Apolipoprotein A-I, A-II, and H mRNA and protein accumulation sites in the developing lung in late gestation

Abstract

Background: Expression of apolipoprotein A-I (apoA-I), A-II, and H was previously observed at 16 to 50-fold higher levels in the fetal than the adult mouse lung. Here, sites of apoA-I, A-II, and H mRNA and protein accumulation were determined in mouse fetal lungs by in situ hybridization and immunohistochemistry in late gestation.

Results: Expression sites vary for the three genes and change for the distal epithelium before the end of the canalicular stage, thus where and when the surge of surfactant synthesis occurs. Messenger of apoH, but not those of apoA-I and A-II, was also observed in the proximal epithelium and smooth muscles surrounding arteries. In contrast to apoC-II protein, none of the three studied apolipoproteins accumulated within secretory granule-like structures. Immunohistochemistry revealed that apoA-I and apoH accumulated mainly in capillaries. Three different positive signals with the anti-apoA-II antibody were found: one transient signal in the nucleus of a portion of mesenchymal cells, a second at lower levels throughout the mesenchyme, and another in capillaries with a specific increase from gestation day 17.5/18.5.

Conclusion: Temporal and geographic co-expression of apoAI, AII, and H genes with surfactant production site suggests that the three apolipoproteins are secreted to play roles supporting the lung-specific surfactant lipid-related metabolism.

Figure 1

Distribution of apolipoprotein A-I mRNA and protein in the mouse fetal lung. Mouse tissue sections are from pseudoglandular (GD 15.5: a-b, f-g) or late canalicular (GD 17.5: c-e, h-k) stages. In situ hybridization (within blue frames) (a-e) was performed with apoA-I anti-sense (a-d) and sense (e) probes. Positive signals (blue) show that the site of apoA-I mRNA synthesis changes according to developmental time. Immunohistochemistry (within red frames) (f-k) was performed using an anti-apoA-I polyclonal antibody (f, h-j) or goat IgG as negative control (g, k). Positive signals (red) were mainly found on capillaries with no change in localization between GD 15.5 and 17.5. GDs are indicated on photographs. Dashed frames, negative controls. Scale bars, 50 μm (a, c, e-h, k) or 20 μm (b, d, i-j). A, artery; C, capillaries; D, distal epithelium; M, mesenchyme; P, proximal epithelium; SM smooth muscle.

Figure 2

Distribution of apolipoprotein A-II mRNA and protein in the mouse fetal lung. Mouse tissue sections are from pseudoglandular (GD 15.5: a-b, g), junction between pseudoglandular and canalicular (GD 16.5: d), late canalicular (GD 17.5: c, e-f, h-k), or early saccular (GD 18.5: l-m) stages. In situ hybridization (within blue frames) (a-f) was performed with apoA-II anti-sense (a-e) and sense (f) probes. Positive signal is blue. A change in sites of apoA-II gene expression was observed according to gestation time. Immunohistochemistry (within red frames) (g-m) was performed using an anti-apoA-II polyclonal antibody (g-h, j-m) or goat IgG as negative control (i). Positive signal (red) was found in the mesenchyme. Some mesenchymal cells presented a stained nucleus between GD 15.5 and GD 17.5. Positive signals were also observed on capillaries. GDs are indicated on photographs. Dashed frames, negative controls. Scale bars, 50 μm (a, c-d, f-i, k-l) or 20 μm (b, e, j, m). C, capillaries; D, distal epithelium; M, mesenchyme; N, positive nuclei; P, proximal epithelium.

Figure 3

Distribution of apolipoprotein H mRNA and protein in the mouse fetal lung. Mouse tissue sections are from pseudoglandular (GD 15.5: f-h) or late canalicular (GD 17.5: a-e, i-l) stages. In situ hybridization (within blue frames) (a-e) was performed with apoH anti-sense (a, c-e) and sense (b) probes. Positive signals (blue) were found on GD 17.5 in the distal and proximal epithelia and smooth muscles surrounding large arteries. Immunohistochemistry (within red frames) (f-l) was performed using an anti-apoH polyclonal antibody (f-g, i-k) or goat IgG as negative control (h, l). Positive signals (red) were mainly found on capillaries with no change in localization between GD 15.5 and 17.5. GDs are indicated on photographs. Dashed frames, negative controls. Scale bars, 50 μm (a-b, e-f, h-l) or 20 μm (c-d, g). A, artery; C, capillaries; D, distal epithelium; M, mesenchyme; P, proximal epithelium; SM smooth muscle.