IDENTIFICATION OF THE DEVELOPMENTAL MARKER, JB3-ANTIGEN, AS FIBRILLIN-2 AND ITS DE-NOVO ORGANIZATION INTO EMBRYONIC MICROFIBROUS ARRAYS

The monoclonal antibody JB3 was previously shown to react with a prote in antigen present in the bilateral primitive heart-forming regions an d septation-stage embryonic hearts; in addition, primary axial structu res at primitive streak stages are JB3-immunopositive (Wunsch et al. [ 1994] Dev. Biol. 165:585-601). The JB3 antigen has an overlapping dist ribution pattern with fibrillin-1, and a similar molecular mass (Galla gher et al. [1993] Dev. Dyn. 196:70-78; Wunsch et al. [1994] Dev. Biol . 165:585-601). Here we present immunoblot and immunoprecipitation dat a showing that the JB3 antigen is secreted into tissue culture medium by day 10 chicken embryonic fibroblasts, from which it can be harveste d using JB3-immunoaffinity chromatography. A single polypeptide (M-r = 350,000), which was not immunoreactive with an antibody to fibrillin- 1, eluted from the affinity column. Mass spectroscopy peptide microseq uencing determined the identity of the JB3 antigen to be an avian homo logue of fibrillin-2. Live, whole-mounted, quail embryos were immunola beled using a novel microinjection approach, and subsequently fixed. L aser scanning confocal microscopy indicated an elaborate scaffold of f ibrillin-2 filaments encasing formed somites. At more caudal axial pos itions, discrete, punctate foci of immunofluorescent fibrillin-2 were observed; this pattern corresponded to the position of segmental plate mesoderm. Between segmental plate mesoderm and fully-formed somites, progressively longer filamentous assemblies of fibrillin-2 were observ ed, suggesting a developmental progression of fibrillin-2 fibril assem bly across the somite-forming region of avian embryos. Extensive filam ents of fibrillin-2 connect somites to the notochord. Similarly, fibri llin-2 connects the mesoderm associated with the anterior intestinal p ortal to the midline. Thus, fibrillin-2 fibrils are organized by a div erse group of cells of mesodermal or mesodermally derived mesenchymal origin. Fibrillin-2 microfilaments are assembled in a temporal and spa tial pattern that is coincident with cranial-to-caudal segmentation, a nd regression of the anterior intestinal portal. Fibrillin-2 may funct ion to impart physical stability to embryonic tissues during morphogen esis of the basic vertebrate body plan. Dev. Dyn. 1998;222:461-471. (C ) 1998 Wiley-Liss,Inc.