The zfh-2 gene displays a limited expression pattern, largely restricted to the CNS of late embryos (Lai, 1991).
Serotonin has been implicated as a stimulatory modulator of locomotion in invertebrates. Although eagle is expressed in lateral and medial serotonin cells, the eg loss of-function mutants often affect the development of only one serotonin cell of each pair. Would the development of one specific cell in each pair be consistently affected? In grasshopper, the two sister cells have slightly different growth patterns and projections, but these are impossible to discern in Drosophila. The two cells can be distinguished from each other by differential expression of zfh-2 (Lundell, 1994). In a wild-type CNS, both zfh-2 and pdm-1 are selectively expressed in the more lateral of two serotonin cells but not in the more medial cell. Both antigens are also expressed in the midline dopamine cells and by the entire midline. The remaining single serotonin cells in eg mutants consistently express both zfh-2 and pdm-1, characteristic of the more lateral serotonin cell (Lundell, 1998).
The Dichaete gene of Drosophila encodes a group B Sox protein related to mammalian Sox1, -2, and -3. Like these proteins, Dichate is widely and dynamically expressed throughout embryogenesis. In order to unravel new Dichaete functions, the organization of the Dichaete gene was characterized using a combination of regulatory mutant alleles and reporter gene constructs. Dichaete expression is tightly controlled during embryonic development by a complex of regulatory elements distributed over 25 kb downstream and 3 kb upstream of the transcription unit. A series of regulatory alleles which affect tissue-specific domains of Dichaete were used to demonstrate that Dichaete has functions in addition to those during segmentation and midline development that have been previously described. (1) Dichaete has functions in the developing brain. A specific group of neural cells in the tritocerebrum fails to develop correctly in the absence of Dichaete, as revealed by reduced expression of labial, zfh-2, wingless, and engrailed. (2) Dichaete is required for the correct differentiation of the hindgut. The Dichaete requirement in hindgut morphogenesis is achieved, in part, via regulation of dpp, since ectopically supplied dpp can rescue Dichaete phenotypes in the hindgut. Taken together, there are now four distinct in vivo functions described for Dichaete that can be used as models for context-dependent comparative studies of Sox function (Sanchez-Soriano, 2000).
To look for defects in the development of Dichaete mutant brains the expression of Zfh-2, a transcription factor expressed in many neuronal lineages, was examined. In comparison to the wild type, in Dr8 stage 16 mutant embryos, a reduction in Zfh-2-positive is seen in cells in the posterior part of the brain, most likely the tritocerebrum. Similarly, ming-LacZ expression, another marker abundant in the CNS, is reduced in this region at stage 12 and 16 when analyzed in null and Dr8 mutant backgrounds. However, anti-Fasciclin II stainings of wild-type and mutant embryos reveal no detectable differences in the location or arrangement of the major brain commissures and longitudinal connectives. Thus, despite widespread expression, Dichaete requirement is mainly restricted to a specific region of the embryonic brain and has no apparent function in the development of the major identified axon tracts in the brain (Sanchez-Soriano, 2000).
A GAL4 insertion within the zfh-2 transcription unit, MS209, (zfh-2MS209) and antisera against Zfh-2 have been used to monitor the expression of zfh-2. 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 (Whitworth, 2003).
wg expression, monitored by a lacZ reporter, is initiated in the early second instar (L2) in an approximately anterior-ventral domain. A lacZ reporter driven by zfh-2MS209 is expressed in a very similar pattern. 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. It is apparent that, although the wg-lacZ reporter gene shows wg expression induced in a narrow domain, the protein can be detected at some distance outside of this region. This is a measure of the mobility of Wg protein. Consistent with this, Zfh-2 nuclear expression is at high levels in the wedge-like domain of wg-lacZ, but is also detectable away from this region at lower levels. 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. The expression of Wg at this stage is proposed to determine the differentiation of the presumptive 'wing primordium' as opposed to notum. However, since Zfh-2 is also widely expressed at this time, it suggests that the 'wing primordium' has not been further subdivided into proximal or distal domains. At the onset of L3, Zfh-2 begins to decline in the center of the disc, suggesting differentiation of more distal fates here. At this time, Wg is still expressed throughout the 'wing primordium' but becomes upregulated at the D/V boundary, where it plays a central role in defining the wing margin. This is also the time when the vg quadrant enhancer (vgQE) is activated on either side of the D/V boundary, marking the establishment of the wing pouch. During mid-L3, the pattern of Wg expression is refined further, becoming upregulated at the periphery of the wing pouch and at the D/V boundary, the presumptive wing margin. Zfh-2 is also refined and is now only present in a ring around the wing pouch overlapping the rings of Wg (Whitworth, 2003).
zfh-2 gene is located on the fourth chromosome and encodes a large Zinc Finger Homeodomain protein. It is expressed in the CNS throughout embryonic and larval life (Lai, 1991; Lundell and Hirsh, 1992) and specifically in the wing imaginal disc. A set of P-elements inserted in the 5' region of the gene has been identified; one of these, zfh-2MS209, expresses GAL4 in the wing imaginal disc in a pattern indistinguishable from anti-Zfh-2 antisera. Significantly, zfh-2MS209 homozygotes and transheterozygotes between zfh-2MS209 and two other independently isolated P-elements (M390.R and M707.R) have a proximal wing deletion phenotype, suggesting that it is required for proximal wing development. Using zfh-2 as a specific marker for proximal wing fate, it has been shown that the P/D axis of the wing imaginal disc is sequentially elaborated from proximal notum to distal wing blade in a temporal sequence that is mediated by a set of differential responses to the signalling molecule Wg (Whitworth, 2003).
zfh-2MS209 homozygotes, while poorly viable, display a recessive proximal wing phenotype. The phenotype consists of deletion of both anterior and posterior wing structures, including the medial costa, parts of the radius, and the alula. The P-element insertion in zfh-2MS209 was mapped to the first intron of the zfh-2 transcription unit. Evidence that the insertion causes the wing phenotype is twofold; the phenotype can be reverted by loss of the P-element, and independently isolated Pelement insertions in the same region of the zfh-2 gene have similar phenotypes. M390.R and M707.R were isolated in a fourth chromosome P-element screen and, like zfh-2MS209, these insertions are poorly viable. Homozygous escapers and transheterozygotes with zfh-2MS209 display similar proximal wing phenotypes (Whitworth, 2003).
When examined for wg expression, the L3 wing discs of zfh-2MS209 homozygotes show a loss of tissue between the rings of wg expression that demarcate the proximal wing; there are no effects on the expression of wing pouch markers, such as nub or vg, or the notum marker tsh. Although null mutations in zfh-2 have not been isolated, the fact that at least three independently isolated P-element insertions show similar phenotypes strongly suggests that, consistent with its expression pattern, zfh-2 is required for the correct development of the proximal wing (Whitworth, 2003).
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date revised: 10 March 2004
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