PIE-1 Translation in the Germline Lineage Contributes to PIE-1 Asymmetry in the Early Caenorhabditis elegans Embryo

Abstract

In the C. elegans embryo, the germline lineage is established through successive asymmetric cell divisions that each generate a somatic and a germline daughter cell. PIE-1 is an essential maternal factor that is enriched in embryonic germline cells and is required for germline specification. We estimated the absolute concentration of PIE-1::GFP in germline cells and find that PIE-1::GFP concentration increases by roughly 4.5 fold, from 92 nM to 424 nM, between the 1 and 4-cell stages. Previous studies have shown that the preferential inheritance of PIE-1 by germline daughter cells and the degradation of PIE-1 in somatic cells are important for PIE-1 enrichment in germline cells. In this study, we provide evidence that the preferential translation of maternal PIE-1::GFP transcripts in the germline also contributes to PIE-1::GFP enrichment. Through an RNAi screen, we identified Y14 and MAG-1 (Drosophila tsunagi and mago nashi) as regulators of embryonic PIE-1::GFP levels. We show that Y14 and MAG-1 do not regulate PIE-1 degradation, segregation or synthesis in the early embryo, but do regulate the concentration of maternally-deposited PIE-1::GFP. Taken together, or findings point to an important role for translational control in the regulation of PIE-1 levels in the germline lineage.

Keywords: C. elegans; PIE-1; asymmetric cell division; exon junction complex; germline.

Figure 1

Quantification of the increase in PIE-1::GFP concentration in the P lineage. A. Schematic of PIE-1 (gray) localization from the 1-cell to the 4-cell stage. Maternally deposited PIE-1 segregates asymmetrically to the germline blastomeres P1 and P2 during the first two rounds of cell division. PIE-1 is also degraded in somatic cells. Sister cells are connected by a line. B. Top panel: Coomassie stained SDS-PAGE gel of recombinant GFP and BSA, which was used as a loading standard. Bottom panels: Images of N2 and PIE-1::GFP embryos bathed in 300 nM GFP. Images were pseudocolored using the CyanHot lookup table in ImageJ (scale at the bottom). In order to highlight the dimmer PIE-1::GFP signals, the nuclear signal in the main image of the 4-cell embryo is saturated. The image normalization was adjusted equivalently in the 2 and 4-cell embryo insets such that the nuclear signal is not saturated. PIE-1::GFP concentration in the 1-cell embryo was determined using a 150 nM GFP bath, but is shown in a bath of 300 nM GFP to allow comparison with the later stage embryos. C. Estimates of PIE-1::GFP concentration in P0, P1 and P2. For P1 and P2, concentration estimates are shown for the entire cell (Tot), the cytoplasm (Cyt) and for the nucleus (Nuc). Mean concentrations and the number of embryos analyzed are indicated below the graph. Error bars represent 95% confidence intervals. Statistical significance was determined using unpaired t-tests with Welch’s correction for comparisons between embryos (P0 vs. P1; P1 vs. P2) and using paired t-tests for comparisons between cytoplasmic and nuclear concentrations in either P1 or P2. In this and subsequent figures: * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001, n.s. = not significant. D. Estimates of the volume of each cell from the 1 to 4-cell stage, determined using embryos expressing GFP::PH^PLCδ1 (Audhya, Hyndman et al. 2005), which marks the plasma membrane. E. The relative concentration of PIE-1::GFP in germline and somatic daughter cells (P1 and AB; P2 and EMS) just after the division of P0 and P1. Statistical significance was determined using an unpaired t-test with Welch’s correction. Error bars represent 95% confidence intervals.

Figure 2

PIE-1::GFP is synthesized in P1 and P2. A. Images of PIE-1::GFP embryos from the 1-cell to the 4-cell stage. PIE-1::GFP fluorescence was bleached in the embryo on the right. Scale bar = 5 μm. PNM, pronuclear meeting in the 1-cell embryo. Time is indicated relative to PNM. B. Quantification of the average PIE-1::GFP fluorescence in embryos that were photobleached at the 1-cell stage. Note the increase in fluorescence in P1 and P2. Values were normalized to the pre-bleach values (n = 6 embryos). Statistical significance (Student’s t-test) comparing the final timepoint for each cell to the initial postbleach timepoint is indicated. Error bars represent SEM. Vertical dotted lines indicate cell divisions. C. Change in PIE-1::GFP concentration in P1 and AB in both wild-type and zif-1(egx5) embryos. Embryos were imaged following the division of P0. The concentration was normalized to the mean PIE-1::GFP intensity in the entire control embryo. Error bars indicate SEM. D. Zygotic transcription of PIE-1::GFP was tested with the indicated crossing scheme. Cross progeny were identified by the presence of male sperm-derived mitochondria that were labeled with MitoTracker Red (indicated with white arrows in the top panel). No PIE-1::GFP fluorescence was observed in the cross progeny embryos (n = 4).

Figure 3

PIE-1::GFP concentration is reduced in P2 in spn-4(RNAi), Y14(RNAi), and mag-1(RNAi) embryos. A. Images of PIE-1::GFP in 4-cell embryos of the indicated genotypes. Note that the decrease in PIE-1::GFP levels in spn-4(RNAi) embryos depends on ZIF-1 (indicated by white arrows). Images were pseudocolored using the CyanHot lookup table in ImageJ (scale at the right). Scale bar = 5 µm. B, C. Mean PIE-1::GFP concentration in P2 in wild-type (panel B) and zif-1(egx5) (panel C) embryos treated with the indicated RNAi. All values were normalized to the mean of the control RNAi. Error bars indicate 95% confidence intervals. Statistical significance was determined using a Student's t test.

Figure 4

PIE-1::GFP segregation and synthesis in Y14(RNAi) and mag-1(RNAi) embryos. A. PIE-1::GFP localization in polarized zygotes of the indicated genotype. B. The ratio of PIE-1::GFP concentration in posterior and anterior cytoplasm of zygotes of the indicated genotype at nuclear envelope breakdown. C. Change in the average PIE-1::GFP concentration in P1 in embryos of the indicated genotype. The concentration values were normalized to the entire control embryo, including both AB and P1. D. Change in the average cytoplasmic and nuclear PIE-1::GFP concentration in P1 in embryos of the indicated genotype. The same embryos were analyzed in panels C and D. Error bars indicate SEM in panels B, C and D.

Figure 5

Quantification of the levels of maternally deposited proteins in Y14(RNAi) and mag-1(RNAi) embryos. A. PIE-1::GFP expression in the germline of adult hermaphrodites treated with the indicated RNAi. B. PIE-1::GFP and MEX-1::GFP fluorescence in P0 and P2 of embryos treated with the indicated RNAi. Values were normalized to the control at both stages. C. MEX-6::GFP fluorescence in 1 and 4-cell embryos treated with the indicated RNAi. Values were normalized to the control for each cell. D. MEG-3::meGFP and PGL-1::GFP fluorescence in 1-cell embryos treated with the indicated RNAi. Values were normalized to the control for each cell. For panels B – D, the number of embryos is indicated in parentheses and error bars indicate 95% confidence intervals.

Figure 6

Effects of Y14(RNAi) and mag-1(RNAi) on PIE-1::GFP localization and embryonic viability. A. PIE-1::GFP localization from the 4-cell to ∼100-cell stage. The nuclear PIE-1::GFP levels are saturated in the main images. Each of the insets in the bottom right corner are normalized equivalently such that the nuclear signals are not saturated. The dotted ellipses outline the embryos. B. Embryonic viability of embryos of the indicated genotype. Error bars indicate SEM.