Further delineation of mechanisms by which 6-aminonicotinamide (6-AN) induces micromelia in the chick embryo was investigated by studies on rates of incorporation of thymidine, proline, glucosamine and sulfate as precursors to DNA, collagen and mucopolysaccharide, respectively. Twenty-four hours after in ovo administration of the vitamin antagonist, 6-AN, to day-4 chick embryos, hind limbs from experimental and control groups were excised and incubated for 1 h in medium containing 3 × 10−6m radioactive precursor. Molar incorporation of precursors into the TCA-precipitable fraction showed, in isolated limb buds, (a) that 6-AN enhanced incorporation of thymidine, (b) that 6-AN inhibited utilization of sulfate, and (c) that 6-AN did not significantly alter utilization of glucosamine and proline.
Rates of incorporation of thymidine, glucosamine and proline indicate that 6-AN is not cytotoxic to the isolated limb bud. Enhanced incorporation of thymidine suggests expression of compensatory change 24 h after initial effects of 6-AN on DNA synthesis. Rate of incorporation of proline suggests that, under the influence of 6-AN, tropocollagen was synthesized in normal quantities by limb cells. Similarly, rate of incorporation of glucosamine suggests that under the influence of 6-AN normal amounts of hexosamine sugars were being attached to the nascent core-protein of chondroitin. Inhibition of sulfation and failure to complete the chondroitin sulfate molecule seem to account for 6-AN-induced micromelia. This suggests that sulfation depends upon specific NAD-dependent dehydrogenase reactions. As far as can be established by rates of incorporation of labeled precursors, 5-day limb buds, at 24 h after exposure to teratogenic levels of 6-AN, synthesize matrix proteins and hexosamine polysaccharides at normal rates.
The combination of limb-bud removal and in ovo electroporation techniques: A new powerful method to study gene function in motoneurons undergoing lesion-induced cell death