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

Abstract

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.

Figure 1

Physical and molecular anterior-posterior polarity requires emb-8 function. Images of emb-8(+) (A-D) and emb-8(hc69) mutant embryos (E-K). Representative DIC images of two cell embryos (A, E) and immunolocalization of PAR-3 (B, F, and I), PAR-2 (C, G, and J), and germline granules (D, H, and K) in one-cell embryos. All embryos are oriented with anterior to the left and each image represents a different embryo. All fluorescent images and data represent analysis of one-cell embryos between pronuclear meeting through anaphase of the first cell cycle when PAR-3, PAR-2, and germline granules are normally asymmetrically distributed. All Wild-type embryos (A) divide asymmetrically and restrict (B) PAR-3 and (C) PAR-2 to the anterior and posterior cortices, respectively. (D) Germline granules are segregated to the posterior cytoplasm. (E) Half of emb-8(hc69) mutant embryos divide symmetrically. Some emb-8 mutant embryos show (F) uniform cortical PAR-3 localization; 5/13; (G) reduced PAR-2 domains at the posterior; 5/15; and (H) centrally dispersed germline granules; 6/22. Other emb-8 mutant embryos show (I) reduced, lateral localization of cortical PAR-3; 5/13 ; (J) mislocalization of PAR-2 to the posterior-lateral cortex; 6/15; (K) and mistargeted segregation of germline granules to posterior-lateral corners; 5/22.

Figure 2

The emb-8 locus is encoded by K10D2.6, the C. elegans NADPH-cytochrome P450 reductase homolog. (A) Schematic representation of mapping results which place emb-8 in the dpy-17 – unc-32 interval on chromosome III. Sequencing of emb-8 alleles hc69 and t1462 revealed two missense mutations in the predicted gene K10D2.6 which encodes the C. elegans NADPH-cytochrome P450 reductase. Sequencing of two independent clones from wild-type cDNA libraries confirmed the predicted gene structure. RNA-interference of K10D2.6 phenocopies the emb-8(hc69) mutant (B-D). RNAi of the K10D2.6 sequence causes embryos to (B) divide symmetrically at the first division, 41/59; and mislocalization of PAR-2 to (C) reduced posterior domains, 10/31 or (D) posterior-lateral regions of the cortex 14/31. (E) Results of time course experiments for K10D2.6 RNAi using synchronized mid-L4 staged hermaphrodites. Bars represent average proportion of embryos dividing symmetrically from 2–4 experiments with 8 hermaphrodites per experiment per time point (on average 10–15 embryos per time point).

Figure 3

pod-2 encodes acetyl-CoA carboxylase (ACC) which, along with fatty acid synthase (FAS), is required for anterior-posterior polarity. (A) Schematic representation of the POD-2/ACC protein indicating regions homologous with ACC of other species. The number below each box represents the percent amino acid identity between POD-2 and human ACC1 in that domain. (B) RNA-interference of W09B6.1/ACC phenocopies the symmetric division of pod-2(ye60) mutant embryos. (C) Overview of the fatty acid biosynthetic pathway. ACC and FAS catalyze the first two committed steps in fatty acid biosynthesis. Supplementary fatty acids can feed into this pathway where they become interconverted through the action of elongases and desaturases. Ultimately fatty acids can become incorporated into lipids or can be catabolized for metabolic energy. (D-H) Images of embryos depleted of fatty acid synthase (FAS) by RNA-interference. Loss of FAS function causes embryos to (D) divide symmetrically at the first division (17/19 embryos) and results in mislocalization of PAR-3 (E and G) and germline granules (F and H). RNAi of FAS causes PAR-3 to either lose cortical localization (E; 15/33 embryos) or to become more symmetric about the cortex (G; 10/33 embryos). Germline granules are missegregated to the center of the embryo (F; 14/33 embryos) or to a lateral location (H; 2/33).

Figure 4

Loss of emb-8 or pod-2 impairs cortical interaction by the paternal pronucleus which correlates with loss of polarity. Representative wild-type (A-D), emb-8(RNAi) (E-H), and pod-2(ye60) embryos (I-L). In wild type, (A) the paternal pronucleus initially appears in the posterior cytoplasm and (B) moves to the posterior where it closely associates with the cortex. This cortical interaction is maintained for approximately 5 minutes before (C) the pronucleus dissociates and (D) migrates to meet the maternal pronucleus in the posterior cytoplasm. In 8/16 emb-8(RNAi) embryos, (E) the paternal pronucleus appears in the posterior cytoplasm and some posterior movement occurs; however (F-G) contact with the cortex is not made and (H) the paternal and maternal pronuclei meet near the center of the embryo. In pod-2(ye60) mutants, (I-K) the paternal pronucleus does not become juxtaposed with the cortex. Subsequently, (L) the pronuclei meet in the center of the embryo. (M) Correlation between impaired cortical interaction and symmetric division in emb-8(RNAi) embryos. Relative size of the anterior blastomere (cross-sectional area) of individual embryos is indicated on the y-axis. Duration of pronucleus-cortex interaction is indicated along the x-axis. For 8 embryos, no interaction occurred ("no contact"). Symmetric division was defined as two-cell embryos in which the anterior blastomere comprised less than 53% of the total area. Blue bars = symmetric division. Red bars = asymmetric division.