Amphibian Pleurodeles waltl Fibronectin: cDNA Cloning and Developmental Expression of Spliced Variants

Partial cDNA clones encoding approximately the carboxy terminal half of Pleurodeles fibronectin (FN) were isolated. They account for 4.7 Kbp of the 3′ region of the FN mRNA. The cDNA nucleotide sequence comprises all three alternatively spliced segments designated EIIIA, EIIIB and V-segment, respectively. All three segments are included in FN mRNA synthesized during early embryogenesis whereas, from the tailbud stage onward the V-region was partially excluded. The isolation of Pleurodeles cDNA clones including the three different spliced EIIIA, EIIIB and V segment raises new possibilities for the study of the precise role of specific regions of FN in early amphibian development.     

Fibronectin Isoform Distribution in the Mouse I. The Alternatively Spliced EIIIB,EIIIA, and V Segments Show Widespread Codistribution in the Developing Mouse Embryo

Fibronectins (FNs) are extracellular matrix glycoproteins that are essential for embryonic development. In order to gain clues to possible developmental roles played by the particular isoforms of FN, we used indirect immunofluorescence microscopy to examine and compare the distributions of the alternatively spliced EIIIB, EIIIA, and V segments, as well as the total pool of FNs, in serial sections from mouse embryos. Antibodies to each of these segments produced staining patterns that colocalized during gastrulation (E7.5) and during early morphogenesis of somites and notochord (E9.5). During the period of continuing organogenesis in the latter half of gestation (E10.5 to E16.5), the antibodies generally continued to produce similar staining patterns localized to epithelial basement membranes, stromal connective tissues, blood vessel walls, and muscles. However, as development proceeded, there was a gradual decline in the intensity of staining for the spliced segments relative to the total pool of FN, with a particularly noticeable decline in staining for EIIIB and EIIIA segments in certain glandular organs, including the liver. A specific reduction in expression of these latter two segments was also evident in the uterus and placenta at early timepoints in gestation. However, the most dramatic difference in the expression of the spliced segments occurred in developing hyaline cartilage, which showed a selective reduction in staining for the EIIIA segment that was evident in the axial skeletal precursors by E12.5 and complete throughout the embryo by E15.5. Our findings suggest that the alternatively spliced EIIIB, EIIIA, and V segments are included in the FN that is required for the morphogenesis of “FN dependent” structures, including somites, notochord, and the vasculature. Conversely, these segments would appear to play divergent, and sometimes exclusive, biological roles in specific tissues such as liver, cartilage, and placenta.     

Spatial expression of the alternatively spliced EIIIB and EIIIA segments of fibronectin in the early chicken embryo

Using domain-specific antibodies, we have analyzed the tissue distribution of fibronectins (FNs) containing the alternatively spliced EIIIB and EIIIA segments relative to total FN in early chicken embryos. The results show a selective loss of EIIIA+ FN staining in the notochordal sheath and in cartilaginous structures between 4.5 and 7.0 days of development. In other regions, EIIIB+ and EIIIA+ FNs are extensively codistributed in and around mesoderm-derived structures (somites, notochord, heart, and blood vessels), in basal laminae of endoderm and ectoderm-derived structures, as well as within the vicinity of neural crest formation and migration. We also noted that EIIIA staining overlaps with spatial patterns of distribution that have previously been described for the alpha4 integrin subunit, a component of the EIIIA receptor alpha4beta1.     

Fibronectin Isoform Distribution in the Mouse I. The Alternatively Spliced EIIIB, EIIIA, and V Segments Show Widespread Codistribution in the Developing Mouse Embryo

Fibronectins (FNs) are extracellular matrix glycoproteins that are essential for embryonic development. In order to gain clues to possible developmental roles played by the particular isoforms of FN, we used indirect immunofluorescence microscopy to examine and compare the distributions of the alternatively spliced EIIIB, EIIIA, and V segments, as well as the total pool of FNs, in serial sections from mouse embryos. Antibodies to each of these segments produced staining patterns that colocalized during gastrulation (E7.5) and during early morphogenesis of somites and notochord (E9.5). During the period of continuing organogenesis in the latter half of gestation (E10.5 to E16.5), the antibodies generally continued to produce similar staining patterns localized to epithelial basement membranes, stromal connective tissues, blood vessel walls, and muscles. However, as development proceeded, there was a gradual decline in the intensity of staining for the spliced segments relative to the total pool of FN, with a particularly noticeable decline in staining for EIIIB and EIIIA segments in certain glandular organs, including the liver. A specific reduction in expression of these latter two segments was also evident in the uterus and placenta at early timepoints in gestation. However, the most dramatic difference in the expression of the spliced segments occurred in developing hyaline cartilage, which showed a selective reduction in staining for the EIIIA segment that was evident in the axial skeletal precursors by E12.5 and complete throughout the embryo by E15.5. Our findings suggest that the alternatively spliced EIIIB, EIIIA, and V segments are included in the FN that is required for the morphogenesis of “FN dependent” structures, including somites, notochord, and the vasculature. Conversely, these segments would appear to play divergent, and sometimes exclusive, biological roles in specific tissues such as liver, cartilage, and placenta.     

Multiple cardiovascular defects caused by the absence of alternatively spliced segments of fibronectin

Alternatively spliced variants of fibronectin (FN) containing exons EIIIA and EIIIB are expressed around newly forming vessels in development and disease but are downregulated in mature vasculature. The sequences and patterns of expression of these splice variants are highly conserved among vertebrates, suggestive of their biological importance; however the functions of EIIIA and EIIIB-containing FNs are unknown. To understand the role(s) of these splice variants, we deleted both EIIIA and EIIIB exons from the FN gene and observed embryonic lethality with incomplete penetrance by embryonic day 10.5. Deletion of both EIIIA and EIIIB exons did not affect synthesis or cell surface deposition of FN, indicating that embryonic lethality was due specifically to the absence of EIIIA and EIIIB exons from FN. EIIIA/EIIIB double-null embryos displayed multiple embryonic cardiovascular defects, including vascular hemorrhage, failure of remodeling embryonic and yolk sac vasculature, defective placental angiogenesis and heart defects. In addition, we observed defects in coverage and association with dorsal aortae of alpha-smooth-muscle-actin-positive cells. Our studies indicate that the presence or absence of EIIIA and EIIIB exons alters the function of FN and demonstrate the requirement for these alternatively spliced exons in cardiovascular development.     

Fibronectin Isoform Distribution in the Mouse II. Differential Distribution of the Alternatively Spliced EIIIB,EIIIA, and V Segments in the Adult Mouse

The alternatively spliced EIIIB, EIIIA. and V segments of fibronectin (FN) show widespread codistribution in the mouse embryo, suggesting that EIIIB+, EIIIA+, and V+ isoforms serve to facilitate morphogenesis and organogenesis (Peters, JH, and Hynes, RO, 1996, this issue). To gain further clues to functions of these segments, we have used segment-specific anti-FN antibodies to perform immunofluorescence microscopy on tissue sections obtained from mice aged 9 to 15 weeks. Staining for each of the three spliced segments, relative to that for the total FN pool, was reduced in the adult as compared with the embryo. Anti-V antibodies produced patterns which were most similar to those obtained with anti-total FN antibodies, localizing to basement membranes, connective tissues subjacent to epithelia, walls of blood vessels, and cartilage. Anti-EIIIA antibodies produced the next most widespread pattern. which included prominent staining of the walls of blood vessels of all sizes, the lung inter-stitium. and smooth muscle associated with the gastrointestinal (GI), genitourinary (GU), and respiratory tracts. Although anti-EIIIB antibodies produced the faintest and most restricted pattern of staining, EIIIB+ FN could be detected in the walls of some smaller blood vessels, smooth muscle of the GI, GU, and respiratory tracts, as well as within cartilaginous structures, and eye. There were quantitative and/or qualitative differences in the staining patterns produced by the three segment-specific antibodies in a variety of tissues, including liver, cartilage, synovium, cornea, muscle, peripheral nerve, and lymph node. These findings suggest that each of the spliced segments of the FN molecule may occupy unique physical or functional positions within the extracellular matrix of the adult mouse.     

Fibronectin Isoform Distribution in the Mouse II. Differential Distribution of the Alternatively Spliced EIIIB, EIIIA, and V Segments in the Adult Mouse

The alternatively spliced EIIIB, EIIIA. and V segments of fibronectin (FN) show widespread codistribution in the mouse embryo, suggesting that EIIIB+, EIIIA+, and V+ isoforms serve to facilitate morphogenesis and organogenesis (Peters, JH, and Hynes, RO, 1996, this issue). To gain further clues to functions of these segments, we have used segment-specific anti-FN antibodies to perform immunofluorescence microscopy on tissue sections obtained from mice aged 9 to 15 weeks. Staining for each of the three spliced segments, relative to that for the total FN pool, was reduced in the adult as compared with the embryo. Anti-V antibodies produced patterns which were most similar to those obtained with anti-total FN antibodies, localizing to basement membranes, connective tissues subjacent to epithelia, walls of blood vessels, and cartilage. Anti-EIIIA antibodies produced the next most widespread pattern. which included prominent staining of the walls of blood vessels of all sizes, the lung inter-stitium. and smooth muscle associated with the gastrointestinal (GI), genitourinary (GU), and respiratory tracts. Although anti-EIIIB antibodies produced the faintest and most restricted pattern of staining, EIIIB+ FN could be detected in the walls of some smaller blood vessels, smooth muscle of the GI, GU, and respiratory tracts, as well as within cartilaginous structures, and eye. There were quantitative and/or qualitative differences in the staining patterns produced by the three segment-specific antibodies in a variety of tissues, including liver, cartilage, synovium, cornea, muscle, peripheral nerve, and lymph node. These findings suggest that each of the spliced segments of the FN molecule may occupy unique physical or functional positions within the extracellular matrix of the adult mouse.     

Alternative splicing of fibronectin: three variants, three functions.

Fibronectin (FN) is a multi-functional extracellular matrix protein required for cell adhesion and migration, blood clotting, wound healing, and oncogenic transformation. The functional complexity is paralleled by structural diversity in that multiple forms of FN are generated by cell type-specific alternative splicing. In the rat, up to 12 different combinations of the three alternatively spliced segments (EIIIA, EIIIB, and the V region) are produced. What effects do these segments have on FN function? Recently, progress has been made in the identification of specific activities for the three Variants of the V region, V120, V95, and V0. FN-mediated cell adhesion, FN synthesis and secretion, and incorporation into blood clots are differentially affected by these isoforms. These results suggest that cellular behavior is modulated by environmental cues provided by different types and proportions of alternatively spliced FN variants.     

Embryonic fibronectins are up-regulated following peripheral nerve injury in rats

Fibronectin mRNAs that include the alternatively spliced exons EIIIA, EIIIB, and V are prevalent during embryogenesis, and EIIIA and EIIIB reappear during wound healing. Using ribonuclease protection analyses, we found an up-regulation of V120 (containing the α₄β₁ integrin binding site), as well as EIIIA, and EIIIB in fibronectin mRNAs in the crush-injured adult rat sciatic nerve. In situ hybridization using splice variant-specific probes revealed that cells within endoneurial tubes of the injured nerve synthesize these embryonic forms of fibronectin. Our results suggest that embryonic fibronectins synthesized within the nerve contribute to the permissiveness of the peripheral nervous system to axon regrowth and a mechanism by which alternative splicing of the V region in fibronectin mRNA could enhance nervous system regeneration. © 1995 John Wiley & Sons, Inc.     

Cellular fibronectin is induced by epidermal growth factor, but not by dexamethasone or cyclic AMP in rat liver epithelial cells

Epidermal growth factor (EGF) induces fibronectin (FN) and FN mRNA in rat liver epithelial cells, under conditions where the factor also induces the cells to migrate. Newly synthesized protein is secreted into the medium and deposited as substratum-bound extracellular matrix. The levels of mRNA and the amount of protein synthesized are not influenced by cyclic AMP or dexamethasone, factors that have been found to modulate FN expression in other cells. However, the cells are sensitive to the factors, suggesting a cell-specific regulation. The EGF-induced RNA contains the sequences EIIIA and EIIIB characteristic of cellular fibronectin.     

Structural insights into fibronectin type III domain-mediated signaling

The alternatively spliced type III extradomain B (EIIIB) of fibronectin (FN) is expressed only during embryogenesis, wound healing and tumorigenesis. The biological function of this domain is unclear. We describe here the first crystal structure of the interface between alternatively spliced EIIIB and its adjacent FN type III domain 8 (FN B-8). The opened CC' loop of EIIIB, and the rotation and tilt of EIIIB allow good access to the FG loop of FN-8, which is normally hindered by the CC' loop of FN-7. In addition, the AGEGIP sequence of the CC' loop of EIIIB replaces the NGQQGN sequence of the CC' loop of FN-7. Finally, the CC' loop of EIIIB forms an acidic groove with FN-8. These structural findings warrant future studies directed at identifying potential binding partners for FN B-8 interface, linking EIIIB to skeletal and cartilaginous development, wound healing, and tumorigenesis, respectively.     

Expression of variant fibronectins in wound healing: cellular source and biological activity of the EIIIA segment in rat hepatic fibrogenesis

We have examined the cell-specific expression of two fibronectin isoforms, EIIIA and EIIIB, during experimental hepatic fibrosis induced by ligation of the biliary duct. AT the mRNA level, EIIIA and EIIIB were undetectable in normal liver but expressed early injury, preceding fibrosis. The cellular sources of these changes were determined by fractionating the liver at various time points after bile duct ligation into its constituent cell populations and extracting RNA from the fresh isolates. EIIIA-containing fibronectin mRNA was undetectable in normal sinusoidal endothelial cells but increased rapidly within 12 h of injury. By contrast, the EIIIB form was restricted to hepatic lipocytes (Ito or fat-storing cells) and appeared only after a lag of 12-24 h: it was minimal in sinusoidal endothelial cells. Both forms were minimal in hepatocytes. At the protein level, EIIIA-containing fibronectin was markedly increased within two days of injury and exhibited a sinusoidal distribution. Secretion of this form by endothelial cells was confirmed in primary culture. Matrices deposited in situ by endothelial cells from injured liver accelerated the conversion ("activation") of normal lipocytes to myofibroblast-like cells, and pretreatment of matrices with monoclonal antibody to the EIIIA segment blocked this response. Finally, recombinant fibronectin peptide containing the EIIIA segment was stimulatory to lipocytes in culture. We conclude that expression of EIIIA fibronectin by sinusoidal endothelial cells is a critical early event in the liver's response to injury and that the EIIIA segment is biologically active, mediating the conversion of lipocytes to myofibroblasts.     

Multiple sites of alternative splicing of the rat fibronectin gene transcript

We describe analyses of the structure and expression of the rat fibronectin gene with particular attention to the 40-kb stretch from the center of the gene which encodes 17 type-III repeating units. Each repeat is precisely separated from its neighbors by introns and most are encoded by pairs of exons. Three repeats are encoded precisely by single exons and two of these (EIIIA and EIIIB) are alternatively spliced in a cell type-specific fashion. A third site of alternative splicing (EIIIB) reported here is similar in expression to the previously described EIIIA segment. Both are excluded from mRNA in liver cells and are, therefore, absent from plasma fibronectin. These two alternative splices, plus a third one (V) reported previously, can occur in all possible combinations giving 12 fibronectin mRNAs from a single gene. These splicing variations account for most but not all of the known fibronectin subunit variants. We report investigations designed to detect other regions of alternative splicing. We also show that the pattern of alternative splicing is somewhat altered on oncogenic transformation.