Drosophila Rx is expressed in the embryo in the procephalic region and in the clypeolabrum from stage 8 on and later in the brain and the central nervous system. Compared with eyeless, Rx expression in the embryo starts earlier, similar to the pattern in vertebrates, where Rx expression precedes Pax-6 expression. Because the vertebrate Rx genes have a function during brain and eye development, it was proposed that Drosophila Rx has a similar function. The Rx expression pattern argues for a conserved function, at least during brain development, but no expression was detected in the embryonic eye primordia or in the larval eye imaginal discs. Therefore Rx could be considered as a homolog of vertebrate Rx genes. The Rx genes might be involved in brain patterning processes and specify eye fields in different phyla (Eggert, 1998).
Drosophila embryos were examined for Rx expression by whole mount in situ hybridization. During the early stages of embryonic development, the syncitial and cellular blastoderm stage, no signals were detected. With the onset of gastrulation and germ-band extension at early stage 8, the first expression is seen in two dorsolateral spots in the procephalic region. At the end of stage 8, an additional signal is visible in a dorsal region that later on will gives rise to the clypeolabrum. The Rx expression becomes more pronounced at stage 9, when the dorsolateral spots are increasing in size. During extended germ-band stage, when the clypeolabrum becomes a distinct structure of the procephalon, cells expressing DRx are moving closer to the midline, and an additional expression in cells of the central nervous system is detected. During stage 12, when the germ-band retracts and metamerization is clearly visible, the optic lobe starts to invaginate. The cells expressing Rx in the procephalon move even closer together, and the expression pattern splits at this stage and the clypeolabrum expression extends more laterally. Because of the morphogenetic movements during head involution, DRx-positive cells in the clypeolabrum move inside the embryo. At this stage expression is observed in the antennomaxillary complex. Staining in the medial edges of the two brain lobes, in the clypeolabrum, and in the antennomaxillary complex is then seen until the end of embryogenesis. Rx expression in the brain is similar to that of eyeless, but the expression patterns are not completely overlapping. However, in contrast to eyeless, no staining of the eye disc primordia per se is observed, when they become distinct structures during stage 16, nor is DRx expressed in imaginal discs of third-instar larvae (Eggert, 1998).
To characterize the Rxex8 deletion, Southern and PCR analyses was performed using reagents derived from the region. Sequences between RxP(3A2) and act57BP(F5) are deleted in line Rxex8. In contrast, sequences proximal to RxP(3A2) and distal to act57BP(F5) are present. Moreover, the coding regions of CG9235 and act57B are intact, indicating that the deletion specifically affects Rx coding sequences. To confirm that the deletion abolishes Rx expression, immunohistochemistry was performed on adult brains using Rx antibodies. To test the specificity of the antiserum, Rx antibodies were incubated with fly embryos and the staining pattern was compared with previous reports of Rx RNA expression (Eggert, 1998; Mathers, 1997). The antibodies stain both the developing embryonic brain and clypeolabral bud, which is an expression pattern characteristic of Rx. Since Rx is expressed in the embryonic brain, third-instar larval brains were stained and Rx expression was observed in multiple cell clusters. In contrast, Rx was not detected in the eye-antennal, leg, or wing imaginal discs. To confirm that Rx is not expressed in Rxex8 mutants, adult brains from Rxex8 heterozygotes and homozygotes were stained. In Rxex8 heterozygotes, Rx antibody stains locations on the dorsal and posterior brain. In contrast, in Rxex8 homozygotes, no staining was detected. Together, these results demonstrate that Rxex8 is a molecular null allele of Rx (Davis, 2003).
To test whether Rx is required for Drosophila visual development, Rxex8 mutants were analyzed for defects in compound eye development. Scanning electron microscopy of Rxex8 mutants reveals no gross abnormalities in adult eye size, shape or pattern compared to controls. In addition, the ocelli are present in the mutants. Similarly, analysis of thin plastic sections of Rxex8 mutant eyes demonstrates no gross defects in ommatidial structure or organization. To determine whether Rx plays a role in the development of the larval visual system, a negative phototaxis assay was performed on Rxex8 mutant larvae. The percentage of heterozygotes and homozygotes found on the dark quadrants was similar, indicating that there are no detectable abnormalities in phototactic behavior of Rxex8 mutants. Since Rxex8 is a null allele, these data demonstrate that there is no apparent role for Rx in the development of the compound eye or function of the larval visual system (Davis, 2003).
Previous reports demonstrate that Rx overexpression induces ectopic retinal tissue in Xenopus and zebrafish [Andreazzoli, 1999, Chuang, 2001; Mathers, 1997). To test whether Rx is sufficient to induce ectopic eyes, the GAL4/UAS system was used to overexpress Rx in imaginal tissues using the dpp-GAL4 driver. Members of the retinal determination pathway, eyeless, eyes absent, and dachshund, induce ectopic eyes when overexpressed using this GAL4 driver. In contrast, neither Rx nor Xrx1 overexpression resulted in ectopic eye formation, but instead produces loss of adult structures in regions where the driver is active, including the eye, antenna, leg, and wing. Thus, Rx overexpression is insufficient to induce compound eye development and suggests that the effects of Rhjmuller/rxrx1 overexpression in the fly are nonspecific and toxic (Davis, 2003).
Although the eyes of Rxex8 mutants are normal, defects were revealed in the development of another anterior head structure. Examination of Rxex8 heads demonstrates a missing clypeus. Externally, the clypeus is an inverted U-shaped cuticle element located between the antennae and maxillary palps. To determine whether loss of Rx function is the cause of abnormal clypeus development, the ability of the Rx minigene BSKK to rescue the phenotype was tested. While Rxex8 homozygotes fail to eclose and lack a clypeus, Rxex8, BSKK/Rxex8 mutants can successfully eclose and exhibit a rescued clypeus (Davis, 2003).
Genetic analysis of the Rxex8 culture phenotype indicates that more than one gene is affected by this deletion. While Rxex8 homozygotes die as pharate adults, Rxex8/Df(2R)E2 animals die before the pupal phase. In addition, although the Rx minigene rescues pharate adult lethality in Rxex8 homozygotes, the minigene fails to rescue prepupal lethality in Rxex8/Df(2R)E2 animals. Together, these results indicate that the Rxex8 deletion creates a hypomorphic lesion in another gene. Since no known coding sequences, other than Rx, are deleted in the line, regulatory elements of another gene may have been removed (Davis, 2003).
One candidate gene disrupted in Rxex8 mutants is act57B, since the deletion breakpoint is upstream of act57B exon1. Two genomic rescue transgenes were constructed, BH and 7BBH, which contain the entire coding region of act57B but have ~1.0 kb and ~8.2 kb of upstream sequences, respectively. To test these transgenes, it was determined whether they could rescue prepupal lethality in Rxex8/Df(2R)E2 transheterozygotes. While BH and 7BBH fail to rescue the pharate lethal phenotype in Rxex8 homozygotes, Rxex8/Df(2R)E2 mutants carrying either act57B transgene survive prepupal lethality but die as pharate adults. Dissection of these animals from the pupal case reveals that they lack a clypeus. These data indicate that Rxex8 is a hypomorphic act57B allele, and that act57B is required for prepupal survival in Rxex8/Df(2R)E2 transheterozygotes (Davis, 2003).
To determine whether act57B plays a role in adult brain development, the ability of the act57B transgenes to rescue the c232 EB phenotype in Rxex8 homozygotes was analyzed. The 7BBH act57B transgene was recombined onto the Rxex8 chromosome, which was then used to assemble a UCG;act57B rescue, Rxex8/CyO;c232 tester stock. The tester stock was then crossed to w;Rxex8/CyO flies, and the brains from non-CyO progeny were analyzed. Similar to the Rx minigene, the act57B transgenes can rescue the Rxex8 EB phenotype. In addition, when the Rx and act57B transgenes are combined in the same animal, their effect on rescue is additive. Compared with either transgene alone, doubly rescued animals exhibit an increase in the percentage of "wild-type" and "ventral" defective EBs and corresponding reductions in the percentage of "elongated" and "unfused" EBs. This additive effect is specific to the EB phenotype, since the act57B transgene does not significantly improve the ability of the Rx transgene to rescue pharate lethality. Thus, these data indicate that both Rx and act57B are required for normal c232 EB development (Davis, 2003).
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date revised: 2 November 2003
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