Identification of transferrin as one of multiple EDTA‐extractable extracellular proteins involved in early chick heart morphogenesis

Abstract

It was demonstrated previously that a polyclonal antibody (ES1) raised against EDTA extractable proteins from embryonic chicken heart blocks cardiac endothelial-mesenchymal transformation in a culture bioassay and stains extracellular matrix at sites of embryonic inductive interactions, e.g., developing heart, limb buds, and neural crest forming region [Krug et al., 1987, Dev Biol 120:348-355; Mjaatvedt et al., 1991, Dev Biol 145:219-230). In the present study, by using an antiserum (ES3) to a similar immunogen, we affinity purified four major EDTA-soluble proteins. These proteins migrated as 27, 44, 63, and 70 kD molecules under reduced conditions and 27, 41, 52, and 59 kD under nonreduced conditions, respectively, on SDS-PAGE. Based on several criteria, the protein migrating at 70/59 kD (reduced/nonreduced) was indistinguishable from chicken transferrin (conalbumin): 1) amino acid sequencing showed that eight N-terminal residues were identical to those of chicken transferrin, 2) acid hydrolysates of both proteins had nearly identical compositions, 3) the protein co-migrated exactly with chicken transferrin under both reduced and nonreduced conditions, and 4) ES3 IgG recognized both the 70/59 kD protein and chicken transferrin by western blot analysis of nonreduced samples, but not with reduced samples. Immunohistochemistry of chicken embryonic heart with antibodies against transferrin demonstrated that anti-transferrin immunoreactivity is present in myocardium but absent in cardiac endothelium before the initiation of cardiac endothelial-mesenchymal formation. However, both cardiac endothelium and migrating mesenchymal cells became immunoreactive with anti-transferrin at the time transformation occurred. These findings suggest a possible involvement of transferrin in the inductive process of cardiac endothelial-mesenchymal transformation.

Late Deposition of Elastin to Vertical Microfibrillar Fibers in the Presumptive Dermis of the Chick Embryonic Tarsometatarsus

“…1) in ED8-17 were excised from the aldehyde-fixed embryos and used to prepare frozen sections and conventional paraffin sections. Specimens for frozen sections were cryoprotected as described previously (Isokawa et al, 1994). Some embryos at ED13 and 17 were fixed with 4% PA containing 0.5% glutaraldehyde in PBS for 2 hr at 48C and processed for immunoelectron microscopy.…”

Development of the Tarsometatarsal Skeleton by the Lateral Fusion of Three Cylindrical Periosteal Bones in the Chick Embryo (Gallus gallus)

“…Specimens of PFA-fixed TMT at ED7 and 8 were cryoprotected, embedded in OCT Compound (Tissue-Tek TM ; Miles) and frozen in liquid nitrogen-cooled 2-methyl butane (Isokawa et al, 1994). PFA-fixed specimens at ED10, 13, and 17 were embedded in 5% carboxymethyl cellulose (CMC), according to the procedure by Kawamoto and Shimizu (2000).…”

Subectodermal microfibrillar bundles are organized into a distinct parallel array in the developing chick limb bud

“…Zygopodial cartilage was dissected from the chicken leg bud at stages 34 -36 and homogenized at 4°C in PBS (pH 7.4) containing a cocktail of protease inhibitors (Isokawa et al, 1994). The supernatant clarified by centrifugation at 14,000 g was emulsified with an equal volume of Freund's complete (first injection) and incomplete (subsequent injections) adjuvant.…”

“…In some cases, the residual supernatant was reused for one or more cycles of immunoprecipitation. The precipitates thus obtained were washed five times (3 min each) in 500 l of TNE, solubilized in a SDS-containing sample buffer (63 mM Tris-HCl, pH 6.8, 2% SDS, 6 M urea, 60 mM dithiothreitol), and resolved by SDS-PAGE as described previously (Isokawa et al, 1994). The gel was fixed in 25% isopropanol/65% acetate for 20 min, incubated in Amplify™ fluorographic reagent (Amersham) for 15 min, dried, and then exposed to Kodak X-OMAT film at -80°C.…”