UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Studies on the roles of the various differentiation inducing factors (DIFS) in Dictyostelium discoideum Xie, Jennifer Yinjuan 1989

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1990_A6_7 X53.pdf [ 3MB ]
Metadata
JSON: 831-1.0098326.json
JSON-LD: 831-1.0098326-ld.json
RDF/XML (Pretty): 831-1.0098326-rdf.xml
RDF/JSON: 831-1.0098326-rdf.json
Turtle: 831-1.0098326-turtle.txt
N-Triples: 831-1.0098326-rdf-ntriples.txt
Original Record: 831-1.0098326-source.json
Full Text
831-1.0098326-fulltext.txt
Citation
831-1.0098326.ris

Full Text

STUDIES ON THE ROLES OF THE VARIOUS DIFFERENTIATION INDUCING FACTORS (DBFS) LN DICTYOSTELIUM DISCOLDEUM By JENNIFER YINJUAN XIE B.M.Sc. China Medical School, 1982  A THESIS SUBMITTED LN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Microbiology)  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUBIA December  1989  © Jennifer Yinjuan Xie, 1989  In  presenting  degree  this  at the  thesis  in  partial  fulfilment  of  University of  British  Columbia,  I agree  freely available for reference copying  of, this  department  or  publication of  thesis by  this  and study.  for scholarly  his  or  her  Department The University of British Columbia Vancouver, Canada  DE-6 (2/88)  Vi£-.>1.  <7$ ?  requirements that  I further agree  purposes  representatives.  may be It  thesis for financial gain shall not  permission.  Date  the  that  the  advanced  Library shall make it  by the  understood be  an  permission for extensive  granted  is  for  allowed  that without  head  of  my  copying  or  my written  ii  ABSTRACT  The effects of stalk cell differentiation inducing factor (DIF) on stalk cell induction from vegetative cells and prestalk cells of Dictyostelium  discoideum were investigated. It was found that the  major DDF component DEF-1 is a poor inducer of stalk cell formation from prestalk cells, but a minor component, DIF-2 is a more important inducer for the conversion of prestalk to stalk cells. Evidence is presented that DIF-2 synergizes the activity of the other DIF components for prestalk to stalk cell conversion. In contrast DDF-5 inhibits the activity of DIF-1 and DIF-3/4. A model is proposed to explain those results.  Table o f Contents Page Abstract  ii  List of Figures  v  List of Tables Acknowledgement Introduction Materials and Methods  vi vii 1 14  1. Organism and culture conditions  14  2. Crude D B F preparation  14  3. DBF separation by H P L C  15  4. L o w density monolayer assay for D B F activity  16  5. Separation of prestalk and prespore cells on Percoll gradients 6. C y c l i c A M P removal experiments Results  17 18 19  1. Assay of D B F activity  19  2. Requirments of D I F for prestalk-stalk conversion  19  3. Crude D I F separation by H P L C  22  4. Bioassay of combination of active fractions  27  5. Effect of c A M P on the conversion of prestalk cells to stalk cells  28  iv 6. Sequential treatment of monolayers with and D I F  34  7. Inhibitory effect of c A M P Discussion References  cAMP  35 ,  37 49  List of Figures Page Figure 1. Figure 2.  Life cycle of Dictyostelium discoidium The chemical structure of DIF-1, DIF-2 and DIF-3  Figure 3. Assay for DTF Figure 4.  3 9 20  Comparison of stalk cell formation induced by crude DTF and synthetic DIF-1  21  Figure 5.  HPLC resolution of crude DDF  23  Figure 6.  DIF assay of HPLC fractions  24  vi  List of Tables Page Table 1.  Table 2.  Table 3.  Table 4.  Table 5.  Table 6.  Comparison of stalk cell formation from vegetative and prestalk cells induced by various preparations of DIF  26  Stalk cell formation from prestalk cells induced by combinations of DIF species from the preparation  29  Stalk cell formation from prestalk cells induced by chemically synthesized DIF-1 and DIF-2  31  Stalk cell formation from vegetative cells induced by combinations of DIF species  32  Stalk cell formation from prestalk cells, induced by DIF species in the presence and absence of cAMP  33  Stalk cell formation induced by sequential treatment of cAMP and DTF  36  Acknowledgement I would like to thank Dr. Weeks for his guidance and support throughout this work. I am grateful to L . Kwong for her assistance.  1  INTRODUCTION  Developmental biology is a multidisciplinary science concerned with the analysis of the progressive acquisition of specialized structure (morphogenesis)  and function (differentiation)  by  organisms and their various cellular components (Browder, 1984). This subject has been studied in diverse organisms such as moulds, worms, flies, frogs and mice. Dictyostelium discoideum provides a very useful system for studies in developmental biology because it exhibits important characteristics of embryonic development on a relatively simple scale, i.e. from an originally homogeneous population of amebae, two cell types, stalk and spore cells differentiate. A characteristic feature of diffferentiation in this system is the formation of a migrating pseudoplasmodium in which there is a simple, but tightly regulated developmental pattern consisting of prestalk cells at the anterior end and prespore cells at the posterior end. This linear arrangement of two cell types provides the simplest possible model for studies on pattern regulation during  development.  It was observed that by varying the composition of the nutrient broth in which the cells are grown, or by purifying cells at defferent stages in the cell cycle, it is possible to generate populations of cells that selectively sort to different parts of the slug or that will develop to give a skewed prestalk-to-prespore, or stalk-to-spore, ratio (Leach et al.. 1973; Tasaka & Takeuchi, 1981; McDonald & Durston, 1984; Weijer et al.. 1984; Blaschke et al..  2  1986). It seems plausible to suggest, therefore, that cells entering development are weakly predetermined (for review see Williams, 1988). In addition, certain diffusible molecules can influence the interconversion of prespore and prestalk cells, suggesting the possibility that these molecules are morphogens: molecules capable of regulating pattern formation during development (for review see Williams, 1988). A central problem  in developmental biology is to understand  how morphogenetic fields are created and how they act to direct regionalized cellular defferentiation. Using Dictyostelium discoideum as a tractable system, four such molecules so far have been identified, cyclic AMP, the stalk cell Differentiation Inducing Factor (DIF), adenosine and ammonia and the properties of each of these will be discussed  subsequently.  The Dictyostelium Life Cycle During vegetative stage of growth, Dictyostelium discoideum exists as solitary amoebae (Figure 1, A). Upon starvation, the amoebae enter the developmental pathway by signaling to each other with pulses of  low concentrations of cAMP until a signalling  centre arises randomly, and eventually entrains up to 1 0  5  amoebae  (for review see Bloom & Kay, 1988). Cells respond to the receipt of the oscillatory emissions of the chemotactic signal by migrating towards the signalling centre (Figure 1, B and C). As the aggregation proceeds, a tip at the center of the aggregate becomes more and more evident (Figure 1, D). It has been suggested that emissions of  Figure 1. Life cycle of Dictyostelium discoidium adapted from Williams (1988).  4  cAMP are propagated from the tip and direct cell movement (for review see Schaap, 1986). The tip then elongates to form a structure known as first finger (Figure 1, E), which collapses onto the substratum and becomes an elongated migratory pseudoplasmodia or slug (Figure 1, F). The slug is of great value because of its anatomy. The anterior one fifth of the slug is composed of prestalk cells and the posterior four fifths is predominantly composed of prespore cells but approximately 1015% of the cells in this region are called anterior-like cells because they display the morphological and biochemical characteristics of prestalk cells (Sternfeld & David, 1981, 1982; Devine & Loomis, 1985; Jermyn et al.. 1989). These prestalk and prespore cells have distinct characteristics and they are important intermediate cell types in the process of differentiation. Since they are readily separable by centrifugation on Percoll gradients (Tsang & Bradbury, 1981; Ratner & Borth, 1983; Sobolewski, 1987) and by microdissection, the migratory slug certainly provides a ready source of these cells for developmental studies. At culmination, the anterior of the slug rises off the substratum (Figure 1, G). Prestalk cells at the tip move downwards through the prespore zone to form stalk cells, which act to lift the spore cell mass off the substratum (Figure 1, H), eventually forming a mature fruiting body (Figure 1, I). Under laboratory conditions, the whole process of development takes 24-28 hours. Approximately 80% of the cells that enter development form spores and the remainder form stalk cells. The stalk is composed of highly vacuolated,  J  cellulose-ensheathed dead cells, and spores are ellipsoidal cells, resistant to environmental stress. The differentiated cell types therefore show a remarkable 'homeostasis' (for review see Bloom & Kay, 1988). By using immunological techniques and Nothern blot analysis, it is now possible to estimate when prestalk and prespore cell differentiation occurs, and it is now known that the first presporespecific markers appear late in aggregation at, or just prior to, the time of tip formation (Muller & Hohl, 1973; Hayashi & Takeuchi, 1976; Forman & Garrod, 1977; Tasaka et al.. 1983; Morrissey et al.. 1984; Dowds & Loomis, 1984; Krefft et al.. 1984; Williams et al.. 1987). Due to confusion over the validity of some stalk cell markers the initiation of stalk cell formation is less well defined; pDd63 mRNA and acid phosphatase II are highly stalk cell specific and they first appear at, or just after, the tipped aggregate stage of development,  approximately two hours after the prespore cell  components first appear (Oohata, 1983; Williams et al.. 1988). The regulation of prestalk and prespore cell differentiation has been extensively studied over the past two decades and four potential morphogens have so far been identified, cyclic AMP, DIF, adenosine and ammonia. Cyclic A M P As stated by Bloom and Kay (1988), "Cyclic AMP is a virtuoso performer throughout Dictyostelium development, with several important roles already established and hints of others on the way".  5  6  Cyclic AMP was identified initially as the chemoattractant which is responsible for aggregation. Amoebae aggregate because the cyclic AMP sent out periodically by one starving cell causes nearby cells to move toward the signal source and to secrete cyclic AMP themselves, resulting in coordinated streaming toward an aggregation centre. The tip of the migrating slug has some of the classical properties of an embryonic organizer and emissions of cyclic AMP from the tip have been proposed to direct cell movement in the slug and are possibly responsible for tip activation (for review see Schaap, 1986). Cyclic AMP also induces the expression of genes during aggregation, including those for cyclic AMP-dependent phosphodiesterase,  cyclic AMP surface  receptors and the cell cohesion molecule contact site A (Bloom & Kay, 1988). Cyclic AMP induces the expression of many presporespecific genes and stabilizes prespore specific mRNA (Chung et al.. 1981; Mangiarotti et al.. 1983). Cyclic AMP is also known to be required for stalk cell formation (Bonner, 1970) to bring cells to a DIF-responsive state (Sobolewski et al.. 1983). But recently, Berks and Kay (1988) reported that later in development cyclic AMP becomes inhibitory to stalk cell differentiation indicating a possible pathway-specific role for cyclic AMP for cell differentiation. Furthermore, it has also been suggested that extracellular cyclic AMP may play a role in cell type proportioning because it inhibits the conversion of prespore to prestalk cells (Weijer & Durston, 1985).  7  Adenosine The hydrolysis product of cyclic AMP, adenosine, appears to function as an antagonist to cyclic AMP. Schaap and Wang observed that exposure to a high external concentration of adenosine increased the average size of slug twofold (1986). Newell and Ross (1982) found that during early aggregation, adenosine acts to increase the average size of aggregation territories by reducing the number of signalling centers; at late stages of aggregation, adenosine was found to act as an inhibitor of the binding of cyclic AMP to its cell surface receptor and reduce cyclic AMP relay (Newell, 1982; Theibert & Devreotes, 1984; Van Lookeren Campagne et al.. 1986). Therefore, it has been suggested that adenosine might be responsible for tip inhibition probably by counteracting tip activation by cyclic AMP (Schaap & Wang, 1986). In addition, adenosine was found to competively inhibit the induction of prespore differentiation by cyclic AMP (Schaap & Wang, 1986). When endogenous adenosine is enzymatically removed from slugs, prespore cells are formed throughout the prestalk region. Presumably adenosine again exerts its effect by reducing cyclic AMP binding to the cell surface receptor (Schaap & Wang, 1986). Furthermore, adenosine was also found to inhibit the conversion of prestalk to prespore cells and stabilize prestalk cells (Weijer & Durston, 1985). DIF  Studies on cell differentiation in monolayers, where normal morphogenesis is suppressed led to the discovery of DIF. After  8  Bonner had found that a small proportion of starved amoebae plated on to agar containing cyclic AMP formed clumps of stalk-like cells (Bonner, 1970). Town, Gross & Kay (1976) found that isolated amoebae cells plated at low density on agar containing cyclic AMP do not differentiate, but can be induced to do so by a layer of high density helper cells from which they are separated by a thin cellophane membrane. The dialyzable factor released by the high density cell population was named differentiation inducing factor (DIF) and was extracted from the cell culture supernatant by organic solvents (Town & Stanford, 1979; Brookman et al.. 1987). This crude DIF preparation is capable of inducing isolated cells to differentiate into stalk cells on agar in the presence of cyclic AMP. This assay had been modified (Gross et al.. 1981; Kay & Jermyn, 1983) and is now performed using tissue culture plates and this" modification has proven to be very useful for studying the effects of potential biological effectors of differentiation. Purification by high pressure liquid chromatography (HPLC) of the crude DIF preparation revealed a family of five active components, called DIFs 1 to 5 (Kay et al.. 1983). DEF-1 was found to be the major bioactive component comprising 95% of the total activity of crude DIF (Kay et al.. 1983). So far DLF-1, DIF-2 and DIF3 have been identified using a combined microchemical and spectroscopic  approach (Morris et al.. 1987) and their structures are  shown in Figure 2.  (a) DIF-1 O H  O  O H  O  (b) DIF-2  CI  (c) DIF-3 O H  O  Figure 2. The chemical structure of DIF-1, DIF-2 and DIF-3.  The chemical structure of DIF-1 was determined as l-(3,5dichloro-2,6-dihydroxy-4-methoxy-phenyl)-l -hexanone.  The  structure of DIF-2 was determined as l-(3,5-dichloro-2,6dihydroxy-4-methoxy phenyl)-1-pentanone. Although a number of naturally occurring chlorinated phenols are known (Suida & DeBernardis, 1973; Niedleman & Geigert, 1986), none have an additional alkyl ketone substitution on the benzene ring. Therefore, DIF molecules are unusual among biologically active substances and may represent a new class of biological effector molecules (Morris et al.. 1987).  DIF-1 and DIF-2 have now been chemically  sythesized. Work to date has mostly concentrated on DIF-1 because it is the major activity detected using monolayers of vegetative cells, and because the molecule has been chemically sythesized, providing a convenient source of material for further studies. Little DIF can be detected during aggregation, and mutants that are defective in DIF accumulation aggregate fairly normally (Brookman et al.. 1982; Sobolewski et al.. 1983; Kopachik et al.. 1983) Thus, there is no evidence for an essential role for DIF in aggregation (Kay et al.. 1988). DIF-1 is not made in substantial amounts until the mound stage of normal development, some hours after cyclic AMP levels first rise (Brookman et al.. 1987). In other words, the DIF-1 level rises just as the prestalk/prespore pattern is being established in the aggregate (Brookman et al.. 1987). Mutants making only trace  11 amounts of DIF (e.g. H M 44) are blocked at this stage , but the cells can become stalk cells if given DIF-1 (Kopachik et al.. 1983). In addition to inducing the conversion of vegetative cells to stalk cells (Kay & Jermyn, 1983), DIF is also required for prestalk cells to differentiate to stalk cells (Kwong et al.. 1988b). Furthermore, Percoll-gradient purified prespore cells were induced to form stalk cells in the presence of DIF-1 (Kay & Jermyn, 1983). At the molecular level, DIF-1 was found to be able to induce the transcription of the pDd63 mRNA, the most prestalk-specific mRNA so far discovered, within 15 min of its addition to HM44, a 'DIF-less' mutant (Williams et al.. 1987). and the accumulation of D19, a prespore-specific mRNA sequence, is repressed at the transcriptional level by DIF-1 (Early & Williams, 1988). Therefore, the role of DIF-1 in cell differentiation has been clearly identified as an inducer of prestalk cell gene expression and is essential for stalk cell formation in monolayers (Kay et al.. 1988). In addition it might control the choice of differentiation into prestalk or prespore cells to establish the prestalk zone in the aggregate (for review see Bloom & Kay, 1988). Ammonia A large amount of ammonia is produced during differentiation as the end product of the degradation of a major fraction of cellular protein and RNA. Ammonia has been shown to be an antagonist to DIF action during cellular differentiation in several ways (Gross et al.. 1983) .  Ammonia was found to be inhibitory to stalk cell differentiation (Gross et al.. 1983; Town, 1984) and it was found that stalk cells can be induced to differentiate within the slug by localized exposure to DIF and an enzyme cocktail that depletes ammonia (Wang & Schaap, 1989). On the other hand, ammonia stimulates spore cell formation. When large clumps of D. discoideum cells were transferred to fresh medium no spore cells differentiated, but ammonia showed significant stimulation of spore cell formation when added to the medium; when ammonia concentration was reduced about 50-fold, almost the entire stimulation was lost (Sternfeld & David, 1979). Gross et al (1983) also observed that when cells from a sporogenous mutant were incubated at high density in the submerged culure, a mixture of stalks and spores was formed. But in presence of ammonia the proportion of cell differentiating into spores is increased. Furthermore, ammonia might be complementary to cyclic AMP in stabilizing prespore mRNA because when the cyclic AMP relay was inhibited by rapid shaking cells in highly hypertonic medium, ammonia was necessary for maintaining the expression of several prespore genes (Oyama & Blumberg, 1986). Ammonia also prevents fruiting because it causes newly formed aggregates to develop into migrating slugs under conditions which otherwise would permit them to construct fruiting bodies directly. Culmination can also be induced when ammonia is enzymaticlly depleted (Schindler & Sussman, 1977).  13  Other  Factors  In addition to these four substances, of known structure, a number of other activities have been detected which stimulate prespore cell differentiation, such as: spore cell inducing factor (SPIF) (Wilkinson et al.. 1985) and spore inducing factor (PIF) (Kumagai & Okamoto, 1986). The fact that sporogenous mutants were able to differentiate into spores in the absence of any factor other than cyclic AMP and the fact that there is no evidence for the rapid induction of prespore gene expression by any of these factors suggest that these structurally uncharacterizied factors may act as permissive factors, facilitating cellular differentiation but not having a directly inductive effect (for review see Williams, 1988). Studies in this thesis have focused mainly on the effects of DIF on stalk cell formation. DDF-1 constitutes 95% of the total DIF activity and is able to perform all the functions of total DIF thus far studied. The functions of other DBF species are unknown (Morris et al.. 1987). In this thesis I show that DIF-1 is a poor inducer of prestalk to stalk cell conversion and that DDF-2 is more efficient in performing this function.  MATERIALS & METHODS: Organism and Culture Conditions Dictyostelium discoideum. strain V12-M2, was grown in association with Enterobacter aerogenes on rich nutrient plates at 2 2 ° C until the bacterial lawn had visibly cleared, about 48 hours (Sobolewski  et al.. 1983). The vegetative cells were then harvested  and bacteria removed by suspension and centrifugation in Bonner's salts (Bonner, 1949) for three times before they were either set up at low density for stalk cell formation or set up at high density for DIF production. They were also be allowed to differentiate on nonnutrient agar (2%) containing 5% Bonner's salts until the migrating pseudoplasmodial stage of development  (16-18 hours).  Crude DIF Preparation DIF preparation was obtained using the method of Town & Stanford (1979) modified by Kay et al (1983). Washed vegetative cells (2 x 10 ) were spread on a layer of thin cellophane at a density of 9  about  1.5xl0 /cm 9  2  supported by a nylon mesh standing in a plastic  tray. The undersurface of the cellophane was bathed in medium containing 10 mM KC1, 2 mM NaCl, 1 mM CaCh, 100 mg/L streptomycin, 1 mM cyclic AMP and 7 grams washed XAD-2 beads, which absorb the DIF (Kay et al.. 1983) that passes through the cellophane from the cells. Cells were covered with another sheet of cellophane to prevent them from drying out. The plastic tray was then  15 covered with aluminum foil and shaken at 22°C at about 16 rev./min. for 4 days. After this incubation, the white XAD-2 beads, now turned yellow with absorbed materials were collected and washed thouroghly with water. DIF was eluted into the ethanol, by soaking the beads several times with absolute ethanol. About 100 ml ethanol eluate from 2xl0  9  cells was concentrated to about 5 ml by rotary evaporation, and  resuspended in 100 ml water. The aqueous solution was again concentrated by rotary evaporation to remove most of the ethanol Enough 2 N KOH was added to a final concentration of 0.1 N. This mixture was then extracted three times with an equal volume of hexane to remove many neutral and basic non-polar impurities. The aqueous phase was adjusted to pH 5.1 with 2 N HC1 and DIF was then extracted three times with an equal volume of fresh hexane. 100 u.1 of BHT (10 mg/ml in ethanol) was added to this hexane extract of DIF, which was then dried down to small volume by rotary evaporation and taken up in 3 mis ethanol. A final volume of 1 ml was obtained by further evaporation. This crude DIF preparation is now ready for bioassay or for further separation by HPLC. DIF Separation by HPLC A Waters 501 HPLC machine was used fitted with a Whatman reverse-phase 50 cm CI8 column connected to a model 441 ultraviolet absorbance detector (280 nm wavelength). The chromatography conditions were exactly as described by Kay (1987). About 400 u.1 crude DIF preparation in ethanol was loaded on the column. The column was eluted at 2.5 ml/min using increasing concentrations of 98% methanol and 2% acetic acid (solvent) in water.  16 The column was eluted initially with a gradient from 40% to 80% solvent over the first 80 min.; then a gradient of 80% to 100% solvent for the next 20 min and finally 100% solvent for the last 20 min. The methanol and acetic acid were HPLC grade from BDH. All solvents were degassed by filteration through a nylon 66 membrane filter from Fisher Scientific (mesh size 47mm). Fractions were collected every minute by a FRAC-100 fraction collector (Pharmacia). All the fractions were stored at -20°C before they were assayed using low density monolayers of either vegetative cells or prestalk cells. Synthetic DIF-1 and DIF-2 were kindly supplied by R. Kay. Low density Monolayer Assay for DIF Activity The low density monolayer assays technique was first described by Town et al (1976), then modified by Kay & Jermyn (1983). This system has the advantage that the response of cells to added factors can be monitored in the absence of endogenously produced factors and direct cell-cell contact. Washed vegetative cells, strain V12-M2, were diluted to a concentration of 10 cells/ml in a buffered Bonner's salts 4  solution (BBS) which comprised 10 mM NaCl, 10 mM KC1, 2 mM CaCh, 5 mM 4-morpholineethanesulfonic  acid (MES), pH 6.2, 100 u.g/ml  streptomycin sulphate, 1 mM cyclic AMP. 2 ml aliquots of this suspension were added to 5 cm Nunc tissue culture dishes and 1 or 2 u,l of DDF: (crude DDF, pure DIF-1,  pure DIF-2 or fractions from the  HPLC column). A cell monolayer at a density of 10  3  cells/cm was 2  obtained and incubated at 22°C for 48 hours. Stalk cell formation was assessed using a model Leitz SM-LUX phase contrast microscope. Cells  were scored positive for stalk cell formation if greater than 50% of the cell volume was vacuolated. For each assay 200 cells were scored and each assay was performed in duplicate. The average value of the duplicates was plotted against the amount of added DIF. DIF activity was determined from the initial portion of the curve where there was a linear response to added DIF. Using the same definition of Brookman et al (1982), 1 unit of DIF activity was defined as the amount of DIF necessary to induce 1% stalk cells in the standard assay. Separation of Prestalk and Prespore cells on Percoll Gradients The separation of prestalk and prepore cells was performed using a modification of the Ratner & Borth (1983) method devised by Sobolewski (1987). Pseudoplasmodia were harvested with 50 ml 20 mM potassium phosphate buffer, pH 6.0 and filtered through a fine nylon sieve (mesh size 20 jim) to remove slime sheath and undifferentiated cells. Pseudoplasmodial cells were resuspended in 10 ml of potassium phosphate buffer and separated by vortexing vigorously. The suspension was centrifuged at l,000xg for 5 min. The cell pellets were washed once more by resuspension and contrifugation. The cells were then resuspended in 0.5 ml of 5 mM MES, pH 6.2, 0.06% B-mercaptoethanol and 1 mg/ml Sigma type XIV protease and incubated at room temperature for 15 min. with constant trituration through a 23G needle. Aliquots (0.25 ml) containing approximately 10  8  cells were layered onto the surface of  an ice-cold discontinuous Percoll gradient and centrifuged in a Beckman SW41 rotor at 12,000 rpm for 5 min. at 4 ° C . The discontinuous gradient was made up of 2.5 ml of 45% Percoll in the  1 8 bottom layer; 2.5 ml of 30% Percoll in the middle layer and 2.5 ml of 15% Percoll in the top layer, each diluted in 20 mM MES, pH 6.2, 20 mM EDTA, pH 7.0 (Sobolewski, 1987). Cells containing the prestalkspecific isozyme, acid phosphatase II, were recovered as a band at the interface between the top and middle layers of the gradient, whereas cells containing the prespore-specific enzyme UDPgalactosyl: mucopolysaccharide transferase (Newell et al.. 1971) were recovered between the middle and bottom layers. Cells were collected with a Pasteur pipette, diluted with ice-cold Bonner's salts and centrifuged at 1,000 x g for 5 min. Cell pellets were washed once in Bonner's salts by resuspension and recentrifugation. These cells were then set up in low density monolayers for the DIF bioassay as described  above.  Cyclic AMP Removal Experiments Cells were plated at low density (10 /cm ) as described above. 3  2  After incubation of 16, 20 or 24 hours at 2 2 ° C ,  supernatants were  removed. The monolayers were then washed with 2 ml of Bonner's salts three times and replenished with 2 ml of Bonner's salts buffer supplemented with varying amount of crude DIF, synthetic DIF-1 or synthetic DIF-2 in the presence of absence of 1 mM cyclic AMP. Incubation was continued until for 48 hours from the begining of the initial incubation period and then the plates were scored microcopically for stalk cell formation, as described above.  RESULTS Assay of DIF Activity DIF is a stalk cell-specific morphogen which when added to low density monolayers of vegetative Dictyostelium  discoideum cells, is  able to induce stalk cell formation in the presence of cyclic AMP (Town, 1976). Serial dilutions of DIF were made and assessed for stalk cell formation (Figure 3). From linear portion of the graph, the activities of the stock solutions of crude DIF, DIF-1 and DIF-2 were determined to be 906, 714 and 448 units/u.1  respectively.  Requirments of DIF for Prestalk to Stalk Conversion Interestingly, when equal activities of crude DIF and synthetic DIF-1 were added to low density monolayers of prestalk cells, DIF-1 was found a much poorer inducer for stalk cell formation compared with crude DIF (Figure 4) Since it had been shown previously that some stalk cell formation occurred from prestalk cells in the absence of cyclic AMP (Kwong et al.. 1988), the assay was repeated in the absence of cyclic AMP. In the absence of cyclic AMP stalk cells were formed in the presence of crude DIF, but there was no stalk cell formation in the presence of DIF-1. These differences suggested that there was some other component(s) in the crude DIF preparation that were more active for prestalk cell conversion to stalk cells than DIF-1.  40  c  jo cu  E L. o LL  <0  o  0  1/128  1/64  1/32  Concentration  1/16  of  DIF  Figure 3. Assay for D I F . Stalk cell formation from vegetative cells in response to D I F concentration. Washed vegetative cells o f strain V 1 2 M 2 were plated at 1 0 . c m 2 in 2 m l buffered Bonner's salts solution ( B B S ) containing I m M c A M P and the indicated relative amounts of D I F . A stock solution of D I F was serially diluted to provide the indicated relative amounts of D I F in a 1 u.1 final volume. C e l l s were then incubated at 2 2 ° C . S t a l k - c e l l formation was assessed after 48 hours by phase contrast microscopy, (a) Induction by crude D I F ; ( • ) induction by synthetic D I F - 1 ; (•) induction by sythetic D I F - 2 . 3  _  50  1200 Amount  of  DIF (units)  Figure 4. Comparison of stalk c e l l formation induced by crude D I F and synthetic D I F - 1 i n monolayers of prestalk cells. Prestalk cells were plated at 1 0 . c m i n tissue culture dishes i n 2 m l B B S containing the indicated units o f crude D I F i n presence of 1 m M c A M P ( • ) , or absence of c A M P (•); or containing the indicated units of synthetic D I F - 1 i n presence of 1 m M c A M P (•), or absence of c A M P (•). The dishes were incubated at 2 2 ° C for 48 hours. Stalk c e l l formation was assessed by phase contrast microscope. 3  _ 2  22  Crude DIF separation by high-pressure liquid chromatography Crude DIF preparations were then fractionated by HPLC (Figure 5) and all the eluted fractions were assayed for their ability to mediate stalk cell formation from both vegetative and prestalk cells (Figure 6). Figure 6A and 6B show HPLC of different preparations of crude DIF. The elutions are slightly different, but this variability was often observed. The only active fractions detected were qualitatively identical for the two different bioassays. The four active components were identified by comparing their activities and elution times with those reported earlier (Brookman et al.. 1987) for DIF-1, DIF-2, DEF-3/4 and DIF-5. DIF-3 and DLF-4 are not separated by this procedure (Brookman et al.. 1987). DLF-1 was the major active peak for stalk cell formation from vegetative cells (over 95% of the total) consistent with the earlier results. Other components exhibited very low activity (Figure 6). However, for the bioassays with prestalk cells, the activity of DIF-1 dropped dramatically consistent with the results presented earlier (Figure 4), whereas DIF-2 was slightly more active when assayed with prestalk cells than with vegetative cells (Figure 6). However, since some of the high activities observed with the column fractions were outside the linear portion of the DIF assay, the activities of DIF-1 and DIF-2 for stalk cell formation from vegetative and prestalk cell were more precisely compared (Table 1). The results revealed that DIF-1  was approximately 7 fold better at converting vegetative  cells to stalk cells than in converting prestalk cells to stalk cells, whereas DIF-2 was only 60% as efficient when comparing  Fig .5 HPLC resolution of crude DBF. Crude DIF was loaded onto a Whatman 50 cm Partisil M9 ODS-3 column and eluted as described under Materials and Methods. The eluate was analized by a Waters model 441 absorbance detector (wave length = 280 nm) coupled to 3390A intergrator from Hewlett-Packard. Eluted fractions were collected every minute with a FRAC-100 fraction collector from Pharmacia and stored at -20°C prior to the bioassay. A and B were separations of two independent crude DIF preparations. A: 400 ul of crude DIF were loaded onto the column; B: 900 \il of crude DIF were loaded onto the column.  A  B  DIF-1  (V)  DIF-5  P  DIF-2 DIF-3/4  20  40 Fraction  60 80 Number  100  20  40 60 80 100 Fraction Number  120  60 50 -  (P)  (P)  40 30 DIF-3/4 DIF-5  DIF-1  DIF-3/4  20  DIF-1  DIF-2 10  DIF-5  DIF-2  L  0 J4»44J44JJ44J4JJ4ljJ JJJJJJJT^QjlJJ.g JIJJNJJJ ll lj i  20  40 Fraction  60 80 Number  1 00  0  20  <  <  40 60 80 100 Fraction Number  i  120  Figure 6. D I F assay of H P L C fractions. A : W a s h e d vegetative cells ( V ) , or prestalk cells (P) were plated at 1 0 . c m i n monolayers i n 2 m l of B B S containing 1 m M c A M P and 1 or 2 j i l of each fraction of the eluate from the H P L C column. Cells were incubated at 2 2 ° C for 48 hours before stalk-cell formation was assessed by phase contrast m i c r o s c o p y . B : Details are the same as Figure 6 A except that a different preparation of crude D I F was used. 3  Table 1. Comparison of stalk-cell formation from vegetative and prestalk cells induced by various preparations of DIF. Stalk Crude DIF  Cell  Formation ( % ) DIF-2  DIF-1 Purified  Synthetic  Purified  32  54  36.5  3.75  5.75  Prestalk  13.75  7.5  4.75  5.75  8.25  p/yb  0.43  0.14  0.14  1.53  1.32  Vegetative  28  56  46.5  1  8.5  Prestalk  17  8  5.5  P/V*>  0.6  0.14  0.12  Experiment  l  a  Vegetative  Experiment  2  Synthetic  a  1.5 1.5  1 1 1.29  Experiments 1 and 2 are independent assays of the two independent DIF preparations described in Figure 6A and 6B respectively. P/V ratio is the number of stalk cells induced from prestalk cells (P) to the number of stalk cells induced from vegetative cells (V) by the same amount of the specified DIF preparation.  vegetative with prestalk cell conversion to stalk cells (Table 1). Results were similar for HPLC purified and chemically synthesized material (Table 1). Clearly DIF-1 and DIF-2 display an appreciable difference in their abilities to induce the conversion of prestalk cells to stalk cells relative to their activities with vegetative cells. The difference between the two is most dramatically seen in the ratio of activities of the two assays (P/V). DIF-1 was an relatively poorer inducer as observed before (P/V=0.14), whereas DIF-2 (P/V=1.4), was approximately 10 fold better than DIF-1 for stalk cell formation from vegetative cells. Bioassay of combinations of active fractions Using the vegetative cell assay, the recovery of activity from the column was 83% and 77% respectively for two independent crude DIF preparations. In contrast, with the prestalk cell assay the recovery of activity for the two experiments was 13.5% and 8%. Thus a considerable amount of the prestalk cell to stalk cell activity present in the crude DIF preparation is not recovered from the column. The column was exhaustively washed with the more nonpolar solvent, acetone, but this did not elute any additional activity (data not shown). A possible explanation for the low recovery from the column is that other components in the crude DIF preparation synergize the DIF activities. We therefore assayed various combinations of the column fractions in an extensive series of experiments to test for possible synergy. DIF-2 was the only fraction with synergizing  28  activity and it synergized each of the other DIF species (Table 2). In most instances the activities obtained when mixtures of DIF-2 and other DIF species were used were about 2 fold greater than the expected values. None of the fractions eluting between the DIF species had any effect on the activitiy of any other fraction (data not shown). Surprisingly, DIF-5 was found to inhibit the activity of DIF-1 and DLF-3/4. The inhibition occured without any cell lysis. It had no effect on the activity of DIF-2 (Table 2). Synergy between DIF-1 and DIF-2 activities was also observed with the chemically synthesized components (Table 3). When the various combinations were tested for their ablity to induce stalk cell formation from vegetative cells, no synergy with DIF-2 or inhibition with DIF-5 was observed (Table 4), which is consistent with a previous observation reported by Masento et al. (1987). It is apparent that despite the synergy effect of DIF-2, there is still considerable loss of prestalk to stalk cell inducing activity on the HPLC column, suggesting the possibility of an as yet undetected additional activity. Effect of cyclic AMP on the conversion of prestalk cells to stalk cells Previous work in this laboratory has shown that part of the prestalk population is independent of cyclic AMP for stalk cell formation (Kwong et al.. 1988b) and this was confirmed for a crude DIF preparation (Figure 3, Table 5). However, DIF-1 was unable to induce the conversion of prestalk cells to stalk cells in the absence  29  Table 2 A. Stalk cell formation from prestalk cells induced by combinations of DIF species from the preparation described in Figure 6 A. DLF Components  Stalk cell Formation(%) observed 3  1 ul  2 ul  Stalk cell formation(%) expected b  2 ul  11.0 + 0.5  21.3 ± 1 . 5  9.7 ± 0.6  1+3/4  3.810.8  8.0 ± 2.2  9.0 ± 1.0  1+5  0.7 ± 0 . 8  1.2 ± 1 . 3  7.2 ± 0.6  2+3/4  6.7 ± 1 . 5  11.0 ± 1 . 7  5.7 ± 1 .  4.7 ± 1 . 0  8.0 ± 1 . 7  3.8 ± 0.6  0  0.2 ± 0.3  3.2 ± 2  1+2+3/4  9.5 ± 0.5  16.8 ± 1 . 0  .,8.2 ± 0 . 4  1+2+5  6.5 ± 0.5  11.0 ± 1 . 0  6.9 ± 2  2+3/4+5  5.3 ± 2.1  9.2 ± 3.8  4.4 ± 1 . 2  1+3/4+5  0  0  6.4 ± 1.2  1+2  2+5 3/4+5  1+2+3/4+5  9.0 ± 1 . 0  17.3 ± 3 . 2  6.5 ± 0.8  The stalk cell formation obtained when a mixture of equal volumes of each of the indicated DIF fractions was assayed. Under these conditions: 1 n l of DIF-1 yielded 6.5 ± 0.5% stalk cells; 1 ul of DIF-2, 3.2 ± 0 . 3 % stalk cells; 1 ul of DIF-3/4, 2.5 ± 1.3% stalk cells and 1 ul of DIF-5, 0.7 ± 0.8% stalk cells. Expected values are the sum of the activities that would have been obtained if each of the components had been assayed independently under the same conditions. The values are the means ± the standard deviation for three experiments, and are all within the linear portion of the assay curve. a  b  Table 2 B . Stalk c e l l formation from prestalk cells induced by combinations of D I F species from the preparation described i n F i g u r e 6 B.  DIF components  Stalk cell formation(%) observed 3  Stalk cell formation(%) expected 0  1 ul  2 ul  2|il  11.3 ± 0 . 8  23.0 ± 0  10.5 ± 0.5  5.5 ± 0  10.0 ± 0  9.5 ± 0.5  1+5  0.5 ± 0.5  1.3 ± 0.8  9.0 ± 0  2+3/4  5.0 ± 1 . 0  10.0 ± 3 . 0  3.0 ± 0  3/4+5  0  0  1.5 ± 0.5  2+5  3.5 ± 1 . 5  6.3 ± 2.8  2.5 ± 0.5  1+2+5  4.8 ± 0.3  8.5 ± 0.5  7.3 ± 0.5  1+2+3/4  6.5 ± 1.5  12.0 ± 3.0  7.7 ± 0  1+3/4+5  0  1.0 ± 0  6.7 ± 0 . 5  2+3/4+5  3.5 ± 1 . 5  7.8 ± 3.3  2.4 ± 2.3  1+2+3/4+5  4.3 ± 0.3  7.8 ± 0.8  6.0 ± 0  1+2 1+3/4  T h e stalk c e l l formation obtained when a mixture of equal volumes of each of the indicated D I F fractions was assayed. U n d e r these conditions 1 u l of DIF-1 yielded 8.5 ± 0.5% stalk cells; 1 u l of D I F - 2 , 2 ± 0% stalk cells; 1 u l of DIF-3/4, 1 ± 0% stalk cells and 1 u l of D I F - 5 , 0.5 ± 0.5% stalk cells. a  E x p e c t e d values are the sum of the activities that w o u l d have been obtained i f each of the components had been assayed independently under the same conditions. The values are the means ± the standard error o f the mean for two experimemts, and are a l l within the linear portion of the assay curve.  b  31  Table 3. Stalk c e l l formation from prestalk cells induced by c h e m i c a l l y synthesized D I F - 1 and D I F - 2 .  DIF Component  Stalk c e l l Formation observed 1 Hi 3  DIF-1  5.8 ± 0.5  DIF-2  13.8 ± 0.5  DIF-1 + DIF-2  19.3 + 0.5  Stalk c e l l formation expected 1 ul b  9.8 ± 0.5  The values were the means + the standard error o f two separate experiments. Expected values are the sum of the activities that have been obtained when each of the components had been assayed independently under the same conditions. a  b  Table 4. Stalk c e l l formation from vegetative cells induced by combinations of D I F species. Stalk cell formation (%) DIF component  observed  Stalk cell formation(%) expected  0  1 u.1  2 ul  2 ul  1+2  13.3 ± 0.8  26.5 ± 3.5  27.5 ± 3.5  1+3/4  12.5 ± 1 . 0  25.0 ± 3.0  27.5 ± 2.5  1+5  11.3 ± 3 . 3  24.5 ± 6.5  27.0 ± 4 . 5  3.0 ± 0  6.0 ± 0.5  5.0 ± 0  2+5  2.5 ± 0.5  5.3 ± 0.8  5.0 ± 0.5  3/4+5  1.5 ± 0.5  4.0 ± 1.0  4.5 ± 0  1+2+3/4  13.0 ± 1 . 0  21.3 ± 0.8  19.5 ± 2 . 5  1+2+5  2.5 ± 0.5  5.3 ± 0.3  5.8 ± 0 . 8  1+2+3/4+5  11.5 ± 1 . 0  19.0 ± 1 . 0  16.0 ± 2 . 0  2+3/4  3  0  Fractions from the H P L C separations described i n Figure 6 A . T h e stalk c e l l formation obtained when a mixture of equal volumes of each of the indicated D I F fractions was assayed. Under these conditions 1 u l of D I F - 1 yielded 25 ± 1% stalk cells; 1 u l of D I F - 2 , 2.5 ± 0.5% stalk cells; 1 n l of DIF-3/4, 2.5 ± 0.5% stalk cells and 1 u l of D I F - 5 , 2 ± 0.3% stalk cells. The values are the means ± the standard error for two experiments, and are a l l w i t h i n the linear portion of the assay curve. Expected values are the sum of the activities that w o u l d have been obtained i f each o f the components had been assayed independently under the same conditions. a  b  c  Table 5. Stalk c e l l formation from prestalk cells, induced by D E F species i n the presence and absence of c y c l i c A M P . Stalk  Cell  Formation  3  (%)  DBF  - cAMP  + 1 m M cAMP  crude D I F  3.3 ± 0.5  26.511.8  DIF-1  0  5.8±0.5  DIF-2  0  13.810.5  DIF-1 + DIF-2  0  19.310.5  T h e values are the means 1 the standard error of the mean for two separate experiments. a  of c y c l i c A M P (Figure 3, Table 5). A l t h o u g h D I F - 2 was a better inducer of prestalk to stalk c e l l conversion, it too failed to induce i n the absence of c y c l i c A M P and the conbination of both D I F - 1 and D L F - 2 also failed to induce i n the absence of c y c l i c A M P . These results  show that the c y c l i c A M P independent  cells are insensitive  to both D I F - 1 and D I F - 2 and suggest the possibility of an additional factor i n crude D I F that is essential for terminal differentiation. Sequential treatment of cells i n monolayers w i t h c y c l i c A M P followed by crude D I F . D I F - 1 or D I F - 2 Since D I F - 1 was more active i n the conversion of vegetative cells to stalk cells and D I F - 2 was more active i n the conversion of prestalk cells to stalk cells, we postulated that D I F - 1 and D I F - 2 might act sequentially i n i n d u c i n g stalk c e l l formation, i.e. induction by D I F - 1 preceeds induction by D I F - 2 . It was shown previously i n this laboratory ( S o b o l e w s k i et a l . . 1983; K w o n g et a l . . 1988a) that stalk c e l l formation can be induced by a sequential treatment w i t h c y c l i c A M P and D I F . T o try to compare the timing of DIF-1 induction w i t h the t i m i n g of D I F - 2 induction, vegetative cells were plated at l o w density i n tissue culture dishes under buffered  Bonner's  salts  containing c y c l i c A M P and after different lengths of time (16, 20 and 24 hours respectively), the c y c l i c A M P was removed and crude D I F , D I F - 1 or D I F - 2 was added either i n the presence or absence of c y c l i c A M P . Despite the obvious variability i n the activities obtained i n different  experiments,  some  general  conclusions can be  drawn.  either  Under these conditions, crude DIF is a better inducer than DIF-1 and DIF-1 is better than DIF-2. In the presence of cyclic AMP, increasing the preincubation period from 16 to 24 hours results in a decrease in stalk cell formation for crude DIF, DIF-1 and DIF-2. In the absence of cyclic AMP there was essentially no change in stalk cell formation with increasing preincubation time. Finally, when cells were preincubated with cyclic AMP for 16 hours, addition of cyclic AMP during the subsequent incubation slightly stimulated stalk cell formation with all these DIF preparations. In contrast, when cells were incubated for 24 hours, addition of cyclic AMP during the subsequent incubation period was inhibitory. Inhibitory Effect of Cyclic AMP during sequential induction in monolayers In previous studies from this laboratory, cyclic AMP always stimulated stalk cell formation when present during the final incubation period (Sobolewski et al.. 1983; Kwong et al.. 1988a). This stimulation might due to DIF accumulation stimulated by cyclic AMP (Kwong & Weeks, 1988). In the experiment described in Table 6, cyclic AMP was only slightly stimulatory when the initial incubation period was 16 hours. When the initial incubation period was 24 hours, cyclic AMP was inhibitory. The effect of cyclic AMP during the final incubation period changes from being stimulatory to inhibitory when the initial incubation period is increased from 16 to 24 hours (Table 6).  Table 6. Stalk cell formation induced by sequential treatment of cyclic AMP and DIF.  Initial incubation period  Stalk  Crude DIF  Cell  a  Formation (%)b  DIF-1 +cAMP  DIF-2  +cAMP  -cAMP  -cAMP  +cAMP  -cAMP  16 Hours  27.9±5.7(3)  20.3±3.0(3)  4.4±2.3(3)  4.1±2.5(3)  1.3+1.8(2)  0.8±1.1(2)  20 Hours  21.4±10.1(5)  24.5±11.1(5)  4.5±4.0(3)  4.1+1.7(3)  1.0±0.8(4)  0.7±0.8(4)  24 Hours  12.3±3.3(3)  24.7±9.3(3)  1.0±0.1(2)  4.7±0.8(2)  0.2±0.3(2)  0.7±0.9(2)  Low density monolayers of vegetative cells were initially incubated in BBS and ImM cyclic AMP for the indicated time period. After preincubation, cells were washed and 2 ml BBS containing crude DIF, DIF-1 or DIF-2 in the presence or absence of 1 mM cyclic AMP. Incubation was continued until 48 hours from the begining of the initial incubation period. The actual experimental values were not shown. They were obtained using variable amounts of the DIF preparations that yielded significant stalk cell formation, but were still linear for added DIF. These values have been normalized to a constant amount of added DIF (50 units) to allow direct comparison between the different DIF fractions and different experiments. The values are the means ± standard deviations for the number of experiments indicated in the brackets. a  b  DISCUSSION  DIF is one of the four morphogens so far identified that are believed to be responsible for the spore-stalk pattern formation of the cellular slime mould Dictyostelium discoideum. Ammonia stimulates spore cell differentiation (Sternfeld & David, 1979) and inhibits stalk cell differentiation (Gross et al.. 1983; Town, 1984), whereas DIF has the opposite effects. Adenosine produces a decrease in the proportion of prespore cells in migrating slugs (Schaap & Wang, 1986), but there is no evidence that it is a direct inducer of stalk cell formation. The effects of cyclic AMP are complex. Spore cell formation is stimulated by cyclic AMP and it is also essential for stalk cell formation initially, but there are several reports now that suggest that it inhibits some subsequent step in stalk cell formation (Berks & Kay, 1988; Weijer & Durston, 1985). The work described in this thesis has concentrated on the effects of DIF on stalk cell formation. Since DIF-1 was able to induce cells to differentiate all the way from vegetative cells to stalk cells, it had been assumed that DIF-1 would fulfil all the DIF requirments for prestalk to stalk cell conversion. It was surprising to find that DIF-1 was a poor inducer of stalk cell formation from prestalk cells. DIF-2 was a more active molecule for the conversion of prestalk to stalk cells and in addition, it synergized the activity of the other DIF species including DIF-1, for this conversion.  DIF-2 is only 40% as efficient as DIF-1 for the conversion of vegetative cells to stalk cells (Masento et al.. 1988), but it is about 10 times more efficient than DIF-1 for the conversion of prestalk cells to stalk cells (Table 1). These results suggest that DIF-2 is more important than DIF-1 at later stages of differentiation. DIF-2 is a lower homologue of DIF-1, having a C4 rather than a C5 alkyl side chain (Figure 2). We are yet not aware of why this small difference in structure should make DIF-2 only 40% as active as DIF-1 for stalk cell formation from vegetative cells (Masento et al.. 1988), nor why DIF-2 is more efficient in converting prestalk to stalk cells and is able to synergize the effect of DIF-1 for this latter conversion. The sequential requirments for DIF-1 and DIF-2 probably reflect the changes in cell structure that occur during the formation of prestalk cells form aggregation competent cells, i.e. DIF-1 is specific for prestalk precursor cells (aggregation competent cells present at the mound stage of aggregation); whereas DIF-2 is specific for prestalk cells. The comparison of the efficiency of stalk cell conversion from prestalk cells induced by DIF-1 and DLF-2 (Table 1), together with the evidence that the DIF-1 inducible pDd63 mRNA is not detectably expressed until the tipped aggregate stage and only expressed in the prestalk zone (Williams et al.. 1987) is consistent with this assumption. The reason for the decrease in the activity of DIF-1 as cells progress from vegetative to prestalk cells is as yet unknown.  Recently, the receptor of DIF-1 has been isolated (Kay, personal communication). Further work is required to determine whether the receptor is intracellular or on the cell surface, which would help understanding the mechanisms of the functions of DIF-1 and its analogues. It is possible that the DIF-1 receptor is lost during the transition from the aggregation phase cells to prestalk cells. However, we do not yet have evidence at the molecular level to support this idea, nor the idea that receptors to DIF-2 increase as differentiation progresses. An alternative possibility, the appearance of a DIF-1 specific inhibitor, such as DIF-5 can not be excluded (Table 2). Although prestalk cells were washed before they were plated at low density, the action of intracellular inhibitors or extracellular inhibitors produced after the Percoll gradients purification remains possible. It was surprising that DIF-2 synergized the effect of other DIF components including DIF-1 on stalk cell formation from prestalk cells. This effect is specific to the properties of prestalk cells because no synergy was observed on stalk formation from vegetative cells (Masento, et al.. 1988; Table 4). We are yet not aware of the mechanism of the synergy, but it is possible that the combination of DIF-1 and DIF-2 stimulates DIF-2 production better than DIF-1 alone and DLF-2 is a better inducer of stalk cell formation from prestalk cells as described above. It is also possible that the induction by DIF2 involves the production of an additional molecule that is necessary for the conversion of prestalk cells to stalk cells and this  presumptive inducer is made in larger quantities in the presence of both DEF-1 and DIF-2. Another possibility is that prestalk cells dedifferentiate during the cell separation procedure or during the early stages of the monolayer incubation period (Kwong et al.. unpublished). DIF-1 would be required to convert the dedifferentiated cells back to prestalk cells and DIF-2 would be required for the conversion of prestalk cells to stalk cells. The inhibitory effect of DIF-5 was also unexpected since DIF-5 alone induces stalk cell formation. DIF-1 is a potent molecule (at 10 M, it induces >90% of vegetative amoebae to differentiate into _9  stalks in presence of cyclic AMP) (Bloom & Kay, 1988) and is freely diffusible. There must be some way of restricting its action and it is attractive to suppose the existence of such inhibitors. Gross et al. (1983) searched for such an inhibitor and showed that ammonia could effectively  inhibit stalk cell differentiation in monolayers of  vegetative cells of strain V12M2. Recently Berks and Kay (1988) reported the inhibitory effect of cyclic AMP after a cyclic AMPdependent period. Here we present another possible inhibitor for the prestalk-stalk pathway of differentiation (Table 2). However these results need to be comfirmed with synthetic DIF-5, which is not yet available. There was no antagonism among DIF analogues on vegetative cells (Table 4), which is in agreement with the observation by Morris et al (1988), and probably indicates  considerable intrinsic regulation by the cells when the inducers and inhibitors are present throughout the whole period of development. Previous work in this laboratory had revealed a subpopulation of prestalk cells that was cyclic AMP-independent for stalk cell formation (Kwong et al.. 1988b). When cells from the D. discoideum pseudoplasmodial stage were separated on Percoll gradients into prestalk and prespore cells, and the requirements for stalk cell formation in low density monolayers from the two cell types were tested, it was found that the isolated prespore cells required both cyclic AMP and DIF for stalk cell formation. In contrast, only part of the isolated prestalk cell population required both DIF and cyclic AMP, the remainder requiring DIF only, suggesting that there were two populations of prestalk cells (Kwong et al.. 1988b). This has been confirmed as shown by the results presented in Table 5 and Figure 4. Cyclic AMP stimulated the level of stalk cell formation from prestalk cells induced by crude DIF, but there was still appreciable stalk cell formation in the absence of cyclic AMP. However, in the absence of cyclic AMP there was no stalk cell induction by DIF-1 or DIF-2 either alone or in combination. These results suggest that there is some component in crude DIF, other than DIF-1 and DIF-2 that is capable of inducing stalk cell formation from cyclic AMP independent prestalk cells. The results discussed thus far suggest a possible sequential role for DIF-1 and DEF-2 in the differentiation process, that is: DIF-1 functions early during the process, during the formation of prestalk  cells in the pseudoplasmodia, and then DIF-2 is responsible for the conversion of prestalk cells to stalk cells. To obtain additional evidence for this model, monolayer experiments were performed. It was hoped that with increasing preincubation time that DIF-2 would be more effective than DIF-1 when added to low density monolayers of vegetative cells that had been preincubated with cyclic AMP. In fact DIF-2 was a poorer inducer of stalk cell formation under these conditions than DIF-1 and crude DEF was much better than both DLF1 and DIF-2. Under these monolayer conditions it is probable that DIF-2 can not induce stalk cell formation unless cells are first preincubated with DIF-1. Since crude DIF is better than DIF-1 and DIF-2, these results further suggest that crude DIF may contain an additional inducing component(s). It should be pointed out that in vitro differentiation do not precisely reproduce the normal differentiation process. Sobolewski and Weeks (1988) observed a difference in the timing of in vitro and in vivo cell differentiation by detecting the expression of the prestalk cell-specific isozyme acid phosphatase II and stalk cell-specific 41,000 M antigen (ST 41). The two components were expressed r  within 2 hours of each other during differentiation in vitro, whereas during development in vivo the expression of ST41 was detected 812 hours later than the expression of acid phosphase II. Furthermore, no one has ever observed the transient appearance during monolayer differentiation of a cell that morphologically resembles prestalk cells in the pseudoplasmodia. Perhaps in vitro conditions do not exactly mimic the in vivo development. Therefore,  the failure to detect large changes in DIF-1 and DIF-2 activity that are observed in vivo might reflect the fact that monolayer differentiation is not normal. The inhibitory effects of cyclic AMP in the monolayers were unexpected in view of previously published results from my laboratory showing stimulatory effects of cyclic AMP (Sobolewski et al., 1983; Kwong et al.. 1988a) regardless of the length of the initial incubation period. In the present study, cyclic AMP became inhibitory rather than stimulatory as the length of the initial incubation period increased. The central role of cyclic AMP in inducing aggregative, postaggregative and prespore cell differentiation has been clearly established during the past two decades (Darmon et al.. 1975; Gerisch et al.. 1975; Town & Gross, 1978;  Kay et al.. 1978). Cyclic AMP is also  necessary for stalk cell formation (Bonner, 1970) to bring vegetative amoebae to a fully DIF-responsive state (Sobolewski et al.. 1983). However, after the cyclic AMP-dependent period, cyclic AMP actually inhibits at least one essential step during stalk cell differentiation (Berks & Kay, 1988), and my monolayer results are consistent with this observation. Evidence so far available show that cyclic A M P inhibits expression of an antigen recognized by the monoclonal antibody JAb1 (Berks & Kay, 1988). This antigen appears to be the ST310 protein identified by Morrissey et al (1984), the gene product of pDd56 mRNA (McRobbie, et al.. 1988), which is maximally expressed at  midculmination in stalk cells, though low levels of the protein and its cognate mRNA are present  in the prestalk cells of the  pseudoplasmodia (Jermyn et al.. 1987; McRobbie et al.. 1987). A possible role for the inhibition by cyclic AMP may be as a repressor of stalk cell maturation at culmination, which could then be triggered by an abrupt drop in the effective cyclic AMP in the prestalk region (Berks & Kay, 1988). Such a drop in cyclic AMP levels in the stalk region of culminating organisms has not yet been detected (Merkle et al., 1984), but would be consistent with the high levels of cyclic AMP phosphodiesterase  and low levels of adenylate cyclase present in this  tissue at this time (Brown & Rutherford, 1980; Merkle & Rutherford, 1984). It is possible that adenosine antagonizes the effects of cyclic AMP during aggregation and later in development (Newell, Weijer & Durston, 1985;  1982;  Schaap & Wang, 1986). Adenosine might  promote stalk cell formation by relieving the inhibitory effects of cyclic AMP (Schaap & Wang, 1986). Although I have confirmed the inhibitory effect of cyclic AMP at later stages of monolayer development (Table 6), I have also shown that cyclic AMP is absolutely required for stalk cells formation from prestalk cells induced by DIF-1 or DIF-2, and that it enhances the induction by crude DIF (Table 5). It will be important to determine precisely which steps in the stalk cell differentiation pathway are stimulated and which steps are inhibited by cyclic AMP. Several models of pattern formation have been postulated (for reviews see Williams, 1988; MacWilliams & Bonner, 1979): the 'cellcontact model' of McMahon, in which pattern is created by  interactions of cells with their immediate neighbors (1973), the activator-inhibitor model based upon position-dependent  signals  (MacWilliams & Bonner, 1979; Gross et al.. 1981), and the positionindependent, double negative feedback model of Weijer & Durston (1985). It was proposed that cyclic AMP was the prespore to prestalk inhibitor, and adenosine, the prestalk to prespore inhibitor in the position independent model (Weijer & Durston, 1985), whereas DIF was proposed to be the activator, and ammonia the inhibitor in the position dependent model (Gross et al.. 1981). However, none of these models take into account all of the potential morphogens that have been described and the effects of DIF-2 and DIF-5 discribed in this thesis. I would like to propose the following hypothetical scheme for differentiation and cell patterning in Dictyosteleum. Proposed Model: 1. Cyclic AMP signalling among amoebae cells is triggered by food exhaustion, which stimulates chemotactic movement of the amoebae towards the signalling centre. 2. DIF-1 is made when the concentration of cyclic AMP increases to a threshold level, probably by those cells at the centre where the theoretical concentration of cyclic AMP is highest. Also, cell contact may be required for DIF-1 sythesis. 3. DIF-1 induces certain number of amoebae cells to form prestalk cells in the presence of cyclic AMP. Only those cells exposed  to sufficiently high concentrations of D I F - 1 and c y c l i c A M P form prestalk cells. Therefore the prestalk region is around the tip where c y c l i c A M P and D I F - 1 might be at the highest concentration. 4. D I F - 1 only diffuses a certain distance so that only 20% of the pseudoplasmodia becomes prestalk cells. The remaining 80% of the population under the influence of c y c l i c A M P alone, become prespore cells. 5. D I F - 1 also induces the precursor prestalk amoeboid cells to produce D I F - 2 and D I F - 5 and the balance between D I F - 2 and D I F - 5 determines  stalk  cell  maturation.  6. The roles of ammonia and adenosine might be homoestatic, modulators of the effects of c y c l i c A M P and DTF and might also influence the levels of D I F - 2 and D I F - 5 . These events are summarized i n Figure 7.  as  cAMP* DII -2  ^PRESTALK cAMP / DIF-1 / / ammonia AMOEBAE  >STALK cAMP * ammonia DIF-5  > AGGREGATING CELLS  ^PRESPORE DIF-1 adenosine  >SPORE  Figure 7 Summary of the preposed roles of cyclic AMP, DIF, ammonia and adenosine on differentiation thoughout Dictyostelium development. Cell differetiation is viewed as a bifurcating pathway with prespore cell differentiation proceeding prestalk cell differentiation (for reviews see Williams 1988; Bloom & Kay, 1988). Development is triggered by starvation, and the initial common pathway and prespore differentiation are driven by cAMP signals. Prestalk cell differentiation is brought about by the localized activity of DIF-1 in the aggregate. Prestalk to stalk conversion may depend upon the balance between DIF-2 and DIF-5. Adenosine and ammonia may assist cell-type diversification as indicated. Morphogens in bold letter favour the pathway indicated; those in italic form inhibit. * During the conversion of prestalk cells to stalk cells there appear to be a step(s) that is stimulated by cAMP and a step(s) that is inhibited by cAMP.  REFERENCES  Berks, M . & Kay, R. R. (1988). Cyclic AMP is an inhibitor of stalk cell differentiation in Dictvostelium discoideum. Devi. Biol. 126, 108-114. Blaschke, A., Weijer, C & MacWilliams, M . (1986). Dictvostelium discoideum: cell type proportioning; cell-differentiation preference, cell fate and the behaviour of anterior-like cells in HS1, HS2 and G+, G- mixtures. Differentiation 32, 1-9. Bloom, L . & Kay, R. R. (1988). The search for morphogens in Dictvostelium. Bioessays 9, 187-191. Bonner, J. T. (1970). Induction of stalk cell differentiation by cyclic AMP in the cellular slime mould Dictvostelium discoideum. Proc. Natl. Acad. Sci. U. S. A. 65, 110-113. Bonner, J. T. & Slifkin, M.K. (1949). A study of the control of differentiation. The proportions of stalk and spore cells in the slime mould Dictvostelium discoideum. Am. J. Bot. 36, 727-734. Brookman, J. J., Jermyn, K. A. & Kay, R. R. (1987) Nature and distribution of the morphogen DIF in the Dictvostelium slug. Development 100 (1), 119-124. Brookman, J. J., Town, C. D., Jermyn, K. A. & Kay, R. R. (1982). Developmental regulation of a stalk-cell differentiation-inducing factor in Dictvostelium discoideum. Devi. Biol. 91, 191-196. Browder, L . W. (1984). Developmental biology, second edition, part one. Saunders College Publishing. Brown, S. S. & Rutherford, C. L . (1980). Localization of cyclic nucleotide phosphodiesterase in the multicellular stages of Dictvostelium discoideum. Differentiation 16, 173-184. Ceccarelli, A., McRobbie, S. J., Jermyn, K. A., Duffy, K., Early, A. E. & Williams, J. G. (1987). Structural and functional characterization of a Dictvostelium gene encoding a DIF inducible, prestalk-enriched mRNA sequence. Nucl. Acids Res. 15, 7463-7476.  Chung, S., Landfear, S. M . Blumberg, D. D., Cohen, N. S. & Lodish, H. F. (1981). Sythesis and stability of developmentally regulated Dictyostelium mRNAs are affected by cell-cell contact and cAMP. Cell 24, 785-797. Darmon, M., Brachet, P. & Pereira Da Silva, L . H. (1975). Chemotactic signals induce cell differentiation in Dictyostelium discoideum. Proc. Natl. Acad. Sci. U.S.A. 72, 3163-3166. Devine, K. M . & Loomis, W. F. (1985). Molecular characterization of anterior like cells in Dictyostelium discoideum. Devi. Biol. 107, 364-372. Dowds, B. C. A. & Loomis, W. F. (1984). Cloning and expression of a cDNA that comprises part of the gene coding for a spore coat protein of Dictyostelium discoideum. Mol. Cell. Biol. 4, 2273-2278. Early, A. E . & Williams, J. G. (1988) A Dictyostelium presporespecific gene is transcriptionally repressed by DIF in vitro. Development 103, 519-524 (1988). Forman, D. & Garrod, D. R. (1977). Pattern formation in Dictyostelium discoideum. I. Development of prespore cells and its relationship to the pattern in the fruiting body. J. Embryol. exp. Morph. 40, 215-228. Gerisch, G., Fromm, H., Huegsen, A. & Wick, U. (1975). Control of cell-contact sites by cyclic AMP pulses in differentiating Dictyostelium cells. Nature (Lond.) 225, 547-549. Gross, J. D., Bradbury, J., Kay, R. R. & Peacey, M. J. (1983). Intracellular pH and the control of cell differentiation in Dictyostelium discoideum. Nature, Lond. 303, 244-245. Gross, L. D., Town, C. D., Brookman, J. J., Jermyn, K. A., Peacy, M. J. & Kay, R. R. (1981). Cell patterning in Dictyostelium discoideum. Phil. Trans. R. Soc. Lond. B 295, 497-508. Hayashi, M . & Takeuchi, I. (1976). Quantitative studies on cell differentiation during morphogenesis of cellular slime mould, Dictyostelium discoideum. Devi. Biol. 50, 302-309.  51 Jermyn, K. A., Berks, M., Kay, R. R. & Williams, J. G. (1987). Two distinct classes of prestalk-enriched mRNA sequences in Dictyostelium discoideum. Development 100, 745-755. Jermyn, K. A., Duffy, K. T. I. & Williams, J. G. (1989). A new anatomy of the prestalk zone in Dictyostelium. Nature Lond. 340, 144-146. Kay, R. R. (1987). Cell differentiation in monolayer and the investigation of slime mold morphogens. Methods in cell Biol. 28, 433-448. Kay, R. R., Berks, M., Traynor, D., Taylor, G. W., Masento, M. S. & Morris, H. R. (1988). Signals controlling cell differentiation and pattern formation in Dictyostelium. Devi, genetics. 9, 579-587. Kay, R. R., Dhokia, B. & Jermyn, K. A. (1983) Purification of stalkcell-inducing morphogens from Dictyostelium discoideum. Eur. J. Biochem. 136, 51-56. Kay, R. R., Garrod, D. & Tilly, R. (1978). Requirements for cell differentiation in Dictyostelium discoideum. Nature Lond. 271, 58-60. Kay, R. R. & Jermyn, K. A. (1983). A possible morphogen controlling differentiation in Dictyostelium. Nature, Lond. 303, 242-244. Kopachik, W., Oohata, A., Dhokia, B., Brookman, J. J. & Kay, R. R. (1983) . Dictyostelium Mutants lacking DIF, a putative morphogen. Cell 33, 397-403. Krefft, M., Voet, L., Gregg, J. H. & Mairhofer, H. & Williams, K. L . (1984) . Evidence that positional information is used to establish the prestalk-prespore pattern in Dictyostelium discoideum aggregates. EMBO J. 3, 201-206. Kumagai, A. & Okamoto, K. (1986). Prespore inducing factors in Dictyostelium discoideum: Developmental regulation and partial purification. Differentiation 31, 79-84. Kwong, L . , Sobolewski, A., Atkinson, L . & Weeks, G. (1988b). Stalk cell formation in monolayers from isolated prestalk and prespore  cells of Dictvostelium discoideum: evidence for two populations of prestalk cells. Development 104, 121-127. Kwong, L . , Sobolewski, A. & Weeks, G. (1988a). The effect of cAMP on differentiation inducing factor (DIF)-mediated formation of stalk cells in low-cell-density monolayers of Dictvostelium discoideum. Differentiation 37, 1-6. Kwong, L . & Weeks, G. (1989). Studies on the acdumulation of the differentiation-inducing factor (DIF) in high-cell-density monolayers Dictvostelium discoideum. Devi. Biol 132, 554-558. Leach, C. K., Ashworth, J. M . & Garrod, D. R. (1973). Cell sorting our during the differentiation of mixtures of metabolically distinct populations of Dictvostelium discoideum. J. Embryol. exp. Morph. 29, 647-661. MacWilliams, H. K. & Bonner, J. T. (1979). The prestalk and prespore pattern in cellular slime moulds. Differentiation 14, 1-22. Mangiarotti, G., Ceccarelli, A. & Lodish, H. F. (1983). Cyclic AMP stabilizes a class of developmentally regulated Dictvostelium discoideum mRNAs. Nature Lond. 301, 616-618. Masento, M . S., Morris, H. R., Taylor, G. W. Johnson, S. J., Skapsk, A. C. & Kay, R. R. (1988). Differentiation-inducing factor from the slime mould Dictvostelium discoideum and its analogues. Biochem J. 15, 256: 23-28. McDonald, S. A. & Durston, A. J. (1984). The cell cycle and sorting behavour in Dictvostelium discoideum. J. Cell Sci. 66, 195-204. McMahon, D. (1973). A cell-contact model for cellular position determination in development. Proc. Natl. Acad. Sci. U.S.A. 70, 2396-2400. McRobbie, S. J., Tilly, R., Blight, K , Ceccarelli, A. & Williams, J. G. (1987) Identification and localization of proteins encoded by two DIF-inducible genes of Dictvostelium. Devi. Biol. 125, 59-63.  Merkle, R. K., Cooper, K. K. & Rutherford, C. L . (1984). Localization and levels of cyclic Amp during development of Dictvostelium discoideum. Cell Differ. 14, 257-266. Merkle, R. K. & Rutherford, C. L . (1984). Localization of adenylate cyclase during development of Dictvostelium discoideum. Differentiation 26, 23-29. Morris, H. R., Taylor, G. W., Masento, M. S., Jermyn, K. & Kay, R. R. (1987). Chemical structure of the morphogen differentiation inducing factor from Dictvostelium discoideum. Nature, Lond. 328, 811-814. Morrissey, J. H., Devine, K. M . & Loomis, W. F. (1984). The timing of cell-type specific differentiation in Dictvostelium discoideum: Devi. Biol. 103, 414-424. Muller, U. & Hohl, H. R. (1973). Pattern formation in Dictvostelium discoideum: Temporal and spatial distribution of prespore vacuoles. Differentiation 1, 267-276. Newell, P. C. (1982). Cell surface binding of adenosine to Dictvostelium and inhibition of pulsatile signalling. FEMS Microbiol. Lett. 37, 221-226. Newell, P. C. & Ross, F. M . (1982) Inhibition by adenosine of aggregation centre initiation and cyclic AMP binding in Dictvostelium. J. gen. Microbiol. 128, 2715-2724. Newell, P. C , Telser, A. & Sussman, M . (1971). Alternative developmental pathways determined by environmental conditions in the cellular slime mould Dictvostelium discoideum. J. Bact. 100, 763-778. Niedleman, S. L . & Geigert, J. (1986) Biohalogenation: Principles, Basic Roles and Applications, Ellis Horwood, Chichester Oohata, A. (1983). A prestalk cell specific acid phosphatase in Dictvostelium discoideum. J. Embryol. exp. Morph. 74, 311-319. Oyama, M . & Blumberg, D. D. (1986). Cyclic AMP and NH4+ both regulate cell type specific mRNA accumulation in the cellular slime mould Dictvostelium discoideum. Devi. Biol. 117, 557-566.  Ratner, D. & Borth, W. (1983). Comparison of differentiating in Dictyostelium. Nucl. Acids Res. 13, 8853-8866. Schaap, P. (1986). Regulation of size and pattern in the cellular slime moulds. Differentiation 33, 1-16. Schaap, P., Van Lookern Campagne, M . M., Van Driel R., Spek, W., Van Haastert, P. J. M . & Pinas, J. (1986). Postaggregative differentiation induction by cyclic AMP in Dictyostelium: intracellular transduction pathway and requirement for additional stimuli. Devi. Biol. 118, 52-63 (1986). Schaap, P. & Wang, M . (1986). Interactions between adenosine and oscillatory cAMP signalling regulate size and pattern in Dictyostelium. Cell 45, 137-144. Schindler, J. & Sussman, M . (1977). Ammonia determines the choice of morphogenetic pathways in Dictyostelium discoideum. J. Mol. Biol. 116, 161-170. Sobolewski, A. (1987), Ph. D. thesis. The role of cyclic AMP and differentiation-inducing factor in stalk cell differentiation during the development of the cellular slime mold Dictyostelium discoideum. University of British Columbia. Sobolewski, A., Neave, N. & Weeks, G. (1983). The induction of stalk cell differentiation in submerged monolayers of Dictyostelium discoideum. Differentiation. 25, 93-100. Sternfeld, J. & David, C. N. (1979). Ammonia plus another factor are necessary for differentiation in submerged clumps of Dictyostelium. J. Cell Sci. 38, 181-191. Suida, J. F. & DeBernardis, J. F. (1973) Lloydia 36, 107-143 Tasaka, M . & Takeuchi, I. (1981). Role of cell sorting in pattern formation in Dictyostelium discoideum. Differentiation 18, 191196. Tasaka, M . , Noce, T. & Takeuchi, I. (1983). Prestalk and prespore differentiation in Dictyostelium as detected by cell type-specific  monoclonal antibodies. Proc. Natl. Acad. Sci. U. S. A. 80, 53405344. Theibert, A. & Devreotes, P. N. (1984). Adenosine and its derivatives inhibit the cAMP signalling response in Dictyostelium discoideum. Devi. Biol. 106, 166-173. Town, C. D. (1984). Differentiation of Dictyostelium discoideum in monolayer cultures and its modification by ionic conditions. Differentiation 27, 29-35. Town, C. D., Gross, J. D. & Kay, R. R. (1976). Cell differentiation withour morphogenesis in Dictyostelium discoideum. Nature, Lond. 262, 717-719. Town, C. D. & Stanford, E . (1979). An oligosaccharide-containing factor that induces cell differentiation in Dictyostelium discoideum. Proc. Natl. Acad. Sci. U. S. A. 76, 308-312. Tsang, A. & Bradbury, J. M . (1981). Separation and properties of prestalk and prespore cells of Dictyostelium discoideum. Expl. Cell Res. 132, 433-441. Van Haastert, P. J. M. (1983). Binding cAMP and adenosine derivatives to Dictyostelium discoideum cells. Relationship between binding, chemotactic and antagonistic activities. J. Biol. Chem. 258, 9643-9648. Van Lookern Campagne, M . M . V., Schaap, P. & Van Haastert, P. J. M . (1986). Specificity of adenosine inhibition of cAMP-induced responses in Dictyostelium resembles that of the P site of higher organisms. Devi. Biol. 117, 245-251. Wang, M . & Schaap, P. (1989), Ammonia depletion and DIF trigger stalk cell differentiation in intact Dictyostelium discoideum slugs. Development 105, 569-574. Weijer, C. J. & Durston, A. J. (1984). Dependence of cell type proportioning and sorting on cell cycle phase in Dictyostelium discoideum. J. Cell Sci. 70, 133-145.  Weijer, C. J. & Durston, A. J. (1985). Influence of cAMP and hydrolysis products on cell type regulation in Dictvostelium discoideum. J. Embryol. exp. Morph. 86, 19-37. Wilkinson, D. G., Wilson, J. & Hames, B. D. (1985). Spore coat protein synthesis during development of Dictvostelium discoideum requires a low molecular weight inducer and continued multicellularity. Devi. Biol. 107, 38-46. Williams, J. G. (1988). The role of diffusible molecules in regulating the cellular differentiation of Dictvostelium discoideum. Development 103, 1-16. Williams, J. G., Berks, M. M., Kay, R. R. & Jermyn, K. A. (1987). Direct induction of Dictvostelium prestalk gene expression by DIF provides evidence that DIF is a morphogen. Cell 49, 185-192.  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0098326/manifest

Comment

Related Items