The a subunit isoforms of vacuolar-type proton ATPase exhibit differential distribution in mouse perigastrulation embryos

Abstract

Vacuolar-type H⁺-ATPases (V-ATPases) are large multi-subunit complexes that play critical roles in the acidification of a variety of intracellular or extracellular compartments. Mammalian cells contain four isoforms of the membrane integral subunit a (a1-a4); these isoforms contain the information necessary to target the enzyme to different cellular destinations. They are also involved in regulating the efficiency of ATP hydrolysis and proton transport. Previously, we showed that early embryogenesis requires V-ATPase function, and the luminal acidic endocytic and lysosomal compartments in the visceral endoderm of mouse embryos at the pre-gastrulation stage (E6.5) are essential for both nutrition and signal transduction during early embryogenesis. In this study, we examined the expression and distribution of a subunit isoforms in mouse embryos at E6.5. We found that all four isoforms expressed and exhibited differential distribution in the E6.5 embryo. At this developmental stage, the embryos establish highly elaborate endocytic compartments called apical vacuoles, on which the a3 isoform specifically accumulated.

Figure 1

Whole-mount in situ hybridization analysis of a subunit isoform expression in mouse E6.5 embryos. Whole-mount in situ hybridization assays for a1, a2, a3, and a4 mRNA were carried out with anti-sense (a,c,e,g) or control sense (b,d,f,h) probes using E 6.5 mouse embryos. All assays were performed using at least three embryos. Specific labelling was observed with anti-sense probes, and no specific staining above background was observed with the negative control probes. An illustration of the E6.5 embryo is shown on the right. VE, visceral endoderm; ExE, extraembryonic ectoderm; epi, epiblast. Scale bar, 100 µm.

Figure 2

Localization of a1 subunit isoforms in mouse E6.5 embryos. Wild-type E6.5 embryos (n = 8) were isolated and stained with antibodies against a1 (red) and lamp2 (green). Nuclear DNA is also labelled (blue). The control embryos (n = 5) labelled without the primary antibodies against a1 (w/o primary Ab to a1) was shown in (f–j) and (n–p). The secondary antibody alone controls in which the embryos were labelled without primary antibodies against the a1 isoform were performed in parallel and the images were obtained under the same microscopic setup. The boxed region in (b,g) is magnified and shown as (k–m,n–p), respectively. Scale bars represent 50 µm and 5 µm in (a–j,k–p), respectively.

Figure 3

Localization of a2 subunit isoforms in mouse E6.5 embryos. Wild-type E6.5 embryos (n = 9) were isolated and stained with antibodies against a2 (red) and lamp2 (green). Nuclear DNA is also labelled (blue). The control embryos (n = 4) labelled without the primary antibody against a2 (w/o primary Ab to a2) was shown in (f–j,n–p). The secondary antibody alone controls in which the embryos were labelled without primary antibodies against the a2 isoform were performed in parallel and the images were obtained under the same microscopic setup. The boxed region in (b,g) is magnified and shown as (k–m,n–p), respectively. Scale bars represent 50 µm and 5 µm in (a–j,k–p), respectively.

Figure 4

Localization of a3 subunit isoforms in mouse E6.5 embryos. Wild-type E6.5 embryos (n = 10) were isolated and stained with antibodies against a3 (red) and lamp2 (green). Nuclear DNA is also labelled (blue). Arrows indicate the apical vacuole. The control embryos (n = 5) labelled without the primary antibodies against a3 (w/o primary Ab to a3) was shown in (k–j,n–p). The secondary antibody alone controls in which the embryos were labelled without primary antibodies against the a3 isoform were performed in parallel and the images were obtained under the same microscopic setup. The boxed region in (b,g) is magnified and shown as (k–m,n–p), respectively. Scale bars represent 50 µm and 5 µm in (a–j,k–p), respectively.

Figure 5

Localization of a4 subunit isoforms in mouse E6.5 embryos. Wild-type E6.5 embryos (n = 8) were isolated and stained with antibodies against a4 (red) and lamp2 (green). Nuclear DNA is also labelled (blue). The control embryos (n = 5) labelled without the primary antibodies against a4 (w/o primary Ab to a4) was shown in (k–j,n–p). The secondary antibody alone controls in which the embryos were labelled without primary antibodies against the a4 isoform were performed in parallel and the images were obtained under the same microscopic setup. The boxed region in (b,g) is magnified and shown as (k–m,n–p), respectively. Scale bars represent 50 µm and 5 µm in (a–j,k–p), respectively.