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 TGF beta family of genes has been shown to play an important role in regulating various aspects of development, although the mechanisms by which TGF beta exerts its effects have not yet been clarified. Growth and differentiation of both murine embryonic palate mesenchymal (MEPM) cells and palatal epithelium can be regulated by the TGF beta s. We therefore examined the expression of mRNAs encoding TGF beta 1, TGF beta 2, and TGF beta 3 in developing embryonic palatal tissue as well as factors that modulate their levels of expression. Northern blot analysis of RNA isolated from murine embryonic palatal tissue on gestational days (GD) 12, 13, and 14 demonstrated the presence of one mRNA transcript for TGF beta 1 (2.5 kb), two transcripts for TGF beta 2 (4.4 kb, 6.0 kb), and one transcript for TGF beta 3 (3.5 kb). Although steady-state levels of TGF beta 1 mRNA showed no changes during development of the palate, TGF beta 2 mRNA levels were maximal on both GD13 and GD14 and TGF beta 3 mRNA levels transiently increased on GD 13. In addition, levels of TGF beta 3 mRNA seemed much higher than either TGF beta 1 or TGF beta 2. both TGF beta 1 and TGF beta 2 were able to increase, in a dose-related manner, the expression of TGF beta 3 mRNA in murine embryonic palate mesenchymal cells in vitro. In contrast, epidermal growth factor (EGF) down-regulated the expression of TGF beta 3 mRNA even in the presence of TGF beta 1 or TGF beta 2.(ABSTRACT TRUNCATED AT 250 WORDS)

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Fibronectin, a component of the extracellular matrix in a variety of tissues, participates in many critical cellular processes, including differentiation, adhesion, and migration. A positive correlation exists between the presence of fibronectin and the onset of chondrogenesis, the differentiation of mesenchyme into cartilage. 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. We have previously shown that the fibronectin mRNA splicing patterns change during chondrogenesis (Bennett et al. [1991] J. Biol. Chem. 266:5918–5924). All of the fibronectin mRNAs from prechondrogenic chick limb mesenchyme contain exons IIIB, IIIA, and V (B + A + V +), whereas all of the fibronectin mRNAs from chick cartilage contain exons IIIB and V but do not contain exon IIIA (B + A − V +). In this study, we show that fibronectin mRNAs containing exon IIIA (FN‐A) and/or the mRNAs containing exon IIIB (FN‐B) are expressed in a specific and different spatiotemporal manner in the developing chick limb in vivo, as well as in limb mesenchymal cells undergoing chondrogenesis in vitro. Specifically, in situ hybridization reveals that FN‐B mRNAs are present throughout the various stages (HH 20–30) of limb cartilage development in vivo, whereas FN‐A mRNAs disappear following the condensation phase of chondrogenesis and are absent from the resulting cartilage. Chick limb cartilage fibronectin mRNAs are therefore B + A −, as in other embryonic cartilage tissues. Furthermore, limb mesenchymal cells undergoing chondrogenesis in vitro lose FN‐A mRNAs immediately following condensation, recapitulating the events that occur during chondrogenesis in vivo. These results suggest an important role for fibronectin mRNA alternative splicing during chondrogenic differentiation. © 1996 Wiley‐Liss, Inc.