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. 2012 Jun;241(6):1111-24.
doi: 10.1002/dvdy.23786. Epub 2012 May 2.

ATP-binding Cassette (ABC) Transporter Expression and Localization in Sea Urchin Development

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Free PMC article

ATP-binding Cassette (ABC) Transporter Expression and Localization in Sea Urchin Development

Lauren E Shipp et al. Dev Dyn. .
Free PMC article

Abstract

Background: ATP-binding cassette (ABC) transporters are membrane proteins that regulate intracellular concentrations of myriad compounds and ions. There are >100 ABC transporter predictions in the Strongylocentrotus purpuratus genome, including 40 annotated ABCB, ABCC, and ABCG "multidrug efflux" transporters. Despite the importance of multidrug transporters for protection and signaling, their expression patterns have not been characterized in deuterostome embryos.

Results: Sea urchin embryos expressed 20 ABCB, ABCC, and ABCG transporter genes in the first 58 hr of development, from unfertilized egg to early prism. We quantified transcripts of ABCB1a, ABCB4a, ABCC1, ABCC5a, ABCC9a, and ABCG2b, and found that ABCB1a mRNA was 10-100 times more abundant than other transporter mRNAs. In situ hybridization showed ABCB1a was expressed ubiquitously in embryos, while ABCC5a was restricted to secondary mesenchyme cells and their precursors. Fluorescent protein fusions showed localization of ABCB1a on apical cell surfaces, and ABCC5a on basolateral surfaces.

Conclusions: Embryos use many ABC transporters with predicted functions in cell signaling, lysosomal and mitochondrial homeostasis, potassium channel regulation, pigmentation, and xenobiotic efflux. Detailed characterization of ABCB1a and ABCC5a revealed that they have different temporal and spatial gene expression profiles and protein localization patterns that correlate to their predicted functions in protection and development, respectively.

Figures

Fig. 1
Fig. 1. Developmental stages surveyed and relative ABC transporter gene expression
(A) DIC micrographs depict the developmental stages included in the gene expression survey. (B) Heat map of quantitative real-time PCR data expressed as fold change from the reference stage (i.e. earliest detectable stage). Reference stage is egg in all transporters except ABCC5a, ABCG2b, ABCG11, and ABCG12. Reference stage is hatching blastula for ABCC5a, ABCG11, and ABCG12. Reference stage is early prism for ABCG2b. All data represent the average of progeny from four females (N=4).
Fig. 2
Fig. 2. Relative ABC transporter gene expression during sea urchin development
Data depicted in Fig. 1 is presented as individual graphs on a logarithmic scale. Expression profiles are shown in three color-coded groups. Group 1 is shown in blue, Group 2 in orange, and Group 3 in green. N = 4, error bars represent standard error.
Fig. 3
Fig. 3. Number of ABC transporter transcripts per egg/embryo
Number of mRNA copies per egg/embryo is shown. Transcript copies were determined using a standard curve to quantify the reference point (egg for ABCB1a, ABCB4a, ABCC1, and ABCC9a; hatching blastula for ABCC5a; early prism for ABCG2b), and by applying fold change values (Fig. 1, Fig. 2, Supplemental Table 1) to quantify all other stages. Dashed lines (D, F) indicate where transcripts were below the threshold of detection using qPCR (i.e. <100 copies per egg/embryo). N=4, bars represent standard error.
Fig. 4
Fig. 4. Spatial expression of ABC transporter genes
Whole mount in situ hybridization depicts endogenous expression of ABCB1a and ABCC5a. (A–D) For controls, DIG-labeled sense probe was used. (E–H) ABCB1a is expressed in all cells of embryos. (I) ABCC5a mRNA is not detected prior to hatching, after which it is detected (J) in the vegetal pole of hatched mesenchyme blastulae, and (K, L) in mesodermal cells in later embryos. (A, E, I) 16-cell embryos were fixed at 7 hpf, (B, F, J) hatched mesenchyme blastulae at 36 hpf, (C, G, K) early gastrulae at 42 hpf, and (D, H, L) late gastrulae at 48 hpf.
Fig. 5
Fig. 5. Localization of ABCB1a and ABCC5a proteins
Micrographs show exogenous ABCB1a and ABCC5a localization from expression of fluorescent-protein fusions. Representative embryos are shown. B is an inset from the embryo shown in A. mRNA from ABCB1a-mCitrine (ii, green) and ABCC5a-mCherry (iii, red) were injected into fertilized eggs, then embryos were grown to mesenchyme blastulae for imaging. (iv) Merged channel includes Histone H2B-CFP (blue), injected as a nuclear marker. ABCB1a-mCitrine protein localizes to the apical surface of the embryo, while ABCC5a-mCherry localizes on the basolateral cell surfaces.
Fig. 6
Fig. 6. Model of endogenous ABCB1a and ABCC5a protein localization
(A) A cartoon represents WMISH transcript localization shown in Fig. 4F, 4J. By combining this with FP-fusion ABCB1a and ABCC5a protein localization data shown in Fig. 5A, we propose (B) a model for endogenous ABCB1a (green) and ABCC5a (red) expression. ABCB1a is primarily expressed on the apical membrane of polarized epithelial cells, while ABCC5a is expressed on the basolateral membranes of secondary (non-skeletogenic) mesenchyme cells.

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