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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. 相似文献
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The phylogeny of Greya Busck (Lepidoptera: Prodoxidae) was inferred from
nucleotide sequence variation across a 765-bp region in the cytochrome
oxidase I and II genes of the mitochondrial genome. Most parsimonious
relationships of 25 haplotypes from 16 Greya species and two outgroup
genera (Tetragma and Prodoxus) showed substantial congruence with the
species relationships indicated by morphological variation. Differences
between mitochondrial and morphological trees were found primarily in the
positions of two species, G. variabilis and G. pectinifera, and in the
branching order of the three major species groups in the genus. Conflicts
between the data sets were examined by comparing levels of homoplasy in
characters supporting alternative hypotheses. The phylogeny of Greya
species suggests that host-plant association at the family level and larval
feeding mode are conservative characters. Transition/transversion ratios
estimated by reconstruction of nucleotide substitutions on the phylogeny
had a range of 2.0-9.3, when different subsets of the phylogeny were used.
The decline of this ratio with the increase in maximum sequence divergence
among taxa indicates that transitions are masked by transversions along
deeper internodes or long branches of the phylogeny. Among transitions,
substitutions of A-->G and T-->C outnumbered their reciprocal
substitutions by 2-6 times, presumably because of the approximately 4:1
(77%) A+T-bias in nucleotide base composition. Of all transversions,
73%-80% were A<-->T substitutions, 85% of which occurred at third
positions of codons; these estimates did not decrease with an increase in
maximum sequence divergence of taxa included in the analysis. The high
frequency of A<-->T substitutions is either a reflection or an
explanation of the 92% A+T bias at third codon positions.
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