Interacting signaling pathways controlling multicellular development in Dictyostelium

545

Interacting signaling pathways controlling multicellular development in Dictyostelium Richard

A Firtel

CAMP functions

Dictyostehm

controlling classic

as the key extracellular

is controlled

more continuous differentiation

micromolar

mechanisms

functions

to differentially

This review

signal controls

extracellular

control

aggregation

differentiation

controlling

including

signaling

cell-type cascades

3 and CAMP-dependent

transcription

differentiation

regulation mediated protein

enable

Whereas

multicellular via

pathway. CAMP followed

by

are discussed.

new findings

in this organism, kinase,

synthase

key regulators

and newly

identified

factors.

Address Department of Biology, Center for Molecular Genetics, University of California at San Diego, La Jolla, California 92093-0634, USA; e-mail: [email protected] Current Opinion

in Genetics

& Development

1996, 6:545-554

0 Current Biology Ltd ISSN 0959-437X Abbreviations CARS CAMP receptors CRAC cytosolic regulator of adenylyl cyclase differentiation-inducing factor DIF guanine exchange factor GEF G-box binding factor GBF GSK3 glycogen synthase kinase 3 mitogen-activated protein MAP PM CAMP-dependent protein kinase

Introduction Formation of the multicellular organism in Dic/yoste/ium occurs by a process that is quite distinct from that of metazoans. In Dic~ostelium, multicellularity is the result of chemotactic aggregation of up to 105 individual cells

into a mound to extracellular

the

and spatial

signal transduction

pathways

that

organism

to regulate

cell-type

differentiation

patterning

over these

enormous

size ranges.

In this review, I discuss our present understanding of how signal transduction pathways control multicellular development in this organism. Understanding the development of Dhyosfelium should provide insight into how signaling pathways function to control cell movement, morphogenesis

and gene

expression.

elucidating

by glycogen

in metazoans,

Dic~ostdium has evolved

of CAMP, a

cascade

by which

also summarizes

pathways

of cell-type

pulses

a transcriptional

and cell-type

molecule

through

but non G-protein-coupled

Potential

morphogenesis

acting

receptors.

by nanomolar

by activating

a receptor-mediated

kinase

development

G-protein-coupled/serpentine

aggregation

signaling

[l,Z’]. The aggregation CAMP that is sensed

is in response by cell surface

serpentine/G-protein-coupled receptors. As the organism is forming the mound, extracellular CAMP then functions to induce cell-type differentiation and subsequent morphogenesis. [Jr-dike metazoans, the size of the multicellular aggregate in Dic~os~ehun can range over three orders of magnitude, from
Formation

of the multicellular

organism

Over the past few years, significant insights have been obtained into how Dic~ostelium cells aggregate to form a multicellular organism. As more extensive reviews have recently examined this in detail, only the basic pathways will be discussed, as this information is essential for understanding signaling pathways controlling multicellular differentiation. Aggregation in Dictyostelium is mediated by nanomolar pulses of extracellular CAMP that interact with cell surface serpentine/G-protein-coupled receptors (CARS) [1,2*]. The production of CAMP is controlled by a biological oscillator that regulates the kinetics and activation of adenylyl cyclase. Addition of CAMP to aggregation-stage cells results in the rapid activation of adenylyl cyclase, required for relaying the cAhlP signal, activation of guanylyl cyclase that is coupled to chemotactic movement, and the induction of aggregation-stage gene expression [3-61. These genes include those encoding the coupled Gcx protein subunit Ga2, the CAMP receptor cAR1, adenylyl cyclase, catalytic subunit of cAhlP-dependent protein kinase (PKA), and contact sites A (gp80; a cell surface adhesion molecule). hlany of these are components of the signaling pathway itself. Cells within the developing of cAhIP with a periodicity Cyclic cyclase

aggregation center emit pulses of about once every 6 minutes.

ARIP, acting through cAR1, stimulates producing cAhlP, which is then relayed

adenylyl outward

from the aggregation center to stimulate additional cells. Cells chemotactically move inward, resulting in the formation of a multicellular organism. Both the activation of adenylyl cyclase and guanylyl cyclase adapt rapidly after responding to cAhlP. This rapid adaptation ensures that the CAMP signal moves outward from the aggregation center and that cells move inward toward the center. Addition of a continuous cAhlP signal results in a single activation of both cyclases, followed by adaptation of the pathways. Further activation of these pathways cannot be initiated until cAhlP is removed from the extracellular

546

Differentiation and gene regulation

environment

occurs

-5-6

and the signaling pathways de-adapt, which minutes after removal of the cAhlI? The roles

of many of the components defined by biochemical and knockout bination,

in aggregation have physiological studies

of essential aggregation-stage genes. ERKZ is rapidly activated in response to CAMP with kinetics very similar

been using

to that of adenylyl cyclase [1.5’]. Interestingly, activation, like that of cAhlP-stimulated Caz+

mutants obtained either by homologous recomby insertional mutagenesis or by expression of

mutated gene products. of the aggregation-stage

is receptor-dependent G-protein-independent

Figure 1 details the basic outline signaling pathways.

ERKZ influx,

but functions via an apparently mechanism [15’,16]. In addition, a

putative Ras guanine nucleotide exchange factor (GEF) is also required for both proper chemotactic movement and the activation of adenylyl cyclase but not for the activation of ERKZ [17’].

Activation of adenylyl and guanylyl cyclases im e+~o requires the G protein containing the Ga2 subunit, which is coupled to the cAhlP receptors c.4Rl and cAR3 [7-91. In Dictyostelium, the GP subunit regulates adenylyl cyclase directly and requires a pleckstrin domain containing protein, cytosolic regulator of adenylyl cyclase (CRAC), but a subunits do not participate directly in adenylyl cyclase stimulation [lO,ll,lZ”]. In contrast, guanylyl cyclase is thought to be regulated through Ga2 [13]. The hlAP kinase ERKZ activity is also essential for adenylyl cyclase activation [14-l, although it is not known whether this is a direct effect on the pathwa) or an indirect effect mediated through the regulation

As stated above, adaptation of these signaling pathways also plays an essential role in controlling aggregation. The adaption pathways are probably regulated, at least in part, through Ga2, as expression of a GTPase deficient (constitutive) Ga2 subunit in wild-type cells yields cells that are fully adapted for aggregation-stage processes [ 131. It is also possible that each of the individual pathways may have its own adaptation mechanisms. This is known to be the case for the activation of the hIAP kinase ERKZ, which shows a significantly extended period of activation and delayed adaptation in the Ras GEF or PKA null

Figure 1

(b)

(a)

(c) Continuous

CAMP pulses

Contmuous

CAMP

CAMP pulses

CAMP

V Ca2+ Influx and ERKZ actlvatlon are G protein Independent CAMP deoendent protein k,n& (PKA)

factor GEF

(AutoregulatIon)

n

speclflc

n P

P

P

P

“P

+

PKA

P

SIgnalrelay chemotaxls Spec. Induction of pulse-Induced genes (cARI, GaP, 02, gp80, ERK7)

(GBF;

Pnmary late genes LagC, rasQ Gtr4, PTP1)

Prespore-speclflc gene expressjon

& 1996 Current Oplmon

I” Genetics & Development

Signaling pathways mediated by the CAMP-receptor during aggregation and later development. (a) Pathways controlling aggregation-stage responses to nanomolar pulses of CAMP. It is probable that many of the same components of this pathway are also involved in controlling

cell

movement during morphogenesis In the multicellular stages. (b) Induction of GBF activation of postaggregative gene expression. The pathway functions through CAMP receptors cAR1 and cAR3 and is G-protein independent. There is an autoregulatory loop, as low levels of GBF are thought to be necessary to induce high levels of GBf expression in response to CAMP. This pathway is controlled through a continuous CAMP signal. (c) Model for induction of prespore gene expressiorl. Both GBF-dependent pathways and pathways mediated by ERK2 and PKA are required. Prespore genes such as SP60 require GBF and a prespore-specific regulatory element. It is possible that the PKA and ERKP pathways are controlled by the cAR4 CAMP receptor, although this is not known as car4 null cells express prespore genes. As ERKP and PKA are required, it is thought that an oscillatory CAMP signal is probably needed. by, G protein subunits p and y; CAR, CAMP receptor; ERKP, MAP kinase ERKP; Ga2, Ga subunit; GC, guanylyl cyclase; PLC, phospholipase C (see text for further detail).

Multicellular development in Dictyostelium Firtel

cells (L Aubry

et al., unpublished

data).

In addition,

both

Classic

promoter

analysis

on a number

547

of the postaggrega-

Ras and the GCX subunit Gal play a role in modulating the adaptation of guanylyl cyclase [l&19]. In viva, an

tive and &acting

extracellular, membrane-bound and secreted form of phosphodiesterase removes CAMP from the extracellular environment. Disruption of the phosphodiesterase gene PDE results in cells that are unable to aggregate-because CAMP accumulates and cannot be removed from the

rich element) [23,24]. The element is a repeated domain containing G/T residues on one strand, with a short spacer region between the two domains. These elements bind a developmentally regulated nuclear factor, G-box binding

system-and the pathways remain in the adapted state [ZO]. Overexpression of PDE also results in cells that are unable to aggregate, as the CAMP is degraded too rapidly [Zl].

Initiation of the developmental switch from aggregation to multicellular development As the mound forms, there is a developmental switch in which aggregation-specific processes-including the expression of aggregation-stage genes -are repressed, while genes specific for the multicellular aggregate (postaggregative genes) are induced. This is followed several hours later by the initiation of cell-type differentiation (Fig. 2). This developmental switch from aggregation to multicellular differentiation is activated by a rise in extracellular CAMP within the mound. Reconstruction experiments using cells in suspension show that high continuous CAMP results in the adaptation and down-regulation of the aggregation-stage signaling pathways [ 1,2’]. Similarly, a high continuous level of cAMP, but not oscillatory pulses that activate aggregate-stage responses, induces postaggregative gene expression. The initial postaggregative genes that are induced as the mound forms and CAMP levels rise include genes encoding the transcription factor GBF (see below), two homeobox-containing proteins, several Ga protein subunits, Ga2 and cAR1 expressed from specific late promoters, additional CAMP receptors, RasD, and a cell surface signaling molecule LagC. Differentiation of prestalk and prespore cells, which is initiated several hours later, requires the expression of several of this first wave of postaggregative genes [1,2*,22].

cell-type-specific genes identified sequence designated a G-box

factor (GBF). Mutations that affect the ability of the G-box to bind the factor in mobility gel shifts in vitro proportionally affect the ability of the &-acting region to confer developmental and cAhIP-induced gene expression in viva. GBF contains two zinc fingers, a glutamine-rich amino-terminal domain, but no other regions of homology to other known proteins [ZS]. DNA-binding activity is directly proportional to the amount of protein quantitated by Western blot (SK0 Mann et a/., unpublished data). Although the protein is phosphorylated, in vitro and in vivo expression studies suggest that modifications in the protein are not involved in modulating binding activity (C Briscoe, RA Firtel, unpublished data).

Disruption of the GBF gene confirms that GBF is an essential component of the developmental switch required for multicellular differentiation [2.5]. Cells that are gbfnull aggregate normally and express aggregation-stage genes properly. Upon mound formation, aggregation-stage gene expression is suppressed but there is no induction of postaggregative genes, either under standard developmental conditions or when the cells are exposed to extracellular CAMP in culture. The fact that aggregation-stage genes are repressed properly indicates that GBF-mediated processes are not essential for this down-regulation, consistent with the down-regulation being caused by an adaptation of the aggregation-stage signaling pathways. GBF is regulated developmentally, being induced late during aggregation. In suspension assays in response to high (micromolar) CAMP, GBF is the first postaggregation gene to be induced, with transcripts detected within 15 minutes of stimulation. These

kinetics

of induction

are consistent

Figure 2 Diagram

of multicellular

showing

the cell types and expression

pattern

development

of some of the genes.

The genes

ecmA and ecmB

prestalk-specific

markers

are

whereas

a degenerate or CAE (C/A

SP60

is a prespore-specific marker. (Adapted from [2’,60] and references therein.) ALC, anterior-like ceils; ecm, extracellular matrix gene; SP60, 60 kDa spore coat protein.

Late culminant

with

GBF’s

548

Differentiation and gene regulation

function as the primary regulator of other and cell-type-specific gene expression. The

phenotype

of the gbfnull

postaggregative

cells is more complex

than a

cells that constitutively express GBF preferentially induce either aggregation-stage gene expression in response to pulses or postaggregative gene expression in response to high

continuous

levels

of CAMP

(A Nomura,

RA Firtel,

simple mound arrest and the analysis of the phenotype has provided insights into the different functions of oscillatory

unpublished ferentially

and continuous CAMP signals. Cells that are gbf null form mounds with normal developmental timing; however, after several hours, the mounds disperse, whereupon they reaggregate [25]. This process is repeated in a cyclical

exists as nanomolar oscillatory pulses or a micromolar continuous signal. These studies emphasize that two very different signaling pathways mediate early and late responses through the same CAMP receptors, cAR1 and

fashion several times before the cells eventually die. The high CAMP level in the mounds results in a turning

cAR3 [4,9]. The aggregation-stage mediated by oscillatory signals-are,

off (adaptation) of the aggregation-stage pathways. The subsequent dispersion of the mounds appears to be caused by the inability of the organism to continue through morphogenesis, as these cell movements are dependent on GBF. Mound dispersion results in a lowering of the CAMP in the mounds, reinducing aggregation. This conclusion is supported by the observation that aggregation-stage genes are reinduced as the cells start to reaggregate and are repressed again as the new mounds form.

part G-protein-dependent, whereas those mediated by a continuous signal are G-protein-independent. Although the mechanism of GBF activation is not known, GBF is phosphorylated and it is reasonable to postulate that a receptor-mediated pathway leading to differential phosphorylation of GBF potentiates GBF-mediated gene expression.

A G-protein-independent cAMP signal transduction pathway is required for induction of postaggregate development Whereas GBF is regulated developmentally and induced by extracellular cAh,lP, expression of GBF alone is not sufficient to induce post-aggregative gene expression. Reconstruction experiments show that a novel cAh4P receptor-mediated signal transduction pathway is essential for inducing postaggregative gene expression [26**]. Vegetative cells that constitutively express GBF do not express postaggregative genes but will induce them in response to extracellular cAh,lP. Analysis using strains lacking the CAMP receptor cAR1 and a related receptor, cAR3 -which is also expressed in the early stages of development-indicate this pathway is, as expected, mediated through CAMP cell surface receptors. In contrast to many of the aggregation-stage pathways, GBF-mediated gene expression does not require the only known GB subunit, the coupled GUY protein subunit Ga2, or any of the other seven known Ga protein subunits. This analysis has led to the model that receptor-mediated induction of GBF is independent of heterotrimeric G proteins (Fig. lb). Induction of postaggregate either the hlAP kinase C Briscoe,

RA Firtel,

genes also does not require ERKZ or PKA (G Schnitzler,

unpublished

data).

A single receptor mediates two independent signaling pathways The aggregation-stage pathways that mediate the relay of CAMP and chemotactic movement require oscillatory CAMP stimulation. A continuous signal leads to adaptation and the inability to activate adenylyl and guanylyl cyclases and the MAP kinase ERKZ. In contrast, GBF-mediated pathways require high continuous levels of CAMP This difference in the pathways activated by pulsitile and continuous signals is highlighted by experiments in which

data). A single cell can thus respond difto CAMP depending on whether the signal

pathways thus,

-which for the

are most

G proteins are thought to interact with ligand-bound receptor in response to a conformational change in the receptor. A similar conformational change is thought to promote the interaction of receptors with regulatory kinases, such as mammalian BARK or rhodopsin kinase with their respective receptors [27,28]. This interaction of the kinases with receptors is ligand-dependent, as is G protein coupling, but is G-protein-independent. It is therefore probable that ligand-bound receptors interact with other signaling components ultimately leading to GBF activation. We envision that serpentine receptors (as with receptor tyrosine kinases) function as docking sites for components other than heterotrimeric G proteins and that such a mechanism mediates activation of GBF-controlled

transcription.

The late development conundrum - how does cAMP mediate oscillatory and continuous response pathways? During the multicellular stages of Dictyostelium development, CAMP functions to control gene expression, celltype differentiation, and morphogenesis. Elegant timelapse, dark-field video microscopy shows that cAh4P waves continue to be produced from the apical tip throughout multicellular development [29,30”]. Disruption of this signal by placing CAMP in the underlying substratum or by overexpressing the extracellular phosphodiesterase from a prestalk promoter leads to aberrant morphogenesis, consistent with cAhlP functioning as the morphogen controlling patterning [31,32]. In addition, CAMP must be synthesized at high levels in the developing aggregate to initiate GBF-mediated gene expression and cell-type differentiation. In order to produce CAMP waves and morphogenetic movements, an oscillatory cAhlP signal is needed, yet GBF-mediated functions require a continuous cAh4P signal. Dictyostehm appears to solve this conundrum by expressing two classes of CAMP receptors, of high and low affinity, on the surface during later development.

Multicellular development in Dictyostelium Firtel

The

receptors

cAR1

and

cAR3 -high-affinity

the promoter

receptors

that respond to nanomolar oscillatory pulses of CAMP to mediate the activation of adenylyl and guanylyl cyclases during aggregation-are fully saturated and are in the adapted conformation at micromolar concentrations of CAMP in the mound [9,33,34]. cAR2 and cAR4 are low-affinity receptors that cannot respond to nanomolar pulses [33,35,36]. These receptors, which are induced in the multicellular aggregate, however, respond to micromolar concentrations of CAMP. It is thought that, during the multicellular stages, micromolar oscillations of CAMP are produced from the anterior oscillator at the tip and are propagated posteriorly. In this model, cAR1 and cAR3 would mediate GBF-regulated responses, as these receptors are in the fully phosphorylated/adapted configuration. cAR2 and cAR4 presumably detect CAMP oscillation as cAR1 does during aggregation by responding to micromolar oscillations of CAMP to mediate the activation of adenylyl cyclase and morphogenetic movements. A single cell might thus be able to respond to pulses in the micromolar range to mediate the oscillatory pathways controlling cell movement and activation of adenylyl cyclase, as well as continuous CAMP stimulation pathways inducing GBF function through two different receptors simultaneously.

Control of cell-type differentiation -distinct signaling pathways control prestalk and prespore pathways Prestalk and prespore pathways, although both require GBF function, are regulated by distinct mechanisms. The morphogen DIF (differentiation-inducing factor), a chlorinated alkylphenone, was identified as a factor capable of inducing isolated cells to differentiate into stalk cells and of inhibiting spore differentiation assays [37-391. Mutant cells that are unable

in similar to produce

sufficient quantities of DIF arrest at the mound stage and do not induce the prestalk pathway, which includes the expression of the prestalk-specific genes ecmA and ecmB [40]. These cells can be complemented, however, by the addition of exogenous DIE In contrast, extracellular CAMP is sufficient in such assays to induce prespore gene expression, whereas high levels of CAMP repress the expression expressed

of the prestalk-specific preferentially in prestalk

gene ecmB, which B cells (Fig. 3).

is

in vivo. Induction

of prestalk

549

and prespore

cell-type differentiation in cell culture systems requires the presence of CAMP for a period of time. Removal of CAMP and addition of DIF results in prestalk cell differentiation whereas maintenance of the CAMP level results predominantly in prespore differentiation. The period of continuous CAMP is required for the induction of GBF-mediated postaggregative gene expression, as some of these gene products are essential for cell-type differentiation. For example, LagCwhich is induced by GBF -encodes a developmentally regulated cell surface signaling molecule that is essential for both prestalk and prespore

differentiation

[Zl].

Although both prestalk and prespore pathways require some common elements, such as GBF and LagC, there are independent signaling pathways controlling the differentiation of both cell types. New insights into this process have resulted from the analysis of PKA, the cAR4P-dependent protein kinase and glycogen synthase kinase 3 (GSK3), the serine/threonine protein kinase that is encoded by the shaggv/Zeste-white 3 locus in Drosophifa. Both kinases have been shown to play important regulatory roles in cell-type differentiation in Dicoostelium and metazoans. Disruption of GSK3 results in presumptive prespore cells being induced to form prestalk B cells [47**] -defining GSK3 as an essential gene for prespore cell differentiation and suggesting that the ground state pathway may be toward prestalk cell differentiation. What is particularly interesting is that gsR3 null cells have lost the inhibition by CAMP to differentiate to prestalk B cells, indicating that GSK3 may not only be an activator of the prespore pathway but an inhibitor of prestalk pathways (Fig. 3a,b).

Regulation of cell-type differentiation GSK3 and PKA

by

PKA and ERKZ are also essential for the prespore pathway. Cells that are pka null express GBF but to a lower level than that of wild-type cells ([51]; SK0 Mann et al., unpublished data). In response to CAMP, pka null cells (which do not aggregate) express the prestalk-specific gene ecmA at a low level in suspension culture; however, this level of expression increases to -50% of that of wild-type cells in pka null cells that constitutively express GBE

induces the a significant

Regardless of GBF expression levels, no prespore-specific gene expression is detected. Moreover, expression of a dominant negative PKA regulatory subunit in prespore

understanding of the signaling pathways controlling prestalk and prespore gene expression. Promoters for both prestalk and prespore genes have been identified and dissected using classic promoter analysis. The SP6U and SP70 prespore-specific and ecmA (prestalk A/O) and ecmB (prestalk B) genes (Fig. 2) all have GBF binding sites that are required for expression [41--45,46”]. None of these genes are expressed in gbf null cells and disruption of the GBF binding sites in the promoter regions of these genes results in a loss of activity of

cells blocks prespore differentiation, whereas expression of the catalytic subunit in prespore cells results in accelerated development and an immediate induction of newly formed prespore cells into spores [49-511. Interestingly, PKA also appears to regulate prestalk cell to stalk differentiation during culmination [52]. ERKZ is also required for prespore but not ecmA expression. Cells that are e&Z null are unable to aggregate because of their inability to activate adenylyl cyclase [14’]. By using chimeras of wild-type and e&’ null cells expressing

Although prestalk

the mechanism pathway is not

by which known, we

DIF have

550

Differentiation

Figure

and gene

regulation

3

(a) Hiah cAMP

Prestalk /‘u

Q

4

n

Q3

celk?

Stalk r

n

Q3

0-d CAMP

a

C 1996 Current Op~man I” Geneics

& Development

Flow diagram of cell-type differentiation. (a) A high level of CAMP mediates the inductton of post-aggregative genes that include new CARS, additional Ga subunits, two homeobox-containing genes, a Ras gene, the cell surface signaling molecule LagC, among a large number of other identified gene products. Prestalk and prespore cell-type differentiation requires GBF, LagC, and a number of other postaggregative gene products [22]. In addition, the roles of other signaling molecules are shown that, in some cases, differentially regulate the prestalk and prespore pathway. From the car4 null phenotype, we know that cAR4 is required for prestalk cell differentiation and results In a significant increase in prespore gene expression. As stated in the legend to Figure 1, however, cAR4 may play a role in mediating CAMP signaling during later development. The relative roles of cAR2 and cAR4 in these pathways are not known. cAR4 is included as a stimulator of the prestalk pathway. It is possible that it functions by inhibiting the action of GSK3 (see Fig. 4). (b) Differences in the prestalk pathways are outlined. (CAMP refers to extracellular

CAMP.)

/acZ under the control of cell-type-specific has been shown that erk.? null cells express

promoters, it prestalk but

not prespore genes [53’]. A temperature-sensitive ERKZ mutation was then used to bypass the inability of erk2 null cells to aggregate [53-l; ERKZts cells that are allowed to aggregate at a permissive temperature and then shifted to a non-permissive temperature induce ecmA but not prespore genes. Whether GSK3, PKA, and ERKZ function in common or parallel pathways or how these pathways may interact is unknown presently but it is expected that they function through an identified prespore-specific element

[54] (Fig.

lc).

Other signaling pathways, including those mediated by other Ga subunits and CAMP receptors, appear to regulate Dic~ostelium morphogenesis and cell-type differentiation. The Ga protein subunit Ga4-which couples to folate/pterin receptors -is expressed in anterior-like cells (a regulatory cell type) and is required for morphogenesis past the finger stage and normal expression of prestalk and prespore markers [55,56]. Cells that are gal null show a very extended stalk and a normal anterior population of prestalk AB cells in migrating slugs: overexpression of the Gal subunit results in very abnormal morphogenesis and an apparent loss of anterior prestalk AB cells,

suggesting that Gal this pathway [19].

functions

as a negative

regulator

of

The low affinity CAMP receptors cARZ and cAR4 are expressed in the multicellular stages and control both morphogenesis and prespore gene expression. Cells that are car2 null arrest at the mound stage, with some cells continuing in development [36]. The cAR2 receptor is preferentially expressed in prestalk cells and is required for tip formation [57], probably functioning to activate adenylyl cyclase in the anterior oscillator. Unexpectedly, car2 null cells display a significant overexpression of prespore-specific markers with no significant increase in the number of prespore cells. The effect of deleting cAR4, which is expressed in both prestalk and prespore cells, is a similar increase in prespore gene expression [35]. In addition, these cells show a reduced expression of prestalk cell markers and have an impairment in culmination. One expects that these phenoytpes may be caused by blocking two distinct receptor-dependent pathways: first, the activation of adenylyl cyclase; and second, a pathway that modulates the level of prespore gene expression. Pathways may be controlled by cARZ/cAR4 in prestalk cells that inhibit GSK3 function in prespore cells, possibly

Multicellular development in Dictyostelium

through

the

production

of a soluble

negative

regulator

(Fig. 4). Experiments in which cAR4 or cAR2 are also disrupted in cells lacking the adenylyl cyclase A, ACA, should allow further insight into the mechanisms by which these expression.

receptors

Regulation

regulate

the level of prespore

gene

A common

factor

thus

appears

activation and repression the activity of this factor

to

regulate

The anterior -15~20% portion of the slug comprises the prestalk domain and contains at least three distinct cell types that show different patterns of spatial localization and origin in the developing mound [S&60,61’] (Fig. 2). Ttvo prestalk-specific genes, ecmA and ermB, have been used to dissect an intriguing regulatory pathway that controls gene expression in multiple classes of prestalk cells. Recent data suggest that a common transcription factor may control the expression of ecnrA and ecmB differentially in multiple cell types [46”]. There is no ermB expression in the slug prestalk A population but this gene is expressed in developing stalk cells that arise from the prestalk A population as they enter the stalk tube [Ml. The stalk-specific ermB promoter is not expressed in the slug because of a &-acting repressor element that contains an inverted tetranucleotide repeat: when this element is deleted, ecmB is expressed in prestalk A cells during the later stages of development. hloving this repressor element into the ermA gene causes the suppression of ern]A expression in the same prestalk A cells, indicating that this regulatory element can regulate prestalk A gene expression in multiple genes [62]. The prestalk-A-specific promoter of the ecmA gene contains a direct repeat of the same tetranucleotide found in the inverted repeat of the ermB repressor; both elements bind the same developmentally regulated nuclear factor [46**].

both

the

of prestalk-specific genes is dependent on whether

and the

element is found in a direct or inverted repeat. DIF may function through this nuclear factor, possibly by regulating its induction and/or mediating its function as a transcription factor. Studies using PKA indicate that the ermB gene is turned on during stalk differentiation through

in differentiating stalk this factor (Fig. 3a).

cells

directly

by phosphorylating

A GATA/zinc finger transcription factor and a homeobox-containing gene involved in cell-type specification Raper first demonstrated over fifty years ago [63] that differentiation of cell types in Dicoostelium is plastic until the culmination stage. When the anterior prestalk and posterior prespore regions are separated, both sections are capable of forming a new migrating slug that, over time, has the same cell-type proportions as the original slug. Recent experiments (e.g. [59]) have shown that various cell type populations dedifferentiate and redifferentiate into different cell types during normal slug migration. In addition, slugs can be dissociated and placed in growth medium and the cells will re-enter the cell cycle and initiate vegetative growth. Such mechanisms lend the organism significant flexibility in maintaining cell-type ratios as the slug migrates and celis are lost from the posterior either in response to environmental stresses or when a food source becomes available. R:hat maintains the proportions of the different cell types within a migrating slug is not known: however, a new

Figure 4 Model for the interactions of prestalk and prespore cells and the role of the is modeled GSK3

CAMP

Here, cAR2/cAR4

to inhibit the function

in prestalk

n of

cells. In addition,

cell

autonomous experiments suggest that a possible diffusible factor (A) may lie downstream

from the cAR2/cAR4

receptors

in prestalk

modulate

GSK3

cells functioning

prespore

gene expression

activity

to

and the level of in prespore

cells. ERK2 and PKA are shown to be essential activators of the prespore pathway which is thought to be activated through cARli receptors. As modeled in Figure lc, cAR4, or another low affinity receptor, may also function in medtating CAMP levels in prespore

551

the action of PKA functioning to block repressor activity [62]. PKA may alleviate the repression of ecmB expression

of prestalk cell differentiation

CAMP receptors.

Firtel

cells.

Prestalk cell

Prespore cell

552

Differentiation

and gene

regulation

insight into possible mechanisms has come from the analysis of a developmentally regulated homeobox-containing gene. Cells carrying a disruption of the gene Ddhbx-2 aggregate normally (2 Han, RA Firtel, unpublished data); some of the aggregates arrest at the mound stage, whereas the others continue to develop with near normal morphology. When cell-type-specific /acZ reporters were used to examine spatial patterning of the cell types, there was seen to be a reduction in the size of the prespore domain and an increase in the prestalk domain with the majority of this increase being in the prestalk 0 region. Prestalk 0 cells are known to generate anterior-like cells and vice versa and it is thought that anterior-like cells may transdifferentiate into prespore cells and vice versa. It is possible that this homeobox-containing gene controls the equilibrium of this reaction. In its absence, the equilibrium may be slanted in the direction of the prestalk pathway. A transcription factor encoded by the Stalky gene appears to play an essential role in the terminal differentiation of prespore and prestalk cells [64-l. Stalky was identified many years ago by classic mutant analysis. Cells that are stalky null produce a slug which contains prestalk and prespore cells but, during culmination, the prespore cells all differentiate into stalk cells. Stalky encodes a GATAlike transcription factor that is preferentially-although not exclusivelyexpressed in prespore cells. Expression of Stalky is required to maintain the prespore pathway and in its absence prespore cells transdifferentiate into prestalk and then stalk cells. Stalky has no apparent effect on the initial cell choice mechanism and may thus be involved in the homeostasis between the cell types during later development. The analysis of S’taLky and GSK3 suggest that the prestalk pathway may be the ground state pathway and that, in an organism in which CAMP and DIF are present, specific regulatory circuits are required to maintain the prespore state; from a teleological perspective, this is unexpected. One might expect the ground state pathway to be toward spore differentiation, as this is essential for maintaining the next generation.

Conclusions

Acknowledgements I

would like to thank Alan Kimmel, hlarianne Gamper, Jason Brown, and Sean Buchanan for valuable comments. This work was supported by National Institutes of Health USPHS grants to RA Firtel.

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reading

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and future analysis

The ability to identify important developmental mutations and to isolate and clone the relevant genes has permitted a thorough examination of various developmental pathways in Dictyostelium. In addition to isolating genes that are homologs to proteins found in other systems, these approaches have already provided exciting new findings, including the identification of pioneer genes with no known homology to previously identified genes or the identification of novel functions for genes previously identified in other systems. If one considers the evolutionary conservation of signaling pathways, we can expect that insights derived from the analysis of Dic~ostehm development should have a significant impact on our understanding of basic cellular processes during growth and development in a variety of systems.

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Multicellular

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16.

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