BIOLOGICAL OVERVIEW

The Drosophila rotund (rn) gene is required in the wings, antenna, haltere, proboscis and legs. Previously identified in the rotund region was a member of the Rac family of GTPases, denoted the RacGAP84C or rotund racGAP gene (see Raymond, 2001). However, rotund racGAP is not responsible for the rotund phenotypes. The rotund gene has now been isolated. It is a member of the Krüppel family of zinc finger genes. The adjacent roughened eye locus specifically affects the eye and is genetically separable from rotund. However, roughened eye and rotund are tightly linked, and thanks to this connection, the roughened eye transcript was isolated. Intriguingly, roughened eye is part of the rotund gene but is represented by a different transcript. The rotund and roughened eye transcripts result from the utilization of two different promoters that direct expression in non-overlapping domains in the larval imaginal discs. The predicted Rotund and Roughened Eye proteins share the same C-terminal region, including the zinc finger domain, but differ in their N-terminal regions. Each cDNA can rescue only the corresponding mutation and show negative effects when expressed in each other's expression domain. These results indicate that in addition to the differential expression of rotund and roughened eye, their proteins have distinct activities. rotund and roughened eye act downstream of early patterning genes such as dachshund and appear to be involved in Notch signaling by regulating Delta, scabrous and Serrate (St Pierre, 2002).

The Drosophila rotund locus is recessive viable causing male and female sterility as well as defects in adult body structures (Cavener, 1986). These defects include those in the antennae, wing, haltere and proboscis as well as fusion of all five leg tarsi into one fused tarsal-like segment. Analysis of third instar larvae imaginal discs revealed localized cell death in the regions giving rise to the affected adult structures (Kerridge, 1988). The rn locus has previously been molecularly analyzed (Agnel, 1989) and a cDNA encoding a member of the Rac family of GTPase-activating proteins (GAP) was isolated from this genomic region (Agnel, 1992b). Since this gene was located in the rn genomic region it was denoted the rotund racGAP (rnracGAP), but molecular analysis of multiple rn alleles have indicated that the rnracGAP is not responsible for the rn phenotypes (Agnel, 1992a). In fact, all studies to date instead point to an uncharacterized larger transcript (Agnel, 1992a; Hoemann, 1996) as the likely candidate for the rn gene (St Pierre, 2002).

The closely linked roughened eye (roe) locus affects a late step in the development of the eye, and roe mutants display rough eye morphology and reduction of photoreceptors (Renfranz, 1989). The roe gene is genetically separable from rn, but the two genes show complex complementation (Brand, 1990; Kerridge, 1988; Ma, 1996). This finding previously led to the suggestion that rn and roe may be 'two classes of mutation of the same gene, each of them disrupting a subfunction' (Ma, 1996). To address the tight link between these two adjacent loci, the rn and roe genes have been isolated. Intriguingly, roe is part of the rn gene. Each cDNA can rescue only the corresponding mutation and misexpression in one another's domain of expression has negative effects. These results indicate that these two loci are genetically separable not only because of their differential expression but also because of distinct activities of the Rn and Roe proteins (St Pierre, 2002).

Regarding the function of the rnracGAP, both this work and previous studies argue against any involvement of rnracGAP in the rn or roe phenotypes (Agnel, 1989; Agnel, 1992a; Hoemann, 1996). In situ studies indicate that rnracGAP is only expressed at low levels in the imaginal discs during pupal stages (Agnel, 1989; Agnel, 1992a; Hoemann, 1996). In addition, there is no obvious difference in the severity of rn and roe phenotypes whether or not the rnracGAP is simultaneously removed. For instance, no significant difference was found in the severity of rn leg phenotypes in rn²⁰/rn²⁰ (that removes rn, roe and rnracGAP) compared to rn¹⁹/rn²⁰ (rn¹⁹ does not remove rnracGAP). Similarly, roe³/rn²⁰ (roe³ has a premature stop codon in the roe-specific exon) displays as severe an eye phenotype as rn²⁰/rn²⁰. Furthermore, rn and roe mutants can be rescued with the rn and roe cDNAs. Recent studies may indicate an involvement of rnracGAP specifically in male fertility, and high levels of rnracGAP expression have been observed in the adult testis (Agnel, 1989; Agnel, 1992a; Hoemann, 1996). The rn⁸⁹ and rn^GAL4#5 P-element insertions may provide useful starting materials for the generation of mutations specifically affecting the rnracGAP by local P-element mobilization (St Pierre, 2002).

Little is known about the genetic cascades within which roe and rn are acting. The results from eye-antennal imaginal discs indicate that roe acts at the morphogenetic furrow, as evident both from its expression and from the effects on Delta and Scabrous expression in roe mutants. Both Dl and sca play roles in spacing the array of ommatidial preclusters in the morphogenetic furrow, and it is interesting to note that the expression of roe at the furrow is not evenly distributed and appears stronger in clusters of cells. Genetic screens for modifiers of the N^spl mutation have identified roe as an enhancer, and sca and Dl as suppressors of the N^spl eye phenotype (Brand, 1990). Given the dynamics of N signaling, these results support models where Roe acts to either positively or negatively regulate Dl and Sca. A genetic interaction screen for enhancers of glass also identified roe (Ma, 1996), an interesting finding given that ectopic expression of roe using GMR-GAL4 leads to a glass-like phenotype with a loss of bristles and pigment cells (St Pierre, 2002).

In the leg, rn expression is the earliest marker known for tarsal development (Couso, 1998). rn is required for the development of this region and for its subsequent patterning, as observed by the loss of Ser expression. Thus, the transient expression of rn in the leg might reveal that the intercalation of the presumptive tarsal region between the distal tip and medial leg regions occurs during early third instar (St Pierre, 2002).

It is increasingly common, even in invertebrates, to find genes that utilize two or more promoters. Although this may lead to the generation of different proteins, it is often unclear whether the proteins have distinct activities. In fact, this issue is not easily resolved by traditional forward genetics and subsequent molecular analysis, since even if a locus can be genetically dissected into different subfunctions, this does not identify whether the different proteins have distinct activities. Perhaps the best way to test whether the variant proteins are interchangeable in vivo, is by cross-rescue in each others domain of expression. The rn gene is a clear example of a locus that utilizes both tissue-specific promoters and functionally distinct proteins to achieve functional diversity, a scenario likely to be observed more and more frequently in the post-genomic era (St Pierre, 2002).

GENE STRUCTURE

The roe gene shows complex complementation with rn and a number of mutant roe alleles also exhibit a rn phenotype (Agnel, 1989; Brand, 1990; Kerridge, 1988; Ma, 1996). The rn gene structure together with previous molecular work on rn alleles gave some initial insight into the identity of roe. Particularly informative were the rn^Delta2-2 and rn¹⁹ alleles. The rn^Delta2-2 P-element excision allele contains a deletion in the rn 5' region removing the first and part of the second exon of rn. Complementation analysis of rn^Delta2-2 shows that it is a null allele of rn but that it does not cause roe phenotypes. Furthermore, the rn¹⁹ allele, shown to contain a larger deletion in the rn 5' region (Agnel, 1989), acts as a rn null allele and, although it removes at least one other lethal complementation group, does not cause roe phenotypes. These results indicate the existence of roe-specific functions encoded in the genomic region proximal to the breakpoint of rn¹⁹. One model could be the existence of roe specific exon(s) that are spliced and utilized specifically in the eye. However, the fact that rn¹⁹ extends further distally, uncovering other complementation group(s), but does not produce roe phenotypes argues against eye-specific splicing of a long transcript originating from a promoter in the rn region. Instead, a more likely scenario would be the existence of an eye-specific promoter and exon(s). This notion was further supported by analysis of P-element insertions in the rn 5' area that result in the rn phenotype and matching expression but not in the roe phenotype or eye disc expression. These results prompted a search for additional exons that could explain the molecular nature of the roe gene. By screening a larval cDNA library with a rn 3' probe and by subsequent PCR analysis the roe cDNA was isolated. The roe gene utilizes the same two 3' exons as rn but contains a different 5' exon. As a result the predicted Roe protein shares the C-terminal region (including the ZF domain except the first finger) with Rn but differs in the N-terminal region. It is interesting to note that the rn genomic structure was not revealed by the analysis of the sequences carried out by the Drosophila Genome Project. Although parts of the rn coding regions were identified (CG14600, CG14601, CG14603 and CG10040), the rn transcript was not predicted, probably because rn has several small exons spread over 50 kb. In contrast, the roe transcript was accurately predicted, short of one aa error in the splice junction between exons 1 and 2 (CG10040). At the submission of this study, the rn and roe cDNAs had not been isolated in the BDGP or RIKEN expressed sequence tag (EST) projects (St Pierre, 2002).

The proposed genomic structure of the rn locus fits well, both with previous studies as well as with molecular analysis of rn and roe alleles. (1) rn¹⁶ and rn²⁰ are deletions that show both rn and roe phenotypes, while the rn¹⁹ deletion only shows rn phenotypes (Agnel, 1989). In agreement, rn¹⁶ deletes both the common ZF coding exons and roe-specific exons, rn²⁰ deletes the whole region, and rn¹⁹ removes most of the rn-specific exons. (2) roe³, a strong roe-specific allele, was sequenced and shown to be the result of a nonsense mutation in the roe-specific exon. This mutation does not affect the common 3' exons and explains why roe³ acts as a roe null allele but does not show rn phenotypes. (3) rn⁸⁹, a lacZ-containing P-element transposon allele was shown to be inserted within the 5' region of the rn gene. This explains why it only displays rn and not roe phenotypes. In addition, imprecise excision of rn⁸⁹ yielded rn^Delta2-2, which contains a deletion of the first and part of the second rn exon. As expected, rn^Delta2-2 displays a rn null phenotype but no eye phenotype. In agreement with this, in situ hybridization failed to detect any rn transcript in rn^Delta2-2 mutant discs. rn^GAL4#5 was generated by P-element conversion of rn⁸⁹. rn^GAL4#5 displays a stronger leg phenotype than rn⁸⁹, possibly due to differences in the structure of the P element, but again no aberrant eye phenotype. Wild-type revertants of rn⁸⁹ and rn^GAL4#5 were generated that complement other rn alleles, verifying that in both cases the rn phenotype was caused by the P-element insertion (St Pierre, 2002).

cDNA clone length - roughened eye: 2444; rotund: 3354

Bases in 5' UTR - rotund: 522

Bases in 3' UTR - rotund: 304

PROTEIN STRUCTURE

Amino Acids - Roughened eye: 692;Rotund: 943

Structural Domains

The cDNA sequence indicates that rn encodes a Krüppel-type zinc finger (ZF) protein and contains six C₂H₂ ZFs. The predicted Rn protein has a high degree of homology to the predicted protein of Drosophila gene CG5557, and to C.elegans Lin-29 (Rougvie, 1995). Over the ~150 aa ZF domain these two proteins display 84%-90% identity to Rn. Among mammalian proteins, a recently identified rat cDNA, Cas-Interacting Zinc finger (CIZ) (Nakamoto, 2000), displays the highest homology (59% in the ZF) to Rn. Rn and CG5557 also share a short C-terminal domain of high homology not found in the other proteins. In line with the complex genetics of this area, the alignment of the rn cDNA with the genomic sequence reveals that rn spans ~50 kb and extends on both sides of the rnracGAP (St Pierre, 2002).

date revised: 15 May 2002

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