MAP kinase signaling antagonizes PAR-1 function during polarization of the early Caenorhabditis elegans embryo

Abstract

PAR proteins (partitioning defective) are major regulators of cell polarity and asymmetric cell division. One of the par genes, par-1, encodes a Ser/Thr kinase that is conserved from yeast to mammals. In Caenorhabditis elegans, par-1 governs asymmetric cell division by ensuring the polar distribution of cell fate determinants. However the precise mechanisms by which PAR-1 regulates asymmetric cell division in C. elegans remain to be elucidated. We performed a genomewide RNAi screen and identified six genes that specifically suppress the embryonic lethal phenotype associated with mutations in par-1. One of these suppressors is mpk-1, the C. elegans homolog of the conserved mitogen activated protein (MAP) kinase ERK. Loss of function of mpk-1 restored embryonic viability, asynchronous cell divisions, the asymmetric distribution of cell fate specification markers, and the distribution of PAR-1 protein in par-1 mutant embryos, indicating that this genetic interaction is functionally relevant for embryonic development. Furthermore, disrupting the function of other components of the MAPK signaling pathway resulted in suppression of par-1 embryonic lethality. Our data therefore indicates that MAP kinase signaling antagonizes PAR-1 signaling during early C. elegans embryonic polarization.

Figure 1.—

Characterization of the allele par-1(zu310). The allele par-1(zu310) exhibits phenotypes previously described for other alleles of par-1 and bears a lesion in the par-1 coding sequence. (A and B) Images taken from timelapse recordings, showing embryos undergoing the first division. Note the less pronounced asymmetry in cell size of the two daughter cells generated in par-1(zu310) mutant embryos when compared to wild-type embryos. (C and D) Images from time lapse recordings showing two-cell embryos undergoing asynchronous division (wild type) or synchronous division (par-1(zu310)). Asterisks mark spindle poles, circles mark centrosomes before nuclear envelope breakdown. (E and F) Two cell-embryos stained with anti-PIE-1 antibodies. PIE-1 is enriched in the posterior daughter cell in wild-type embryos and is more evenly distributed in par-1(zu310) mutants. Embryo length is ∼50 μm. (G) Sequence alignment of a part of the kinase domain in PAR-1 and human KP78, rat MARK2, mouse EMK, rat AMPK, and yeast SNF1 and KIN1 according to Guo and Kemphues (1995). par-1(zu310) contains a lesion in the kinase domain leading to an I306N exchange. All orthologs carry nonpolar residues in this position as opposed to par-1(zu310). The alignment was generated with ClustalW software. BoxShade backgrounds show identical residues as solid; similar residues are shaded. Borders of catalytic loop and activation segment are assigned according to Panneerselvam et al. (2006).

Figure 2.—

Genetic interaction between par-1 and the MAP kinase pathway. (A) Schematic representation of genes involved in MAP kinase signaling required for vulva formation in C. elegans (adapted from Sundaram 2006). For clarity, only the core components are depicted. Human homologs of C. elegans genes are indicated in parentheses. (B) Reduction of function of several genes in the MAP kinase pathway increases viability in a par-1(zu310) mutant background. Values correspond to average viability of the progeny as percentage of total progeny ± standard deviation (n = 9 plates in three independent assays, except for let-23(RNAi), which corresponds to three plates in one assay). Values significantly different from par-1(zu310) control animals are marked with a circle (P ≤ 0.03).

Figure 3.—

Asymmetric cell division and asynchrony of cell cycle timing in par-1 and mpk-1; par-1 mutant embryos. (A) Top panel: Asymmetry in cell division was determined as the size of the AB cell relative to the whole embryo length. For this, the ratio between the distance from the anterior pole to the cleavage furrow and the total length of the embryo was determined at the time of cleavage completion. Bottom panel: The anterior cell in par-1(zu310) is significantly smaller than in wild type. mpk-1(ga111); par-1(zu310) is not significantly different from par-1(zu310). (B) Top panel: Cell cycle timing was assessed by measuring the time from completion of the first cleavage furrow until nuclear envelope breakdown in the anterior (AB) and posterior (P₁) blastomeres. Bottom panel: Quantification of cell cycle timing in AB (light shading) and P₁ (dark shading) daughter blastomeres. In par-1(zu310) both cells divide synchronously as compared to wild type. This phenotype is restored in mpk-1(ga111); par-1(zu310) animals. All values shown are averages ± standard deviation (n ≥ 20). Values that significantly differed (P < 0.05) from wild type are marked with a circle, values different from par-1 are marked with an asterisk.

Figure 4.—

Cell fate specification is partially restored in mpk-1; par-1 double mutant embryos. (A–L) Localization of endogenous MEX-5 (top panel) and PIE-1 (middle panel) in two-cell stage embryos and localization of PAR-1 (red) and P granules (green) in one-cell stage embryos during centration (bottom panel). Wild-type embryos and mpk-1 embryos show asymmetric localization of MEX-5, PIE-1, and P granules. par-1 embryos exhibit a less pronounced gradient of MEX-5 and PIE-1 between the two daughter blastomeres and P granules are absent in one-cell stage embryos. mpk-1; par-1 animals show partially restored asymmetries for MEX-5 and PIE-1. P granules are present, but not localized to the posterior. Embryo length is ∼50 μm. (M and N) Quantification of fluorescence intensity ratio between the cytoplasm of AB and P₁ on MEX-5 or PIE-1 stained embryos. Circles indicate values different from wild type and asterisks mark values different from par-1 mutants (P < 0.05).

Figure 5.—

PAR-1 localization in one- and four-cell embryos (A) Quantification of PAR domain sizes in one-cell embryos stained for either PAR-3 or PAR-1 at anaphase or telophase. PAR-3 domain size (green bars) and PAR-1 domain size (red bars) were determined in independent experiments and are plotted together in a bar diagram for comparison. Zero percent corresponds to the anterior of the embryo, while 100% corresponds to the posterior pole. Values shown correspond to domain size averages ± standard deviation. Values that are significantly different from wild type are marked with a circle and values significantly different from par-1 with an asterisk. Both the anterior and the posterior domains are extended in par-1(zu310) animals. (B) Confocal images of embryos double stained for both PAR-3 and PAR-1 to visualize domain extension in the same embryos. As for the single-stained embryos quantified in A, the domains of PAR-3 and PAR-1 extend further in par-1 mutant embryos compared to wild type (n = 6). Quantifications were done using mid-plane confocal sections (top panels). (C–G) Dorsoventral PAR-1 asymmetry is not restored in mpk-1(ga111); par-1(zu310) mutant embryos. (C–F) Confocal images of anti-PAR-1 staining (red) of late four-cell stage embryos. Anti-tubulin staining is shown in green, DAPI staining shown in blue. (C, E, and G) In wild-type embryos and in mpk-1(ga111) mutants, PAR-1 localizes asymmetrically along the cortex of P₂ (cell marked with an asterisk), with more protein localizing between P₂/EMS than between P₂/ABp. (D, F, and G) In par-1(zu310) or in mpk-1(ga111); par-1(zu310) animals, PAR-1 localizes symmetrically along the dorsoventral axis. Embryo length is ∼50 μm. (G) Top panel: Schematic representation of a four-cell stage embryo. The cortices that were measured for the quantifications shown in the graph below are depicted in red. (G) Bottom panel: Quantification of ratio of the fluorescence intensities along the cortex between P₂/EMS and the cortex between P₂/ABp. Values shown correspond to average intensity ± standard deviation. Values significantly different (P < 0.05) from wild type are marked with a circle.

Figure 6.—

Models depicting possible genetic interactions between mpk-1 and polarity regulators. mpk-1 could act upstream of (A), downstream of (B), or parallel to (C) par-1 to antagonize its function.