Breakdown of the reciprocal stabilization of QBRICK/Frem1, Fras1, and Frem2 at the basement membrane provokes Fraser syndrome-like defects

Abstract

An emerging family of extracellular matrix proteins characterized by 12 consecutive CSPG repeats and the presence of Calx-beta motif(s) includes Fras1, QBRICK/Frem1, and Frem2. Mutations in the genes encoding these proteins have been associated with mouse models of Fraser syndrome, which is characterized by subepidermal blistering, cryptophthalmos, syndactyly, and renal dysmorphogenesis. Here, we report that all of these proteins are localized to the basement membrane, and that their basement membrane localization is simultaneously impaired in Fraser syndrome model mice. In Frem2 mutant mice, not only Frem2 but Fras1 and QBRICK/Frem1 were depleted from the basement membrane zone. This coordinated reduction in basement membrane deposition was also observed in another Fraser syndrome model mouse, in which GRIP1, a Fras1- and Frem2-interacting adaptor protein, is primarily affected. Targeted disruption of Qbrick/Frem1 also resulted in diminished expression of Fras1 and Frem2 at the epidermal basement membrane, confirming the reciprocal stabilization of QBRICK/Frem1, Fras1, and Frem2 in this location. When expressed and secreted by transfected cells, these proteins formed a ternary complex, raising the possibility that their reciprocal stabilization at the basement membrane is due to complex formation. Given the close association of Fraser syndrome phenotypes with defective epidermal-dermal interactions, the coordinated assembly of three Fraser syndrome-associated proteins at the basement membrane appears to be instrumental in epidermal-dermal interactions during morphogenetic processes.

Fig. 1.

Localization of Frem2 at the basement membrane zone. (A) Schematic view of the protein family that contains 12 CSPG repeats and variable Calx-β motifs. Underlined are the regions used as immunogenic epitopes. (B and C) Immunolocalization of Frem2 in the skin of E17.5 mice. In the dorsal epidermal basement membrane, which is counterstained for laminin-γ1 (red), Frem2 (green) was detected by antibodies recognizing the ectodomain (B) but not the cytoplasmic tail (C). (Scale bar, 20 μm.) (D and E) Secretion of Frem2 and Fras1 after expression in 293F cells. 293F cells were transfected with Frem2 expression vector or empty vector, followed by immunoblotting of the conditioned medium (M) and cell lysates (C) with an anti-Myc antibody (to detect Frem2 containing an N-terminal 3xMyc tag) or an antibody against the cytoplasmic tail of Frem2 (CP). Adequate separation of the medium and the cell fraction was monitored by measuring immunoreactivity to cellular Erk (D). Similarly, 293F cells were transfected with Fras1 expression vector or empty vector, followed by immunoblotting with anti-HA (to detect Fras1 containing an N-terminal 3xHA tag) and anti-Erk (E).

Fig. 2.

Expression profiles of Fraser syndrome-associated proteins in my mutant mice. (A) Northern blot analyses of Frem2, Fras1, Qbrick/Frem1, and GAPDH expression. Two independent probes were used to detect Frem2 transcripts. (B–L) Comparative immunohistochemical analyses of dorsal skin cryosections. In each image, Upper and Lower represent representative immunofluorescence seen in my/+ and my/my littermates, respectively. Basement membranes were counterstained (red) with antibodies against laminin-γ1 chain (B–J, L) or EHS-laminin (K). (B–D) Frem2 immunofluorescence (green) in E13.5 (B), E17.5 (C), and newborn (D) my/+ and my/my mice. Frem2 immunoreactivity was scarce in my/my animals. (E–G) Fras1 immunofluorescence (green) in E13.5 (E), E17.5 (F), and newborn (G) my/+ and my/my mice. Fras1 immunoreactivity was considerably reduced in my/my animals. Collagen-VI (H), NG2 (I), collagen-IV (J), perlecan (K), and GRIP1 (L) were expressed at equal levels in my/+ and my/my animals at E13.5 (green). (Scale bar, 20 μm.)

Fig. 3.

Impaired expression of Frem2 and Fras1 in eb mutant mice. Frem2 (A and B), Fras1 (C and D), and other ECM proteins (E, perlecan; F, collagen-IV; G, collagen-VI; and H, NG2) were localized by immunofluoresccence (green) in cryosections of dorsal skin isolated from E14.5 (A, C, E–H) and newborn (B and D) eb mutant mice. Basement membranes were counterstained (red) with antibodies against laminin-γ1 (A–D, F–H) or EHS-laminin (E). In each image, Upper and Lower were taken from eb/+ and eb/eb animals, respectively. In eb/eb animals, Frem2 immunoreactivity was reduced at E14.5; this reduction became more prominent in newborn mice. Fras1 immunoreactivity is almost absent in eb/eb animals. Asterisks indicate nonspecific binding of antibodies to the cornified epithelium. (Scale bar, 20 μm.)

Fig. 4.

Impaired expression of QBRICK/Frem1 in my and eb mutant mice. (A–C) QBRICK/Frem1 immunofluorescence (green) in my/my and my/+ mice at E13.5 (A), E17.5 (B), and at birth (C). QBRICK/Frem1 immunoreactivity is reduced in my/my mice at E13.5; this reduction becomes more prominent at later developmental stages. (D and E) QBRICK/Frem1 immunofluorescence (green) in E14.5 (D) and newborn (E) eb/eb and eb/+ mice. The immunoreactivity was reduced at E14.5 and almost absent at birth. Asterisks represent nonspecific binding of antibodies to the cornified epithelium. (Scale bar, 20 μm.)

Fig. 5.

Reduced expression of Frem2 and Fras1 in Qbrick/Frem1−/− mice. (A) Schematic representation of the targeted disruption of Qbrick/Frem1. Open boxes represent exons. The targeting construct was designed to replace exon 2, which contains the initiation codon, with a neomycin-resistance gene (shaded box). The probe used for Southern blotting in B is indicated as a bold line. E, EcoRV restriction site. (B) Southern blot analysis of genomic DNA from wild-type, heterozygous, and homozygous offspring after digestion with EcoRV. Detection of a 13.7-kbp fragment indicates targeted disruption of the Qbrick/Frem1 gene. (C–E) Qbrick/Frem1−/− animals exhibit cryptophthalmos (C), syndactyly in the hindlimb (D), and subepidermal blistering at E14.5 (arrowheads in E). (F) Electron microscopic observation of the blebs of Qbrick/Frem1−/− embryos demonstrated that blistering occurred between the lamina densa of the basement membrane (arrowheads) and the underlying dermis. A blister cavity is indicated by an asterisk. Epi, epidermal cell. (G–M) Immunohistochemistry of dorsal skin cryosections of Qbrick/Frem1+/− and Qbrick/Frem1−/− animals taken at E14.5 (G, H, J, and L–O) and at birth (I and K). Basement membranes were counterstained (red) with antibodies against laminin-γ1 (G–K, M–O) or EHS laminin (L). In addition to the absence of QBRICK/Frem1 immunoreactivity at the epidermal BM in Qbrick/Frem1−/− animals (G), Frem2 (H and I) and Fras1 (J and K) immunoreactivities (green) were reduced in comparison to heterozygotes, whereas staining for laminin-γ1 (red) remained constant in the same area. Perlecan (L), collagen-IV (M), NG2 (N), and collagen-VI (O) were expressed at equal levels in Qbrick/Frem1+/− and Qbrick/Frem1−/− animals at E14.5 (green). Asterisks indicate nonspecific binding of antibodies to the cornified epithelium. (Scale bar, 20 μm.)

Fig. 6.

A model for the reciprocal stabilization of the Fras1, Frem2, and QBRICK/Frem1 through complex formation at the epidermal basement membrane. (A) HA-tagged Fras1 protein was immunoprecipitated by using anti-HA mAb in the absence or presence of HA peptide. The absence of Frem2 resulted in a failure of QBRICK/Frem1 to coprecipitate with Fras1. Coprecipitation of QBRICK/Frem1 with Fras1 was abolished in the presence of the antigenic peptide, confirming the specificity of immunoprecipitation. (B) Fras1 and Frem2, expressed in epidermal cells, are transported to the plasma membrane in a GRIP1-dependent manner and shed from the cell surface by proteolytic processing. The secreted Fras1/Frem2 complex is deposited in the basement membrane at the epidermal–dermal interface after secretion of QBRICK/Frem1 from dermal mesenchymal cells. Stable basement membrane deposition of QBRICK/Frem1 also depends on the secretion of Fras1 and Frem2 from epidermal cells. This reciprocal stabilization of the three Fraser syndrome-associated proteins is due to their macromolecular complex formation, which secures the stable anchoring of the dermis to the basement membrane. (C) Abortion of the interactions between the three Fraser syndrome-associated proteins, which can arise from deficiency or mutation in any individual gene, results in their failure to stably deposit at the basement membrane, leading to impaired anchoring of the dermis to the basement membrane and subsequent blister formation.