Fibronectin in the extracellular matrix of tissues acts as a substrate for cell adhesion and migration during development. Heterogeneity in the structure of fibronectin is largely due to the alternative splicing of at least three exons (IIIB, IIIA, and V) during processing of a single primary transcript. Fibronectin mRNA alternative splicing patterns change from B+A+V+ to B+A-V+ during chondrogenesis. In this report, immunohistochemical analysis demonstrates that while fibronectin protein containing the region encoded by exon IIIB is present throughout the limb at all stages of development, fibronectin protein containing the region encoded by exon IIIA disappears from cartilaginous regions just after condensation in vivo and in high-density mesenchymal micromass cultures in vitro. Treatment of mesenchymal micromass cultures prior to condensation with an antibody specific for the region encoded by exon IIIA disrupts the formation of cellular condensations and inhibits subsequent chondrogenesis in a dose- and time-dependent manner. Furthermore, microinjection of the exon IIIA antibody into embryonic chick limb primordia in vivo results in malformations characterized by smaller limbs and loss of limb skeletal elements. These results strongly suggest that the presence of the region encoded by exon IIIA in mesenchymal fibronectin is necessary for the condensation event that occurs during chondrogenesis.

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The alternative exon EIIIA of the fibronectin gene is included in mRNAs produced in undifferentiated mesenchymal cells but excluded from differentiated chondrocytes. As members of the SR protein family of splicing factors have been demonstrated to be involved in the alternative splicing of other mRNAs, the role of SR proteins in chondrogenesis-associated EIIIA splicing was investigated. SR proteins interacted with chick exon EIIIA sequences that are required for exon inclusion in a gel mobility shift assay. Addition of SR proteins to in vitro splicing reactions increased the rate and extent of exon EIIIA inclusion. Co-transfection studies employing cDNAs encoding individual SR proteins revealed that SRp20 decreased mRNA accumulation in HeLa cells, which make A+ mRNA, apparently by interfering with pre-mRNA splicing. Co-transfection studies also demonstrated that SRp40 increased exon EIIIA inclusion in chondrocytes, but not in HeLa cells, suggesting the importance of cellular context for SR protein activity. Immunoblot analysis did not reveal a relative depletion of SRp40 in chondrocytic cells. Possible mechanisms for regulation of EIIIA splicing in particular, and chondrogenesis associated splicing in general, are discussed.

Our goals have been to define the biochemical characteristics of megakaryocytes during maturation that are critical for platelet assembly and release into the circulation and to introduce biochemical markers for megakaryocytes. To achieve these goals, we have studied fibronectin (FN) and von Willebrand factor (vWF), which are large adhesive proteins that are synthesized by megakaryocytes, stored in alpha granules, and thought to have a fundamental role in hemostasis. The study demonstrated that vWF is primarily synthesized in mature megakaryocytes, which synthesized 7.5 times more vWF than immature megakaryocytes. Brefeldin A, which blocks the exit of proteins from the rough endoplasmic reticulum (RER), inhibited the formation of vWF multimers but did not affect the synthesis of monomers and dimers in mature megakaryocytes. These data are consistent with the formation of vWF dimers in the RER and the assembly of vWF multimers in the trans- and post-golgi. The synthesis of both the 260-kD and 275-kD pro-vWF was detected. However, the synthesis of 275-kD pro-vWF and 220-kD mature vWF was only evident after 2 hours, suggesting that the transit time of nascent vWF through the RER is about 2 hours. Constitutive secretion of vWF was demonstrated in megakaryocytes. About 14.5% and 4.6% of synthesized vWF was secreted by mature and immature megakaryocytes, respectively. In contrast, the synthesis of FN monomers and dimers was established in immature megakaryocytes, and their synthesis in mature megakaryocytes was very similar. Constitutive secretion of FN was not seen in megakaryocytes. Brefeldin A did not inhibit the synthesis of FN dimers; thus, formation of FN dimers occurs in the RER. The demonstration that vWF and FN are synthesized at different phases of megakaryocyte maturation and that only vWF is constitutively secreted by megakaryocytes provides new information relevant to alpha granule formation and possibly bone marrow matrix assembly.