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Signal transduction and the fate of the R7 photoreceptor in Drosophila
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T h e developmental fate of an individual cell is influenced by the signals it receives from its environment. These inputs can range from long-range signals mediated by hormones to short-range signals mediated by direct cell-cell contact. The biochemical nature of such signals and their receptors, the mechanisms employed to ensure that the signal is transmitted only at the correct time and to the appropriate recipient cell, and the intracellular machinery used to interpret and implement the instruction(s) conveyed by the signal are being actively investigated in a wide variety of developing organisms and tissues. The developing Drosophila eye has proven to be well suited for genetic, molecular and cellular experiments aimed at elucidating these issues. The adult eye is made up of a simple array of -800 20-cell units, called ommatidia. Each ommatidium contains eight photoreceptor cells, R1-R8, as well as four lenssecreting cone cells and eight other accessory cells. The cells that make up each ommatidium are thought to be recruited by a series of local cell-cell interactions, with differentiating cells instructing their immediate neighbors to adopt particular fates. Developmen t of the Drosophila eye has been extensively reviewed (see Refs 1--4). This article focuses on recent progress in understanding one inductive event, the recruitment of the R7 photoreceptor.
The choice The R7 photoreceptor is the last of the eight photoreceptors to be recruited to the developing ommatidium (Fig. 1). Mutations in three genes - sevenless (sev; Ref. 5), bride of sevenless (boss; Ref. 6) and Boss EXPRESSION IN R8
Signal transducti0nand the fate of the R7 ph0t0recept0r in
Drosophila
GERALD M. RUBIN The seve~less protein tyrosine kinase receptor plays a central role in the pathway of cellfate induction that determitws the development of the R7 photoreceptor in the Drosoplrila eye. In the last year we have learned much about the probable ligand for sevealess and have begun to dissect the sigm~l transduction pathway that relays the information from the sevenless kinase. Studies of the mechanisms governing the specificity of signal transmission and reception suggest that the sevenless signal directs a bipotential cell towards a neuronal rather than a cone cellfate.
seven-in-absentia (sina; Ref. 7) - have been shown to disrupt this recruitment. In each of these mutants, the presumptive R7 cell - the cell that finds itself in the pocket created by the differentiating R8, R1 and R6 cells - develops into a non-neuronal, lens-secreting cone cell. Thus, the presumptive R7 cell appears to face a simple choice between two alternative cell fates: it will develop into an R7 photoreceptor if it receives appropriate instructions, but will adopt a non-neuronal cone cell fate if these instructions are disrupted by Boss INTERNALIZED INTO R7 R7 CELL COMMITS TO CONE CELL FATE IN THE ABSENCE OF Sev FUNCTION
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Sev EXPRESSION -'I-'D Sev EXPRESSION IN R7 ~ IN R3 & R4 R7 NUCLEUS RISES APICALLY
hours (at 25°C)
R7 NUCLEUS FALLS BASALL~'
FIGII Summary of the time-course of events involved in R7 cell recruitment. The time line is calibrated both in hours of developmental time at 25°C and in columns of ommatidial development. The developmental times given are approximate; the rate of development (columns per hour) varies slightly between strains and is not uniform, being slower in the posterior portions of the eye disk 16. The times at which cells begin neural differentiation (see Ref. 1) are indicated in the diagrams of assembling ommatidia shown above the time line, beginning with R8 at column 1 and adding R2 and R5 at column 3, R3 and R4 at column 6, R1 and R6 at column 8, and R7 (darker shading) at column 12. The times are indicated at which sev expression is first observed in R3, R4 and R7 (Ref. 10), and boss expression in R8 (Ref. 8), as well as the times at which the nucleus of the presumptive R7 cell begins its apical and basal migrations 1. Also shown are the 6 h period during which sev activity appears to be required 17 (shaded region), and the overlapping period during which boss internalization into R7 is observed ~. Finally, the time at which the presumptive R7 cell commits to an alternative developmental fate in the absence of sev activity~vis shown. "FIGNOVEMBER/DECEMBER1991 VOL.7 NO. 11/12 ©1~)1 Elsevier S c i e n c e Publishers Ltd (UK) (HOg - 9479/91/$02.00
[]~EVIEWS A
B
The 8 photoreceptors & 4 cone cells
C
sevenless (sev)
bride of sevenless (boss)
seven-up (svp)
seven in absentia (sina)
D
wild type
mutant for either sev, boss, or sina
ligand-independent Sev activity -or- ubiquitous BOSS expression
mutant for svp
FIG~
Summary of expression patterns and genetic data used to infer the roles of sevenless (sev), bride ofsevenless (boss), seven-up (svp) and seven in absentia (sina) during ommatidial development. (A) An ommatidium showing the cell contacts made by the R1-R8 cells (1-8) and the four cone cells (PC, EC, AC and PLC). (B) The expression patterns of sev, boss, svp and sina. Black, strong expression; gray, internmdiate expression; white, no detectable expression. (C) The cells in which the function of sev, boss, svp and sina is required, as determined by mosaic analysis. (D) Cells that adopt an R7-1ike cell fate in the indicated genotypes are shown by black shading. mutation of sev, boss or sina. A variety of genetic 6 and molecular s data indicate that boss encodes a cell surface ligand on the R8 cell that activates a sev-encoded transmembrane protein tyrosine kinase receptor9 on the surface of the R7 cell 5,1°. The s i n a gene encodes a nuclear protein required in the R7 cell v.
The ligand The product of the boss gene is a -100 kDa glycoprotein with seven putative membrane-spanning regions and a 498 amino acid extracellular domainS, H. The boss protein is expressed exclusively on the surface of the R8 cell 8 (see Fig. 2) and this expression in R8 is essential for R7 cell determination 6. Kr~imer et al. ~ demonstrated a direct interaction between the boss and sev proteins by showing that boss- and sevexpressing cell lines form heterotypic aggregates. Two lines of evidence suggest that this interaction also occurs in vivo. First, sev immunoreactivity is concentrated on the membranes of sev-expressing cells where they contact R8 (Ref. 10; Fig. 3A, B). Second, boss immunoreactivity is internalized into the R7 cell in a sev-dependent manner 8 (Fig. 3C). Taken together, these observations strongly support the hypothesis that boss functions as the ligand for the sev receptor.
The receptor The sevenless protein (sev) is synthesized as a 280 kDa glycoprotein precursor that is subsequently
cleaved into 220 kDa amino-terminal and 58 kDa carboxy-terminal subunits that remain associated by noncovalent interactions .2. The carhoxy-terminal subunit contains the transmembrane domain and a cytoplasmic
domain highly homologous to known tyrosine kinases9. The sev protein has been shown to have protein tyrosine kinase activity in vitro ~2 and this activity appears to be essential for sev function in vivo 13. Although sev activity is only required in the R7 cell 5, sev is expressed on the apical surface of many cells in the developing retina (Figs 1, 2) in a highly dynamic pattern 1° that is controlled at the level of transcription of the sev gene v'. Temperature-inducible hsp70-sev fusion genes 15,16 and temperature-sensitive sev mutations Ir have been used to show that sev activity is required only to initiate R7 cell development and not for its subsequent differentiation or function. However, the sev-mediated signal must be maintained for several hours to be effective 17 (Fig. 1).
The signaltransductionpathway Activation of the sev tyrosine kinase must result in intracellular changes in the presumptive R7 cell that cause it to adopt the R7 cell fate rather than the cone cell fate. The mechanisms by which receptor tyrosine kinases effect changes in cell physiology are still poorly understood. Biochemical studies with mammalian protein tyrosine kinase receptors (PTKs) have led to the identification of proteins that bind to or are phosphorylated by tyrosine kinases18,19. While some of these interactions suggest potential mechanisms of signal transmission, their role in vivo is still unclear. Two features make the sev pathway ideal for genetic approaches to understanding signaling by receptor tyrosine kinases. First, both the R7 cell and the sev protein are dispensable for viability and fertility. Second, the functioning of this signaling pathway can be inferred from the presence of the R7 cell in a
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FIGII
Electron micrographs illustrating sev and boss expression. (A, B) sev immunoreactivity at the early (A) and late (B) symmetrical cluster stage (see Ref. 10 for details). Note that the staining on the membranes of R3, R4 and R7 is clustered where they contact R8, and the sev immunoreactivity is seen in large vesicular structures in the cytoplasm (arrow in A). (C) boss immunoreactivity at the late symmetrical cluster stage (photograph courtesy of R. Cagan and S.L Zipursky; see Ref. 8 for details). Note the large vesicular structure containing boss in the cytoplasm of R7. The boss protein is synthesized only by the R8 cell; the staining in R7 is the result of sev-mediated internalization~. live, anesthetized fly. Screens for mutations that alter the strength of sev signaling can be used to identify other elements in the pathway. Rogge et al. 2° carried out a screen for dominant suppressors of sev and isolated a mutation in a gene they named Son o f sevenless (Sos). This gain-of-function mutation, SosJ2, does not bypass the requirement for sev function as it cannot suppress a sev null mutation. Rather, SosJ2 appears to increase the efficiency of transduction of a weak sev-mediated signal. Simon et al. 21 carried out a systematic genetic screen for mutations that decrease the effectiveness of signaling by the sevenless PTK, by looking for dominant enhancers of sev. By adjusting the temperature at which flies carrying a temperature-sensitive allele of sev were grown, Simon et al. could adjust sevenless PTK activity to a level barely above the threshold necessary for R7 cell formation. Small reductions in the abundance or activity of other elements of the pathway might then be expected to lower signal strength sufficiently to cause a sevenless phenotype in flies grown at this threshold temperature. This sensitivity allowed Simon et al. to identify genes encoding putative downstream elements of the pathway, by screening for genes in which inactivation of only one copy of the gene - which would be expected to reduce the level of gene product by half resulted in the absence of the R7 cell. Since the other copy of the gene remained functional, they were able to identify these loci even though their functions were essential for viability. Seven genes were identified in this screen; one of these corresponded to So~; in which several loss-of-function mutations were isolated. The products of four of the seven loci 21 also appear to be involved in transduction of signals from another PTK, the Drosophila homolog of the EGF receptor (DER), as judged by the ability of mutations in these loci to suppress Ellipse, a mutation that results in DER hyperactivity22. These results suggest that these four loci encode proteins that participate in the transduction of signals from at least two different PTKs.
One of the four loci corresponds to the R a s l gene, which encodes a p21 ras protein 2i. Several previous studies have suggested a role for Ras activity in PTK function. Injection of monoclonal antibodies that inhibit Ras activity blocked the ability of PTKs to induce mitosis, and a dominant-negative ras gene was able to interfere with oncogenic transformation by several tyrosine kinase-encoding oncogenes 23. Furthermore, recent studies of vulval development in the nematode Caenorbabditis elegans place the ras gene downstream of the PTK encoded by the let-23 gene (see article by Sternberg and Horvitz in this issue). Moreover, the product of the Sos locus shows sequence similarity to the Saccharomyces cerevisiae CDC25 protein 21, which activates yeast RAS by facilitating exchange of GTP for GDP. These results support a model in which the stimulation of Ras activity is a key element in signaling by PTKs, and suggest that this stimulation may be achieved by activating the exchange of GDP for GTP by Ras proteins. A model incorporating this hypothesis is shown in Fig. 4. While the sina gene product is required for R7 determination, its role within the presumptive R7 cell remains unclear. Modification of sina activity may be one end-point for the signal transduction pathway initiated by sevenless. Alternatively, sina may function in parallel to the sevenless pathway.
Specificity The ability of cells that develop in close proximity to adopt distinct fates implies a high level of precision in cell-cell signaling. In principle, two mechanisms could provide specificity in the sevenless signaling pathway. First, the distributions in time and space of the ligand (boss) and the receptor (sev) could be tightly controlled; in the extreme case they would be coincident only when appropriate to specify the R7 cell fate. Second, the developmental history of individual cells could limit their ability to receive or be influenced
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by the sevenless-mediated signal. In the extreme case of this mechanism, only the presumptive R7 cell would E R8 be capable of responding to a ubiquitous signal. Data from a number of studies have clearly established that a SEVENLESS combination of these mechanisms ..... operates to ensure that one, and only GDP GTP 7/ /! one, R7 cell is specified in each ommatidium. To test the first possible mechanism, the temporal and spatial distributions of sev and boss have been ACTIVE ".. ? altered by expressing tl{ese proteins ? from heat-inducible hsp70-sev (Refs 15, 16) or hsp70-boss (Ref. 24) fusion genes. The distribution of sev, R7 ? despite being highly dynamic in space and time 1°, does not appear to V play an important role in determining R7 Determination specificity. The sev protein needs to Pi be present in the R7 cell at the critical FIG~[~ time, but its ectopic presence in other Model for the sev-mediated signal transduction pathway. Data for the involvement cells does not obviously decrease the and structure of the indicated proteins can be found in the following references: specificity of R7 cell determination. boss, Refs 6, 8, 11, 24; sev, Refs 5, 9, 10, 12, 13; Sos, Refs 20, 21; Ras, Ref. 21; Rapl, In contrast, the restriction of boss Ref. 31. Note that it is not yet clear whether the amino-terminal region of the larger expression to the R8 cell does play a sev subunit spans the membrane; that region is shown in gray to indicate this major role. Ubiquitous expression of uncertainty, The involvement of phosphatases, GAP and effectors for Ras is based boss causes the cone cells to differen- on analogy to other signal transduction systems; these components have not yet tiate as R7 cells 24 (Fig. 2). Normally, been identified in the R7 cell. the cone cells express sev, but do not contact R8. Thus restricting boss expression to R8 can determined, leading to an early disruption of ommafully account for the cone cells not becoming R7 cells tidial assembly 27. More than one R7-1ike cell whose during normal development. However, the limited presence depends on sev are often observed 28. presentation of boss cannot account for the fact that Van Vactor el al. 24 have suggested that the inability the R1-R6 cells do not b e c o m e R7 cells. All these cells of the R1-R6 photoreceptors to respond to the sevencontact R8 and four of them normally express sev. less signal may be connected with boss binding or Expression of sev can be experimentally induced in the internalization. The sev-mediated internalization of other two, yet these cells do not become R7 cells, indi- boss, as observed in the R7 cell, does not occur in any cating that their developmental history has made them of the R1-R6 cells, even though R3 and R4 express s o m e h o w unreceptive to the sev-mediated signal. high levels of sev and contact R8 (Ref. 8). In contrast, Consistent with this view, a constitutively active, in N'P t (another mutation that disrupts normal ommaligand-independent form of the sevenless receptor 2s tidial assembly) and rough flies, ectopic internalization can cause the cone cells to differentiate as R7-1ike of boss is seen 24. Arguing against ligand internalization cells, but does not appear to be able to induce R7 being an essential step in the response to sev, howdevelopment in R1-R6 cells. ever, is the observation that a ligand-independent, If the developmental history of the R1-R6 cells activated sev can induce cone cells to differentiate as the inputs they have received and the resultant restric- R7-1ike cells 2s. Moreover, the apparent block in the tions that have been placed on their developmental sev-mediated pathway in R3 and R4 does not appear potentials - is responsible for their lack of response to to be simply at the level of ligand internalization, since sev, then mutations that disrupt R1-R6 cell fate detereven activated sev cannot induce R3 and R4 to mination might be expected to permit additional cells become R7-1ike cells zs. to adopt the R7 fate. This prediction was first shown to Alternatively, it is possible that reception of the be true for the seven-up mutation z6. The seven-up gene sev-mediated signal is not blocked in R3 and R4; encodes a steroid receptor superfamily m e m b e r that is rather, the sev-mediated signal may simply be redunexpressed and required in R1, R3, R4 and R6 (Ref. 26; dant in these cells. Suppose, for example, that actiFig. 2). In the absence of seven-up function, these four vation of Ras is required in each photoreceptor precurcells develop as R7-1ike cells. Surprisingly, only two of sor for it to adopt a neural fate, the type of neuron these cells appear to require sev function for their formed being determined by a Ras-independent pathdevelopment as R7-1ike cells; as discussed below, this way. The sev protein may indeed be capable of actiobservation implies that it is possible to bypass the vating Ras in R3 and R4, but by the time sev is activated requirement for the sev-mediated signal. The phein these cells, Ras activation, and neural development, n o m e n o n of supernumerary R7 cells is also seen in may already have been triggered by another signal. rough mutant flies, where R2 and R5 are incorrectly The lack of boss internalization in R3 and R4 might
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The role of the sev-mediated signal is to distinguish between two cell fates: R7 and cone cell. The developmental histories of the presumptive R7 cell and the cone cells may be very similar. For example, their transcriptional programs are related in that they both express high levels of sev and sin& Moreover, the cone cells, unlike the R1-R6 cells, can respond to the sev-mediated signal by differentiating as R7 cells. Thus, it may not be necessary to attribute much information content to the sev-mediated signal. Indeed, the instruction to become an R7 cell, as opposed to one of the R1-R6 cells, may not be conveyed by sev at all, but may have already been established by the developmental history of the R7 cell. Thus, in the extreme view, activation of sev may simply tip the balance in favor of neuronal development rather than nonneuronal development. Three observations argue that such an extreme view is tenable. First, ectopic expression of rough in a developing R7 cell can cause it to differentiate as an R1-R6-1ike cell; however, the decision to become a neuron still requires sev function 29,30. Thus it is possible to uncouple the sevmediated decision of whether to become a neuron from the choice of what type of neuron to become. Second, several genes have been identified in screens for enhancers 21 and suppressors 20 of sev; these genes encode products that are likely to function in the transduction of signals initiated by sev. However, these genes are not dedicated to the sevenless pathway; they can function in conjunction with other PTKs and they are required for the development of other photoreceptors that do not depend on sev. It seems unlikely that such a general signal could, by itself, convey photoreceptor subtype identity. Third, in certain unusual circumstances, such as in svp- sev- double mutants, it is possible for R7-1ike cells to differentiate without sev function. This would be more difficult to explain if sev were required to specify R7 cell type identity. In the simplest model, two decisions would be required to determine the R7 cell fate. The first decision would establish a bipotential cell that could become either an R7 cell or a cone cell. The acquisition of this fate rather than that of R1-R6 could be as simple as the expression of a single additional transcription factor, since we know that the ectopic expression of rough in the presumptive R7 cell achieves the opposite effect, that is, directs it towards a fate resembling R1-R6. Then the second decision, directed by the sevenless signal, need simply steer this bipotential cell towards the R7 fate.
Other inputs The pathway proposed for the sev-mediated signal (Fig. 4) suggests many steps at which additional inputs could be integrated with the sev-initiated signal. Some, like the tyrosine phosphatase and Ras GTPaseactivating protein, have not yet been identified. Interestingly, the Roughened mutation, which appears
to affect R7 formation, has been shown to be a dominant gain-of-function mutation in a homolog of the human rapl/Krev gene31. The mammalian Rap proteins are small GTPases that have opposing effects to Ras in cell transformation32. Roughened flies lack R7 cells in most ommatidia, consistent with the notion that Drosophila Rasl and Rap1 gene products have opposing effects in R7 cell determination 31, perhaps by competing for a common effector (Fig. 4).
Concludingremarks Over the past few years, studies of R7 cell determination in the developing Drosophila eye have taught us much about the molecular mechanisms of induction and signal transduction by protein tyrosine kinase receptors. The experimental advantages of being able to examine mutant phenotypes at single cell resolution in a developing tissue that is dispensable for viability and fertility ensure that new insight will continue to emerge from studies of the sev-mediated signal transduction pathway. In particular, the utility of genetic screens for enhancers and suppressors of sevenless to identify components of the pathway has been established and should encourage similar endeavors starting from other points in the pathway. This approach could be used to identify some of the most elusive players in Ras-mediated signal transduction: the effector molecules through which Ras acts to alter cell function. Finally, the identification of Ras as an important component in both the sev and EGF receptor pathways in Drosophila suggests that signal transduction pathways acting downstream of protein tyrosine kinase receptors may be shared by many different kinases and may be highly conserved in organisms as diverse as flies, worms and humans.
Acknowledgements I am grateful to Mike Simon and Iswar Hariharan; many of the ideas expressed in this review were developed and clarified in conversations with them. I thank S.L. Zipursky and his co-workers for communicating their results before publication.
References I 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Tomlinson, A. (1988) Development 104, 183-189 Ready, DF. (1989) Trends Neurosci. 12, 102-110 Rubin, G.M. (1989) Cel157, 519-520 Banerjee, U. and Zipursky, S.L. (1990) Neuron 4, 177-187 Tomlinson, A. and Ready, D.F. (1987) Dev. Biol. 123, 264-275 Reinke, R. and Zipursky, S.L. (1988) Cell 55, 321-330 Carthew, R.W. and Rubin, G.M. (1990) Cell 63, 561-577 Kr~imer,H., Cagan, R.L. and Zipursky, S.L. (1991) Nature 352, 207-212 Hafen, E., Basler, K., Edstroem, J-E. and Rubin, G.M. (1987) Science 236, 55-63 Tomlinson, A., Bowtell, D.D.L., Hafen, E. and Rubin, G.M. (1987) Cell 51,143-150 Hart, A.C. et al. (1990) Genes Dev. 4, 1835-1847 Simon, M.A., Bowtell, D.D.L. and Rubin, G.M. (1989) Proc. Natl Acad. Sci. USA 86, 8333-8337 Basler, K. and Hafen, E. (1988) Cell 54, 299-311 Bowtell, D.D.L., Kimmel, B.E., Simon, M,A. and Rubin, G.M. (1989) Proc. Natl Acad. Scl. USA 86, 6245--6249 Bowteli, D.D.L., Simon, M.A. and Rubin, G.M. (1989) Cell 56, 931-936
11G NOVEMBER/DECEMBER1991 VOt. 7 NO. 11/12 ~,-(~
[]~EVIEWS 16 Basler, K. and Hafen, E. (1989) Development 107, 723-731 17 Mullins, M.C. and Rubin, G.M. Proc. Natl Acad. Sci. USA
27 Tomlinson, A., Kimmel, B.E. and Rubin, G.M. (1988) Cell
(in press) Cantley, L.C. et al. (1991) Cell 64, 281-302 Koch, C.A. etal. (1991) Science 252, 668-674 Rogge, R.D., Karlovich, C.A. and Banerjee, U. (1991) Cell 64, 39-48 Simon, M.A., Bowtell, D.D.L., Dodson, G.S., Laverty, T.R. and Rubin, G.M. Cell (in press) Baker, N.E. and Rubin, G.M. (1989) Nature 340, 150-153 Fieg, L.A. and Cooper, G.M. (1988) Mol. Cell. BioL 8, 3235-3243 Van Vactor, D.L., Jr, Cagan, R.L., Kr~imer, H. and Zipursky, S.L. Cell (in press) Basler, K, Christen, Bt and Hafen, E. (1991) Cell64. 1-20 Mlodzik, M. et al. (1990) Cell60, 211-224
28 Heberlein, U., Mlodzik, M. and Rubin, G.M. (1991) Development 112, 703-712 29 Kimmel, B.E., Heberlein, U. and Rubin, G.M. (1990) GenesDev. 4, 712-727 30 Basler, K., Yen, D., Tomlinson, A. and Hafen, E. (1990) Genes Dev. 4, 728-739 31 Hariharan, I., Carthew, R. and Rubin, G.M. Cell (in press) 32 Kitayama, H. et al. (1989) Cell 56, 77-84
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A l l metazoan cells, whether in culture or in the organism, must be able to make decisions about cell division or cellular differentiation based, in part, on environmental cues. Accordingly, cells express receptor systems, either intracellular or cell surface, that allow them to detect the presence of hormones, growth factors and other signalling molecules that manipulate the regulatory processes of the cell. Such molecules will influence the decision to proliferate or differentiate by stimulating the activation of a cascade of events that ultimately impinges on the genetic apparatus. In turn, this leads to altered transcription of a defined set of genes 1,2. But what happens when a cell is exposed to both differentiation and proliferation factors? Are both networks independently activated, or is there a cellular mechanism that integrates these two signal transduction pathways to avoid their antagonistic effects? This review describes recent evidence for the presence of an intracellular 'checks and balance system' that allows these distinct regulatory pathways to modulate each other's activities. In one branch of the signalling pathway, steroid, retinoid and related hormones typically function as differentiation factors that alter cell fate and function during embryonic development and regulate organ physiology in the adult. These hormones exert their effects by binding to intracellular receptors that function as hormone-dependent transcription factors. All intracellular receptors are structurally related, forming a superfamily of regulatory proteins that have broadly conserved functional domains for transcriptional activation, nuclear localization, DNA binding and hormone binding 1. Ligand binding modifies the receptor so that it can interact with high-affinity sites in target DNA, termed hormone response elements (reviewed in Refs 1-4). While this is a common view for the action of steroid hormones, recent evidence reveals a novel pathway of regulation that does not involve binding to target DNA5-v. The second signalling pathway involves cell surface receptors that must transmit an external signal via the production of intracellular second messengers including small molecules like cyclic AMP, Ca 2÷ or diacylglycerol. In turn, these second messengers serve as allosteric triggers that activate intracellular protein TIG N O ' ~ M B E R / D E C E M B E R ~"1991 Elsevier Sc'ien~.t' Publislters Ltd (1 K) Ol(xg 94"Tg/91,$O2,(RI
55, 771-784
G.M. RUBIN IS IN THE HOWARD HUGHES MEDICAL INSTITUTE AND DEPARTMENT OF MOLECULAR AND CELL BIOLOGY, UNIVERSITY OF CALIFORNIA AT BERKELEY, BERKELEY,
CA 94720, USA.
Cr0ss-c0upling of signal transducti0n pathways: zinc finger meets leucine zipper ROLAND SCHOI~ AND RONALD M. EVANS
Cellproliferation and cellular differentiation are often thought of as opposing phenomena. The molecular mechanisms by which steroid, retinoid and thyroid hormones inhibit cellular proliferation and by which growth factors stimulate this process are poor~F understood We discuss recent evidence suggesting that these two signal transduction pathways converge through a process referred to as 'cross-coupling', which involves a possible interaction between steroid hormone receptors and the oJun oncoproteitt kinase systems, which continue to propagate the signal. In some cases, the cell surface receptor is itself a protein kinase that becomes activated via the binding of the external ligand (reviewed in Refs 8, 9). Of particular interest to this review are the converging lines of evidence indicating that the Jun and Fos protooncogene products are part of a signalling pathway that transmits stimuli from the cell surface to the nucleus. It has been shown that Jun and Fos directly regulate gene expression in response to stimulation of growth factor receptorsl°,lL Both Jun and Fos are members of a class of nuclear proteins that has been widely implicated in diverse aspects of cell growth and differentiation. Mutant forms of both Jun and Fos are oncogenic, causing formation of foci in vitro and tumors in vivo. Jun was originally defined as interacting with the control regions of genes containing promoter elements responsive to the phorbol ester TPA (12-Otetradecanoylphorbol-13-acetate). The binding sequence is referred to as an AP-1 site, which is recognized by c-Jun homodimers as well as by c-Jun--c-Fos heterodimers. Homodimer and heterodimer formation is mediated through noncovalent interactions facilitated 1991 vot. 7 NO. 11/12
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T h e developmental fate of an individual cell is influenced by the signals it receives from its environment. These inputs can range from long-range signals mediated by hormones to short-range signals mediated by direct cell-cell contact. The biochemical nature of such signals and their receptors, the mechanisms employed to ensure that the signal is transmitted only at the correct time and to the appropriate recipient cell, and the intracellular machinery used to interpret and implement the instruction(s) conveyed by the signal are being actively investigated in a wide variety of developing organisms and tissues. The developing Drosophila eye has proven to be well suited for genetic, molecular and cellular experiments aimed at elucidating these issues. The adult eye is made up of a simple array of -800 20-cell units, called ommatidia. Each ommatidium contains eight photoreceptor cells, R1-R8, as well as four lenssecreting cone cells and eight other accessory cells. The cells that make up each ommatidium are thought to be recruited by a series of local cell-cell interactions, with differentiating cells instructing their immediate neighbors to adopt particular fates. Developmen t of the Drosophila eye has been extensively reviewed (see Refs 1--4). This article focuses on recent progress in understanding one inductive event, the recruitment of the R7 photoreceptor.
The choice The R7 photoreceptor is the last of the eight photoreceptors to be recruited to the developing ommatidium (Fig. 1). Mutations in three genes - sevenless (sev; Ref. 5), bride of sevenless (boss; Ref. 6) and Boss EXPRESSION IN R8
Signal transducti0nand the fate of the R7 ph0t0recept0r in
Drosophila
GERALD M. RUBIN The seve~less protein tyrosine kinase receptor plays a central role in the pathway of cellfate induction that determitws the development of the R7 photoreceptor in the Drosoplrila eye. In the last year we have learned much about the probable ligand for sevealess and have begun to dissect the sigm~l transduction pathway that relays the information from the sevenless kinase. Studies of the mechanisms governing the specificity of signal transmission and reception suggest that the sevenless signal directs a bipotential cell towards a neuronal rather than a cone cellfate.
seven-in-absentia (sina; Ref. 7) - have been shown to disrupt this recruitment. In each of these mutants, the presumptive R7 cell - the cell that finds itself in the pocket created by the differentiating R8, R1 and R6 cells - develops into a non-neuronal, lens-secreting cone cell. Thus, the presumptive R7 cell appears to face a simple choice between two alternative cell fates: it will develop into an R7 photoreceptor if it receives appropriate instructions, but will adopt a non-neuronal cone cell fate if these instructions are disrupted by Boss INTERNALIZED INTO R7 R7 CELL COMMITS TO CONE CELL FATE IN THE ABSENCE OF Sev FUNCTION
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Sev EXPRESSION -'I-'D Sev EXPRESSION IN R7 ~ IN R3 & R4 R7 NUCLEUS RISES APICALLY
hours (at 25°C)
R7 NUCLEUS FALLS BASALL~'
FIGII Summary of the time-course of events involved in R7 cell recruitment. The time line is calibrated both in hours of developmental time at 25°C and in columns of ommatidial development. The developmental times given are approximate; the rate of development (columns per hour) varies slightly between strains and is not uniform, being slower in the posterior portions of the eye disk 16. The times at which cells begin neural differentiation (see Ref. 1) are indicated in the diagrams of assembling ommatidia shown above the time line, beginning with R8 at column 1 and adding R2 and R5 at column 3, R3 and R4 at column 6, R1 and R6 at column 8, and R7 (darker shading) at column 12. The times are indicated at which sev expression is first observed in R3, R4 and R7 (Ref. 10), and boss expression in R8 (Ref. 8), as well as the times at which the nucleus of the presumptive R7 cell begins its apical and basal migrations 1. Also shown are the 6 h period during which sev activity appears to be required 17 (shaded region), and the overlapping period during which boss internalization into R7 is observed ~. Finally, the time at which the presumptive R7 cell commits to an alternative developmental fate in the absence of sev activity~vis shown. "FIGNOVEMBER/DECEMBER1991 VOL.7 NO. 11/12 ©1~)1 Elsevier S c i e n c e Publishers Ltd (UK) (HOg - 9479/91/$02.00
[]~EVIEWS A
B
The 8 photoreceptors & 4 cone cells
C
sevenless (sev)
bride of sevenless (boss)
seven-up (svp)
seven in absentia (sina)
D
wild type
mutant for either sev, boss, or sina
ligand-independent Sev activity -or- ubiquitous BOSS expression
mutant for svp
FIG~
Summary of expression patterns and genetic data used to infer the roles of sevenless (sev), bride ofsevenless (boss), seven-up (svp) and seven in absentia (sina) during ommatidial development. (A) An ommatidium showing the cell contacts made by the R1-R8 cells (1-8) and the four cone cells (PC, EC, AC and PLC). (B) The expression patterns of sev, boss, svp and sina. Black, strong expression; gray, internmdiate expression; white, no detectable expression. (C) The cells in which the function of sev, boss, svp and sina is required, as determined by mosaic analysis. (D) Cells that adopt an R7-1ike cell fate in the indicated genotypes are shown by black shading. mutation of sev, boss or sina. A variety of genetic 6 and molecular s data indicate that boss encodes a cell surface ligand on the R8 cell that activates a sev-encoded transmembrane protein tyrosine kinase receptor9 on the surface of the R7 cell 5,1°. The s i n a gene encodes a nuclear protein required in the R7 cell v.
The ligand The product of the boss gene is a -100 kDa glycoprotein with seven putative membrane-spanning regions and a 498 amino acid extracellular domainS, H. The boss protein is expressed exclusively on the surface of the R8 cell 8 (see Fig. 2) and this expression in R8 is essential for R7 cell determination 6. Kr~imer et al. ~ demonstrated a direct interaction between the boss and sev proteins by showing that boss- and sevexpressing cell lines form heterotypic aggregates. Two lines of evidence suggest that this interaction also occurs in vivo. First, sev immunoreactivity is concentrated on the membranes of sev-expressing cells where they contact R8 (Ref. 10; Fig. 3A, B). Second, boss immunoreactivity is internalized into the R7 cell in a sev-dependent manner 8 (Fig. 3C). Taken together, these observations strongly support the hypothesis that boss functions as the ligand for the sev receptor.
The receptor The sevenless protein (sev) is synthesized as a 280 kDa glycoprotein precursor that is subsequently
cleaved into 220 kDa amino-terminal and 58 kDa carboxy-terminal subunits that remain associated by noncovalent interactions .2. The carhoxy-terminal subunit contains the transmembrane domain and a cytoplasmic
domain highly homologous to known tyrosine kinases9. The sev protein has been shown to have protein tyrosine kinase activity in vitro ~2 and this activity appears to be essential for sev function in vivo 13. Although sev activity is only required in the R7 cell 5, sev is expressed on the apical surface of many cells in the developing retina (Figs 1, 2) in a highly dynamic pattern 1° that is controlled at the level of transcription of the sev gene v'. Temperature-inducible hsp70-sev fusion genes 15,16 and temperature-sensitive sev mutations Ir have been used to show that sev activity is required only to initiate R7 cell development and not for its subsequent differentiation or function. However, the sev-mediated signal must be maintained for several hours to be effective 17 (Fig. 1).
The signaltransductionpathway Activation of the sev tyrosine kinase must result in intracellular changes in the presumptive R7 cell that cause it to adopt the R7 cell fate rather than the cone cell fate. The mechanisms by which receptor tyrosine kinases effect changes in cell physiology are still poorly understood. Biochemical studies with mammalian protein tyrosine kinase receptors (PTKs) have led to the identification of proteins that bind to or are phosphorylated by tyrosine kinases18,19. While some of these interactions suggest potential mechanisms of signal transmission, their role in vivo is still unclear. Two features make the sev pathway ideal for genetic approaches to understanding signaling by receptor tyrosine kinases. First, both the R7 cell and the sev protein are dispensable for viability and fertility. Second, the functioning of this signaling pathway can be inferred from the presence of the R7 cell in a
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FIGII
Electron micrographs illustrating sev and boss expression. (A, B) sev immunoreactivity at the early (A) and late (B) symmetrical cluster stage (see Ref. 10 for details). Note that the staining on the membranes of R3, R4 and R7 is clustered where they contact R8, and the sev immunoreactivity is seen in large vesicular structures in the cytoplasm (arrow in A). (C) boss immunoreactivity at the late symmetrical cluster stage (photograph courtesy of R. Cagan and S.L Zipursky; see Ref. 8 for details). Note the large vesicular structure containing boss in the cytoplasm of R7. The boss protein is synthesized only by the R8 cell; the staining in R7 is the result of sev-mediated internalization~. live, anesthetized fly. Screens for mutations that alter the strength of sev signaling can be used to identify other elements in the pathway. Rogge et al. 2° carried out a screen for dominant suppressors of sev and isolated a mutation in a gene they named Son o f sevenless (Sos). This gain-of-function mutation, SosJ2, does not bypass the requirement for sev function as it cannot suppress a sev null mutation. Rather, SosJ2 appears to increase the efficiency of transduction of a weak sev-mediated signal. Simon et al. 21 carried out a systematic genetic screen for mutations that decrease the effectiveness of signaling by the sevenless PTK, by looking for dominant enhancers of sev. By adjusting the temperature at which flies carrying a temperature-sensitive allele of sev were grown, Simon et al. could adjust sevenless PTK activity to a level barely above the threshold necessary for R7 cell formation. Small reductions in the abundance or activity of other elements of the pathway might then be expected to lower signal strength sufficiently to cause a sevenless phenotype in flies grown at this threshold temperature. This sensitivity allowed Simon et al. to identify genes encoding putative downstream elements of the pathway, by screening for genes in which inactivation of only one copy of the gene - which would be expected to reduce the level of gene product by half resulted in the absence of the R7 cell. Since the other copy of the gene remained functional, they were able to identify these loci even though their functions were essential for viability. Seven genes were identified in this screen; one of these corresponded to So~; in which several loss-of-function mutations were isolated. The products of four of the seven loci 21 also appear to be involved in transduction of signals from another PTK, the Drosophila homolog of the EGF receptor (DER), as judged by the ability of mutations in these loci to suppress Ellipse, a mutation that results in DER hyperactivity22. These results suggest that these four loci encode proteins that participate in the transduction of signals from at least two different PTKs.
One of the four loci corresponds to the R a s l gene, which encodes a p21 ras protein 2i. Several previous studies have suggested a role for Ras activity in PTK function. Injection of monoclonal antibodies that inhibit Ras activity blocked the ability of PTKs to induce mitosis, and a dominant-negative ras gene was able to interfere with oncogenic transformation by several tyrosine kinase-encoding oncogenes 23. Furthermore, recent studies of vulval development in the nematode Caenorbabditis elegans place the ras gene downstream of the PTK encoded by the let-23 gene (see article by Sternberg and Horvitz in this issue). Moreover, the product of the Sos locus shows sequence similarity to the Saccharomyces cerevisiae CDC25 protein 21, which activates yeast RAS by facilitating exchange of GTP for GDP. These results support a model in which the stimulation of Ras activity is a key element in signaling by PTKs, and suggest that this stimulation may be achieved by activating the exchange of GDP for GTP by Ras proteins. A model incorporating this hypothesis is shown in Fig. 4. While the sina gene product is required for R7 determination, its role within the presumptive R7 cell remains unclear. Modification of sina activity may be one end-point for the signal transduction pathway initiated by sevenless. Alternatively, sina may function in parallel to the sevenless pathway.
Specificity The ability of cells that develop in close proximity to adopt distinct fates implies a high level of precision in cell-cell signaling. In principle, two mechanisms could provide specificity in the sevenless signaling pathway. First, the distributions in time and space of the ligand (boss) and the receptor (sev) could be tightly controlled; in the extreme case they would be coincident only when appropriate to specify the R7 cell fate. Second, the developmental history of individual cells could limit their ability to receive or be influenced
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by the sevenless-mediated signal. In the extreme case of this mechanism, only the presumptive R7 cell would E R8 be capable of responding to a ubiquitous signal. Data from a number of studies have clearly established that a SEVENLESS combination of these mechanisms ..... operates to ensure that one, and only GDP GTP 7/ /! one, R7 cell is specified in each ommatidium. To test the first possible mechanism, the temporal and spatial distributions of sev and boss have been ACTIVE ".. ? altered by expressing tl{ese proteins ? from heat-inducible hsp70-sev (Refs 15, 16) or hsp70-boss (Ref. 24) fusion genes. The distribution of sev, R7 ? despite being highly dynamic in space and time 1°, does not appear to V play an important role in determining R7 Determination specificity. The sev protein needs to Pi be present in the R7 cell at the critical FIG~[~ time, but its ectopic presence in other Model for the sev-mediated signal transduction pathway. Data for the involvement cells does not obviously decrease the and structure of the indicated proteins can be found in the following references: specificity of R7 cell determination. boss, Refs 6, 8, 11, 24; sev, Refs 5, 9, 10, 12, 13; Sos, Refs 20, 21; Ras, Ref. 21; Rapl, In contrast, the restriction of boss Ref. 31. Note that it is not yet clear whether the amino-terminal region of the larger expression to the R8 cell does play a sev subunit spans the membrane; that region is shown in gray to indicate this major role. Ubiquitous expression of uncertainty, The involvement of phosphatases, GAP and effectors for Ras is based boss causes the cone cells to differen- on analogy to other signal transduction systems; these components have not yet tiate as R7 cells 24 (Fig. 2). Normally, been identified in the R7 cell. the cone cells express sev, but do not contact R8. Thus restricting boss expression to R8 can determined, leading to an early disruption of ommafully account for the cone cells not becoming R7 cells tidial assembly 27. More than one R7-1ike cell whose during normal development. However, the limited presence depends on sev are often observed 28. presentation of boss cannot account for the fact that Van Vactor el al. 24 have suggested that the inability the R1-R6 cells do not b e c o m e R7 cells. All these cells of the R1-R6 photoreceptors to respond to the sevencontact R8 and four of them normally express sev. less signal may be connected with boss binding or Expression of sev can be experimentally induced in the internalization. The sev-mediated internalization of other two, yet these cells do not become R7 cells, indi- boss, as observed in the R7 cell, does not occur in any cating that their developmental history has made them of the R1-R6 cells, even though R3 and R4 express s o m e h o w unreceptive to the sev-mediated signal. high levels of sev and contact R8 (Ref. 8). In contrast, Consistent with this view, a constitutively active, in N'P t (another mutation that disrupts normal ommaligand-independent form of the sevenless receptor 2s tidial assembly) and rough flies, ectopic internalization can cause the cone cells to differentiate as R7-1ike of boss is seen 24. Arguing against ligand internalization cells, but does not appear to be able to induce R7 being an essential step in the response to sev, howdevelopment in R1-R6 cells. ever, is the observation that a ligand-independent, If the developmental history of the R1-R6 cells activated sev can induce cone cells to differentiate as the inputs they have received and the resultant restric- R7-1ike cells 2s. Moreover, the apparent block in the tions that have been placed on their developmental sev-mediated pathway in R3 and R4 does not appear potentials - is responsible for their lack of response to to be simply at the level of ligand internalization, since sev, then mutations that disrupt R1-R6 cell fate detereven activated sev cannot induce R3 and R4 to mination might be expected to permit additional cells become R7-1ike cells zs. to adopt the R7 fate. This prediction was first shown to Alternatively, it is possible that reception of the be true for the seven-up mutation z6. The seven-up gene sev-mediated signal is not blocked in R3 and R4; encodes a steroid receptor superfamily m e m b e r that is rather, the sev-mediated signal may simply be redunexpressed and required in R1, R3, R4 and R6 (Ref. 26; dant in these cells. Suppose, for example, that actiFig. 2). In the absence of seven-up function, these four vation of Ras is required in each photoreceptor precurcells develop as R7-1ike cells. Surprisingly, only two of sor for it to adopt a neural fate, the type of neuron these cells appear to require sev function for their formed being determined by a Ras-independent pathdevelopment as R7-1ike cells; as discussed below, this way. The sev protein may indeed be capable of actiobservation implies that it is possible to bypass the vating Ras in R3 and R4, but by the time sev is activated requirement for the sev-mediated signal. The phein these cells, Ras activation, and neural development, n o m e n o n of supernumerary R7 cells is also seen in may already have been triggered by another signal. rough mutant flies, where R2 and R5 are incorrectly The lack of boss internalization in R3 and R4 might
INACTIVE~
I
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U~EVIEWS simply reflect downregulation of an endocytosis pathway in these cells as they begin their terminal differentiation. Information content
The role of the sev-mediated signal is to distinguish between two cell fates: R7 and cone cell. The developmental histories of the presumptive R7 cell and the cone cells may be very similar. For example, their transcriptional programs are related in that they both express high levels of sev and sin& Moreover, the cone cells, unlike the R1-R6 cells, can respond to the sev-mediated signal by differentiating as R7 cells. Thus, it may not be necessary to attribute much information content to the sev-mediated signal. Indeed, the instruction to become an R7 cell, as opposed to one of the R1-R6 cells, may not be conveyed by sev at all, but may have already been established by the developmental history of the R7 cell. Thus, in the extreme view, activation of sev may simply tip the balance in favor of neuronal development rather than nonneuronal development. Three observations argue that such an extreme view is tenable. First, ectopic expression of rough in a developing R7 cell can cause it to differentiate as an R1-R6-1ike cell; however, the decision to become a neuron still requires sev function 29,30. Thus it is possible to uncouple the sevmediated decision of whether to become a neuron from the choice of what type of neuron to become. Second, several genes have been identified in screens for enhancers 21 and suppressors 20 of sev; these genes encode products that are likely to function in the transduction of signals initiated by sev. However, these genes are not dedicated to the sevenless pathway; they can function in conjunction with other PTKs and they are required for the development of other photoreceptors that do not depend on sev. It seems unlikely that such a general signal could, by itself, convey photoreceptor subtype identity. Third, in certain unusual circumstances, such as in svp- sev- double mutants, it is possible for R7-1ike cells to differentiate without sev function. This would be more difficult to explain if sev were required to specify R7 cell type identity. In the simplest model, two decisions would be required to determine the R7 cell fate. The first decision would establish a bipotential cell that could become either an R7 cell or a cone cell. The acquisition of this fate rather than that of R1-R6 could be as simple as the expression of a single additional transcription factor, since we know that the ectopic expression of rough in the presumptive R7 cell achieves the opposite effect, that is, directs it towards a fate resembling R1-R6. Then the second decision, directed by the sevenless signal, need simply steer this bipotential cell towards the R7 fate.
Other inputs The pathway proposed for the sev-mediated signal (Fig. 4) suggests many steps at which additional inputs could be integrated with the sev-initiated signal. Some, like the tyrosine phosphatase and Ras GTPaseactivating protein, have not yet been identified. Interestingly, the Roughened mutation, which appears
to affect R7 formation, has been shown to be a dominant gain-of-function mutation in a homolog of the human rapl/Krev gene31. The mammalian Rap proteins are small GTPases that have opposing effects to Ras in cell transformation32. Roughened flies lack R7 cells in most ommatidia, consistent with the notion that Drosophila Rasl and Rap1 gene products have opposing effects in R7 cell determination 31, perhaps by competing for a common effector (Fig. 4).
Concludingremarks Over the past few years, studies of R7 cell determination in the developing Drosophila eye have taught us much about the molecular mechanisms of induction and signal transduction by protein tyrosine kinase receptors. The experimental advantages of being able to examine mutant phenotypes at single cell resolution in a developing tissue that is dispensable for viability and fertility ensure that new insight will continue to emerge from studies of the sev-mediated signal transduction pathway. In particular, the utility of genetic screens for enhancers and suppressors of sevenless to identify components of the pathway has been established and should encourage similar endeavors starting from other points in the pathway. This approach could be used to identify some of the most elusive players in Ras-mediated signal transduction: the effector molecules through which Ras acts to alter cell function. Finally, the identification of Ras as an important component in both the sev and EGF receptor pathways in Drosophila suggests that signal transduction pathways acting downstream of protein tyrosine kinase receptors may be shared by many different kinases and may be highly conserved in organisms as diverse as flies, worms and humans.
Acknowledgements I am grateful to Mike Simon and Iswar Hariharan; many of the ideas expressed in this review were developed and clarified in conversations with them. I thank S.L. Zipursky and his co-workers for communicating their results before publication.
References I 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Tomlinson, A. (1988) Development 104, 183-189 Ready, DF. (1989) Trends Neurosci. 12, 102-110 Rubin, G.M. (1989) Cel157, 519-520 Banerjee, U. and Zipursky, S.L. (1990) Neuron 4, 177-187 Tomlinson, A. and Ready, D.F. (1987) Dev. Biol. 123, 264-275 Reinke, R. and Zipursky, S.L. (1988) Cell 55, 321-330 Carthew, R.W. and Rubin, G.M. (1990) Cell 63, 561-577 Kr~imer,H., Cagan, R.L. and Zipursky, S.L. (1991) Nature 352, 207-212 Hafen, E., Basler, K., Edstroem, J-E. and Rubin, G.M. (1987) Science 236, 55-63 Tomlinson, A., Bowtell, D.D.L., Hafen, E. and Rubin, G.M. (1987) Cell 51,143-150 Hart, A.C. et al. (1990) Genes Dev. 4, 1835-1847 Simon, M.A., Bowtell, D.D.L. and Rubin, G.M. (1989) Proc. Natl Acad. Sci. USA 86, 8333-8337 Basler, K. and Hafen, E. (1988) Cell 54, 299-311 Bowtell, D.D.L., Kimmel, B.E., Simon, M,A. and Rubin, G.M. (1989) Proc. Natl Acad. Scl. USA 86, 6245--6249 Bowteli, D.D.L., Simon, M.A. and Rubin, G.M. (1989) Cell 56, 931-936
11G NOVEMBER/DECEMBER1991 VOt. 7 NO. 11/12 ~,-(~
[]~EVIEWS 16 Basler, K. and Hafen, E. (1989) Development 107, 723-731 17 Mullins, M.C. and Rubin, G.M. Proc. Natl Acad. Sci. USA
27 Tomlinson, A., Kimmel, B.E. and Rubin, G.M. (1988) Cell
(in press) Cantley, L.C. et al. (1991) Cell 64, 281-302 Koch, C.A. etal. (1991) Science 252, 668-674 Rogge, R.D., Karlovich, C.A. and Banerjee, U. (1991) Cell 64, 39-48 Simon, M.A., Bowtell, D.D.L., Dodson, G.S., Laverty, T.R. and Rubin, G.M. Cell (in press) Baker, N.E. and Rubin, G.M. (1989) Nature 340, 150-153 Fieg, L.A. and Cooper, G.M. (1988) Mol. Cell. BioL 8, 3235-3243 Van Vactor, D.L., Jr, Cagan, R.L., Kr~imer, H. and Zipursky, S.L. Cell (in press) Basler, K, Christen, Bt and Hafen, E. (1991) Cell64. 1-20 Mlodzik, M. et al. (1990) Cell60, 211-224
28 Heberlein, U., Mlodzik, M. and Rubin, G.M. (1991) Development 112, 703-712 29 Kimmel, B.E., Heberlein, U. and Rubin, G.M. (1990) GenesDev. 4, 712-727 30 Basler, K., Yen, D., Tomlinson, A. and Hafen, E. (1990) Genes Dev. 4, 728-739 31 Hariharan, I., Carthew, R. and Rubin, G.M. Cell (in press) 32 Kitayama, H. et al. (1989) Cell 56, 77-84
18 19 20 21 22 23 24 25 26
A l l metazoan cells, whether in culture or in the organism, must be able to make decisions about cell division or cellular differentiation based, in part, on environmental cues. Accordingly, cells express receptor systems, either intracellular or cell surface, that allow them to detect the presence of hormones, growth factors and other signalling molecules that manipulate the regulatory processes of the cell. Such molecules will influence the decision to proliferate or differentiate by stimulating the activation of a cascade of events that ultimately impinges on the genetic apparatus. In turn, this leads to altered transcription of a defined set of genes 1,2. But what happens when a cell is exposed to both differentiation and proliferation factors? Are both networks independently activated, or is there a cellular mechanism that integrates these two signal transduction pathways to avoid their antagonistic effects? This review describes recent evidence for the presence of an intracellular 'checks and balance system' that allows these distinct regulatory pathways to modulate each other's activities. In one branch of the signalling pathway, steroid, retinoid and related hormones typically function as differentiation factors that alter cell fate and function during embryonic development and regulate organ physiology in the adult. These hormones exert their effects by binding to intracellular receptors that function as hormone-dependent transcription factors. All intracellular receptors are structurally related, forming a superfamily of regulatory proteins that have broadly conserved functional domains for transcriptional activation, nuclear localization, DNA binding and hormone binding 1. Ligand binding modifies the receptor so that it can interact with high-affinity sites in target DNA, termed hormone response elements (reviewed in Refs 1-4). While this is a common view for the action of steroid hormones, recent evidence reveals a novel pathway of regulation that does not involve binding to target DNA5-v. The second signalling pathway involves cell surface receptors that must transmit an external signal via the production of intracellular second messengers including small molecules like cyclic AMP, Ca 2÷ or diacylglycerol. In turn, these second messengers serve as allosteric triggers that activate intracellular protein TIG N O ' ~ M B E R / D E C E M B E R ~"1991 Elsevier Sc'ien~.t' Publislters Ltd (1 K) Ol(xg 94"Tg/91,$O2,(RI
55, 771-784
G.M. RUBIN IS IN THE HOWARD HUGHES MEDICAL INSTITUTE AND DEPARTMENT OF MOLECULAR AND CELL BIOLOGY, UNIVERSITY OF CALIFORNIA AT BERKELEY, BERKELEY,
CA 94720, USA.
Cr0ss-c0upling of signal transducti0n pathways: zinc finger meets leucine zipper ROLAND SCHOI~ AND RONALD M. EVANS
Cellproliferation and cellular differentiation are often thought of as opposing phenomena. The molecular mechanisms by which steroid, retinoid and thyroid hormones inhibit cellular proliferation and by which growth factors stimulate this process are poor~F understood We discuss recent evidence suggesting that these two signal transduction pathways converge through a process referred to as 'cross-coupling', which involves a possible interaction between steroid hormone receptors and the oJun oncoproteitt kinase systems, which continue to propagate the signal. In some cases, the cell surface receptor is itself a protein kinase that becomes activated via the binding of the external ligand (reviewed in Refs 8, 9). Of particular interest to this review are the converging lines of evidence indicating that the Jun and Fos protooncogene products are part of a signalling pathway that transmits stimuli from the cell surface to the nucleus. It has been shown that Jun and Fos directly regulate gene expression in response to stimulation of growth factor receptorsl°,lL Both Jun and Fos are members of a class of nuclear proteins that has been widely implicated in diverse aspects of cell growth and differentiation. Mutant forms of both Jun and Fos are oncogenic, causing formation of foci in vitro and tumors in vivo. Jun was originally defined as interacting with the control regions of genes containing promoter elements responsive to the phorbol ester TPA (12-Otetradecanoylphorbol-13-acetate). The binding sequence is referred to as an AP-1 site, which is recognized by c-Jun homodimers as well as by c-Jun--c-Fos heterodimers. Homodimer and heterodimer formation is mediated through noncovalent interactions facilitated 1991 vot. 7 NO. 11/12
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