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par-1

REGULATION

DEVELOPMENTAL BIOLOGY

Par-1 transcripts are expressed in both the germline and somatic follicle cells throughout oogenesis, but appear to be uniformly distributed at all stages. However, stainings with the Par-1 antisera reveal that the protein localizes to a number of specific sites in both the germ cells and the follicle cells. Par-1 localizes cortically in the follicle cells at early stages, and is restricted to the basolateral membrane domain of the columnar epithelium in stage 10 egg chambers, similar to the distribution of mammalian Par-1 homologs in cultured epithelial cells (Bohm, 1997). The earliest Par-1 staining in the germline is localized to the fusome, a branched structure extending through the ring canals that functions to orient mitotic spindles during the cell divisions in the germarium. As the cysts develop further, Par-1 localizes to the ring canals and the cortical cytoskeleton of the nurse cells. No asymmetric localization has been detected within the oocyte during stages 1-8, but in early stage 9 egg chambers, Par-1 is transiently enriched at the anterior of the oocyte. It then starts to accumulate at the posterior of the oocyte, and becomes progressively more concentrated at the posterior pole during stages 9-10. The posterior localization of Par-1 is very similar to that of the first components of the pole plasm, OSK mRNA and Staufen (Stau) protein, which also show a transient anterior localization before moving to the posterior cortex during stage 9. Indeed, Par-1 colocalizes with a GFP:Stau fusion protein at the posterior of the oocyte. Thus, like its counterpart in C. elegans, Drosophila Par-1 is one of the earliest markers for the posterior pole. The localization of Par-1 cannot be reliably followed at later stages in oogenesis because the chorion blocks antibody penetration, but the protein is not enriched at the posterior of early embryos (Shulman, 2000).

Par-1 and the polarization of the oocyte

After its specification, the Drosophila oocyte undergoes a critical polarization event that involves a reorganization of the microtubules (MT) and relocalization of the determinant Orb within the oocyte. This polarization requires Par-1 kinase and the PDZ-containing Par-3 homolog, Bazooka (Baz). Par-1 has been observed on the fusome, which degenerates before the onset of oocyte polarization. How Par-1 acts to polarize the oocyte has been unclear. Par-1 is shown to become restricted to the oocyte in a MT-dependent fashion after disappearance of the fusome. At the time of polarization, the kinase itself and the determinant BicaudalD (BicD) are relocalized from the anterior to the posterior of the oocyte. Par-1 and BicD are interdependent and require MT and the minus end-directed motor Dynein for their relocalization. baz is required for Par-1 relocalization within the oocyte and the distributions of Baz and Par-1 in the Drosophila oocyte are complementary and strikingly reminiscent of the two PAR proteins in the C. elegans embryo. It is proposed that, through the combined actions of the fusome, MT, and Baz, Par-1 is selectively enriched and localized within the oocyte, where, in conjunction with BicD, Egalitarian (Egl), and Dynein, it acts on the MT cytoskeleton to effect polarization (Vaccari, 2002).

In Drosophila, early oogenesis involves formation of a germline cyst consisting of an oocyte surrounded by 15 nurse cells. The cyst develops in region 1 of the germarium, where a germline stem cell-derived cystoblast divides four times with incomplete cytokinesis and produces a cyst of 16 cells interconnected by the fusome, a continuous membrane-skeletal organelle. The fusome is asymmetrically partitioned into the sibling cells and acts at multiple steps in early oogenesis. During the cyst divisions, it is connected to one spindle pole and guides the cells through an invariant division pattern. Later on, it acts as a scaffold for the migration of the cyst centrosomes into the oocyte, resulting in the organization of a polarized microtubule (MT) network in the cyst. During oocyte determination, the fusome progressively disassembles. In region 2a, the presumptive oocyte, which contains the most fusomal remnants, accumulates determinants such as Bicaudal D (BicD), Egalitarian (Egl), and oo18 RNA binding protein (Orb). Between germarial region 2b and region 3 (also referred to as stage 1 of oogenesis), the oocyte itself becomes polarized, as revealed by the relocalization of Orb from the anterior to the posterior of the oocyte (Vaccari, 2002 and references therein).

Genetic analysis of Drosophila par-1 function has revealed a requirement for the kinase during oocyte polarization; in par-1 null alleles, Orb fails to relocate to the posterior of the oocyte and eventually disappears, after which the oocyte loses its fate and becomes a nurse cell. In addition to Orb, two other oocyte determinants, BicD and Egl, also fail to relocalize within par-1 null oocytes. It has been unclear how Par-1, reported to localize exclusively to the fusome during early oogenesis, could affect the polarization process, which takes place several hours after disappearance of the fusome. D. melanogaster par-1 encodes several isoforms, which are composed of three alternative N termini (N-ter 1–3), a constant kinase, and a spacer domain; they also may include or exclude a C-terminal (C-ter) domain. It was reasoned that the reported Par-1 localization, as revealed by a pan-Par-1 antibody raised against the spacer domain (Par-1 spacer antibody), might only partially reflect the distribution of the kinase. Germline expression of an isoform (N1S) consisting of N-ter 1, the kinase, and the spacer domains and lacking the C-ter allows normal development of par-1 null oocytes. A peptide antibody was raised specifically against N-ter 1 (N1 antibody) and compared the Par-1 signals detected by this antibody and those detected by the original Par-1 spacer antibody. In contrast to the spacer antibody, which detects Par-1 on the fusome, the N1-specific antibody first detects Par-1 in the cyst as the fusome disappears, with the signal becoming restricted to the cytoplasm of the oocyte during its determination in region 2b. This novel Par-1 signal colocalizes with the oocyte marker Orb. The fact that the N1 antibody does not detect Par-1 on the fusome might be explained by a selective accessibility of different domains of the protein. In fact, the N1-specific antibody, which detects Par-1 in the early oocyte, does not recognize fusome-associated Par-1, even in ovaries in which the protein is overexpressed and visible as a GFP fusion on the fusome (Vaccari, 2002).

During oocyte specification, localization of the determinants BicD, Egl, and Orb to the early oocyte relies on the asymmetric distribution of microtubules in the cyst, evident as a dense focus of MT in the oocyte. Depolymerization of the MT by colchicine abolishes the localization of BicD, Egl, and Orb and results in egg chambers with 16 nurse cells and no oocyte. It was therefore asked if the restriction of Par-1 to the oocyte during the transition from region 2a to region 2b is also MT dependent. Ovaries of flies fed with colchicine for 12 hr fail to localize Par-1 and Orb to the oocyte, indicating that Par-1 restriction to the oocyte is indeed MT dependent. This is in contrast to the localization of Par-1 to the fusome, which occurs independently of MT (Vaccari, 2002).

The distribution of Par-1 within the oocyte was further examined by focusing on the transition between regions 2b and 3, when par-1-dependent polarization of the oocyte occurs. In germarial region 2b, Par-1 is enriched anterior to the oocyte nucleus. In region 3, the protein is mainly detected at the posterior of the oocyte, where it remains. During this relocalization, Par-1 colocalizes completely with BicD in the germline. Because par-1 is required for BicD relocalization within the oocyte, the distribution of Par-1 was examined in BicD hypomorphs that allow differentiation of an oocyte. Par-1 is detected but mislocalized in an anterior dot within the BicD mutant oocytes. Hence, Par-1 and BicD are interdependent for their relocalization to the posterior of the oocyte region 3b (Vaccari, 2002).

To assess whether the MT cytoskeleton mediates relocalization of Par-1 and BicD within the oocyte, wild-type ovaries were dissected a short time after treatment with colchicine. A screen was carried out for region 3 egg chambers in which the focus of oocyte MT was destroyed. In these, BicD and Par-1 remain anterior to the oocyte nucleus, indicating that MTs are required for oocyte polarization (Vaccari, 2002).

The MT motor Dynein has been reported to influence development of the germline cyst. Loss-of-function mutants in dhc64C, encoding the heavy chain of the minus end-directed molecular motor Dynein, fail to develop an egg chamber because of mitotic failure in the germarium. However, hypomorphic dhc64C mutants develop an oocyte and 15 nurse cells. In a high percentage of such egg chambers, both Par-1 and BicD remain at the anterior of the oocyte in region 3. Hence, after its initial requirement in cyst formation, the minus end-directed motor Dynein is involved in the relocalization of Par-1 and BicD to the posterior of the oocyte (Vaccari, 2002).

It has been reported that, like Par-1, the D. melanogaster homolog of Par-3 Bazooka (Baz) is necessary for oocyte maintenance. Germline clones of a baz null allele are defective in oocyte polarization, establishing a functional parallel with C. elegans, in which the PAR genes polarize the early embryo. In the worm, restriction of Par-1 activity to the posterior cortex of the embryo crucially depends on Par-3, but the reverse is not the case. In the Drosophila germarium, Par-1 localization to the fusome appears to be independent of Baz. To assess whether the subsequent localization of Par-1 in the oocyte requires Baz, Par-1 distribution was further evaluated in baz germline clones. Par-1 is initially detected at the anterior of the oocyte in region 2b, but the protein disappears in region 3, revealing that Par-1 relocalization is baz dependent (Vaccari, 2002).

The relative distributions of Par-1 and Baz were investigated during later stages of oogenesis in wild-type ovaries. Par-1 is detected at the posterior of the oocyte as of germarial region 3 and becomes tightly associated with the cortex between oogenesis stages 3 and 5. Concomitantly, Baz becomes transiently localized to the anterior of the oocyte. The anterior localization of Baz is specific; it is not observed in germline clones of a baz null allele. Interestingly, maximal expression of Baz at the anterior coincides with the apparent tightening of Par-1 signal at the posterior cortex, suggesting a role for Baz in sharpening Par-1 localization. At this stage, the localization of the two proteins appears to be mutually exclusive. The anterior enrichment of Baz between stages 2 and 5 is absent in germline clones of the par-1 null allele (Vaccari, 2002).

The mutually exclusive distribution of Par-1 and Baz in the Drosophila oocyte is strikingly reminiscent of that observed in the C. elegans embryo. The ability to visualize the two proteins has allowed their respective roles in achieving this distribution to be genetically evaluated. The seeming dependence of Baz localization on par-1 is in contrast to results in C. elegans, in which localization of Par-3 is independent of Par-1. However, the absence of Baz in par-1 germline clones may well reflect the loss of oocyte fate and the onset of its degeneration that occurs at stage 1. Nonetheless, the anterior localization of Baz during stages 2–5 suggests that, after acting in oocyte polarization in region 2b/3, baz may be required again during oogenesis. The existence and nature of such a second requirement for baz after oocyte polarization is not yet clear (Vaccari, 2002).

Research has shown that the fusomal localization of Par-1 is unaffected in baz mutants, suggesting a difference between the Drosophila oocyte and the C. elegans embryo, in which Par-1 localization depends on par-3. Remarkably, at the time when oocyte polarization takes place, baz is in fact required for Par-1 localization within the oocyte. Hence, it appears that a similar relationship exists between Par-1 and Baz at the time when their activities are critical in the two organisms (Vaccari, 2002).

Impairment of the MT cytoskeleton and mutations in BicD and dhc64C also affect Par-1 relocalization within the oocyte. Conversely, in par-1 mutants, the MT cytoskeleton is not focused in the oocyte, BicD fails to relocalize, and Dynein is not enriched in the oocyte. The mutual interdependence of these genes and the MT suggests that all these components cooperate to form a polarization complex in the oocyte. Interestingly, the N1 antibody begins to detect Par-1 only when its function is genetically required, suggesting that, in region 2, the kinase may undergo a change in conformation or in its association with other factors (Vaccari, 2002).

The presence and localization of Par-1 in the oocyte at the time of its determination and polarization complements the previously reported localization of Par-1 on the fusome prior to oocyte determination and establishes Par-1 as a unique oocyte marker, for at least two reasons. (1) Absence of any one of the oocyte determinants, BicD, Egl, or Orb, prevents the concentration of the two other determinants in this cell. In contrast, in the absence of Par-1, it is the relocalization of the determinants within the oocyte that is specifically affected. (2) BicD, Egl, and Orb are not present on the fusome, and the observed enrichment of these determining factors in the oocyte is the result of the enrichment of their RNAs in this cell during its specification. In contrast, no par-1 RNA is detected in the germline at such early stages. The idea that Par-1 is initially loaded on the fusome, where it perdures during the cyst divisions, and that it is later preferentially inherited by the oocyte, is favored. Taken together, the facts that par-1 mutants show no fusomal defects and that accumulation of Par-1 itself in the oocyte requires MT suggest that Par-1 does not affect the oocyte MT cytoskeleton from its fusomal location. It is proposed that, through the combined actions of the fusome, MT, and Baz, Par-1 is selectively enriched and localized within the oocyte, where it acts in conjunction with BicD, Egl, and Dynein to effect polarization (Vaccari, 2002).

Effects of Mutation or Deletion

Given the analogous posterior localization of PAR-1 in the C. elegans zygote and the Drosophila oocyte, an examination of whether Drosophila PAR-1 has a similar role to the nematode gene in the localization of the germline determinants and the polarization of the A/P axis was undertaken. The null allele, par-1^W3, is homozygous lethal, and germline clones arrest oogenesis at about stage 5, preventing analysis of its effect on axis formation. The maternal-effect phenotypes were examined of the homozygous viable mutant combinations par-1⁶⁸²¹/par-1⁶³²³, par-1⁶³²³, and par-1⁶³²³/par-1^W3, all of which which reduce the levels of PAR-1 protein without disrupting the function of mei-W68. The progeny of mothers of all three genotypes show typical posterior group phenotypes: the embryos lack abdominal segments, and pole cells fail to form, giving rise to a grandchildless phenotype, in which the adult 'escapers' have agametic gonads. Furthermore, these embryos show little or no Vasa staining at the posterior pole, indicating that the phenotypes arise from a defect in pole plasm formation. The severity of these phenotypes correlates well with the reduction in the levels of Par-1 protein in the mutant ovaries. The strongest mutant combination, par-1⁶³²³/par-1^W3, produces a completely penetrant posterior group phenotype and has the lowest amounts of maternal Par-1, whereas the other mutant combinations express more protein and produce weaker phenotypes. In addition to its requirement for posterior patterning, loss of par-1 causes other less penetrant maternal-effect phenotypes, such as defects in the organization of the head skeleton and telson, and a low frequency of death prior to cuticle deposition, but these appear to be unrelated to its function in A/P axis formation (Shulman, 2000).

A similar ectopic mislocalization of OSK mRNA is observed in mutants such as gurken (grk), Egfr, Pka, and Notch. These mutations disrupt signaling between the posterior follicle cells and the oocyte, resulting in the formation of a symmetric microtubule cytoskeleton that localizes OSK mRNA to the oocyte center and BCD mRNA to the posterior as well as the anterior pole. However, in par-1 oocytes, BCD mRNA localization is indistinguishable from wild type. Furthermore, whereas the oocyte nucleus frequently fails to migrate to the anterior in grk or Notch, it always moves to the correct position in par-1 egg chambers. The localization of GRK mRNA to the dorsal-anterior margin of the oocyte is also unaffected, consistent with the normal dorsal-ventral patterning of par-1 eggs and embryos. Thus, the par-1 mutants cause a novel polarity phenotype, in which the posterior transport of OSK mRNA is redirected to the center of the oocyte, whereas BCD and GRK mRNAs and the oocyte nucleus localize normally at the anterior (Shulman, 2000).

Since the posterior localization of OSK mRNA is dependent on a polarizing signal from the posterior follicle cells, an examination was carried out to see whether these cells are correctly specified in par-1 mutants, and whether Par-1 function is required in the germline or somatic cells of the egg chamber. Unlike grk mutants, in which the 'posterior' follicle cells adopt an anterior fate and express the enhancer-trap line L53b, this line is expressed solely at the anterior in par-1⁶³²³/par-1^W3 egg chambers, indicating that Par-1 is not required for Gurken signaling to the posterior follicle cells. Since germline clones of the par-1 null allele block oogenesis, GFP-marked germline clones of the hypomorphic allele, par-1⁶⁸²¹, were generated and it was found that Stauffen is mislocalized to the center of the oocyte. In addition, a par-1(N1S) transgene expressed from the germline-specific alpha4-tubulin promoter efficiently rescues the par-1⁶³²³/par-1^W3 OSK mRNA localization and embryonic patterning defects. Thus, par-1 is required autonomously in the germline for the posterior localization of OSK mRNA, and this function must occur after stage 2, when the transgene is first expressed (Shulman, 2000).

The Drosophila homolog of C. elegans PAR-1 becomes asymmetrically localized as polarity is established mutations affecting its expression perturb posterior patterning, germ-line development and posterior localization of pole-plasm components. It is proposed that par-1 has an important and conserved function in the establishment of polarity and development of the germ-line lineage in both of these species (Tomancak, 2000).

Analysis of the distribution of Drosophila PAR-1 in ovaries reveals that it is present in the spectrosome and early fusome of the germarium and becomes enriched in the oocyte during early oogenesis. During stages 5-8 of oogenesis, the protein is uniformly distributed along the oocyte cortex, and at stage 9, it is enriched at the posterior pole, although it can be detected at a low level throughout the oocyte cortex. PAR-1 is also expressed in follicle cells. To determine the role of Drosophila PAR-1, P-element insertions were identified within the genomic region encompassing the par-1 locus and the overlapping mei-w68 locus. Two insertions, l(2)k06323 (par-1^9A) and l(2)k05603 (par-1⁵⁷⁴) affect expression of PAR-1 isoforms. Both P elements cause reductions in levels of isoforms of M_r ~110K and lead to increased levels of an isoform of M_r ~116K. Excision of the par-1^9A P element restores the wild-type expression pattern, demonstrating that these changes are caused by insertion of the P element and confirming the identity of the affected proteins as PAR-1 isoforms. In par-1-mutant egg chambers, distribution of PAR-1 was found to be altered. In stage-9 oocytes, PAR-1 enrichment at the posterior pole is reduced in these mutants; instead the protein appears evenly distributed along the cortex. These changes in PAR-1 distribution presumably reflect changes in the amounts of the various PAR-1 isoforms, as detected by Western blotting (Tomancak, 2000).

Females harboring the par-1^9A/par-1^9A and par-1^9A/par-1⁵⁷⁴ mutations are fertile; however, 20% of embryos produced by these individuals died, exhibiting defects ranging from disturbances in posterior patterning, such as fusion or bifurcation of segments, to complete absence of abdominal segments. Abdominal patterning in Drosophila is initiated by assembly of the posterior pole plasm, which is also required for formation of the germ-line precursors, the pole cells. In the majority of par-1-mutant embryos, the number of pole cells is significantly reduced, indicating that pole-plasm formation may be affected by par-1 mutations (Tomancak, 2000).

Pole-plasm assembly is induced by localization and translational derepression of oskar messenger RNA at the posterior pole of the oocyte, creating a localized source of Oskar protein, which in turn recruits further pole plasm components. In 70% of par-1^9A-mutant egg chambers at stages 8-10, oskar mRNA is either fully or partially mislocalized to the middle of the oocyte. This aberrant distribution of oskar mRNA was mirrored by similar defects in localization of Staufen, another pole-plasm component. Moreover, in 20% of stage 8-9 egg chambers, some or all of the Oskar protein detected is found in the middle of the oocyte, indicating that pole-plasm mispositioning may be a cause of defects in abdominal patterning and pole-cell formation (Tomancak, 2000).

Thus, Drosophila PAR-1, like its counterpart in C. elegans, has a function in establishing embryonic polarity, and becomes asymmetrically distributed during the period when polarity is specified. Mutations that alter its expression lead to alterations in the distribution of pole-plasm components, reduced numbers of pole cells and defective posterior patterning. Given the nature of the changes in expression, these defects could result from reduced levels of a critical isoform, inappropriate levels or distribution of an isoform, or a combination of the above (Tomancak, 2000).

Drosophila PAR-1 may influence polarity through the cytoskeleton. Localization of Oskar mRNA to the posterior pole requires both microtubule-dependent and microfilament-dependent processes. The idea that Drosophila PAR-1 regulates microtubules is supported by the fact that mammalian PAR-1 homologs exhibit microtubule-destabilizing activities. In C.elegans, however, there is no evidence of a role for microtubules in par-1-dependent processes, and PAR-1 protein has been shown to interact with non-muscle myosin, an actin motor. Furthermore, proper asymmetric localization of P granules is resistant to microtuble-depolymerizing drugs but sensitive to microfilament-depolymerizing agents. It is therefore also possible that Drosophila PAR-1 acts through a microfilament-dependent mechanism, such as anchoring oskar mRNA and/or OSKAR protein to the posterior pole. Indeed, maintenance of pole-plasm components at the posterior pole of the Drosophila embryo depends on an intact actin cytoskeleton. Previous studies have shown fundamental differences in the early development of Drosophila and C. elegans with respect to establishment of embryonic polarity. In spite of these marked differences, PAR-1 and Drosophila PAR-1, members of the PAR-1/MARK/KIN1 family of Ser/Thr kinases, are distributed asymmetrically at the cell periphery in oocytes and in early embryos of Drosophila and C. elegans, respectively. This finding raises the possibility that at the heart of these two widely divergent systems lies a conserved mechanism to initiate cell polarity and germ-line development (Tomancak, 2000).

Drosophila oocyte determination involves a complex process by which a single cell within an interconnected cyst of 16 germline cells differentiates into an oocyte. This process requires the asymmetric accumulation of both specific messenger RNAs and proteins within the future oocyte as well as the proper organization of the microtubule cytoskeleton, which together with the fusome provides polarity within the developing germline cyst. In addition to its previously described late oogenic role in the establishment of anterior-posterior polarity and subsequent embryonic axis formation, the Drosophila par-1 gene is required very early in the germline for establishing cyst polarity and for oocyte specification. Germline clonal analyses, for which a protein null mutation was used, reveal that Drosophila par-1 is required for the asymmetric accumulation of oocyte-specific factors as well as the proper organization of the microtubule cytoskeleton. Similarly, somatic clonal analyses indicate that par-1 is required for microtubule stabilization in follicle cells. The Par1 protein is localized to the fusome and ring canals within the developing germline cyst in direct contact with microtubules. Likewise, in the follicular epithelium, Par1 colocalizes with microtubules along the basolateral membrane. However, in either case Par1 localization is independent of microtubules. It is concluded that Drosophila par-1 plays at least two essential roles during oogenesis: it is required early in the germline for organization of the microtubule cytoskeleton and subsequent oocyte determination, and it has a second, previously described role late in oogenesis in axis formation. In both cases, par-1 appears to exert its effects through the regulation of microtubule dynamics and/or stability, and this finding is consistent with the defined role of the mammalian Par1 homologs (Cox, 2001).

To investigate the role of Par1 in oogenesis, a polyclonal antibody was generated against a portion of the linker region that is shared by all predicted protein isoforms. Consistent with previous studies, staining with the Par1 antisera reveals that Par1 is expressed in both the germline and the soma. Par1 germline expression is first detectable in the germarium, where it is localized to the fusome, a highly branched structure rich in membrane skeletal proteins such as the Adducin-like hu-li tai shao (hts) gene product. Par1 colocalizes with the fusome component Hts. Subsequently, during cyst development, Par1 colocalizes with actin to the ring canals. In the soma, Par1 is asymmetrically localized to the basolateral membrane of the follicular epithelium. In apical cross section, Par1 is cortically localized in follicle cells, and in transverse sections Par1 is enriched along the basolateral membranes of follicular epithelia. This pattern is highly similar to that observed for the mammalian Par1 homolog MARK, which is also localized basolaterally in cultured epithelial cells. To control for the specificity of the affinity-purified Par1 antisera, Par1 expression was analyzed in homozygous mutant germline and somatic cell clones of a protein null allele (par-1^delta16. Par1 basolateral staining is completely eliminated in the mutant follicle cell clones, and fusome and ring canal expression is similarly lost in mutant germline clones (Cox, 2001).

Previous studies of hypomorphic alleles of par-1 have revealed a role in establishing anterior-posterior polarity in egg chambers and subsequent embryonic axis formation. To investigate whether par-1 function is required earlier in oogenesis, a protein null allele was generated in par-1 (par-1^delta16). The par-1^delta16 allele originates from the imprecise excision of the l(2)k06323 line and is homozygous embryonic lethal. This allele contains a 16 kb deletion removing most of the coding sequences 3' of the P-element insertion; the deletion includes the entire kinase domain and the majority of the linker region. The fact that this allele is embryonic lethal indicates an additional function for par-1 during zygote development. Therefore, to determine the role of par-1 in early oogenesis, mosaic egg chambers were generated whose germline cyst was homozygous mutant for par-1^delta16. The FLP/DFS technique was used to generate both par-1 mutant and wild-type germline clones. The germline phenotype of par-1 mutant clones is largely indistinguishable from that of control ovo^D females when analyzed by DAPI staining to reveal nuclear morphology. In either case, there are no vitellogenic oocytes observed, and mutant females fail to produce any eggs. These results indicate that par-1 is required early in the germline for oocyte development (Cox, 2001).

To further characterize the germline requirement for par-1 function during oocyte specification, the mosaic analysis was repeated in the absence of ovo^D. The FLP/FRT technique was used to generate par-1 mutant germline clones, which are marked by the absence of nuclear GFP expression. Mosaic egg chambers containing homozygous par-1 germline clones were counterstained with the chromatin marker propidium iodide to determine the number and size of the nuclei. Mosaic egg chambers lacking par-1 in germline cells were compared with control egg chambers, which retain par-1 expression in both germline and follicle cells. Control egg chambers invariably contain 15 nurse cells and a single oocyte. In contrast, par-1 mosaic egg chambers contain the normal number of 16 cells; however, all of these cells develop as nurse cells, as indicated by their polyploid nuclei. Mosaic egg chambers that contain wild-type germline cells surrounded by a monolayer of par-1 mutant follicle cells give rise to normal germline cysts of 15 nurse cells and a single oocyte. Phenotypic analysis of germline mosaic egg chambers further reveals a par-1 dependent block at stage 5 to stage 6 of oogenesis. These results taken together clearly demonstrate that par-1 is required in the germline to regulate oocyte differentiation (Cox, 2001).

To analyze how oocyte determination is blocked in par-1 mutant egg chambers, an examination was carried out to see how removing Par1 affects the formation or structure of the fusome and ring canals to which Par1 localizes within the germline. The morphology of fusomes was examined in par-1 mutant cysts by double labeling germaria with nuclear GFP and monoclonal anti-Hts antibody 1B1, a marker for the fusome. In wild-type germaria, a spherical fusome or spectrosome (Sp) is present in germline stem cells and cystoblasts. Following cystoblast division, the fusome (Fu) undergoes a morphogenesis from a spherical fusome in the cystoblast into a polarized, branched structure in the 2-, 4-, 8-, and 16-cell cysts. Analysis of par-1 mutant clones reveals that par-1 is not required for formation of the fusome and that, furthermore, fusome morphology is indistinguishable from wild-type clones within the same germaria. Therefore, while par-1 is not required for fusome formation, the defects in cyst development and oocyte determination suggest that par-1 may nevertheless play an important role as a component of the fusome in establishing and/or maintaining cyst polarity (Cox, 2001).

The ring canals are stable intercellular bridges that maintain the connection between all cells within the 16-cell germline cyst. In wild-type germline cysts, the pattern of ring canal connections is highly invariant; one of the two cells with four ring canals invariably differentiates to form the oocyte, while the remaining 15 cells become nurse cells. Therefore, the pattern of ring canal connections reflects the spatial pattern of the cystoblast mitotic divisions. To analyze the size, formation, and spatial distribution of ring canals in par-1 mutant clones, wild-type and mutant germline cysts and egg chambers stained with rhodamine-conjugated phalloidin were compared to visualize the filamentous actin component of ring canals. In wild-type germaria, each 16-cell germline cyst displays a stereotypic pattern of ring canal connections. In region 2b, when the germline cyst flattens out across the width of the germarium, the spatial distribution of ring canals is manifest as an orderly, linear arrangement across the width of the germline cyst. Similarly, in region 3 of the germarium, the spatial distribution of ring canals remains highly ordered. However, in par-1 mutant germline cysts, the arrangement of ring canals in both region 2b and region 3 of the germarium is disrupted. In region 2b, the wild-type linear distribution of ring canals is clearly disrupted; likewise, in region 3, the arrangement of ring canals appears less ordered and more unevenly distributed. In mosaic egg chambers, the size and location of the ring canal connections is also disrupted. The most posterior germline cell in the egg chamber did not display the typical cluster of the four largest ring canals characteristic of the oocyte. In contrast, in wild-type egg chambers the oocyte is invariantly positioned at the posterior and is easily identified by its four large ring canals at the anterior margin of the cell. Thus, par-1 function is required early in germline cyst formation to establish cyst polarity. Furthermore, the lack of cyst polarity correlates with the failure in oocyte determination that results in the differentiation of 16 polyploid nurse cells (Cox, 2001).

While the precise mechanism of oocyte determination is unknown, one hallmark of this process is the differential accumulation of specific oocyte differentiation factors within a single cell of the germline cyst. A number of these molecules, including Bic-D, Egl, Orb, and Dhc64C, appear to be directly involved in this process since they are required for determination of the oocyte. To further define the role of par-1 in oocyte determination, the expression pattern of these proteins was examined in par-1 mutant germline clones during early and late stages of oogenesis and that expression was compared to wild-type control germaria and egg chambers. In contrast to wild-type germline cysts, par-1 mutant cysts fail to maintain the accumulation of Bic-D protein in a single cell. In region 2b of the germarium, Bic-D protein appears to localize to a single cell; however, this differential localization is not maintained in region 3 of the germarium. Later in oogenesis, par-1 mosaic egg chambers also fail to accumulate Bic-D in a single cell, but rather they display diffuse expression of Bic-D throughout the egg chamber. Similarly, par-1 mutant cysts and egg chambers fail to accumulate Orb, Dhc64C, and Staufen proteins within a single cell. This failure leads to diffuse expression throughout the germline cyst or egg chamber. To investigate the integrity of the microtubule network, oskar mRNA expression was examined in par-1 mosaic egg chambers by in situ hybridization. oskar localization to the future oocyte has previously been shown to require a stable MTOC and microtubule network. In wild-type ovarioles, oskar mRNA is first detected in region 2b, where it begins to accumulate in a single cell. Subsequently, oskar mRNA is localized posteriorly in the oocyte of each developing egg chamber. In contrast, par-1 mosaic egg chambers fail to accumulate oskar mRNA posteriorly. These results indicate that par-1 is required for the differential accumulation of oocyte differentiation factors and further suggests that par-1 may function in establishing and/or maintaining polarity of the microtubule network (Cox, 2001).

In addition to its role in the establishment of axial polarity and the localization of morphogens within the developing oocyte, the microtubule cytoskeleton has a very important role in the early events of oocyte determination. The microtubule cytoskeleton is critical in many aspects of oocyte determination since microtubule depolymerizing drugs disrupt the accumulation of oocyte differentiation factors, such as Bic-D and oskar mRNA, and lead to the development of cysts with 16 nurse cells. Given that par-1 is also required for the differential accumulation of oocyte factors and for oocyte determination, the organization of the microtubule cytoskeleton was analyzed in par-1 mutant clones. In wild-type germaria, the oocyte is characterized by the presence of an active MTOC which organizes the microtubule network within the developing germline cyst. In region 3 of the germarium, the MTOC is clearly present in a single posterior cell. However, in par-1 mutant cysts there is no apparent posterior MTOC formation; rather, the microtubules appear evenly distributed within region 3 of the germarium. Similarly, in stage 2 mutant egg chambers, there is no apparent posterior accumulation of microtubules, but rather the microtubules remain evenly distributed within the mutant cyst. Finally, in later-stage par-1 mosaic egg chambers there remains no posterior accumulation of microtubules or visible MTOC formation. These results indicate that par-1, like Bic-D and Egl, is required for the formation and/or maintenance of an active MTOC within the future oocyte. The even distribution of microtubules within the mutant germline cysts and egg chambers and the failure to maintain an active posterior MTOC may account in part for the defects observed in cyst polarity and, ultimately, oocyte determination (Cox, 2001).

To better understand the relationship between par-1 function and microtubule organization, a detailed analysis was conducted of microtubule structure in par-1 mutant germline clones. To examine the dependence of Par1 localization on an intact microtubule cytoskeleton, adult females were fed with the microtubule depolymerizing drug colchicine. To assay the effect of the drug on disrupting the microtubule cytoskeleton, the production was monitored of egg chambers containing 16 nurse cells and no oocyte and microtubule structure was analyzed by immunofluorescence. In a wild-type germarium, Par1 is expressed on the spectrosome and the fusome, and the microtubules display a highly organized network originating from the posterior MTOC. Following microtubule depolymerization, no disruption was observed in the localization of Par1 to the spectrosome or fusome. This indicates that Par1 localization within the germline is independent of microtubules (Cox, 2001).

Finally, the orientation and attachment of mitotic spindle poles to the fusome during cystocyte divisions is believed to ensure the proper pattern of cell divisions necessary to generate a polarized cyst. Therefore, spindle orientation was examined in dividing par-1 mutant germline cysts. par-1 mutant germline cysts were stained with antibodies to alpha-tubulin and the Hts-related antigen 1B1 to label microtubules and fusomes, respectively. No apparent defect in spindle orientation or attachment to the fusome was found. These results taken together suggest that par-1 regulates microtubule dynamics during cyst formation and oocyte determination by establishing and/or maintaining polarization of the microtubule network (Cox, 2001).

Given that Par1 functions to regulate microtubule dynamics and cyst polarity in the germline, attempts were made to test whether par-1 plays similar roles in the somatic follicle cells. par-1^delta16 follicle cell clones were generated and egg chambers were stained to assay both microtubule and epithelial organization as well as follicle cell polarity. par-1 mutant follicle cell clones were examined by double labeling with anti-alpha-tubulin and anti-alpha-Spectrin antibodies to assay microtubule organization and epithelial integrity, respectively. In wild-type follicle cells, alpha-tubulin is normally localized to the basolateral membrane in a pattern very similar to that observed for Par1. However, in par-1 mutant clones, the basolateral alpha-tubulin expression is completely abolished. The effect on alpha-tubulin expression of removing par-1 function is specific since alpha-Spectrin expression is unaffected in par-1 mutant follicle cell clones. Moreover, anti-Spectrin staining reveals defects in epithelial shape and organization. Wild-type follicle cells at this stage form a columnar monolayer that surrounds the oocyte; however, par-1 mutant follicle cells appear to disrupt this monolayer. By using anti-Spectrin staining as a cortical marker, two defects were observed in monolayer organization. (1) It was found that follicle cells lacking Par1 function appear to lose their characteristic columnar shape and appear irregular in size and shape. (2) A disruption was observed in the monolayer such that two follicle cells appear to be stacked on top of one another rather than in a monolayer. This result suggests that disruption in the monolayer may lead to a reorientation or randomization of the plane of division in par-1 mutant follicle cells. An alternative and equally probable interpretation could be that mutant follicle cell division occurs normally; however, following division there may be a reorientation of the mutant follicle cells perpendicular to the wild-type orientation (Cox, 2001).

Since par-1 is required in follicle cells to organize and/or stabilize the microtubule cytoskeleton as well as to maintain the integrity of the epithelial monolayer, the potential role of par-1 in the establishment and/or maintenance of follicle cell polarity was examined. Mutant follicle cell clones were labelled with anti-alpha-tubulin and anti-Armadillo (Arm) antibodies to assay microtubule organization and epithelial polarity, respectively. Armadillo displays a polarized accumulation within follicle cells with intense staining apically in the adherens junctions as well as laterally between cell surfaces. In contrast to the polarized pattern of Arm accumulation in wild-type follicle cells, par-1 mutant follicle cells display an even and slightly weakened distribution of Arm protein. Furthermore, in an apical cross section, it is clear that par-1 mutant follicle cells display a highly disrupted pattern of Arm expression along the cell cortex. These defects in Arm accumulation suggest a loss of polarity within the par-1 mutant follicle cells and further indicate that Par1 may function to organize and restrict the basolateral membrane domain within follicle cells since removing par-1 function leads to a loss of this apical Arm restriction. To further assay the role of par-1 in organizing the basolateral membrane domain, mosaic egg chambers were stained with anti-Neurotactin to assay both apical and lateral membrane integrity. Neurotactin is a transmembrane glycoprotein expressed in epithelial tissues along the apical and lateral membranes. Similar to the effects of par-1 on microtubule stabilization, a dramatic destabilization was observed of both apical and lateral Neurotactin expression in par-1 mutant follicle cell clones. These results taken together strongly suggest that Par1 is required to organize the microtubule cytoskeleton as well as maintain polarity within the follicular epithelium (Cox, 2001).

The PAR-1 kinase is required for the posterior localization of the germline determinants in C. elegans and Drosophila, and localizes to the posterior of the zygote and the oocyte in each case. Drosophila PAR-1 is also required much earlier in oogenesis for the selection of one cell in a germline cyst to become the oocyte. Although the initial steps in oocyte determination are delayed, three markers for oocyte identity, the synaptonemal complex, the centrosomes and Orb protein, still become restricted to one cell in mutant clones. However, the centrosomes and Orb protein fail to translocate from the anterior to the posterior cortex of the presumptive oocyte in region 3 of the germarium, and the cell exits meiosis and becomes a nurse cell. Furthermore, markers for the minus ends of the microtubules also fail to move from the anterior to the posterior of the oocyte in mutant clones. Thus, PAR-1 is required for the maintenance of oocyte identity, and plays a role in microtubule-dependent localization within the oocyte at two stages of oogenesis. PAR-1 localizes on the fusome, and provides a link between the asymmetry of the fusome and the selection of the oocyte (Huynh, 2001a).

To examine the phenotype caused by the complete lack of PAR-1 activity in the germline, the ovo^D technique was used to generate clones of par-1^W3, a null mutation that deletes most of the par-1-coding region. This technique provides a powerful selection for homozygous mutant clones in the germline, because the ovo^D transgene blocks oogenesis at an early stage, so only the mutant clones that have lost the transgene survive. However, no late stage egg chambers were recovered, suggesting that cysts that lack PAR-1 arrest their development before stage 6. Therefore germline clones were induced that were marked by the loss of nuclear GFP, so that the mutant egg chambers could be identified at all stages of oogenesis. Egg chambers with homozygous mutant germlines do develop, but they remain small, and never reach the stage where the oocyte is larger than the nurse cells. The oocyte can be distinguished from the nurse cells early in oogenesis, because it is arrested in meiosis and condenses its chromosomes into a hollow sphere called the karyosome, whereas the nurse cells endoreplicate their DNA to become polyploid. In the mutant egg chambers, none of the cells forms a karyosome, and instead all 16 germ cells become polyploid. The oocyte can also be identified by staining for Orb protein, which localizes in a crescent at the posterior of the cell. Orb does not localize to any of the cells in par-1 mutant egg chambers after stage 3, indicating that mutant cysts develop 16 nurse cells and no oocyte (Huynh, 2001a).

The 16 nurse cell phenotype of par-1 null germline clones is very similar to that produced by mutations in egl and BicD. These mutants disrupt the earliest known step in the selection the oocyte, which is the formation of the synaptonemal complex (SC) in the two pro-oocytes. In egl mutants, all cells in the cyst enter meiosis and reach the pachytene stage, but then lose the SC and revert to the nurse cell pathway of development. In contrast, none of the cells enters meiosis and forms any SC in BicD null germline clones. To see whether par-1 is also required for these early steps, the behavior of the SC was examined in par-1 mutant clones. In contrast to wild type, mutant cysts contain up to 16 cells with SC in early region 2a, indicating that all of the cells have entered meiosis. Unlike egl mutant cysts, however, the SC does not disappear from all cells. Instead, the SC becomes progressively restricted to two cells, and then to one cell in region 3 (also called stage 1). However, this restriction is delayed compared with wild-type cysts, where the SC is always restricted to the oocyte by region 2b. In addition, the SC disappears prematurely from mutant oocytes. Wild-type oocytes retain a compacted SC until stage 3-4, whereas the SC can no longer be detected in mutant stage 2 egg chambers that have just left the germarium (Huynh, 2001a).

This behavior of the SC suggests that par-1 acts at two stages in the germarium: (1) it plays a role in the selection of the pro-oocytes and the oocyte, since too many cells enter meiosis in early region 2a, and the choice of the oocyte is delayed; (2) par-1 is required to keep the oocyte in meiosis after stage 1, since the SC is not maintained (Huynh, 2001a).

The restriction of the SC to one cell suggests that at least some of the initial steps in oocyte determination still occur in par-1 clones. The behavior of other early oocyte markers was examined: the oocyte-specific cytoplasmic protein Orb and the centrosomes. In wild-type cysts, Orb protein accumulates in the pro-oocytes in region 2a of the germarium, and then concentrates at the anterior of the oocyte in late region 2a. As the cyst enters region 3, Orb moves from the anterior to the posterior of the oocyte, and forms a crescent at the posterior pole. The same movement has been reported for a BicD-GFP fusion protein. In par-1 mutant cysts, Orb protein still accumulates in one cell, but this localization is delayed until region 3 (stage 1), like the restriction of the SC. Furthermore, Orb never translocates to the posterior of the oocyte, and the protein disappears from the oocyte by stage 2 (Huynh, 2001a).

The migration of the centrosomes from the nurse cell to the oocyte can be followed by staining for gamma-tubulin. Like Orb, the centrosomes localize to the anterior of the oocyte in region 2b. They then move from the anterior to the posterior of the oocyte in region 3, and coalesce to form a bright dot at the posterior pole. In par-1 mutant clones, the centrosomes accumulate at the anterior of the oocyte in late region 2b or early region 3, indicating that the first phase of centrosome migration occurs normally. The centrosomes remain at the anterior, however, and never translocate to the posterior (Huynh, 2001a).

These results indicate that the loss of par-1 blocks oocyte determination at a novel step. The initial selection of the oocyte occurs normally, since three independent oocyte markers, the SC, Orb and the centrosomes, still accumulate in one cell, but the identity of this cell is not fixed, and it soon reverts to the nurse cell pathway of development. This reversion to the nurse cell fate correlates with a defect in the migration of Orb and the centrosomes from the anterior to the posterior of the oocyte, suggesting that this PAR-1-dependent A-P movement is required for the maintenance of oocyte identity (Huynh, 2001a).

PAR-1 is required for the correct organization of microtubules in the oocyte at stage 9, whether this early phenotype in oocyte determination is also a consequence of a defect in the microtubule cytoskeleton was also examined. In a wild-type germarium, the microtubule cytoskeleton becomes progressively polarized in region 2 and is clearly focused on one cell in late region 2b. par-1 mutant clones stained for alpha-tubulin show a slight delay in the focusing of the microtubules to one cell in region 2b, which is similar to the delay in Orb restriction. However, the overall organization of the microtubules in region 2 appears essentially normal (Huynh, 2001a).

Since it is difficult to interpret the organization of the microtubules within the oocyte in anti-tubulin stainings, markers for the minus-ends of the microtubules were used to examine the polarity of these microtubules in wild type and par-1 mutant cysts. Although the centrosomes lie at the minus ends of the microtubules in most cells, this is not necessarily the case in the germline, since they appear to be inactivated when the cyst leaves region 1, and a Nod-GFP transgene was therefore used as an alternative marker for the minus ends. Nod-GFP localization in wild-type cysts reveals that the minus ends are first focussed at the anterior of the oocyte, before switching to the posterior as the cyst buds off the germarium. In par-1 mutant cysts, Nod-GFP still becomes restricted to the anterior of one cell, but never re-localizes to the posterior (Huynh, 2001a).

Furthermore, the minus end-directed motor, dynein, shows an identical behavior as that of Nod-GFP: it localizes to the anterior of one cell in mutant cysts, but fails to switch to the posterior in region 3. Thus, par-1 is required for a reorganization of the microtubule cytoskeleton of the oocyte in region 3, and this may account for the failure of Orb and the centrosomes to move from the anterior to the posterior of the cell (Huynh, 2001a).

The par-1 null allele is also mutant for mei-W68, which shares a promoter with par-1 and lies entirely within its first intron. Since Mei-W68 is required for the dsDNA breaks that initiate meiotic recombination, some or even all of the phenotypes of par-1 clones could therefore be due to this meiotic defect rather than the loss of par-1 itself. To distinguish between these possibilities, the Gal4/UAS system and a Nanos-Gal4-VP16 driver were used to express the N1S isoform of par-1 in the germarium as a GFP-fusion protein, to determine whether it could rescue the phenotype of par-1^W3 germline clones. The fusion protein localizes to a branched structure in regions 1 and 2 of the germarium, which is presumably the fusome, as the endogenous protein is a component of this structure. More importantly, PAR-1N1S completely rescues the delay in Orb localization to the oocyte in par-1^W3 homozygous cysts, and also restores the anterior-posterior movement of Orb within the oocyte in region 3. Furthermore, this construct rescues the posterior movement of the centrosomes, and the maintenance of oocyte identity. Thus, these phenotypes are specific to par-1, and are not due to the reduction in Mei-W68 activity or any other mutations on the par-1^W3 chromosome (Huynh, 2001a).

The N1L isoform of PAR-1, which differs only from N1S by the exclusion of five amino acids in an alternative exon in the linker region of the protein and the presence of the conserved C-terminal par-1 domain, was also expressed. The PAR-1N1L fusion protein also localizes to the fusome, but is unable to rescue any of the early defects in par-1^W3 germline clones, although it is functional because it rescues the par-1 phenotype at other stages of development. This suggests that the par-1 domain inhibits the early function of the kinase, and provides the first example where different PAR-1 isoforms have different activities (Huynh, 2001a).

The GFP-PAR-1 localization in the germarium is intriguing, since it has been suggested that the fusome provides the initial cue for the determination of the oocyte. To confirm that par-1 is recruited to the fusome, wild-type germaria were stained for PAR-1, using an antibody that recognises all isoforms of the protein, and Hu li tai shao (Hts), an integral component of the fusome. PAR-1 and Hts co-localize on the spectrosome, which is the precursor of the fusome in the germline stem cells and cystoblasts, and on the fusome itself. This co-localization is most apparent in region 1, but some PAR-1 persists on the fusome in region 2. In addition, the PAR-1 antibody labels small particles that do not contain Hts, but this is due to a cross-reacting antigen, because these are not seen with the GFP-PAR-1 fusions, and do not disappear in par-1 null germline clones (Huynh, 2001a).

Mutants in other components of the fusome, such as Hts and alpha-Spectrin, produce cysts with fewer than 16 nurse cells, because the precise pattern of cyst divisions is disrupted. However, all par-1^W3 mutant egg chambers contain 16 cells, indicating that par-1 is not required for this function of the fusome. To rule out the possibility that the lack of an effect on the pattern of divisions in the cyst is due to the perdurance of wild-type PAR-1 protein in mutant clones, mutant egg chambers derived from persistent stem cell clones that were induced 10 days earlier, were also analysed and the same result was obtained. To test if par-1 affects the formation or morphogenesis of the fusome, mutant cysts were stained for alpha-Spectrin, which is a component of the spectrosome and the fusome at all stages of its development. The spectrosome is asymmetrically partitioned during stem cell division, so that two-thirds remain in the daughter stem cell, and one-third in the cystoblast, and this asymmetry is unaffected in par-1^W3 clones. Furthermore, the fusome is asymmetrically partitioned at each subsequent division, as in wild type: when a mutant cystoblast divides, one cell still inherits more fusome than the other; this asymmetry is maintained throughout the next three mitoses, and can be seen in four-cell cysts and in 16-cell cysts that have stopped dividing. Thus, PAR-1 localizes on the fusome, but is not required for its formation or its asymmetric segregation (Huynh, 2001a).

The re-localization of cytoplasmic markers in region 3 reveals the earliest known A-P polarity within the oocyte. It is very intriguing that PAR-1 is required both for this polarization, and the later polarization of the oocyte at stage 9. This raises the question of whether the two are linked. For example, it is possible that the altered organization of the microtubules and mislocalization of Oskar mRNA at stage 9 in par-1 hypomorphs is a consequence of earlier problems in the polarization of the oocyte in the germarium. This seems unlikely, however, for several reasons. (1) The localization of all known markers for oocyte polarity changes during stage 7, suggesting that the early polarization of the oocyte is erased when it re-polarizes in response to a signal from the posterior follicle cells. (2) In the vast majority of egg chambers that are mutant for par-1 hypomorphs, Orb protein moves normally from the anterior to the posterior of the oocyte, and is maintained there as it is in wild type. Furthermore, the Oskar mRNA localization defect of these mutants can be rescued by a par-1 transgene that is only expressed after the cysts have left the germarium. The view that PAR-1 plays a role in the anterior-posterior polarization of the oocyte at two stages of oogenesis is therefore favored. It will be interesting to determine whether these processes are related in other ways, and whether PAR-1 serves a common function in oocyte determination and Oskar mRNA localization (Huynh, 2001a).

The anterior-posterior axis of C. elegans is defined by the asymmetric division of the one-cell zygote, and this is controlled by the PAR proteins, including PAR-3 and PAR-6 (see Drosophila par-6), which form a complex at the anterior of the cell, and PAR-1, which localizes at the posterior. PAR-1 plays a similar role in axis formation in Drosophila: the protein localizes to the posterior of the oocyte and is necessary for the localization of the posterior and germline determinants. PAR-1 has recently been shown to have an earlier function in oogenesis, where it is required for the maintenance of oocyte fate and the posterior localization of oocyte-specific markers. The homologs of PAR-3 (Bazooka) and PAR-6 are also required to maintain oocyte fate. Germline clones of mutants in either gene give rise to egg chambers that develop 16 nurse cells and no oocyte. Furthermore, oocyte-specific factors, such as Orb protein and the centrosomes, still localize to one cell but fail to move from the anterior to the posterior cortex. Thus, PAR-1, Bazooka, and PAR-6 are required for the earliest polarity in the oocyte, providing the first example in Drosophila where the three homologs function in the same process. Although these PAR proteins therefore seem to play a conserved role in early anterior-posterior polarity in C. elegans and Drosophila, the relationships between them are different, since the localization of PAR-1 does not require Bazooka or PAR-6 in Drosophila, as it does in the worm (Huynh, 2001b).

The determination of the oocyte can be followed by the migration of the centrosomes, which move along the fusome to cluster at the anterior of the oocyte in region 2b, and then translocate to the posterior of the oocyte in region 3. In baz mutant cysts, the centrosomes accumulate at the anterior of the oocyte but never move to the posterior cortex. Furthermore, alpha-tubulin stainings of mutant cysts indicate that microtubules remain focused on the anterior of the oocyte and fail to rearrange. These phenotypes are identical to those produced by par-1 mutants, indicating that Bazooka and PAR-1 act in the same step in oocyte determination. Neither is necessary for the initial selection of the oocyte, because Orb and the centrosomes still become restricted to one cell, but they are required for the maintenance of oocyte fate, since the oocyte soon dedifferentiates and becomes a nurse cell. This failure to maintain oocyte identity correlates with a block in the movement of oocyte-specific factors and the centrosomes from the anterior to the posterior of the cell, suggesting that this early polarization is important for the further development of the oocyte (Huynh, 2001b).

PAR-6 has been shown to localize to the same protein complex as PAR-3 in C. elegans, Drosophila, and mammalian cells and is essential both for the localization and the function of this complex. In Drosophila, Bazooka and PAR-6 colocalize to the apical side of the embryonic ectoderm, where they are necessary for the maintenance of epithelial polarity, and both proteins are also inherited by the neuroblasts when they delaminate and are required for the basal localization of cell fate determinants during their asymmetric divisions. To test if Drosophila PAR-6 also functions with Bazooka during oogenesis, germline clones were generated of the par-6^Delta226 allele, which is a deletion of the promoter, the start codon, and the first 121 amino acids of the protein and is therefore a strong loss of function mutation if not a null. The majority of mutant egg chambers appear small, oval-shaped, and contain 16 polyploid nurse cells and no oocyte, indicating that PAR-6 is also required for oocyte determination. Furthermore, Orb and the centrosomes accumulate in one cell at the posterior of the cyst, although with a slight delay compared to wild-type. Both remain at the anterior of the oocyte, however, and fail to translocate to the posterior pole. Thus, the loss of PAR-6 from the germline gives an identical phenotype to Bazooka and PAR-1. As is the case for bazooka germline clones, some of the par-6 mutant egg chambers escape the early arrest and go on to produce normal eggs. When the females are scored 2 days after eclosion, half of the egg chambers form a normal oocyte, and about a quarter still do so after 10 days. This increase in the penetrance of the phenotype with age shows that PAR-6 protein perdures for many days after the clones are produced. Consistent with this, PAR-6 appears to be unusually stable in the embryo; the protein can be detected throughout embryogenesis in zygotic par-6 null embryos, at levels that are only slightly lower than in wild-type. However, the continued presence of escapers after 10 days suggests that PAR-6 may not be essential for oocyte determination in all cases and that there may be redundant pathways that can partially compensate for its absence (Huynh, 2001b).

To investigate the relationships between Bazooka, PAR-6, and PAR-1 during oocyte determination, their localizations were analyzed in both wild-type and mutant germaria. In region 2a to region 3 of the germarium, Bazooka localizes around the ring canals, in a ring that is about twice the diameter of that formed by actin. This localization is very similar to that of the adherens junction components Shotgun (E-cadherin) and Armadillo. A double staining was therefore performed for Arm and Baz. Although Arm localizes to these rings before Bazooka in early region 2a, the two proteins colocalize from the middle of region 2a until region 3, when they both disappear. Bazooka also colocalizes with Shotgun and Armadillo in the zonula adherens of the embryonic epithelium, which provides a boundary between the apical and basolateral membrane domains. This raises the possibility that the Shotgun, Armadillo, and Bazooka rings in the germarium perform a similar function by marking the separation between an anterior and a posterior domain within the oocyte. It is unclear whether PAR-6 also localizes to these rings, since none of the available antibodies give any significant staining that disappears in par-6 null germline clones

These results show that PAR-1, Bazooka, and PAR-6 act in the same step in oocyte determination, providing the first example in Drosophila where these three homologs of C. elegans PAR proteins participate in the same process. Furthermore, mutants in all three genes disrupt the movement of oocyte-specific proteins and the centrosomes from the anterior to the posterior of the oocyte, which is the earliest visible sign of polarity within the oocyte. Given the role of these PAR proteins in other systems, it seems very likely that their primary function in the germarium is in the anterior-posterior polarization of the oocyte, and that the failure to maintain oocyte fate is a consequence of this defect (Huynh, 2001b).

It is intriguing that this very early anterior-posterior polarity of the Drosophila oocyte requires three of the PAR proteins that mediate the anterior-posterior polarization of the first cell division in C. elegans. Although this suggests that these proteins act in a conserved pathway for generating cell polarity in these two systems, the relationships between the localizations of these proteins are quite different in the Drosophila oocyte and C. elegans zygote. Thus, at least some aspects of their function are not conserved, and it will therefore be interesting to determine whether the downstream pathways that generate other cellular asymmetries in response to this polarity are related (Huynh, 2001b).

PAR-1 regulates the polarised microtubule cytoskeleton in the Drosophila follicular epithelium

The PAR-1 kinase plays a conserved role in cell polarity in C. elegans, Drosophila and mammals. The role of PAR-1 in epithelial polarity was investigated by generating null mutant clones in the Drosophila follicular epithelium. Large clones show defects in apicobasal membrane polarity, but small clones induced later in development usually have a normal membrane polarity. However, all cells that lack PAR-1 accumulate spectrin and F-actin laterally, and show a strong increase in the density of microtubules. This is consistent with the observation that the mammalian PAR-1 homologs, the MARKs, dramatically reduce the number of microtubules, when overexpressed in tissue culture cells. The MARKs have been proposed to destabilize microtubules by inhibiting the stabilizing activity of the Tau family of microtubule-associated proteins. This is not the case in Drosophila, however, since null mutations in the single tau family member in the genome have no effect on the microtubule organization in the follicle cells. Furthermore, PAR-1 activity stabilizes microtubules: microtubules in mutant cells depolymerize much more rapidly after cold or colcemid treatments. Loss of PAR-1 also disrupts the basal localization of the microtubule plus ends, which are mislocalized to the center of mutant cells. Thus, Drosophila PAR-1 regulates the density, stability and apicobasal organization of microtubules. Although the direct targets of PAR-1 are unknown, it is suggested that it functions by regulating the plus ends, possibly by capping them at the basal cortex (Doerflinger, 2003).

Although par-1 mutants produce similar polarity phenotypes in the follicle cells to mutants in the components of the Bazooka/PAR-6/aPKC complex, they are not identical. par-1 clones cause a complete disruption of polarity only when induced early in the follicle cell lineage. The smaller clones that arise later in oogenesis often show little or no reduction in the localization of apical and basolateral markers, and usually remain as a single layer of cells. By contrast, even late clones of bazooka or aPKC produce penetrant epithelial defects. This difference is also apparent in the embryo, where loss of zygotic bazooka, PAR-6 or aPKC disrupts epithelial organization. par-1 homozygous embryos, however, display no obvious epithelial polarity phenotype, although it is not possible to remove the maternal PAR-1 completely. The reason for low penetrance of polarity defects in smaller par-1 follicle clones is unclear, but similar differences between early large clones and smaller late clones have been observed for crumbs and lkb1 (the Drosophila par-4 homolog - required for the early A-P polarity of the oocyte and for the repolarization of the oocyte cytoskeleton that defines the embryonic A-P axis). One possibility is that these genes are required for the initial formation of the follicular epithelium, but not for its maintenance. This seems unlikely to be the case for par-1, however, because small clones containing only one or two cells can sometimes show strong apicobasal polarity defects. A more likely explanation is that the low penetrance of this phenotype in small clones is due to the perdurance of the PAR-1 that was present at the time when the clones were induced. In support of this, the penetrance of the polarity defects of par-1 clones increases with clone size and the stage of oogenesis, as one would expect if the protein is gradually degraded over time, and is diluted out by cell division (Doerflinger, 2003).

In contrast to apicobasal membrane polarity, the density, the stability and the organization of MTs are disrupted in all par-1 clones, regardless of their size or the stage of oogenesis. This is likely to represent a distinct function of the kinase from its other roles in cell polarity, because it is much more sensitive to a reduction in activity. The effects of PAR-1 on MT density are consistent with results on the mammalian PAR-1 homologs, MARK1 and MARK2. These experiments show that removal of PAR-1 causes an increase in the density of MTs in each cell, whereas overexpression of MARK1 or MARK2 in unpolarized tissue culture cells causes most MTs to disappear (Doerflinger, 2003).

The MARKs have been proposed to regulate the MT cytoskeleton by phosphorylating Tau family MAPs, thereby inhibiting them from binding to and stabilizing MTs. Although PAR-1 does phosphorylate Drosophila Tau in vitro, tau null mutations are viable and fully fertile and have no effect on the arrangement of MTs in either the follicle cells or the oocyte. Therefore, this mechanism cannot account for its function in organizing the MTs in the follicle cells. The viability of tau mutants is surprising, given the many functions ascribed to Tau in human neurons. It does have a precedent, however, since tau knockout mice are viable, have a morphologically normal nervous system, and display only defects in MT stability and organisation in small-caliber axons. The mild phenotype of tau in mice has been proposed to be due to functional redundancy with the closely related MAP2, but this cannot be the case in Drosophila, which does not contain a MAP2 homolog. It may be redundant with other types of MAP, however, and the best candidate is Futsch, which has significant structural and functional homology to mammalian MAP1B. MAP1B appears to have functional overlap with both Tau and MAP2 in mammals, because mice that are homozygous for null mutations in map1b and tau, or map1b and map2, show defects in axonal elongation, neuronal migration and MT organization that are much more severe than in mice lacking only one of these genes (Doerflinger, 2003).

Another compelling argument that PAR-1 regulates MTs by a different mechanism from that proposed for the MARKs is that it is required to stabilize rather than destabilize MTs, at least in epithelial cells. The MTs in follicle cells are among the most stable in nature, because they are almost completely resistant to cold or to prolonged colchicine treatments. By contrast, the MTs in par-1 mutant cells appear to be highly dynamic because: (1) they disappear after brief colchicine treatments; (2) they depolymerize at 4°C, but re-grow in a few minutes after return to 25°C; (3) they are lost during fixation, if the fixative is too weak (Doerflinger, 2003).

The opposite effects of PAR-1 and the MARKs on MT stability may indicate that these closely related kinases have evolved to fulfil distinct functions in invertebrates and mammals. It is also possible, however, that this reflects the different experimental approaches and cell-types that have been used to examine their activities. The MARKs have been assayed by over-expressing them in CHO cells, which are a transformed line of rapidly dividing, undifferentiated and unpolarized cells. By contrast, the loss-of-function phenotype of PAR-1 was examined in post-mitotic follicle cells, highly polarized and differentiated epithelial cells. The two cell types also have very different microtubule cytoskeletons. In CHO cells, microtubules are nucleated from a central centrosome, and are presumably reasonably dynamic, because they disassemble at each mitosis, whereas the follicle cells lose their centrosomes when they form a columnar epithelium, and nucleate a very stable apicobasal array of microtubules. It would therefore be interesting to test the effects on MT stability of disrupting the function of PAR-1 homologs in more similar mammalian cell-types, such as polarized MDCK cells (Doerflinger, 2003).

In addition to its effect on stability, PAR-1 is required to maintain the normal organization of the MTs. The MT arrangement in the follicle cells is typical of a polarized epithelium, with the minus ends associated with the apical membrane, and the plus ends at the basal side of the cell. The arrangement of minus ends appears to be largely unchanged in par-1 mutant cells, but a marker for the plus ends, Kinesin:ß-gal, accumulates in the center rather than the basal region of the cell. This phenotype is very similar to that observed in par-1 mutant oocytes, in which the plus ends become abnormally focussed in the center of the oocyte, rather than at the posterior, and there is an increase in the density of MTs around the cortex. Thus, PAR-1 may regulate the MTs in the same way in the two cell types, and most probably acts primarily on the plus ends. PAR-1 may also have some effect on the distribution of the minus ends of MT in the oocyte, as bicoid mRNA, which is believed to be transported towards minus ends, is found around the lateral cortex of mutant oocytes, rather exclusively at the anterior. Although the possibility that there is also an effect on the minus ends in mutant follicle cells cannot be ruled out, this is not detectable using Nod:ß-gal (Nod is a kinesin-related protein) as a marker (Doerflinger, 2003).

It seems paradoxical that the loss of PAR-1 should increase the density of MTs in follicle cells, while decreasing their stability, but one possible explanation is suggested by studies in mammalian cells on populations of stable MTs that are marked by detyrosinated alpha-tubulin. These MTs are resistant to nocadazole-induced depolymerization, and fail to incorporate new tubulin subunits, leading to the proposal that they are capped at their plus ends in a way that prevents both the addition and loss of tubulin. Thus, it is possible that PAR-1 stabilises the MTs in the follicle cells by capping plus ends when they reach the basal cortex, thereby preventing them from either growing or shrinking. If the conditions inside the cell favour MT polymerization, the loss of the PAR-1-dependent cap would allow the plus ends to continue to grow once they reach the basal cortex. This could account for both the increase in MT density and the redistribution of plus ends to the center of mutant cells. However, the uncapped MTs would rapidly shrink under conditions that favor MT depolymerization, such as cold or colchicine treatment, explaining why the MTs disappear in mutant cells (Doerflinger, 2003).

par-1 null clones also show fully penetrant and cell-autonomous increases in the recruitment of ß-spectrin and actin to the lateral cortex. Like the microtubule phenotype, these effects appear to be independent of the defect in apicobasal membrane polarity, since the latter is much less penetrant. These phenotypes may therefore reflect a third distinct function of the kinase. It is also possible, however, that the MT defects are a consequence of the changes in actin organization or vice versa. In this context, it is interesting to note that Rho family GTPases, major regulators of the actin cytoskeleton, have also recently been found to control the capping of MT plus ends at the leading edge of migrating cells. The Rac and Cdc42 effector, IQGAP, interacts with the plus end binding protein, CLIP170, to stabilize MTs transiently, whereas the Rho effector, mDia, leads to the formation of more stable MTs, perhaps through the plus-end binding protein EB1. Given that PAR-1 does not appear to function through the obvious candidate, Tau, it would be interesting to test whether this kinase acts through either of these pathways to regulate MT organization in epithelial cells (Doerflinger, 2003).

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par-1: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 10 July 2004

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