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. 2002 May 14;99(10):6816-21.
doi: 10.1073/pnas.092130099. Epub 2002 Apr 30.

Epidermolysis bullosa and embryonic lethality in mice lacking the multi-PDZ domain protein GRIP1

Friedhelm Bladt  1 , Anna Tafuri, Sigal Gelkop, Lowell Langille, Tony Pawson
Affiliations

Epidermolysis bullosa and embryonic lethality in mice lacking the multi-PDZ domain protein GRIP1

Friedhelm Bladt et al. Proc Natl Acad Sci U S A. .

Abstract

Glutamate receptor-interacting protein 1 (GRIP1) is an adaptor protein composed of seven PDZ (postsynaptic density-95/Discs large/zona occludens-1) domains, capable of mediating diverse protein-protein interactions. GRIP1 has been implicated in the regulation of neuronal synaptic function, but its physiologic roles have not been defined in vivo. We find that elimination of murine GRIP1 results in embryonic lethality. GRIP1(-/-) embryos develop abnormalities of the dermo-epidermal junction, resulting in extensive skin blistering around day 12 of embryonic life. Ultra-structural characterization of the blisters (or bullae) revealed cleavage of the dermo-epidermal junction below the lamina densa, an alteration reminiscent of the dystrophic form of human epidermolysis bullosa. Blisters were also observed in the lateral ventricle of the brain and in the meninges covering the cerebral cortex. These genetic data suggest that the GRIP1 scaffolding protein is required for the formation and integrity of the dermo-epidermal junction and reveal the importance of PDZ domains in the organization of supramolecular structures essential for mammalian embryonic development.

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Figures

Figure 1
Figure 1
Generation of a GRIP1-deficient mouse strain. (A) Schematic representation of GRIP1 gene-targeting strategy. Restriction map of the wt GRIP1 locus, targeting vector, and targeted locus. The targeting vector contains a tau–LacZ reporter gene fused in-frame with the exon encoding the first PDZ domain. Antibodies used for Western blot analysis are indicated. (B) Genotypic analysis of E12 embryos by PCR. To identify wt and targeted loci, primers were designed in the deleted wt locus and the β-galactosidase region, respectively. (C) Expression of GRIP1 was assessed by Western blot analysis of protein extracts from E12 embryos. The antibody used to detect GRIP1 expression is specific for the C-terminal portion of GRIP1 and does not cross-react with GRIP2 (18). The 100-kDa cross-reactive band bound by the C-terminal peptide-specific antibody was not recognized by antisera raised to the PDZ4 or the PDZ7 domains of GRIP1 (data not shown) and is therefore not GRIP1 specific.
Figure 2
Figure 2
GRIP1 is expressed in the developing nervous system and epidermis. Whole-mount staining of GRIP1+/− E16 embryos shows GRIP1tau-LacZ expression in the olfactory nerve (a). GRIP1 is also expressed in the optic nerve (b, on), olfactory epithelium (b, oe), cochlea (c, co), motor axons (d, ma), dorsal root ganglia (d, drg), nerve of the dorsal root ganglia (e, dn), and motor neurons (f, mn), as assessed by β-galactosidase staining on transverse (b–d) and sagittal (e and f) sections. Notably, β-galactosidase activity was also present in the skin (f, s). Immunostaining with antibodies specific for GRIP 1 (g) and pan-cadherin (h) of skin sections from wt embryos revealed GRIP1 expression in the basal epithelial cells (g). The expression of cadherin in the BMZ (h) colocalizes with that of GRIP1 in the BMZ (arrowheads), as shown by overlapping staining of the GRIP1 and pan-cadherin antibodies (i). Sections were obtained from GRIP1+/− E16 (b and c), E14 (d–f), and wt E12 (g–i) embryos. (Magnifications: a, ×3; b, ×7; c and d, ×15; e and f, ×20; and g–i, ×60.)
Figure 3
Figure 3
Development of a severe bullous disorder in GRIP1−/− embryos. (A) Gross morphology of GRIP1−/− E12 embryos, showing large serous bullae on the surface of the head (a and b, arrowheads), upper and lower limbs (a) and with a large haemorrhagic bulla in the right lateral ventricle of the brain (b, arrowhead). In GRIP1−/− E14 embryos (shown inside the yolk sac), haemorrhagic bullae were found in similar locations: head (d and e), upper limbs (d). wt littermate controls are shown for comparison (c and f). (Magnifications: a–c, ×4; d–f, ×25.) (B) Frequency of serous (black bars) and haemorrhagic (gray bars) bullae in the head (H), upper limbs (UL), lower limbs (LL), and back bone (Bb) of GRIP1−/− embryos at different stages of embryonic development. The data derive from the cumulative observation of 35, 31, and four GRIP1−/− embryos at E12, E14, and E16, respectively.
Figure 4
Figure 4
Cleavage at the dermal side of the cutaneous BMZ is responsible for skin blistering in GRIP1−/− mutants. (A) Hematoxylin-eosin-stained sections of lower limbs of wt (a and b) and GRIP1−/− (c and d) E12 embryos. Serous blisters are clearly visible on both sides of the limb (c). At higher magnification, the epidermis appears diffusely detached from the underlying dermis in blistered (arrowhead) and nonblistered areas (arrow). (B) Immunostaining of limb sections of wt (a–c) and GRIP1−/− (d–f) E12 embryos with antibodies recognizing laminin (a and d), collagen type VII (b and e), and PECAM (c and f; counterstaining with Hoechst 33258). Laminin staining segregates on the roof of the bulla (d, arrowheads), whereas collagen type VII is present on both sides of the tissue cleft (e, arrowheads). (Magnifications: A a and c, ×20, b and d, ×60; B, a–f, ×60.)
Figure 5
Figure 5
Development of bullae in the lateral ventricle of the brain. (A) Hematoxylin-eosin staining of brain sections from wt (a–d) and GRIP1−/− (e–h) E12 embryos. Large haemorrhagic bulla (e, arrowhead) and blister on the surface of the meninges (e, arrow). A large serous blister (f, arrowhead; g, higher magnification) and extensive detachment of the neural epithelium from the underlying mesenchymal tissue (f, arrow; h, higher magnification) can be observed in the lateral ventricle, in close proximity to the developing choroid plexus. (B) PECAM immunostaining of a large bulla (arrowhead) protruding into the cerebral ventricle. Lack of PECAM staining on the surface of the bulla rules out its vascular origin. (Magnifications: A a, b, e, and f, ×20; c, d, g, and h, ×50; B, ×20.)
Figure 6
Figure 6
GRIP1−/− embryos develop dystrophic EB. (A) Schematic representation of the ultrastructure of the dermo-epidermal junction (as described in the text). (B) Ultrastructural analysis of skin from wt (a and b) and GRIP1−/− mutants (c and d). In GRIP1−/− mutants, nonblistered areas exhibit a large gap between epidermis and dermis (c), a feature absent in the skin of littermate controls. Analysis of the roof of the tissue cleft at higher magnification (d) reveals the presence of the LD. This type of lesion is typical of the dystrophic form of EB. (Magnifications: a and b, ×5,500; c, ×44,500; d, ×45,000.)
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