Localization of swallow-Green Fluorescent Protein in Drosophila oogenesis and implications for the role of swallow in RNA localization.

The localization of a hybrid protein composed of swallow and Green Fluorescent Protein (GFP) during Drosophila oogenesis is reported. I constructed a hybrid gene with GFP inserted into an internal position of swallow. This gene was integrated into the Drosophila genome and provides full swallow+ function, as assayed by the complete rescue of strong swallow mutants. Swallow-GFP is localized at all points along the oocyte cortex from vitellogenic stages of oogenesis through the end of oogenesis. Higher concentrations of swallow-GFP are present at the anterior oocyte cortex than at the lateral and posterior oocyte cortices at Stages 10 and 11, when bicoid and htsN4 mRNA transport from nurse cells and localization in the oocyte are most active. At Stage 9 and at Stages 12-14 swallow-GFP is equally distributed at the anterior, lateral, and posterior oocyte cortices. The position of swallow-GFP in vitellogenic stages is identical to the position of endogenous swallow protein determined by indirect immunofluorescence using an anti-swallow antibody. At the oocyte cortex, swallow-GFP is present in particulate structures that lie within or just internal to the dense cortical actin meshwork. These particles show little or no movement, suggesting that they are attached to or embedded in the oocyte cortex. These observations are most easily interpreted in the context of mRNA anchoring or microtubule organizing functions for the swallow protein.

Identification of hypomorphic and null alleles of swallow via molecular and phenotypic analyses.

The swallow gene of Drosophila is required for the localization of two messenger RNAs, bicoid and hu-li tai shao, to the anterior pole of oocytes during the later stages of oogenesis. In addition, swallow appears to play a role in early embryogenesis, as swallow mutant embryos have defects in early nuclear cleavage and migration. In an effort to identify regions of the Swallow protein that are essential for function, we have initiated a molecular characterization of seven existing alleles of swallow. All seven alleles have been sequenced, and comparison to wild-type swallow indicates that the seven alleles include single amino acid substitutions that identify critical residues, as well as lesions that result in truncated proteins. Western blots using affinity-purified antibodies agree well with the DNA sequence data, and identify a probable null protein. In order to determine the extent to which each allele affects swallow function, females homozygous or hemizygous for each allele were tested for the range and abundance of (1) RNA localization defects, and (2) embryonic cuticular defects. Swallow alleles can be grouped into two categories: those that retain partial function, and those indistinguishable from the putative null allele. Some swallow mutant alleles partially rescue the dominant female sterility of mutations in the atypical 67C alpha-tubulin gene, supporting other studies that suggest a link between RNA localization and the microtubule cytoskeleton.

Oocyte and embryonic cytoskeletal defects caused by mutations in the Drosophila swallow gene.

The maternal effect gene swallow ( swa) of Drosophila is required for bicoid and htsN4 mRNA localization during oogenesis. Swallow is also required for additional, poorly understood, functions in early embryogenesis. We have examined the cytoskeleton in swa mutant oocytes and embryos by immunocytochemistry and confocal microscopy. Mid- and late-stage swaoocytes have defective cytoplasmic actin networks. Stage-10 oocytes have solid actin clumps and hollow actin spheres in the subcortical layer, and late-stage oocytes have uniformly distributed hollow actin spheres in the subcortical layer and in deeper cytoplasm. Swa preblastoderm embryos have uneven and irregularly distributed actin at the cortex, and defective subcortical actin networks that contain hollow and solid spheres. In swa syncytial blastoderm embryos, the abnormal actin cytoskeleton is associated with defects in nuclear distribution, migration and cleavage. Actin cytoskeletal defects correlate with spindle defects, suggesting that the abnormal organization of the actin cytoskeleton allows interaction of mitotic spindles, which induces defective nuclear divisions and loss of nuclei from the surface of the embryo.