Cell differentiation in Dictyostelium under submerged conditions.

Abstract

Hitherto it has not been possible to obtain spore and stalk cell differentiation of the cellular slime molds in submerged cultures. It is shown here that cells when elaced in roller tubes under an atmosphere of oxygen, will form clumps and differentiate in 48-72 hr into mature spores and stalk cells. Although differentiation occurs without the normal morphogenetic movements, there is the appearance of an anteroposterior polarity of the cells in the clump. In addition to oxygen we examined a number of other factors that affect differentiation. It has always been assumed that differentiation in the cellular slime molds could not occur under water, for development would invariably stop at aggregation (1), and that an air-water or an oil-water interface was required. The success of an oilwater interface, which was first demonstrated by Potts (2), has been ascribed to-the fact that mineral oil can be saturated with 10 times more oxygen than can water. As will be shown, if oxygen is added to roller tubes containing Dictyostelium discoideum cells suspended in a physiological salt solution, then clumps of cells will form that show both mature spore and stalk cell differentiation. This work was first presented at a meeting in May, 1976 at Cold Spring Harbor; at the same meeting I. Takeuchi reported similar results in roller tubes using different methods. Ultimately, the findings of both these studies will increase our understanding of the mechanism of cell differentiation in the cellular slime molds. MATERIALS AND METHODSAll the experiments reported here were done with Dictyosteltum discoideum NC-4 grown on Escherichia colt B/r on 1% peptone-dextrose buffered agar. The amebae were washed three times by centrifugation at 75 X g in 10-2 M salt solution (1). RESULTSTime course description of differentiation One of the most interesting aspects of this study has been the morphological events that accompany the differentiation of the clumps in the roller tubes. The following description, which is also shown in diagrammatic sequence in Fig. 1

Prolyl 4-hydroxylase-1 mediates O2 signaling during development ofDictyostelium

“…Another function for O 2 in Dictyostelium, cytochrome oxidase subunit VII switching (Sandona et al, 1995), does not involve P4H1 based on DNA microarray studies (L. Eichinger and C.M.W., unpublished). The role of P4H1 in O 2 -dependent slug polarization and guidance of slug migration (Sternfeld and Bonner, 1977;Sternfeld and David, 1981) remains to be examined. Because hypoxic regulation is connected with sporulation, Dictyostelium offers a unique genetic opportunity to investigate the mechanism of P4H1 action, including its role as a direct O 2 sensor and the identification of upstream and downstream regulatory genes.…”

The Skp1 Prolyl Hydroxylase from Dictyostelium Is Related to the Hypoxia-inducible Factor-α Class of Animal Prolyl 4-Hydroxylases

“…Cytochrome oxidase subunit VII is encoded by two genes whose expression is oppositely regulated by O 2 tension (60). Culmination, the final morphogenetic step in sporulation, and terminal cell differentiation also require nearly normal O 2 tension (60,61). The polarity and direction of migrating slugs appear to be influenced by O 2 levels (62).…”

Cell Sorting daring Pattern Formation in Dictyostelium

“…Lack of sufficient oxygen is the principal factor limiting development in clumps of cells in submerged culture [3,4]. If the oxygen concentration is increased, development occurs in submerged cultures and the cells in clumps differentiate into stalk cells and spores.…”

“…While these results indicate that prestalk-prespore interconversion requires a relatively long time compared to the rapid sorting of anterior and anterior-like cells, they do not preclude the existence of more rapid differentiation events. For example, Gregg [37] observed that, within 15-30 min after isolation of the prestalk zone, some cells incorporate 3 An interesting feature of the sorting mechanism is the localization of an extracellular signal -in this case the chemotactic agent -in one region of the slug. Such localization of a signal could be used by the organism to stimulate differentiation of cells in that region without affecting cells elsewhere in the slug.…”

“…However, current information does not permit a choice between models and there is controversy about a central issue, the role of sorting-out. In an attempt to bring new experimental methods to bear on the problem, we have investigated the formation of the prestalk-prespore pattern in slugs and in clumps of cells in a submerged culture System [3]. To identify the pattern we have used vital dyes which stain prestalk cells but do not stain prespore cells.…”