The extracellular matrix gene Frem1 is essential for the normal adhesion of the embryonic epidermis

Abstract

Fraser syndrome is a rare recessive disorder characterized by cryptophthalmos, syndactyly, renal defects, and a range of other developmental abnormalities. Because of their extensive phenotypic overlap, the mouse blebbing mutants have been considered models of this disorder, and the recent isolation of mutations in Fras1 in both the blebbed mouse and human Fraser patients confirms this hypothesis. Here we report the identification of mutations in an extracellular matrix gene Fras1-related extracellular matrix gene 1 (Frem1) in both the classic head blebs mutant and in an N-ethyl-N-nitrosourea-induced allele. We show that inactivation of the gene results in the formation of in utero epidermal blisters beneath the lamina densa of the basement membrane and also in renal agenesis. Frem1 is expressed widely in the developing embryo in regions of epithelial/mesenchymal interaction and epidermal remodeling. Furthermore, Frem1 appears to act as a dermal mediator of basement membrane adhesion, apparently independently of the other known "blebs" proteins Fras1 and Grip1. Unlike both Fras1 and Grip1 mutants, collagen VI and Fras1 deposition in the basement membrane is normal, indicating that the protein plays an independent role in epidermal differentiation and is required for epidermal adhesion during embryonic development.

Fig. 1.

Phylogenetic analysis and domain organization of CSPG-containing proteins. (A) Phylogenetic trees were constructed from aligned CSPG domain-containing regions of each protein by using neighbor-joining (NJ), maximum parsimony (MP), and minimum evolution (ME) methods. (Mm, Mus musculus; Hs, Homo sapiens; Fr, Fugu rubripes; Dm, Drosophila melanogaster; Ce, C. elegans; and Lv, Lytechinus variegatus. Black spots indicate tree nodes with >95% bootstrap support for all three methods, and the gray spot indicates a node supported to 89% by NJ and MP and 60% by ME. The remaining node was not consistently resolved by these methods. Fr.Fras1 and Mm.Cspg5 are incomplete at their N termini (*). The three lightly shaded CSPG domains only partially match the full domains as defined in B. (B) Multiple sequence alignment of CSPG domains with a cadherin of known structure (16) presented by using chroma (33). The line below the alignment shows the consensus structural features of cadherins aligned to the CSPG domains. E indicates β-strand residues. Residues shown to directly interact with chelated Ca²⁺ ions are shaded; red indicates physical–chemical properties that are >60% conserved between cadherins and CSPGs and gray indicates where they are not conserved. The consensus properties are summarized by the symbol – for negative and by the letters p for polar and h for hydrophobic residues.

Fig. 2.

bat mice display cryptophthalmos and embryonic blebbing. (A and B) bat mice display cryptophthalmos in adults and embryonic blebs from ≈13.5 dpc, which invariably affect the developing eyes (B, arrowheads). (C) Sectioning at 16.5 dpc illustrates the formation of a pseudoblepharon (arrowheads), which severely affects development of the underlying eyelids. (D) The epidermis (ep) in the blebs of bat embryos separates from the dermis below the lamina lucida (ll) and the lamina densa (ld) of the basement membrane. The resulting blister cavity (bc) contains residual anchoring fibrils on the subepidermal face.

Fig. 3.

The bat and heb phenotypes result from mutation of Frem1. (A) Sequencing of all 36 Frem1 coding exons identified an ENU induced T-C mutation in the splice donor site of exon 25 (Left). RT-PCR analysis shows that this mutation leads to skipping of exon 25 in bat RNA compared with C57BL/6J mice on which the mutation was induced. (B) Southern blot analysis of heb DNA with a probe spanning exon 17 detected a genomic rearrangement not present in the parental AKR/J strain. Inverse PCR identified the insertion of a LINE1 element 41 bp from the end of the exon. The BglII sites and primers used for inverse PCR (Pr, Pf, Dr, and Df) are indicated. (C) Position of the heb and bat mutations relative to the protein domains of Frem1 (CSPG, light gray; Calx, black; LectinC, dark gray).

Fig. 4.

Frem1 is expressed dynamically during embryogenesis in regions of epithelial/mesenchymal interaction. (A) Frem1 is expressed from ≈10.5 dpc and becomes pronounced in the differentiating eyelids and sensory vibrissae (white arrowheads) by 12.5 dpc. At this stage, low levels of epithelial expression are noted over the head and trunk. (B) By 14.5 dpc, expression is maintained in the vibrissae and eyelids (white arrowhead) and is initiated in the epithelium of the developing ear (*) and in the mammary glands (black arrowheads). Transcripts are also detected in the hair follicle placodes, initiating on the trunk. Follicle expression is initially detected in both epidermis and condensing mesenchyme at ≈14 dpc, but as development progresses, it becomes restricted to cells of the future dermal papillae. (C) By 16.5 dpc, Frem1 is expressed in the differentiating pelage follicles, limbs, and paw pads as well as in the meibomian glands. Section in situ hybridization at 15.5 dpc indicated strong dermal/mesenchymal expression of Frem1 in the developing eyelids (el, D), limb (E and G), and mammary gland (F). Hybridization signal is shown in yellow and red. The position of the basement membrane is indicated by a dashed line. (H) Frem1 expression was also detected in the developing caecum (black arrowhead) and is restricted to the mesenchyme (data not shown). At this stage, apical and basal dermal limb expression is detected (white arrowheads). (I) Frem1 is expressed in the kidney at 12.5 dpc in the mesenchyme surrounding the growing ureteric bud in a pattern complementary to Fras1. (Left) In situ expression and virtual optical projection tomography sections. (Right) α-Fras1 immunostaining (red) and Frem1 in situ hybridization (brightfield). (J) At 13.5 dpc, Frem1 and Fras1 expression also colocalizes with α-Wilms tumor suppressor 1 (Wt1) immunostaining (green) in the podocytes (as in I).

Fig. 5.

bat mice display normal localization of Fras1 and collagen VI (ColVI) in the epidermal basement membrane. Immunolocalization of Fras1, collagen V (COLV), and collagen in both the head and blister epidermis of bat mice appeared indistinguishable from that in wild-type embryos. Staining for different proteins is indicated in red, and sections have been counterstained with 4′,6-diamidino-2-phenylindole (blue). High-magnification images of blister roofs are shown (Insets).