Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Overexpression of activated RAS alters cell fate determination during the development of Dictyostelium… Jaffer, Zahara M. 2000

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

Item Metadata

Download

Media
831-ubc_2000-565661.pdf [ 18.68MB ]
Metadata
JSON: 831-1.0089875.json
JSON-LD: 831-1.0089875-ld.json
RDF/XML (Pretty): 831-1.0089875-rdf.xml
RDF/JSON: 831-1.0089875-rdf.json
Turtle: 831-1.0089875-turtle.txt
N-Triples: 831-1.0089875-rdf-ntriples.txt
Original Record: 831-1.0089875-source.json
Full Text
831-1.0089875-fulltext.txt
Citation
831-1.0089875.ris

Full Text

OVEREXPRESSION O F A C T I V A T E D RAS A L T E R S C E L L F A T E D E T E R M I N A T I O N D U R I N G T H E D E V E L O P M E N T O F DICTYOSTELIUM DISCOIDEUM by Z A H A R A M . JAFFER B. Sc., The University of Western Ontario A THESIS SUBMITTED IN P A R T I A L F U L F I L L M E N T O F T H E REQUIREMENTS FOR T H E D E G R E E OF D O C T O R OF PHILOSOPHY in T H E F A C U L T Y O F G R A D U A T E STUDIES (Department of Microbiology and Immunology) We accept this thesis as conforming to the required standard T H E UNIVERSITY OF BRITISH C O L U M B I A August 2000 © Zahara M . Jaffer, 2000 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of M ^ ^ f f l^mu^l^j The University of British Columbia Vancouver, Canada Date OS - I T DE-6 (2/88) 11 ABSTRACT The development of Dictyostelium discoideum provides an attractive model for studying the role of Ras in cell fate determination since amoebae differentiate into one of only two cell types, stalk or spore. RasD is the predominant Ras expressed during development and it is expressed in both cell types. It was previously demonstrated that a transformant overexpressing activated RasD (Ddras-Thrl2) forms multi-tipped aggregates, is blocked from further morphogenesis, and exhibits marked changes in cell type specific gene expression. The morphological phenotype was reproduced in rasD::rasG(G12T) transformants. It was not known if the developmental defects induced by RasD(G12T) resulted from its expression in prestalk or prespore cells. Transformants expressing RasG(G12T) from the prestalk specific ectnAO promoter formed single-tipped aggregates that produced outwardly normal slugs and almost normal cell type specific gene expression. However, prestalk cells were mislocalized and terminal morphology was abnormal: stalk cells were present in basal cell masses and there were no spore cells or stalk tubes. Thus, the formation of multi-tipped aggregates and deregulated cell type specific gene expression is not a consequence of activated Ras expression in prestalk cells. Transformants expressing RasG(G12T) from the prespore specific psA promoter formed mounds with multiple tips and cell type specific gene expression was markedly altered. However, the tips extended to form finger-like and then slug-like structures which then formed stalks. Therefore, the formation of multiple tips and the deregulation of cell type specific gene expression, but not the block in morphological development, results from expression of activated Ras in prespore cells. Since limiting overexpession of activated Ras to either of the cell types did not completely recapitulate the phenotype of the Ddras-lhxll transformant, cotransformants were generated which expressed both ecmAO::rasG(G12T) and psA::rasG(G12T). These cotransformants formed aggregates with multiple protruding tips that were blocked from further morphogenesis. Thus, when activated Ras is expressed from the rasD promoter, the developmental aberrations result from expression in both cell types. TABLE OF CONTENTS iv Abstract. . . . . . . . . . . i i Table of Contents . . . . . . . . . i v List of Tables . . . . . . . . . v i i i List of Figures . . . . . . . . . ix List of Abbreviations . . . . . . . . xi i Acknowledgements . . . . . . . . xiv Dedication . . . . . . . . . . xv CHAPTER ONE INTRODUCTION 1 1.1 Pathways Regulating Development . . . . 1 1.2 Repetitive Use of Signaling Pathways . . . . 6 1.3 Ras P r o t e i n s . . . . . . . . 13 1.4 Development of Dictyostelium discoideum . . . 22 1.5 Effects of Dictyostelium Ras on Development. . . 43 1.6 Rationale and Research Objective . . . . 47 CHAPTER TWO EXPERIMENTAL PROTOCOLS . . . . . . 49 2.1 D N A Preparation for Plasmid Construction . . . 49 2.2 Transformation of Bacterial Cells . . . . 49 2.3 Vector Construction . . . . . . 50 2.4 Growth of Dictyostelium discoideum cells. . . . 57 2.5 Development of Dictyostelium discoideum cells . . 57 2.6 Spore Formation . . . . . . 2.7 Calcofluor Staining . . . . . 2.8 Transformation of Dictyostelium discoideum cells . 2.9 Colony Lift for P-Galactosidase Expression 2.10 In Situ Detection of (3-Galactosidase Expression 2.11 cDNA Probe Preparation . . . . . 2.12 R N A Isolation and Northern Analysis. . 2.13 Protein Isolation and Western Analysis 2.15 Phototaxis Assay . . . . . . CHAPTER THREE OVEREXPRESSION OF RASG(G12T) IN PRESTALK CELLS 3.1 Background . . . . . . . 3.2 Developmental Phenotype of ecmAO::rasG(G12T) Transformants . . . . . . 3.3 Terminal Differentiation of ecmAO::rasG(G12T) Transformants . . . . . . 3.4 Cell Type Specific Gene Expression During Development of ecmAO::rasG(G12T) Transformants . 3.5 PstA Cell Localization in ecmAO::rasG(G12T) Transformants . . 3.6 PstO Cell Localization in ecmAO::rasG(G12T) Transformants . . . . . . 3.7 PstB Cell Localization in ecmAO::rasG(G12T) Transformants . . . . . . 3.8 Prespore Cell Localization in ecmAO::rasG(G12T) Transformants. . . . . . . vi 3.9 Spore Formation in Chimaeras of Wild Type and ecmAO::rasG(G12T) Transformants . . . . 86 3.10 Cell Sorting in Chimaeras of Wild Type and ecmAO::rasG(G12T) Transformants . . . . 88 3.11 Phototaxis of ecmAO::rasG(G12T) Slugs . . . 9 1 3.12 Discussion . . . . . . . . 93 CHAPTER FOUR OVEREXPRESSION OF RASG(G12T) IN PRESPORE CELLS . . 102 4.1 Background . . . . . . . . 102 4.2 Developmental Morphology of psA::rasG(G12T) Transformants . . . . . . 103 4.3 Terminal Differentiation of psA::rasG(G12T) Transformants . . . . . . . 108 4.4 Cell Type Specific Gene Expression in psA::rasG(G12T) Transformants . . . . . I l l 4.5 Prestalk Cell Localization in Developing psA::rasG(G12T) Organisms . . . . . . . . 116 4.6 Prespore Cell Localization in Developing psA::rasG(G12T) Organisms . . . . . . . . 119 4.7 Chimaeric Development with Wild Type Cells . . 121 4.8 Cell Sorting in Chimaeras . . . . . 121 4.9 Overexpression of RasG(G12T) in Prespore and Prestalk Cells 124 4.10 Discussion . . . . . . . . 129 CHAPTER FIVE PERSPECTIVES 136 REFERENCES 149 APPENDIX I Materials Used for This Thesis APPENDIX II. Culture Media and Buffer Recip V l l l LIST OF TABLES Table I. Proteins that regulate differentiation during Dictyostelium development. . . . . . . . 30 Table II. Oligonucleotide primers used for PCR and sequencing reactions. . . . . . . . . 52 Table III. Strains used for the investigations described in this thesis.. 62 Table IV. Spore formation by wild type cells and ecmAO::rasG(G12T) and ecmAOr.rasG transformant cells. . . . . 74 Table V. Spore formation by chimaeras of wild type and ecmAO::rasG(G12T) transformant cells. Table VI. Spore formation by wild type cells and psA::rasG(G12T) and psA::rasG transformant cells. Table VII. Spore formation by chimaeras of wild type and psA::rasG(G12T) transformant cells 89 109 122 ix LIST OF FIGURES Figure 1-1. Spitz and Argos during eye development in Drosophila. . 10 Figure 1-2. Asymmetric cell division of a sensory organ precursor cell during Drosophila neurogenesis. 12 Figure 1-3. Model for RTK-mediated activation of Ras. . 15 Figure 1-4. The developmental program of Dictyostelium discoideum. 24 Figure 1-5. Diagram of the cell types within the slug. 27 Figure 1-6. Amino acid sequence of RasG. . . . . . 44 Figure 2-1. Sequence of the 5' end of the rasG cDNA. . 51 Figure 2-2. Partial cloning strategy for the construction of the psA::rasG(G12T) vector D N A . . . . . . 53 Figure 2-3. Second half of cloning scheme for the psA::rasG(G12T) construct. . . . . . . . 56 Figure 2-4. Cloning strategy for the ecmAO::rasG(G12T) construct. 59 Figure 3-1. Western blot of cell lysates harvested at 16 hours of development and probed with an antibody specific for RasG.. . . 70 Figure 3-2. Developmental morphologies of psAr.lacZ control transformant cells. . . . . . . 71 Figure 3-3. Developmental structures formed by ecmAO::rasG(G12T) transformants at different stages. . . . . 72 Figure 3-4. Calcofluor staining of wild type and ecmAO::rasG(G12T) structures . . . . . 76 Figure 3-5. Northern blots of R N A of ecmAOr.lacZ transformed control cells and of ecmAO::rasG(G12T) cells at various developmental time points. . . . . . 77 Figure 3-6. PstA cell localization in developing wild type and ecmAO::rasG(G12T) structures. . . . . . 79 Figure 3-7. PstO cell localization during wild type and ecmAO::rasG(G12T) development. . . . 81 Figure 3-8. PstB cell localization in wild type and ecmAO::rasG(G12T) structures. . . . . . Figure 3-9. Localization of cells expressing the STr.lacZ construct during wild type and ecmAO::rasG(G12T) development. . Figure 3-10. Prespore cell localization in wild type and ectnAO::rasG(G12T) structures. . . . . . Figure 3-11. Cell sorting in chimaeras of Ax2 and ecmAO::rasG(G12T) cells. . . . . . . Figure 3-12. Results of phototaxis assay. . . Figure 3-13. A schematic diagram depicting the differentiation of the various prestalk cell types during development and the effects of ecmAO::rasG(G12T) expression. . . . . Figure 4-1. Western blots of lysates harvested at 0 hours and 16 hours of development and probed with an antibody specific for RasG. Figure 4-2. Developmental structures formed by psA::rasG(G12T) cells. Figure 4-3. Culminants of wild type, psA::rasG(G12T), and psAr.rasG cells . . . . . . . Figure 4-4. Culminants of Ax2 cells and psA::rasG(G12T) cells stained with calcofluor . . . . . . Figure 4-5. Culminants of Ax2 and rasD::rasG(G12T) cells stained with calcofluor. . . . . . . Figure 4-6. Northern blots of R N A isolated from psAr.lacZ transformed control and psA::rasG(G12T) cells . . . . Figure 4-7. Northern blots of R N A isolated from psAr.lacZ transformed control and psA::rasG(G12T) cells. . . . . Figure 4-8. Developmental structures of wild type and psA::rasG(G12T) cells carrying the ecm.AOr.lacZ reporter. Figure 4-9. Developmental structures of wild type and psA::rasG(G12T) cells carrying the STr.lacZ reporter. . . . . Figure 4-10. Developmental structures of wild type and psA::rasG(G12T) cells carrying the psA::(his)lacZ reporter. Figure 4-11. Cell sorting during chimaeric development. . Figure 4-12. Cell sorting during chimaeric development. . Figure 4-13. Developmental morphologies of cotransformants. Figure 4-14. Culminants of Ax2 cells and ecmAO::rasG(G12T)/ psA::rasG(G12T) cells stained with calcofluor.. X l l 1 1 S T OF ABB^^VTATIONS ALC Anterior like cells BME (3-mercaptoethanol BMP-4 Bone morphogenetic protein-4 BS Bonner's salts starvation buffer cAMP Cyclic adenosine monophosphate cAR cAMP receptor cDNA Complementary deoxyribonucleic acid [35S]-a-dATP [35S]-labeled deoxyadenosine triphosphate [32P]-oc-dCTP [32P]-labeled deoxycytosine triphosphate DIF-1 Differentiation inducing factor-1 DMF Dimethyl formamide D N A Deoxyribonucleic acid dNTP Deoxynucleotide triphosphate ECL Enhanced chemiluminescence EDTA Ethylenediaminetetaacetic acid (disodium EGFR Epidermal growth factor receptor GAP GTPase activating protein GEF Guanine nucleotide exchange factor GSK3 Glycogen synthase kinase 3 HBS Hepes-buffered saline HRP Horseradish peroxidase KK2 A potassium phosphate starvation buffer LB Luria broth M A P K Mitogen activated protein kinase MOPS Morpholinoprapanesulfonic acid m R N A Messenger ribonucleic acid NAPS Nucleic acid processing service PCR Polymerase chain reaction PEG Polyethylene glycol PI3K Phosphoinositol 3-kinase PKA Protein kinase A R N A Ribonucleic acid rpm Revolutions per minute RTK Receptor tyrosine kinase SDS Sodium dodecyl sulphate PAGE Polyacrylamide gel electrophoresis SM V A N A nutrient rich growth medium SSC Sodium chloride/sodium citrate buffer TBE Tris-buffered EDTA TBST Tris-buffered saline with tween-20 TGF(3 Transforming growth factor (3 Tris Tris(hydroxymethyl)aminomethane Tween-20 Polyoxyethylene-20-sorbitan monolaurate U V Ultraviolet X-gal 5-bromo-4-chloro-3-indolyl-p-D-galactoside xiv ACKNOWLEDGEMENTS I take this opportunity to express my gratitude to my supervisor, Dr. Gerry Weeks for his guidance and support. Thanks for always finding time and answers for my seemingly never-ending questions! I am also grateful to my co-supervisor, Dr. George Spiegelman for his direction and insight. I would also like to thank my advisory committee, Dr. Doug Kilburn, Dr. John Schrader, and Dr. Mike Gold, for their critical thinking and for providing interesting ideas and perspectives. Also, I would like to thank the departmental administrative staff for their assistance throughout the course of this thesis. Many members of the Weeks Lab deserve to be acknowledged. Patrick, Kathy, Sharon, I thank you for getting me started in the world of graduate research. Meenal, Emily, Shiv, Sam, Brent, James, David, and Rujun, thank you for your help and suggestions and most importantly for the comic relief that made the tough times not-so-tough. This page is not long enough for me to mention all the people whose friendship has meant so much and wil l continue to wherever I am. Angela, Nureen, and Helen, thanks for always being there and for always listening. To Bilkis Aunty, I thank you for being my family away from home and my friend (and for all the great food)! Karim, I am grateful to you for always knowing what to say, even at times when I thought no words would help. To Mom and Dad, I thank you for always believing in me and for helping me to do the same. X V To my mother and father, Karim, and Ma, for your love and your unwavering faith in me. 1 CHAPTER ONE INTRODUCTION "...[one must have the ... patience to] 'hear what the material has to say to you, [the openness to] let it come to you, ... a feeling for the organism." Barbara McClintock (from Keller, 1983) 1.1 Pathways Regulating Development Every aspect of multicellular development including cell growth and death, axis specification, cell and tissue differentiation, morphogenesis, healing, and regeneration is regulated by signal transduction. John Gerhart (1998) counted at least seventeen different pathways that orchestrate the development of multicellular organisms. Many components of pathways and even the interactions between components are well conserved amongst evolutionarily divergent species. It is this strong conservation that validates the use of model eukaryotic organisms in our attempts to understand the signaling pathways that specify developmental decisions. Some of the best understood of these pathways are briefly described below. 1.1.1 The Wnt Pathway The Wnt signal transduction pathway (Wodarz and Nusse, 1998) is utilized by diverse organisms for varied developmental decisions such as the establishment of parasegment boundaries and cuticle pattern in the Drosophila embryo. A fruitfly Wnt signal, Wingless (Wg), is received by a 7-span transmembrane receptor of the 2 Frizzled family. Disheveled (Dsh) is a cytoplasmic protein required for Wnt signaling. The function of Dsh is not known but the protein is recruited to the plasma membrane in response to the Wg signal and contains protein interacting domains indicating that it may serve as an adaptor protein. Downstream of Dsh is Zeste-white 3 (Zw3) the homologue for the serine/threonine kinase, glycogen synthase kinase 3 (GSK3). Wg signaling represses Zw3 which otherwise constitutively represses Arm/p-catenin by shortening its half-life. With GSK3 activity suppressed, P-catenin accumulates, and forms a transcription factor complex with LEF-1 to regulate gene expression. 1.1.2 The TGFp Pathway The TGFP superfamily signals have been shown to be involved in various developmental processes such as mesoderm ventralization in Xenopus. A distinguishing feature of this pathway (reviewed by Whitman, 1998) is that the TGFP family ligands bind and induce dimerization of two distinct receptor serine/threonine kinases (Cho and Blitz, 1998). The type II receptor phosphorylates and activates the type I receptor which then relays the signal by phosphorylating specific receptor-activated members of the Smad family of proteins. Once phosphorylated, the specific Smads (e.g. Smadl, Smad2) complex with other Smad proteins that are shared amongst TGFP pathways (e.g. Smad4). The Smad complex then translocates to the nucleus and regulates the expression of target genes. A third group of inhibitory Smad proteins (Smad6 and Smad7) antagonize TGFP signaling, either by preventing the type I receptor-mediated phosphorylation of Smadl and Smad2 proteins or by preventing the association of phosphorylated Smadl and Smad2 proteins with Smad4. In some cases, the-inhibitory Smad proteins are induced by the signal they antagonize resulting in a negative feedback loop that limits signal duration. TGFf} signaling is also negatively regulated by factors secreted by the dorsal mesoderm. Noggin, Chordin and other factors in Xenopus bind to TGF(3 family signals and prevent their interaction with receptors and consequently permit dorsal development. 1.1.3 The Hedgehog Pathway Signaling via the Hedgehog (Hh) pathway has a demonstrated involvement in the patterning of various structures including the Drosophila wing and the chick limb. Two different transmembrane proteins, Smoothened (Smo) and Patched (Ptc), are part of the Hh signaling pathway (reviewed in Johnson and Scott, 1998). It appears that Ptc inhibits Smo, possibly through a physical association. This inhibition is relieved in the presence of the Hh signal, allowing Smo to relay the signal. The endpoint of the Hh pathway is the control of a transcription factor of the Gl i family. The Drosophila transcription factor, Cubitus interruptus, C i , exists in a complex and is released by Hh signaling. Activation of C i results in the transcription of specific genes including ptc, as well as genes encoding members of other signaling pathways. 1.1.4 The Notch Pathway The Notch pathway (reviewed in Weinmaster, 1998) acts in neurogenesis, myogenesis, and hematopoiesis to prevent progenitor cells from differentiating. In 4 addition, signaling via Notch can promote the specification of certain cell types. Notch is a cell surface receptor and is activated upon interaction with another cell expressing a specific ligand (Jagged 1 or 2, or Delta 1, 2, or 3). Once activated, Notch is proteolytically cleaved such that the intracellular portion of the protein is released. Truncated Notch protein then interacts with a transcription factor of the CSL group. This association results in the conversion of the CSL family protein from a transcriptional repressor to a transcriptional activator. Surprisingly, disrupting the Notch domain required for interaction with the CSL protein does not necessarily disrupt Notch-mediated signaling, indicating the presence of an alternate Notch pathway, independent of the CSL proteins. 1.1.5 The Receptor Tyrosine Kinase Pathway Pathways downstream of receptor tyrosine kinases (RTK) (Gerhart, 1998) are involved in numerous developmental processes such as eye, limb, and muscle development. Ligand binding to the extracellular domain induces dimerization of receptor molecules and cross phosphorylation of their cytoplasmic tails. The activated receptors can then promote signaling via downstream pathways such as the Ras and the phosphoinositol-3 kinase (PI3K) pathways. The RTK-Ras pathway is addressed in more detail later in Section 1.3. 1.1.6 Signaling Networks That Organize Development In recent years, much progress has been made in dissecting the various pathways, such as the ones described above, that are utilized to transmit extracellular signals to the nucleus. It has become apparent, however, that these 5 signaling pathways do not work in isolation, rather developmental decisions are made in response to a combination of various positive and negative signals transduced through multiple pathways. The process of muscle development provides one example of how signaling pathways converge to regulate development. The MyoD family of proteins are basic helix-loop-helix transcription factors that interact with transcription factors of the MADS family and are essential for the specification and differentiation of vertebrate skeletal muscle (reviewed in Brand-Saberi and Christ, 1999; Chen and Goldhamer, 1999). In mice, two members of the MyoD family, MyoD and Myf-5 are required for muscle lineage determination and their overexpression can convert many cells to the myogenic fate. The other two family members, Myogenin and MRF4 are required for the differentiation of committed cells. Investigations into the regulation of expression of the genes of the myoD family revealed the presence of complex signaling interactions. Wnt signals from the neural tube and the dorsal ectoderm function to induce the expression of myoD and myf-5. It is possible that different Wnt signals may induce the different myoD genes. Sonic hedgehog (Shh) signals from the notochord and the ventral neural tube also positively regulate myogenic specification. Ectopic shh expression results in an increased domain of rayoD-expressing cells whereas shh knockout mice exhibit a block in myf-5 expression. MyoD-dependent muscle cell specification is also regulated by negative signals such as Bone Morphogenetic Protein-4, BMP-4 (a TGFfi family signal), produced by the lateral plate mesoderm. A further complexity is that the negative influence of BMP-4 is antagonized by Noggin and Noggin is downstream of both Wnt and Shh. These results indicate that the process of muscle development (and probably all other developmental processes) is coordinated by a complex network of signals that are only just beginning to be understood. 1.2 Repetitive Use of Signaling Pathways Although numerous signal transduction pathways operate during multicellular development, these are used repeatedly by the same organism to specify very different outcomes. The Drosophila epidermal growth factor receptor (EGFR) homolog provides an excellent example to illustrate this point (Perrimon and Perkins, 1997; Freeman, 1998). In the embryo EGFR is involved in several processes including the establishment of ventral cell fates, the specification of cells of the nervous system, and the production of cuticle. It appears that in all instances, EGFR-mediated effects are dependent on signal transduction through a single signaling pathway, that involving Ras/Raf/MAPK. (This pathway wil l be discussed later in Section 1.3). Consequently, the question arises as to how one signaling pathway can specify such a diverse set of outcomes. In the next few sections, I wil l briefly present some of the common mechanisms by which various signaling pathways ensure the specificity of downstream effects. 1.2.1 Negative Feedback Mechanisms In a recent review, Perrimon and McMahon (1999) discussed how negative feedback of incoming signals can regulate a cell's response either by regulating the level of induced activation, by spatially limiting the field of cells responding to the signal, or by modulating the effects of responding cells according to signal 7 concentration. Negative feedback can be cell autonomous, limiting an activated cell's response to a signal, or non-cell autonomous, also limiting the response of neighboring cells. RTK signaling in Drosophila is subjected to cell autonomous negative feedback control by the cytoplasmic protein Sprouty (Sty). Sty was first observed to function as a negative regulator of FGFR signaling during tracheal development (Hachohen et al., 1998). Subsequently, Sty was also shown to inhibit EGFR signaling (Casci et al., 1999). Loss of sty results in eyes with too many photoreceptor, cone, and pigment cells whereas over expression of sty is correlated with a decrease in the number of these eye cells. In the same study, Sty was also found to antagonize the effects of activated Torso (Tor) and Sevenless (Sev), two other Drosophila RTKs. Sty appears to regulate RTK signal transduction by preventing transmission of the signal to Ras. Sty binds to the Drk adaptor protein and to Gap 1 indicating inhibitory mechanisms whereby Sty may bind Drk and prevent the recruitment of SOS. Alternatively, Sty may bind and recruit Gapl to the plasma membrane to inactivate Ras. Since sty expression is dependent on the pathway it consequently inhibits (Hachohen et al., 1998; Casci et al., 1999) it functions as a cell autonomous negative regulator modulating the level of activity of the RTK/Ras pathway. The importance of controlling the level of signal activity is illustrated by the work of Greenwood and Struhl (1997). Torso (Tor) is involved in specifying the terminal parts of the Drosophila embryo. Active Tor signals through the Ras pathway to induce expression of the genes for the Tailless (Til) and Huckebein (Hkb) transcription factors. The level of Tor/Ras activity determines the expression of these two genes and the differentiation of terminal structures (Greenwood and 8 Struhl, 1997). A high level of Ras activity results in the expression of both til and hkb and the consequent specification of terminal structures such as the anal pad and tuft. Lower Ras activity results in the expression of til but not hkb and the specification of less terminal structures such as abdominal parts. How the intensity of Tor-mediated Ras activity is regulated is not known. It is possible that the level of Tor activation may itself be regulated by a gradient of the activating ligand. In light of the above-mentioned findings, it is conceivable that Sty or another negative regulator may fine-tune the level of Ras activity to specify different developmental structures. Drosophila RTK signaling is also subject to non-cell autonomous negative feedback regulation. As described above, the EGFR functions in the specification of several cell fates. Argos (Aos) has been shown to modulate the level of EGFR signaling to specify several distinct outcomes (Freeman, 1996; Golembo et al., 1996a; Wasserman and Freeman, 1998). Aos contains an EGF motif (Howes et al., 1998) and is a secreted inhibitor of EGFR signaling (Schwietzer et al., 1995). The expression of aos is dependent on EGFR signaling (Golembo et al., 1996b) and this thus constitutes a negative feedback loop that influences EGFR signaling in neighboring cells. In the developing eye, the EGFR is required for the specification of all cell types in successive waves of induction (Freeman, 1996). First, there is recruitment of the photoreceptor neurons, followed by recruitment of the cone cells, and finally recruitment of the pigment cells. (The requirement for EGFR in R8 photoreceptor cell differentiation is not known). Inhibition by Aos establishes the necessary waves of induction to ensure that in a particular subset of cells, EGFR is activated when the cells are competent to differentiate into the correct cell type. Initially, the EGFR 9 ligand, Spitz (Spi), is secreted by the central cells (R8, R2, R5). This results in the differentiation of neighboring cells into photoreceptor cells (R3, R4, R l , R6, R7). As the cells differentiate, they express and secrete Aos which diffuses outward to prevent more distal cells from assuming the photoreceptor fate. Later, these distal cells overcome Aos inhibition and produce Spi (Fig. 1-1). Aos thus spatially restricts the activation of the EGFR, enabling specific patterning during development. 1.2.2 Morphogen Gradients Spatial concentration gradients in morphogen distribution can invoke different developmental decisions on a field of cells. One way in which such gradients are established is by regulating the spatial distribution of the receptor for a secreted morphogen (Perrimon and McMahon, 1999). Ptc is the transmembrane protein proposed to bind Hh (Chen and Struhl, 1996). Ptc expression is induced by the presence of Hh. The absence of Ptc correlates with an increase in the domain size of Hh-activated cells. Also, high levels of Ptc restrict the spread of Hh. Thus, by activating the expression of its own receptor, Hh uses a negative feedback inhibition mechanism to limit its ability to move through developing tissue. Variations in the ability of different cells to induce ptc expression in response to Hh may facilitate the establishment of different distribution ranges for the signal. 1.2.3 Asymmetric Cell Division One mechanism that enables a diversity of cell fates to be established from a 1 si I si U 1 C J T u t t CD O O o o N CO < CO o 5-H *H as cc T3 '3 u QJ as CO "a! a, 0 ) ^ SH G o ns - M . QJ O u rj QJ H 0 ) o U o u OH-- 3 to OHT3 en o 60 " • ^H 3 0 ) Ki co 13 a» C o u CD "al T 3 QJ 0 ) QJ aj co O O T3 CJ ^ cu OJ o * co " £ ! G a 8 u O 3 SH OJ J-H ^ • i"H UJ OJ co C O OH CO O OJ 13 •£ > H OJ ^ g OJ o to OJ cS •5 x T3 £ c C3 N 00 .s 3 OH . QJ co U O Si < g OH £ < OH G C3 J-l +-> c QJ u QJ 1-4 re CO 0 ) u H-> r£> G OJ CO O 0 J-l re £ 14 <o 00 <~> 0\ •s a» cr\ OH^ 0 0 "5 QJ OH^ 2 OH . co box ^ Si £ OJ QJ u QJ CO K r l '— i £ OJ u QJ U S c^2 u O OH OH CC 11 single progenitor cell is that of asymmetric cell division (Rhyu and Knoblich, 1995). The result of such cell division is the generation of two daughter cells that are unequal with respect to some fate-specifying agent. For example, during embryonic Drosophila development, a sensory organ precursor (SOP) cell divides asymmetrically generating a neuron and glia precursor (NGP) cell and a shaft and socket precursor (SSP) cell. Another round of asymmetric cell division results in four distinct daughter cells: a neuron, a glia, a socket, and a shaft (hair) cell (Okano, 1995). One of the genes regulating this process is numb (Uemura et al., 1989; Rhyu et al., 1994). Loss-of-function numb mutants prevent the first asymmetric division and instead generate two SSP cells. Consequently, the differentiation of neuron and glia cells does not occur. Overexpression of numb results in the opposite phenotype and two NGPs are formed (Rhyu et al., 1994). Numb is a plasma membrane protein and its distribution is in the shape of a crescent that is limited to half of the SOP and overlies one of the centrosomes (Rhyu et al., 1994; Knoblich et al., 1995). Since cell division is asymmetric, only one daughter cell, the NGP, inherits the Numb protein indicating that it is the differential acquisition of Numb that distinguishes the lineage (Fig. 1-2). During the second round of asymmetric cell division, Numb is again localized to only half the cell membrane and is consequently only bequeathed to the neuron cell. The SSP cell that did not inherit Numb from the SOP, also divides asymmetrically. Before mitosis, Numb is synthesized and localized to half the cell membrane. Upon division, the daughter cell inheriting Numb becomes a shaft cell (Wang et al., 1998). The repetitive use of Numb indicates that it is a 12 TJ .55 <u S lo j £ rt g ~ O o .2 w w ej 55 ~ .5 TJ CS ^ _ & H a 7! tfi A cu 3 O O J-i t-H -t-> O <s w j £ co cS I-H TJ 1 ) CU CJ O g cu cj o co TJ C cS CS X, co O u w cu cu CJ -cu C ^ • — | OH g o & H s i - 3 Pi 2 CO CD CS CO cS Of VH cu CO CU CS CJ cy C v a» CH M-I •2 xi CO 11 % o co X J-H j g CU £3 * cu ^3 <-» < n cu .2 tb TJ r3 co cu CJ CU cu co el o > • i—I TJ IE! u u • I—I VH +-> CU B CU M-H 45 O ' el o .g "cu u CU CU I-H o CO 3 ^_ (-H O CO S-H CJ CU S-H CIH CO CU +-> cS . y o tH CU C CO bp cs <N CU es HH CS TJ el cs >< § S-H cy ^ X CU CU TJ ^ CO CU (S N XI CU CO •y cu C H-J 2 CO -J3 cu el tH CU O n M-l CU "55 s 00 C?\ O N CS -t-> CU CO 3 c^ 6 2 o . 1 tH TJ ^ c & CS OH ^ cS CO ^ H 13 common mediator for distinguishing two different daughter cell fates following cell division (Lu et a l , 1998). 1.3 Ras Proteins A key signaling intermediate involved in multiple cell fate decisions is Ras. The Ras proteins are small monomeric guanine nucleotide binding proteins (G proteins) associated with the inner surface of the cytoplasmic membranes of all eukaryotic cells. Ras was originally identified as the transforming oncogene in animal tumor viruses. Furthermore, the human Ras proteins, H-Ras, N-Ras, and K-Ras were found to be mutated in approximately 15% of all tumors (Bos, 1989). Wild type Ras proteins cycle between a GTP-bound 'on' state and a GDP-bound 'off state in their function as molecular switches (Satoh et al., 1992). They are involved in the transduction of diverse signals from cell surface receptors via various effectors to regulate cell growth, differentiation, and apoptosis (reviewed in Khosravi-Far and Der, 1994; Denhardt, 1996; Campbell et a l , 1998). Ras proteins possess intrinsic GTPase activity which is very low but which is enhanced by GTPase activating proteins or GAPs (Wittinghofer, 1998; Boguski and McCormick, 1993). The GAPs (pl20GAP and NF1) are thus negative regulators of Ras activity. Amino acid substitutions at positions 12, 13, 61 interfere with GAP-stimulated GTP hydrolysis and thus render the mutant Ras protein constitutively active. The guanine nucleotide exchange factors (GEFs), including SOS1, SOS2, CDC25, RasGRF, postitively regulate Ras activity by promoting the exchange of bound GDP for GTP (Boguski and McCormick, 1993; Quilliam et al., 1995). 14 1.3.1 Role of Ras in Cell Proliferation and Transformation Once activated, Ras induces proliferation in diverse cell types and constitutively active Ras has transforming potential (Lowe and Skinner, 1994). Ras proteins are activated by a variety of signals that bind to and activate receptor tyrosine kinases (RTKs), receptor-associated tyrosine kinases, as well as heterotrimeric G protein-coupled serpentine receptors (Khosravi-Far and Der, 1994). The best characterized signaling pathway that links a cell surface receptor to Ras is downstream of activated RTKs (reviewed in Schlessinger, 1993; Khosravi-Far and Der, 1994) (see Fig. 1-3). In response to ligand binding, individual RTK molecules dimerize and transphosphorylate their cytoplasmic tails. The adaptor protein, She, binds to the tyrosine phosphorylated receptor via Src homology 2 (SH2) domains (Mayer and Baltimore, 1993; Feig, 1994). She is then phosphorylated and directs the binding of another adaptor protein, Grb2 via its SH2 domain. She is not required for Grb2 recruitment since Grb2 can also interact directly with activated EGFR. Grb2 is associated with SOS; consequently then, the association of Grb2 with She (or with an activated RTK) effectively results in the recruitment of SOS to the plasma membrane where it can activate Ras (Feig, 1994; Khosravi-Far and Der, 1994). A n important downstream effector of active Ras is the serine/threonine kinase Raf, a mitogen activated protein kinase kinase kinase (MAPKKK or MEKK), (Moodie and Wolfman, 1994). When bound to GTP, Ras can interact with Raf which results in the translocation of Raf to the membrane where it is activated (Morrison and Cutler, 1997). Raf then phosphorylates a M A P K kinase, MEK1 or MEK2 (Crews and Erickson, 1993). The MEKs then phosphorylate tyrosine and threonine motifs in two MAPKs, ERK1 and ERK2 (Crews et a l , 1992). These 8 "8 43 bO 3 bo 8 4 > O 1 8 -g g C 41 P 41 4) 04 o c * U O 0 g U Pi 4) T J 0 1 c .H £ <D cu h w £ T J $ x5 g u co 3 M-j T J C cu 5 O co £ P - 3 co "SI'S 43 a, ,_, 73 o 2 C 4 H C L u 4) 4» CO -t-> CO 4) 5 - 1 CO 2 o % O S S 3 « 2 j2 P-i Q O T J <*> C 4=1 ^ •5 43 i-* M - l T J 0 41 41 6 l o x U 41 CO 4) c N ^ 43 4) 41 13 ro U & 9 i 8 2 cc 41 "^ 3 >~> o 6 % a « 2 •2 7L Cu . £ >^4* x, cu •Q U Cu"£ ST3 5 3 ^ T J ^ 43 D C u 6 -2 x O Cu S ^ co Cu ^ 4^ O T J n +j O Cu Cu ^ 4» nj ^ 43" T J a >^  4) *Q 4^ T J H | 3T • O cC u CD -2 -~ u a IH 4) O 4^ ^ I: 41 > .•tt 41 CO C bO g .S 43 TJ g • 1—I 43 4) 6 CO 41 1-1 o • cfl 4) co 43! 8 -2 4) W co en 16 activated M A P K s then translocate to the nucleus where they activate kinases such as p90RSK (Blenis, 1993), or transcription factors such as Elk-1 (Marais et al., 1993), and thereby mediate the regulation of gene expression. This linear Ras/Raf /MAPK signaling cascade has been shown to be conserved through evolution. However, it is only one of several signaling pathways downstream of Ras (Campbell et al., 1998). Effector domain mutants of activated Ras that are unable to interact with Raf (White et al., 1995) still retain some properties of Ras-mediated transformation (Khosravi-Far et al., 1996) indicating that additional effectors must function downstream of active Ras. It was subsequently shown that GTP-bound Ras activates two other M A P K cascades - the J N K / S A P K and the p38/HOG cascades (Kyriakis and Avruch, 1996) that are dependent on MEKK1 (Johnson et al., 1996) but independent of Raf (Minden et a l , 1994; Olson et a l , 1995). Thus, MEKK1 is likely another direct effector of Ras. Although the Ras GAPs, pl20 GAP and NF1 negatively regulate Ras activity (Boguski and McCormick, 1993), there is evidence supporting effector roles for both proteins (McCormick, 1995; Tocque et al., 1997). pl20GAP knockouts (Henkenmeyer et al., 1995) and NF1 knockouts (Bollag, et a l , 1996) are both embryonic lethal in mice. In addition, the N-terminal domain of pl20GAP disrupts the actin cytoskeleton, possibly via an interaction with the GAP for another small GTPase, Rho (McGlade et al., 1993). NF1 probably also has additional signaling roles. Although it is unable to stimulate GTPase activity of activated Ras, NF1 inhibits transformation by v-Ras (Johnson et al., 1994). Another enzyme that functions as an effector of Ras is phosphatidylinositol 3-kinase (PI3K) which catalyzes the phosphorylation of the 3' position of the inositol 17 ring of phosphoinositides (Carpenter and Cantley, 1996a). Ras interacts with and activates multiple isoforms of PI3K (Rodriguez-Viciana, et al., 1997). Ras can activate PI3K in vivo and expression of activated Ras in COS cells results in an increase in the levels of PIP 3 (Rodriguez-Viciana et al., 1994). The pathways downstream of PI3K include the small GTPase Rac, p70S6 kinase, protein kinase B (PKB/Akt), and several atypical isoforms of PKC (Carpenter and Cantley, 1996b). Evidence has recently been accumulating for the GEFs of another small GTPase, Ral, as effectors for Ras (Feig et al., 1996). Transient expression of Ras in COS cells has been shown to increase RalGEF activity (Urano et al., 1996). Furthemore, overexpression of the Ras-interacting domain (RID) of the RalGEFs inhibits Ras-mediated NIH 3T3 transformation and Raf activity. However, RID overexpression did not reduce Raf-mediated transformation, indicating that RID specifically binds to Ras and prevents transduction of the signal from Ras to Raf (Okazaki et al., 1996; Peterson et al., 1996). In addition, dominant negative Ral decreases Ras-mediated focus formation (Urano, et al., 1996). The existence of a several downstream effectors for Ras raises a number of interesting scenarios (Campbell, et al., 1998). It is possible that some effectors may mediate the functions of normal Ras where as others are activated only in response to oncogenic Ras. Some of these putative effectors may actually be negative regulators that prevent productive interactions with signal transducing effectors. Other effectors may confer tissue specific consequences for Ras signaling. Perhaps different Ras proteins can activate different pathways via specific effectors. Also, given the very high degree of conservation amongst the proteins of the Ras family, 18 it is possible that some of the putative Ras effectors may actually function as effectors for Ras-related proteins under physiological conditions. 1.3.2 Roles for Ras in Development One of the first indications for a role for Ras in differentiation came from experiments conducted with the PC12 phaeochromocytoma cell line. In this cell line, activated Ras can replace the requirement for nerve growth factor (NGF) for induction of neuronal differentiation (Szeberenyi et al., 1990). Since then, Ras has been shown to be involved in the regulation of developmental decisions in several organisms. Although the pathways mediating these decisions are not fully understood, it appears that the pathways defining Ras-mediated differentiation are the same as those that mediate proliferation. 1.3.2.(a) Invasive Growth of Saccharomyces cerevisiae Haploid S. cerevisiae cells respond to nitrogen starvation by initiating a program of invasive filamentous growth (Roberts and Fink, 1994). Mosch et al. (1999) studied the role of Ras in this differentiation process. The S. cerevisiae Ras homologues, Raslp and Ras2p (see Broach, 1991 for a review) are independently dispensable for growth but a double knock out is lethal. However, ras2 null cells cannot induce filamentous growth. Clearly, endogenous Raslp cannot substitute for Ras2p but, overexpression of rasl does rescue the loss-of-function ras2 defect. It was found that Ras2p induces the invasive growth response by activating both the M A P K and P K A pathways. Thus, in addition to regulating the mitosis (Morishita et 19 al., 1995) and longevity (Sun et al., 1994) of S. cerevisiae cells, Ras2p also regulates the ability of these cells to undergo starvation-induced differentiation. 1.3.2.(b) Embryonic Axis Formation in Xenopus laevis During Xenopus embryogenesis, basic fibroblast growth factor (bFGF), activin, and BMP-4 have been identified as inducers of mesoderm development. A l l three mediate their effects via Ras (Whitman and Melton, 1992; MacNicol et a l , 1993; Xu et a l , 1996). BMP-4 is required for ventral mesoderm development, activin is required for dorsoanterior development, and bFGF is required for posterior and lateral development. Thus, Ras activity is implicated in multiple developmental pathways during Xenopus embryogenesis. Interestingly, although activin and BMP-4 are both proteins of the TGF-p family, BMP-4 requires both Ras and Raf function for its effects (Xu et al., 1996) while activin does not require Raf (Whitman and Melton, 1992). This distinction indicates that diverse Ras signaling pathways may lead to different developmental effects. 1.3.2.(c) Roles for Ras during Caenorabhditis elegans Development The best characterized role for Ras during the development of C. elegans is vulval cell specification (Kayne and Sternberg, 1995; Sternberg and Han, 1998). Normally, six multipotent vulval precursor cells (VPCs) can differentiate into one of three fates (Kenyon, 1995; Sundaram and Han, 1996). Above the VPCs lies an anchor cell (AC) which induces vulval development. The VPC that lies beneath the A C generates the 1° lineage and the two adjacent VPCs generate the 2° lineage. Each of the 1° and 2° VPCs divide three times and produce vulval cells. The 1° VPC 20 produces 8 vulval descendents that become attached to the A C . The 2° VPCs produce 7 vulval cells each. In the 3° lineage, each of the three remaining VPCs divide to produce two epidermal cells (Sulston and White, 1980). Without induction, all 6 VPCs assume the 3° epidermal fate and no vulva forms. Overinduction of the vulval signaling pathway results in a multivulva phenotype. The C. elegans homolog of Ras, Let-60, is involved in this pathway of vulval cell specification (Han and Sternberg, 1990). Dominant-negative let-60 alleles were found to suppress a multivulva phenotype. Gain-of-function let-60 alleles were found as multivulva mutants in an otherwise wild type background or as suppressors of a vulvaless mutant. The nematode homologue for the EGFR, Let-23, was shown to be upstream of Let-60 Ras. Downstream of Ras, this signaling pathway involves the C. elegans homologs of Raf (Han et al., 1993), MEK2 (Wu et a l , 1995), and M A P K (Lackner et al., 1994; Wu and Han, 1994). Thus the RTK/Ras /Raf /MAPK pathway described earlier is conserved in C. elegans and regulates the decision of vulval fate specification. There is evidence for a combined role of Ras and HOX genes in vulval fate specification. Let-60 Ras mediated vulval specification requires the product of the HOX gene lin-39 (Maloof and Kenyon, 1998). lin-39 is expressed at a low level in the VPCs (Clark et al., 1993). In response to Ras induction, lin-39 expression is upregulated (Maloof and Kenyon, 1998). If Lin-39 is absent during Ras signaling, the VPCs do not divide and vulval cells are not formed. In addition to permitting Ras-mediated differentiation, HOX genes can confer specificity. Ras signaling is also involved in the differentiation of the pre-anal ganglion (PAG). The P A G precursors express the HOX gene mab-5 (Salser et al., 1993). If lin-39 is expressed in mab-5' 21 animals, induced P A G precursor cells adopt vulval characteristics. Similarly, when mab-5 was expressed in a lin-39 deficient organism (using a temperature sensitive lin-39 allele), the VPCs adopted the P A G fate. The results of this study indicate that one way in which specificity can be achieved downstream of Ras signaling is by the HOX gene product active in a given cell. C. elegans Let-60 Ras is involved in other fate specification choices during development. The first let-60 mutations identified were recessive lethal (Clark et al., 1988; Rogalski et al., 1982). Homozygotes carrying a null allele die by the fourth larval stage and exhibit a fluid-filled morphology (Han and Sternberg, 1991). A recent mosaic analysis has indicated that death does not result from a defect in cell proliferation at this stage but from the absence of excretory duct differentiation (Yochem et al., 1997). Normally one of two competent cells responds to a signal, activates Ras, adopts the excretory duct cell fate and at the same time, laterally inhibits the second cell from adopting the same fate. Gain-of-function Let-60 Ras can promote both cells to adopt the excretory duct cell fate. Thus, Ras is critical in establishing the excretory duct that is essential for viability. 1.3.2.(d) Roles for Ras During Drosophila melanogaster Development The role for Ras during the development of photoreceptors in the Drosophila compound eye has been studied extensively. The eye imaginal disc, a set of undifferentiated multipotent cells differentiates to form the eye which consists of 800 ommatidia (reviewed in Wolf and Ready, 1993). Each ommatidium contains 8 photoreceptor cells (R1-R8), 4 cone cells, and 8 accessory cells. During the establishment of this complex structure, Drosophila Rasl plays an integral role in 22 the establishment of cell fates (reviewed in Wassarman et al., 1995). Differentiation of the R7 photoreceptor is triggered by the activation of the Sevenless (Sev) RTK. Subsequently, Rasl is activated via the SOS GEF and the Drk adaptor protein. Rasl is upstream of a Raf homologue which is, in turn, upstream of a M A P K homologue. In addition, the Rasl signaling cascade, which is activated by the EGFR, is required for the differentiation of all cell types in the eye (Freeman, 1996). Rasl also functions downstream of other RTKs during Drosophila development. The Drosophila FGFR1, the product of the breathless gene, is required for migration of tracheal cells (Klambt et al., 1992). Activated forms of Rasl or Raf suppress the migration defect in btl' animals (Reichman-Freid et al., 1994). I have already mentioned that Rasl functions downstream of the Tor RTK in the specification of terminal structures of the developing embryo (Greenwood and Struhl, 1997). In addition, the Drosophila EGFR is involved in multiple developmental pathways, some of which have been mentioned previously. Ras signaling mediates EGFR-mediated differentiation in all instances (Freeman, 1998). The multitude of differentiation processes in Drosophila that require Ras signaling indicate the importance and versatility of Ras as a regulator of developmental decisions. 1.4 Development of Dictyostelium discoideum Dictyostelium discoideum is a unicellular slime mold for which the processes of growth and differentiation are largely separate events. In the wild, vegetative amoebae feed on bacteria within the upper layers of the soil and divide by binary fission. When the nutrient source has been depleted, an interactive mode of 23 development is initiated (reviewed in Loomis, 1993). In response to pulses of cAMP, up to 105 cells aggregate to form a mound. A tip forms at the apex of the mound and then elongates to form a finger-like structure that can fall to the substratum to form a migrating slug. The slug sits on end to initiate the process of culmination which results in the formation of a fruiting body that consisting of a spore-filled sorus supported on a column of stalk cells (Fig. 1-4). 1.4.1 Cell Type Differentiation and Pattern Formation As the aggregating cells compact into a tight mound, they assume the characteristics of either one of two cell types: prestalk or prespore. There is evidence that a cell's decision to become either prestalk or prespore is biased by an inherent heterogeneity in the vegetative cell population resulting from the cell's position in the cell cycle at the time of starvation (Maeda, 1993). It has been shown that cells starved late in the cell cycle preferentially differentiate as prespore cells and those starved early in the cell cycle preferentially differentiate as prestalk cells (Ohimori and Maeda, 1987). In addition, it is likely that the cell cycle position biases cell fate by affecting the speed with which cells enter the aggregate (Araki et al., 1997). Thus, cells starved early in the cell cycle enter the aggregate late and consequently, respond to positional cues directing their differentiation as prestalk cells. Although such biases exist in a natural population of starving amoebae, if cells are synchronized prior to starvation, both prestalk and prespore cells are formed suggesting that other factors exist to regulate differentiation (Maeda, 1997). A key feature of Dictyostelium development is that cell type differentiation is regulative. Differentiation occurs such that by the slug stage, 20% of the cells are 2 4 Figure 1-4: The developmental program of Dictyostelium discoideum. Development progresses clockwise in the figure beginning with the mound stage at the bottom right and culminating with formation of the fruiting body, top right. The structure in the bottom left corner is the migrating slug. 25 prestalk cells and 80% are prespore cells. This proportionality is maintained regardless of the size of the developing organism (Loomis and Cann, 1982). In fact, once differentiation has occurred, if the prestalk cells are removed, prespore cells wil l transdifferentiate to prestalk cells until the correct proportions are reestablished (Raper, 1940; Sakai, 1973). The mechanism by which the cells sense and maintain proportion homeostasis is not known. It has been suggested, however, that lateral inhibitors are employed by the differentiated cell types to maintain their proportions within the organism (MacWilliams and Bonner, 1979; Meinhardt, 1983; Loomis, 1993). Although the prestalk population only comprises 20% of the cells in the developing organism, it is complex. As the aggregate forms, the different prestalk subtypes become identifiable based on the differential expression of prestalk specific genes, as detected by coupling the promoters of these genes to lacZ. Two classes of cells express the gene ecmA which encodes a prestalk cell specific extracellular matrix protein (Williams et al., 1987; McRobbie et al., 1988). The proximal region of the ecmA promoter directs expression in PstA cells and the distal region directs a comparatively lower level of expression in PstO cells (Jermyn and Williams, 1991; Early et al., 1993). In the multicellular structure, PstA and PstO cells are labeled with reporter constructs using the appropriate promoter element. PstA cells are first visible at the periphery of the mound while PstO cells are first detected scattered in the mound (Early et al., 1995). As the mound compacts and a tip emerges, the PstA cells move from the periphery to the apex and populate the tip while the PstO cells sort to the region immediately below to the tip (Early et al., 1995). The differential sorting of the two cell types has been hypothesized to involve 26 differential chemotaxis with the PstA cells chemotaxing towards cAMP twice as fast as PstO cells (Early et al., 1995). During subsequent morphogenesis, as the mound elongates to form a finger and then a migrating slug, the PstA cells remain at the tip with the PstO cells forming a collar just behind them. Together, these two cell populations make up most of the anterior prestalk region of the developing organism (Fig 1-5). Also during aggregate formation, another prestalk cell type, PstB, is detected. These cells are characterized by the expression of ecmB, which encodes another extracellular matrix protein (McRobbie et al., 1988; Ceccarelli et al., 1987). The PstB cells are initially scattered throughout the mound and then accumulate at the base (Jermyn et al., 1996). If the slug culminates in situ, these PstB cells form part of the basal disc (Jermyn et al., 1996). If the slug migrates, PstB cells form a rearguard region at the posterior from which cells are shed during migration (Sternfeld, 1992; Jermyn et al., 1996). A second distinct region of ecmB expression is detected in the anterior tip of the slug in a central cone of cells (Jermyn and Williams, 1991; Sternfeld, 1992). Since the cells in the cone express both the ecmA and ecmB genes, they have been referred to as PstAB cells (Jermyn and Williams, 1991). These cells lie at the position where the stalk tube formation is initiated. A final group of prestalk cells is found scattered throughout the prespore region of the developing slug. These cells, referred to as Anterior Like Cells (ALC), vary in their expression patterns of the ecmA and ecmB genes such that individual A L C may express either one or both of these genes. 27 Figure 1-5: Diagram of the cell types within the slug. The horizontal lines in the anterior tip represent the PstA compartment and the diagonal lines represent the PstO collar. The triangle in the PstA zone represents the PstAB cells that form in the tip during migration. The scattered cells throughout the slug are the anterior like cells and the cluster of cells at the posterior are the PstB rearguard cells. 28 As the slug migrates, the PstB cells at the posterior are left behind in the slime trail (Sternfeld, 1992; Jermyn et al., 1996) and are replaced by the posterior movement of PstAB cells from the anterior cone (Sternfeld, 1992, Abe et al., 1994). The PstAB lost from the cone are replaced by PstA cells that initiate expression of the ecmB gene (Sternfeld, 1992; Abe et al., 1994). PstA cells are replaced by PstO cells, which are in turn replaced by A L C and these are subsequently replaced by the transdifferentiation of prespore cells (Sternfeld, 1992; Abe et al., 1994). Thus, the relative cell type proportions are maintained during slug migration, despite the loss of cells from the posterior of the slug. As the slug rounds up for culmination, PstAB cells initiate stalk tube and stalk cell formation at the tip (Jermyn and Williams, 1991) and the PstA cells express ecmB as they enter the stalk tube (Jermyn and Williams, 1991). The stalk cells and stalk tube pass to the rear through the posterior prespore cells and the leading edge of the stalk embeds into the rearguard cells. The rearguard cells also differentiate into stalk cells to form part of the basal disc of the fruiting body (Jermyn and Williams, 1991; Sternfeld, 1992). Some ALC's move to the anterior region of the prespore cell mass to form the upper cup, and some move to the basal region of the prespore cell mass to form the lower cup and part of the basal disc (Ceccarelli et al., 1991; Jermyn and Williams, 1991; Jermyn et al., 1996). As the remaining PstA and PstO cells gradually enter the stalk tube and terminally differentiate into stalk cells, the nascent stalk elongates, lifting the prespore cell mass up. At this point, the prespore cells terminally differentiate into spores. The tip region of the developing Dictysotelium organism is extremely important and has been referred to as the 'organizer' of development (Raper, 1940; 29 Rubin and Robertson, 1975; Schaap, 1986; Siegert and Weijer, 1995). Once a mound elongates to form a finger, the tip cells sense cues from the surrounding environment and respond either by initiating culmination in situ or by inducing the formation of a slug (Smith and Williams, 1980). During slug migration, the tip cells sense gradients of light and temperature and direct the migration of the slug accordingly (Fisher, 1997). The cells in the tip also initiate the process of culmination. 1.4.2 Regulators of Cell Type Differentiation in Dictyostelium Although only a few of cell types are formed during Dictyostelium development, the regulation of their differentiation is complex. A number of gene products have been identified that promote the differentiation one cell type or the other (Aubry and Firtel, 1999) and many others probably await discovery. A n understanding of how these various proteins, along with other low molecular weight factors, affect differentiation should enable us to appreciate how starving amoebae chose their fate. In this section, I wil l briefly describe some of the key regulators of differentiation in Dictyostelium (Table I). 1.4.2(a) DIF-1 Differentiation Inducing Factor-1 (DIF-1) is the most active member of a family of chlorinated alkyl phenones (Morris et al., 1987) and was identified based on its ability to induce stalk cell differentiation in isolated amoebae (Town et aL, 1979). DIF-1 induces the transcription of prestalk genes and suppresses the transcription of prespore genes (Williams et aL, 1987; Early and Williams, 1988). 30 Table I. Proteins that regulate differentiation during Dictyostelium development. Protein Effect on Dictyostelium differentiation ERK2 Required for induction and maintenance of prepsore gene expression. M E K K a Required for induction and maintenance of prespore gene expression and correct cell type distribution. RasGAPl Possibly required for correct sorting and terminal differentiation. Warai Required for maintaining correct proportions of prespore and PstO cells. Stalky Required for prespore to spore differentiation. TagB Required for efficient prestalk gene expression and terminal differentiation of both cell types. G a l Involved in PstB differentiation and cell type sorting. Ga2 Involved in prestalk to stalk differentiation. Ga4 Required for prespore gene expression. Spalten Required for prespore and prestalk gene expression. cAR2 Involved in limiting prespore differentiation. cAR3 Required for cAMP-mediated inhibition of stalk cell differentiation, via GSK3. cAR4 Involved in differentiation and sorting of both cell types; antagonizes GSK3 activity. GSK3 Required for prespore gene expression and for inhibition of ecmB expression and stalk cell differentiation. P K A Required for expression of genes in both cell types and for terminal differentiation. rZIP Involved in prespore gene activation and prespore to prestalk transdifferentiation. STATa. Required for inhibition of ecmB expression and stalk cell differentiation. 31 It can also cause isolated prespore cells to form stalk cells instead of spore cells (Kay and Jermyn, 1983). These and additional findings led to the proposal that DIF-1 is the prestalk morphogen. However, the inductive properties of DIF-1 may not be straight-forward. There is evidence that within the multicellular organism, DIF-1 concentration is the highest in the prespore region (Brookman et al., 1987). Also, not all prestalk genes are induced by DIF-1 (Saxe et al., 1996; Shaulsky and Loomis, 1996). It is possible that DIF-1 cooperates with other extracellular morphogens to regulate differentiation. The mechanism by which DIF-1 promotes stalk cell formation has not been elucidated. A cytoplasmic protein with affinity for DIF-1 has been identified (Insall and Kay, 1990) but not purified. Williams' group searched for targets of DIF-1 induction in the promoters of the ecmA and ecmB genes, both of which are rapidly induced by DIF-1 (Jermyn et al., 1987; Ceccarelli et al., 1987). They identified repeats of the TTGA sequence that constitute a DIF-l-response element (Kawata et al., 1996). 1.4.2(b) PSI Recently, a novel glycoprotein factor has been identified that influences prespore differentiation (Oohata et al., 1997; Nakagawa et al., 1999). Prespore-inducing-factor (PSI or VF) is a factor purified from medium conditioned by the growth of amoebae at high density. PSI factor induces the differentiation of isolated amoebae into prespore cells and also induces cell division in prespore cells. The presence of DIF-1 antagonizes the activity of ¥ factor as it induces stalk cell formation even in the presence of *F factor. The importance of *F factor during multicellular development has not yet been ascertained. 32 1.4.2(c) G Protein Subunits The Dictyostelium genome contains single genes encoding GP and Gy subunits. Deletion of the gene encoding GP blocks the onset of development. Using temperature sensitive variants of this protein (Jin et al., 1998), it was found that GP is required for proper morphogenesis at all stages of development. However, it is dispensable for the expression of cell type specific genes. Nine different G a subunits (Gal — Ga9) have been identified and these are expressed at various stages of the life cycle. Only those that have a demonstrated effect on cell type determination are discussed here. The G a l subunit has been shown to be involved in the regulation of ecmB expression and stalk cell formation (Dharmawardhane et al., 1994). Cells in which gal has been disrupted do not display visible defects in the expression of ecmB but form stalks that are longer and skinnier than wild type stalks. Cells overexpressing gal do not express ecmB in the core of the anterior tip. These cells develop to form culminants with thick, short stalks. Cells overexpressing constitutively active G a l display defects in mound tip formation which has been correlated with the inefficient sorting of prestalk cells to the anterior of the mound (Rietdorf et al., 1997). The Ga2 subunit is required early in development for aggregation. Later during development, expression of gal occurs in prestalk cells. The expression of a dominant negative Ga2 in prestalk cells resulted in abnormal stalk differentiation at culmination indicating a role for Ga2 in stalk cell maturation (Carrel et al., 1994). 33 The Ga4 subunit appears to be expressed in the A L C population (Hadwiger et al., 1994) and is involved in morphogenesis and the differentiation of prespore cells (Hadwiger and Firtel, 1992). ga4 null cells have reduced prespore gene expression and form few spores during development. The defect is partially rescued by the presence of wild type cells, indicating that mutant cells are deficient for a signal required for prespore differentiation. The multidomain protein, Spalten, is not a true G protein subunit. However, the amino terminal domain is closely related to Goc subunits while the carboxy terminal domain contains a functional protein phosphatase (Aubry and Firtel, 1998). Spalten is required for development past the mound stage and for the expression of both prestalk and prespore genes. Nul l mutant cells are able to undergo prespore differentiation when developed in mixtures with wild type cells. Prestalk differentiation of the null is not corrected under this condition. 1.4.2(d) Stalky Chang et al. (1996) reported the cloning of a putative transcription factor, STKA, which is required for the terminal differentiation of spore cells. Early development of stkA' cells appears normal although expression of the ecmB gene is upregulated. However, late in development, prespore cells appear to lose their specification and the culminant that forms is composed entirely of stalk cells. The defect is probably not due to the absence of an extracellular sporulation signal since the presence of wild type cells cannot induce the mutant cells to sporulate. Constitutive activation of P K A does not override the spore defect in the mutant, indicating either that STKA is downstream of P K A in a pathway promoting spore 34 formation or that STKA is a positive regulator of a prespore pathway not involving PKA. Alternatively, STKA may indirectly promote spore formation by inhibiting the expression of ecmB. 1.4.2(e) Warai One of two Dictyostelium proteins with a homeodomain, Warai is postulated to function in a pathway regulating cell type proportions (Han and Firtel, 1998). A null mutation in wri results in an enlarged PstO zone at the expense of the prespore zone. Proportions of the other cell types are unaffected. Fruiting bodies of wri null cells have larger basal discs, consistent with the presence of additional stalk cells. Since wri is expressed in PstA cells, the results indicate a non-cell autonomous role in the regulation of PstO cell differentiation. 1.4.2(f) RasGAP Faix and Dittrich (1996) isolated a Dictyostelium GAP homolog, DGAP1, with similarities to the IQGAPs. A null mutation in the corresponding gene resulted in the formation of multi-tipped aggregates during development. These aggregates proceeded to make abnormal fruiting bodies with thick, short stalks and irregular clusters of spore cells. Lee et al. (1997) independently isolated the same protein and called it RasGAPl. Cells in which the ddrasgapl gene is disrupted exhibit apparently normal development till the migrating slug stage. At this time, the normal patterning of prestalk and prespore cells is lost and scattered localization of the two cell types is observed. No terminal fruiting bodies are constructed and the abnormal terminal 35 structures formed contain no stalk cells and few spore cells. Given the conflicting results, the role of this protein has yet to be resolved. 1.4.2 (g) TagB The TagB protein contains a serine protease domain and a domain with sequence identity to the ABC transporters (Shaulsky et al., 1995). tagB is expressed in prestalk cells and knock out mutants arrest morphogenesis at the mound stage before tip formation. The tagB' cells are able to express prespore genes but prestalk gene expression is reduced. Mutant cells do not form stalk or spore cells during multicellular development. However, the mutant can differentiate into spore cells in the presence of wild type cells. Another protein, TagC, contains significant homology to TagB. Disruption of tagC results in a very similar phenotype to that of tagB. 1.4.2(h) M A P K Cascade Components Slugs of null mutants in the gene encoding the putative MEK kinase homolog, mekka, exhibit an increased PstO cell zone, a decreased prespore cell zone, and a less defined boundary between prestalk and prespore cells (Chung et al., 1998). MEKKcc is required for the induction and maintenance of prespore differentiation and when developed in a chimaera with wild type cells, mekka" cells preferentially differentiate into prestalk cells. Additional results indicate that the amount of M E K K a determines the fate of the cell; cells overexpressing MEKKoc preferentially form prespore cells in mixtures with wild type cells. 36 A Dictyostelium M A P K , ERK2 has been isolated and shown to be required for aggregation (Segal et al., 1995). Later in development, ERK2 is required for the induction and maintenance of prespore gene expression but not for prestalk gene expression (Gaskins et al., 1996). ERK2 does not appear to be downstream of M E K K a (Chung et a l , 1998). 1.4.2(i) Role of Membrane Bound cyclic A M P Receptors The effects of extracellular cAMP are mediated by the 7-span transmembrane cAMP receptors, cARl-4. The receptors have varied affinities for cAMP; c A R l has the highest affinity, cAR3 has intermediate affinity, and cAR2 and cAR4 have low affinities (Saxe et al., 1991; Kim et al., 1998). During post-aggregative development, carl expression is enriched in prestalk cells whereas car3 expression is observed only in prespore cells (Gollup and Kimmel, 1997; Yu and Saxe, 1996). Development of car3 null cells is delayed but is otherwise normal (Johnson et al., 1993). The cAR2 receptor is expressed preferentially in anterior prestalk cells (Ginsberg et al., 1995; Saxe et al., 1996). Cells deficient in cAR2 are blocked at the mound stage before tip formation (Saxe et al., 1993). Nul l cells overexpress prespore genes indicating that the binding of cAMP to the cAR2 receptor in prestalk cells may laterally control the differentiation of prespore cells. Expression of the ecmA gene is normal in carl' cells but expression of ecmB is reduced, possibly due to the inability of these cells to culminate. cAR4 is expressed in both cell types, but expression is enriched in the prestalk population (Louis et al., 1994; Ginsberg et al., 1995). car4 null cells overexpress prespore genes and underexpress prestalk genes. Patterning of the cell types is also 37 affected with prespore cells present in the anterior prestalk region (Ginsberg and Kimmel, 1997). When developed in chimaera with wild type cells, car4~ cells can be induced to efficiently form prestalk cells. Moreover, wild type cells in these mixtures were not prevented from prestalk differentiation. Thus, the prestalk defect in the car4~ null cells does not appear to result from a defect in either the production or reception of an extracellular signal. Similar experiments revealed that car4' prespore cells were capable of normal pattern formation in a chimaeric mixture containing a majority of wild type cells but, wild type prespore cells were mislocalized in a chimaeric mixture containing a majority of car4~ cells. Thus, prespore cell localization is determined non-cell autonomously by the major population of cells in the mixture. Evidence was also presented for the existence of an extracellular DIF modulation factor (DMF) that appears to decrease the sensitivity of prespore cells to DIF. This factor is not produced by the car4~ cells. 1.4.2(j) Role of GSK The Dictyostelium GSK3 homolog is involved in regulating the differentiation of prespore cells and PstB cells (Harwood et al., 1995). Developing gskA null cells produce an enlarged basal disc supporting a short stalk and usually no sorus. They also exhibit drastically reduced prespore gene expression and elevated ecmB expression. In addition, the normal cAMP-mediated induction of prespore gene expression and repression of ecmB gene expression and stalk cell formation is not observed. These results indicate that GSK activation stimulates spore formation and inhibits stalk cell formation. When wild type cells are treated 38 with LiCl , an inhibitor of GSK3 activity, the gsk3~ phenotype is mimicked (Sakai, 1973; Van Lookeren Campagne, 1988). In car3~ cells, GSK activity is not elevated at the time of cell type determination as it is in wild type cells (Plyte et al., 1999). In addition, car3~ cells do not exhibit cAMP-mediated repression of stalk cell formation (Plyte et al., 1999). Also, stimulation of wild type cells with cAMP results in an increase in GSK3 activity; this increase does not occur in cells lacking cAR3 (Plyte et al., 1999). However, multicellular development of car3~ cells is essentially normal (Johnson et al., 1993) whereas gskA' cells form aberrant structures. It is possible that loss-of-function cAR3 may be compensated by another cAMP receptor. c A R l is a good candidate since early in development, cAR3 can compensate for the absence of c A R l indicating that some functional redundancy exists amongst these receptors (Insall et al., 1994). cAR4 has also been implicated in the regulation of GSK3 activity (Ginsburg and Kimmel, 1997). At the level of gene expression, the phenotypes of car4~ cells and gskA' cells are opposite. The treatment of developing car4' cells with L i C l to inhibit GSK3 activity results in a correction of the defects in cell type specific gene expression. cAMP binding to cAR4 may negatively modulate the activity of GSK3 and in the absence of cAR4, GSK3 is hyperactive and prespore differentiation increases. Thus cAMP signaling through cAR4 might antagonize the effect of cAMP signaling through cAR3 with respect to the regulation of GSK3 activity. GSK3 activity is also regulated by a novel tyrosine kinase, ZAK1 (Kim et al., 1999). cAMP-mediated ZAK1 activation is defective in car3 null cells and GSK3 activity is reduced in zakl null cells. In addition, zakT cells have reduced ability to 39 form spores and do not exhibit cAMP-mediated repression of stalk cell differentiation, properties characteristic of gskA null cells. Also, ZAK1 phosphorylates and activates mammalian GSK3(3 in vitro. Together, these results place ZAK1 downstream of cAR3 in the pathway regulating GSK3 activity. 1.4.2(k) Role of P K A The Dictysotelium cAMP-dependent protein kinase, P K A is required at multiple stages during development and plays an integral role in the differentiation of both spore and stalk cells (Loomis, 1998). When P K A is inactivated in prespore cells, using the psA promoter to drive expression of a dominant inhibitory form of the P K A regulatory subunit (PKA-Rm), spore formation does not occur (Hopper et al., 1993a). Also, as prespore cells accumulate sufficient PKA-Rm protein, the level of transcription of the spore coat genes becomes greatly reduced. This result is consistent with the fact that most prespore genes require P K A for maximum expression (Fosnaugh and Loomis, 1991; Hopper et al., 1995). However, the expression of psA is unaffected in this psA::PKA-Rm strain indicating that P K A does not regulate the expression of all prespore specific genes (Hopper et al., 1993a; Hopper and Williams, 1994). Prestalk specific expression of the PKA-Rm protein, driven by the ecmAO promoter, results in the formation of defective slugs unable to culminate (Zhukovskaya et al., 1996). Expression of ecmA and ecmB are reduced, indicating a requirement for P K A in prestalk cells for efficient prestalk gene expression. It is believed that ecmB expression is repressed during the slug stage and that the repression is relieved by P K A activity during culmination (Loomis, 1998). 40 P K A activity is also required for the terminal differentiation of both cell types. During development, the catalytic subunit of P K A is preferentially expressed in prestalk cells but once culmination is initiated, expression is induced in the rising spore mass (Mann et al., 1994). Constitutive activation of P K A results in rapid development and a sporogenous phenotype suggesting that control of P K A activity is required to prevent premature sporulation. The signal pathway thought to activate P K A during culmination is described later in this chapter. 1.4.2(1) Role of rZIP rZIP is a multidomain protein containing a RING-type zinc-binding domain, a leucine zipper, and an SH3 domain (Balint-Kurti et al., 1997). rZIP has similarities to the Cbl family proteins, that have been proposed to negatively regulate RTK-mediated signaling pathways (Yoon et al., 1995). When rZIP is overexpressed, prestalk gene expression is elevated and prespore gene expression is repressed, the opposite phenotype of an rzpA knock out (Balint-Kurti et al., 1997). Slugs of rzpA null cells are less motile than wild type slugs and mutant prespore cells are unable to transdifferentiate to prestalk cells. When mutant cells are developed in a chimaera with an excess of wild type cells, the mutant cells display a gradient of expression of spore coat genes (cotB and cotC) such that expression is highest in the anterior of the prespore zone and decreases towards the posterior (Balint-Kurti et al., 1998). This gradient is corrected by the activation of P K A (using the membrane permeable cAMP analogue, 8-Br-cAMP, or the ectopic expression of the catalytic subunit of PKA). Based on these results, it was suggested that within the prespore zone of the slug, P K A activation 41 responds to an anterior to posterior graded signal and that rzpA' cells have a reduced capacity to respond to this signal. 1.4.2(m) Role of STATa Analysis of the DIF-inducible ecmA gene revealed a promoter element required for DIF induction (Kawata et al., 1996). It was found that the necessary sequence is bound by the Dd-STATa protein (Kawata et al., 1997). Mammalian STAT (signal transducer and activator of transcription) proteins are activated in response to cytokine stimulation via tyrosine phosphorylation by Janus kinases (JAKs). Phosphorylated STAT proteins dimerize via C-terminal SH2 domains and translocate to the nucleus (reviewed in Ihle and Kerr, 1995). The Dictyostelium STATa is activated in response to extracellular cAMP (Araki et al., 1998) and is required early in development for proper aggregation (Mohanty et al., 1999). STATa binds to a TTGA direct repeat in the ecmA promoter and inverted repeat repressor element in the ecmB promoter (Kawata et al., 1997). Deletion of the repressor element and insertional inactivation of the STATa gene both result in precocious expression of ecmB in the entire anterior prestalk domain (Kawata et a l , 1997; Mohanty et a l , 1999). Slugs of STATa null cells do not form fruiting bodies and do not produce stalk cells during multicellular development. Spore cells are formed but with reduced efficiency compared to wild type. The STATa null cells are more sensitive to DIF than wild type cells and require a lower DIF concentration for the induction of prestalk genes. The level of ecmA expression in STATa null cells is normal, indicating that while STATa can bind to the ecmA promoter, it is not required for induction of the gene. Precocious ecmB expression 42 in the null cells indicates that STATa is a negative regulator of ecmB expression and stalk cell differentiation. Although STATa is a repressor of stalk cell formation, its absence does not result in precocious stalk differentiation. This is most likely a consequence of the increased sensitivity of mutant cells to cAMP mediated inhibition of stalk cell formation. 1.4.3 Signals Regulating Terminal Differentiation of Dictyostelium Results obtained over recent years have yielded insights into the regulation of terminal differentiation during culmination (reviewed in Loomis, 1998; Aubry and Firtel, 1999; Thompson et al., 1999). Late in the developmental program, when the slug ceases migration and rounds up prior to culmination, the prestalk cells at the apex secrete a sporulation factor (Anjard et al., 1998). This factor, SDF-2 (spore differentiation factor), is secreted via TagB and TagC and has an autocrine effect on prestalk cells triggering a burst of SDF-2 release. In prespore cells, SDF-2 activates DhkA, a membrane-bound histidine kinase (Wang et al., 1996). DhkA then inhibits RegA in a manner dependent on the M A P K , ERK2 (Shaulsky et al., 1998). RegA is a cAMP specific phosphodiesterase so its inhibition leads to a rise in intracellular cAMP and a concomitant rise in P K A activity which is required for prespore cells to become encapsulated. Stalk cell differentiation may also be initiated in response to activation of P K A as a consequence of RegA inhibition. In the basal disc, however, stalk cell formation may occur via a separate mechanism. In this case, low levels of cAMP (due to the physical distance from the anterior cAMP oscillator) lead to low levels of GSK-3 activity which in turn, induces stalk cell differentiation (Thompson etal., 1999). 43 1.5 Effects of Dictyostelium Ras on Development At least six different ras sub-family genes are expressed by Dictyostelium, each with its own unique temporal expression pattern (Reymond et al., 1984; Robbins et a l , 1989; Robbins et al., 1991; Daniel et al., 1993a; Daniel et al., 1993b). The protein products of these genes, RasD, RasG, RasB, RasC, and RasS, are related to the mammalian H-Ras whereas Rapl is related to mammalian Rap. The RasG and RasD proteins are the most closely related to H-Ras, sharing 69% and 62% identity with the mammalian protein, respectively (Robbins et al., 1991). These two Dictyostelium Ras homologues are 82% identical and only differ in 3 positions over the first 100 amino acids (Robbins et al., 1989). Hence, both RasG and RasD have identical effector and effector-proximal domains (Figure 1-6). RasG and RasD have been studied the most intensely and activated forms of both have been shown to inhibit the normal developmental program. 1.5.1 Dictyostelium RasG The rasG gene is expressed during vegetative growth and during the first few hours of development (Robbins et al., 1989). Transformants in which the activated rasG(G12T) is driven by the folate-repressible discoidin promoter fail to aggregate under all starvation conditions tested (Khosla et a l , 1996). The aggregation defect occurs even when cells are grown in the presence of folate, indicating that a low level of the induced protein is sufficient to impair the onset of development (Khosla et al., 1996). The activated RasG transformants are impaired in the 44 MTEYKLVIVVGGGVGKSALTIQLIQNHFTDEYDPTIEDSYRKQVTIDEET I I S D • ••••• CLLDILDTAGQEEYSAMRDQYMRTC I LRVKDKDRVPMIVVGNKCDLESDRQVTTGEGQDLAKSFGSPFLETSAK I I I I I I I I I I I I I I I I I L L A DHE SVN E DYSLS H S IRVNVEEAFYSLVREIRKDKKGDSKPEKGKKKRPLKACTLL I I I I I I I I I I I I S I E QS G A Q K K Q - - LI Figure 1-6: Amino acid sequence of RasG. The residues in RasD that differ from RasG are indicated below the RasG sequence. RasG has two additional amino acids relative to RasD at the C-terminal tail. These are indicated by dashes below the sequence. Residues involved in guanine nucleotide binding are labeled with dots above the sequence and residues involved with interactions with effector proteins are indicated with asterisks above the sequence. (Adapted from Robbins et a l , 1989) 45 induction of two genes, carl and pde, both of which are expressed soon after the initiation of starvation (Khosla et al., 1996). When transformants are pulsed with cAMP while shaking in starvation buffer, the expression of these early response genes is increased and cells pulsed for 4 hours are able to complete the developmental program when plated on a solid substratum (Khosla et al., 1996). Since the pVEII-rasG-G12T transformants are capable of responding to cAMP pulses, the defect in aggregation could be due to a failure to generate cAMP. Consistent with this, the pVEII-rflsG-G12T transformants produce only very small amounts of cAMP in response to a pulsatile stimulus but in the presence of wild type cells, they are able to participate in development (Khosla et al., 1996). 1.5.2 Dictyostelium RasD The rasD gene was initially isolated as a prestalk specific gene (Reymond et. a l , 1984) and the staining pattern for developing slugs expressing a rasDr.lacZ reporter construct resembles the expression pattern for the prestalk cell specific ecmA gene (Esch and Firtel, 1991). This supports the prestalk specific designation for rasD. However, when prestalk and prespore cell populations are separated and assayed for gene expression by northern blot analysis, rasD mRNA is found to be only 3-fold enriched in prestalk cells, whereas both ecmA and ecmB mRNA's are more than 10-fold enriched in the prestalk cells (Jermyn et. a l , 1987). Moreover, when (3-galactosidase is assayed over longer time periods in slugs expressing rasDr.lacZ, the prespore region also stained, confirming that prespore cells do express rasD but at a significantly lower level than prestalk cells (Jermyn and Williams, 1995). It was proposed that rasD is initially expressed in a non-cell type-specific 46 manner and then expression decreases in the prespore cells and as such, expression becomes prestalk enriched (Jermyn et. al., 1987). Consistent with this suggestion, the prespore region stains less intensely when slugs express a labile (3-galactosidase (Jermyn and Williams, 1995). A transformant overexpressing activated rasD (Drfras-Thrl2) forms aggregates with multiple tips that do not proceed to form fruiting bodies (Reymond et al., 1986). Only 0.2% of the original population was found to form viable spores and upon disaggregation, stalk cells were not observed (Louis et al., 1997a). It was also found that the mRNA levels for 3 prestalk specific genes, ecmA, ecmB, and tagB, were dramatically elevated relative to control cells, while prespore specific cotC mRNA levels were reduced to barely detectable levels (Louis et al., 1997a). Consistent with this northern blot data, there was an increased expression of an ecmAr.lacZ prestalk reporter and the entire aggregate gained the ability to express the P-galactosidase (Louis et al., 1997a). RasG and RasD have very similar sequences and when activated RasG was expressed from the rasD promoter, the rasD::rasG(G12T) transformants produced the same multi-tipped phenotype exhibited by the Ddras-Thrl2 transformant (M. Khosla, unpublished observations). When the Ddras-Thrl2 transformant cells were mixed with wild type Ax3 cells in a 1:3 ratio and induced to develop, the wild type cells formed normal fruiting bodies that excluded the transformant cells (Louis et al., 1997). These results indicated that signals from the wild type cells were incapable of rescuing the development of the transformant and that the transformant cells did not negatively affect the development of the wild type cells. In the reverse scenario, when Ax3 cells were mixed with Ddras-Thrl2 cells in a 1:3 ratio, multi-tipped aggregates 47 formed that did not proceed further and the wild type cells were found to be trapped within the deformed aggregates (Louis et a l , 1997a). Thus, an excess of the transformant cells inhibited development of Ax3 cells. 1.6 Rationale and Research Objective It had been hypothesized that the defect in the development of the Ddras-Thrl2 transformant was due to expression of activated RasD in the prestalk cell population (Esch and Firtel, 1991). However, since rasD expression also occurs in the prespore cell population, there were several plausible alternative explanations that were consistent with the available data (Louis et al., 1997a). Activated RasD might exert its effects only in prespore cells, inhibiting prespore gene expression and enhancing prestalk gene expression, but blocking the conversion of prestalk cells to stalk cells. Alternatively, activated RasD might have effects in both cell types: preventing gene expression in prespore cells and enhancing gene expression in prestalk cells, but again inhibiting the conversion of prestalk cells to stalk cells. Finally, even if the defect in the Ddras-Thrll transformant was due to an effect only in prestalk cells, as suggested by Esch and Firtel (1991), then this event must lead to the lateral inhibition of prespore gene expression. To distinguish between these possibilities, it was necessary to express the activated protein in a cell type specific manner, and this has been accomplished in the work described in this thesis. To accomplish this task, I set out to express the activated rasD from the ecmAO and psA promoters. Considerable problems were encountered in attempting to construct the ecmAO::rasD(G12T) and psA::rasD(G12T) transformation plasmids. Since RasD(G12T) and RasG(G12T) have identical developmental consequences, when expressed from the rasD promoter (M. Khosla, unpublished observations), I attempted to construct transformation plasmids containing ecmAO::rasG(G12T) and psA::rasG(G12T) and this was successful. CHAPTER TWO 49 EXPERIMENTAL PROTOCOLS A list of materials used to conduct these experiments, along with names of the suppliers is provided in Appendix I. Recipes for media and buffers are detailed in Appendix II. 2.1 DNA Preparation for Plasmid Construction Small scale preparations of D N A were isolated from Escherichia coli cells using the miniprep protocol detailed in Sambrook et al. (1989). Large scale preparations of D N A for cloning and for Dictyostelium transformations were isolated by the CsCl 2 density gradient centrifugation method, except for the actl5::lacZ, psAr.lacZ, psA::(his)lacZ, and psA::rasG(G12T) plasmid DNAs which were isolated by the PEG precipitation method. Both methods are described in Sambrook et al. (1989). Recovery of D N A from agarose gels was accomplished using the Qiagen Gel Extraction Kit, except for DNAs used in the cloning of the psA::rasG(G12T) construct which were recovered using the GeneCleanll Kit. A l l kits were used according to their manufacturer's protocols. 2.2 Transformation of Bacterial Cells E. Coli XL-1 cells were used for all transformations. Cells were made competent by the rubidium chloride technique (Sambrook et al., 1989) and then transformed using the CaCl 2 protocol (Sambrook et al., 1989). Selection for transformants was carried out on LB agar plates supplemented with 100 jj,g/ml 50 ampicillin. Rapid screening of transformants to detect successful cloning was performed by the Slot Lysis method (Sekar, 1987). 2.3 Vector Construction 2.3.1 psA::rasG(G12T) and psA::rasG Constructs Using ptZ19R-rasG(G12T) (Khosla et al., 1996) as a template, the 5' portion of the activated rasG cDNA was modified by PCR to delete a stop codon located 5' to the start codon (Fig. 2-1). (This was done to prevent any negative effect the stop codon may have had on translation). The Ras-5' and Ras-3' primers (Table II) were used in the amplification reactions. VentR® D N A polymerase was used for the PCR reactions along with 10 ng of template D N A , 20 pmol of each primer, and 400 m M of each dNTP per reaction. The conditions used for the PCR reactions were as follows: denaturation at 95°C for 30 seconds, annealing at 47°C for 50 seconds, extension at 72°C for 21 seconds, and cycles were repeated 25 times. The PCR product from the rasG(G12T) template was digested with Bglll and AccI and ligated with a similarly digested ptZ19R-rasG (Khosla et al., 1996) vector fragment in which a second AccI site in the polylinker had been destroyed by digesting with Sail, which recognizes an overlapping site, blunting the ends, and religating (Fig. 2-2). From this construct, ptZ19R-rasG(G12T)ASalIAstop, the rasG(G12T) cDNA was released by digesting with Bglll and Kpnl and ligated to the vector fragment of the Bglll/Kpnl digested psA-DdPK2 plasmid (Hopper et al., 1993b). The construct thus generated, psA::rasG(G12T)3'PK2 contained the rasG(G12T) cDNA linked to the psA promoter but also contained a 3' portion of the 51 11 I I I I lATATMTTTTTAAATTTTATA^ 6 5 ******xxx AAAAAAAAAAAMCCAMTCAMTTATAAMCCCACACATAmATATATMC^ 130 VVV A C T AAAAAMTGA(^GMTACAMTTAGTTATTGTTGTTGrGTGGTGGTGTCGCT 195 CATTCMTTMTCCAAMCCATTTCATTGA^ 26 0 MCMGTTACCATTGATGMGAMC^^ 32 5 • • • • • • TACTCTGCMTGAGAGA(XMTATATG^ 39 0 Figure 2-1: Sequence of the 5' end of the rasG cDNA. The BglH and AccI restriction sites are indicated by asterisks and diamonds above the recognition sequence, respectively. The sequence for the stop codon is underlined and labeled from above with crosses. The sequence for the start codon is underlined and indicated with check marks from above. Numbers indicate the position of the rightmost nucleotide of that row. Table II. Oligonucleotide primers* used for PCR and sequencing reactions. Ras-5' G T C T A G A T C T T T T A A A A A A A T G A C A G Ras-3' C T T A G A G T T A A G G A T A A G G A S17N G T C G G T A A A A G T G C C T T A A C C JD5 G A T A C T G C T G G T C A A G SR3 C T T A G A G T T A A G G A T A A G G A Sequences are given in 5' to 3' order. 53 3' Figure 2-2: Partial cloning strategy for construction of the psA::rasG(G12T) vector DNA. The ptzl9R-rasG vector, with the Sail site destroyed, was digested with Bglll and AccI and the vector fragment isolated. This was ligated to a similarly digested PCR fragment of the 5' end of the rasG(G12T) cDNA to generate the ptz!9R-rasG(G12T)ASalIAStop vector. 54 DdPK2 gene which was removed by digesting with Kpnl and Xhol, blunting the termini, and religating (Fig. 2-3). To clone the wild type rasG downstream of the psA promoter, the ptZ19R-rasG vector was used as a template in a PCR reaction identical to the one described above. The PCR product was digested with Bglll and AccI and ligated to the vector fragment from a Bglll/AccI digestion of the psA::rasG(G12T) construct. This cloning resulted in replacing the region containing the activating mutation with wild type coding sequence and thus generated the psAr.rasG construct. The generated vectors were isolated by the CTAB D N A preparation protocol (Del Sal et al., 1987) (for psA::rasG(G12T)) or using the Nucleobond Ax-20 D N A extraction kit (for psA::rasG) and the construction was verified by sequencing with the dideoxy chain termination method using the SN17, JD5, and SR3 primers (Table II). Reactions were carried out using the Sequenase2® kit according to manufacturer's protocol except that the D N A and primer were boiled for 5 minutes prior to the addition of the Sequenase buffer (Andersen et al., 1992). DNAs were labeled with [35S]-oc-dATP and electrophoresed on a 6% acrylamide gel buffered with TBE. The gel was then vacuum dried and exposed to X-ray film. 2.3.2 ecmAO::rasG(G12T) and ecmAOr.rasG Constructs The psA::rasG(G12T) and psAr.rasG constructs were digested with Bglll and ligated to a Bglll fragment from the ecmAOr.lacZ vector. This fragment contained the ecmAO promoter DNA. After verifying the orientation of the ligated promoter by restriction analysis, the generated constructs were digested with Kpnl and Xbal to 55 Figure 2-3: Second half of cloning scheme for the psA::rasG(G12T) construct. The ptzl9R-rasG(G12T)ASalIAStop construct was digested with BgUI and Kpnl and the rasG(GUT) cDNA isolated. The psA-Dd(PK2) vector was also digested with BgUI and Kpnl and the vector fragment was isolated. These were ligated to generate the psA::rasG(G12T)3'PK2 vector which was digested with Kpnl and Xhol, blunted and religated to create the psA::rasG(G12T) vector. 57 remove the psA promoter, blunt ended and religated to create the ecmAO::rasG(G12T) and ecmAOr.rasG constructs (Fig. 2-4). The promoter/gene fusions were verified by sequencing. D N A was isolated using the Nucleobond Ax-20 kit and sequenced by the NAPS facility using the SN17, JD5, SR3 primers. 2.4 Growth of Dictyostelium discoideum Cells Dictyostelium discoideum Ax2 cells were grown at 22°C in HL5 culture medium (Watts and Ashworth, 1970), containing 50 | ig /ml streptomycin, either in shaking flasks (at 180 rpm) or in Nunclon™ (9 cm) dishes. Culture density was determined by counting cells using a hemocytometer. For clonal selection of transformants, Dictyostelium amoebae were seeded at low density in association with Klebsiella oxytoca on nutrient rich S M - V A N agar plates (modified from Sussman, 1987) and grown at 22°C. Vegetative amoebae from individual plaques were picked and transferred to HL5 medium for continued growth. For spore germination, a wire loop, prewetted with HL5 medium, was used to pick spores from fruiting bodies on S M - V A N plates and to transfer them to HL5 medium. 2.5 Development of Dictyostelium discoideum Cells To initiate development, exponentially growing Dictyostelium cells were harvested and washed twice in Bonner's Salts (BS) by centrifugation for 5 minutes at 1200 rpm. Cell pellets were then resuspended in BS and 2.5xl07 cells were plated onto white Millipore nitrocellulose filters (4.7 cm diameter) (Bonner, 1947). Black filters were used instead of white filters when unstained developing structures were Figure 2-4: Coning strategy for the ecmAO::rasG(G12T) construct. The ecmAr.lacZ vector was digested with Bglll to release the ecmAO promoter which was ligated with a Bglll digested psA::rasG(G12T) vector. After determining the orientation of the inserted ecmAO promoter by restriction analysis, the psA promoter was removed by digesting with Kpnl and Xbal, blunting, and religating. A n identical strategy was used to generate the ecmAOr.rasG construct using the psAr.rasG vector. 59 Bglll Kpnl Neo-R CPI ten sA Promoter 'gill Neo-R Act 6 promoter Act 6 Promoter psA::rasG(G12TM rasG(Gi2T) fa 8 Amp-R term • Digest with Bglll isolate promoter Digest with Bglll isolate vector CPI term psA Promoter gill Kpnl Neo-R Act 6 Promoter ecmA promoter iglll rasG(G12T) Digest with Kpnl & Xbal, isolate vector, blunt, religate Neo-R Act 6 Promoter kct8 Amp-R term 60 to be photographed. Filters were placed atop BS-saturated pads in 4 cm Falcon® petri plates and incubated in a moist chamber at 22°C. Structures were viewed with a WILD M3Z dissecting microscope. Unless otherwise indicated, unstained structures were photographed with a DAGE-MTI CCD100 digital camera using Scion Image 1.62a software. 2.6 Spore Formation To determine the spore formation, cells were allowed to develop for 36 to 48 hours to ensure fruiting body formation was complete. Cells were then washed off the filters in 5 ml of BS with 1% Triton X-100 and incubated for 30 minutes. Detergent-resistant spores were counted using a hemocytometer. 2.7 Calcofluor Staining To label cellulose in stalk cell walls, cells were stained with calcofluor by a modification of the method described in Springer et al. (1994). Filters were incubated until development was complete. Sections of filters with several terminal structures were cut, and inverted onto a drop of calcofluor (5 |ig/ml) on a glass slide to allow the structures to float off the filters and into the stain. The piece of filter was then removed and a coverslip placed on the drop containing the submerged structures. Stained structures were viewed with a Zeiss Azioplan2 fluorescence microscope and images captured with a digital camera (Spot Diagnostics Instruments, Inc.). 61 2.8 Transformation of Dictyostelium discoidum cells Ax2 cells were transformed by the previously described CaP0 4 DNA precipitation and transformation method (Early and Williams, 1987). Between 2 x 106 and 5 x 106 Ax2 amoebae, passaged no more than twice after spore germination, were plated on Nunclon™ (9 cm) dishes. Cells were allowed to adhere to the plastic for approximately 30 minutes following which time the medium was replaced by bis-HL5 (supplemented with streptomycin). DNA was precipitated in an Eppendorf tube by mixing 400 ul dH20,10 |ig plasmid DNA, 100 ul CaCl 2, and 500 |il 2X HBS. The mixture was incubated at room temperature for 30 minutes and then added to the cells in one dish in a dropwise manner. The cells were incubated at 22°C for 4-5 hours, the bis-HL5 removed, and the cells then subjected to a glycerol shock (15% glycerol in HBS) for 2 minutes at 22°C The cells were then refed HL5 supplemented with streptomycin, and incubated overnight at 22°C. The following day, G418 was added at a concentration of 10 {ig/ml. Growth medium was changed every 2-3 days until colonies appeared, usually after about 7 tolO days. Isolated colonies were picked and transferred to HL5 medium containing 10 |J.g/ml G418 in 4 cm wells of 6-well Nunclon™ plates. Once confluent, cells were transferred for continued growth, to 9 cm Nunclon™ plates or in shake culture. When the transforming plasmid DNA contained lacZ, f3-galactosidase expressing colonies were selected for by the colony lift method described below. A complete list of the transformants generated for the investigations described in this thesis is provided in Table III. Table III. Strains used for the investigations described in this thesis. Ax2 Parental axenic strain, the wild type control actl5::lacZ Wild type strain, constitutive expression of lacZ in all cells ecmAO::lacZ Wild type strain, expression of lacZ in PstA/PstO cells ecmAr.lacZ Wild type strain, expression of lacZ in PstA cells ecmOr.lacZ Wild type strain, expression of lacZ in PstO cells ecmBr.lacZ Wild type strain, expression of lacZ in PstB cells STr.lacZ Wild type strain, expression of lacZ in cells of the stalk and basal disc psA::(his)lacZ Wild type strain, expression of labile lacZ in prespore cells ecmAOr.rasG Transformants expressing wild type RasG in PstA/PstO cells ecmAO::rasG(G12T) Transformants expressing activated RasG in PstA/PstO cells ecmAO::rasG(G12T)/ actl5::lacZ Transformants expressing activated RasG in PstA/PstO cells and constitutively expressing lacZ in all cells ecmAO::rasG(G12T)/ ecmAr.lacZ Transformants expressing activated RasG in PstA/PstO cells and expressing lacZ in PstA cells ecmAO::rasG(G12T)/ ecmOrlacZ Transformants expressing activated RasG in PstA/PstO cells and expressing lacZ in PstO cells ecmA0::rasG(G12T)/ ecmBr.lacZ Transformants expressing activated RasG in PstA/PstO cells and expressing lacZ in PstB cells ecmAO::rasG(G12T)/ STr.lacZ Transformants expressing activated RasG in PstA/PstO cells and expressing lacZ in stalk and basal disc cells ecmAO::rasG(G12T)/ psA::(his)lacZ Transformants expressing activated RasG in PstA/PstO cells and expressing labile lacZ in prespore cells Table III. Strains used for the investigations described in this thesis (continued). psAr.rasG psA::rasG(G12T) psAr.rasG(GllT)/ actl5::lacZ psA::rasG(G12T)/ ecmAO r.lacZ psArrasG(GUT)/ ecmBr.lacZ psA::rasG(G12T)/ ST::lacZ psA::rasG(G12T)/ psA::(his)lacZ ecmAO::rasG(G12T)/ psA::rasG(G12T) rasD::rasG(G12T)* Transformants expressing wild type RasG in prespore cells Transformants expressing activated RasG in prespore cells Transformants expressing activated RasG in prespore cells and constitutively expressing lacZ in all cells Transformants expressing activated RasG in prespore cells and expressing lacZ in PstA/PstO cells Transformants expressing activated RasG in prespore cells and expressing lacZ in PstB cells Transformants expressing activated RasG in prespore cells and expressing lacZ in stalk and basal disc cells Transformants expressing activated RasG in prespore cells and expressing labile lacZ in prespore cells Transformants expressing activated RasG in PstA/PstO cells and in prespore cells Transformants expressing activated RasG from the rasD promoter (expression in both cell types) *Strain obtained from M . Khosla. 64 2.9 Colony Lift for [3-Galactosidase Expression Dictyostelium cells were transformed with one of several reporter plasmids containing the lacZ gene. Once colonies became visible, the transformant cells were pooled and transferred to 4 cm wells in 6-well Nunclon™ plates to continue growth. A sample of the pooled cells was diluted and an aliquot, containing about 75 cells, was spread in association with Klebsiella oxytoca onto S M - V A N plates. After 4-5 days incubation, plaques from single Dictyostelium cells had formed and cells in the center of the plaques had begun differentiation. Clones were screened for expression of (3-galactosidase as described by Buhl et al. (1993). Cells were adsorbed to Hybond-N+ nitrocellulose filters, the filters were fixed for 10 minutes in Z buffer containing 0.5% gluteraldehyde and then washed 4 times for 5 minutes each in Z buffer containing 2% Tween-20. The filters were then immersed in fj-Galactosidase Staining Solution and incubated in a dark chamber overnight at 37°C. Positive clones with wild type developmental morphology were selected and cells from the corresponding plaques were transferred to HL5 medium for growth. A n identical protocol was followed to screen for cotransformants expressing both lacZ and ras constructs except that selected clones were positive for P-galactosidase expression and exhibited the developmental phenotype characteristic of the transforming ras construct. 2.10 In Situ Detection of fj-Galactosidase Expression Dictyostelium transformants expressing P-galactosidase were washed free of nutrients and set up for development on white Millipore filters using KK2 instead 65 of BS. At various times after aggregate formation, portions of the filters were cut and immersed for 10 minutes in Z buffer containing 1% gluteraldehyde to fix the cell masses (Dingermann et al., 1989). Samples were then washed twice with Z buffer and then incubated in p-galactosidase staining solution at 37°C overnight. Stained organisms were again washed with Z buffer and then photographed with a Nikon 35 mm camera and Kodak Ektachrome Color Slide Film. 2.11 cDNA Probe Preparation Plasmid D N A containing the cDNA of the gene of interest was digested with the appropriate restriction enzymes to release the cDNA fragments. The digested DNAs were size fractionated by electrophoresis in a 0.7% agarose gel buffered with TBE. Electrophoresed D N A was stained with ethidium bromide and viewed on a U V transilluminator. The band containing the desired cDNA fragment was excised from the gel and purified using the Qiaquick Gel Extraction Kit. Approximately 100 ng of the cDNA, estimated by U V fluorescence relative to a standard concentration of a D N A mixture fractionated on the same gel, was labeled with [32P]-a-dCTP by the random oligonucleotide primer method (Feinberg and Vogelstein, 1983). The labeled cDNA probes were purified from unincorporated nucleotides by passage through a Sephadex G-25 spin column (Sambrook et al., 1989). 2.12 RNA Isolation and Northern Analysis The guanidinium isothyocyanate method (Chomczynki and Sacchi, 1987) was used to extract total R N A from Dictyostelium cells. Each 20 |ig sample of R N A was 66 resuspended in R N A sample buffer and electrophoresed on a 1.25% agarose gel (containing 2.2 M formaldehyde). Ethidium bromide fluorescence of rRNA was used to ensure equal loading of R N A samples. The gel was then washed 4 times for 5 minutes each with distilled water. After washing, the gel was soaked in 20X SSC and then transferred by the capillary method to a Hybond-N+ nitrocellulose membrane (Sambrook et al., 1989). Transfer was permitted to occur overnight after which the R N A was fixed to the membrane by baking at 80°C for 2 hours. Blots were hybridized with radiolabeled cDNA probes in Hybridization Solution overnight at 42°C. Blots were washed first with 2X SSC, 0.1% SDS at room temperature and then with 0.5X SSC, 0.1% SDS at 60°C for 20 minutes before being exposed to x-ray film for varying times depending on signal intensity. 2.13 Protein Isolation and Western Blot Analysis Dictyostelium cells were harvested and washed in BS and then lysed in 1% SDS in BS. A small volume was used to determine the protein concentration by measuring optical density at wavelengths 260 nm and 280 ran. The remainder of the lysate was mixed with an equal volume of 2X Protein Sample Buffer. Protein samples of 20 |ig of each were then boiled for 3 minutes and subjected to SDS-PAGE analysis (Laemmli, 1970). A test gel for each set of protein samples was stained with Coomassie to confirm equal loading. For western analysis, following electrophoresis, proteins were transferred to Hybond-P PVDF membranes (Towbin et al., 1979). Prior to antibody probing, the blots were blocked with TBST (50mM Tris-HCl p H 7.5, 150 m M NaCl, 5% Tween-20) containing 5% non-fat dry milk for 1 hour at room temperature. Blots were then washed twice briefly in TBST and then 67 incubated with antibody specific for RasG (Khosla et al., 1994) (1:1000) in TBST containing 1% non-fat dry milk overnight. Probed blots were then washed 4 times for 5 minutes each in TBST and then incubated with secondary antibody, HRP-conjugated donkey-a-rabbit antibody (1:10,000) in TBST containing 1% non-fat dry milk for 1 hour. Blots were again washed 4 times for 5 minutes each with TBST. To detect bound secondary antibody, blots were treated with ECL reagents and exposed to X-ray film. 2.15 Phototaxis Assay Cells were harvested and washed twice with BS, resuspended in BS at a density of 1x10s cells/ml and spotted at one side of a 1% water agar plate (Dormann et al., 1996). The plates were then placed in a chamber lined with moist towels in an orientation such that the cells were placed furthest away from a single slit cut into one side of the chamber to allow light entry, and exposed to room light. Under these conditions, developing structures are maintained at the slug stage. Plates were incubated for 2 days and then examined for evidence of slug migration. CHAPTER THREE OVEREXPRESSION OF RASG(G12T) IN PRESTALK CELLS 68 3.1 Background As described in the Introduction, wild type starving amoebae aggregate to form single tipped mounds which elongate and fall to the substratum to form migrating slugs. Starving amoebae of the Ddras-Thrll transformant are different: they aggregate to form multi-tipped mounds that do not proceed to form slugs (Reymond et al., 1986). rasD was originally identified as a prestalk specific gene (Reymond et al., 1984) and its expression has been shown to be enriched in the ecmA-expressing subset of prestalk cells (Esch and Firtel, 1991; Jermyn and Williams, 1995). It has also been determined that the Ddras-Thrll cells exhibit a defect in the desensitization of guanylyl cyclase which is involved in the chemotaxis to cAMP (Van Haastert et al., 1987). Given these results, and the fact that the ecraA-expressing cells sort to the tip (Early et al., 1995), which is the region responsible for generating the cAMP pulses that orchestrate development (Schaap, 1986), it was postulated that the overexpression of activated RasD in these prestalk cells was responsible for the developmental aberrations in the Ddras-ThxYl transformant (Esch and Firtel, 1991). I tested this possibility using activated RasG, which phenocopies activated RasD when expressed from the rasD promoter (M. Khosla, unpublished observations). Activated RasG was expressed in prestalk cells and the generated transformants characterized. 3.2 Developmental Phenotype of ecmAO::rasG(G12T) Transformants The rasG(G12T) cDNA was cloned downstream of the ecmAO promoter (the complete promoter for the ecmA gene) (Early et al., 1993) in order to direct RasG(G12T) overexpression in ecraA-expressing prestalk cells. The construct was 69 transformed into Dictyostelium Ax2 cells. Multiple transformants were selected and maintained in 10ug/ml G418. Western blot analysis was used to detect overexpression of RasG(G12T) during development and the result for one transformant is given in Fig. 3-1. The transformant (Fig. 3-1B) contained a higher level of RasG relative to the wild type Ax2 control (Fig. 3-1 A). To examine the developmental characteristics of these ecmAO::rasG(G12T) transformants relative to wild type Dictyostelium, cells were starved on nitrocellulose filters to induce development. Wild type cells (transformed with a prespore specific lacZ construct as a control for transformation) aggregated to form a mound with a single apical tip (Fig. 3-2A) which first elongated to form a finger shaped structure (Fig. 3-2B) and then fell to the substratum to form a slug (Fig. 3-2C). Development culminated in the formation of a fruiting body. Fig. 3-2D shows a wild type culminating structure with the prespore mass raised above the substratum by a stalk. When ecmAO::rasG(G12T) transformants were starved under similar conditions, the cells aggregated and formed mounds at approximately the same time as wild type cells. However, the subsequent development of each of the transformants studied was slow, asynchronous, and terminated with the formation of varied abnormal culminants. Due to the variation in developmental phenotypes exhibited by each of the transformants, only the most commonly observed morphologies are described. Some aggregates did not progress beyond the mound stage. Other mounds produced a single small but distinct tip (Fig. 3-3A). In many cases, the tipped mounds formed by ecmAO::rasG(G12T) cells elongated vertically to form a finger (Fig. 3-3B) which subsequently fell to the substratum to form a slug (Fig. 3-3C). These early structures were morphologically similar to the wild type structures (Fig. 3-2A, B, C). However, by the end of the developmental program, no wild type fruiting bodies 70 B Figure 3-1: Western blot of cell lysates harvested at 16 hours of development and probed with an antibody specific for RasG. The lysate in lane (A) was made from wild type Ax2 cells. The lysate in lane (B) was made from an ecmAO::rasG(G12T) transformant. Figure 3-2: Developmental morphologies of psAr.lacZ control transformant cells. Cells were starved on filters to induce development and were photographed at various stages. Cells formed a single-tipped mound (A), an elongated finger, (B), a slug (C), and a culminating fruiting body (D). 72 B Figure 3-3: Developmental structures formed by ecmAO::rasG(G12T) transformants at different stages. Transformants were plated on filters for development and photographed at various stages. A mound of transformant cells formed a single tip (A). Some tipped mounds elongated to form a finger (B), which fell to the substratum to form a slug (C). Culminants were of various irregular morphologies (D) and (E). The culminant in (E) contained a cloudy and yellow sorus-like cell mass. Culminants of an ecmAO ::rasG transformant are also shown (F). 73 were observed. Instead, a variety of morphologically abnormal culminants were produced by each of the ecmAO::rasG(G12T) transformants. Those that most resembled normal fruiting bodies consisted of a spherical sorus-like structure supported on a short irregular cell mass (Fig. 3-3D). However, other more aberrant culminant morphologies were also observed (Fig. 3-3E). The ecmAO::rasG(G12T) transformants required 36 to 48 hours to form these terminal structures whereas most wild type cells had completed development in 24 hours. Although the developmental morphology of the ecmAO::rasG(G12T) transformants was varied, no multi-tipped mounds were observed at any stage. This indicates that the expression of activated RasG (and by implication, RasD) in prestalk cells did not cause multi-tip formation. Transformants that expressed wild type rasG from the ecmAO promoter were also generated. Developmental morphologies of these ecmAO::RasG transformants were also varied and irregular. But, as shown in Fig. 3-2F, these transformants were able to form fruiting bodies with sori raised on stalks. 3.3 Terminal Differentiation of ecmAO::rasG(G12T) Transformants Although the terminal structures formed by the ecmAO::rasG(G12T) transformants were abnormal, some of the sorus-like cell masses were cloudy and yellow (Fig. 3-3E), indicating the possibility that they contained mature spores (Staples and Gregg, 1967). However, when filters of developed transformant cells were harvested in Bonner's salts containing Trition-X-100, the number of spores observed for the transformants was far less than for wild type strain. Table IV lists spore formation values for wild type (Ax2) cells and for one ecmAO::rasG(G12T) transformant. The transformant produced a very low number of spores. Spore formation data for an ecmAOr.msG transformant is also given. These cells also 74 Table IV. Spore formation by wild type cells and ecmAO::rasG(G12T) and ecmAOr.rasG transformant cells. Strain Spore Formation (as % of cells induced to develop)* Ax2 ecmAO::rasG(G12T) ecmAOr.rasG 114 ± 10 0.5 ± 0.4 66 ± 7 *Spore formation values are the average (and range) from two separate samples in one experimental trial. 75 produced fewer spores than the wild type but numbers were higher than for the ecmA0::rasG(G12T) transformants. The ecmAO::rasG(G12T) culminants were also examined for the presence of stalk cells. Culminants were stained with calcofluor, which binds cellulose in spore coats and stalk cell walls (Harrington and Raper, 1968), and were then viewed by fluorescence microscopy. In a wild type culminant, the cell walls of cells in the basal disc and in the emerging stalk tube stained with calcofluor (Fig. 3-4A). Similarly stained cells were also observed in an ecmAO::rasG(G12T) culminant (Fig. 3-4B). The results indicate that these transformants were capable of differentiating into mature stalk cells. However, the structures formed were unlike the long, slender stalks formed by wild type cells (Fig. 3-4A) and stalk tubes were not observed. The arrow in Fig. 3-4B points to an apparently unstained mass of cells in the ecmAO::rasG(G12T) culminant that corresponded to a sorus-like cell mass. The lack of stain suggests the absence of spores, consistent with the fact that the transformants were defective in spore formation. In the wild type culminant shown in Fig. 3-4A, the spore head was outside the field of view but scattered, stained spore cells were visible. 3.4 Cell Type Specific Gene Expression During Development of ecmAO::rasG( G12T) Transformants To further investigate the developmental characteristics of the ecmAO::rasG(G12T) transformants I examined the expression patterns of cell type specific genes. Northern blots of R N A isolated from ecmAO::rasG(G12T) transformant cells and control cells (transformed with the ecmAOr.lacZ vector) (Early et al., 1993) at various developmental time points were probed with a labeled cDNA fragment of the prespore specific cotC gene (Fosnaugh et al., 1989). Compared to the cotC expression Figure 3-4: Calcofluor staining of wild type and ecmAO::rasG(G12T) structures. Culminants of wild type Ax2 cells (A) and ecmA0::rasG(G12T) transformant cells (B) were stained with calcofluor and viewed by fluorescence microscopy to identify cellulose in stalk cell walls. The arrow in (A) points to a wild type stalk tube. The arrow in (B) points to a sorus-like cell mass in the transformant culminant. 10 12 14 24 36 cote Control 77 B 10 12 14 24 36 y ^ ' M r w W i n i i i i l i l l i i i i in naM ; ! M H I RasG(G12T) 10 12 14 16 24 36 D RasG(GlZT) 16 18 24 36 ecmB Control 16 18 24 36 RasG(G12T) Figure 3-5: Northern blots of R N A of ecmAO::/acZ transformed control cells and of ecmAO::rasG(G12T) cells at various developmental time points. R N A was isolated, electrophoresed, and blotted as described in Chapter Two. The numbers above the lanes refer to the hour of development at which R N A was isolated. The blots in (A, C , E) are from ecmAOr.lacZ control transformant cells. The blots in (B, D, F) are from ecmAO::rasG(G12T) cells. Blots in (A, B) were probed with the prespore specific cotC cDNA. Blots in (C, D) were probed with the prestalk specific ecmA cDNA. Blots in (E, F) were probed with the prestalk specific ecmB cDNA. 78 pattern in the control cells (Fig 3-5A), expression in the transformant was delayed by 2 hours but mRNA levels were only slightly reduced (Fig. 3-5B). I also probed northern blots with two prestalk specific labeled cDNA's, ecmA and ecmB (Williams et al., 1987; Ceccarelli et al., 1987). Relative to the expression of ecmA in the control cells (Fig. 3-5C), the expression in the ecmAO::rasG(G12T) cells was delayed by 2 hours and the mRNA levels were slightly elevated (Fig. 3-5D). For the ecmB gene also, compared to the wild type cells (Fig. 3-5E), the transformant exhibited an increase in expression levels (Fig. 3-5F). These results demonstrate that the expression of activated RasG in prestalk cells resulted in a slight reduction of prespore gene expression and a slight enhancement of prestalk gene expression. In the Ddras-Thrl2 transformant, prespore gene expression was drastically reduced and prestalk gene expression was drastically enhanced (Louis et al., 1997a). Thus, the ecmAO::rasG(G12T) transformant exhibited far less pronounced effects on cell type specific gene expression than those observed in the Ddras-Thxl2 transformant. 3.5 PstA Cell Localization in ecmAO::rasG(G12T) Transformants The slugs formed by the ecmAO::rasG(G12T) cells appeared morphologically similar to wild type slugs but did not go on to complete normal morphogenesis. One possible explanation was that the positioning of the various cell types within the slugs was unusual. I used cell type specific lacZ reporter constructs to determine the localization of different cell types in the developing ecmAO::rasG(G12T) organisms. The ecmAr.lacZ construct incorporates the proximal domain of the ecmAO promoter and drives expression of the linked gene in the PstA subset of prestalk cells (Early et a l , 1993). When Ax2 cells transformed with this construct were developed, stained cells in mounds were concentrated in the center of the mound (as viewed from above) but were also observed scattered throughout the mound (Fig. 3-6A). Figure 3-6: PstA cell localization in developing wild type and ecmAO::rasG(G12T) structures. A wild type mound (A), finger (B), slug (C), and culminant (D), and an ecmAO::rasG(G12T) mound (E), finger (F), slug (G), and culminant (H) were stained to detect the expression of p-galactosidase from the ecmA promoter. The arrows in (B, F) point to the tips of the fingers. The arrows in (C, G) point to the tips of the slugs. 80 Subsequently, PstA cells were strongly localized to the tips of the fingers (Fig 3-6B) and slugs (Fig. 3-6C) that were formed. As developing structures culminated, PstA cells were detected in the anterior tip of the culminant as well as in the stalk tube (Fig. 3-6D). This staining pattern is similar to that previously observed (Early et al., 1993 and 1995). When mounds of an ecmAO::rasG(G12T)/ecmA::lacZ cotransformant were viewed from above, cells staining for |3-galactosidase expression were observed to be concentrated in the center (Fig. 3-6E), similar to the pattern seen in wild type mounds (Fig. 3-6A)). However, the central stained cells did not rise upward to localize to the tip of elongating fingers (Fig. 3-6F). Transformant slugs varied with respect to the amount of anterior staining, but the tips of the slugs contained relatively few PstA cells (Fig. 3-6G) compared to wild type slugs (Fig. 3-6C). The majority of PstA cells in ecmAO::rasG(G12T) slugs were ectopically localized to the posterior (Fig. 3-6G). Cells expressing the PstA marker were also shed from the posterior of some slugs and expression was detected in scattered cells throughout the slug (Fig. 3-6G). In the culminants that contained a sorus-like structure, the irregular mass of supporting cells stained for lacZ expression (Fig. 3-6H). These results indicate that activated RasG expression in PstA cells resulted in their mislocalization in the developing organism. 3.6 PstO Cell Localization in ecmAO::rasG(G12T) Transformants A fusion between the distal portion of the promoter for the ecmA gene that had been linked to lacZ, was used to identify PstO cells (Early et al., 1993). In slugs of Ax2 cells carrying the ecmOr.lacZ marker, /acZ-expressing cells were located in an anterior collar just posterior to the tip and in A L C throughout the prespore region (Fig. 3-7A). During culmination, /acZ-expressing cells were observed mostly in the region forming the upper cup and some were also present in the lower cup (Fig. 3-7B). These results 81 Figure 3-7: PstO cell localization during wild type and ecmAO::rasG(G12T) development. A wild type slug (A) and culminant (B) and an ecmAO::rasG(G12T) slug (C) and culminant (D) stained to detect expression of (3-galactosidase from the ecmO promoter. The arrows in (A, C) point to the tips of the slugs, are similar to staining patterns previously observed for PstO cells (Early et al., 1993 and 1995). 82 When cells cotransformed with ecmAO::rasG(G12T) and ecmOr.lacZ were induced to develop, the usual collar of PstO cells was not observed in the slugs; only a low level of expression occurred and this was limited to a few scattered cells (Fig. 3-7C). Although P-galactosidase expression increased slightly as development progressed in culminants, the p-galactosidase expression was limited to a small region at the posterior of the organism (Fig. 3-7D). These results indicate that activated RasG expression inhibited the differentiation and localization of PstO cells. 3.7 PstB Cell Localization in ecmAO::rasG(G12T) Transformants The promoter for a second prestalk gene, ecmB, was also used to drive lacZ expression in order to identify the location of PstB cells (Jermyn and Williams, 1991). PstB cells were observed to be scattered throughout wild type mounds (Fig. 3-8A) In slugs, PstB cells were found in the posterior rearguard region of the slug, as anterior like cells scattered throughout the prespore region of the slug, and in a group of cells in the anterior prestalk region (Fig. 3-8B). At culmination, lacZ expression was observed in the stalk, the basal disc, and in the upper and lower cups (Fig. 3-8C). The wild type staining patterns observed for P-galactosidase expression were consistent with those reported previously (Ceccarelli et al., 1991; Jermyn and Williams, 1991; Jermyn et a l , 1996). In ecmAO::rasG(G12T) mounds, PstB cells were observed scattered throughout the mound and concentrated in the center (Fig. 3-8D), a pattern that was identical to the mound stage localization of PstA cells (Fig. 3-6E). This staining pattern is unusual since in wild type mounds PstB cells were not concentrated in the center (Fig. 3-6A). The central PstB cells did not rise to the tip and in transformant slugs the anterior 83 Figure 3-8: PstB cell localization in wild type and ecmAO::rasG(G12T) structures. A wild type mound (A), slug (B), and culminant (C), and an ectnAO::rasG(G12T) mound (D), slug (E), and culminants (F, G) stained to detect |3-galactosidase expression from the ecmB promoter. The arrows in (B, E) point to the slug tips. The arrows in (F, G) point to the tips of the culminants. 84 region remained free of stained cells (Fig. 3-8E). p-galactosidase expression in the slugs was observed in the posterior rearguard cells and in scattered cells in the prespore region (Fig. 3-8E). By the time culminants were formed, some structures had gained the ability to express ecmBr.lacZ in anterior tips. In the culminant depicted in Fig. 3-8F, PstB cells were localized in the tip as well as in the posterior of the structure and in the discarded sheath. In other culminants such as those in Fig. 3-8G, PstB cells were localized exclusively at the posterior in the irregular shaped cell mass that supports the sorus-like structure. The ecmB promoter has been dissected and a region termed ST has been shown to be responsible for directing expression only in the cells that form the stalk and the basal disc (Ceccarelli et al., 1991). Ax2 slugs expressing P-galactosidase from this promoter fragment exhibited staining in a few cells within the anterior tip region (Fig. 3-9A). During culmination, expression was detected in the extending stalk in and the basal disc (Fig. 3-9B). In the developing ecmAO::rasG(G12T) cells marked with the STr.lacZ construct, P-galactosidase expression was delayed relative to expression in wild type cells and stained cells were only observed in the posterior of the developing slugs (Fig. 3-9C). During the subsequent morphogenesis of these transformants, lacZ expression remained restricted to the posterior of the organism. Culminants expressed P-galactosidase in the irregular cell mass that supported the sorus-like structure (Fig. 3-9D). There was no indication of a developing stalk tube within the sorus-like structures of the transformants. The pattern of ecmBr.lacZ staining was consistent with that of calcofluor staining since both indicate that stalk cells were formed in the basal regions of ecmAOr.rasG(GHT) culminants. • 85 Figure 3-9: Localization of cells expressing the STr.lacZ construct during wild type and ecmAO::rasG(G12T) development. A wild type slug (A) and culminant (B) and an ecmAO::rasG(G12T) slug and culminant stained to detect P-galactosidase expression from the ST promoter fragment. The arrows in (A, C) point to the tips of the slugs. 86 3.8 Prespore Cell Localization in ecmAO::rasG(G12T) Transformants To determine prespore cell localization, I used the prespore specific psA promoter (Early et al., 1988) to drive expression of lacZ. Since prespore gene expression was slightly reduced in the ecmAO::rasG(G12T) transformants (see Section 3.4), it was possible that the number of prespore cells within multicellular structures decreased during the developmental time course. To examine this possibility, I used a psA::(his)lacZ construct encoding a labile P-galactosidase (with a half-life of approximately 3 hours (Detterbeck et al., 1994)) so that a loss of prespore cells would correlate with a decrease in the proportion of stained cells. When wild type slugs expressing this labile P-galactosidase from the psA promoter were fixed and stained, the majority of the slug was stained blue except for the prestalk region in the anterior of the slug and the posterior rearguard region (Fig. 3-10A). In culminating wild type structures, the developing sorus was intensely stained for lacZ expression while the anterior tip and developing stalk remained unstained (Fig. 3-10B). These results are consistent with the expression pattern previously reported (Detterbeck et al., 1994). In ecmAO::rasG(G12T) slugs, psA-driven lacZ expression was observed in the prespore region and also in the posterior rearguard region while the anterior region remained unstained (Fig. 3-10C). Culminants formed by the ecmAO::rasG(G12T) transformants contained small patches of stained cells amongst irregular masses of unstained cells (Fig. 3-10D). These results demonstrate that initial prespore cell localization was essentially normal in the ecmAO::rasG(G12T) slugs. The results also indicate that by the end of development, the proportion of prespore cells was reduced in the transformant structures. 3.9 Spore Formation in Chimaeras of Wild Type and ecmAO::rasG(G12T) Cells Since prespore cell specific gene expression and slug stage prespore cell Figure 3:10: Prespore cell localization in wild type and ecmAO::rasG(G12T) structures. A wild type slug (A) and culminant (B) and an ecmAO::rasG(G12T) slug (C) and culminant (D) stained to detect expression of a labile P-galactosidase from the psA promoter. The arrows in (A, C) point to the slug tips. 88 localization were both relatively normal in the ecmAO::rasG(G12T) transformants, I reasoned that the lack of spore formation likely resulted from a defect at a late stage of development. It was possible that the transformant cells were defective in the production of a sporulation signal, in which case the phenoypte might be rescued by providing the wild type signal. To test this hypothesis, I mixed ecmAO::rasG(G12T) cells and wild type cells, induced them to develop in chimaera, and assayed for spore formation. Harvested spores were grown in medium with or without G418 selection to determine their respective genotype. As shown in Table V , the number of spores formed during development of chimaeric mixes was less than the number of spores formed during development of wild type cells alone. However, of the spores formed by the chimaeras, the majority were produced by the ecmAO::rasG(G12T) transformant cells. These results indicate that the transformant prespore cells were capable of terminally differentiating into spore cells and that during ecmAO::rasG(G12T) development, a signal inducing this differentiation was absent. 3.10 Cell Sorting in Chimaeras of Wild Type and ecmAO::rasG(G12T) Cells The number of spores formed by chimaeric mixtures of wild type and ecmAO::rasG(G12T) cells was much less than would be expected if the fraction of wild type cells in the chimaera were induced to develop alone. This indicates that the transformant cells may have an inhibitory effect on wild type spore formation. Alternatively, wild type cells may preferentially form the prestalk and stalk cell regions of a mixture. To test this possibility, ecmAO::rasG(G12T) cells were mixed in a 9:1 ratio with wild type cells marked with the actl5::lacZ construct. In these chimaeras, wild type cells expressed the lacZ gene throughout development. As shown in Fig. 3-11 A , in chimaeric slugs, wild type cells were preferentially localized to the tip and Table V . Spore formation by chimaeras of wild type and ecmAO::rasG(G12T) transformant cells. 89 Chimaera Spore Formation (as % of cells induced to develop)* ecmAO::rasG(G12T) Spores (as % of total spores formed) 100% Ax2 90 ± 1 1 0 25% Ax2 75% ecmAO::rasG(G12T) 19 ± 2.7 61 10% Ax2 90% ecmAO::rasG(G12T) 2 ± 0 . 3 83 100% ecmAO::rasG <1 100 *Spore formation values are the average (and range) from two separate samples in one experimental trial. 90 Figure 3-11: Cell sorting in chimaeras of Ax2 and ecmAO::rasG(G12T) cells. A slug (A) and culminants (B) formed by mixtures of 10% actl5::lacZ labeled Ax2 cells mixed with 90% unlabeled ectnAO::rasG(G12T) cells and a slug (C) and culminant (D) formed by 10% actl5::lacZ labeled ecmAO::rasG(G12T) cells mixed with 90% unlabelled Ax2 cells. 91 were also found scattered in the prespore region. In chimaeric culminants (Fig. 3-11B), the tips and stalk tubes were stained indicating that wild type cells constructed stalks during mixed development. Often, a mound of unstained cells was observed at the base of the chimaeric culminant, indicating that the ecmAO::rasG(G12T) cells were either forming an enlarged basal disc or being excluded from the developing structure (Fig. 3-11B). When unmarked wild type cells were mixed in a 9:1 ratio with actl5::lacZ labeled ecmAO::rasG(G12T) transformants, stained cells were localized to the rearguard region and throughout the prespore region of the slug, but very few were present in the anterior (Fig. 3-1 IC). This result indicated that again the tip of the chimaeric slug was formed by wild type cells. Thus, the ecmAO::rasG(G12T) transformants are inherently defective in the formation of normal tip cells. In culminants, ecmAO::rasG(G12T) cells were observed in the basal disc and in the prespore region, but seldom in tip or in the stalk tube (Fig. 3-1 ID). 3.11 Phototaxis and Motility of ecmAO::rasG(G12T) Slugs It has been shown that the tip region is responsible for directing slug motility and phototaxis (reviewed by Fisher, 1997). Given that the ecmAO::rasG(G12T) transformant slugs exhibited mislocalization of PstA cells which normally occupy the tip, I examined the ability of the ecmAO::rasG(G12T) slugs to migrate towards light. After two days of exposure to unidirectional light, Ax2 slugs had clearly migrated away from the point of deposition and towards the light (Fig. 3-12A). In contrast, ecmAO::rasG(G12T) slugs had migrated in random directions for only very short distances (Fig. 3-12B). These results indicated that the ecmAO::rasG(G12T) slugs were not phototactic and exhibited reduced motility compared to wild type slugs. When chimaeric slugs with 10% Ax2 cells and 90% ecmAO::rasG(G12T) cells were exposed to 92 Figure 3-12: Results of phototaxis assay. Slugs of Ax2 cells (A), ecmAO::rasG(G12T) cells (B), and chimaeric mixtures of 10% Ax2 and 90% ecmAO::rasG(G12T) cells were tested for their ability to migrate towards a unidirectional light source. The arrows indicate the direction of light entry. Cells were deposited on the right side of the rectangle on the bottom of each plate. 93 unidirectional light, some slugs migrated towards the light, indicating that the presence of wild type cells was able to partly rescue the phototactic defect (Fig. 3-12C). 3.12 Discussion As discussed previously, overexpression of activated Ras from the rasD promoter deranges normal morphogenesis and cell type differentiation (Reymond et al., 1986; Louis et al., 1997a). Since rasD expression is enriched in the prestalk cells, it was hypothesized that the overexpression of activated RasD in this population of cells caused the abnormal development (Esch and Firtel, 1991). The results presented in this chapter indicate otherwise. The developmental abnormalities seen in the ectnAO::rasG(G12T) transformants were varied, however, the transformants did not produce mounds with multiple tips. Although some mounds produced no tip, those that did produced single tips that were able to elongate and form slugs. In addition, although cell type specific gene expression was slightly altered in the ecmAO::rasG(G12T) transformants, the drastic deregulation that occurs in the Ddras-Thrl2 transformants (Louis et al., 1997a) was not observed. I cannot dismiss the possibility that activated RasG and activated RasD would induce different developmental phenotypes if expressed from the ecmAO promoter. However, expression of either protein from the rasD promoter induces the same developmental phenotype (M. Khosla, unpublished observations). Therefore, the results obtained with the ecmAO::rasG(G12T) transformants suggest that the overexpression of activated RasD in the ecmA-expressing prestalk cells was not solely responsible for the developmental phenotypes of the Ddras-Thrl2 transformants. The normal progression of prestalk differentiation is summarized in Fig. 3-13. Progenitor amoebae differentiate into four distinct prestalk subtypes, PstA, PstB, PstO, and A L C . The majority of PstA cells in the tip become PstAB cells prior 94 T ip ^ T i P -PstA P s t A B * Stalk non-Tip > PstAB a m o e b a e - 1 ^ P s t B ^ Basal Disc PstO • Upper Cup Figure 3-13: A schematic diagram depicting the differentiation of the various prestalk cell types during development and the effects of ecmAO::rasG(G12T) expression. Cross bars indicate differentiation steps that are inhibited in the transformant cells. The dotted line indicates a differentiation step that does not normally occur but is favored in the transformant cells. Wild type amoebae that differentiate into PstO cells in the mound form the upper cup of the mature culminant. The differentiation of this cell type is largely inhibited in the ecmAO::rasG(G12T) transformants. Wild type PstA cells sort to the tip where they subsequently form PstAB cells and initiate formation of the stalk. This differentiation pathway was also inhibited in the ecmAO::rasG(G12T) transformants. Some wild type PstAB cells move to the posterior during slug migration and subsequently participate in basal disc formation during culmination- In the ecmAO::rasG(G12T) transformants, posterior PstAB cells were observed and believed to contribute to the formation of an extended basal disc. However, these cells did not arise from anterior cells. Wild type PstB cells at the posterior of the slug contribute to basal disc formation. Although PstB cell differentiation and localization was affected in the transformants, this normal pathway was still observed. The A L C , which contribute to the lower cup formation, have not been depicted for the sake of simplicity. 95 to terminal differentiation, at which point they form stalk cells and build the stalk tube (Jermyn and Williams, 1991; Sternfeld, 1992). A subset of tip PstA cells become PstAB cells during slug migration. These become localized to the posterior of the slug, and contribute to the formation of the basal disc (Sternfeld, 1992). PstB cells are localized mostly in the posterior of the slug and terminally differentiate as stalk cells and also contribute to the formation of the basal disc, although some are discarded during slug migration (Jermyn and Williams, 1991; Sternfeld, 1992). Cells that differentiate as the PstO subtype are ultimately found in the upper cup at culmination (Early et al., 1993; Abe et al., 1994). Some progenitor cells differentiate into A L C , but this conversion is not shown in Fig. 3-17 since it is not relevant to the following discussion. Overexpression of activated RasG from the ecmAO promoter induced a number of dramatic changes in prestalk differentiation. In developing ecmAO::rasG(G12T) transformants, most PstA cells were not positioned in the tip of the slug, but instead were localized in the posterior. PstO cells were reduced in number and of these, most were scattered throughout the slug rather than localized in an anterior collar. The proportion of PstB cells appeared to be greater and these cells were found in the posterior of the slugs and later in development, were often found in the anterior regions of culminants also. Although stalk cells were formed, they were confined to the posterior region of the terminally differentiated organism and no stalk tube was observed. I propose that the major direct effect of Ras(G12T) in these transformants is the mislocalization of PstA cells and that the remaining developmental aberrations are consequences of this mislocalization. The tip cells have been referred to as the . organizers of multicellular development in Dictyostelium (Raper, 1940; Rubin and Robertson, 1975; Durston, 1976; Schaap, 1986; Siegert and Weijer, 1995) and the tip is 96 normally comprised of PstA cells (Jermyn and Williams, 1991; Abe et a l , 1994; Early et al., 1995). Since the tip is responsible for directing slug migration (Fisher, 1997), initiating formation of the stalk tube (Jermyn and Williams, 1991; Sternfeld, 1992), and orchestrating the movements necessary for culmination (Dormann et al., 1996; Chen et al., 1998), it is not surprising that the slugs of the ecmAO::rasG(G12T) transformant exhibited abnormalities in these processes. M y results show that the outwardly normal slugs formed by these transformants were compromised in both phototaxis and migration, could not initiate stalk tube synthesis, and formed a variety of disorganized terminal structures. During wild type development, the PstA cells arise at the periphery of the mound and then sort to the tip (Early et al., 1995). The mechanism of PstA cell sorting has not been fully elucidated but differences in the chemotactic response of the cells may be involved. The PstA cells chemotax towards c A M P more quickly than the PstO or prespore cells and this difference may facilitate PstA cell migration from the mound periphery to the mound apex (Early et al., 1995). Activated RasD has previously been shown to affect desensitization of guanylate cyclase (Van Haastert et al., 1987) which is involved in chemotaxis. However, cells expressing activated Ras still chemotax towards cAMP. In addition, PstA cells in the ecmAO::rasG(G12T) transformants were observed in the mound center from where the tip would rise. Thus, the cells did initially sort in the expected manner. In addition, the localization defect of the ecmAO::rasG(G12T) transformants was cell autonomous. When a small number of labeled transformant cells were mixed with wild type cells, the transformants were excluded from the tip. This indicates that the localization defect of the transformants was not a consequence of the lack of a localization signal. Also, the presence of ecmAO::rasG(G12T) cells did not prevent the localization of wild type cells to the tip in 97 a chimaeric slug indicating that the transformant slugs did not produce a signal that interferes with this process. I propose that RasG(G12T) induces a more rapid conversion of PstA to PstAB cells and as a consequence, the PstA cells do not localize in the tip. In support of this hypothesis is the observation that wild type anterior PstA cells that have become PstAB move to the posterior region of the slug from where they are discarded during slug migration (Sternfeld, 1992). In addition, there are notable similarities between the staining patterns for ecmAr.lacZ and ecmBr.lacZ expression in the ecmAO::rasG(G12T) transformants at the slug stage and even earlier at the mound stage. A l l the regions that stain for ecmA expression also stain for ecmB expression. I suggest that RasG(G12T) induces PstA cells to express ecmB and hence become PstAB cells. As a result, these cells migrate to the posterior region of the organism. Further support for this hypothesis comes from the observation that although ecmAO::rasG(G12T) slugs do not migrate extensively, they discard large clumps of ecmA and ecmB expressing cells from the posterior. Although all the regions of the ecmAO::rasG(G12T) slug that stained for ecmA expression also stained for ecmB expression, there were cells in the tip region of some structures that only stained for ecmB expression and were therefore classified as PstB cells. Localization of PstB cells in the anterior is not observed during normal development and the ectopic localization of PstB cells in the anterior of the transformant may also be a consequence of the mislocalization of PstA cells. During normal development, the PstA cells at the tip are believed to be the source for c A M P oscillations. Since ecmAO::rasG(G12T) slugs did not have a normally functioning tip, c A M P oscillations from the tip would likely have been significantly altered. c A M P inhibits expression from the ecmB promoter (Ceccarelli et al., 1987), so the absence of PstA cells at the tip may result in the expression of ecmB in that region. 98 A role for Ras in modulating cell migration during development has been described in other systems. In C. elegans, Ras has been implicated in the migration of sex myoblasts. A pair of sex myoblasts (SMs) migrate anteriorly during the second larval stage to the mid-body to become positioned on either side of the gonad. In loss of function let-60-ras mutants and in gonad-ablated organisms, the SMs still migrate anteriorly but their final position is altered such that a broad range of positions were observed (Sundaram, et al., 1996). Constitutively1 active let-60-ras transgenes displayed no phenotype in a wild type background but allowed proper positioning of the SMs in gonad-ablated organisms, suggesting that Ras alters the S M signal response (Sundaram, et al., 1996). In Drosophila, a group of anterior follicle cells, called border cells, migrate from the anterior tip of the egg chamber to the nurse cell - oocyte border during stage 9 of oogenesis. Expression of dominant negative Ras prior to the migration period induces early migration whereas activated Ras expression inhibits migration. When expressed during the migration period, both dominant negative and activated Ras inhibit migration (Lee et a., 1996; Lee and Montell, 1997). Increasing Ras activity during early oogenesis induces expression of posterior markers in the anterior follicle cells indicating an alteration of cell fate that may consequently affect the migration of these cells (Lee and Montell, 1997). This situation is similar to that observed in Dictyostelium where activated Ras changes PstA cells to PstAB cells and consequently alters their localization. The PstB cells observed in the tips of some structures were clearly not able to rescue normal tip functions. Notably, they were unable to initiate synthesis of a stalk tube. Hence, staining for neither the STr.lacZ reporter nor calcofluor was observed in the anterior region of the ecmAO::rasG(G12T) organisms. These results also indicated that the anterior prestalk cells did not create the upper cup, which normally appears 99 after stalk formation. Thus, it appears that the anterior PstB cells did not terminally differentiate. Cells expressing ecmB at the posterior of the organisms were able to terminally differentiate. Based on the intense expression of both ecmA and ecmB reporter constructs in this region, these cells were most likely PstAB cells. Normally, this rearguard region participates in formation of the basal disc (Jermyn and Williams, 1991; Sternfeld, 1992). Cells in this region were the only ones to express lacZ from the ST promoter and to stain with calcofluor, suggesting that the stalk cells formed during ecmAO::rasG(G12T) development were extended basal disc structures. Spore formation was also inhibited during ecmAO::rasG(G12T) development. Since prespore cell gene expression and prespore cell localization was not drastically altered in these transformants, prespore differentiation was probably normal. I conclude that the lack of spore formation is a result of a block in the conversion of prespore to spore cells at culmination. Prestalk cells have been shown to release factors that signal prespore cells to induce sporulation (Anjard et al., 1998). It is believed that the signal is generated from the tip since sporulation occurs progressively from the anterior to the posterior of the sorogen (Richardson et al., 1994). The absence of spore formation in ecmAO::rasG(G12T) transformants was therefore likely to be another consequence of the defective tip. Additional evidence supporting the idea that mislocalization of tip cells was the primary defect in the ecmAO::rasG(G12T) transformants comes from the results of chimaeric development. When mixed with a majority of transformant cells, Ax2 cells preferentially sorted to the tip region. The presence of wild type cells in the tip enabled chimaeric organisms to sense and respond to a light gradient, construct a stalk tube, and form spores. 100 Since activated RasG would be expected to be simultaneously expressed in PstA cells and PstO cells, I cannot rule out the possibility that Ras(G12T) had a primary effect in PstO cell differentiation which in turn lead to the later developmental defects. Since very few PstO cells were formed during ecmAO::rasG(G12T) development it is unlikely that activated RasG stimulated the production of an inhibitor in PstO cells. However, lack of PstO cells could have contributed to the abnormal phenotypes. It is more likely that activated RasG simply shut off expression from the ecmO specific regions of the ecmAO promoter and cell autonomously inhibited PstO differentiation. Alternatively, it is also possible that PstA cells expressed activated RasG and laterally inhibited the differentiation of PstO cells. Lateral regulation of PstO specification by PstA cells has been previously observed for the warai null cells (Han and Firtel, 1998). The Dictyostelium H O X (hox) gene, warai, is expressed in PstA cells, warai null cells exhibit an enlarged PstO compartment, indicating that PstA cells regulate the proportion of PstO cells. In Chapter One, I presented a few examples of Ras-mediated H O X gene regulation. It is possible that Ras(G12T) could be a positive inducer of Warai or of another homeotic gene specifying PstAB or PstB cell fate. The Work presented in this chapter was intended to determine if expression of activated RasG in the ecraA-expressing prespore cells could recapitulate the Ddras-Thrl2 phenotype. The results illustrate that RasG(G12T) expression from the ecmAO promoter resulted in the formation of single-tipped aggregates and only slightly altered cell type specific gene expression patterns. The ecmAO::rasG(G12T) transformants did exhibit alterations in prestalk cell localization and consequently the tips formed lacked normal organizing capacity. Thus, it is unlikely that activated RasD expression in the prestalk population induces the multi-tipped aggregates and 101 the drastically deregulated cell type specific gene expression patterns characteristic of the Ddras-Thrl2 transformant. CHAPTER FOUR OVEREXPRESSION OF RASG(G12T) IN PRESPORE CELLS 102 4.1 Background I expected that overexpression of activated RasG in the ecraA-expressing prestalk cells would recapitulate the phenotype of the Ddras-ThxYl transformant. However, as described in Chapter Three, this was not the case. The ecmAO::rasG(G12T) transformants did not form multi-tipped mounds, were not blocked from slug formation, and did not exhibit drastic alterations in the expression of cell type specific genes. Given these results, it was important to determine if the overexpression of activated RasG in prespore cells would produce a phenotype similar to that of the Ddras-Thrl2 transformant. Although rasD was originally identified as a prestalk specific gene (Reymond et al., 1984), its expression is not limited to the prestalk population. Whereas ecmA expression is ten-fold enriched in prestalk cells over prespore cells, rasD expression is only three-fold enriched (Jermyn et al., 1991). Using a labile p-galactosidase expressed from the rasD promoter, Jermyn and Williams (1995) demonstrated that an appreciable level of rasD expression occurs in the prespore cells of the aggregate. Although prespore specific expression decreases as development progresses beyond the tipped mound stage, it is conceivable that the overexpression of activated RasD in the prespore population causes the developmental abnormalities observed by Reymond et al. (1986). To test this possibility, I used a prespore specific promoter to overexpress RasG(G12T) specifically in prespore cells and characterized the resulting transformants. 103 4.2 Developmental Morphology of psA::rasG(G12T) Transformants PsA is a cell surface glycoprotein expressed only in prespore cells (Early et al., 1988). The promoter for the psA gene was used to construct a vector to drive overexpression of rasG(G12T) exclusively in prespore cells. The psA::rasG(G12T) construct was transformed into Dictyostelium Ax2 cells and stable transformants were selected. Overexpression of activated RasG during development was demonstrated in several independently isolated transformants by western blot analysis and data for two of the transformants is shown in Fig. 4-1. Both psA::rasG(G12T) transformants clearly overexpressed RasG protein compared to the vector control transformant at 16 hours of development. The level of overexpressed protein was higher in clone A than in clone B. The overexpressed protein in the transformant was slightly larger than the endogenous RasG because the transgene encodes additional amino acids derived from the 5' end of the psA gene. Upon starvation, wild type cells formed mounds with single tips such as the one shown in Fig. 3-2A, whereas all of the psA::rasG(G12T) transformants formed mounds with multiple tips (Fig. 4-2A). The tips extended to form finger-like projections that then fell to the substratum and appeared to attempt migration, as would be typical of wild type slugs. However, the slug-like structures remained anchored to the basal mound. Many of the mounds formed by the developing psA::rasG(G12T) transformant had two or three emerging protrusions (Fig. 4-2B) while some produced more protrusions (Fig. 4-2C). When the further differentiation of these transformants was monitored by time-lapse video microscopy, it was observed that additional tips continued to emerge from the mounds (data not shown). These structures were formed at the same time that wild type cells had formed slugs (Fig. 3-2C). 104 Figure 4-1: Western blots of lysates from cells harvested at 0 hours (lanes A , C , E) and 16 hours (lanes B, D, F) of development and probed with an antibody specific for RasG. Lanes A and B contain lysates from a control strain transformed with psAr.lacZ. Lysates from two independent psA::rasG(G12T) clones were separated in lanes C , D (clone A) and E, F (clone B). 105 Figure 4-2: Developmental structures formed by psA::rasG(G12T) cells. Cells formed mounds with multiple tips (A) which elongated to form finger-like structures (B, C). Transformant culminants contained a basal mound with emerging stalk-like protrusions and multiple small tips (D). 106 The psA::rasG(G12T) transformants formed aberrant culminants at about the same time that wild type cells formed normal fruiting bodies. The aberrant culminants consisted of a basal mound to which stalk-like structures were attached (Fig. 4-2D). Time lapse photography indicated that the stalk-like structures were formed from the protrusions that were produced initially at the mound stage. Multiple small tips were also visible on basal mound (Fig. 4-2D). Time lapse photography indicated that these tips emerged from the mound late in development and did not extend further. Although most of the stalk-like structures lacked a sorus, a few did produce an apical pseudo-sorus, which was very small and translucent compared to the wild type sorus (Fig. 3-2C). Thus, the prespore specific expression of activated RasG did result in multi-tip formation, similar to that observed for the Ddras-Thrl2 transformant. However, the multiple tips of the psA::rasG(G12T) mounds were not blocked from further morphogenesis. These results suggest that in the Ddras-Thrl2 transformant, expression of activated RasD in the prespore cells causes multiple tips to form but is not sufficient to inhibit elongation of the tips. Transformants overexpressing wild type RasG in prespore cells were also generated and examined for their developmental morphology. Fig. 4-3 shows culminant morphologies for wild type cells, psA::rasG(G12T) cells, and psAr.rasG cells. Wild type fruiting bodies consisted of a cloudy sorus raised on a stalk (Fig. 4-3A). The psA::rasG(G12T) culminants contained mounds with multiple emerging stalk-like structures (many of which had fallen to the substratum) and no sori (Fig. 4-3B). The psAr.rasG culminants contained some basal cell masses and translucent sori raised on stalks (Fig. 4-3B). B Figure 4-3: Culminants of wild type cells (A), psA::rasG(G12T) cells (B), and psAr.rasG cells (C). The arrow in (A) points to a cloudy sorus whereas the arrow in (C) points to a translucent sorus. Photographs were taken with a Nikon 35 mm camera and Kodak TMAX-400 black and white film. 108 4.3 Terminal Differentiation of psA::rasG(G12T) Transformants Given that the psA::rasG(G12T) transformants produced infrequent and small sori, it seemed unlikely that they were capable of producing mature spore cells. Several of the psA::rasG(G12T) transformants were tested and although they varied slightly in their ability to sporulate, spore formation was always much less than for the controls. Values for spore formation for wild type cells and two psA::rasG(G12T) transformants are given in Table VI. The two psA::rasG(G12T) transformants assayed for spore formation were the same for which western blot results are given in Fig. 4-1. I observed that the transformant that expressed a higher level of RasG protein produced fewer spore cells. In addition, as indicated in Table VI, cells expressing psA::rasG (wild type RasG) were also defective in spore formation. Since the culminants formed by the psA::rasG(G12T) transformants contained stalk-like structures, I examined them for the presence of mature stalk cells by staining with calcofluor. Wild type culminants contained calcofluor stained stalk cells in the basal disc and the emerging stalk tube (Fig. 4-4A). When viewed by phase contrast microscopy, these cells were observed to be highly vacuolated (Fig. 4-4B) as is characteristic of stalk cells. When culminants of psA::rasG(G12T) cells were stained with calcofluor, the entire basal mounds and the emerging stalk-like structures were composed of fluorescing cells (Fig. 4-4C). When these structures were viewed by fluorescence microscopy, the stalk-like structures were observed to contain vacuolated cells (Fig. 4-4D), similar to the wild type stalk tube (Fig. 4-4B). However, since the mound cell mass was densely packed, it was difficult to determine if individual cells within the mass stained with calcoflour and were vacuolated. I therefore tapped the coverslip on the microscope slide in order to break apart the structures. Once psA::rasG(G12T) culminant mounds were disrupted, I observed that cells within the mounds were labeled with calcofluor (Fig. 4-4E) and were highly vacuolated in 109 Table VI . Spore formation by wild type cells and psA::rasG(G12T) and psA::rasG transformant cells. Strain Spore Formation* (as % of cells induced to develop) Ax2 128 ± 39 psA::rasG(G12T), clone A 0 psA::rasG(G12T), clone B 0.8 ± 0.2 psAr.rasG 3.8 ± 3 . 2 *Spore formation values are the average (and range) of three separate samples from one experiment. Figure 4-4: Culminants of Ax2 cells (A, B), and psA::rasG(G12T) cells (C, D, E , F) were stained with calcofluor and viewed by fluorescent microscopy (A, C , E) or by phase contrast microscopy (B, D, F). Images in (E, F) are from cells released after a transformant culminant was smashed apart. appearance (Fig. 4-4F). Thus, the majority of cells in the psA::rasG(G12T) culminant terminally differentiated into stalk cells. In Chapter Three, I presented results demonstrating that stalk cell formation was not inhibited when activated RasG was overexpressed in prestalk cells. Together with the data presented here, these results contradicted expectations based on an earlier report that stalk cell formation did not occur in the Ddras-Thxll transformants (Louis et al., 1997a). I therefore examined cells that express RasG(G12T) from the rasD promoter (M. Khosla, unpublished results) by staining culminants with calcofluor. Fig. 4-5 shows data comparing a wild type culminant (Fig 4-5A, B) with a rasD::rasG(G12T) culminant (Fig. 4-5 C , D, E , F). The rasD::rasG(G12T) culminant emitted fluorescence throughout the mound and the multiple protruding tips (Fig. 4-5C). Although this indicates the presence of stalk cells, the density of the structure prevented the observation of cells within the mound by either fluorescent microscopy (Fig. 4-5C) or phase contrast microscopy (Fig. 4-5D). Therefore, the structures were broken apart as described above. Cells released from the rasD::rasG(G12T) culminant mounds were stained with calcofluor (Fig. 4-5E) and were vacuolated (Fig. 4-5F). Thus, overexpression of activated RasG from either the psA or rasD promoters resulted in stalk cell differentiation in the majority of cells in the population. 4.4 Cell Type Specific Gene Expression in psA::rasG(G12T) Transformants To further investigate the aberrant development of psA::rasG(G12T) cells, northern blots of m R N A isolated from one of the transformants and from control cells (transformed with psAr.lacZ) at various times of development were probed with cell type specific cDNA's. As seen in Fig. 4-6A, expression of the prestalk specific gene, ecmA (Williams et al., 1987), was markedly elevated during the development of the 112 D E Figure 4-5: Culminants of Ax2 cells (A, B), and rasD::rasG(G12T) cells (C, D, E , F) were stained with calcofluor and viewed by fluorescence microscopy (A, C , E) or by phase contrast microscopy (B, D, F). Images in (E, F) are from cells released after a transformant culminant was smashed apart. 113 Figure 4-6: Northern blots of m R N A isolated from psA::lacZ transformed control cells (A) and psA::rasG(G12T) cells (B) at the times (in hours) during development indicated above the lanes. Blots were probed with a labeled c D N A fragment of the ecmA gene. The message indicated 'a' corresponds to ecmA m R N A and the message indicated 'b' corresponds to the cross-hybridized ecmB mRNA. 114 psA::rasG(G12T) cells. In control cells, ecmA m R N A was first observed at 12 hours of development, increased to a maximal level at 16-18 hours and declined thereafter (Fig. 4-6A). In the psA::rasG(G12T) cells, the ecmA m R N A expression pattern was at first similar to the wild type pattern but there was no decrease in ecmA m R N A late in development (Fig. 4-6B). The ecmA probe cross-hybridized with a similar prestalk specific m R N A , ecmB (Ceccarelli al., 1987), and this allowed for the coincident determination of ecmB expression patterns. In both control cells and psA::rasG(G12T) cells, ecmB expression was detected by 14 hours of development (Fig. 4-6A, B). However, the psA::rasG(G12T) cells expressed a considerably higher level of ecmB m R N A by 18 hours than the control cells and this elevated level persisted throughout development (Fig. 4-6A, B). Northern blots were also probed for prespore specific mRNA's. As illustrated in Fig. 4-7B, expression of cotC m R N A (Fosnaugh and Loomis, 1989) was delayed by 2 hours during the development of psA::rasG(G12T) cells, and the level of expression was dramatically reduced relative to wild type cells (Fig. 4-7A). Additionally, cotC m R N A levels in the transformant rapidly decreased as development progressed and transcripts were barely detectable by 20 hours post-starvation (Fig. 4-7B). Panels C and D of Fig. 4-7 compare the expression of another prespore specific gene, psA. The level of psA expression was also lower in the transformant cells relative to the control cells. In addition, psA expression in the transformants rapidly declined to undetectable levels. The northern blot analysis clearly demonstrated that in a population of developing psA::rasG(G12T) cells, prestalk gene expression was greatly enhanced while prespore gene expression was significantly reduced. This is similar to the phenotype of the Ddras-Thrl2 transformant which also overexpresses prestalk genes and underexpresses prespore genes (Louis et al., 1997a). Figure 4-7: Northern blots of m R N A isolated from psA::lacZ transformed control cells (A, C) and psA::rasG(G12T) cells (B, D) at the times (in hours) during development indicated above the lanes. Blots were probed with a labeled c D N A fragments for the cotC gene (A, B) and the psA gene (C, D). 116 4.5 Prestalk Cell Localization in Developing psA::rasG(G12T) Organisms The increase in prestalk gene expression and the decrease in prespore gene expression during development could be due to an increase in the number of prestalk cells and a concomitant decrease in the number of prespore cells within developing psA::rasG(G12T) structures. To test this possibility, Ax2 cells were cotransformed with the psA::rasG(G12T) construct and with one of several constructs containing lacZ expressed under the control of cell type specific promoters. When wild type cells were transformed with the ecmAOr.lacZ construct and induced to develop, staining was observed in the anterior region of the slugs that were formed (Fig. 4-8A). In culminants, stained cells were observed in the stalk tube, basal disc, and upper and lower cups (Fig. 4-8B). When psA::rasG(G12T) cells expressing ecmAO-lacZ were induced to develop and stained with x-gal, the tips of the multiple finger-like projections and a few scattered cells within the mound stained blue (Fig. 4-8C). As the multiple tips extended, stained cells were observed throughout the lengths of the finger like structures (Fig. 4-8D) and as development progressed, an increasing proportion of psA::rasG(G12T) cells expressed p-galactosidase and entire culminant structures stained blue (Fig. 4-8E). The ST region of the ecmB promoter drives expression in cells that either enter the stalk tube or those that form the basal disc. Wild type slugs stained for ST::lacZ expression in few scattered cells within the anterior of the slug (Fig. 4-9A). The level of anterior staining had increased in structures initiating culmination (Fig. 4-9B) and in culminants, staining was evident in the stalk tube and basal disc (Fig. 4-9C). In developing psA::rasG(G12T) structures containing the ST-lacZ construct, lacZ expression was first detected in scattered cells within the basal mound (Fig. 4-9D). As development progressed, more of the mound cells and some cells in the finger-like projections expressed the ST-lacZ marker (Fig. 4-9E). The pattern of the stained cells Figure 4-8: Developmental structures of wild type and psA::rasG(G12T) cells carrying the ecmAO-lacZ reporter. A wild type slug (A) and culminant (B) and a psA::rasG(G12T) multi-tipped mound (C) intermediate stage mound (D) and culminant (E) stained with x-gal. Figure 4-9: Developmental structures of wild type and psA::rasG(G12T) cells carrying the ST-lacZ reporter. A wild type slug (A), a structure initiating culmination (B) and a culminant (C) and a psA::rasG(G12T) mound with elongated fingers (D), an intermediate stage (E) and a culminant (F) stained to detect (j-galactosidase expression. 119 within the finger-like projections resembled that of a wild type slug (Fig. 4-9A). In psA::rasG(G12T) culminants, the mounds were almost completely blue (Fig. 4-9F). The protruding stalk-like structures also expressed the ST-lacZ reporter. This staining pattern is consistent with the observation that in terminally developed psA::rasG(G12T) organisms, the mound mass, as well as the protruding stalk-like structures, are composed mainly of terminally differentiated stalk cells. 4.6 Prespore Cell Localization in Developing psA::rasG(G12T) Organisms In view of the fact that psA m R N A levels rapidly declined during the development of psA::rasG(G12T) transformant cells (Fig. 4-6B), a labile lacZ reporter construct was used to determine the spatial expression from the psA promoter. Wild type slugs stained for expression of psA:(his)lacZ in the prespore region while the anterior and posterior regions remained unstained (Fig. 4-10A). In wild type culminants, p-galactosidase expression was observed in the sorus region (Fig. 4-10B). psA::rasG(G12T) cells containing the psA::(his)lacZ construct expressed P-galactosidase in the basal mounds but not in the finger-like projections (Fig. 4-10C). As the development of these transformants continued, there was a decrease in the proportion of psA-positive cells so that by the time culminants were formed, the level of psA::(his)lacZ expression had declined dramatically and only a few scattered cells stained positive for p-galactosidase (Fig. 4-10D). These results are consistent with the idea that in developing p$A::rasG(G12T) structures, most cells in the mound initially assumed a prespore fate but subsequently lost the prespore specification and transdifferentiated to the prestalk lineage. 120 Figure 4-10: Developmental structures of wild type and psA::rasG(G12T) cells carrying the labile psA::(his)lacZ reporter. A wild type slug (A) and culminant (B) and a psA::rasG(G12T) mound with elongated fingers (C) and culminant (D) stained with x-gal. 121 4.7 Chimaeric Development with Wild Type Cells I wished to test whether the presence of wild type cells would enable the psA::rasG(G12T) transformants to complete the spore development and produce viable spore cells. psA::rasG(G12T) cells were mixed in various proportions with Ax2 cells and starved to induce development. Development was allowed to progress for 48 hours and spore formation was then assessed. The number of spores produced by the chimaeric culminants reflected the number of Ax2 cells in the mix (Table VII). Furthermore, all of the germinated spores exhibited a wild type developmental phenotype. From these results, I concluded that the presence of wild type cells during development was not sufficient to enable spore formation. These results also indicated that the psA::rasG(G12T) induced sporulation defect was cell autonomous since the presence of the transformants did not hinder the ability Ax2 cells to make spores. This suggests that the psA::rasG(G12T) transformants did not produce a negative signal that inhibited sporulation. 4.8 Cell Sorting in Chimaeras The results presented in Table VII indicated that psA::rasG(G12T) cells could not contribute to the spore population during chimaeric development. To test if they were capable of contributing prestalk cells to chimaeric organisms, chimaeras of Ax2 cells and psA::rasG(G12T) cells, in which one of the strains was carrying a lacZ reporter construct, were created. Ax2 cells were transformed with a construct in which the actl5 promoter constitutively drives lacZ expression in all cells and these cells were mixed with psA::rasG(G12T) cells in a 1:9 ratio. When the mixture was induced to develop, the wild type cells sorted away from the transformant cells (Fig. 4-11 A) and progressed through development to form a culminant, leaving behind a mound of 122 Table VII. Spore formation by chimaeras of wild type and psA::rasG(G12T) transformant cells.. Columns 1 and 2 indicate the proportion of Ax2 and psA::rasG(G12T) cells in the chimaera. Column 3 indicates the spore formation of the cell mixture as a percentage of total cells. Column 4 presents the spore formation as a percentage of Ax2 control cells in the chimaera. % Ax2 Cells % Ras Cells Spore Formation* (as % of cells induced to develop) Ax2 Spore Formation (as % of Ax2 cells induced to develop) 100 0 154 154 75 25 118 157 50 50 74 148 25 75 34 137 0 100 2 — *Spore formation values are the average of three separate samples from one experiment. 123 Figure 4-11: Cell sorting during chimaeric development. Mixtures of 10% actl5::lacZ labeled wild type cells and 90% unlabeled psA::rasG(G12T) cells were induced to develop and stained with x-gal at intermediate (A) and culminant (B) stages. 124 psA::rasG(G12T) cells (Fig. 4-11B). The fact that the mound was for the most part devoid of stained cells indicated that very few wild type cells had been trapped. Although I cannot rule out the possibility that a few psA::rasG(G12T) cells were carried along with the developing wild type cells (since unstained cells would not be readily visible within a majority of stained cells), it was clear that the majority of wild type cells had segregated away from the majority of psA::rasG(G12T) cells. psA::rasG(G12T) cells containing the actl5-lacZ construct were mixed with unlabelled Ax2 cells in a 1:9 ratio. In this case, the psA::rasG(G12T) cells were able to participate in development and sorted to the prestalk regions of the chimaeric organisms. By the slug stage, the majority of the psA::rasG(G12T) cells were found in the anterior prestalk zones although some remained at the posterior and as the slug migrated, some psA::rasG(G12T) cells were shed onto the substratum (Fig. 4-12A). At culmination, the psA::rasG(G12T) cells were present in the basal disc, the stalk, and the upper and lower cups (Fig. 4-12B). I also created similar chimaeras in which the psA::rasG(G12T) cells contained the prestalk specific lacZ reporter constructs, ecmAO::lacZ (Fig. 4-12C) or ecmBr.lacZ (Fig. 4-12D). In these cases, I found similar staining patterns to that just described which verifies that the psA::rasG(G12T) cells that sort to the prestalk regions in chimaeras do in fact express prestalk markers. 4.9 Overexpression of RasG(G12T) in Prespore and Prestalk Cells It had been hypothesized that the defects in the Drfras-Thrl2 transformants could be accounted for by the fact that the majority of rasD-expressing cells are prestalk (Esch and Firtel, 1991). However, as I have shown, expression of activated RasG in the prestalk cell population alone was not sufficient to induce the defects observed in the Ddras-Thrll transformants. Although the ecmAO::rasG(G12T) transformants exhibited defects in PstA cell localization, slug behavior, culmination, Figure 4-12: Cell sorting during chimaeric development. Mixtures of 10% actl5::lacZ labeled psA::rasG(G12T) and 90% unlabeled wild type cells were induced to develop and stained with x-gal at the slug (A) and culminant (B) stages. Culminants of similar mixes using ecmAOr.lacZ labeled psA::rasG(G12T) cells (C) or ecmB::lacZ labeled psA::rasG(G12T) cells (D) are also shown. 126 and spore formation, the transformants did not form multi-tipped mounds nor did they exhibit drastic deregulation of cell type specific gene expression. However, the psA::rasG(G12T) transformants did form multi-tipped mounds in which cell type specific gene expression was grossly distorted and spore formation was inhibited. However, the finger-like projections were capable of morphogenetic movements and formed stalk tubes. In combination, these results suggested that the developmental phenotype of the Drfras-Thrl2 transformant resulted from the combined overexpression of activated Ras in both prestalk and prespore cells. To test this possibility, I cotransformed Ax2 cells with the psA::rasG(G12T) and the ecmAO::rasG(G12T) constructs. When starved to induce development, the cotransformants formed mounds with multiple protruding tips (Fig. 4-13A) that did not form finger-like projections or stalk tubes (Fig. 4-13B) and thus were reminiscent of the mounds formed by either Ddras-Thrl2 (Reymond et a l , 1986) or rasD::rasG(G12T) (M. Khosla, unpublished observations) transformants. These cotransformants were also defective for spore formation (only 5.2 ± 0.8% of cells induced to develop produced spores). Although no stalk tubes were observed in the culminants of the ecmAO::rasG(G12T)/psA::rasG(G12T) cotransformants, based on the results.in Section 4.3 of this thesis, I expected that stalk cells would be present. When stained with calcofluor, the mounds and protruding tips of the cotransformant culminants were observed to fluoresce. Again, due to the density of these structures, neither calcofluor staining nor phase contrast microscopy was sufficient to establish whether the structures in fact contained stalk cells (Fig. 4-14C, D). (For comparison, a wild type culminant is shown in Fig. 4-14A, B). Once the structures were broken apart, individual cells that stained with calcofluor (Fig. 4-14E) and were vacuolated (Fig. 4-14F) were observed, indicating that the cotransformant cells differentiated into stalk 127 Figure 4-13: Developmental morphologies of cotransformants. ecmAO::rasG(G12T)/psA::rasG(G12T) cells formed multi-tipped mounds (A) which did not extend to form finger or slug like structures (B). Figure 4-14: Culminants of Ax2 cells (A, B), and ecmAO::rasG(G12T)/psA::rasG(G12T) cells (C, D, E, F) were stained with calcofluor and viewed by fluorescence microscopy (A, C, E) or by phase contrast microscopy (B, D, F). Images in (E, F) are from cells released after a cotransformant culminant was smashed apart. 129 cells. Thus, based on the developmental characteristics of the ecmAO::rasG(G12T)/psA::rasG(G12T) cotransformants, I conclude that the developmental defects in the Ddras-lhxYl cells are the result of the combined expression of activated Ras in both prestalk and prespore cells. 4.10 Discussion In Chapter Three, I presented results demonstrating that the overexpression of activated RasG in prestalk cells did not result in the developmental aberrations characteristic of the Ddras-Thxll transformant. In this chapter, I have presented results illustrating that it is the overexpression of activated RasG in prespore cells that mimic the Ddras-Thrll induced defects. The psA::rasG(G12T) transformant mounds resemble those of the Ddras-Thrl2 transformants in that both produce multiple tips. A distinction between the two transformants is that the psA::rasG(G12T) transformant mound tips did extend to form finger and slug like structures. The psA::rasG(G12T) transformants also exhibited drastic deregulation of cell type specific gene expression similar to that observed with the Ddras-Thrl2 transformant (Louis et al., 1997a). These results indicate that during the development of Ddras-Thrll transformant, it is the presence of activated Ras in the prespore cells that makes a major contribution to the observed phenotype. However, the psA::rasG(G12T) transformants were not morphogenetically inhibited at the multi-tipped mound stage, indicating that the overexpression of activated Ras in prespore cells is not sufficient to induce all of the defects exhibited by the Ddras-Thrll strain. It is therefore likely that the block at the tipped mound stage is the result of the presence of activated Ras in both cell types. The phenotype of the ecmAO::rasG(G12T)/psA::rasG(G12T) transformants is consistent with this prediction. 130 Cotransformant cells produced multiple tipped mounds and were blocked at that stage. The deregulation of cell type specific gene expression is consistent with the fact activated Ras induced transdifferentiation of the prespore cells to prestalk cells in the psA::rasG(G12T) and Diiras-Thrl2 transformants. The formation of multi-tipped aggregates and the alterations in gene expression patterns could be the consequence of activated Ras interfering with a single pathway. However, it is more likely that the two phenotypes are due to two separate pathways since the concurrent overexpression of rasD(G12T) and the rapl gene resulted in a correction of cell type specific gene expression, although multiple tip formation persisted (Louis et al., 1997b). Thus, the effect of RasD(G12T) on cell type specific gene expression was antagonized by Rap, whereas the misregulation of tip formation was independent of Rap. Tip formation is a pivotal event during Dictyostelium development since the tip is believed to orchestrate the remainder of the developmental program. However, the process of tip formation is complex and poorly understood. Characterization of mutant strains deficient in tip formation has indicated that proper tip formation involves extracellular cAMP-mediated signaling, cell movement, cell sorting, and cell adhesion (Saxe et al., 1993; Stege et a l , 1997; Tsujioka et a l , 1999; Jiang et a l , 1998; Vasiev and Weijer, 1999). There is evidence that the tip acts as an oscillating center, responsible for the generation of c A M P waves. As the mound compacts, the c A M P waves are observed as spirals or concentric circles originating from a single center that then becomes the tip (Siegert and Weijer, 1995; Rietdorf et al., 1998). Multiple competing oscillating centers can occasionally arise in the same mound but eventually one center dominates and gives rise to the single tip. It is possible that the expression 131 of activated Ras in the prespore cells of either Ddras-ThxYl or psA::rasG(G21T) may alter the c A M P signaling in the mound and consequently, multiple tips arise. A number of other Dictyostelium strains exhibit a multi-tip phenotype. A double knockout of two of the PI3K isoforms (Zhou et al., 1995), as well as the disruption of a ubiquitin-conjugating enzyme (Clark et al., 1997), or the overexpression of a phosphotyrosine phosphatase (Howard et al., 1992) all compromise the ability to control tip number. In addition, multi-tipped mounds are formed during the development of strains with disruptions in any one of four tip genes that encode proteins with as yet no homologues in the data base (Stege et al., 1997; Stege et al., 1999). Finally, the scrA null strain also forms multi-tipped aggregates during development (Bear et al., 1998). The scrA gene was isolated as a suppressor of the carB null strain (Saxe et al., 1993) that lacks cAR2, one of the four c A M P receptors. The fact that there is no obvious connection between the disrupted or deregulated proteins in the various multi-tipped strains, indicates that the suppression of multiple tips in the wild type mound depends upon a number of signaling pathways. It seems unlikely that activated Ras simply downregulates any of the proteins that are required to suppress multiple tip formation because the subsequent development of the activated Ras expressing strains is unlike that of any of the null strains. During chimaeric development of psA::rasG(G12T) and wild type cells, only the wild type cells produced spores. This indicates that wild type signals were unable to correct the sporulation defect of the psA::rasG(G12T) transformants. This also indicates that the transformant cells did not produce an inhibitory signal preventing sporulation of the wild type cells. When a small number of psA::rasG(G12T) cells were mixed with wild type cells, the transformants were able to participate in development and formed prestalk cells. When the proportions were reversed and a small number of wild type 132 cells were mixed with transformants, the wild type cells formed culminants without the participation of the transformants. These results are different from those in which wild type cells were mixed with Ddras-Thrl2 cells. Expression from the rasD promoter occurs earlier than from the psA promoter and it is possible that this temporal difference results in the altered ability of the transformants to participate in chimaeric development. As mentioned in Chapter One, differentiation in Dictyostelium is regulated such that the relative proportions of the various cell types are maintained. Cell type proportions were deregulated by the overexpression of activated Ras in prespore cells. Although the majority of psA::rasG(G12T) transformant cells initially adopted a prespore fate, they transdifferentiated into prestalk cells. If we assume that lateral inhibitors function to maintain cell type proportion homeostasis during normal development, several possible perturbations of such a system could potentially explain the observed psA::rasG(G12T) phenotype. Activated Ras in prespore cells could either interfere with the production of a prespore activator within the prespore cells or alter the cells' response to a prespore activator or inhibitor produced by the prestalk cells. Either of these effects could potentially induce prespore transdifferentiation into prestalk cells. However, if the psA::rasG(G12T) cells were deficient in activator production, then the presence of wild type cells during chimaeric development should provide activator and rescue the prespore development of the transformants. Since this was not the case, it is unlikely that Ras(G12T) inhibited the production of a prespore activator, but rather that the prespore cells were altered in their response to signals from prestalk cells. Transdifferentiation has been shown to occur during the normal course of development as a consequence of slug migration (Sternfeld, 1992; Abe et al., 1994). As slugs migrate, PstB cells are lost from the posterior. These are replaced by anterior 133 PstA cells in which expression of the ecmB gene is induced. These cells are referred to as PstAB cells. The PstA cells are in turn replaced by the conversion of PstO cells to PstA cells. The decrease in PstO cells is compensated for by the recruitment of anterior like cells (ALC's, another prestalk subtype). Finally, ALC's are replaced by prespore cells that transdifferentiate into A L C prestalk cells. Thus the sequence of transdifferentiation during normal development has a specific directionality: from prespore to A L C to PstO to PstA to PstAB. However, in wild type cells, these cell type conversions must be reversible since, as mentioned previously, isolated prestalk compartments can reestablish correct proportions and form complete slugs (Raper, 1940). Given the number of prestalk cell subtypes, a complex network of activators and inhibitors regulating the relative proportions of each of the cell types must exist. Since the transdifferentiated psA::rasG(G12T) cells expressed both the ecmA and ecmB genes, I conclude that the cells became PstAB cells. As discussed above, one possible explanation for the psA::rasG(G12T) phenotype is that expression of activated Ras in the prespore cells eliminated their sensitivity to inhibition of transdifferentiation. A n alternative possibility is that activated Ras is a positive inducer of the PstAB cell fate and that the observed transdifferentiation does not involve lateral inhibitors. In fact, I have shown in Chapter Three that in the ecmAO::rasG(G12T) transformants, activated Ras induced the transdifferentiation of PstA cells to PstAB cells. Activated Ras may therefore be a positive inducer of the PstAB cell fate regardless of which cell type it is expressed in. However, I cannot discount the possibility that in the ecmAO::rasG(G12T) transformants, the PstA to PstAB conversion may be a consequence of a Ras-G12T mediated loss of inhibition rather than a positive induction of PstAB differentiation. Since differentiation to PstAB is a commitment step prior to terminal stalk cell differentiation, it is possible that Ras(G12T) actually induces terminal stalk cell 134 formation. This conclusion is supported by the fact that the entire population of psA::rasG(G12T) cells appeared to differentiate into stalk cells, based on the expression of STr.lacZ and staining with calcofluor. Further support is provided by the finding that culminants of the rasD::rasG(G12T) transformant cells also contained terminally differentiated stalk cells. In addition, during the development of psA::rasG(G12T) transformants, the cells in the basal mound expressed STr.lacZ before expression was detected in the extended slug-like structures. Since the cells in the basal mound expressed activated Ras whereas the cells in the slug-like structures did not, as determined by the psA::(his)lacZ expression pattern, it appears that activated Ras accelerates terminal stalk cell differentiation. The psA::ra$G(G12T) transformants exhibited a notable difference from cells overexpressing RasG(G12T) from either the rasD or ecmAO promoters. Only when activated Ras was expressed from the prespore promoter was the formation of stalk cells encased in stalk tubes observed. Wild type stalk formation is initiated in the tip of a culminating organism and extends basipetally. When activated Ras is overexpressed from the ecmAO promoter, the tip that forms during development is not a functional developmental organizer. The ecmAO::rasG(G12T) tip cells do not initiate stalk tube synthesis; cells that differentiate as stalk cells are at the posterior of the organism and may represent enlarged basal disc structures. In the Diiras-Thrl2 transformants activated Ras was expressed in both cell types and it is therefore likely that the expression of activated Ras in the prestalk cells prevented the formation of functional tips. As a result, no stalk tubes were observed. The stalk cells in the terminal mounds of Dtiras-Thrl2 transformants also probably represent extended basal disc structures. In contrast, during the development of psA::rasG(G12T) aggregates, two populations of prestalk cells were specified. The first was the population of prestalk cells that was specified at the time of initial cell type divergence and which did not express the psA-driven rasG(G12T) transgene. These prestalk cells formed the initial multi-tips and since these tip cells were in essence wild type, they were able to organize the later developmental processes. As a result, these tips extended to form finger-like and slug-like structures that even attempted migration. The tip cells were also able to initiate the synthesis of a stalk tube during the later stages of differentiation. The second population of prestalk cells arose as a result of transdifferentiation of prespore cells. These cells expressed activated Ras during their short period as prespore cells and were therefore defective as organizers due to the presence of residual activated Ras. Thus, the tips that formed late during the development of psA::rasG(G12T) transformants, did not have the organizing capacity of wild type tips and were thus morphogenetically blocked. 136 CHAPTER FIVE PERSPECTIVES Data was presented by Reymond et al. (1986) indicating that activated RasD overexpression resulted in the formation of multi-tipped mounds that were morphogenetically inhibited at that stage. The transformant was examined in greater detail by Louis et al. (1997a), who discovered that the transformant cells underexpressed prespore cell specific genes, overexpressed prestalk cell specific genes, and did not form either terminally differentiated stalk or spore cells. It had originally been suggested that it was the expression of RasD(G12T) in prestalk cells which induced these developmental phenotypes (Esch and Firtel, 1991). However, endogenous rasD expression was demonstrated to occur in prespore cells as well (Jermyn and Williams, 1995), leading to other possible hypotheses to explain the consequences of RasD(G12T) overexpression (Louis et al., 1997a). A l l of the hypotheses suggested by Louis et al. (1997a) required an explanation that included a block in prestalk to stalk cell formation. Since I have found that stalk cells were produced by the rasD::rasG(G12T) transformants, the number of possible hypotheses was reduced. It was possible that in the Ddras-Thvll transformant, activated Ras might have exerted its effects in prestalk cells, activating prestalk gene expression and repressing prespore gene expression by a lateral mechanism. Alternatively, activated RasD might have acted in the prespore population, decreasing prespore gene expression and enhancing prestalk gene expression. Finally, it is possible that activated RasD may have functioned in both cell types to produce the developmental defects of the Diras-Thrl2 transformant. 137 The experiments described in this thesis were designed to dissect these effects and attempt to determine which of the possible scenarios, if any, was correct. The hypothesis that activated RasD functioned in prestalk cells was tested by examining the developmental characteristics of the ecmAO::rasG(G12T) transformants. During development, these transformants did exhibit a slight increase in prestalk gene expression and decrease in prespore gene expression as assessed by northern blot analysis. However, the effects on gene expression were minimal compared to the drastic alterations exhibited during the development of the Ddras-Thrll transformant (Louis et a l , 1997a). These results indicate that activated RasG expression, and by analogy activated RasD expression, in prestalk cells does not have a major effect on prestalk gene expression or prespore gene expression. In addition, these transformants did not form multi-tipped aggregates, indicating that the morphological defect of the Ddras-Thrl2 transformant did not result from RasD(G12T) expression in prestalk cells. However, the ecmAO::rasG(G12T) transformants were not able to construct stalk tubes, suggesting that the absence of stalk tubes in the Ddras-Thrll culminants results from activated Ras expression in prestalk cells. Also, the ecmAO::rasG(G12T) transformants exhibited some morphogenetic defects; some structures were blocked at the mound stage and the slugs were impaired in motility. These results suggest that the morphogenetic block in the Ddras-Thrll transformant might have resulted in part from the expression of activated RasD in prestalk cells. Since most of the developmental defects in the Ddras-Thrl2 transformants were not recapitulated in the ecmAO::rasG(G12T) transformants, it was important to 138 test the second hypothesis. Prespore specific expression of activated RasG in the psA::rasG(G12T) transformants did result in drastic alterations in cell type specific gene expression. These transformants greatly underexpressed prespore genes and overexpressed prestalk genes, similar to the Ddras-Thrll transformant. In addition, by culmination, most psA::rasG(G12T) cells no longer expressed a prespore specific reporter construct and instead expressed a prestalk reporter. Evidence of such prespore to prestalk transdifferentiation was also observed during the development of the Ddras-Thrll transformant (Louis et al., 1997a). These results suggest that in the Ddras-Thrll transformant, activated RasD expression in prespore cells induced their transdifferentiation to prestalk cells and as a consequence, resulted in altered patterns of cell type specific gene expression. The psA::rasG(G12T) transformants also formed multi-tipped aggregates during development, suggesting that this morphological defect during Ddras-Thrll development was also a result of prespore specific RasD(G12T) expression. However, the psA::rasG(G12T) transformants were not blocked at this stage. The tips extended to form finger-like and slug-like structures and finally formed stalk tubes. The most likely explanation for this is the fact that during the development of the psA::rasG(G12T) cells, the originally determined prestalk population never expressed activated RasG and was able to organize tip elongation and stalk tube construction. Thus, prespore specific expression of activated RasG was not sufficient to recapitulate all of the phenotypic features of the Ddras-Thrll transformant. Since expression of RasG(G12T) in either cell type alone did not explain the full phenotype of the Ddras-Thrll cells, it was likely that activated RasG expression in both cell types was necessary. This possibility was examined by characterizing the 139 developmental morphology of the cotransformants. Indeed, the ecmAO::rasG(G12T)/psA::rasG(G12T) cotransformants developed to produce mounds with multiple tips. These mounds were morphogenetically inhibited and no stalk tubes were constructed. Hence, the developmental phenotype of the Ddras-Thrl2 transformant was most likely the combined result of RasD(G12T) expression in both cell types. In Chapter One, I briefly described some known regulators of cell type determination in Dictyostelium. Although few biochemical or genetic interactions between the known regulators of cell type determination in Dictyostelium have been demonstrated, some of the Dictyostelium proteins induce developmental consequences similar to those produced by activated RasD or activated RasG expression and possible interactions are therefore worth considering. Cells disrupted in gskA exhibit a developmental phenotype (Harwood et al., 1995) that is similar to that of the psA::rasG(G12T) transformants. Both strains form large mounds with emerging stalks (only one stalk in the gskA null strain). In both cases, the cells in the mounds differentiate as stalk cells and spore cell formation is severely compromised. Similarities between the two strains also exist at the level of gene expression. However, the psA::rasG(G12T) cells overexpress both the ecmA and ecmB genes where as the gskA' strain overexpressed the ecmB gene without altering the expression of ecmA. So, while psA::rasG(G12T) cells appear to differentiate as PstA cells as an intermediate step in their transdifferentiation from prespore to PstAB cells, the gsk null cells appear to differentiate as PstB cells without inducing expression of the ecmA gene. Thus, although the majority of cells differentiate as stalk cells in both cases, the molecular events leading to terminal 140 differentiation are likely to be different. In addition, the gskA' cells do not exhibit cAMP-mediated repression of stalk cell formation. The Drfras-Thrl2 transformant cells were prevented from stalk cell formation in the presence of c A M P (Louis et al., 1997a). Therefore, stalk cell formation mediated by activated RasD most likely occurs through a pathway that does not involve GSK3 inhibition. Slugs of STATa null cells (Mohanty et al., 1999) resemble those of the ecmAO::rasG(G12T) transformants in some respects. Developing STATa' cells overexpress ecmB and in mutant slugs, PstB cells are observed throughout the whole anterior prestalk region. ecmAO::rasG(G12T) cells exhibit a slight increase in ecmB expression and in some transformant slugs, PstB cells were observed in the anterior. However, the PstB cell localization varied and many slugs exhibited no anterior expression of the ecmBr.lacZ marker indicating that important differences exist between the two strains in the regulation of PstB cell differentiation. In addition, PstA cell differentiation is also dissimilar in the two strains since the cell type was mislocalized in the ecmAO::rasG(G12T) slugs but normally localized in the STATa null slugs. STATa has the properties of a negative regulator of stalk cell formation but in the multicellular organism, null cells do not differentiate as stalk cells, probably because of increased sensitivity to cAMP-mediated inhibition. It is possible that activated RasG may act upstream of STATa in a pathway regulating ecmB gene expression. However, other pathways must exist downstream of RasG(G12T) which cause mislocalization of the prestalk cell types and allow for stalk cell differentiation to occur. Prespore specific expression of the dominant inhibitor of P K A results in the inability of cells to maintain prespore gene expression and the absence of spore 141 formation (Hopper et al., 1993a). Although this phenotype resembles that of the psA::rasG(G12T) transformants, it is unlikely that activated RasG signals only via P K A . In the psA::PKA-Rm transformants, expression of the spore coat genes is drastically reduced but expression of psA is not. The psA gene does not depend on P K A for expression whereas the spore coat genes require P K A activity for maximum expression. Thus, in this transformant, the PKA-sensitive prespore gene expression pathways are effected. In contrast, in the psA::rasG(G12T) transformants, expression levels of both the spore coat gene, cotC, and of psA are were drastically reduced. This indicates that in the strain expressing activated RasG, the inhibition of prespore gene expression is a more general effect than in the strain expressing P K A - R m . Thus, although PKA-sensitive gene expression is reduced in the psA::rasG(G12T) transformants, the defect in prespore cells likely occurs at a step upstream of P K A and affects multiple pathways. N P K A is also required for terminal differentiation. Presumably, P K A can still be activated in activated RasG or activated RasD expressing cells since all transformants tested form stalk cells and P K A activity is required for stalk cell differentiation (Zhukovskaya et a l , 1996). In addition, in the ecmAO::rasG(G12T) transformants, it is apparent that P K A activity is regulated in the prespore cells. Although these transformants do not form spore cells on their own, they are capable of being induced to form spores by signals from wild type cells. Thus, within the prespore cells, this response pathway is functional. These results indicate that the ecmAO::rasG(G12T) transformants may be defective in the production of a sporulation signal. SDF-2 has been shown to induce spore formation in isolated cells and is most likely produced from the prestalk cells during culmination (Anjard 142 et al., 1998). Thus, activated RasG expression in prestalk cells may interfere with the production of SDF-2 or another such factor. The existence of a M A P K cascade downstream of Ras has not yet been demonstrated in Dictyostelium. A M A P K , ERK2 has been identified and shown to be required early in development for aggregation (Segal et al., 1995) and later in development for the induction and maintenance of prespore gene expression but not for prestalk gene expression (Gaskins et al., 1996). In wild type cells, ERK2 has been shown to be transiently activated by c A M P (Maeda et a l , 1997; Knetsch et al., 1996; Kosaka et al., 1998). In addition, during starvation, ERK2 is required for the activation of adenylyl cyclase and therefore, for the production of c A M P (Segall et a l , 1995). The role of Ras in the activation of ERK2 has been investigated by several laboratories. In one report, the presence of constitutively activated RasD(G12T) was shown to increase the basal level of ERK2 activity (Knetsch et al., 1996). However, Maeda et al. (1997) determined that activated RasD(T61Q) inhibited the c A M P -induced activation of ERK2 and a similar result was obtained by Kosaka et al. (1998) using RasG(G12T). Also, the expression of activated RasG inhibits the production of c A M P (Khosla et al., 1996). While the discrepancy between the positive and negative effects of activated Ras on ERK2 activation remains to be resolved, these results indicate the possibility that Ras is a negative regulator of the M A P K cascade or that activated Ras is acting as a dominant negative in the cascade. If activated Ras is a negative regulator of ERK2 activity during aggregation, it also follows that RasD may negatively regulate ERK2 during later development. Consistent with this idea, activated RasG, expressed in prespore cells drastically 143 reduces prespore gene expression and loss of ERK2 function during development also reduces prespore gene expression levels. It is possible that activated RasG or activated RasD inhibits the expression of prespore genes by inhibiting ERK2 activation. However, since loss of ERK2 during development does not result in increased prestalk gene expression, activated Ras must be having additional effects that do not involve the regulation of ERK2 activity. Alternatively, if activated Ras is a positive regulator of ERK2 (Knetsch et al., 1996), this could explain the multi-tipped phenotype induced by activated RasD or activated RasG. Wild type aggregates treated with c A M P produce multiple tips (Nestle and Sussman, 1972) and erkl null cells are defective in the activation of adenylyl cyclase (Gaskins et al., 1995). Therefore, if activated Ras stimulates ERK2 activity, the downstream effect could be an overstimulation of adenylyl cyclase and consequently, an overproduction of c A M P . Thus, the multi-tip formation induced by activated RasD or activated RasG could be a consequence of ERK2 activation. The putative Dictyostelium M E K kinase homolog, M E K K a , is required for maintaining cell type proportions in the developing organism. Nul l mutants exhibit an enlarged PstO zone and a reduced prespore zone (Chung et al., 1998). In addition, M E K K a is required for induction and maintenance of prespore gene expression. Although the requirement for prespore gene expression is similar to that of ERK2, there is no evidence linking the two proteins in a single pathway. Since activated RasG and activated RasD have negative effects on prespore differentiation, if Ras and M E K K a lie in the same pathway, Ras-GTP would be expected to negatively regulate M E K K a . This is the opposite of what has been observed in other systems. But, given the results indicating a negative regulation of 144 ERK2 by activated Ras (Maeda et al., 1997; Kosaka et al., 1998), it is possible that in Dictyostelium, Ras may inhibit activation of M A P K cascade components or that activated Ras has a dominant negative effect on the signaling cascade. It is difficult to imagine that a R a s / M A P K cascade does not exist in Dictyostelium since it is such a well conserved signaling circuit amongst many other organisms including lower organisms such as S. cerevisiae. However, most of the available data do not suggest that active Ras postitively regulates any of the M A P K cascade components. The M A P K pathway components thus far identified in Dictyostelium do not constitute a single pathway, indicating that multiple such pathways exist and that the remaining components have yet to be isolated. Perhaps when these pathways are better characterized, we will be better able to assess the role of Ras proteins in their regulation. The PI3K1 isoform of Dictyostelium interacts with activated RasG and activated RasD (Lee et al., 1999). However, a double knockout of the PI3K1 and PI3K2 isoforms results in the formation of multi-tipped mounds (Zhou et al., 1995) suggesting that Ras might be a negative regulator of PI3K. In contrast, in mammalian systems, activated Ras positively regulates PI3K activity. Although a large number of small GTPases have been described in Dictyostelium, limited interactions with GEFs or GAPs have been identified. Several putative Ras GEFs have been identified (Insall et a l , 1996; www.csm.biol.tsukuba.ac.jp/cDNAproject.html) but interactions with Ras family GTPases have not been demonstrated. Nul l mutations in aimless, which encodes a Ras G E F homologue, result in cells that are impaired in chemotaxis and do not aggregate (Insall et al., 1996). Although proper G D P / G T P cycling of RasG is required 145 for aggregation, both cells expressing activated RasG and cells with null mutations in rasG are able to chemotax (Khosla et al., 1996; Tuxworth et al., 1997). It is possible that the activity of a Dictyostelium Ras protein other than RasG is regulated by Aimless. A n IQGAP-related protein has been identified independently by two groups and found to have different developmental consequences when knocked out (Faix and Dittrich, 1996; Lee et a l , 1997; Faix et al., 1998). Although Lee et al. (1997) detected an interaction between the putative IQGAP homologue and RasD, the developmental phenotypes of loss-of-function G A P and gain-of-function RasD are very different. Faix and Dittrich (1996) demonstrated that loss-of-function G A P resulted in cells with multi-tipped fruiting bodies. However, they did not detect an interaction between the IQGAP homologue and RasD (Faix et al., 1998). Instead, a biochemical interaction was detected with Dictyostelium Racl but no increase in GTPase activity was observed (Faix et al., 1998). A second IQGAP related protein, G A P A , has been identified by Adachi et al. (1997). A n interaction between G A P A and a Dictyostelium GTPase has not yet been demonstrated. Since the mammalian IQGAPs do not interact preferentially with Ras subfamily proteins, it is likely that the Dictyostelium homologues will not have G A P activity for the Dictyostelium Ras proteins. The possible effects of activated Ras signaling in Dictyostelium appear to contradict what is known from other systems. However, the putative interactions are well worth investigating. Although multiple Ras pathways have been identified in mammalian systems, it is clear that additional Ras-mediated interactions and effects have yet to be uncovered. It is possible that in an organism such as 146 Dictyostelium, if the effectors for Ras are different, or if the interactions between Ras and its effectors are different from those identified in other organisms, such findings would reveal novel aspects of Ras signaling. The work described by Reymond et al. (1986), Louis et al. (1997a), and in this thesis illustrates that the expression of either activated RasD or activated RasG induces significant defects during the development of Dictyostelium. Recently, mutants have been created in which the rasD gene was ablated (Wilkins et al., 2000). These rasD' mutants exhibit normal development with no observable defects in morphology, patterning, cell type proportions, or terminal differentiation. Mutant slugs were, however, compromised with regards to their phototactic and thermotactic responses. The mechanisms by which slugs detect and respond to gradients in temperature and light have not been elucidated. However, one of the second messengers involved in regulating these processed is cGMP. Activated RasD was shown to affect the desensitization of guanylyl cyclase during chemotaxis (Van Haastert et al., 1987). Recently, a Ras Interacting Protein (RIP3) has been identified that interacts with activated RasG and, to a lesser extent, with activated RasD (Lee et al., 1999). RIP3 is necessary for proper activation of guanylyl cyclase during aggregation. It is possible that during normal development, RasD regulates guanylyl cyclase activity and consequently enables normal slug migration, but RasD is not the Ras protein that directly regulates aggregation since aggregation is apparently normal in rasD null slugs. The finding that rasD null cells exhibit normal differentiation brings into question the previously proposed significance of RasD as a regulator of essential developmental processes. Is the regulation of slug migration RasD's only function? 147 Since there are a large number of Ras homologs in Dictyostelium, it is possible that RasD does have a function during normal development but this function is compensated for by the presence of other Ras proteins. It is also possible that RasD(G12T) and RasG(G12T) interfere with the normal function of other Ras or Ras-superfamily proteins that are essential for pattern regulation. To establish which of these possibilities is correct will require the construction of other ras null strains and an analysis of upstream and downstream Ras pathway components in order to identify the level of cross talk between the various Ras proteins. With the results in this thesis, I have shown that activated RasG expression in prespore cells results in the formation of multi-tipped aggregates, the transdifferentiation of prespore cells to prestalk cells, and the terminal differentiation, of most cells into stalk cells. Also, the overexpression of activated RasG in prestalk cells prevents the correct localization of prestalk cells and although these cells differentiate into stalk cells, the differentiation of prespore cells to spore cells is inhibited. Although the phenotypes of these two transformants are quite different with respect to morphology and gene expression patterns, they are similar with respect to terminal differentiation. It appears that all cells expressing activated Ras are induced to differentiate as stalk cells regardless of whether they initially were of the prestalk or prespore specification. Ras has been shown to regulate choice of cell fate in other systems, some of which are described in the Introduction to this thesis. In Dictyostelium, stalk cell formation is under tight control by multiple regulators including GSK3, STATa, and P K A . It is possible that the pathway(s) downstream of activated Ras may constitute another mechanism whereby stalk cell formation is specified. Further investigation into activated Ras signaling and elucidation of downstream effectors should shed more light on the compli question of how cells choose their fate. REFERENCES Abe, T., Early, A. , Siegert, F., Weijer, C , and Williams, J. (1994). Patterns of cell movement within the Dictyostelium slug revealed by cell type-specific surface labeling of living cells. Cell 77,687-699. Adachi, H . , Takahashi, Y., Hasebe, T., Shirouzu, M . , Yokoyama, S., and Sutoh, K. (1997). Dictyostelium IQGAP-related protein specifically involved in the completion of cytokinesis. J. Cell. Biol. 137,891-898. Andersen, A . S., Pettersson, A . F., and Kjeldsen, T. B. (1992). A fast and simple technique for sequencing plasmid D N A with SequenaseR using heat denaturation. BioTechniques 13,678-679. Anjard, C , Chang, W. T., Gross, J., and Nellen, W. (1998). Production and activity of spore differentiation factors (SDFs) in Dictyostelium. Development 125,4067-4075. Araki, T., Abe, T., Williams, J. G. , and Maeda, Y. (1997). Symmetry breaking in Dictyostelium morphogenesis: evidence that a combination of cell cycle stage and positional information dictates cell fate. Dev. Biol. 192, 645-648. Araki, T., Gamper, M . , Early, A. , Fukuzawa, M . , Abe, Y., Kawata, T., Kim, E. , Firtel, R. A. , and Williams, J. G. , (1998). Developmentally and spatially regulated activation of a Dictyostelium STAT protein by a serpentine receptor. E M B O J. 17,4018-4028. Araki, T., Nakao, H . , Takeuchi, I., and Maeda, Y. (1994). Cell-cycle-dependent sorting in the development of Dictyostelium cells. Dev. Biol. 162,221-228. Aubry, L. , and Firtel, R. A . (1998). Spalten, a protein containing Got-protein-like and PP2C domains, is essential for cell-type differentiaion in Dictyostelium. Genes Dev. 12, 1525-1538. Aubry, L . , and Firtel, R. (1999). Integration of signaling networks that regulate Dictyostelium differentiation. Annu. Rev. Cell. Dev. Biol. 15,469-517. Balint-Kurti, P., Ginsburg, G. , Rivero-Lezcano, O., and Kimmel, A . R. (1997). rZIP, a RING-leucine zipper protein that regulates cell fate determination during Dictyostelium development. Development 124,1203-1213. Balint-Kurti, P., Ginsburg, G. T., Liu, J., and Kimmel, A . R. (1998). Non-autonomous regulation of a graded, PKA-mediated transcriptional activation signal for cell patterning. Development 125,3947-3954. Bear, J. E . , Rawls, J. F., and Saxe, C. L. Ill (1998). SCAR, a WASP-related protein isolated as a suppressor of receptor defects in late Dictyostelium development. J. Cell Biol. 142,1325-1335. Blenis, J. (1993). Signal transduction via the M A P kinases: proceed at your own RSK. Proc. Natl. Acad. Sci. USA. 90,5889-5892. 150 Boguski, M . S., and McCormick, F. (1993). Proteins regulating Ras and its relatives. Nature 366,643-654. Bollag, G. , Clapp, D. W., Shih, S., Adler, F., Zhang, Y. Y., Thompson, P., Lange, B. J., Freedman, M . H . , McCormick, F., Jacks, T., and Shannon, K. (1996). Loss of NF1 results in activation of the Ras signaling pathway and leads to aberrant growth in haematopoietic cells. Nature Genet. 12,144-148. Bonner, J. T. (1947). Evidence for the formation of cell aggregates by chemotaxis in the development of the slime mold Dictyostelium discoideum. J. Exp. Zool. 106,1-26. Bos, J. L. (1989). ras oncogenes in human cancer: a review. Cancer Res. 49, 4682-4689. Brand-Saberi, B., and Christ, B. (1999). Genetic and epigenetic control of muscle development in vertebrates. Cell Tissue Res. 296,199-212. Broach, J. R. (1991). RAS genes in Saccharomyces cerevisiae: signal transduction in search of a pathway. Trends Genet. 7, 28-33. Brookman, J., Jermyn, K., and Kay, R. (1987). Nature and distribution of the morphogen DIF in the Dictysotelium slug. Development 100,119-124. Buhl, B., Fischer, K., and MacWilliams, H . K. (1993). Cell sorting within the prespore zone of Dictyostelium discoideum. Dev. Biol. 156,481-489. Campbell, S. L. , Khosravi-Far, R., Rossman, K. L . , Clark, G. J., and Der, C. J. (1998). Increasing complexity of Ras signaling. Oncogene. 17,1395-1413. Carpenter, C. L. , and Cantley, L. C. (1996a). Phosphoinositide kinases. Curr. Op. Cell. Biol. 8,153-158.-Carpenter, C. L. , and Cantley, L. C. (1996b). Phosphoinositide 3-kinase and the regulation of cell growth. Biochim. Biophys. Acta, M11-M16. Carrel, F., Dharmawardhane, S., Clark, A . M . , Powell-Coffman, J. A. , and Firtel, R. A . (1994). Spatial and temporal expression of the Dictyostelium discoideum Goc protein subunit Goc2: Expression of a dominant negative protein inhibits proper prestalk to stalk differentiation. Mol. Biol. Cell 5, 7-16. Casci, T., Vinos, J., and Freeman, M . (1999). Sprouty, an intracellular inhibitor or Ras signaling. Cell 96,655-665. Ceccarelli, A . , Mahbubani, H . , and, Williams, J. G. (1991). Positively and negatively acting signals regulating stalk cell and anterior-like cell differentiation in Dictyostelium. Cell 65,983-989. Ceccarelli, A . , McRobbie, S. J., Jermyn, K. A . , Duffy, K., Early, A . , and Williams, J. G. (1987). Structural and functional characterization of a Dictyostelium gene encoding a DIF inducible, prestalk-enriched m R N A sequence. Nuc. Acids Res. 15, 7463-7476. 151 Chang, W.-T., Newell, P. C , and Gross, J. D. (1996). Identification of the cell fate gene Stalky in Dictyostelium. Cell 87,471-481. Chen, J. C. J., and Goldhamer, D. J. (1999). Transcriptional mechanisms regulating MyoD expression in the mouse. Cell Tissue Res. 296,213-219. Chen, Y., and Struhl, G. (1996). Dual roles for Patched in sequestering and transducing Hedgehog. Cell 87,553-563. Chomczynki, P., and Sacchi, N . (1987). Single-step method of R N A isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162,156-159. Cho, K. W. Y., and Blitz, I. L. (1998). BMPs, Smads and metalloproteases: extracellular and intracellular modes of negative regulation. Curr. Op. Genet. Dev. 8,443-449. Chung, C. Y., Reddy, T. B. K., Zhou, K., and Firtel, R. A . (1998). A novel, putative M E K kinase controls developmental timing and spatial patterning in Dictyostelium and is regulated by ubiquitin-mediated protein degradation. Genes Dev. 12, 3564-3578. Clark, A. , Nomura, A. , Mohanty, S., and Firtel, R. A . (1997). A ubiquitin-conjugating enzyme is essential for developmental transitions in Dictyostelium. Mol. Biol. Cell 8, 1989-2002. Clark, S. G. , Chisholm, A . D., and Horvitz, H . R. (1993). Control of cell fates in the central body region of C. elegans by the homeobox gene lin-39. Cell 74,43-55. Clark, D. V. , Rogalski, T. M . , Donati, L. M . , and Baillie, D. L. (1988). The unc-22(IV) region of Caenorhabditis elegans: genetic analysis of lethal mutations. Genetics 119, 345-353. Crews, C. M . , Alessandrini, A . , and Erikson, R. L . (1992). Erks: their fifteen minutes has arrived. Cell Growth Differ. 3,135-142. Crews, C. M . , and Erikson, R. L. (1993). Extracellular signals and reversible protein phosphorylation: what to M E K of it all. Cell 74,215-217. Daniel, J., Bush, J., Cardelli, J., Spiegelman, G . B., and Weeks, G. (1993a). Isolation of two novel ras genes in Dictyostelium discoideum: evidence for a complex, developmentally regulated ras-gene subfamily. Oncogene 9,501-508. Daniel, J., Spiegelman, G. B., and Weeks, G. (1993b). Characterization of a third ras gene, rasB, that is expressed throughout the growth and development of Dictyostelium discoideum. Oncogene 8,1041-1047. DelSal, G. , and Schneider, C. (1987). The CTAB-dna Precepitation Method: A common mini-scale preparation of template D N A from phagemids, phages, or plasmids suitable for sequencing. Biotechniques 7,514. Denhardt, D. T. (1996). Signal-transducing protein phosphorylation cascades mediated by Ras/Rho proteins in the mammalian cell: the potential for multiplex signalling. Biochem. J. 318, 729-747. 152 Detterbeck, S., Morandini, P., Wetterauer, B., Bachmair, A . , Fischer, K., and MacWilliams, H . K. (1994). The 'prespore-like cells' of Dictyostelium have ceased to express a prespore gene: analysis using short-lived P-galactosidases as reporters. Development 120,2847-2855. Dharmawardhane, S., Cubitt, A . B., Clark, A . M . , and Firtel, R. A . (1994). Regulatory role of the G a l subunit in controlling cellular morphogenesis in Dictyostelium. Development. 120,3549-3561. Dingermann, T., Reindl, N . , Werner, H . , Hildebrandt, M . , Nellen, W., Harwood, A . , Williams, J., and Nerke, K. (1989). Optimization and in situ detection of Escherichia coli P-galactosidase gene expression in Dictyostelium discoideum. Gene 85, 353-362. Dormann, D., Siegert, F., and Weijer, C. J. (1996). Analysis of cell movement during the culmination phase of Dictyostelium development. Development 122, 761-769. Early, A . , Abe, T. and Williams, J. (1995). Evidence for positional differentiation of prestalk cells and for a morphogenetic gradient in Dictyostelium. Cell 83, 91-99. Early, A . E. , Gaskell, M . J., Traynor, D., and Williams, J. G . (1993). Two distinct populations of prestalk cells within the tip of the migratory Dictyostelium slug with differing fates at culmination. Development 118, 353-362. Early, A . E. , and Williams, J. G. (1987). Two vectors which facilitate gene manipulation and a simplified transformation procedure for Dictyostelium discoideum. Gene 59,99-106. Early, A . E. , and Williams, J. G. (1988). A Dictyostelium prespore-specific gene is transcriptionally repressed by DIF in vitro. Development 103,519-524. Early, A . E. , Williams, J. G. , Meyer, H . E. , Por, S. B., Smith, E. , Williams, K. L . , and Gooley, A . A . (1988). Structural characterization of Dictyostleium discoideum prespore-specific gene D19 and of its product, cell surface glycoprotein, PsA. Mol . Cell. Biol. 8, 3458-3466. Esch, R. K. and Firtel, R. A . (1991). c A M P and cell sorting control the spatial expression of a developmentally essential cell-type-specific ras gene in Dictyostelium. Genes Dev. 5,9-21. Faix, J., Clougherty, C , Konzok, A. , Mintert, U . , Murphy, J., Albrecht, R., Muhlbauer, B., and Kuhlmann, J. (1998). The IQGAP-related protein DGAP1 interacts with Rac and is involved in the modulation of the F-actin cytoskeleton and control of cell motility. J. Cell Sci. I l l , 3059-3071. Faix, J., and Dittrich, W. (1996). DGAP1, a homologue of rasGTPase activating proteins that controls growth, cytokinesis, and development in Dictyostelium discoideum. FEBS Lett. 394,251-257. Feig, L. A . (1994). Guanine-nucleotide exchange factors: a family of positive regulators of Ras and related GTPases. Curr. Op. Cell. Biol. 6, 204-211. 153 Feig, L. A . , Urano, T., and Cantor, S. (1996). Evidence for a Ras/Ral signaling cascade. Trends Biochem. Sci. 21,438-441. Feinberg, A . P., and Vogelstein, B. (1983). A technique for radiolabeling D N A restriction endonuclease fragments to high specific activity. Anal. Biochem. 132,6-13. Fisher, P. R. (1997). Genetics of phototaxis in a model eukaryote, Dictyostelium discoideum. BioEssays 19,397-407. Fosnaugh, K. L. , and Loomis, W. F. (1989). Spore coat genes SP60 and SP70 of Dictyostelium discoideum. Mol. Cell. Biol. 9, 5215-5218. Fosnaugh, K. L. , and Loomis, W. F. (1991). Coordinate regulation of the spore coat genes in Dictyostelium discoideum. Dev. Genet. 12,123-132. Freeman, M . (1996). Reiterative use of the E G F receptor triggers differentiation of all cell types in the Drosophila eye. Cell 87,651-660. Freeman, M . (1998). Complexity of E G F receptor signalling revealed in Drosophila. Curr. Op. Genet. Dev. 8,407-411. Gaskins, C , Clark, A . M . , Aubry, L. , Segall, J. E. , and Firtel, R. A . (1996). The Dictyostelium M A P kinase ERK2 regulates multiple, independent developmental pathways. Genes Dev. 10,118-128. Gerhart, J. (1998). Signaling Pathways in Development. Teratology 60, 226-239. Ginsburg, G. T., Gollup, R., Yu, Y. M . , Louis, J. M . , Saxe, C. L . , and Kimmel, A . R. (1995). The regulation of Dictyostelium development by transmembrane signalling. J. Eukaryot. Microbiol. 42, 200-205. Ginsburg, G. T., and Kimmel, A. R. (1997). Autonomous and nonautonomous regulation of axis formation by antagonistic signaling via 7-span c A M P receptors and GSK3 in Dictyostelium. Genes Dev. 11,2112-2123. Golembo, M . , Raz, E. , and Shilo, B.-Z. (1996a). The Drosophila embryonic midline is the site of Spitz processing, and induces activation of the E G F receptor in the ventral ectoderm. Development 122,3363-3370. Golembo, M . , Schweitzer, R., Freeman, M . , and Shilo, B. Z. (1996b). argos transcription is induced by the Drosophila EGF receptor pathway to form an inhibitory feedback loop. Development 122,223-230. Gollup, R., and Kimmel, A . R. (1997). Control of cell-type specific gene expression in Dictyostelium by the general transcription factor GBF. Development 124,3395-3405. Greenwood, S., and Struhl, G. (1997). Different levels of Ras activity can specify distinct transcriptional and morphological consequences in early Drosophila embryos. Development 124,4879-4886. Gregg, J. H . (1965). Regulation in the cellular slime molds. Dev. Biol. 12,377-393. 154 Hachohen, N . , Kramer, S., Sutherland, D., Hironi, Y., and Krasnow, M . A . (1998). sprouty encodes a novel antagonist of FGF signaling that patterns apical branching of the Drosophila airways. Cell 92,253-263. Hadwiger, J. A . , and Firtel, R. A . (1992). Analysis of Ga4, a G-protein subunit required for multicellular development in Dictyostelium. Genes Dev. 6,38-49. Hadwiger, J. A . , Lee, S., and Firtel, R. A . (1994). The Gcc subunit Goc4 couples to pterin receptors and identifies a signaling pathway that is essential for multicellular development in Dictyostelium. Proc. Natl. Acad. Sci. U S A 91,10566-10570. Han, M . , and Sternberg, P. W. (1990). let-60, a gene that specifies cell fates during C. elegans vulval induction encodes a ras protein. Cell 63,921-931. Han, M . , and Sternberg, P. W. (1991). Analysis of dominant-negative mutations of the Caenorhabditis elegans let-60 ras gene. Genes Dev. 5,2188-98. Han, M . , Golden, A . , Han, Y., and Sternberg, P. W. (1993) C. elegans lin-45 ra/gene participates in let-60 ras-stimulated vulval differentiation. Nature 363,133-40. Han, Z. , and Firtel, R. A . (1998). The homeobox-containing gene Warai regulates anterior-posterior patterning and cell-type homeostasis in Dictyostelium. Development 125,313-325. Harrington, B. J., and Raper, K. B. (1968). Use of a fluorescent brightener to demonstrate cellulose in the cellular slime mold. J. Appl . Microbiol. 16,106-113. Harwood, A . J., Plyte, S. E. , Woodgett, J., Strutt, H . , and Kay, R. R. (1995). Glycogen synthase kinase 3 regulates cell fate in Dictyostelium. Cell 80,139-148. Henkenmeyer, M . , Rossi, K. J., Holmyard, D. P., Puri, M . C , Mbamalu, G. , Harpal, K., Shih, T. S., Jacks, T., and Pawson, T. (1995). Vascular system defects and neuronal apoptosis in mice lacking ras GTPase-activating protein. Nature 377, 695-701. Hopper, N . A. , Anjard, C , Reymond, C. D., and Williams, J. G. (1993b). Induction of terminal differentiation of Dictyostelium by cAMP-dependent protein kinase and opposing effects of intracellular and extracellular c A M P on stalk cell differentiation. Development 119,147-154. Hopper, N . A. , Harwood, A . J., Bouzid, S., Veron, M . , and Williams, J. G . (1993a). Activation of the prespore and spore cell pathway of Dictyostelium differentiation by cAMP-dependent protein kinase and evidence for its upstream regulation by ammonia. E M B O J. 12,2459-2466. Hopper, N . A. , and Williams, J. G . (1994). A role for cAMP-dependent protein kinase in determining the stability of prespore cell differentiation in Dictyostelium. Dev. Biol. 163, 285-287. Hopper, N . A. , Sanders, G. , Fosnaugh, K., Williams, J., and Loomis, W. F. (1995). Protein kinase A is a positive regulator of spore coat gene transcription in Dictyostelium. Differentiation 58,183-188. 155 Howard, P. K., Sefton, B. M . , and Firtel, R. A . (1992). Analysis of a spatially regulated phophotyrosine phosphatase identifies tyrosine phosphorylation as a key regulatory pathway in Dictyostelium. Cell 71, 637-647. Howes, R., Wasserman, J. D., and Freeman, M . (1998). In vivo analysis of Argos structure-function: sequence requirement for inhibition of the Drosophila epidermal growth factor receptor. J. Biol. Chem. 273,4275-4281. Ihle, J. N . , and Kerr, I. M . (1995). Jaks and Stats in signaling by the cytokine receptor superfamily. Trends Genet. 11, 69-74 Insall, R. H . , Borleis, J., and Devreotes, P. N . (1996). The aimless RasGEF is required for processing of chemotactic signals through G-protein-coupled receptors in Dictyostelium. Curr. Biol. 6, 719-729. Insall, R., and Kay, R. R. (1990). A specific DIF binding protein in Dictyostelium. E M B O J. 9,3323-3328. Insall, R. H . , Soede, R. D. M . , Schaap, P., and Devreotes, P. N . (1994). Two c A M P receptors activate common signaling pathways in Dictyostelium. Mol. Biol. Cell 5, 703-711. Jermyn, K. A. , Berks, M . , Kay, R. R., and Williams, J. G. (1987). Two distinct classes of prestalk-enriched m R N A sequences in Dictyostelium discoideum. Development 100, 745-755. Jermyn, K., Traynor, D., and Williams, J. (1996). The initiation of basal disc formation in Dictyostelium discoideum is an early event in culmination. Development 122, 753-760. Jermyn, K. A. , and Williams, J. G . (1991). A n analysis of culmination in Dictyostelium using prestalk and stalk-specific cell autonomous markers. Development 111, 779-787. Jermyn, K., and Williams, J. (1995). Comparison of the Dictyostelium rasD and ecmA genes reveals two distinct mechanisms whereby an m R N A may become enriched in prestalk cells. Differentiation 58, 261-267. Jiang, Y., Levine, H . , and Glazier, J. (1998). Possible cooperation of differential adhesion and chemotaxis in mound formation of Dictyostelium. Biophys. J. 75,2615-2625. Jin, T., Soede, R. D. M . , Liu, J., Kimmel, A . R., Devreotes, P. N . , and Schaap, P. (1998). Temperature-sensitive Gb mutants discriminate between G protein-dependent and -independent signaling mediated by serpentine receptors. E M B O J. 17,5076-5084. Johnson, M . R., DeClue, J. E. , Felzman, S., Vas, W. C , Xu, G. , White, R., and Lowy, D. R. (1994). Neurofibromin can inhibit Ras-dependent growth by a mechanism independent of its GTPase-accelerating function. Mol. Cell. Biol. 14, 641-645. 156 Johnson, N . L . , Gardner, A . M . , Diener, K. M . , Lange-Carter, C. , Gleavy, J., Jarpe, M . B., Minden, A. , Karin, M . , Zon, L. I., and Johnson, G. L. (1996). Signal transduction pathways regulated by Mitogen-activated/Extracellular Response Kinase Kinase Kinase induce cell death. J.Biol. Chem. 271, 3229-3237. Johnson, R. L. , Saxe, C. L. Ill, Gollup, R., Kimmel, A . R., and Devreotes, P. N . (1993). Identification and targeted gene disruption of cAR3, a c A M P receptor subtype expressed during multicellular stages of Dictyostelium development. Genes Dev. 7, 273-282. Johnson, R. L . , and Scott, M . P. (1998). New players and puzzles in the Hedgehog signaling pathway. Curr. Op. Genet. Dev. 8, 450-456. Kawata, T., Early, A . , and Williams, J. (1996). Evidence that a combined activator-repressor protein regulates Dictyostelium stalk cell differentiation E M B O J. 15, 3085-3092. Kawata, T., Shevchenko, A. , Fuzukawa, M . , Jermyn, K. A. , Totty, N . F., Zhukovskaya, N . V. , Sterling, A . E. , Mann, M . , and Williams, J. G . (1997). SH2 signaling in a lower eukaryote: A STAT protein that regulates stalk cell differentiation in Dictyostelium. Cell 89,909-916. Kay, R. R., and Jermyn, K. A . (1983). A possible morphogen controlling differentiation in Dictyostelium. Nature 303, 242-244. Kayne, P. S., and Sternberg, P. W. (1995). Ras pathways in Caenorhabditis elegans. Curr. Op. Genet. Dev. 5, 38-43. Keller, E. F. (1983). A Feeling for the Organism: The Life and Work of Barbara McClintock. New York: W. H . Freeman and Company. pl98. Kenyon, C. (1995). A perfect vulva every time: gradients and signalling cascades in C. elegans. Cell 82,171-174. Kim, J. Y., Borleis, J. A . , and Devreotes, P. N . (1998). Switching of chemoattractant receptors programs development and morphogenesis in Dictyostelium: Receptor subtypes activate common responses at different agonist concentrations. Dev. Biol. 197,117-128. Kim, L. , Liu, J., and Kimmel, A . R. (1999). The novel tyrosine kinase Z A K 1 activates GSK3 to direct cell fate specification. Cell 99,399-408. Khosla, M . , Spiegelman, G. B., and Weeks, G . (1996). Overexpression of an activated rasG gene during growth blocks the Initiation of Dictyostelium development. Mol . Cell. Biol. 16,4156-4162. Khosla, M . , Spiegelman, G. B., Weeks, G. , Sands, T. W., Virdy, K. J., and Cotter, D. A . (1994). RasG protein accumulation occurs just prior to amoebae emergence during spore germination in Dictyostelium discoideum. FEMS Micro. Lett. 117, 293-298. 157 Khosravi-Far, R., and Der, C. J. (1994). The Ras signal transduction pathway. Cancer Met. Rev. 13,67-89. Khosravi-Far, R., White, M . A. , Westwick, J. K., Solski, P. A. , Chrzanowska-Wodnicka, M . , Van Aelst, L. , Wigler, M . H . , and Der, C. J. (1996). Oncogenic Ras activation of Raf/Mitogen-Activated Protein Kinase-independent pathways is sufficient to cause tumorigenic transformation. Mol. Cell. Biol. 16,3923-3933. Khwaja, A . , Marte, B. M . , Pappin, D., Das, P., Waterfield, M . D., Ridley, A . , Downward, J. (1997). Role of phosphoinositide 3-OH kinase in cell transformation and control of actin cytoskeleton by Ras. Cell 89, 457-467. Klambt, C , Glazer, L. , and Shilo, B-Z. (1992). breathless, a Drosophila FGF receptor homolog, is essential for migration of tracheal and specific midline glial cells. Genes Dev. 6,1668-78. Knetsch, M . L. W., Epskamp, S. J. P., Schenk, P. W., Wang, Y., Segall, J. E . , and Snaar-Jagalska, B. E. (1996). Dual role of c A M P and involvement of both G-proteins and ras in regulation of ERK2 in Dictyostelium discoideum. E M B O 15, 3361-3368. Knoblich, J. A. , Jan, L. Y., and Jan, Y . -N . (1995). Asymmetric segregation of Numb and Prospero during cell division. Nature 377, 624-627. Kosaka, C , Khosla, M . , Weeks, G. , and Pears, C. (1998). Negative influence of RasG on chemoattractant-induced ERK2 phosphorylation in Dictyostelium. Biochim. Biophys. Acta, 1-5. Kyriakis, J. M . , and Avruch, J. (1996). Protein kinase cascades activated by stress and inflammatory cytokines. BioEssays 18, 567-577. Lackner, M . , Kornfeld, K., Miller, L. , Horvitz, H . R., and Kim, S. K. (1994). A M A P kinase homolog, mpk-1, is involved in ras-mediated induction of vulval cell fates in Caenorhabditis elegans. Genes Dev. 8,160-173. Laemmli, U . K. (1970). Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227, 592-596. Lee, S., Escalante, R., and Firtel, R. A . (1997). A Ras G A P is essential for cytokinesis and spatial patterning in Dictyostelium. Development. 124,983-996. Lee, S., Parent, C. A. , Insall, R., and Firtel, R. A . (1999). A novel Ras-interacting protein required for chemotaxis and cyclic adenosine monophosphate signal relay in Dictyostelium. Mol. Biol. Cell. 10,2829-2845. Lee, T., Feig, L. , and Montell, D. J. (1996). Two distinct roles for Ras in a developmentally regulated cell migration. Development 122,409-418. Lee, T., and Montell, D. J. (1997). Multiple Ras signals pattern the Drosophila ovarian follicle cells. Dev. Biol. 185,25-33. 158 Loomis, W. F., and Cann, R. (1982). Bibliography on Dictyostelium. in The Development of Dictyostelium discoideum., ed. W. F. Loomis, pp. 451-538. New York: Academic. Loomis, W. F. (1993). Lateral inhibition and pattern formation in Dictyostelium. Curr. Top. Dev. Biol. 28,1-46. Loomis, W. F. (1998). Role of P K A in the timing of developmental events in Dictyostelium cells. Microbiol. Molec. Biol. Rev. 62,684-694. Louis, J. M . , Ginsburg, G. T., and Kimmel, A . R. (1994). The c A M P receptor CAR4 regulates axial patterning ad cellular differentiation during late development of Dictyostelium. Genes Dev. 8,2086-2906. Louis, S. A . , Spiegelman, G. B. and Weeks, G. (1997a). Expression of an activated rasD gene changes cell fate decisions during Dictyostelium development. Molecular Biology of the Cell 8,303-312. Louis, S. A . , Weeks, G. , and Spiegelman, G. B. (1997b). Rapl overexpression reveals that activated RasD induces separable defects during Dictyostelium development. Dev. Biol. 190, 273-283. Lowe, P. N . , and Skinner, R. H . (1994). Regulation of Ras signal transduction in normal and transformed cells. Cell. Signal. 6,109-123. L u , B., Jan, L. Y., and Jan, Y . -N . (1998). Asymmetric cell division: lessons from flies and worms. Curr. Op. Genet. Dev. 8,392-399. MacNicol, A . M . , Muslin, A . J., and Williams, L. T. (1993) Raf-1 kinase is essential for early Xenopus development and mediates the induction of mesoderm by FGF. Cell. 73,571-583. MacWilliams, H . K., and Bonner, J. T. (1979). The prestalk-prespore pattern in cellular slime molds. Differ. 14,1-22. Maeda, Y. (1993). Pattern formation in a cell-cycle dependent manner during the development of Dictyostelium discoideum. Dev. Growth. Differentiation 35,609-616. Maeda, M . , Aubry, L. , Insall, R., Devreotes, R., and Firtel, R. A . (1997). The Dictyostelium mitogen-activated prtoein kinase ERK2 is regulated by ras and cAMP-dependent protein kinase (PKA) and mediates P K A function. J. Biol. Chem. 272,3883-3886. Maloof, J. N . , and Kenyon, C. (1998). The H O X gene lin-39 is required during C. elegans vulval induction to select the outcome of Ras signaling. Development 125,181-190. Mann, S. K. O., Richardson, D. L . , Lee, S., Kimmel, A . R., and Firte, R. A . (1994). Expression of cAMP-dependent protein kinase in prespore cells is sufficient to induce spore cell differentiation in Dictyostelium. Proc. Natl. Acad. Sci. U S A 91,10561-10565. 159 Marais, R., Wynne, J., and Treisman, R. (1993). The SRF accessory protein Elk-1 contains a growth factor-regulated transcriptional activation domain. Cell 73,381-393. Mayer, B. J., and Baltimore, D. (1993). Trends Cell. Biol. 3,8-13. McCormick, F. (1995). Ras signaling and NF1. Curr. Op. Genet. Dev. 5,51-55. McGlade, J., Brunkhorst, B., Anderson, D., Mbamalu, G. , Settleman, J., Dedhar, S., Rozakis-Adcock, M . , Chen, L .B., and Pawson, T. The N-terminal region of G A P regulates cytoskeletal structure and cell adhesion. E M B O J. 12,3073-3081. McRobbie, S. J., Jermyn, K. A. , Duffy, K., Blight, K., and Williams, J. G . (1988). Two DIF-inducible, prestalk mRNAs of Dictyostelium encode extracellular matrix proteins fo the slug. Development 104, 275-284. Meinhardt, H . (1983). A model for the prestalk/prespore patterning in the slug of the slime mold Dictyostelium discoideum. Differentiation 24,191-202. Minden, A . , Lin, A . , McMahon, M . , Lange-Carter. C , Derijard, B., Davis, R. J., Johnson, G. L. , and Karin, M . (1994). Differential activation of ERK and JNK mitogen-activated protein kinases by Raf-1 and M E K K . Science 266,1719-1723. Mohanty, S., Jermyn, K. A. , Early, A . , Takefumi, K., Aubry, L. , Ceccarelli, A. , Schaap, P., Williams, J. G. , and Firtel, R. A . (1999). Evidence that the Dictyostelium Dd-STATa protein is a repressor that regulates commitment to stalk cell differentiation and is also required for efficient chemotaxis. Development 126,3391-3405. Moodie, S. A . , and Wolfman, A . (1994) The 3Rs of life: Ras, Raf and growth regulation. Trends Genet. 10,44-48. Morishita, T., Mitsuzawa, H . , Nakafuku, M . , Nakamura, S., Hattori, S., and Anraku, Y. (1995). Requirement of Saccharomyces cerevisiae Ras for completion of mitosis. Science 270,1213-1215. Morris, H . R., Taylor, G. W., Masento, M . S., Jermyn, K. A . , and Kay, R. R. (1987) Differentiation inducing factor from Dictyostelium discoideum: chemical identification of a morphogen. Nature 328,811-814. Morrision, D. K., and Cutler, Jr., R. E. (1997). The complexity of Raf-1 regulation. Curr. Op. Cell Biol. 9,174-179. Mosch, H . -U . , Kubler, E. , Krappmann, S., Fink, G. R., and Braus, G. H . (1999). Crosstalk between the Ras2p-controlled Mitogen-activated protein kinase and c A M P pathways during invasive growth of Saccharomyces cerevisiae. Mol. Biol. Cell. 10, 1325-1335. Nakagawa, M . , Oohata, A . A . , Tojo, H . , and Fujii, S. (1999). A prespore-cell-inducing factor in Dictyostelium discoideum: its purification and characterization. Biochem J. 343, 265-271. 160 Nestle, M . , and Sussman, M . (1972). The effect of c A M P on morphogenesis and enzyme accumulation in Dictyostelium discoideum. Dev. Biol. 28,545-554. Ohimori, R., and Maeda, Y. (1987). The developmental fate of Dictyostelium discoideum cells depends greatly on the cell-cycle position at the onset of starvation. Cell Differ. 22,11-18. Okano, H . (1995). Two major mechanisms regulating cell-fate decisions in the developing nervous system. Develop. Growth Differ. 37, 619-629. Okazaki, M . , Kishida, S., Murai, H . , Hinoi, T., and Kikuchi, A . (1996). Ras-interacting Domain of Ral GDP Dissociation Stimulator Like (RGL) Reverses v-Ras-induced Transformation and Raf-1 Activation in NIH3T3 Cells. Cancer Res. 56,2387-2392. Olson, M . F., Ashwroth, A . , and Hall, A . (1995). A n essential role for Rho, Rac, and Cdc42 GTPases in cell cycle progression through G a . Science 269,1270-1272. Oohata, A . A. , Nakagawa, M . , Tasaka, M . , and Fujii, S. (1997). A novel prespore-cell-inducing factor in Dictyostelium discoideum induces cell division of prespore cells. Development. 124,2781-2787. Perrimon, N . (1996). Serpentine proteins slither into the Wingless and Hedgehog fields. Cell 86,513-516. Perrimon, N . , and Perkins, L. A . (1997). There must be 50 ways to rule the signal: The case of the Drosophila E G F receptor. Cell 89,13-16. Perrimon, N . , and McMahon, A . P. (1999). Negative feedback mechanisms and their roles during pattern formation. Cell 97,13-16. Peterson, S. N . , Trabalzini, L . , Brtva, T. R., Fischer, T., Altschuler, D. L . , Martelli, P., Lapetina, E. G. , Der, C. J., and White II, G. C. (1996). J. Biol. Chem. 271,29903-29908. Plyte, S. E. , O'Donovan, E. , Woodgett, J. R., and Harwood, A . J. (1999). Glycogen synthase kinase-3 (GSK-3) is regulated during Dictyostelium development via the serpentine receptor cAR3. Development 126, 325-333. Quilliam, L. A . , Khosravi-Far, R., Huff, S. Y., and Der, C. J. (1995). Guanine nucleotide exchange factors: activators of the Ras superfamily of proteins. BioEssays 17,395-404. Raper, K. (1940). Pseudoplasmodium formation and organization in Dictyostelium discoideum. J. Elisha Mitchell Sci. Soc. 56,241-282. Reichman-Freid, M . , Dickson, B., Hafen, E. , and Shilo, B. Z . (1994). Elucidation of the role of breathless, Drosophila FGF receptor homolog, in tracheal cell migration. Genes Dev. 8,428-439. Reymond, C. D., Gomer, R., H . , Mehdy, M . C , and Firtel, R. A . (1984). Developmental regulation of a Dictyostelium gene encoding a protein homologous to mammalian ras protein. Cell 39,141-148. 161 Reymond, C. D., Gomer, R. H . , Nellen, W., Theibert, A. , Devreotes, P. and Firtel, R. A . (1986). Phenotypic changes induced by a mutated ras gene during the development of Dictyostelium transformants. Nature 323,340-343. Rietdorf, J., Siegert, F., Dharmawardhane, S., Firtel, R. A. , and Weijer, C. J. (1997). Analysis of cell movement and signalling during ring formation in an activated G a l mutant of Dictyostelium discoideum that is defective in prestalk zone formation. Dev. Biol. 181, 79-90. Rietdorf, J., Siegert, F., and Weijer, C. J. (1998). Induction of optical density waves and chemotactic cell movement in Dictyostelium discoideum by microinjection of c A M P pulses. Dev. Biol. 204,525-536. Rhyu, M . S., Jan, L. Y., and Jan, Y. N . (1994). Asymmetric distribution of numb protein during division of the sensory organ precursor cells confers distinct fates to daughter cells. Cell 76,477-491. Rhyu, M . S., and Knoblich, J. A . (1995). Spindle orientation and asymmetric cell fate. Cell 82, 523-526. Richardson, D. L . , Loomis, W. F., and Kimmel, A . R. (1994). Progression of an inductive signal activates sporulation in Dictyostelium discoideum. Development 120, 2891-2900. Robbins, S. M . , Williams, J. G. , Jermyn, K. A . , Spiegelman, G. B. and Weeks, G . (1989). Growing and developing Dictyostelium cells express different ras genes. Proc. Natl. Acad. Sci. U S A 86,938-942. Robbins, S. M . , Khosla, M . , Thiery, R., Weeks, G. , and Spiegelman, G. B. (1991) Ras-related genes in Dictyostelium discoideum. Dev. Genet. 12,147-153. Roberts, R. L. , and Fink, G. R. (1994). Elements of a single M A P kinase cascade in Saccharomyces cerevisiae mediate two developmental programs in the same cell type: mating and invasive growth. Genes Dev. 8,2974-2985. Rodriguez-Viciana, P., Warne, P. H . , Dhand, R., Vanhaesebroeck, B., Gout, I., Fry, M . J., Waterfield, M . D., and Downward, J. (1994). Phosphoinositol 3-OH kinase as a direct target of Ras. Nature 370,527-532. Rodriguez-Viciana, P., Warne, P. H . , Khwaja, A. , Marte, B. M . , Pappin, D., Das, P., Waterfield, M . D., Ridley, A. , and Downward, J. (1997). Cell 89,457-467. Rogalski, T. M . , Moerman, D. G. , and Baillie, D. L. (1982). Essential genes and deficiencies in the unc-22IV region of Caenorhabditis elegans. Genetics 102, 725-736. Rubin, J., and Robertson, A. (1975). The tip of the Dictyostelium discoideum pseudoplasmodium as an organizer. J. Embryol. Exp. Morph. 33,227-241. Sakai, Y.. (1973). Cell type conversion in isolated prestalk and prespore fragments of the cellular slime mold Dictyostelium discoideum. Dev. Growth. Differ. 15,11-19. 162 Salser, S. J., Loer, C. M . , and Kenyon, C. (1993). Multiple H O M - C gene interactions specify cell fates in the nematode central nervous system. Genes Dev. 7,1714-1724. Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press. Sanger, R., Nicklen, S., and Coulson, A . R. (1977). D N A sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. U S A 74,5463-5467. Satoh, T., Nakafuku, M . , and Kaziro, Y. (1992). Function of Ras as a molecular switch in signal transduction. J. Biol. Chem. 267, 24149-24152. Saxe, C. L. Ill, Johnson, R., Devreotes, P. N . , and Kimmel, A . R. (1991). Multiple genes for cell surface c A M P receptors in Dictyostelium discoideum. Dev. Genet. 12, 6-13. Saxe, C. L. Ill, Ginsburg, G. T., Louis, J. M . , Johnson, R., Devreotes, P. N . , and Kimmel, A . R.(1993). CAR2, a prestalk c A M P receptor required for normal tip formation and late development of Dictyostelium discoideum. Genes Dev. 7, 262-272. Saxe, C. L. Ill, Yu, Y. M . , Jones, C , Bauman, A. , and Haynes, C. (1996). The c A M P receptor subtype cAR2 is restricted to a subset of prestalk cells during Dictyostelium development and displays unexpected DIF-1 responsivenes. Dev. Biol. 174, 202-213. Schaap, P. (1986). Regulation of size and pattern in cellular slime molds. Differentiation 33,1-16. Schlessinger, J. (1993). How receptor tyrosine kinases activate Ras. Trends Biochem. Sci. 18,273-275. Schweitzer, R., Howes, R., Smith, R., Shilo, B.-Z., and Freeman, M . (1995). Inhibition of Drosophila E G F receptor activation by the secreted protein Argos. Nature 376,699-702. Segall, J. E. , Kuspa, A. , Shaulsky, G. , Ecke, M . , Maeda, M . , Gaskins, C , Firtel, R., and Loomis, W. (1995). A M A P kinase necessary for receptor-mediated activation of adenylyl cyclase in Dictyostelium. J. Cell Biol. 128,405-413. Sekar, V. (1987). A rapid screening procedure for the identification of recombinant bacterial clones. Biotechniques 5,11-13. Shaulsky, G. , Fuller, D., and Loomis, W. F. (1998). A cAMP-phosphodiesterase controls PKA-dependent differentiation. Development 125,691-699. Shaulsy, G. , Kuspa, A. , and Loomis, W. F. (1995). A multidrug resistance transporter / serine protease gene is required for prestalk specialization in Dictyostelium. Genes Dev. 9,1111-1122. Shaulsky, G. , and Loomis, W. F. (1996). Initial cell type divergence in Dictyostelium is independent of DIF-1. Dev. Biol. 174,214-240. Siegert, F., and Weijer, C. J. (1995). Spiral and concentric waves organize multicellular Dictyostelium mounds. Curr. Biol. 5,937-943. 163 Smith, E . , and Williams, K. L. (1980). Evidence for tip control of the 'slug/fruit' switch in slugs of Dictyostelium discoideum. J. Embryol. Exp. Morph. 57,233-240. Springer, M . L . , Patterson, B., and Spudich, J. A . (1994). Stage-specific requirement for myosin II during Dictyostelium development. Development 120,2651-2660. Staples, S. O., and Gregg, J. H . (1967). Carotinoid pigments in the cullular slime mold, Dictyostelium discoideum. Biol. Bull. 132,413-422-Stege, J. T., Laub, M . T., and Loomis, W. F. (1999). Tip genes act in parallel pathways of early Dictyostelium development. Dev. Genet. 25, 64-77. Stege, J. T., Shaulsky, G. , and Loomis, W. F. (1997). Sorting of the initial celly tpes in Dictyostelium is dependent on the tip A gene. Dev. Biol. 185,34-41. Sternberg, P. W., and Han, M . (1998). Genetics of Ras signalling in C. elegans. Trends Genet. 14,466-472. Sternfeld, J. (1992). A study of PstB cells during Dictyostelium migration and culmination reveals a unidirectional cell type conversion process. Roux's Arch. Devt. Biol. 210, 354-363. Sulston, J. E. , and White, J. G . (1980). Regulation and cell autonomy during postembryonic development of Caenorhabditis elegans. Dev. Biol. 78, 577-597. Sun, J., Kale, S. P., Childress, A . M . , Pinsadi, C , and Jazwinski, S. M . (1994). Divergent roles of RAS1 and RAS2 in yeast longevity. J. Biol. Chem. 269,18638-18645. Sundaram, M . , and Han, M . (1996). Control and integration of cell signaling pathways during C. elegans vulval development. BioEssays 18, 473-480. Sundaram, M . , Yochem, J., and Han, M . (1996). A Ras-mediated signal transduction pathway is involved in the control of sex myoblast migration in Caenorhabditis elegans. Development 122,2823-2833. Sussman, M . (1987). Cultivation and synchronous morphogenesis of Dictyostelium under controlled experimental conditions. Meth. Cell Biol. 28,9-29. Szeberenyi, J., Cai, H . , and Cooper, G. M . (1990). Effect of a dominant inhibitory Ha-ras mutation on neuronal differentiation of PC12 cells. Mol . Cell. Biol. 10,5324-5332. Thompson, P., Traynor, D., and Kay, R. (1999). Taking the plunge, terminal differentiation in Dictyostelium. Trends Genet. 15,15-19. Tocque, B., Delumeau, I., Parker, F., Maurier, F., Multon, M . - C , and Schweighoffer, F. (1997). Ras-GTPase Activaing Protein (GAP): a putative effector for Ras. Cell. Signal. 9, 153-158. Towbin, H . , Staehelin, T., Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedures and some applications. Proc. Natl. Acad. Sci. USA 79,4350-4354. 164 Town, C , and Stanford, E. (1979). A n oligosaccharide-containing factor that induces cell differentiation in Dictyostelium discoideum. Proc. Natl. Acad. Sci. U S A 76, 308-312. Treisman, R. (1996). Curr. Op. Cell. Bio. 8,3175-3181. Tsujoika, M . , Machesky, L. M . , Cole, S. L . , Yahata, K., and Inouye, K. (1999). A unique talin homologue with a villin headpiece-like domain is required for multicellular morphogenesis in Dictyostelium. Curr. Biol. 9,389-392. Tuxworth, R. I., Cheetham, J. L . , Machesky, L . M . , Spiegelman, G. B., Weeks, G. , and Insall, R. H . (1997). Dictyostelium RasG is required for normal motility and cytokinesis, but not growth. J. Cell Biol. 138,605-614. Uemura, T., Shepard, S., Ackerman, L. , Jan, L. Y., and Jan, Y. N . (1989). numb, a gene required in determination of cell fate during sensory organ formation in Drosophila embryos. Cell 58, 349-360. Urano. T., Emkey, R., and Feig, L. A . (1996). Ral-GTPases mediate a distinct downstream signaling pathway from Ras that facilitates cellular transformation. E M B O J . 15,810-816. VanHaastert, P. J. M . , Kesbeke, F., Reymond, C. D., Firtel, R. A. , Luderus, E.„ and Van Driel, R. (1987). Aberrant transmembrane signal transduction in Dictyostelium cells expressing a mutated ras gene. Proc. Natl. Acad. Sci. U S A 84,4905-4909. Van Lookeren Campagne, M . M . , Wang, M . , Spek, W., Peters, D., and Schaap, P. (1988). Lithium respecifies cAMP-induced cell-type specific gene expression in Dictyostelium. Dev. Genet. 9,589-596. Vasiev, B., and Weijer, C. J. (1999). Modeling chemotactic cell sorting during Dictyostelium mound formation. Biophys. J. 76, 595-605. Wang, N . , Shaulsky, G. , Escalante, R., and Loomis, W. F. (1996). A two-component histidine kinase gene that functions in Dictyostelium development. E M B O J. 15,3890-3898. Wang, S., Yonger-Shepherd, S., Jan, L. Y., and Jan, Y . -N . (1998). Only a subset of the binary cell fate decisions mediated by Numb/Notch signaling in Drosophila sensory organ lineage requires Suppressor of Hairless. Development 124,4435-4446. Wasserman, J. D., and Freeman, M . (1998). A n autoregulatory cascade of EGF receptor signaling patterns the Drosophila egg. Cell 95,355-364. Wassserman, D. A. , Therrien, M . , and Rubin, G. M . (1995). The Ras signaling pathway in Drosophila. Curr. Op. Genet. Dev. 5,44-50. Watts, D., and AshworthJ. (1970). Growth of myxamoebae of the cellular slime mold Dictyostelium discoideum in axenic culture. Biochem. J. 119,171-174. 165 Weinmaster, G. (1998). Notch signaling: direct or what? Curr. Op. Genet. Dev. 8, 436-442. White, M . A . , Nicolette, C , Minden, A . , Polverino, A . , Van Aelst, L. , Karin, M . , and Wigler, M . H . (1995). Multiple Ras functions can contribute to mammalian cell transformation. Cell 80,533-541. Whitman, M . (1998). Smads and early developmental signaling by the TGFp superfamily. Genes Dev. 12,2445-2462. Whitman, M . , and Melton, D. A . (1992). Involvement of p21 ras in Xenopus mesoderm induction. Nature 357, 252-254. Wilkins, A. , Khosla, M . , Fraser, D. J., Spiegelman, G. B., Fisher, P. R., Weeks, G. , and Insall, R. H . (2000). Dictyostelium RasD is required for normal phototaxis, but not differentiation. Genes Dev., in press. Williams, J. G. , Ceccarelli, A. , McRobbie, S., Mahbubani, H . , Kay, R. R., Early, A . , Berks, M . , and Jermyn, K. A . (1987). Direct induction of Dictyostelium prestalk gene expression by DIF provides evidence that DIF is a morphogen. Cell 49,185-192. Wittinghofer, A. , (1998). Signal Transduction via Ras. Biol. Chem. 379, 933-937. Wodarz, A . , and Nusse, R. (1988). Mechanisms of Wnt signaling in development. Annu. Rev. Cell. Dev. Biol. 14, 59-88. Wolf, T., and Ready, D. F. (1993). Pattern formation in the Drosophila retina. (In) The development of Drosophila melanogaster. Ed. Bate, M . , and Martinez-Arias, A . Cold Spring Harbor, NY: Cold Spring Harbor Press. pl277-1325. Wu, Y., and Han, M . (1994). Suppression of activated Let-60 Ras protein defines a role of Caenorhabditis elegans Sur-l/mpk-1 M A P kinase in vulval differentiation. Genes Dev. 8,147-159. Wu, Y., Han, M . , and Guan, K. L . , (1995). MEK-2, a Caenorhabditis elegans M A P kinase kinase, functions in Ras-mediated vulval induction and other developmental events. Genes Dev. 9, 742-755. Xu, R.-H. , Song, Z. , Maeno, M . , Kim, J., Suzuki, A . , Ueno, N . , Sredni, D., Colburn, N . H . , and Kung, H.-F. (1996). Involvement of Ras/Raf/AP-1 in BMP-4 signaling during Xenopus embryonic development. Proc. Natl. Acad. Sci. U S A 93, 834-838. Yan, Z . , Deng, X., Chen, M . , Ahram, M . , Sloane, B. F., and Friedman, E. (1997). J. Biol. Chem. 272,27902-27907. Yochem, J., Sundaram, M . , and Han, M . (1997). Ras is required for a limited number of cell fates and not for general proliferation in Caenorhabditis elegans. Mol. Cell. Biol. 17,2716-2722. 166 Yoon, C. H . , Lee, J., Jongeward, G. D., and Sternberg, P. W. (1995). Similarity of sli-1, a regulator of vulval development in C. elegans, to the mammalian proto-oncogene, c-cbl. Science 269,1102-1105. Yu, Y. M . , and Saxe, C. L. Ill (1996). Differential distribution of c A M P receptors cAR2 and cAR3 during Dictyostelium development. Dev. Biol. 173,353-356. Zhou, K., Takegawa, K., Emr, S. D., and Firtel, R. A . (1995). A phosphatidyl (PI) kinase gene family in Dictyostelium discoideum: biological roles of putative mammalian p l l O and yeast Vps34p PI3-kinase homologs during growth and development. Mol. Cell. Biol. 15,5645-5656. Zhukovskaya, N . , Early, A . , Kawata, T., Abe, T., and Williams, J. (1996). c A M P -dependent protein kinase is required for the expression of a gene specifically expressed in Dictyostlium prestalk cells. Dev. Biol. 179,27-40. 167 APPENDIX I Materials Used For This Thesis A l l chemicals and reagents were purchased from either Fisher Scientific or Sigma Chemical Co., unless otherwise indicated. Restriction enzymes and D N A modifying enzymes were purchased from Life Technologies unless otherwise indicated. M A T E R I A L S Agar Bacteriological peptone Calcofluor ( a 3 5 S)dATP ( a 3 2 P)dCTP E C L (Enhanced Chemiluminescence kit) G418 (Geneticin) GeneCleanll Kit HRP-conjugated-donkey-a-rabbit IgG Hybond-N+ nitrocellulose Hybond-P PVDF Nucleobond Ax-20 kit N u n c l o n ™ plates Oligonucleotides Qiagen D N A kits Sequenase2® TritonX-100 Vent R® XL-1 E. coli cells X-ray film X-gal Yeast extract SUPPLIER Difco Oxoid Sigma Chemical Co. N E N Scientific N E N Scientific Amersham Life Technologies Bio 101, Inc. Amersham Amersham Amersham Machery-Nagel Nalge Nunc Int. N A P S Qiagen United States Biochemical Fisher Scientific New England Biolabs Stratagene Eastman Kodak Co. Life Technologies Oxoid Additional Materials The oc-RasG-GST fusion protein antibodies were generated by Dr. S. Robbins (Robbins et al., 1989) and by L. Duncan. The rasD::rasG(G12T) transformant was obtained from M . Khosla. The psA::Dd-PK2 was obtained from N . Hopper. The ecmAOr.lacZ, ecmA::lacZ, ecmO::lacZ, ecmBr.lacZ, ST::lacZ, and psAr.lacZ constructs were all obtained from Drs. K. Jermyn and J. Williams. The psA::(his)lacZ was obtained from Dr. H . MacWilliams. 168 APPENDIX II Culture Media and Buffer Recipies ILL Dictyostelium discoideum Growth Media H L 5 Medium: 14.3 g Neutralized bacteriological peptone 7.15 g Yeast extract 0.96 g N a 2 H P 0 4 0.48 g K H 2 P 0 4 Per liter of distilled water (The medium was supplemented with 3 ml of 30.8% glucose after autoclaving.) S M - V A N Agar: 10 g Glucose 10 g Neutralized bacteriological peptone 1 g Yeast extract 1 g MgSO 4-7H 20 1.55 g NaH 2 -PO 4 H 2 0 1 g K 2 H P 0 4 10 g Agar Per 980 ml of distilled water Il.ii. Dictyostelium discoideum Starvation Media 1% Water Agar Plates: 1 g Agar Per 100 mis of distilled water Bonner's Salts (BS): (For lOx stock solution) 6 g N a C l 7.52 g KC1 3 g CaCl 2-2H 20 Per liter of distilled water KK2: (For lOx stock solution) 200 m M K 2 H P 0 4 200 m M K H 2 P 0 4 (The potassium phosphate dibasic is titrated with potassium phosphate monobasic until the solution reaches a p H of 6.0.) Il.iii. Dictyostelium discoideum Transformation Buffers Bis-HL5: 2.1 g Bis-Tris 10 g Peptone 5 g Yeast extract Per liter of distilled water (The medium was supplemented with 3 ml of 30.8% glucose after autoclaving.) 2X HBS: 0.27 M N a C l 10 m M KC1 1.4 m M N a 2 H P 0 4 42 m M Hepes 10 m M Glucose p H to 7.1 with 0.5 N N a O H Filter sterilize and store at ~20°C Il.iv. Bacterial Media LB Agar: 10 g Bacteriological peptone 10 g NaCl 1 g Yeast extract 10 g Agar Per liter of distilled water II.v. Buffers for Northern Analysis TBE: (For 10X stock solution) 108 g Tris base 7.4 g E D T A 55 g Boric acid Per liter of distilled water R N A Sample Buffer: (For each 20 Ug R N A sample) 2.0 ul 5x R N A Gel Running Buffer 3.5 ul Formaldehyde 10.0 ul Deionized formamide 2.0 ul R N A loading dye 0.5 ul Ethidium bromide (10mg/ml) 4.5 ul Distilled water 5X R N A Gel Running Buffer: 0.2 M MOPS (pH 7.0) 50 m M Sodium acetate 5 m M E D T A (pH 8.0) R N A Loading Dye: SSC: Hybridization Solution: 50X Denhardt's Reagent: 50% Glycerol 1 m M E D T A (pH 8.0) Bromphenol blue and xylene cyanol to color (For 20X stock solution) 175.25 g NaCl 88.25 g Sodium citrate p H to 7.0 with 10 N N a O H 5X SSC IX Denhardt's Reagent 50 m M Sodium phosphate buffer, p H 6.5 0.5% SDS 30% Deionized formamide 250 ug /ml s sDNA 30 ug /ml polyA for 100 ml 1 g Ficoll (type 400) 1 g Polyvinylpyrrolidone 1 g Bovine serum albumin (fraction V) H.vi. Buffers for Western Analysis 2X Protein Sample Buffer: TBST: 0.5% B M E 0.5% SDS 50 m M Tris p H 6.8 12.5% Glycerol 0.04% Bromphenol blue 50 m M Tris-HCl p H 7.5 150 m M N a C l 5% Tween-20 H.vii. Solutions for fj-galactosidase Assays Z Buffer: 60 m M N a 2 H P 0 4 40 m M N a H 2 P 0 4 10 m M KC1 1 m M MgSO, 2 m M M g C l 2 fj-Galactosidase Staining Solution: (Made in Z buffer) 1 m M X-gal (in DMF) 5 m M K 3[Fe(CN) 6] 5 m M K 4[Fe(CN) 6] 1 m M E G T A 

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:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0089875/manifest

Comment

Related Items