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Address correspondence to Francesco Ramirez, Department of Biochemistry and Molecular Biology, Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1020, New York, NY 10029. Tel.: (212) 241-1757. Fax: (212) 722-5999. E-mail: ramirf01{at}doc.mssm.edu
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Abstract |
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Key Words: BMP; fibrillin; limb patterning; morphogenesis; syndactyly
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Introduction |
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The extracellular matrix consists of a highly heterogeneous mixture of insoluble and soluble molecules that orchestrate a variety of cellular processes and developmental programs. Extracellular microfibrils, alone or associated with elastin in the elastic fiber, confer critical properties to the connective tissue of the developing and adult organism (Ramirez, 1996). Microfibrilelastic fiber networks vary greatly in length, thickness, and tridimensional organization in order to accommodate the strength and direction of mechanical forces experienced by individual tissues (Mecham and Davis, 1994). It is widely believed that the morphological diversification of the insoluble matrix may also reflect nonstructural roles. For example, transient appearance of an elastic scaffold in the limb bud of the chick embryo was interpreted to suggest that this specialized matrix may physically delineate the location and size of cartilaginous structures (Hurlé et al., 1994). Indeed, ectopic stimulation of chondrogenesis at interdigital sites was shown to be preceded by disruption of the elastic scaffold and substantial matrix reorganization (Hurlé et al., 1994). However, the role of extracellular macroaggregates in demarcating specific areas in developing tissues has never been formally or genetically proven.
Another evolving concept is that the insoluble matrix may also modulate intercellular signaling by guiding the diffusion, sequestration, activation, or presentation of soluble factors to the surrounding cells (Flaumenhaft and Rifkin, 1991). A case in point is the postulated regulation by microfibrils of transforming growth factor-ß activity through binding of the large latent complex (Taipale and Keski-Oja, 1997). The glycoproteins fibrillin 1 (Fbn1) and fibrillin 2 (Fbn2) are the major, if not the only, structural components of extracellular microfibrils (Handford et al., 2000). Fbn gene expression and microfibrillar deposition precede elastic fiber formation, which in most tissues occurs between midgestation and early postnatal life (Mecham and Davis, 1994). Mutations in the human Fbn1 and Fbn2 genes are responsible for the pleiotropic manifestations of Marfan syndrome and the transient phenotype of congenital contractural arachnodactyly (CCA), respectively (Ramirez, 1996). Mice harboring targeted mutations in the Fbn1 gene have demonstrated that Marfan syndrome severity is determined by the degree of functional impairment of extracellular microfibrils (Pereira et al., 1997, 1999; Gayraud et al., 2000). Moreover, the longer bones of Fbn1-deficient mice lend credence to the hypothesis that microfibrils play a role in skeletal development (Pereira et al., 1999).
In contrast to the emerging information about Fbn1 biology, little is known about the role of Fbn2 during embryonic development. Here we report the creation of a null allele of the mouse Fbn2 gene. Mutant homozygotes recapitulate the human CCA phenotype, and also display bilateral syndactyly of forelimbs and hindlimbs. The patterning abnormality appears early in autopod formation, and before apoptotic cells are observed in the interdigital tissues. We show that Fbn2 deficiency is associated with disorganized microfibrils, and provide genetic evidence for interaction between Fbn2 and BMP-7. Altogether, the results demonstrate for the first time that specific intercellular signaling events during limb morphogenesis depend on proper supramolecular assembly of the insoluble extracellular matrix.
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Results and discussion |
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Fbn2 deficiency alters microfibrillar organization and BMP signaling
Two distinct experiments were performed to validate the above hypothesis. In the first, we examined the morphology of microfibrillar aggregates during autopod development. Analysis of Fbn1 and Fbn2 expression documented that both transcripts accumulate predominantly, but not exclusively, in the interdigital rays (Fig. 3
a). Indirect immunofluorescence using specific antisera for Fbn1 and Fbn2 proteins, as well as for the cartilage-specific type II collagen, provided an overview of the arrangement of these extracellular macroaggregates in the wild-type and mutant autopods (Fig. 3 b). Whereas the Fbn-rich network of the wild-type embryo is distinctly arranged in the digital and interdigital rays (Fig. 3 b, top), that of the mutant is significantly disorganized with little or no Fbn immunoreactivity in the interdigital rays (Fig. 3 b, bottom). This last finding implies that Fbn2 is required for proper microfibrillar assembly; the requirement is tissue specific, in that networks of other organs are morphologically normal (Fig. 3 c).
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Summary
This study identifies Fbn2 as the first insoluble matrix component to regulate limb patterning. This conclusion is based on two lines of evidence. First, altered microfibrillar morphology in mutant autopods implies a causal relationship between the organization of the insoluble macroaggregate and the patterning defect. Second, defective digit morphogenesis in double heterozygous Fbn2+/-/Bmp7+/- mice indicates functional interaction between the matrix component and the growth factor. Hence, interactions with the microfibrillar network apparently control the distribution and/or activity of intercellular signals in the developing limb. The effect is highly specific in that it is restricted to a specific tissue and morphogenetic program.
A variety of extracellular modulators are known to be involved in intercellular signaling, including extracellular proteins that control signal availability, such as proteoglycans and tissue proteases (Christian, 2000). To the best of our knowledge, this is the first instance in which evidence has been presented for the involvement of an insoluble matrix macroaggregate in the control of a specific patterning event. Thus more generally, the insoluble matrix may provide the structural scaffold that arranges morphogenetic clues in the intercellular space of the developing organism. This function could be exerted by binding directly to inactive growth factors (such as in the case of the latent transforming growth factor-ß complex), indirectly through interaction with other matrix components (such as proteoglycans), or by a combination of both mechanisms (Flaumenhaft and Rifkin, 1991; Taipale and Keski-Oja, 1997). Irrespectively, our study demonstrates that the tridimensional organization of insoluble extracellular macroaggregates is critically important in establishing morphogenetic gradients during vertebrate embryogenesis.
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Materials and methods |
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RNA analyses
Northern blot analysis was performed on 40 µg of total RNA purified from newborn lungs (Pereira et al., 1997); probes included the 3' untranslated regions of Fbn1 and Fbn2, and the control GAPDH (Zhang et al., 1995). In situ and whole-mount hybridizations were performed as described previously using the same Fbn probes (Sumiyoshi et al., 2001). Additional in situ hybridization probes included the 3' untranslated regions of Msx1, Msx2, Bmp-4, and Fgf-8 (Marazzi et al., 1997; Semba et al., 2000). RT-PCR screening of the mutant Fbn2 transcript was performed using overlapping oligonucleotides that cover the entire gene (Zhang et al., 1995).
Immunoanalyses
The anti-Fbn1 antibody, pAb9543, was prepared using the Fbn1 NH2-terminal half-recombinant polypeptide rF11 (Reinhardt et al., 1996). The mouse Fbn2 antibody was raised in the rabbit against the recombinant NH2-terminal peptide rF37. The antibody was tested by ELISA against the immunizing peptide, and specificity was determined by Western blot analysis. The type II collagen monoclonal antibody was obtained from the Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA). Western blot analyses were performed on conditioned media of primary dermal fibroblasts established from newborn Fbn2-/- and syfp/syfp mice (Gayraud et al., 2000). Antibodies were visualized with nitro blue tetrazolium/5-bromo-4-chloro-3' indoylphosphate (Pierce Chemical Co.). For indirect immunofluorescence, fresh limbs were fixed in 2% paraformaldehyde, 0.75 M lysine, and 0.01 M sodium periodate in 1x PBS for 4 h at 4°C, washed in 1x PBS for 1 h at 4°C, and stabilized in 40% sucrose in 1x PBS for 24 h at 4°C. The tissue was then embedded in OCT compound (Tissue Tek) and frozen in liquid nitrogen. 5 µm sections were generated using a Leica cryostat and diamond-coated disposable blades (C. L. Sturkey) and mounted on Fisherbrand Superfrost glass slides. Sections were dried at room temperature for 1 h and stored at 4°C. Immunostaining was performed as described previously (Gayraud et al., 2000), and fluorescence was monitored using a Zeiss Axiophot microscope using 10x or 20x Plan-Neofluar objective. Images were taken with a Spot camera (Diagnostic Instrument) and processed using Photoshop 5.5.
Morphological analyses
Adult mice were anesthetized by intraperitoneal injection of 0.017 ml of 25% Avitin per gram weight, and embryos were fixed in Bouin's fixative. Whole-mount pictures were taken using a Nikon SMZ-U dissecting scope and a Sony digital photo camera (model DKC-5000); images were processed using Photoshop 5.5. For whole-mount skeletal analysis, mice were fixed overnight in 95% ethanol after removing soft tissue. Cartilage was stained in a fresh solution of 80 ml 95% ethanol, 20 ml glacial acetic acid, and 1530 mg alcian blue (Sigma-Aldrich) for 1248 h. Skeleton was washed twice in 95% ethanol, and soft tissue was dissolved overnight in 2% KOH. Bones were stained overnight in 1% KOH, 75 µg/ml alizarin red S (Sigma-Aldrich), destained in 20% glycerol and 1% KOH for 37 d, and cleared first in 20% glycerol and 20% ethanol, and then in 50% glycerol and 50% ethanol. Images were prepared as described above. X-ray radiographs were performed on anesthesized mice using a Micro 50 (Microfocus Imaging) at 30 kV for 2030 s. In situ TUNEL assay on paraffin sections was performed using the Apoptosis Detection System (Promega).
Bead implantation
Affi-Gel blue agarose beads (Bio-Rad Laboratories) at 100200 mesh (5075 µm) were soaked in 100 ng/µl human recombinant BMP-4 (Genetics Institute) or in BSA, and implanted into interdigital tissue of E13.5 hindlimbs as described previously (Semba et al., 2000). After 6 h incubation at 37°C in 5% CO2, limbs were processed for in situ hybridization.
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Footnotes |
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Acknowledgments |
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This work was supported by grants from the National Institutes of Health (AR42044, GM18511), the National Marfan Foundation, and the Shriners Foundation.
Submitted: 8 May 2001
Revised: 6 June 2001
Accepted: 8 June 2001
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