Limb Bud Tissue Research Articles

Chlorambucil-induced cell death in embryonic mouse limb buds

A high frequency of congenital limb defects after maternal treatment with the nitrogen mustard chlorambucil suggested that cytotoxic effects could be analyzed as correlates of teratogenic action of this alkylating agent. Limb buds from ICR DUB mouse embryos were exposed in vivo or in vitro to teratogenic doses of chlorambucil. At 0.5- to 72-hr intervals following exposure, the limbs were processed for observation with the light and electron microscopes. Light microscopy of limb buds exposed to the drug in vivo or in vitro showed evidence of cellular involvement within 4 hr following treatment. The effects were manifested in the form of cytoplasmic inclusions which increased in number as the time following exposure increased, and, in vivo, reached a maximum at 24 hr. Increased doses of the drug also produced increased numbers of affected cells. In limb buds exposed to chlorambucil in vitro, the number of affected cells reached a plateau at 48 hr after treatment and decreased significantly by 72 hr, at which time exposure to a second dose of the drug had little or no effect. Affected cells were restricted almost exclusively to regions of undifferentiated mesenchymal cells. Electron microscopic examination of the cytoplasm of affected cells demonstrated the presence of membrane-bound vacuoles containing cellular organelles in various stages of degeneration. The number and size of these vacuoles increased with time following exposure until finally the cells fragmented. Nuclear material was not involved until fragmentation of the cells occurred, at which time the nucleus became pyknotic. Following cellular fragmentation and destruction, macrophages were observed in association with the cellular debris. Acid phosphatase was demonstrated within the vacuoles (by the presence of lead phosphate precipitates) with the electron microscope after incubating limb bud tissue in a buffered solution of β-glycerophosphate/lead nitrate. The amount of demonstrable enzyme was found to increase as the degree of breakdown within vacuoles increased. These results and the histological information provided evidence consistent with a process of autophagocytosis.

Enzymatic events during cartilage differentiation in the chick embryonic limb bud

Chondroitin sulfate and an unidentified sulfated mucopolysaccharide insensitive to hyaluronidase are synthesized by the prospective limb region of stage 15–16 chick embryos and stage 17–18 limb buds more than 2 days before the first appearance of metachromatic material. Sulfate-binding mucopolysaccharide material with the same electrophoretic mobility as chondroitin sulfate becomes entirely sensitive to hyaluronidase in stage 19 and older limb buds. The activity of four enzymes presumed to be involved in the pathway of chondroitin sulfate biosynthesis has been demonstrated before histological evidence of cartilage in both cell-free extracts of stage 19 chick limbs and in cultured aggregates of dissociated cells from the same source. The developmental kinetics of three of these enzymes, UDPG dehydrogenase, UDPGNAc-4-epimerase, and the sulfate-activating enzymes, have been examined; the first two enzymes are involved in the synthesis of the polysaccharide backbone of chondroitin sulfate, and the last system is responsible for sulfation of the polysaccharide chain. Although all three enzymes are present in low levels in limb bud tissue before chondrogenic differentiation, the rise in their specific activity is related to the development of cartilage both in vivo and in vitro. The fourth and most specific enzyme catalyzing the formation of a polysaccharide from UDPGNAc and UDPGA has merely been identified in both fresh stage 20 limb buds and aggregates cultured for more than 6 days. The products of the four enzymatic reactions have been identified. The relationship of these results to a theory of biochemical differentiation based upon restriction or enhancement of preexisting metabolic potentialities has been discussed.