BIOLOGICAL OVERVIEW

homeless, now termed spindle E, was initially detected in a P element insertion screen: a female sterile line was obtained in which the insertion mapped at 80A5-6. spindle E mutants contain mislocalized oocytes in a small percentage of their vitellogenic egg chambers. Ovaries dissected from mutants contain a range of late-stage phenotypes. A wild-type egg chamber at stage 14 of oogenesis possesses two dorsal eggshell respiratory appendages, just lateral to the dorsal midline. Ninety to ninety-five percent of the mutant egg chambers show aberrant appendage formation: the majority possess only one appendage or fused appendages emerging from one base on the dorsal midline. The dorsal appendage phenotype suggests that spn-E plays a role in dorsalization of the oocyte, while the mislocalization phenotypes suggest that spn-E is involved in an even earlier role in egg polarity (Gillespie, 1995).

spindle E encodes a DEAD box protein that is likely to function as an RNA helicase (an RNA unwinding function). The similarity of Spn-E to proteins that function through binding RNAs suggests a possible role for Spn-E in RNA processing, transport, or stabilization. The localization patterns of seven mRNAs known to be localized during oogenesis were examined. These transcripts fall into three classes: (1) those that fail to be transported or localized correctly in some fraction of spn-E egg chambers; (2) those that are localized correctly but are reduced in amount, and (3) those that remain unaffected in spn-E mutants. Gurken mRNA is localized appropriately in the majority of stage 9 and stage 10 spn-E mutant egg chambers. However, about 30% of hls mutant chambers reveal a defective pattern. Some show no GRK mRNA localization, some show an anterior ring, and some show a dorsal patch that is broader than normal. Although Oskar mRNA is transported to the oocyte normally in these early stages, later localization to the posterior pole is defective. In the majority of S10 egg chambers, OSK mRNA is diffuse throughout the oocyte. In spn-E mutant egg chambers clearly aberrant Bicoid mRNA distribution is observed in stage 8. In most egg chambers, some BCD mRNA is transported to the oocyte, but the majority remains in the nurse cells, concentrated at the apical cortex. In addition, much of the BCD message within the oocyte is not transported laterally toward the periphery but instead remains centrally located. The failure to transport BCD and OSK mRNAs is the earliest defect in RNA transport observed in spn-E mutants (Gillespie, 1995).

Examination of two other anteriorly localized transcripts, the K10 and ORB mRNAs, identifies a second class of messages. In spn-E mutants, K10 transcripts are transported to the oocyte and later localized to the anterior edge, but the level of transcript is greatly decreased relative to wild type, specifically in vitellogenic stages. In spn-E mutants ORB mRNA accumulates normally in oocytes, but the anterior localization, while present, is significantly weaker in stage 8 to stage 10 oocytes. Bic-D and HTS mRNAs are unaffected in spn-E mutants (Gillespie, 1995).

Because the Spn-E protein has homology to RNA-binding proteins of the DE-H family, it seems likely that Spn-E is acting directly to localize RNAs in oogenesis. It is possible, however, that mutations in the gene are affecting RNA localization by disrupting the microtubule architecture that is a common component of the RNA's localization mechanism. Hence, the localization of a Kinesin heavy chain:beta-Galactosidase fusion protein was examined in a spn-E mutant background to analyze one aspect of microtubule structure and function. In wild-type stage 8 and stage 9 egg chambers, microtubule organizing centers (MTOCs) are located at the anterior of the oocyte and direct the formation of a gradient of microtubules whose plus ends extend toward the posterior pole. The fusion protein is thus propelled by the plus-end-directed kinesin motor function and is present in a band at the posterior of the oocyte, as detected by beta-Gal activity on an X-Gal substrate. In spn-E mutants, the fusion protein is detected in the center of S8 oocytes and in central and lateral portions of S9 oocytes, suggesting that microtubule structure or function is disrupted. Microtubule organization was examined directly in spn-E mutant egg chambers by comparing anti-alpha-Tubulin immunofluorescence in wild-type and mutant ovaries. In a majority of stage 8 spn-E chambers, a dense network of microtubules is present throughout the oocyte in place of the normal anterior MTOC localization. In all cases, the oocyte nucleus is correctly positioned at the dorsal anterior corner of the oocyte. Despite the presence of an aberrant network in stage 8 and 9 egg chambers, a normal rearrangement was observed in stage 10b spn-E oocytes. Thus the inappropriate formation of an extensive microtubule meshwork is confined to stage 8 and stage 9 egg chambers (Gillespie, 1995).

Spn-E could be functioning in one of at least two ways: (1) Spn-E could act downstream of the signaling pathways that induce correct microtubule reorganization during stages 8 and 9, actively interacting in microtubule reorganization, or, (2) Spn-E may be required for efficient transcription, pre-mRNA processing, localization, or translational regulation of products that control the kinetics of microtubule assembly, or to direct the reorganization of microtubule structures. The similarity of Spn-E protein to members of the DE-H family makes this second hypothesis attractive (Gillespie, 1995).

Subsequent studies reveal that spn-E phenotypes resemble a family of Drosophila mutants, all involved in a number of polarity determining steps during oogenesis. During wild-type oogenesis, the two cells in each germline cyst appear to be equivalent: these are the progeny of the first division of the cystoblast, derived from asymmetric division of a germ-line stem cell. Both cells enter meiosis to become pro-oocytes in region 2a of the germarium. In region 2b, one of these two cells is selected to develop as the oocyte and remains in meiosis, while the other exits meiosis and reverts to the nurse cell pathway of development. The event gives rise to the first asymmetry in egg development, the selection of one of two cells to become the oocyte. Later in oogenesis, anterior-posterior polarity originates when the oocyte comes to lie posterior to the nurse cells and signals through the Gurken/Egfr pathway to induce the adjacent follicle cells to adopt a posterior fate. This directs the movement of the germinal vesicle and associated Gurken mRNA from the posterior to an anterior corner of the oocyte, where Gurken protein signals for a second time to induce the dorsal follicle cells, thereby polarizing the dorsal-ventral axis. A group of five genes, the spindle loci, is described which is required for each of these polarizing events. The five spindle genes were originally identified in a screen for maternal-effect mutants on the third chromosome because homozygous mutant females lay ventralized eggs.

Mutations in spn-E give rise to an oocyte displacement phenotype, but also affect the oocyte cytoskeleton and mRNA localization, even when the oocyte is at the posterior of the egg chamber. Double spindle mutants reveal a phenotype even earlier in oogenesis, one where both pro-oocytes develop as oocytes, by delaying the choice between these two cells. spindle mutants inhibit the induction of both the posterior and dorsal follicle cells by disrupting the localization and translation of Gurken mRNA. The transient mislocalization of Gurken mRNA to an anterior ring in spn mutant stage 9 egg chambers is very similar to the mislocalization of Gurken mRNA observed in fs(K10) mutants. However, K10 mutations produce a dorsalization of the egg chamber rather than a ventralisation, because the mislocalization of Gurken mRNA directs Gurken signaling to the follicle cells on all sides of the oocyte. In different spindle mutants, from 19% to 100% of egg chambers show a strong reduction or a complete absence of Gurken protein in the oocyte membrane. The oocyte often fails to reach the posterior of mutant egg chambers and it differentiates abnormally. This analysis of spindle phenotypes suggests that spindle genes are likely to be involved in the localization and/or translation of Gurken mRNA without having any discernible effect on the Gurken mRNA level, yet dramatically reduced amounts of Gurken protein are produced. K10 mutants cause a similar mislocalization of Gurken mRNA without significantly affecting protein expression. Thus, spindle mutants reveal a novel link between oocyte selection, oocyte positioning and axis formation in Drosophila, leading to a proposal that the spindle genes act in a process that is common to several of these events (Gonzalez-Reyes, 1997).

How the asymmetry between the two pro-oocytes arises is unknown, but it has been proposed that it could be generated during the first division of the cystoblast to give rise to a two-cell cyst. During this division, a vesicular structure called the spectrosome (see Drosophila Spectrin) associates with one pole of the mitotic spindle and is asymmetrically partitioned between the two daughter cells. Since each of these cells gives rise to one pro-oocyte and seven nurse cells, this asymmetry might determine which pro-oocyte is fated to become the oocyte. Whatever mechanism generates the initial asymmetry, it seems that the key step in the selection of the oocyte is the accumulation of Bicaudal-D and Egalitarian proteins in a single cell. Null mutants in either gene block the localization of the protein encoded by the other to the presumptive oocyte and prevent all other known steps in oocyte differentiation, such as the formation of an active MTOC in this single cell and the subsequent microtubule-dependent localization of oocyte-specific transcripts, such as Oskar. Although it is unclear at what point in the pathway of oocyte selection the spindle genes act, they must function upstream of the process that results in the localization of Bic-D (and presumably Egl) to a single cell. It is most likely that the spindle proteins are directly involved in this process: (1) the spn double mutant combinations delay but do not block the choice between the two pro-oocytes, suggesting that they do not remove the initial asymmetry, but slow down its expression. (2) A reduction in Bic-D or Egl activity later in oogenesis leads to the same ventralized phenotype that is produced by the single spn mutations. This raises the possibility that the spn gene products interact with Bic-D and Egl at two different stages of oogenesis, first to select the oocyte and then to regulate Gurken expression once the oocyte has formed (Gonzalez-Reyes, 1997).

GENE STRUCTURE

PROTEIN STRUCTURE

Amino Acids - 4359

Structural Domains

Sequence analysis of the spn-E cDNA predicts a protein with amino-terminal homology to members of the DE-H family of RNA-dependent ATPases and putative helicases. Similarity of 51% in the amino-terminal third of the protein was found to two yeast splicing factors, PRP2 and PRP16, and to Drosophila Maleless (Mle) (Kuroda, 1991), which is required for dosage compensation. Within the seven broadly defined domains of these proteins, Spn-E shares 29% identity with PRP2, PRP16 and Mle. The C-terminal two-thirds of the predicted protein reveals no homology to other known protein sequences (Gillespie, 1995).

date revised: 6 February 98

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