Abstract
Peroxidasin (PXDN) is a unique peroxidase containing extracellular matrix motifs and stabilizes collagen IV networks by forming sulfilimine crosslinks. PXDN gene knockout in Caenorhabditis elegans (C. elegans) and Drosophila results in the demise at the embryonic and larval stages. PXDN mutations lead to severe eye disorders, including microphthalmia, cataract, glaucoma, and anterior segment dysgenesis in humans and mice. To investigate how PXDN loss of function affects organ development, we generated Pxdn knockout mice by deletion of exon 1 and its 5′ upstream sequences of the Pxdn gene using the CRISPR/Cas9 system. Loss of both PXDN expression and collagen IV sulfilimine cross-links was detected only in the homozygous mice, which showed completely or almost closed eyelids with small eyes, having no apparent external morphological defects in other organs. In histological analysis of eye tissues, the homozygous mice had extreme defects in eye development, including no eyeballs or drastically disorganized eye structures, whereas the heterozygous mice showed normal eye structure. Visual function tests also revealed no obvious functional abnormalities in the eyes between heterozygous mice and wild-type mice. Thus, these results suggest that PXDN activity is essential in eye development, and also indicate that a single allele of Pxdn gene is sufficient for eye-structure formation and normal visual function.
Highlights
Basement membranes (BM) are sheet-like, cell-adherent extracellular matrices (ECM) consisting of an enmeshed polymer of laminins and collagen IV (Col IV) that are bound to nidogens, agrin, and perlecan [1]
Because PXDN is known to catalyze the formation of the sulfilimine crosslink that stabilizes the Col IV network for mammalian tissue genesis [19], we suspected there would be embryonic lethality or severe impairment of development in Pxdn-null mice
It had been reported that the ENU-induced Pxdn mutation that has a premature stop codon (Cys1272X) leads to severe anterior segment dysgenesis and microphthalmia [16]
Summary
Basement membranes (BM) are sheet-like, cell-adherent extracellular matrices (ECM) consisting of an enmeshed polymer of laminins and collagen IV (Col IV) that are bound to nidogens, agrin, and perlecan [1] They serve as both supportive cell substrata and solid-phase agonists, contributing tissue organization, stability, and differentiation. Col IV is assembled by extracellular oligomerization of triple-helical protomers and is covalently cross-linked by sulfilimine (S=N) bond formation among the C-terminal non-collageneous 1 (NC1) domains that reinforce the structural integrity of the Col IV networks [3] Ablation of both Col IV subunit α1 and α2 in mice causes lethality between E10.5 and E11.5 because of structural deficiencies in the BMs and eventual failure of the integrity of Reichert’s membrane [2]. Col IV α2 mutations in mice and humans cause diseases similar to those resulting from Col IV α1 mutations [8,9]
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