Lectin Binding Sites in Developing Mouse Limb Buds

The binding sites of the following biotinylated lectins were demonstrated in serial paraffin sections of fore- and hindlimb buds from day-9 to day-16 mouse embryos with the Avidin-Biotin-Peroxidase Complex (ABC) procedure: Concanavalin A (Con A), Soybean Agglutinin (SBA), Wheat Germ Agglutinin (WGA), Peanut Agglutinin (PNA), Ricinus Communis Agglutinin I (RCA), Ulex Europaeus I Agglutinin (UEA), and Dolichos Biflorus Agglutinin (DBA). Alternating neighbouring sections were used to compare the distribution of PNA staining, PNA staining after neuraminidase treatment (N-PNA) and the autoradiographic sites of [35S]-sulphate uptake. Unspecific binding sites common to all lectins tested were observed in periderm and chondrocytes. Several lectin affinities were seen in the undifferentiated mesoderm (Con A, WGA, RCA), blood vessels (WGA, PNA, N-PNA, RCA, UEA, DBA) and macrophages (Con A, WGA, N-PNA, RCA). A very selective and mainly extracellular affinity to N-PNA was demonstrated in the condensed preskeletal mesoderm, where it characterizes indistinct prospective chondrogenic, perichondral and pre-articular areas. Comparison with the distribution pattern of [35S]-sulphate uptake and other previously published histochemical data suggests that N-PNA staining occurs at the late blastema stage, i.e. after the stage of cell condensation and before the earliest deposit of stainable matrix in chondrogenic areas. This property later disappears from the chondrifying rudiments, and is maintained in perichondral and pre-articular tissues. Surprisingly, only the pre-articular areas bind PNA without pretreatment with neuraminidase. A transient RCA binding probably related to terminal morphogenesis was detected in the undifferentiated distal part of the predigital columns of day-12 and day-13 limb buds. From the day-13 stage onwards, diverse new lectin affinities appeared in differentiating tissues, such as pretendinous rudiments, perichondrium and prospective periosteum, muscular connective tissue, myotubes, superficial fasciae and prospective dermis. A strong SBA and PNA staining was also detected in the extracellular matrix associated with the epithelial septa separating the roots of the digits in day-15 and day-16 limb buds.