Programmed reduction of ABC transporter activity in sea urchin germline progenitors

Abstract

ATP-binding cassette (ABC) transporters protect embryos and stem cells from mutagens and pump morphogens that control cell fate and migration. In this study, we measured dynamics of ABC transporter activity during formation of sea urchin embryonic cells necessary for the production of gametes, termed the small micromeres. Unexpectedly, we found small micromeres accumulate 2.32 times more of the ABC transporter substrates calcein-AM, CellTrace RedOrange, BoDipy-verapamil and BoDipy-vinblastine, than any other cell in the embryo, indicating a reduction in multidrug efflux activity. The reduction in small micromere ABC transporter activity is mediated by a pulse of endocytosis occurring 20-60 minutes after the appearance of the micromeres--the precursors of the small micromeres. Treating embryos with phenylarsine oxide, an inhibitor of endocytosis, prevents the reduction of transporter activity. Tetramethylrhodamine dextran and cholera toxin B uptake experiments indicate that micromeres have higher rates of bulk and raft-associated membrane endocytosis during the window of transporter downregulation. We hypothesized that this loss of efflux transport could be required for the detection of developmental signaling molecules such as germ cell chemoattractants. Consistent with this hypothesis, we found that the inhibition of ABCB and ABCC-types of efflux transporters disrupts the ordered distribution of small micromeres to the left and right coelomic pouches. These results point to tradeoffs between signaling and the protective functions of the transporters.

Fig. 1.

Sea urchin micromeres and small micromeres accumulate calcein from their appearance through blastulation. (A) Schematic diagrams of the 16-, 28- and 60-cell stage embryos. Each stage is shown from a vegetal view. At the 16-cell stage, the micromeres are green. All cells in the embryo, except the micromeres, progress through one cell cycle to produce the 28-cell embryo. At the 60-cell stage, the micromeres have divided into the large micromeres and the small micromeres. (B) Maximum intensity projections (MIPs) of S. purpuratus embryos showing two developmental switches in calcein accumulation; first, increased in the micromeres (Mics), but not the macromeres (Macs) or the mesomeres (Mes) of the 28-cell stage embryo, and later increased in the large micromeres (Lmic) and small micromeres of the 60-cell embryo; and second, decreased to levels observed in the Mac and Mes for the Lmics in the early blastula. Scale bars: 10 μm.

Fig. 2.

Small micromeres from sea urchin and sand dollar blastulae accumulate multiple ABC transporter substrates. (A) MIPs of blastulae showing small micromeres accumulate more CTRO, BFLVp and BFLVb. (B) Vegetal view of a blastula overexpressing Sp-vasa-mCherry fusion protein. Small micromeres are labeled with intracellular calcein in green and Sp-vasa in red. (C) MIPs of embryos from L. pictus and D. excentricus treated with CAM at the 60-cell stage. Scale bars: 10 μm in A,C; 3 μm in B.

Fig. 3.

The rate of calcein accumulation is faster in micromeres and small micromeres than in large micromeres and the rest of the embryo. (A) MIPs of S. purpuratus embryos accumulating calcein throughout early development. White, green, blue and orange arrowheads indicate each region of interest graphed for the rest of the embryo, micromeres, large micromeres and small micromeres, respectively. Black arrowhead on the graph indicates the start of CAM incubation. Scale bar: 10 μm. (B) Rates of calcein accumulation observed for each region of interest and normalized to basal accumulation of the rest of the embryo. Bars are the average of seven embryos from three females (±s.e.m.) and bars sharing the same letter are not significantly different from one another (ANOVA, P≤0.5), white bars with different letters are significantly different (P≤0.05).

Fig. 4.

ABCB- and ABCC-type transport activity is downregulated in the small micromeres. (A) Average (±s.e.m.) intracellular calcein concentration for the rest of the sea urchin embryo (dashed line) and small micromeres (solid line) after treatment with MK571, an inhibitor of MRP-efflux transporters [n=5×3 (batches × embryos) measured for each concentration] and (B) PSC833, an inhibitor of MDR-efflux transporters (n=4×5). (C) MIP of the vegetal view of a 28-cell and 60-cell embryo treated with the fluorescent ABCG2 substrate 1 μM mitoxantrone. Small micromeres are marked by a white arrow. All of the cells in the embryo accumulate the same amount of mitoxantrone. See also supplementary material Fig. S2. Scale bars: 10 μm.

Fig. 5.

Inhibiting protein tyrosine phosphatases (PTPs) with PAO stops loss of multidrug efflux transport activity in the micromeres. (A) Relative amount of intracellular calcein of the sea urchin micromeres compared with the rest of the embryo [n=3×≥10 (batches × embryos), ±s.e.m.] during treatment with inhibitors of transcription (ActD), Golgi traffic (BfA), endocytosis (CdCl₂ and PAO) and exocytosis (Wort). Asterisk indicates values significantly different from control (ANOVA, P≤0.5). Treatment with PAO stops the loss of multidrug efflux, and postponing PAO treatment until 50 minutes after the appearance of the micromeres does not stop the loss of efflux transport [PAO (50)]. (B) Representative MIPs of calcein accumulation in embryos treated with DMSO, PAO and PAO (50). (C) MIPs showing the cumulative amount of rhodamine dextran and CTB-positive endosomes in micromeres and macromeres for embryos treated with DMSO or PAO for 80 minutes after their appearance. Scale bars: 10 μm.

Fig. 6.

Fluid-phase and GM1 endocytosis is dynamic throughout the cell cycle, peaking at 40-60 minutes after micromere formation. (A) Average relative endosome volume (±s.e.m.) measured in sea urchin micromeres (solid lines) and macromeres (broken lines) using rhodamine dextran [n=3×5×4 (batches × embryos × cells) from each embryo], a marker of fluid-phase endocytosis, and cholera toxin B (n=3×5×4), a marker of GM1 found in lipid rafts. (B) MIPs showing endocytosis of CTB in 16-cell, 28-cell and blastulae after a 5-minute labeling period and a 20- to 30-minute uptake interval. Micromeres are labeled with arrowheads. (C,D) Embryos co-incubated in rhodamine dextran and CTB (C) 20-40 minutes and (D) 40-60 minutes after the appearance of the micromeres showing lack of colocalization of the two markers. See also supplementary material Fig. S1. Scale bars: 10 μm.

Fig. 7.

Inhibiting ABC transporter activity in the whole sea urchin embryos causes small micromeres to become more randomly segregated. (A,B) MIPs of embryos injected with mCherry-SpVasa mRNA to localize the small micromeres in red, and treated with 0.3% DMSO, 5 μM MK571 or 3 μM PSC833 in (A) representative embryos and (B) coelomic pouches. Images combined with DIC channel in 96- to 110-hour-old plutei. White arrowheads indicate the left coelomic pouch. Numbers in B indicate the number of Vasa-positive cells counted in each left coelomic pouch. (C) Number and percent of small micromeres in the left coelomic pouch of mCherry-Sp-Vasa overexpressing embryos treated with DMSO (n=45), MK571 (n=38) or PSC833 (n=44). (D) Average percent (±s.e.m., ≥4 embryos measured per batch) of embryos with left/right coelomic pouch distributions outside of either 3/5 or 4/4 (left pouch/right pouch) from eight batches for the DMSO treatment, six batches for MK571 and seven batches for PSC833. Asterisk indicates values significantly different from the DMSO control (ANOVA, P≤0.5 with square root transformed values). Scale bars: 25 μm in A; 10 μm in B.