Behavioural effects of extracellular matrix protein Fras1 depletion in the mouse.

Fras1 is an extracellular protein of the basement membranes that surround embryonic epithelia, choroid plexuses and meninges in foetal mouse brain. Depletion of Fras1 in knockout mice results in sub-epidermal blistering and fusion of eyelids and digits as well as malformation of lungs and kidneys. Mutations in the human counterpart FRAS1 are responsible for the Fraser Syndrome with clinical manifestations similar to the murine phenotype. In addition, brain deformities or mental impairments have occasionally been reported in patients with Fraser Syndrome. In the present study, we explored the possible involvement of Fras1 in brain function, analysing its expression pattern in mouse brain and investigating aspects of Fras1-/- mice behaviour, related to the function of brain regions expressing Fras1. Transcripts were detected in choroid plexuses and in certain brain regions including cortical, hippocampal and amygdalar areas in juvenile mice. Behavioural tests revealed that Fras1-/- mice exhibit impaired egocentric spatial memory, aberrant olfactory learning and memory, markedly reduced fear memory in an auditory fear conditioning task, as well as reduced anxiety expression in open field and elevated plus maze tests. Moreover, the extracellular matrix organization has been severely affected in cortical and subcortical areas as demonstrated by Wisteria floribunda agglutinin immunolabelling. The widespread detection of Fras1 transcripts in the brain of both pre- and postnatal mice, as well as the behavioural and cellular disturbances exhibited by Fras1-/- adult mice provide evidence for the involvement of Fras1 in brain organization and function.

Identification of a new gene mutated in Fraser syndrome and mouse myelencephalic blebs.

Fraser syndrome is a recessive, multisystem disorder presenting with cryptophthalmos, syndactyly and renal defects and associated with loss-of-function mutations of the extracellular matrix protein FRAS1. Fras1 mutant mice have a blebbed phenotype characterized by intrauterine epithelial fragility generating serous and, later, hemorrhagic blisters. The myelencephalic blebs (my) strain has a similar phenotype. We mapped my to Frem2, a gene related to Fras1 and Frem1, and showed that a Frem2 gene-trap mutation was allelic to my. Expression of Frem2 in adult kidneys correlated with cyst formation in my homozygotes, indicating that the gene is required for maintaining the differentiated state of renal epithelia. Two individuals with Fraser syndrome were homozygous with respect to the same missense mutation of FREM2, confirming genetic heterogeneity. This is the only missense mutation reported in any blebbing mutant or individual with Fraser syndrome, suggesting that calcium binding in the CALXbeta-cadherin motif is important for normal functioning of FREM2.

Endonucleosis mediates internalization of cytoplasm into the nucleus.

Setd8 regulates transcription elongation, mitotic DNA condensation, DNA damage response and replication licensing. Here we show that, in mitogen-stimulated liver-specific Setd8-KO mice, most of the hepatocytes are eliminated by necrosis but a significant number of them survive via entering a stage exhibiting several senescence-related features. Setd8-deficient hepatocytes had enlarged nuclei, chromosomal hyperploidy and nuclear engulfments progressing to the formation of intranuclear vesicles surrounded by nuclear lamina. These vesicles contain glycogen, cytoplasmic proteins and even entire organelles. We term this process "endonucleosis". Intranuclear vesicles are absent in hepatocytes of Setd8/Atg5 knockout mice, suggesting that the process requires the function of the canonical autophagy machinery. Endonucleosis and hyperploidization are temporary, early events in the surviving Setd8-deficient cells. Larger vesicles break down into microvesicles over time and are eventually eliminated. The results reveal sequential events in cells with extensive DNA damage, which function as part of survival mechanisms to prevent necrotic death.

Segmental and restricted localization pattern of Fras1 in the developing meningeal basement membrane in mouse.

The Fras1/Frem family of extracellular matrix proteins consists of Fras1 and its structurally related proteins, Frem1 (Fras1-related extracellular matrix protein 1), Frem2 and Frem3. These are co-localized in embryonic epithelial basement membranes (BMs), where they contribute to epithelial-mesenchymal adhesion. Although Fras1 localization pattern in epithelial BMs has been well defined, it has not yet been comprehensively studied in the central nervous system. Here, we demonstrate the immunohistochemical profile of Fras1 in the developing mouse brain and reveal an exclusively meningeal BM protein deposition. Interestingly, Fras1 displays a segmental localization pattern, which is restricted to certain regions of the meningeal BM. Frem2 protein displays a similar localization pattern, while Frem3 is rather uniformly distributed throughout the meningeal BM. Fras1 and Frem2 proteins are detected in regions of the BM that underlie organizing centers, such as the roof plate (RP) of diencephalon, midbrain and hindbrain, and the RP-derived structures of telencephalon (choroid plexus and hem). Organizing centers exert their activity via the production of bioactive molecules, which are potential Fras1 ligands. The restricted pattern of Fras1 and Frem2 proteins indicates a molecular compartmentalization of the meningeal BM that could reflect, yet unspecified, functional and structural differences.

A direct functional link between the multi-PDZ domain protein GRIP1 and the Fraser syndrome protein Fras1.

Cell adhesion to extracellular matrix (ECM) proteins is crucial for the structural integrity of tissues and epithelial-mesenchymal interactions mediating organ morphogenesis. Here we describe how the loss of a cytoplasmic multi-PDZ scaffolding protein, glutamate receptor interacting protein 1 (GRIP1), leads to the formation of subepidermal hemorrhagic blisters, renal agenesis, syndactyly or polydactyly and permanent fusion of eyelids (cryptophthalmos). Similar malformations are characteristic of individuals with Fraser syndrome and animal models of this human genetic disorder, such as mice carrying the blebbed mutation (bl) in the gene encoding the Fras1 ECM protein. GRIP1 can physically interact with Fras1 and is required for the localization of Fras1 to the basal side of cells. In one animal model of Fraser syndrome, the eye-blebs (eb) mouse, Grip1 is disrupted by a deletion of two coding exons. Our data indicate that GRIP1 is required for normal cell-matrix interactions during early embryonic development and that inactivation of Grip1 causes Fraser syndrome-like defects in mice.

Fras1 deficiency results in cryptophthalmos, renal agenesis and blebbed phenotype in mice.

Loss of tight association between epidermis and dermis underlies several blistering disorders and is frequently caused by impaired function of extracellular matrix (ECM) proteins. Here we describe a new protein in mouse, Fras1, that is specifically detected in a linear fashion underlying the epidermis and the basal surface of other epithelia in embryos. Loss of Fras1 function results in the formation of subepidermal hemorrhagic blisters as well as unilateral or bilateral renal agenesis during mouse embryogenesis. Postnatally, homozygous Fras1 mutants have fusion of the eyelids and digits and unilateral renal agenesis or dysplasia. The defects observed in Fras1-/- mice phenocopy those of the existing bl (blebbed) mouse mutants, which have been considered a model for the human genetic disorder Fraser syndrome. We show that bl/bl homozygous embryos are devoid of Fras1 protein, consistent with the finding that Fras1 is mutated in these mice. In sum, our data suggest that perturbations in the composition of the extracellular space underlying epithelia could account for the onset of the blebbed phenotype in mouse and Fraser syndrome manifestation in human.

Fraser syndrome and mouse blebbed phenotype caused by mutations in FRAS1/Fras1 encoding a putative extracellular matrix protein.

Fraser syndrome (OMIM 219000) is a multisystem malformation usually comprising cryptophthalmos, syndactyly and renal defects. Here we report autozygosity mapping and show that the locus FS1 at chromosome 4q21 is associated with Fraser syndrome, although the condition is genetically heterogeneous. Mutation analysis identified five frameshift mutations in FRAS1, which encodes one member of a family of novel proteins related to an extracellular matrix (ECM) blastocoelar protein found in sea urchin. The FRAS1 protein contains a series of N-terminal cysteine-rich repeat motifs previously implicated in BMP metabolism, suggesting that it has a role in both structure and signal propagation in the ECM. It has been speculated that Fraser syndrome is a human equivalent of the blebbed phenotype in the mouse, which has been associated with mutations in at least five loci including bl. As mapping data were consistent with homology of FRAS1 and bl, we screened DNA from bl/bl mice and identified a premature termination of mouse Fras1. Thus, the bl mouse is a model for Fraser syndrome in humans, a disorder caused by disrupted epithelial integrity in utero.

The Fras1/Frem family of extracellular matrix proteins: structure, function, and association with Fraser syndrome and the mouse bleb phenotype.

Fras1 and the structurally related proteins Frem1, Frem2, and Frem3, comprise a novel family of extracellular matrix proteins, which localize in a similar fashion underneath the lamina densa of epithelial basement membranes. They are involved in the structural adhesion of the skin epithelium to its underlying mesenchyme. Deficiency in the individual murine Fras1/Frem genes gives rise to the bleb phenotype, which is equivalent to the human hereditary disorder Fraser syndrome, characterized by cryptophthalmos (hidden eyes), embryonic skin blistering, renal agenesis, and syndactyly. Recent studies revealed a functional cooperation between the Fras1/Frem gene products, in which Fras1, Frem1 and Frem2 are simultaneously stabilized at the lowermost region of the basement membrane by forming a macromolecular ternary complex. Loss of any of these proteins results in the collapse of the protein assembly, thus providing a molecular explanation for the highly similar phenotypic defects displayed by the respective mutant mice. Here, we summarize the current knowledge regarding the structure, function, and interplay between the proteins of the Fras1/Frem family and further propose a possible scenario for the evolution of the corresponding genes.

Basement membrane localization of Frem3 is independent of the Fras1/Frem1/Frem2 protein complex within the sublamina densa.

The Fraser syndrome protein Fras1 and the structurally related proteins Frem1, Frem2 and Frem3 comprise a novel family of extracellular matrix proteins implicated in the structural adhesion of the embryonic epidermis to the underlying mesenchyme. Fras1, Frem1 and Frem2 have been shown to be simultaneously and interdependently stabilized in the basement membrane by forming a ternary complex located underneath the lamina densa. However, the functional relationships between Frem3 and the other Fras1/Frem proteins remain unknown. Here we show that in the absence of Fras1 the basement membrane localization of Frem3 remains unaffected in contrast to Frem1 and Frem2 which are completely abolished from the basement membrane. This indicates that although Frem3 is localized in the sublamina densa similar to Fras1, Frem1 and Frem2 yet it is anchored in the basement membrane independently. We further demonstrate that loss of Fras1 results in the accumulation of Frem2 within epithelial cells. This finding reveals that Fras1 is not only essential as a component of a macromolecular complex for the extracellular stabilization of Frem2 but it is also required for its proper intracellular trafficking and export from embryonic epithelial cells.

Spatiotemporal distribution of Fras1/Frem proteins during mouse embryonic development.

The Fras1/Frem gene family encodes for structurally similar, developmentally regulated extracellular matrix proteins. Mutations in Fras1, Frem1 and Frem2 have been identified in different classes of mouse bleb mutants, while defects in the human orthologs FRAS1 and FREM2 are causative for Fraser syndrome. The hallmark phenotypic feature of bleb mice is embryonic skin blistering due to dermal-epidermal detachment. The similarity of the phenotypic characteristics among the bleb mouse mutants, together with the fact that Fras1/Frem proteins are co-localized in embryonic epithelial basement membranes, suggest that they operate in a common pathway. Here, we report for the first time the immunofluorescence pattern of Frem3 and provide a comparative analysis of the spatiotemporal localization of all Fras1/Frem proteins during mouse embryonic development. We demonstrate their overall co-localization in embryonic epithelial basement membranes, with emphasis on areas of phenotypic interest such as eyelids, limbs, kidneys, lungs and organs of the gastrointestinal tract and the central nervous system. We further studied collagen VII, impairment of which produces dystrophic epidermolysis bullosa, a postnatal skin blistering disorder. We show that basement membrane levels of collagen VII rise at late embryonic life, concomitant with descending Fras1/Frem immunolabeling.

Ultrastructural localization of Fras1 in the sublamina densa of embryonic epithelial basement membranes.

Fras1 is the first identified member of a protein family comprising Fras1 and the related extracellular matrix proteins Frem1, Frem2 and Frem3. Mutations in Fras1, Frem1 and Frem2 have been associated with the bleb phenotype in mouse, whereas mutations in the human orthologs FRAS1 and FREM2 have been implicated in the pathogenesis of the human Fraser syndrome. Bleb mutant mice are characterized by embryonic sub-epidermal blistering, unilateral or bilateral renal agenesis or dysgenesis, cutaneous syndactyly and fused eyelids. As revealed by immunofluorescence, Fras1 co-localizes with the markers of epithelial basement membranes and is ultrastructurally detected underneath the lamina densa of embryonic mouse epithelia. Since the loss of Fras1 mainly affects the cohesiveness of the embryonic skin basement membrane with its underlying mesenchyme, we compared here the ultrastructural localization of Fras1 in the dermal-epidermal junction and in the basement membrane of other embryonic epithelia that do not show any overt phenotype using preembedding immunocytochemistry. Fras1 immunoreactivity was detected in all epithelia examined, within the sublamina densa adjacent to stromal tissue, as clustered gold/silver enhanced depositions, usually attached to anchoring fibrils. Interestingly, clusters corresponding to Fras1 were frequently detected in close proximity to mesenchymal cells, indicating that Fras1 could serve as a direct link between the sublamina densa and mesenchyme. The localization of Fras1 is consistent with previous results indicating that Fras1 exerts its function below the lamina densa and that Fras1 displays the same localization pattern in all epithelial basement membranes.

The role of Fras1/Frem proteins in the structure and function of basement membrane.

Basement membranes constitute architecturally complex extracellular matrix (ECM) protein networks of great structural and regulatory importance. Recently, a novel group of basement membrane proteins, Fras1 (Fraser syndrome protein (1) and the Fras1-related extracellular matrix proteins Frem1, Frem2 and Frem3, has emerged. They comprise components of the sublamina densa region and contribute to embryonic epithelial-mesenchymal integrity. Fras1/Frem share common polypeptide repetitive motifs with possible interactive and organizing functions. Mutations in genes encoding Fras1, Frem1 and Frem2 are causative for dermal-epidermal detachment in the plane of sublamina densa and have been identified in different classes of mouse bleb mutants, the murine model of human Fraser syndrome, the hallmark phenotypic characteristics of which are embryonic skin blistering, cryptophthalmos and renal agenesis. Indeed, defects in FRAS1 and FREM2 have been identified in Fraser syndrome patients. The phenotypic similarity of mouse bleb mutant strains can be attributed to the fact that Fras1, Frem1 and Frem2 have been experimentally shown to interact, forming a mutually stabilized protein complex, while Frem3, which has not yet been associated with any of the existing known mutations, operates in a more independent fashion. Fras1/Frem have been recently proposed to compensate for the activity of collagen VII, a major anchoring component of the sublamina densa, the levels of which rise only during late embryonic life. By focusing on the aforementioned data, in this review we will summarize the current knowledge about Fraser syndrome proteins and describe their contribution to basement membrane biology.

pH-dependent antigen unmasking in paraformaldehyde-fixed tissue cryosections.

Fras1/Frem family of basement membrane proteins has been associated with the "bleb" phenotype in mouse and the Fraser syndrome in man. Fras1 and Frem2 proteins are known to be colocalized in all epithelial basement membranes during embryonic development. The functional significance of their colocalization has been demonstrated in the corresponding mouse mutants, where the absence of Fras1 results in complete depletion of Frem2 from epithelial basement membranes and vice versa. Nevertheless, under standard immunohistochemical procedures, we were able to detect Fras1, but not Frem2, in the basement membrane of adult mouse tail skin. After reevaluation of our protocol, we established 15-minute acidic buffer treatment to be of critical value upon Frem2 immunodetection, essentially operating as an antigen retrieval process. Testing more polyclonal antibodies revealed no negative effects, but rather reinforced the positive signal, rendering this technique suitable for incorporation to any standard immunohistochemical procedure.

Overlapping and divergent localization of Frem1 and Fras1 and its functional implications during mouse embryonic development.

Frem1 belongs to a family of structurally related extracellular matrix proteins of which Fras1 is the founding member. Mutations in Fras1 and Frem1 have been identified in mouse models for Fraser syndrome, which display a strikingly similar embryonic skin blistering phenotype due to impaired dermal-epidermal adhesion. Here we show that Frem1 originates from both epithelial and mesenchymal cells, in contrast to Fras1 that is exclusively derived from epithelia. However, both proteins are localized in an absolutely overlapping fashion in diverse epithelial basement membranes. At the ultrastructural level, Frem1 exhibits a clustered arrangement in the sublamina densa coinciding with fibrillar structures reminiscent of anchoring fibrils. Furthermore, in addition to its extracellular deposition, around E16, Frem1 displays an intracellular distribution in distinct epidermal cell types such as the periderm layer and basal keratinocytes. Since periderm cells are known to participate in temporary epithelial fusions like embryonic eyelid closure, defective function of Frem1 in these cells could provide a molecular explanation for the "eyes open at birth" phenotype, a feature unique for Frem1 deficient mouse mutants. Finally, we demonstrate loss of Frem1 localization in the basement membrane but not in periderm cells in the skin of Fras1(-/-) embryos. Taken together, our findings indicate that besides a cooperative function with Fras1 in embryonic basement membranes, Frem1 can also act independently in processes related to epidermal differentiation.

Basement membrane distortions impair lung lobation and capillary organization in the mouse model for fraser syndrome.

Fras1 is a putative extracellular matrix protein that has been implicated in the structural adhesion of embryonic epidermis to dermis. Moreover, mutations in Fras1/FRAS1 have been associated with the mouse blebbed phenotype and the human rare genetic disorder Fraser syndrome, respectively. Here we report the mapping of Fras1 within the extracellular space and evaluate the effects of Fras1 deficiency on lung development in the mouse. Expression of Fras1 was detected in the mesothelial cells of the visceral pleura and in the conducting airway epithelia. Immunogold histochemistry identified Fras1 as a component of the extracellular matrix localized below the lamina densa of epithelial basement membranes in the embryonic lung. Embryos homozygous for a targeted mutation of Fras1 exhibited fused pulmonary lobes resulting from incomplete separation during development as well as a profound disarrangement of blood capillaries in the terminal air sacs. We demonstrate that loss of Fras1 causes alterations in the molecular composition of basement membranes, concomitant with local disruptions of epithelial-endothelial contacts and extravasation of erythrocytes into the embryonic respiratory lumen. Thus, our findings identify Fras1 as an important structural component of the sub-lamina densa of basement membranes required for lobar septation and the organization of blood capillaries in the peripheral lung.