The spatial and temporal distribution of polarizing activity in the flank of the pre-limb-bud stages in the chick embryo

The presence of polarizing activity in the limb buds of developing avian embryos determines the pattern of the anteroposterior axis of the limbs in the adult. Maps of the spatial distribution and the strength of the signal within limb buds of different stages are well documented. Polarizing activity can also be found in Hensen's node in the early embryo. We have mapped the distribution of polarizing activity as it emerges from Hensen's node and spreads into the flank tissue of the embryo. There is a clear change in the local pattern of expression of polarizing activity between stage 8 and 18. Almost no activity is measured for stages 8 and 9. More or less uniform levels of around 10% are spread along the flank lateral to the unsegmented somitic mesoderm from somite position 12 to 22 in stage 10 embryos. Some 6 to 8 h later at stage 12, there is a distinct peak of activity at somite position 18, the middle of the wing field. This peak increases at stages 13 to 15 and its position traverses to the posterior edge of the wing field. Full strength of activity is reached shortly before the onset of limb bud formation at stage 16 to 17. Stages 16 to 18 were investigated for polarizing activity in the wing and the leg field. Low levels of polarizing activity are present in the anterior leg field at stages 16 and 17 but have disappeared by stage 18 and all activity is confined to the posterior part of the leg bud.     

The development of handed asymmetry in aggregation chimeras of situs inversus mutant and wild-type mouse embryo

Mutant iv/iv mice develop as if they have no sense of left and right, so the development of asymmetry is random: half normal, half as a mirror-image of normal, situs inversus. We have made aggregation chimeras of 8-cell stage iv/iv and +/+ embryos, transferred them into pseudopregnant mice, and examined their phenotype on day 10 of gestation. The contribution of mutant and wild-type cells to tissues of the embryo was estimated by strain-specific isozyme (GPI-1) analysis. We have also performed reciprocal embryo transfers, iv/iv blastocysts into +/+ mice, and vice versa. These transfers show that the development of handed asymmetry is determined by embryonic genotype, and is unaffected by the maternal environment (at least after day 3), or by the procedures of embryo collection, culture and transfer. Our observations on the development of 21 viable chimeric embryos show that neither iv/iv nor +/+ cells are dominant. All embryos (12) with less than 50% contribution of iv/iv cells to the heart developed with normal situs. Of 9 embryos with greater than 50% iv/iv cells, only 2 developed with inverted situs. These findings suggests that there was partial 'rescue' of embryos by some influence of normal over mutant cells. However, we cannot, statistically, exclude an alternative interpretation that cells are behaving autonomously. Interestingly, the embryos that developed with inverted situs were unique in having greater than two thirds contribution of iv/iv cells to both the heart and the visceral yolk-sac.     

Hydroxamate assay quantitation of ligands covalently bound to affinity chromatography gels

Affinity chromatography which utilizes specific biological interactions in the purification or analysis of a variety of biochemical systems is an exceedingly useful method. The technique was initially limited to the use of immunoadsorbants (1) and since has been broadened in scope largely by the efforts of Cuatrecases, Anfinsen and colleagues (2,3). In principle, a specific ligand interacting with a particular substance, usually a macromolecule, is covalently bound to an insoluble support. Substances with no affinity for the ligand will pass unretarded through a column of the bound support; whereas, the interacting materials will be retarded. The methods for preparing a variety of affinity-chromatographic systems are now readily available (2,3). However, a quantitative measure of the covalently bound ligand in many instances depends on the use of radioactively labelled ligands, access to an automatic amino acid analyser, or the subtraction of the amount of ligand recovered in washings after the binding reaction. The latter method is often inaccurate and the former methods may not be practical for all laboratories.We have employed successfully the hydroxamate assay (4,5) for measuring biotin linked through an amide bond to the ω amino group of a 3,3′-diaminodipropylamine substituted agarose gel. This method with individual modifications should be of general use in measuring ligands bound to solid supports through amide, ester, or thioester bonds.