EXPRESSION OF RETINOL-BINDING PROTEIN AND TRANSTHYRETIN DURING EARLY EMBRYOGENESIS

Previous studies have shown that anterior lateral plate endoderm from stage 6 chicken embryos is necessary and sufficient to enable precardi ac mesoderm to complete its cardiogenic program in vitro, culminating in a rhythmically contractile multicellular vesicle (Sugi and Lough [1 994] Dev. Dyn. 200:155-162). To identify cardiogenic factors, we have begun to characterize proteins that are secreted by endoderm cell expl ants, Fluorography of proteins from endoderm-conditioned medium reveal ed 1-2 dozen bands, the most prominent of which migrated at approximat ely 17 and 25 MD, The bulk of the 17-kD band, which migrates near FGFs and subunits of the transforming growth factor-beta family, was ident ified by N-terminal sequencing as transthyretin (TTR), A component of the 25-kD band was identified by Western blotting as retinol binding p rotein (RBP), RT/PCR analysis revealed that mRNAs for both proteins ar e in the embryo as early as stage 3, In situ hybridization localized t hese mRNAs to the extraembryonic endoderm at stage 6, after which they were detected in endoderm overlying the embryo proper, including the developing heart. Later, REP and TTR mRNA and protein were detected in cells associated with the developing heart. Western blotting of whole embryo proteins revealed the presence of REP by stage 7, followed by sequential increases to stage 25; by contrast, content of REP in isola ted hearts peaked at stage 14, then declined. Immunohistochemistry rev ealed the presence of REP protein in the extracellular matrix subjacen t to lateral plate endoderm beginning at stage 8; upon formation of th e definitive heart, intense staining was observed in the cardiac ''jel ly,'' By contrast TTR was intracellular, first detected as subtle depo sits in stage 6 embryonic endoderm, which by stage 8 were prominent in the dorsally invaginated endoderm subjacent to the precardiac splanch nic mesoderm, At stages 11-14, TTR was detected only in myocardial cel ls. Such localization of REP and TTR may indicate a role in the transp ort and distribution of retinol and thyroid hormone, respectively, fro m yolk to embryo prior to establishment of the circulatory system, and is suggestive of a subsequent role in heart development. Dev. Dyn. 19 98;212:413-422. (C) 1998 Wiley-Liss,Inc.