INTRODUCTION The cellular slime mould Dictyostelium discoideum, has been used as a good model system for morphogenesis and cell differ- entiation because of its simple development. Amoebae of this organism multiply by binary fission, feeding on bacteria, and then aggregate to form multicellular masses when the food source is consumed. The amoebae differentiate into prespore and prestalk cells, which differentiate into spore and stalk cells, respectively, in the subsequent fruiting bodies. In Dictyostelium discoideum, several factors involved in differentiation have been identified and some of them have been purified and characterized (see, Williams, 1988; Oohata, 1995). The first to be identified was cAMP, which controls many essential processes of development by mediating signal transductions (for review see, Gross, 1994; Parent and Devreotes, 1996). In addition, a density-sensing factor, CMF (conditioned medium factor) and differentiation-inducing factor-1, DIF-1 (Gomer et al., 1991; Berks et al., 1991) have been well characterized. CMF, a glycoprotein of 80×10 3 M r , and its breakdown products are considered to function as part of a cell-density- sensing mechanism for cells of strain NC-4 and axenic strains in their normal environment (Gomer et al., 1991). At high cell densities, the extracellular concentration of CMF is sufficient to enable the cells to enter the multicellular stage of the devel- opmental cycle. When present below a threshold concentration, the cells do not initiate the expression of genes required for early development. However, amoebae of strain V12M2 dif- ferentiate into prespore and stalk cells in a salt solution con- taining cAMP even when the cell density is below that at which cell contact is recognised (Oohata, 1992). Thus, V12M2 is a convenient strain to investigate prespore and prestalk cell differentiation, excluding high-cell-density-dependent processes in early development. Using this strain, DIF-1 was discovered and purified from conditioned medium (Town et al., 1976; Kay et al., 1989). DIF-1 is a chlorinated hexaphenone and functions by entering the cells; high concentrations of DIF- 1 induce prestalk and stalk cell differentiation and repress prespore cell differentiation (Kopachik et al., 1985; Early and Williams, 1988; Kay et al., 1989), while low concentrations promote prespore cell differentiation (Oohata, 1995). Therefore, DIF-1 is considered to play a key role in the differ- entiation of this organism. Previous studies have also indicated the existence of dialysable and heat-stable factors that are involved in prespore cell differentiation (Weeks, 1984; Kumagai and Okamoto, 1986; Gibson and Hames, 1988). These findings suggest that DIF-1 and other factors that induce prespore cell differentiation may act mutually to control the prestalk-prespore pattern formation. However, decisive evidence is lacking. In a previous paper, we reported the presence of a prespore- inducing substance(s) in a conditioned medium (CM) (Oohata, 1995). In this study, we further analyze the CM and clearly 2781 Development 124, 2781-2787 (1997) Printed in Great Britain © The Company of Biologists Limited 1997 DEV7550 In Dictyostelium discoideum strain V12M2, at a very low cell density (~10 2 cells/cm 2 ), most amoebae differentiate into prespore cells in a salt solution containing cAMP if an adequately diluted conditioned medium (CM) is provided (Oohata, A. A. (1995) Differentiation 59, 283- 288). This finding suggests the presence of factor(s) released into the medium that are involved in inducing prespore cell differentiation. In the present study, we report the presence of two types of factors that function synergistically in prespore cell induction; one is a heat- stable and dialysable factor(s) and the other is a heat- labile and non-dialysable factor termed psi (ψ) factor (p res pore-i nducing factor). We purified and characterized the psi factor. Its relative molecular mass was determined to be 106×10 3 M r by SDS-PAGE and 180×10 3 M r by gel fil- tration HPLC, respectively. These results indicate that psi factor exists as a dimer under native conditions. In addition to inducing prespore cell differentiation, psi factor induced cell division of prespore cells in submerged culture. Our results suggest that psi factor plays important roles not only in prespore cell differentiation but also in the progress of the cell cycle in the prespore pathway in normal development. Key words: Dictyostelium discoideum, morphogen, differentiation, conditioned medium, cell cycle, cell division SUMMARY A novel prespore-cell-inducing factor in Dictyostelium discoideum induces cell division of prespore cells Akiko A. Oohata 1, *, Manabu Nakagawa 2 , Masao Tasaka 3,† and Shigeru Fujii 2 1 Biological Laboratory and 2 Chemical Laboratory, Kansai Medical University, Hirakata, Osaka 573, Japan 3 National Institute for Basic Biology, Myodaiji, Okazaki 444, Japan *Address for correspondence (e-mail: oohata@makino.kmu.ac.jp) † Present address: Department of Botany, Division of Biological Science, Graduate School of Science, Kyoto University, Kyoto 606-1, Japan