Cellular expression of a mutant gene (cmd/cmd) causing limb and other defects in mouse embryos

The cmd mutation is associated with a systemic defect in all parts of the cartilaginous skeleton. Since limb buds isolated from the embryos and cultured in vitro develop the defect, the gene expression is independent of the humoral factors in the maternal/placental/embryonic environment. The limb development in the mutant progresses normally through early events in morphogenesis including the formation of mesenchymal cell condensations preparatory to chondrogenesis. The deviation first becomes apparent as soon as the chondrogenic cells begin the process of accumulation of extracellular matrix; cmd/cmd chondrogenic cells remain close to each other and lack the abundant extracellular matrix which accumulates between normal cells. Quantitatively normal levels of chondroitin sulfate proteoglycans are synthesized by the mutant limbs during precartilaginous stages. Subsequently, however, the mutant fails to attain the normally high levels of chondroitin sulfate synthesis. Its growth rate also slows down, as judged by the lowered protein synthesis in the mutant cultured limb buds. The lack of at least one species of protein molecules, ie, proteoglycan core-protein, is already known from previous studies on this mutation; an abnormal or a deficient core-protein was shown to lead to a virtually complete shut off of the biosynthesis of cartilage-specific proteoglycans in another mutation in the chick embryo [Goetinck, 1982]. It may be important to note that the cmd mutation does not seem to interfere with the process of determination of cartilage even though it interrupts virtually completely one important biosynthetic aspect of the chondrogenic cell differentiation pathway. The mutant chondrocytes, embedded as they are in an abnormal and proteoglycan-deficient matrix, begin to degenerate prematurely without first undergoing hypertrophy. Also, the process of ossification begins precociously in the shortened cartilage models of the mutant, hence resulting in overall shortening of the limbs. As assessed from the HA:s-GAG ratios during early embryonic limb development, some of the phenotypically normal embryos could be distinguished as recessive carriers of the mutation. Even though these carriers have an intermediate level of chondroitin sulfate proteoglycan synthesis, this does not interfere with their normal development during prenatal stages. It will be of practical importance to follow these carriers through subsequent postnatal stages and adult life to assess any long-term effects.