BIOLOGICAL OVERVIEW

Genetic screens have identified maternal genes involved in anterior-posterior and dorsal-ventral axis formation during oogenesis (Schupbach, 1991 and Wilson, 1996). Gurken (expressed in the oocyte) signals through the Egfr receptor (expressed on somatic follicle cells) to specify posterior and dorsal follicle cell fates during oogenesis, and this signaling determines successively the anterior-posterior and dorsal-ventral axes. Mutations in genes on the second chromosome (okra (okr), deadlock, squash, zucchini, aubergine and vasa, and mutations in genes on the third chromosome (the spindle genes spnA, spnB, spnC, spnD, and spnE) produce ventralized eggshells similar to those produced by mutations in the grk-Egfr pathway. okra, spindle-B, and spindle-D are three members of a group of female sterile loci that produce defects in oocyte and egg morphology, including variable dorsal-ventral defects in the eggshell and embryo, anterior-posterior defects in the follicle cell epithelium and in the oocyte, and abnormalities in oocyte nuclear morphology. Many of these phenotypes can be accounted for by a failure to accumulate wild-type levels of Gurken. okr and spnB have now been cloned. okr encodes the Drosophila homolog of the yeast DNA-repair protein Rad54, and spnB encodes a Rad51-like protein related to the meiosis-specific DMC1 gene. In functional tests of the gene's role in DNA repair, okr behaves like its yeast homolog: it is required in both mitotic and meiotic cells. In contrast, spnB and spnD appear to be required only in meiosis. The finding that defects in genes involved in DNA repair give rise to defects in developmental patterning demonstrates that the progression of meiotic events in the oocyte nucleus is coordinated with and tied to developmental processes that determine egg polarity (Ghabrial, 1998).

Mutations in okra show defects in the morphology of the oocyte nucleus, block genetic recombination during meiosis, resulting from the failure to repair double strand breaks (DSBs) associated with crossing over, and interfere with the ability of mitotic cells to repair DNA damage. Studies on chromosome behavior in wild-type ovaries have shown that during stage 3, the DNA in the oocyte nucleus condenses into a compact spherical structure, the karyosome, which is maintained through the later stages in oogenesis until the onset of metaphase I (Spradling 1993). In ovaries stained with a DNA dye, this structure appears as a bright spot within which there is a spot of even greater intensity. In okr, spnB, and spnD mutant egg chambers, this condensation is aberrant and a variety of defective structures are observed. In some cases, the DNA assumes an ellipsoidal shape that is larger than the normal spot, and in others, the DNA is present in clumps that line the inside of the nuclear membrane. As this defect is not seen in grk mutant egg chambers, it does not arise from a defect in grk-Egfr signaling. These findings corroborate those of a previous study on the spindle genes (Gonzalez-Reyes, 1997; see also spindle E/homeless), and more recent studies on vasa have shown that mutations in this gene have a similar nuclear defect (Styhler, 1998; Tomancak, 1998). Ovaries from females mutant for the remaining loci in this group were examined, and it was found that del, squ, zuc, and aub produce the phenotype as well. Thus, the nuclear defect appears to be a phenotype common to all the genes in this class. In spnB or spnD mutant females heterozygous for X chromosomal markers, the frequency of recombination is 10%-25% of normal levels, whereas for a weak okr allele the frequency of recombination is at 50% of normal levels. There is an ~100-fold increase in X chromosome nondisjunction in both spnB and spnD mutant females, and a 17- to 20-fold increase in the crosses involving okr. In summary, there appears to be a requirement for spnB, spnD and okr in meiotic DSB repair (Ghabrial, 1998).

The predominant phenotype produced by females mutant for okr, spnB, and spnD is a ventralization of the eggshell, reflected in a loss of dorsal appendage material that is similar to the phenotype produced by mutations in the grk-Egfr pathway. However, unlike grk and Egfr alleles, which produce fairly discrete ventralized phenotypes, all alleles of okr, spnB, and spnD produce a broad spectrum of ventralization. The mutant females also produce eggshell phenotypes that are not observed in grk-Egfr mutants, including dorsalized eggs with extra appendage material or multiple appendages as well as small eggs, although these phenotypes are comparatively rare. The majority of the eggs produced by okr, spnB, and spnD mutant females, including those that are only mildly ventralized, do not hatch and show no indication of embryonic development. In addition to the dorsal-ventral patterning defects observed in eggshells, okra mutants share another phenotype with mutants in the Grk-Egfr signaling pathway: they produce eggs that often have a second micropyle at the posterior end. Thus okra mutation effects both dorsal-ventral and anterior-posterior patterning defects. These defects are accompanied by defective distribution of Gurken mRNA. GRK mRNA is normally localized to the oocyte during the early stages of oogenesis, and then, in mid-oogenesis, it is localized within the oocyte, first transiently in an anterior-cortical ring (stage 8), and then to a dorsal-anterior patch overlying the oocyte nucleus (stages 9 and 10). In okr mutant ovaries, GRK mRNA is correctly localized to the oocyte in early stages. However, in mid-oogenesis, instances of persistent localization of the RNA in an anterior-cortical ring are observed. The spnB and spnD mutant phenotypes are more severe. In okr mutant ovaries, levels of Grk are variably reduced throughout oogenesis. Thus defects in dorsal-ventral and anterior-posterior patterning can be explained by defective GRK mRNA distribution in okra, spnB and spnD mutants (Ghabrial, 1998).

One way of accounting for the patterning defects caused by mutations in okr, spnB, and spnD is that defects in DSB repair could lead to a general disorganization of the oocyte nucleus that would affect the organization of the oocyte as a whole. However, general defects in the mutant egg chambers, characteristic of a global misorganization of the cytoskeleton, were not observed. Alternatively, a failure to repair DSBs could result in checkpoint-mediated arrest of meiotic progression, which, in turn, would block certain regulated processes in the cytoplasm. In yeast, mutations in the DSB repair genes, RAD51 and DMC1, lead to a checkpoint-mediated arrest in pachytene. The nature of the nuclear defects in okr, spnB, and spnD has not yet been investigated, but, as discussed above, it is possible that these defects could by explained by an early arrest in meiosis. In yeast and multicellular eukaryotes, it is well established that mitotic and meiotic checkpoint proteins, in addition to their effect on genes involved in DNA metabolism, also regulate various cytoplasmic processes, such as spindle assembly and nuclear envelope breakdown. It is therefore possible that in Drosophila the same factors that regulate meiotic cell cycle targets might also be used in parallel to regulate specific developmental targets. Such targets could include proteins that control translation of developmentally important proteins like Grk and K10 (Ghabrial, 1998 and references).

Effectors for this kind of regulation might be found among the genes described above that produce mutant phenotypes similar to those of okr, spnB, and spnD. Such effectors could act downstream of the DSB-repair checkpoint and regulate translation in response to a signal from the oocyte nucleus. Whereas only two of these genes, vasa and spnE, have been cloned, they both encode RNA helicases and are implicated in the translational regulation of grk (Gillespie, 1995; Gonzalez-Reyes, 1997; Styhler, 1998; Tomancak, 1998). Regulation of genes required for the translation of developmentally important proteins, such as Grk, in response to the status of the oocyte nucleus, could serve to coordinate the timing of progression through meiosis with the developmental program. However, because vasa and spnE also produce nuclear defects, the pathway can not be unidirectional. Thus, information from the cytoplasm (e.g., factors required for chromosome condensation or karyosome formation) contributes to nuclear processes as well (Ghabrial, 1998).

Meiotic prophase in Drosophila oogenesis occurs over an extended time period during which many different developmental events take place. The pachytene stage of meiotic prophase is believed to be achieved as early as region 2a of the germarium (for review, see Spradling 1993). Although the repair of DSBs in wild-type ovaries presumably occurs during pachytene, nevertheless, most of the events of oogenesis appear to proceed normally in these mutants, and only a few specific processes appear to be severely affected. This suggests that for Drosophila, only a subset of the processes occurring within the oocyte cytoplasm are dependent on normal meiotic progression within the oocyte nucleus. Perhaps the processes that are linked to progression through meiosis are those for which precise temporal regulation is of particular importance. It will be interesting to see whether similar effects will be associated with meiotic mutants in other developmental systems (Ghabrial, 1998).

GENE STRUCTURE

PROTEIN STRUCTURE

Amino Acids - 784

Structural Domains

The RAD54 gene of Saccharomyces cerevisiae plays a crucial role in recombinational repair of double-strand breaks in DNA. The putative Dmrad54 protein (coded for by okra) displays 46 to 57% identity to its homologs from yeast and mammals. It is a member of the Snf2 family of DNA helicases. Among the Rad54 homologs, the seven helicase domains are almost identical. At the C-terminus, Dmrad54 has an extra stretch of 35 amino acids not present in other Rad54 homologs (Kooistra, 1997).

date revised: 22 September 98

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