The morphological development of retinal ganglion cells was examined in a tetraploid strain of Xenopus frogs. The enlarged cells of the tetraploid strain facilitate the application of intracellular techniques. Using an in vitro retinal preparation and Nomarski optics, intracellular recording and dye injection were carried out under visual control on ganglion cells in central retina from 2 days of development (stage 24) to metamorphosis (stage 64). We identified three phases in the morphological differentiation of ganglion cells. During the first phase (stages 24-30), all cells were neuroepitheliallike in form and possessed robust resting potentials in the range of -35 to -60 mV, and dye-coupling was occasionally observed between neighboring cells. During the second phase of ganglion cell development (stages 31-45) the neurons had begun to elaborate axons and dendrites. These cells possessing neurites had resting potentials between -15 and -30 mV, and no dye-coupling was observed between neighbors. During the third and final phase of maturation, from stage 46 onward, three distinct morphological types of ganglion cells could be identified. Type I cells had the smallest somata and the smallest-diameter dendritic arborizations. The profusely branched dendrites of these cells ramify extensively throughout the inner plexiform layer. Type II cells had large somata, intermediate-diameter dendritic fields, and a highly elaborate dendritic branching pattern. These cells were seen to arborize within two sublamina in the inner plexiform layer. Type III cells had large somata, the largest-diameter dendritic fields, and a dendritic arbor with long primary branches but little higher-order branching. These large dendritic fields were confined to a single sublamina of the inner plexiform layer, abutting the inner nuclear layer. While most phase 3 cells showed radial axon trajectories from the soma to the optic disc, a minority of cells (1-5%) with erratic and nonradial axon trajectories were also observed. Our data provide a morphological description of ganglion cell maturation in the central retina of Xenopus. We show that very early in development (as early as stage 46) three distinct morphological types of retinal ganglion cells are present, which correspond to the three classes of ganglion cells previously described in adult Xenopus (Chung et al., '75).

Xenopus laevis obtained from indigenous African populations are a rich source of mutants affecting development. Gynogenesis and inbreeding were used to isolate mutants affecting development from wild-caught Xenopus laevis females. Fourteen mutants were recovered from eight females tested. One mutant was recovered from each of two females. This load of 1.875 developmental mutants per female is similar to that found in the axolotl (Ambystoma mexicanum), a urodele amphibian, and is only slightly less than the load of mutants with major developmental effects found in Drosophila and man. These results suggest that the anuran amphibian Xenopus laevis, an ancestrally tetraploid species, has undergone extensive diploidization of developmentally important loci and that gynogenesis and inbreeding of wild-caught animals can provide adequate mutants at diploid loci for developmental genetic studies.

No abstract

Triploid and gynogenetic diploid Xenopus laevis frogs were produced using pressure to suppress the formation of the second polar body. Five to ten minutes after in vitro insemination, eggs were subjected to six minutes of hydrostatic pressure a t 4,800 pounds per square inch. All of the surviving embryos tested were triploid. Application of the same pressure technique to eggs fertilized with ultraviolet irradiated sperm resulted in gynogenetic diploid embryos in every case where the sperm nucleus was inactivated. Triploid and gynogenetic diploid embryos developed normally through the larval period and metamorphosis.Triploid cells are useful cell markers in grafting experiments since amphibian cells and nuclei vary in size in proportion to the number of haploid sets of chromosomes in the nuclei (Fankhauser, '55). Triploid cells have been distinguished from diploid cells in chimerae by cell size, DNA content (Turpen and Volpe, '741, number of nucleoli (Namenwirth, '74), as well as chromosome counts. In these ways, the fates of cells of surgically defined origin in chimaerae can be followed, for triploid cells are as viable as diploid cells.The formation of the second polar body of amphibian eggs can be suppressed in a variety of ways including heat shock, cold shock, and pressure (Smith, '58; Briggs, '47; Fankhauser, '55; Dasgupta, '62). If normal fertilized eggs are so treated a t an appropriate time, triploid embryos are produced. Triploidy has not been used extensively in studies of Xenopus laeuis probably because the method available (Smith, '58) produces triploidy in only slightly more than half the treated embryos.Gynogenetic diploid amphibians have been produced by suppressing second polar body formation in eggs fertilized with sperm whose nuclei are incapable of participating in subsequent development. (Volpe and Dasgupta, '62). Gynogenetic diploids are of interest in studying sex determination, in screening wild populations of animals for mutant genes and mapping mutant genes relative to the centromeres of the chromosomes which bear them (Volpe and Dasgupta, '62; Lindsley et al., '56).In this report methods for producing triploid and gynogenetic diploidXenopus laeuis are de- scribed which are reliable as judged by chromosome counts and genetic analysis. MATERIALS AND METHODSNormal Xenopus laevis were obtained from Nasco, Fort Atkins, Wisconsin. Periodic albino (aP/aP) Xenopus laevis (Hoperskaya, '75; Tompkins, '77) were generously provided by Doctor A. J. Brothers. Ovulation was induced by the method of Gurdon ('67). Artificial insemination by the method of Wolf and Hedrick ('72) yielded poor results, usually less than 10% fertilization. Therefore, the following method was devised. Males were sacrificed and their testes removed and washed in 10% Ringer's solution a t 0°C. One testis was macerated in 10 mi of ice cold 100% Ringer's solution and this suspension was kept on ice until needed. Good fertilization was obtained up to 12 hours after maceration. Eggs were stripped from ovulating females into...

Morphological aspects of four different groups of Golgi impregnated brain cells from a tetraploid strain of Xenopus laevis frogs were compared to analogous cells in comparably sized diploid frogs. The cells examined included neurons from the telencephalon, caudal hypothalamus, and optic tectum, and radial glial cells from the optic tectum. The brains of tetraploid frogs appeared grossly normal and were the same size and contained similar cell types as diploid brains. As observed in previous studies on polyploid amphibia, somal diameters increased significantly in tetraploid cells for each of the four groups of cells examined. Also, the total length of the dendritic arbors in tetraploid brain cells increased significantly by factors ranging from 1.4 to 2.4 times the total length of the analogous processes in diploid cells. Tetraploid neurons in the telencephalon and hypothalamus increased their arbor lengths predominantly by increasing the number of dendritic branches, while maintaining the average distance between branch points in the dendritic segments. In contrast, the tetraploid large pear-shaped neurons in the optic tectum had significantly longer terminal dendritic segments than the analogous diploid neurons, although these tetraploid neurons maintained their average number of dendritic segments per cell. Tetraploid tectal radial glial cells appeared to increase both their number of branches and the lengths of their terminal segments. Thus, the mode by which tetraploid brain cells achieved longer dendritic arbors varied from cell type to cell type. These results suggest a hypothetical basis for possible effects of genomic size on vertebrate brain structure and evolution at the cellular level.