Robert Tompkins

Grafting analysis of the periodic albino mutant of Xenopus laevis.

Abstract The differentiation of normal and mutant (a P /a P ) Xenopus laevis melanophores in chimerae was analyzed to determine the tissues affected by this mutation. Normal melanophores in mutant host tissue differentiate in mutant host tissue prior to those of the mutant host. These normal melanophores were initially normal in appearance, but, after the differentiation of the mutant hosts melanophores, they became indistinguishable from their hosts melanophores. These normal melanophores persist in more than normally punctate form after the disappearance of the mutant hosts melanophores in late larval life. Parabiosis and head transplants between mutant and normal embryos did not affect the character of either type of melanophore developing in tissue of its own genotype, indicating that the hormonal control of melanophore differentiation is not affected by the mutation. Therefore, the periodic albino mutant affects the capacity of the mutant melanophore to differentiate and the ability of the mutant skin to support normal melanophore differentiation.

Chapter 3 Experimentally Induced Homozygosity in Xenopus laevis

Publisher Summary This chapter describes experimentally induced homozygosity in Xenopus laevis . Genomic manipulation of amphibians during meiosis or mitosis is accomplished using primarily physical agents such as cold, heat, and pressure. Chemical methods, such as those involving the use of cytochalasin B, are useful in organisms with small eggs that facilitate removal of the agents by washing, however are difficult to control in larger eggs such as those of amphibia. Under the correct conditions the spindle apparatus is arrested or destroyed so that a restitution nucleus containing all sets of chromosomes destined for the meiotic or mitotic products forms in the cell whose division has been aborted. These experimental strategies have typically limited to second meiotic and first mitotic divisions as their precise timing under controlled condition fosters reliable manipulations. Gynogenetic reproduction occurs naturally in several amphibian species, and it is induced in many species by suppression of second meiotic division in eggs fertilized with irradiated sperm or with other sperm cells that do not contribute nuclear material to the zygote. The chapter describes two methods: (1) propagation of primary homozygous diploid animals, and (2) defining animals homozygous at all loci.

Ontogeny of Amphibian Hemopoietic Cells

The developmental origin of lymphoid cells of vertebrates continues to be a fascinating, perplexing problem. Historically, the disputable issues have revolved around two fundamental aspects: the origin of the precursors of the various types of differentiated blood cells and the lineal relationships among these different cell types. There are special features of amphibians that make them suitable for clarifying the embryogenesis of hemopoietic cells. An array of microsurgical techniques from experimental embryology can be used to marked advantage. In this chapter, we will review the contributions made by investigators who have used the tools of experimental embryology to gain insight into the ontogeny of vertebrate blood cells.

Allograft rejection in Xenopus laevis following larval thymectomy

Xenopus laevis thymectomized at stages 41 through 49 accept first set allografts, while animals thymectomized at stage 51 or older reject allografts in times similar to intact animals. However, thymectomy at progressively earlier stages results in a greater proportion of animals unable to reject second set grafts. In some animals, the allograft response remains deficient even after multiple challenges. The results indicate that alloreactive cells are thymus dependent, and suggest that the thymus processes precursor thymocytes starting upon its formation at around stage 41. The processed cells, competent to respond to alloantigens, are released to the periphery almost immediately. While an increasing pool of processed T cells accumulates during stages 41-49, the persistent defective allograft response displayed by animals thymectomized during these stages suggests that early thymectomy may leave a population of alloreactive cells qualitatively defective in some subpopulation necessary for normal allograft responses, or that any residual cells processed prior to thymectomy are capable of only limited clonal expansion.

Tolerance maintenance depends on persistence of the tolerizing antigen: Evidence from transplantation studies on Xenopuslaevis

In order to assess the role of antigen persistence in the tolerant state, tolerance was induced in Xenopus laevis by the embryonic transplantation of whole eyes or tail tissue. Both types of transplants were seen to heal in and persist, with no signs of immunological incompatibility. At metamorphosis, tail resorption occurred and grafted tail tissue was lost. Eye transplants were maintained through metamorphosis in most eye grafted animals. Eye graft recipients which had maintained the transplant were observed to accept challenge skin allografts from donors of the same genotype as the eye donor in all but one case, while recipients which had lost the eye transplant at metamorphosis or had the eye transplant experimentally removed sometimes did not accept the challenge skin graft. Animals tail grafted as embryos did not accept post metamorphic skin grafts from donors of the same genotype as the tail tissue donor, but rejection was not accelerated. It is proposed that tolerance induction is dependent on the presence of appropriately presented antigen at a time when precursor thymocyte cells are migrating to the thymus, prior to their processing into alloreactive cells, and that tolerance maintenance is dependent upon the persistence of the tolerizing antigen.

Colonization of the thymic primordium by migratory lymphocytoblasts in amphibian embryos.

Abstract The concept that thymic lymphocytes originate directly from cells indigenous to the anlage of the thymus in amphibians is clearly indefensible. The lymphocytes of the anuran thymus owe their origin to migratory stem cells that invade the developing thymic primordium.

Mutants and markers for the study of neurogenesis in

Vertebrate mutations contribute to the analysis of cell interactions in nervous system development. In this symposium, Tompkins will describe grafting of cells with new visualizable genetic markers into histocompatible recipients in Xenopus laevis. This allows the fate of cells to be traced with great precision in relation to connectivity pasterns in developing and mature chimeric brains. Crepel uses mutant mice and x-irradiated rats to explore, with physiological and anatomical methods, the developmental relationships between cerebellar climbing fiber inputs and their Purkinje cell targets. He will describe mechanisms involved in the transition from an initial high innervation ratio to the approximately i:i ratio of the adult, with particular ~mphasis on control by the posts>~aptic cell. The second half of the symposium focuses on the shiverer(shi) mutations in the mouse. The shi locus controls myelin basic protein(MBP) and offers a special opportunity for coordinating results obtained by the methods of molecular genetics and developmental biology. Carson will review his evidence for coordinate genetic control of the several mouse low molecular weight MBPs and will indicate status of efforts to clone the shi gene. Wolf will show through confrontation of mutant and wildtype cells in organotypic culture that the shi locus affects myelinatlon by action in the oligodendrological cell and not in the related axon.

The effects of ploidy interactions on cell shape and size in the optic tectum

Solutions of ammoniacal silver carbonate have been used for the selective staining of cell nuclei. We have now shown that embryonic neural cells engaged in DNA synthesis are differentially stained by this procedure. Three classes of nuclei can be recognized in the neural tube of chick embryos (stages 18-22). Neuronal nuclei show dark clumps of chromatin and a very pale matrix. They are identified by their position close to the marginal layer. The neuroepithelium presents dark and pale nuclei. Both types contain dark clumps of chromatin but only the former show a deep brown staining of their matrix. Both the incorporation of[JH] thymidine and the effect of cytosine arabinoside indicate that it is the dark neuroepithelial nuclei that are engaged in DNA synthesis. There is no such correlation between silver staining and DNA synthesis in the surrounding embryonic tissues.

Duplication of positional information in regeneration of eye-bud fragments results from a specific healing pattern

Fragment cultures of embryonic mouse neural tube were used to study sequential changes in neuronal cell differentiation. We e~amined expression of intermediate filaments (vimentin type, VIF; neurofilaments, NF) and related their patterns to the appearance of other phenotypic markers of neuronal development such as presence of tetanus toxin receptors (TI~, migration of cells, formation of Nissl substance, and the degree of neurite formation. Newly formed neurons, bipolar with scant Nissl substance, were close to the fragment (least migration). Tney were qTR-/NF-/VIF +, with VIF in a perinuclear arrangement e~tending into some processes. Neurons in the middle of the outgrowth zone (average migration) were TTR+/NF+-/VIF~-. Tne morphologically most differentiated neurons, large multipolar cells with abundant Nissl substance, appeared furthest from the fragment (extensive migration) and were TPR+/NF ~rfF-. Between days 7 and i0 of culturing neurons at all stages of differentiation were observable within the outgrowth zone. Neuronal migration appeared limited to the first 10-12 days of culture though neurons continued to differentiate throughout the culture period of 21 days. Tnis system thus provides a model by which mechanisms regulating neuronal proliferation and differentiation in vitro may be studied. Supported by MNC of Canada Grant MT 4235.