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A recessive lethal mutation in axolotls that involves the kidneys, the Mullerian ducts and the limbs was described by Humphrey (1967). In the present experiments, we have examined the structural defects that lead to kidney malfunction and subsequent death in homozygous mutants and compared the defects with those observed in other axolotls lacking this mutant gene. The ultrastructure of the mesonephric kidney was studied in homozygous s/s short-toes axolotls with ascites and/or edema and hemorrhages (group 1a); in s/s short-toes axolotls not yet expressing kidney malfunction symptoms (group 1b); in normal siblings, either +/+ or +/s, without affected limbs but possibly heterozygous for the 's' gene (group 1c); in +/+ animals with malfunctioning kidneys that lack the short-toes gene (group 2a) and in normal +/+ animals from different gene pools (group 2b). In all of the short-toes animals that expressed pathological phenotypes, the proximal tubule showed abnormal morphology. There was no morphological evidence of kidney abnormality in the siblings having normal limbs or in the group 1b axolotls examined. However, mesonephroi from animals (group 2a) of other gene pools that had ascites exhibited a different and more pronounced pathology. On the basis of the dramatically distinct proximal tubule pathology of the sporadic floaters, we conclude that this phenotype is more likely caused by infection than to a variant of the short-toes gene.

In order to examine the role of ependymal cells in the spinal cord regeneration of urodele amphibians, procedures were established to identify and culture these cells. Cell isolation and culture conditions were determined for ependymal cells from larval and adult axolotls (Ambystoma mexicanum). Dissociated cells prepared from intact spinal cords were cultured on fibronectin- or laminin-coated dishes. Dissociated cells attached more rapidly to fibronectin, but attached and spread on both fibronectin and laminin. Essentially pure populations of ependymal cells were obtained by removing 2 week old ependymal outgrowth from lesion sites of adult spinal cords. These ependymal outgrowths attached and grew only on fibronectin-coated dishes. Growth and trophic factors were tested to formulate a medium that would support ependymal cell proliferation. The necessary peptide hormones were PDGF, EGF, and insulin. TGF-beta(1) affected the organization of cell outgrowth. Initially, longterm culture required the presence of high levels of axolotl serum. Addition of purified bovine hemaglobin in the culture medium reduced the serum requirement. Outgrowth from expiants was subcultured by transferring groups of cells. Intrinsic markers were used to identify ependymal cells in culture. The ependymal cells have characteristic ring-shaped nucleoli in both intact axolotl spinal cords and in culture. Indirect immunofluorescence examination of intermediate filaments showed that ependymal cells were glial fibrillary acidic protein (GFAP) negative and vimentin positive in culture. Identification of dividing cells was made using (3)H-thymidine incorporation and autoradiography, and by the presence of mitotic figures in the cultured cells.