Mapping of neural crest pathways in Xenopus laevis using inter- and intra-specific cell markers.

This study examines the pathways of migration followed by neural crest cells in Xenopus embryos using two recently described cell marking techniques. The first is an interspecific chimera created by grafting Xenopus borealis cells into Xenopus laevis hosts. The cells of these closely related species can be distinguished by their nuclear dimorphism. The second type of marker is created by microinjection of lysinated dextrans into fertilized eggs which can then be used for intraspecific grafting. These recently developed fluorescent dyes are fixable and identifiable in both living and fixed embryos. After grafting labeled donor neural tubes into unlabeled host embryos, the distribution of neural crest cells at various stages after grafting was used to define the pathways of neural crest migration. To control for possible grafting artifacts, fluorescent lysinated dextran was injected into a single blastomere which gives rise to a large number of neural crest cells, thereby labeling the neural crest without grafting. By all three techniques, Xenopus neural crest cells were observed along two predominant pathways in the trunk. The majority of neural crest cells were observed along a "ventral" route, between the neural tube and somite, the notochord and somite, and along the dorsal mesentery. A second group of neural crest cells was observed "dorsally" where they populated the dorsal fin. A third minor "lateral" pathway was observed primarily in borealis/laevis chimerae and in blastomere-injected embryos; some neural crest cells were observed underneath the ectoderm lateral to the neural tube. Along the rostrocaudal axis, neural crest cells were not continuously distributed but were primarily located across from the caudal two-thirds of the somite. Fewer than 3% of the neural crest cells were observed across from the rostral third of each somite. When grafted to ventral locations, neural crest cells were not able to migrate dorsally but migrated laterally along the dorsal mesentery. Labeled neural crest cells gave rise to cells of the spinal, sympathetic, and enteric ganglia as well as to adrenal chromaffin cells, Schwann cells, pigment cells, mesenchymal cells of the dorsal fin, and some cells in the integuments and in the region of the pronephros. These results show that the neural crest migratory pathways in Xenopus differ from those in the avian embryo. In avians NC cells migrate as a closely associated sheet of cells while in Xenopus they migrate as individual cells. Both species exhibit a metamerism in the neural crest cell distribution pattern along the rostrocaudal axis.(ABSTRACT TRUNCATED AT 400 WORDS)

Distribution of a putative cell surface receptor for fibronectin and laminin in the avian embryo.

The cell substratum attachment (CSAT) antibody recognizes a 140-kD cell surface receptor complex involved in adhesion to fibronectin (FN) and laminin (LM) (Horwitz, A., K. Duggan, R. Greggs, C. Decker, and C. Buck, 1985, J. Cell Biol., 101:2134-2144). Here, we describe the distribution of the CSAT antigen along with FN and LM in the early avian embryo. At the light microscopic level, the staining patterns for the CSAT receptor and the extracellular matrix molecules to which it binds were largely codistributed. The CSAT antigen was observed on numerous tissues during gastrulation, neurulation, and neural crest migration: for example, the surface of neural crest cells and the basal surface of epithelial tissues such as the ectoderm, neural tube, notochord, and dermomyotome. FN and LM immunoreactivity was observed in the basement membranes surrounding many of these epithelial tissues, as well as around the otic and optic vesicles. In addition, the pathways followed by cranial neural crest cells were lined with FN and LM. In the trunk region, FN and LM were observed surrounding a subpopulation of neural crest cells. However, neither molecule exhibited the selective distribution pattern necessary for a guiding role in trunk neural crest migration. The levels of CSAT, FN, and LM are dynamic in the embryo, perhaps reflecting that the balance of surface-substratum adhesions contributes to initiation, migration, and localization of some neural crest cell populations.

Developmental mutants isolated from wild-caught Xenopus laevis by gynogenesis and inbreeding.

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.

Observations on early and late post-sporozoite tissue stages in primate malaria. II. The hypnozoite of Plasmodium cynomolgi bastianellii from 3 to 105 days after infection, and detection of 36- to 40-hour pre-erythrocytic forms.

Confirmation of the existence of a persistent, uninucleate, dormant pre-erythrocytic stage, the hypnozoite, of the relapsing simian malaria parasite, Plasmodium cynomolgi bastianellii, has been obtained by means of experiments involving the intravenous injection into susceptible monkeys of 48 to 85 x 10(6) sporozoites derived from mosquitoes of a different species and source than employed previously. The development of these hypnozoites was traced from 3 days until 105 days after sporozoite inoculation, employing a sensitive immunofluorescence technique followed by restaining with Giemsa. From an average mean diameter of 4 micrometers at 3 and 5 days, uninucleate hypnozoites grow to 5 micrometers at 7 days, then persist with little change until at least 105 days after infection. Strong evidence for the viability of these persistent forms was obtained by treatment of a host monkey with primaquine, which eliminated all trace of hypnozoites present 2 weeks before. Examination of hepatic tissue from a monkey injected with sporozoites 36 and 40 hours earlier revealed rare uninucleate pre-erythrocytic forms of 2.5-micrometers diameter. These early forms were present in hepatocytes in a density only approximately 1/30th of that expected on the basis of numbers of pre-erythrocytic stages found in the same animal's liver 7 days after infection. Nevertheless, subinoculation experiments appeared to rule out the circulation as a vehicle for dissemination of any putative early intermediate hepatotropic forms from another site.

Observations on early and late post-sporozoite tissue stages in primate malaria. I. Discovery of a new latent form of Plasmodium cynomolgi (the hypnozoite), and failure to detect hepatic forms within the first 24 hours after infection.

Previous work in this laboratory has demonstrated the ability of the immunofluorescence technique to detect pre-erythrocytic stages of the primate malaria parasite, Plasmodium cynomolgi bastianellii, in hepatic tissue obtained as early as 48 hours after sporozoite inoculation. In an attempt to visualize still earlier post-sporozoite stages, hepatic tissue obtained from a rhesus monkey infected with 12,000,000 sporozoites was examined at 2, 12, 24, and 48 hours after inoculation, employing antisera reactive with both invertebrate and vertebrate stages of the parasite. Tissue was also obtained at 7, 50, 102, and 105 days after sporozoite inoculation, and was examined for adequacy of the hepatic infection and for the presence of late exoerythrocytic schizonts. Although a new, previously unrecognized, uninucleate latent stage of 5 micrometer diameter (the "hypnozoite") was detected among large maturing schizonts in the 7-day and later biopsies, no intrahepatic parasites were found in tissue taken at 24 hours or earlier, despite the presence of up to 61 7-day schizonts and eight hypnozoites per 5 X 8 mm section. Pre-erythrocytic forms again were detected at 48 hours, although in far smaller numbers than expected on the basis of the density of parasites at 7 days after infection. The significance of these observations is discussed in the context of previous negative findings.

The 48-hour exoerythrocytic stage of Plasmodium cynomolgi bastianellii.

Detection and specific identification of the 48-hour exoerythrocytic stage of the primate malaria parasite, Plasmodium cynomolgi bastianellii, was accomplished by means of a highly specific indirect immunofluorescence technique (IFA) applied to hepatic tissue fixed in Carnoy's solution. The 48-hour forms appeared as round-to-slightly-oval bodies of average mean diameter 3.0 micrometers (9 parasites) and lying with the cytoplasm of individual hepatic parenchymal cells; each possessed one to three non-fluorescent nuclei or nuclear sections (mean 1.6) within the brightly fluorescent parasitic cytoplasm. In contrast, 72-hour parasites (6) had an average mean diameter of 4.0 micrometers and a mean of 2.2 nuclei. Restaining of IFA preparations with the Giemsa-colophonium method confirmed the plasmodial nature of fluorescent forms, despite some modification of staining characteristics produced by the prolonged exposure of sections to the aqueous phase of the IFA procedure. Exoerythrocytic forms could not be detected in biopsies obtained 24 hours following sporozoite inoculation.

Chromosome replication in Myxococcus xanthus.

The rates of DNA synthesis during the cell-division cycle were measured in Myxococcus xanthus growing in three different media permitting a twofold variation in doubling time. In all three media, simple DNA cycles were observed. Synthesis of DNA occurred during 85% of the cell-division cycle, independent of generation time, from 5 to 11 h. Cells were observed to contain one bacterial nucleoid at birth that later divided synchronously midway through the cell cycle. Nucleoid segregation appeared to begin before chromosome replication was completed. The DNA content of exponential-phase bacteria was determined to be about 20 +/- 3 X 10(-9) microgram per cell; newborn bacteria contained about 14 +/- 2 X 10(-9) microgram of DNA per cell. Exponential-phase bacteria showed about a 50% increase in DNA in the presence of chloramphenicol (50 microgram/ml). The number of randomly segregating chromosomes present in exponential-phase bacteria was determined by following the fate of prelabeled DNA during outgrowth in nonradioactive media. The results are consistent with a model in which cells are born with exactly one complete unreplicated chromosome. The molecular weight of such a chromosome is about 8.4 +/- 1.2 X 10(9).

Cell division in Escherichia coli: analysis of double mutants for filamentation and abnormal septation of deoxyribonucleic acid-less cells.

The link between chromosome termination, initiation of cell division, and choice of division sites was studied in Escherichia coli by preparing double mutants. Hybrid mutants containing div52-ts, a cell division initiation mutation, and min, mutations which affect the choice of division sites resulting in the septation of minicells, were characterized. The mutants produced minicells and normal cells coordinately under all conditions studied, although the fraction of minicells is half that of the parental minicell strain. The mutant gradually stopped dividing at both the median and minicell septation sites when transferred from 30 to 41 C in rich medium. A synchronous cell division of filaments was induced 15 min after addition of chloramphenicol to the medium, even at 41 C. Divisions were observed at both normal and minicell sites. These results indicate that div52-ts and min functions share a common step in a cell division pathway. A double mutant containing div52-ts and div27-ts, a dnaB mutant which divides in the absence of DNA synthesis, was characterized. The mutant continues to divide after a shift to the high temperature, although at a reduced rate. The behavior of this hybrid mutant suggests a hypothesis that the chromosome termination signal and div52-ts division initiation signal act on a single membrane site which is altered in div27-ts strains.