Involvement of fatty acid pathways and cortical interaction of the pronuclear complex in Caenorhabditis elegans embryonic polarity

Background Cell polarity is essential for many decisions made during development. While investigation of polarity-specific factors has yielded great insights into the polarization process, little is known on how these polarity-specific factors link to the basic cellular mechanisms that function in non-polarity aspects of the cell. To better understand the mechanisms that establish embryonic polarity, we investigated genes required for polarity in the one-cell C. elegans embryo that are also required for other non-polarity functions. This has led to the identification of the Pod-class of mutants that are characterized by osmosensitive embryos and defects in anterior-posterior polarity. Results Mutation in either of two loci of this class, emb-8 and pod-2, disrupts embryonic polarization and results in osmotically-sensitive embryos. Loss of emb-8, a previously uncharacterized polarity gene, causes mislocalization of PAR-3 and PAR-2 that molecularly mark the anterior and posterior cortices. emb-8 encodes NADPH-cytochrome P450 reductase, a protein supplying electrons to cytochrome P450-family enzymes, some of which catalyze fatty acid modifications. Cloning of the previously characterized polarity gene pod-2 reveals it encodes acetyl-CoA carboxylase, an enzyme that catalyzes the first step in de novo fatty acid synthesis. Depletion of fatty acid synthase, the next enzyme in the biosynthetic pathway, by RNA-interference (RNAi) also causes similar loss of one-cell polarity. Furthermore, pod-2 polarity defects can be rescued by addition of exogenous fatty acids. By following the behavior of the pronucleus in emb-8 and pod-2 mutant embryos, we demonstrate that loss of polarity correlates with impaired interaction between the pronucleus-centrosome complex and the posterior cortex. Conclusions The characterization of emb-8 and pod-2 mutant embryos suggests that the pronucleus-centrosome complex interaction with the cortex plays a direct role in establishing polarity and that fatty acid pathways are important for this polarizing event.


Background
The formation and function of diverse cell types during development requires the establishment of cell polarity. Cell polarity can provide for the asymmetric localization of cell fate determinants, thereby ensuring their inheritance by only one of the two daughter cells [1]. Prominent examples of this process include ASH1 mRNA localization to the daughter cell in yeast, Numb and Prospero localization during Drosophila neuroblast determination, segregation of germline granules in C. elegans, and Vg1 localization in Xenopus [2][3][4][5][6]. Once differentiated cell types and tissues have been formed, cell polarity continues to play an essential role in their function. For example, directional transport and sorting of molecules to either apical surfaces or basolateral regions are key features of epithelial cells [7,8].
Given the widespread requirement for cell polarity, it is no surprise its establishment involves a conserved set of proteins. Several studies have shown that a conserved protein complex, comprised of PAR-3 (ASIP/bazooka), PAR-6, and atypical PKC, functions in nematodes, fruit flies, and humans and in diverse cells ranging from embryos to epithelia (reviewed in [9,10]). One hallmark of this complex, hereafter referred to as the PAR-3 complex, is its asymmetric localization. In the C. elegans one-cell embryo, the PAR-3 complex becomes restricted to the anterior cortex and another polarity protein, PAR-2, localizes reciprocally to the posterior cortex [11,12]. This spatial distinction between anterior and posterior cortical domains defines the first body axis of the embryo. These molecular landmarks are thought to be translated into the physically asymmetric first division and differential segregation of developmental determinants such as germline granules (reviewed in [13]).
While the asymmetric localization of the PAR-3 complex appears to be essential for downstream polarity processes, the mechanisms underlying the polarization of the complex itself are poorly understood. Part of the difficulty lies in the fact such mechanisms probably involve the more generalized machinery of the cell. In contrast to polarity gene mutants that only affect polarity establishment, impairment of core cellular processes will likely produce more complex phenotypes with both polarity and nonpolarity defects. Yet, in order to better understand the polarization process, the role of these factors will need to be defined.
Research in the C. elegans one-cell embryo is beginning to reveal some of these more general cellular functions required for polarity establishment. In C. elegans, the sperm, probably via its associated centrosome, provides the initial polarizing cue that defines the posterior end [14]. As might be expected, translation of this cue into the final polarization of the zygote requires basic elements of the cytoskeleton. Recent studies have implicated the microtubule cytoskeleton in this process. Mutations in the spd-2 gene attenuate microtubule nucleation from the sperm-derived centrosomes and cause mislocalization of PAR-3 and PAR-2 [15]. Other mutants that create an ectopic microtubule array at the anterior end are sufficient to reverse the localizations of PAR-3 and PAR-2 [16]. These findings suggest that some aspect of the centrosome or microtubule array directs the localization of PAR-3 and PAR-2 to discrete parts of the cell, with PAR-3 adopting a localization opposite that of the microtubule organizing center. Studies with other polarity mutants further suggest that a close association of the sperm-derived centrosome with the cortex provides the polarity-generating cue required for normal polarity [17]. Disruption of actin or the actin-associated proteins, non-muscle myosin (nmy-2) and myosin light-chain (mlc-4), also cause loss of asymmetry at the first division and mislocalization of PAR-3 and PAR-2 although the underlying mechanism is still not understood [18][19][20]. Given the essential nature of the cytoskeleton in general, uncovering its polarity functions required transient disruption or partial protein depletion in order to circumvent other non-polarity phenotypes (e.g., defects in cytokinesis or cell cycle arrest).
Our approach to understanding how the basic cellular machinery is involved in establishing polarity has led to the discovery and characterization of the Pod class of mutants (Polarity and Osmotic Defective; [17]). The inaugural member of this class is an actin-associated protein, POD-1. The loss of POD-1 causes polarity and non-polarity defects including osmotically sensitive (or osmosensitive) embryos, cytokinesis defects, abnormal endocytic structures, and transient appearance of abnormal cell peripheries [21]. The pod genes were defined as a class with the discovery of the pod-2 gene that is also required for polarity and osmotic protection and functions in the same pathway as pod-1 [22]. The osmotic sensitivity of pod-1 and pod-2 mutants likely results from defects in the secreted eggshell that normally protects the embryo from the external environments and make the embryo impermeable to most dyes. These phenotypes and the similarity of POD-1 to coronin, a gene involved in endocytosis and phagocytosis [23,24], suggest polarization of the zygote may involve membrane trafficking processes. Additional Pod class members encode subunits of the anaphase-promoting complex (APC) [17]. These are required to polarize PAR-3 and PAR-2, possibly through separindependent proteolysis of a centrosome/microtubule associated factor normally required to repulse PAR-3 from the posterior cortex.
Here we detail the characterization of two additional Podclass polarity genes: emb-8 and pod-2. We identified emb-8 as a member of the Pod class of polarity genes and show emb-8 is required for establishing anterior-posterior polarity. The emb-8 gene encodes the C. elegans NADPH-cytochrome P450 reductase (NCPR), which transfers electrons to cytochrome P450 monooxygenase enzymes. Our findings implicate NCPR, and thus P450-dependent reactions, in the establishment of cell polarity. The pod-2 locus encodes acetyl-CoA carboxylase, a key enzyme in fatty acid biosynthesis. Furthermore, depletion of fatty-acid synthase, the next enzyme in the pathway, results in similar polarity phenotypes implicating fatty acids in the establishment of polarity. Both emb-8 and pod-2 act upstream of PAR-3 and PAR-2 polarization by enabling association between the paternal pronucleus/centrosome complex with the embryonic cortex. Our data provide important evidence that this association is a key event in polarizing the embryo along the anterior-posterior axis and suggest that the interaction depends on fatty acid pathways.

emb-8, a new gene required for embryonic polarity
We identified emb-8 as a Pod locus by screening through published osmosensitive mutants for those that also divide symmetrically at the first cell division. The emb-8 allele, hc69, was initially isolated as a conditional embryonic lethal mutation required maternally for embryo viability [25]. Initial characterization of hc69 showed mutant embryos were osmotically sensitive [26], and we verified that hc69 mutant embryos placed in hypotonic medium became swollen and arrested similar to other Pod mutants. No polarity defect was reported in these earlier works.
Analysis of emb-8(hc69) mutant embryos in isotonic medium reveals emb-8 mutant embryos have lost polarity in the one-cell embryo. In the wild-type embryo, the first cleavage produces a characteristic asymmetric two-cell embryo with a larger anterior and a smaller posterior blastomere ( Figure 1A). These two cells subsequently divide asynchronously and in orthogonal orientations indicative of their dissimilar identities (only the posterior cell will give rise to germline, for instance). In contrast, half of emb-8(hc69) mutant embryos divide symmetrically, producing two blastomeres of roughly equal size ( Figure 1E, Table 1). In embryos where the first division is symmetric, the second cell cycles are synchronous and the two blastomeres divide with roughly parallel orientations, transverse to the longitudinal axis (data not shown). A second emb-8 mutant, t1462, was isolated in a chromosome IIIwide genetic screen for embryonic lethal mutations [27]. Although emb-8(t1462) mutant embryos have penetrant osmotic defects, they divide asymmetrically (Table 1). Nonetheless, the symmetric first division in emb-8(hc69) mutants coupled with their osmosensitivity (both are also seen in emb-8(RNAi) embryos, see below), places emb-8 in the Pod group of polarity genes. That emb-8(t1462) is fully penetrant for osmosensitivity but has no polarity defect indicates that the two functions are separable, as has been demonstrated for other pod genes to date. Mitotic timing of the first cell cycle in emb-8 mutant embryos is not significantly different from wild-type (data not shown).
To determine if the symmetric division in emb-8(hc69) results from loss of one-cell polarity or from inability to position the first spindle, we examined the distribution of molecular markers of polarity in emb-8(hc69) mutant embryos. In wild-type one-cell embryos, PAR-3 occupies the anterior cortex and PAR-2 adopts a reciprocal location at the posterior cortex ( Figure 1B and 1C, [11,12]). In response to this polarized state, germline granules are segregated to the posterior cytoplasm of the one-cell embryo ( Figure 1D, [6]). The asymmetric localizations of these markers are disrupted in emb-8(hc69) mutant embryos. Reduction of emb-8 function leads to either more uniform cortical localization of PAR-3 ( Figure 1F, 5/13 embryos) or enrichment of PAR-3 along a cortical lateral edge that is sometimes reduced ( Figure 1I, 5/13 embryos). The remaining three embryos had wild-type PAR-3 localization. Although PAR-2 is sometimes present at the posterior in emb-8 mutant embryos, it is usually reduced to a small patch at the cortex ( Figure 1G

emb-8 encodes the NADPH-cytochrome P450 reductase
To gain insight into the role of emb-8 in establishing onecell polarity, we determined the molecular identity of the emb-8 locus. Previous genetic mapping placed emb-8 in a central region of chromosome III [25]. We narrowed the region through three-factor mapping crosses which placed the emb-8 locus between dpy-17 and unc-32 ( Figure 2A); 25 of 28 Uncs and none of 20 Dpy F2 hermaphrodites from dpy-17+unc-32/+emb-8(hc69)+ segregated F3 progeny that produced osmosensitive embryos. Furthermore, the rarity with which we found Unc animals that did not carry the emb-8(hc69) allele suggested that emb-8 was located very close to dpy-17.
We used the osmosensitive phenotype of emb-8 mutants to identify a candidate gene near dpy- 17   The emb-8 locus is encoded by K10D2.6, the C. elegans NADPH-cytochrome P450 reductase homolog  RNAi depletion of one of these, K10D2.6, produced embryos that "lose structural integrity upon dissection" and were placed in a phenotypic category that included the pod-1 locus, an osmosensitive mutant described previously [21,28]. To determine if the K10D2.6 locus contained molecular variations associated with the emb-8 mutants, we sequenced the hc69 and t1462 alleles and identified missense mutations that result in glycine to glutamate changes in residues 608 and 543, respectively, of the predicted protein ( Figure 2A).  Figure 2B). Furthermore, RNAi of K10D2.6 reproduced the mislocalization of PAR-3 and PAR-2 seen in emb-8(hc69) mutants ( Figure 2C and 2D).
Together with the sequencing of alleles, these data indicate that the open reading frame K10D2.6 represents the emb-8 gene and that osmotic sensitivity and symmetric cleavage phenotypes are caused by reduction of EMB-8 function.
K10D2.6 encodes the C. elegans ortholog of the NADPHcytochrome P450 reductase (NCPR). The K10D2.6 encoded protein shows high sequence similarity to NADPH-cytochrome P450 reductase enzymes from a wide range of species; K10D2.6 is 46% identical and 63-64% similar to NCPR proteins from human, mouse, and Drosophila. This similarity extends along the length of the protein and includes the recognizable FMN-, FAD-, and NADPH-binding domains that are involved in transferring electrons from NADPH to cytochrome P450. The only region lacking significant similarity is the first 60 residues which comprises a hydrophobic membrane anchor in NCPR [29,30]. Metazoan species typically possess only one form of NCPR and this appears true in nematodes as well (data not shown). Given the high degree of sequence similarity to NCPR enzymes and the recognizable functional domains of the protein, we conclude that emb-8, encodes the C. elegans NCPR ortholog. Studies in other systems have shown that NCPR catalyzes the transfer of electrons from NADPH to cytochrome P450 enzymes. One of the physiological roles of NCPR-dependent enzymes is to catalyze the modification of fatty acids. This role has interesting implications given the pod-2 gene, identified below.
In the course of our RNAi experiments, we found that only feeding mid-L4 staged larvae reliably produced these polarity defects ( Figure 2E). dsRNA-feeding of late-L3 to early L4-stages produced less penetrant polarity defects although osmosensitive embryos were obtained (data not shown). To date, we have not been able to demonstrate any essential larval functions for emb-8 since dsRNA feeding from the L1 stage does not prevent growth to adulthood.

The pod-2 polarity gene encodes acetyl-CoA carboxylase
The previously characterized Pod-class locus, pod-2, directly implicates fatty acids in the generation of polarity.
Using single-nucleotide polymorphisms, we mapped the pod-2 gene to the region around cosmid W09B6 (data not shown  Figure 3A). W09B6.1 encodes the sole C. elegans homolog of acetyl-CoA carboxylase (ACC) and contains all the domains conserved in ACC from other organisms ( Figure 3A).
To further confirm that the previously observed polarity phenotypes in the pod-2 mutants resulted from loss of ACC-function, we analyzed characteristics of anterior-posterior polarity in embryos depleted of W09B6.1/ACC by RNAi. Wild-type adult hermaphrodites fed control bacteria produce embryos with normal polarity that divide asymmetrically at the first cell division (not shown). In contrast, adult nematodes fed bacteria expressing doublestranded RNA (dsRNA) encoding the ACC gene produce embryos that divide symmetrically at the first cell division ( Figure 3B, Table 1). Feeding of W09B6.1 dsRNA to pod-2(ye60) animals at 15° can increase the number of symmetric embryos to greater than 90% symmetric cleavage (data not shown). As expected, embryos depleted of POD-2/ACC by RNAi display 100% penetrant osmotic sensitivity ( Table 1). RNAi of the other gene with similarity to pod-2 in the C. elegans genome (T28F3.5 that encodes carbamoyl-phosphate synthase) resulted in neither symmetric 2-cell nor osmotically sensitive embryos (data not shown). POD-2/ACC's role in establishing the anterior-posterior (a-p) axis in the embryo appears to be specific. This conclusion is supported by the following observations. First, embryos depleted for POD-2/ACC are healthy and show no overt phenotypes (e.g., cytokinesis defects, loss of cell integrity) apart from loss of polarity and osmotic sensitivity. When placed pressure-free in osmotically balanced medium, these embryos are essentially indistinguishable from other polarity mutants (e.g., par's) and exhibit first cell cycle mitotic timings statistically equivalent to wild type (data not shown). Second, the cold-sensitive pod-2(ye60) allele is a strict maternal affect lethal, and temperature shift studies with this allele suggest that the embryonic defects can be traced to a POD-2 function ~3 hours prior to fertilization [22]. These data suggest the pod-2/ ACC gene can be mutated to uncover a specific role in a-p axis polarization. However POD-2/ACC must also have functions in addition to a-p axis formation since first stage larvae fed pod-2/ACC dsRNA grow slowly and often die at the L3 stage (6/9).

Polarity establishment is linked to fatty acids
The above data suggest that fatty acids, which are synthesized by ACC and potentially modified downstream by EMB-8/NCPR-dependent reactions, play an important role in a-p axis formation. If so, then elimination of the next enzyme in the biosynthetic pathway, fatty acid synthase (FAS; Figure 3C), would also be predicted to lead to loss of a-p polarity. In C. elegans, a single open reading frame, F32H2.5, encodes FAS. Elimination of maternal FAS in embryos by dsRNA feeding results in a loss of a-p polarity in the progeny as judged by symmetric 2-cell embryos ( Figure 3D, Table 1). The polarity phenotype became apparent at 28 hours after the initiation of feeding at 15° C (33%; n = 24) and reached a maximum (89%; n = 19) at 64 hours. FAS(RNAi) embryos also displayed completely penetrant osmotic sensitivity.

(12)
No palmitic 13 0.01 29 (7) No Compounds that rescued polarity defects are shown in bold. 1 Scored by looking at whether embryos shrunk in hypertonic medium. In all cases, >80% osmosensitive embryos were detected. 2 Solvent control. Ethanol alone, the solvent for sphingomyelin and all the fatty acids except stearic acid was spread on the plate. 3 Solvent control. Chloroform alone, the solvent for stearic acid and all lipids but sphingomyelin, was spread on the plate. 4 Glycerol 3-phosphate in water at 1 mg/ml in plate. 5 Stearic and arachidonic acids. 6 Bovine; stearic and nervonic acids. 7 Chicken; palmitic acid. 8 80% lineoleic acid content. 9 Non-hydroxy fatty acid; stearic and nervonic acids. 10 Hydroxy fatty acid. 11 Crude (50%). 12 99% pure; linoleic and palmitic acids. 13 Separate set of experiments studying dose-dependence of fatty acid rescue 14/33 embryos had staining near the middle of the embryos ( Figure 3F), two showed lateral, posterior staining ( Figure 3H), one had staining all over the embryo, and 14 were wild-type in localization. Similar PAR-3 and P granule defects are seen in pod-2(ye60)/ACC mutant embryos [22]. The loss of PAR-3 staining at the cortex observed in pod-2 mutants is intriguing and may be indicative of significant changes in membrane composition. Nonetheless, as indicated below, the primary polarity defect in pod-2 mutant embryos is caused by events that precede PAR-3 localization.
As loss of ACC or FAS function would be expected to impair fatty acid synthesis, dietary supplementation with exogenous fatty acids might be able complement the pod-2/ACC polarity defects. We found that adding the fatty acids palmitic acid, stearic acid, oleic acid, linoleic acid, arachidonic acid, or docosahexaenoic acid to the diet of pod-2(ye60) mothers rescues the a-p polarity defects in their progeny (Table 2). Rescue by palmitic acid was shown to be dose-dependent. Conversely, neither solvents nor glycerol 3-phosphate (required for making phosphatidic acid) rescue. The polarity defect appears to be specific for fatty acids or the fatty acid moieties of lipids as addition of diacylglycerol, the other major component of lipids, to the diet also did not rescue. Although all fatty acids tested rescued the pod-2(ye60) polarity defects, none rescued osmotic sensitivity, confirming that the function of pod-2/ACC required for osmotic protection (i.e., normal eggshell) is separable from its function in a-p polarity. That the addition of fatty acids rescues only a subset of ACC phenotypes has also been seen in yeast [31].
These data demonstrate that fatty acids play an important role in establishing C. elegans polarity. However, since acetyl-CoA is also used in cholesterol biosynthesis, it was possible that the pod-2/ACC polarity defect might be caused by cholesterol deficiencies. To test this, we used RNAi to deplete the function of hydroxylmethylglutanyl (HMG) CoA reductase, an enzyme that uses acetyl-CoA and catalyzes the first committed step in the cholesterol biosynthesis. HMG CoA reductase in C. elegans is encoded by the open reading frame F08F8.2. Injection of F08F8.2 dsRNA into wild-type hermaphrodites resulted in embryos that were osmotically sensitive (100% penetrance 32 hours after injection) but had normal two-cell asymmetry (Table 1). This result confirms previous demonstrations that osmotic sensitivity itself does not lead to polarity defects [17,21,22]. After this time, the mothers became sterile as previously described [32]. Thus the pod-2 polarity defect appears to be specific for defects in fatty acid biosynthesis.

Loss of either emb-8 or pod-2 impairs the interaction between the paternal pronucleus and the posterior cortex
The fact that emb-8 and pod-2 have similar phenotypes and are either implicated in (emb-8) or directly involved in (pod-2) fatty acid biosynthesis, respectively, raises the issue as to whether they function in the same pathway. Although for temperature reasons, an emb-8; pod-2 double mutant is difficult to construct and score (one is temperature sensitive and the other is cold sensitive, including by feeding of dsRNA), we have previously shown that the pod-2 mutant functions genetically in the same pathway as a deletion allele of pod-1. We similarly tested whether emb-8 functions in the same pathway as pod-1 by constructing a pod-1(ye11) emb-8(hc69) double mutant and examining the penetrance of the polarity defect. We found no significant enhancement of the loss of a-p polarity in the double mutant as compared to the single mutants when two cell symmetry was scored (29% symmetric, n = 41 for pod-1(ye11); 42% symmetric, n = 24 for emb-8(hc69); and 29% symmetric, n = 31 for the double mutant). This result, together with the similar polarity phenotypes, suggests that pod-1, pod-2, and emb-8 all function in a similar pathway with regards to polarity.
This finding predicts that both emb-8 and pod-2 mutants should manifest similar defects in the events leading to the polarization of the one-cell embryo. We have previously reported that the APC Pod-class of mutants are defective in the interaction of the pronucleus centrosome complex (PNCC) with the posterior cortex and that our observations, as well as important observations from other laboratories, suggest interaction of the PNCC with the cortex was responsible for repelling PAR-3 from the posterior, thereby establishing anterior-posterior asymmetry. This interaction is characterized as movement of the paternal pronucleus (with associated centrosome) towards the posterior ( Figure 4A) and subsequent tight juxtaposition with the posterior cortex ( Figure 4B,4C). This association lasts for approximately five minutes and coincides temporally with the exclusion of PAR-3 from this region of the cortex [12,33], after which the paternal pronucleus dissociates from the cortex to meet the maternal pronucleus in the posterior cytoplasm ( Figure 4D).
To determine if emb-8 and pod-2 were required for this process, we examined this polarizing interaction in embryos that had been depleted of emb-8 or pod-2 functions. In embryos depleted of emb-8 function by RNAi, the paternal pronucleus initiated posterior movements (Figure 4E) but close association with the cortex was often abbreviated or prevented entirely ( Figure 4F,4G). Lack of interaction was followed by meeting of the pronuclei near the middle of the embryo ( Figure 4H) and symmetric division at first cleavage. Loss of pod-2 function similarly impaired this cortical interaction ( Figure 4I,4J,4K,4L). The variability in the interaction of the pronucleus with the cortex in the emb-8 and pod-2 mutants allowed us to investigate whether the quality and duration of the interaction directly correlated with polarity defects. Indeed, in emb-8(RNAi) embryos, strong correlation was observed between impaired pronucleus-cortex interaction and loss of first division asymmetry ( Figure 4M). Embryos that did not contact the cortex always divided symmetrically (relative area of the anterior blastomere less than 53%) and those that exhibited significant cortical interactions (longer than 2-3 minutes), with one exception, produced asymmetric divisions ( Figure 4M). In emb-8-depleted embryos that subsequently divided symmetrically, posterior-directed cytoplasmic flows were also qualitatively absent (data not shown). Similar correlation between loss of polarity and lack of pronucleus-cortical interaction was also observed in pod-2 mutant embryos (Table 3). Importantly, dietary supplementation of pod-2(ye60) mutants with palmitic acid rescued both the cortical interaction and a-p axis polarization (Table 3). Thus, emb-8 and pod-2 are each required for the cortical interaction by the pronucleus/centrosome complex, and this interaction correlates strongly with the establishment of a-p polarity.

Discussion
Although numerous polarity factors required for anteriorposterior axis formation in C. elegans have been identified, the next challenge facing developmental biologists is connecting these components to the basic cellular machinery. This study reveals two of these central components that are essential for generating asymmetries in the early C. elegans embryo: (1) emb-8 which encodes NADPH-cytochrome P450 reductase (NCPR) and (2) the fatty acid biosynthetic pathway represented by pod-2/ACC and FAS. Depletion of any of these functions causes loss of the asymmetry of the first division and missegregation of germline granules. These defects are not simply a consequence of an inability to translate cell polarity downstream of PAR-3 and PAR-2 but instead result from ineffective polarization of the one-cell embryo as evidenced by mislocalization of PAR-3 and PAR-2 in emb-8 or PAR-3 in FAS mutant embryos. Although PAR-2 still localizes to the cortex in emb-8/NCPR mutant embryos, it is often restricted to a small patch. We speculate that this narrowed PAR-2 domain might reduce the number or quality of the stronger PAR-2-dependent spindle pulling forces exerted on the posterior pole of the one-cell mitotic spindle, thereby preventing its posterior displacement [34]. In emb-8/NCPR mutants and FAS-depleted embryos, germline granule segregation is impaired or mistargeted to lateral regions. This lateral localization of germline granules was also observed in pod-2 mutant embryos [22]. These results indicate emb-8/NCPR and the fatty acid biosynthetic pathway function in establishing polarity in the one-cell embryo.
As additional evidence supporting previous findings with Pod mutants defective in the APC, we demonstrate that the source of the loss of polarity correlates with defects in the interaction between the paternal pronucleus/centrosome complex and the posterior cortex. We found a near perfect correlation between the duration of this interaction and the resultant physical polarity in both emb-8 and pod-2 mutant embryos. These data, together with other research in the one-cell C. elegans embryo field [14][15][16], suggests that contact between short microtubules from the paternal pronucleus-associated centrosome and the cortex provides the cue which polarizes the embryo, most likely via repulsion of PAR-3 from the posterior cortex. Lack of PAR-3 at the posterior cortex permits PAR-2 localization thereby establishing spatial differences in the anterior versus posterior domains.
The similarity in phenotypes shared by the known Pod genes -emb-8/NCPR, pod-1, pod-2/ACC, and APC class (emb-30, pod-3, pod-4, pod-5, and pod-6) suggests each represents a different aspect of a pathway that polarizes the one-cell embryo and that mutates to the Pod phenotype. Indeed, all show defects in the interaction of the paternal pronucleus/centrosome complex and the posterior cortex. We hypothesize that emb-30 and the other APC pod genes represent one aspect of the polarization pathway [17]. Given the APC pathway ultimately regulates separin, which has been shown to affect microtubule stability [35], we propose that the APC pod mutants represent the microtubule aspect of the polarizing interaction between the paternal pronucleus/centrosome complex with the posterior cortex. Although recent data reports that APC-class pod mutants also show failures in second polar body extrusion [36], it is unclear how this would impair the key cortical interaction by the paternal pronucleus at the opposite end of the embryo. Furthermore, polarity defects in an APC mutant do not correlate with defects in meiotic timings, and RNAi of separin, which has disrupted polar- a defined as juxtapositioning of paternal pronucleus with the cell cortex. b in one embryo the spindle initially set up symmetrically but subsequent division was asymmetric. c one embryo divided asymmetrically but with reversed polarity ity, has normal meiotic timings [17]. Although we cannot exclude the possibility that meiosis defects indirectly contribute to the lack of association of the pronucleus with the cortex in Pod mutants, our data suggest otherwise. A second aspect of the POD polarization pathway, represented by pod-1, we hypothesize is indicative of a requirement for the actin cytoskeleton at the cortex or possibly spatial control of membrane trafficking events required for the interaction [21]. A third aspect is represented by EMB-8/NCPR and POD-2/ACC (see below).
Understanding how emb-8/NCPR contributes to the interaction of the pronucleus with the cortex and the subsequent asymmetric localization of PAR-3 and PAR-2 requires dissection of the myriad of functions downstream of NADPH-cytochrome P450 reductase. The best characterized function of NCPR is the transfer of electrons to cytochrome P450 (CYP) family enzymes, although cytochrome b5 can also act as an acceptor [37,38]. General types of CYP-mediated reactions have been identified and include hydroxylation, desaturation, and epoxidation [39]. Most of these P450-dependent reactions pertain to the oxidation of xenobiotics such as drugs and pesticides. Of the few endogenous substrates identified, cytochrome P450 reactions are largely directed towards hydrocarbons such as fatty acids and sterols. For example, mammalian CYP4 and CYP2 subfamily members can promote omegaand omega-1 hydroxylation of laurate and arachidonic acid [40,41] as well as desaturation of laurate [42]. Consistent with this, elimination of yeast NCPR causes changes in the fatty acid profile including chain lengths and levels of unsaturated fatty acids [43].
This function of NCPR-dependent reactions in maintaining proper chain lengths and levels of desaturation of cellular fatty acids is intriguing given the molecular identification of the pod-2 gene as acetyl-CoA carboxylase, the first and rate-limiting step in de novo fatty acid biosynthesis. The fact depletion of another fatty acid biosynthetic enzyme, FAS, shows similar loss of polarity and that dietary supplementation with fatty acids can rescue the polarity defects suggests that polarization of the embryo is linked to a third class of POD components in addition to those mentioned above: fatty acid pathways.
We therefore hypothesize that emb-8/NCPR and pod-2/ ACC influence embryonic polarity via effects on the fatty acid composition of membrane lipids. It is well established that the biochemical properties of membranes are altered by changing the fatty acid composition of the constituent lipids. The fatty acid balance achieved through the combined functions of emb-8/NCPR and pod-2/ACC might be required to (1) establish a particular membrane composition or membrane subdomain (e.g., membrane microdomains [44]) necessary to promote interaction between the pronucleus/centrosome complex and the cortex, or (2) maintain the proper membrane tension or curvature required for this interaction as has been suggested [31]. The importance of lipids and cellular asymmetry is best exemplified by polarized epithelial cells where apical and basolateral domains are characterized by distinct membrane compositions (reviewed in [45]). The fact that both epithelial cells and the C. elegans one-cell embryo require the PAR-3/ASIP complex for polarity suggests additional parallels, including membrane subdomains, may exist between these cell types. Alternatively, fatty acid composition could influence membrane fluidity, which could in turn affect endo-and exocytosis abilities of the cell [46].
The identification of emb-8/NCPR (and presumably cytochrome P450) and fatty acid biosynthesis pathways as necessary players in polarity establishment in the C. elegans embryo highlight the need to better understand the role of basic cellular processes and how they function in developmental contexts. Because of the powerful genetics in C. elegans, nearly all of the identified developmental components represent proteins and protein complexes. The potential link between membrane composition and polarity provided by the emb-8 and pod-2 mutants serves as a reminder that efforts must also be made to understand the contributions of non-protein elements of the cell (i.e. lipids and fatty acids) in the establishment of polarity. Our emb-8/NCPR results also provide evidence that NCPR has important functions beyond the metabolism of exogenous chemicals. The further merging of development with cell biology studies should continue to reveal additional roles for such cellular processes as well as better define the underlying mechanisms used by development to generate complex multicellular organisms and cell polarity.

Conclusions
The characterization of emb-8 and pod-2 mutants suggests that a key event in anterior-posterior axis polarization in the C. elegans embryo is the interaction between the pronucleus/centrosome complex with the posterior cortex. This finding uncovers an important link between the polarization cue provided by sperm entry and the resultant polarization of the anterior and posterior landmarks, PAR-3 and PAR-2, upon which all known downstream asymmetries depend. Furthermore, the molecular identities of these Pod-class polarity genes suggest establishment of polarity requires fatty acid pathways -an unexplored element of the fundamental cellular machinery.
PAR-2 antibodies. For determination of the optimal feeding duration, synchronized wild-type hermaphrodites were plated as early L4-, mid L4-, late L4-, and young adult stages for varying durations of time at 20°C. Hermaphrodites were individually dissected in 100 µl EBGM and two-cell embryos scored for symmetry. A drop (approximately 25 µl) of 5 M NaCl was added to determine osmosensitivity.

Fatty acid and lipid supplementation
Fatty acids and lipids (except for diacylglycerol; BIOMOL, PA) were purchased from SIGMA and resuspended typically at 100 mg/ml (notable exception is DAG at 10 mg/ ml) in the solvent indicated in Table 2. 100 µl of each was spread over a 10 ml NG agar plate. 40 µl of E. coli OP50 were added as food source and ~25 pod-2(ye60) L4 hermaphrodites were plated, grown at 15° for 72 hours, and then dissected to release embryos. In control experiments, 100 µl of either ethanol, chloroform, or water was spread.

Authors contributions
CR carried out the characterization, cloning, and phenotypic analysis of the emb-8 mutants and drafted the manuscript. AT performed the cloning of pod-2, phenotypic characterizations of fatty acid enzyme mutants, and fatty acid feeding analysis. Cloning of pod-2 was aided by unpublished information provided by NL and JA. RA was the primary investigator and coordinated the research efforts. All authors read and approved the final manuscript.
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