Cytoplasmic streaming drifts the polarity cue and enables posteriorization of the Caenorhabditis elegans zygote at the side opposite of sperm entry

Abstract

Cell polarization is required to define body axes during development. The position of spatial cues for polarization is critical to direct the body axes. In Caenorhabditis elegans zygotes, the sperm-derived pronucleus/centrosome complex (SPCC) serves as the spatial cue to specify the anterior-posterior axis. Approximately 30 min after fertilization, the contractility of the cell cortex is relaxed near the SPCC, which is the earliest sign of polarization and called symmetry breaking (SB). It is unclear how the position of SPCC at SB is determined after fertilization. Here, we show that SPCC drifts dynamically through the cell-wide flow of the cytoplasm, called meiotic cytoplasmic streaming. This flow occasionally brings SPCC to the opposite side of the sperm entry site before SB. Our results demonstrate that cytoplasmic flow determines stochastically the position of the spatial cue of the body axis, even in an organism like C. elegans for which development is stereotyped.

FIGURE 1:

The sperm enters only through the proximal side of the oocyte. (A) Schematic of the C. elegans gonad and the definition of the proximal and distal sides. (B) Fluorescence confocal image of zygote expressing GFP::PH, GFP::histone and TBG-1::GFP in a fer-1(hc17) hermaphrodite crossed to a him-5(e1490) male expressing mCherry::histone at a time immediately after fertilization. Asterisks indicate the oocyte-derived chromosomes. The white arrow indicates the position of the SPCC. The distal quartile and proximal quartile of the oocyte or zygote before polarization are defined as the distal and proximal sides, respectively, as shown in the right schematic. Scale bar, 5 μm. (C) Representative examples of the PP and DP-type after SB. Arrowheads indicate the centrosome. White dotted circles indicate the sperm pronucleus. Scale bar, 5 μm. (D–F) The ratios of the zygote of which SPCC is positioned in the distal or proximal side immediately after fertilization and after SB under conditions of (D) mating using CAL0182 (fer-1(hc17) mutant) hermaphrodites and CAL0841 males, (E) self-fertilization (DE90), and (F) mating using CAL1041 hermaphrodites and CAL1651 males are shown; n = 52 and 53 (D), 89 and 84 (E), and 51 and 69 (F) for each time point, respectively.

FIGURE 2:

SPCC stochastically moves within the zygote before cell polarization. (A) Time series images of C. elegans zygotes (DE90 strain) expressing mCherry::histone, GFP::PH, GFP::histone and TBG-1::GFP at fertilization, meiotic anaphase I (Ana I), meiotic anaphase II (Ana II), before pronuclear migration (PNM), and two-cell stage under the condition of self-fertilization. White arrows indicate the position of the SPCC. Representative examples of the DP-type are shown in the bottom panels. The timing of meiotic anaphase I is set to 0 min. Scale bars, 5 μm. (B) The position of SPCC along the long axis of the zygote is evaluated as shown in the diagram and is plotted in C. The proximal pole and the distal pole are set to 0 and 100, respectively. (C) Six representative examples of SPCC dynamics are shown. For details, see the text. (D) Box plots of the position of SPCC along the long axis of the zygote at the timing of fertilization (F), meiotic anaphase I (AI), and meiotic anaphase II (AII), immediately after SB and before pronuclear migration (PNM) under each condition. Asterisks indicate significant differences between F and AII (***P < 0.001). (E) Positional distribution of SPCC along the long axis of the zygote which remained within 25% of the long axis (left, <25%) throughout the observation until SB, or exceeded (right, >25%) 25% at least once before SB. SPCCs detected at more than five of the seven time points (F, AI, mid [5 min after AI], AII, SB, PNM) were plotted. Data include both the self-fertilization and mating conditions; n = 66, 86, 85, 80, 80, 86, and 83 for each time point in <25%; and n = 51, 66, 65, 62, 64, 68, and 66 for each time point in >25%, respectively.

FIGURE 3:

MeiCS moves SPCC. (A) The left two panels show time series of C. elegans zygote labeled yolk granules (VIT-2::GFP) and SPCC (mCherry::histone). The shape of the zygote is outlined by white dotted line. White dotted circles indicate SPCC. The right panel shows projections of sequential images of the zygote during MeiCS (six frames, time interval: 5 s). White arrows indicate flow vectors of yolk granules (>1 μm). The yellow dotted line indicates the position of SPCC at 0 s. Scale bars, 5 μm. (B) Scatter plots of the angle (degree) and velocity (μm/s) of MeiCS and SPCC motion (69 frames from five zygotes). The mean angle and mean velocity of flow vectors of yolk granules within 7 μm radius of SPCC is calculated as the angle and velocity of MeiCS. (C) Box plots of the position of SPCC along the long axis of the zygote under conditions of unc-116 RNAi, yop-1;ret-1 RNAi and nmy-2 RNAi are shown as in Figure 2D. Asterisks indicate significant differences between F and AII (***P < 0.001). NS, not significant. (D) Representative trajectory of SPCC from fertilization to before pronuclear migration in untreated and unc-116 (RNAi) zygote is shown by the colored line. The gray ellipse represents the zygote and the blue and light blue circle indicates the proximal and distal poles, respectively. The blue arrows indicate the position of fertilization. The red circles with red arrows indicate the position of SPCC at the time of SB.

FIGURE 4:

The AP axis can be predicted after MeiCS ceases. (A) The frequency of misprediction in PP- and DP-types. For details, refer to the text. (B) Histograms representing the SPCC positions along the long axis of the zygote at the indicated time points for the PP-(blue) and DP-(orange) types; n = 112 and 9 (F), 139 and 10 (AI), 134 and 11 (mid), 161 and 11 (AII), 170 and 10 (–1 min), 185 and 12 (SB), and 194 and 16 (PNM) for PP- and DP-types, respectively. (C) Representative trajectories of SPCC in the zygote in which SB occurred when SPCC was positioned at 43, 51, and 82% of the long axis are shown as in Figure 3D. (D) Summary of the SPCC dynamics during cell polarization. For details, see the text.