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, 11 (6), 3620-3628
eCollection

Role of Surface Ectoderm-Specific Mitofusin 2 in the Corneal Morphologic Development of Mice

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Role of Surface Ectoderm-Specific Mitofusin 2 in the Corneal Morphologic Development of Mice

Jiao Zhang et al. Am J Transl Res.

Abstract

Mitofusin 2 (Mfn2) mediates the mitochondrial fusion in dynamic balance between mitochondrial fission and fusion. This study aimed to investigate the role of Mfn2 in mice corneal dysplasia with conditional knockout (CKO) technique. The Mfn2 CKO mice model was established with the Cre-loxP system. Each offspring of Le-Cre +/-; Mfn2 fl/fl (Mfn2 CKO) mice and Mfn2fl/fl (Mfn2 WT) mice was identified by polymerase chain reaction (PCR). Macroscopic observation, immunohistostaining and HE staining were used to evaluate the corneal morphologic development in Mfn2 CKO mice and Mfn2 WT mice. The cells proliferation and apoptosis were detected by BrdU labeling and TUNEL assay. Real-time PCR was used to detect mRNA expression of corneal markers (K12, Col1α1, Pax6, keratocan and NSE). Results showed that Mfn2 CKO mice showed increased corneal thickness, small eyeball from E15.5 to P60 and small eye crack after birth. The corneal stromal thickness significantly increased in Mfn2 CKO mice, and the random arrangement fibers of the corneal stroma increased in Mfn2 CKO mice. The proliferative cells in the cornea of Mfn2 CKO mice were less than in Mfn2 WT mice while the apoptotic cells in the cornea of Mfn2 CKO mice were increased. K12 and Pax6 expression decreased in the cornea and the Col1α1 expression increased in Mfn2 CKO mice as compared to Mfn2 WT mice. The expression of corneal stromal marker Col1α1 in the Mfn2 CKO mice was significantly higher than that in the Mfn2 WT mice. Corneal thickness was mainly caused by corneal stroma collagen proliferation. In conclusion, Mfn2 deletion affects corneal development, especially because of collagen hyperplasia in the corneal stroma.

Keywords: Mfn2; apoptosis; corneal development; mitochondria; proliferation.

Conflict of interest statement

None.

Figures

Figure 1
Figure 1
Construction and identification of Mfn2 conditional knockout mice. (A) Mfn2 CKO mice were screened from the offspring after Le-Cre mice mated with Mfn2 fl/fl mice. (B) Genotyping by PCR. Le-Cre is 350 bp in length, mutant Mfn2 is 180 bp in length and wild-type Mfn2 is 145 bp in length. (C) Phenotypes of Mfn2 WT and Mfn2 CKO mice. Compared with Mfn2 CKO mice, the eye crack and eyeball volume reduced, and the iris was adherent to the cornea. Scale bars: 100 μm (e, f), 50 μm (g, h).
Figure 2
Figure 2
Corneal dysplasia in Mfn2 CKO mice. (A) Compared with Mfn2 WT mice, the corneal thickness of Mfn2 CKO mice increased at E15.5. (B) Increased corneal thickness was mainly due to the increased thickness of corneal stroma in postnatal mice. (C) The central corneal thickness of Mfn2 CKO and Mfn2 WT mice. The central cornea of Mfn2 CKO mice was thicker than that of Mfn2 WT mice during embryonic period and after birth. (D, E) The central thickness of corneal epithelium, stroma and endothelium. Corneal stroma thickness was significantly different from the other two layers in two groups at P18 and P60. (n = 3, *P < 0.05, **P < 0.001, ***P < 0.0001, ****P < 0.00001). Scale bars: (A) 100 μm (a-h), 500 μm (i, j), 20 μm (k, l).
Figure 3
Figure 3
BrdU staining of the cornea in Mfn2 CKO mice and Mfn2 WT mice. (A) Cell proliferation of the cornea was mainly concentrated at E16.5 and E17.5. (B) The number of BrdU+ cells in Mfn2 CKO mice was less than in Mfn2 WT mice. Cell apoptosis of the cornea in Mfn2 CKO mice and Mfn2 WT mice. (C) In Mfn2 CKO mice, cell apoptosis of the cornea was observed from E16.5 and it mainly found in the peripheral corneal endothelium (arrow). (D) Cell apoptosis of postnatal cornea was mainly found in the corneal stroma. However, the corneal cell apoptosis was not significantly different between two groups. (n = 3, *P < 0.05). Scale bars: (A) 100 μm (a-f); (C) 50 μm (a-d), 100 μm (e, f).
Figure 4
Figure 4
mRNA and protein expression of cornea related genes in the Mfn2 conditional knockout cornea. (A) Pax6 and K12 mRNA expression was slightly lower in Mfn2 CKO mice than in Mfn2 WT mice. Col1α1 mRNA expression was significantly higher in Mfn2 CKO mice than in Mfn2 WT mice. The expression of K12 and Keratocan was comparable between Mfn2 CKO mice and Mfn2 WT mice. Data are expressed as means ± SD of 3 separate experiments. The expression of K12 and Col1α1 was almost undetectable during embryonic period. (B) Mfn2 expression was mainly located in the corneal epithelium compared to the Mfn2 CKO mice. (C) After birth, the expression of corneal epithelial marker K12 was stained uniformly in Mfn2 WT mice, but the expression of corneal epithelium K12 in Mfn2 CKO mice significantly reduced (c-f). (D) The expression of Col1α1 in Mfn2 CKO mice was significantly higher than in Mfn2 WT mice, and the corneal stroma was thickened (c-f). (n = 3, *P < 0.05, **P < 0.001, ***P < 0.0001, ****P < 0.00001). Scale bars: (B) 20 μm (a, b); (C) 50 μm (a-f); (D) 50 μm (a-f).

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