not. It is shown that hypomorphic alleles of eagle can produce viable adults that have a dramatic reduction in the number of serotonin-producing neurons (Lundell, 1998).
The overlap between Zfh-2 and Wg throughout the larval stages suggests that zfh-2 may be activated by Wg signalling. In order to test this, the effect of ectopic Wg expression on zfh-2 expression was analyzed. dpp-GAL4 was used to drive the expression of a UAS-wg construct along the A/P boundary in all domains along the P/D axis. Under these conditions, Zfh-2 shows a broad expansion into the presumptive notum region but no ectopic expression in the wing pouch. This indicates that ectopic Wg can activate zfh-2 at a distance from its site of expression (Whitworth, 2003).
In the wild type proximal wing disc, Nub overlaps the Zfh-2 domain at the inner ring of Wg. This situation is recapitulated when ectopic Wg is driven by dpp-GAL4, since ectopic Nub is only detected in regions of high Wg expression. This indicates that ectopic Wg is inducing a response similar to the inner ring and suggests that the region expressing ectopic Zfh-2 is now differentiating as proximal wing (Whitworth, 2003).
To assess whether the cells expressing ectopic Zfh-2 have altered their fate, the expression of the notum marker Tsh was analyzed. Tsh is completely repressed throughout the region of ectopic Zfh-2, indicating that cells are no longer fated as notum. Since Wg is an important factor in the development of wing blade, the expression of a wing blade marker, Vg, was also examined. Vg shows no expansion into more proximal regions and is still restricted to the wing pouch (Whitworth, 2003).
These observations support the idea that Wg is able to direct the differentiation of proximal wing fate at the expense of notum. This can also be inferred from an examination of the phenotype of pharate adults of genotype dpp-GAL4/UAS-wg. An outgrowth of tissue is seen with characteristic proximal wing sclerites and a concomitant loss of macrochaete and scutellum normally associated with the notum (Whitworth, 2003).
This experiment to induce ectopic proximal wing was carried out in such a way that the ectopic Wg was expressed in a pattern that intersects the endogenous domain of Wg expression and results in a continuous region of Wg expression. The observed effects could therefore be interpreted as directed overgrowth of the endogenous proximal wing and not differentiation of proximal wing de novo. This is supported by observations that ectopic expression of Wg in the proximal wing anlagen causes disc overgrowth and consequently overgrows proximal wing tissue. In view of this, attempts were made to reproduce the effects of ectopic Wg in a manner that was discontinuous with the endogenous wg domain. To achieve this, clones of wg-expressing cells were induced that were contained entirely within the notal region, outside of the endogenous proximal wing. To prevent diffusion of ectopic Wg, a construct (UAS-Nrt-flu-wg) was used that directs the expression of a membrane-tethered form of Wg marked with a Flu epitope tag. The colocalization of Nrt-Wg bound to the cell surface and GAL4-expressing cells confirms two things: (1) that the only cells expressing GAL4 induce expression of the UAS construct; (2) that the Flu epitope marker is not detectable beyond the site of expression, indicating that the Flu/Wg hybrid molecule is membrane-bound and not detectably diffusible. When stained to reveal Zfh-2, it can be clearly see that Zfh-2 is induced at a distance of several cell diameters from the site of Nrt- Flu-Wg expression, producing a large zone of Zfh-2-expressing cells surrounded by an epithelial fold. This observation is surprising considering that Wg protein is believed to be tethered to the cell membrane. In the wing pouch, the same construct elicits a Wg signal response only in the expressing cell and its immediate neighbors. Although the nature of the long-range induction cannot be explained at present, this does confirm that ectopically expressed Wg is able to induce the expression of Zfh-2 and therefore drive differentiation of proximal wing fate (Whitworth, 2003).
Taken together, these results show Wg is sufficient to direct the differentiation of proximal wing fate. Furthermore, Wg can only induce ectopic Zfh-2, and thereby proximal wing fate, in the more proximal notum tissue and not in the more distal wing pouch (Whitworth, 2003).
To determine whether Wg is required for zfh-2 expression and how this changes through development, a number of methods were employed to remove Wg function at different developmental stages. The temperature-sensitive allele wgIL114 was used in trans to a wg-lacZ insertion line to create a conditional null mutant. When larvae were moved to the restrictive temperature just prior to L2, Zfh-2 expression was no longer detected in the wing primordium. This indicates that Wg function is required at least for initiation of zfh-2 expression in the L2 wing disc. Wg signal transduction can also be antagonized by the expression of a dominant negative TCF (DN-TCF), a component of the Wg signalling pathway. dpp-GAL4 was used to drive expression of DNTCF along the A/P boundary from early larval stages. zfh-2 fails to be activated in the presence of DN-TCF, even into L3. This further supports the findings that Wg signal transduction is absolutely required for initiation of zfh-2 expression during L2 (Whitworth, 2003).
However, when Wg signalling is removed later in L3, under all experimental conditions tested, no effect on zfh-2 expression is seen. Large wg null clones or the expression of a dominant negative form of wg during L3 shows no detectable reduction in Zfh-2 levels. Similarly, no loss of Zfh-2 is observed with clonal expression of DN-TCF. This shows that, after activation in the L2, Wg activity is no longer required during L3 for the maintenance of zfh-2 expression (Whitworth, 2003).
Taken together, these results show that the regulation of zfh-2 by Wg is temporally dynamic. Although Wg is required early to activate zfh-2, when both are extensively coexpressed, Wg appears not to be required later to maintain zfh-2 expression. This raises the possibility that, once activated, zfh-2 might regulate its own expression by an unknown mechanism. This interpretation would also mean that the downstream response to Wg signal is temporally dynamic, since it appears that one set of genes, e.g., those required to determine proximal wing fate, is activated early and later becomes independent of Wg, and then another set of genes is in turn activated, e.g., those delimiting the wing blade (Whitworth, 2003).
Ectopic expression of Wg can induce zfh-2 only in regions outside of the wing pouch. This suggests that some factor has a repressive effect on zfh-2 in the pouch that cannot be overcome by Wg activation. Genes fundamental to wing blade development may be responsible for this repression. Since Vg expression is restricted to the presumptive wing blade and is required for wing blade development, the effects of ectopic expression of vg on the proximal wing region were examined. Using dpp-GAL4 to direct expression of vg along the A/P boundary represses zfh-2 in the proximal wing region. Endogenous wg expression, monitored with the wg-lacZ reporter, also shows complete repression at the point of intersection. Conversely, in vg1 mutant discs, the Zfh-2 expression domain is expanded into the remnant of the wing pouch and shows a greater overlap with Nub expression than in the wild type. In vg1 discs, much of the wing pouch anlagen fails to develop, and this is accompanied by complete loss of Wg expression at the wing margin; however, the two rings of Wg delimiting the proximal wing are maintained. This suggests that derepression of the zfh-2 domain into the pouch region is not caused by ectopic Wg activity (Whitworth, 2003).
Since the loss of vg does not result in complete derepression of zfh-2, it suggests that another repressor must be acting with vg. Nub is also required for wing blade development. Hypomorphic nub alleles display a severely reduced wing phenotype and a transformation of distal structures into proximal ones. nub2 discs show a complete loss of the inner ring of Wg and an expansion of Wg expression at the wing margin. In nub2 mutant discs, Zfh-2 expression is expanded into the wing pouch, along the line of the wing margin. This indicates two things: (1) that Nub normally acts to repress zfh-2 expression, and thus proximal wing fate, within the wing pouch, and (2) that ectopic zfh-2 is induced where Wg is expressed. Therefore, in an environment of reduced Nub, it can be predicted that ectopic Wg would be able to induce ectopic Zfh-2. To test this, ectopic Wg was expressed in a nub mutant background. As in the nub2 background, Zfh-2 is ectopically induced in the wing pouch along the wing margin . In addition, Zfh-2 can now be detected in the wing pouch along the line of dpp-GAL4, where high levels of Wg are ectopically expressed. This demonstrates that, in an environment of reduced Nub, Zfh-2 expression can be induced wherever Wg is expressed and is no longer restricted from the pouch. It is noted that, whereas Wg expression is expanded at the wing margin in nub discs, where ectopic Wg is induced in a nub background, endogenous Wg is expressed normally at the wing margin; however, the reason for this is unknown (Whitworth, 2003).
In nub discs, vg expression is unaffected, but vg is upregulated by high levels of ectopic Wg. Thus, it appears that the increased levels of Vg are not sufficient to repress Zfh-2 in the absence of Nub when Wg is present at high levels. However, further from the source of ectopic Wg, Zfh-2 is not induced in the nub background, and presumably here, Vg alone can repress Zfh-2. Taken together, these data suggest that zfh-2 expression is regulated by a balance between activation by Wg and repression by a combination of Nub and Vg, acting together or independently. The loss of either Nub or Vg is enough to cause only a partial derepression of zfh-2 in the wing pouch, indicating that alone neither Nub nor Vg is sufficient to completely repress proximal wing fate. However, their combined action, as is the case in the wild type, is able to completely repress zfh-2 expression in the wing pouch. Thus, these factors act to restrict zfh-2 expression to the periphery of the wing disc, thereby defining the distal limit of the proximal wing primordium (Whitworth, 2003).
Recent work has indicated that the homeobox gene homothorax (hth) is required for the correct development of the proximal wing by both upregulating Wg expression in the proximal wing and limiting the area of wing blade differentiation. Since loss of Hth function in the proximal wing leads to a dramatic reduction in the level of Wg expression, attempts were made to determine whether Hth is also required for regulation of Zfh-2 expression. In hth- clones, neither the expression pattern nor the level of Zfh-2 is altered compared with neighboring wild type tissue. This is consistent with the observation that late removal of wg does not affect the expression of zfh-2. Similarly, ectopic expression of Hth shows no effect on zfh-2 expression. These data suggest that Hth does not play a role in establishing or regulating the determination of proximal wing fate, since no change in the expression of Zfh-2 was observed. Thus, it appears that the prime functions of Hth in the proximal wing are to maintain Wg expression and define the limits of the wing pouch (Whitworth, 2003).
A 40-bp upstream regulatory region of the DOPA decarboxylase gene (Ddc), that is important for cell-specific expression in the Drosophila CNS, has been investigated. This region contains two redundant elements which when simultaneously mutated result in lowered DDC expression in serotonin neurons. A protein binding site within one of these elements has been uncovered, and a factor has been cloned that binds to the site. This factor is the product of the zfh-2 gene, a complex homeodomain/zinc finger protein previously identified by binding to an opsin regulatory element. The in vivo profile of Zfh-2 in the larval CNS shows intriguing overlap with DDC in specific serotonin and dopamine neurons. Zfh-2 is related to a human transcription factor ATBF1. The multiple homeodomain and zinc finger motifs in these two proteins show a similar linear arrangement that implies coordinate action among the motifs. In addition, the homology defines a new homeodomain subtype (Lundell, 1992).
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