Studies on the differentiation of cranio-visceral cartilage in normal and premature death mutant embryos of Ambystoma mexicanum.

Abstract

The self-differentiative ability of the cranial neural crest cells in the Mexican axolotl, Ambystoma mexicanum was tested at various developmental stages. The processes through which cartilage determination occurs are far from complete; only a very small number of these cultures contained cartilage, although all appeared healthy and contained melanocytes and abundant mesenchyme. At all of these stages, pharyngeal endoderm is the only inductor required for cranial neural crest cells to differentiate into cartilage. Over 90% of cultures containing neural crest and pharyngeal endoderm produced mature cartilage nodules. The time course for chondrogenesis in vitro is similar to that which occurs in situ, which suggests that the culture system provides an accurate assessment of what occurs in vivo. Only those cells which normally contribute to skeletal elements have the capacity to do so. Neither trunk neural crest cells nor the cells of the transverse (anterior-most) neural fold can produce cartilage, even when placed in intimate contact with inductive tissues. Similarly, inductive endoderm is only found in the head, which is the origin of, and ultimate location of, all chondrogenic cells of neural crest origin. A sharp boundary, which occurs at the junction of the pharyngeal wall and the pharyngeal floor, divides the inductive from the non-inductive endoderm. All anterior endoderm is capable of inducing mature cartilage formation in virtually all cases, whereas cartilage formation occurs only rarely in the presence of posterior endoderm. Axolotl embryos which are homozygous for the premature death (p) gene do not develop past stage 37, which is well before cartilage differentiation usually occurs. They also display a variety of abnormalities in the gills, heart, pharynx, and liver, among others. Based on these and other observations, it was suspected that the mutation affected the endoderm. When mutant tissues were tested for the ability to form cartilage, however, the neural crest components of the culture, and not the pharyngeal endoderm, was found to be defective. The lack of cartilage is not caused by an abnormal localization of chondrogenic neural crest, nor does the migration of the cranial neural crest appear to be affected. The normality of the chordamesoderm and the ectoderm of mutant gastrulae was tested through the production of secondary neural structures. These studies determined that the chordamesoderm, which is the initiator of the events leading to neural crest specification, is normal in the mutant; the defect is in the actoderm of mutant embryos. The hypothesis that a number of neural crest functions are defective in the mutant appeared to be supported by the results of rescue attempts involving the transplantation of wild-type neural folds, and by the appearance of wild-type embryos which developed without neural crest cells. Thus, it would appear that the premature death mutation affects a subset of neural crest derivatives, and could therefore prove useful in the study of both the specification and differentiation of neural crest cells. (Abstract shortened by UMI.)