Roles of receptor tyrosine kinases in Drosophila development.

Roles

of receptor

.tyrosine:

in Drosophila

kinases

development

BEN-ZION SHILO Department

of Molecular

Genetics and Vnlog

Wezzmann Institute

of Science,

RTK

2915-2922;

Receptor tyrosine kinases represent a continuously growing family of proteins that are structurally and functionally related (1). The hallmarks of this family are the extremely conserved cytoplasmic kinase domain that carries out the enzymatic activity, the transmembrane domain, and a more divergent extracellular ligand-binding domain. Within the family of RTKs, small subfamilies can be identified in vertebrates, each consisting of two-five members. The basis for this classification are motifs common to each subfamily, such as the structure of the kinase domain (split or continuous), or common structural features of the extracellular domain. In some cases the latter is also reflected by the ability of different receptors to recognize an overlapping set of ligands. The similarity in overall structure within the RTK family results in a common mechanism for the transduction of signals into the cell (2). Biochemical, molecular, and genetic data point to the following mechanism: binding of ligand to free receptors on the cell surface generates a conformational change in the extracellular region, which leads to an increased affinity for the association between receptors. Dimerization of the extracellular domains leads to the juxtaposition of the cytoplasmic kinase domains. The cytoplasmic domains contain sites for tyrosine phosphorylation, and these sites are trans-phosphorylated by the RTK in the course of dimerization. The mechanism by which the close association between the cytoplasmic domains can lead to activation of the kinase remained elusive for several years. Recently, a unifying hypothesis has been presented (3). Many substrates for RTKs contain conserved motifs, termed SH2 domains. The function of these domains is to recognize and associate with phosphotyrosine residues (4). The transphosphorylation process therefore generates sites on the receptor that can be recognized by the substrates using their SH2 domains. Once the substrates bind the receptor they are phosphorylated by it, and thus the respective cellular pathways are triggered. It appears that multiple pathways are activated by the same receptor. For example, the platelet-derived growth factor (PDGF) receptor was shown to associate with the GAP, PLOy, and PT kinase proteins, all containing SH2 domains, as well as with the c-raf serine/threonine kinase (3). The striking similarities in the structure and signal transduction mechanism of RTKs raise a fundamental question as to the specificity of the signal transmitted by each receptor. As the phosphotyrosine recognition specificity of cellular

1992.

Key Words: Drosophila #{149} cell-cell communication

development

receptor

tymsune

kinoses

BETWEEN CELLS DURING development is crucial for determination of cell fate. In most cases the cells are multipotent, and must probe their environment to receive cues directing the developmental decisions they have to make. Cell surface receptors are essential elements in these processes, as they represent the components used by the cell to receive information from its immediate or more distant environment. Once a receptor is identified, it can serve as a starting point to isolate other elements in the signal transduction pathway operating upstream or downstream to it. Genetic screens in Drosophila have uncovered loci participating in a wide variety of fascinating developmental processes. When components of these pathways were cloned, it was rewarding to find that for several different pathways, receptor tyrosine kinases could be identified as pivotal elements. In Drosophila, the reverse approach can also be taken. Receptor tyrosine kinases (RTKs)1 can be isolated on the basis of their homology to known receptors in vertebrates, and mutations in the loci encoding them may be identified. In cases where RTKs are regulating developmental decisions of cells in organs for which there is no convenient genetic screen, this approach can provide the initial genetic definition of elements controlling the development of these organs. COMMUNICATION

0892-6638/92/0006-2915/$01

.50. © FASEB

AND

76100, Israel

ABSTRACT

Communication between cells is a fundamental component of development and morphogenesis. Identification of the molecules mediating cell-cell communication is crucial for elucidation of the molecular basis of these processes. Receptor tyrosine kinases (RTKs) appear to play a central role in this context by transmitting into cells information dictating their fate. The functions of RTKs in Drosophila are extremely diverse, and include maternal determination of embryonic polarity (torso and torpedo), determination of neuroblast identity (faint little ball), and guidance of tracheal celi migration in the embryo (breathless). During compound eye development, RTKs affect the number of photoreceptor clusters (Ellipse) and the determination of photoreceptor R7 identity (seveniess). The phenotypes of mutations in RTK loci serve as a starting point for understanding processes dictating cell identity at the level of the whole organism. Recently, they have also begun to provide a basis for selection of second-site suppressor mutations, encoding additional elements in their signal transduction pathway. Common themes between the functions, regulation, and signal transduction pathways of Drosophila RTKs are drawn. Shilo, B-Z. Roles of receptor tyrosine kinases in Drosophila development. FASEB J. 6:

STRUCTURE

Rehovot

SIGNAL

TRANSDUCTION

‘Abbreviations: CNS, central nervous system; DER, Drosophila EGF receptor homolog; EGF, epidermal growth factor; FGF, fibroblast growth factor; GAP, GTPase-activating protein; MG, midline glial; PDGF, platelet-derived growth factor; P1, phosphatidylinositol; PLOy, phospholipase C’y, RTK, receptor tyrosine kinase; SH2, src homology 2; RTKs, receptor tyrosine kinases.

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ww.fasebj.org by Goteborgs Univ Biomedicinska Biblioteket (130.241.16.16) on November 30, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumb

proteins containing SH2 domains is broad, how does the cell distinguish between the activation of each of the respective RTKs? The distinct cytoplasmic identity or fingerprint of the signal relayed by each of the RTKs may thus be determined by the spectrum of pathways it activates within the cell and by the relative level of activity of each pathway. In addition, each pathway may also use unique downstream elements.

STRUCTURE

OF DROSOPHILA

RTKS

Receptor tyrosine kinases in Drosophila were identified by two strategies. Isolation of several genes, which were studied because of the interesting phenotypes of mutations in their loci, has demonstrated that they encode RTKs. In parallel, the high structural conservation of RTKs was used to isolate the Drosophila counterparts by using cloned vertebrate RTK probes. Several Drosophila RTKs have been described and

sevenless

DER DFGF-R1

(breathless)

torso

100 aa

Figure 1. The structure of Drosophila RTKs. The structure of Drosophila RTKs is schematically illustrated. The cytoplasmic tyrosine kinase domain is drawn as wide black boxes. Transmembrane domains and signal peptides are shown by narrow, small black boxes. For DER (also termed torpedo, faint little ball, and Ellipse), note the presence of two different NH2 termini resulting from alternative splicing (7). The hatched bars at the extracellular domain denote cysteine-rich motifs. For DFGF-R1 (breathless), note the presence of five immunoglobulin-like domains at the extracellular region (drawn as loops). The open box represents a stretch of acidic residues which is also typical to the FGF-receptor class in vertebrates. The sevenless protein is synthesized as a single precursor and is processed to two associated polypeptides (14). The hydrophobic signal peptide, which is found 100 amino acids after the initiator methionine, appears to serve as the transmembrane domain of the NH2-terminal subunit.

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shown to be highly conserved (5-11). The structure of the RTKs that will be discussed in this review is shown in Fig. 1. Several conclusions can be drawn from their sequence. The tyrosine kinase domain is always the most conserved region (showing more than 50% identity to the vertebrate counterpart). The extracellular domains of DER and DFGFRi also show a lower but significant conservation. Each Drosophila RTK appears to represent a different subfamily of receptors in vertebrates, and displays the highest degree of similarity in overall structure and in sequence to that subfamily rather than to the other Drosophila RTKs. This finding indicates that the major classes of RTKs have been generated by gene duplication events that preceded the divergence of chordates and arthropods. Each RTK in Drosophila appears to represent the only member of its subfamily (the exception to this rule being the isolation of a second Drosophila member of the FGF receptor subfamily (M. Zehavi, L. Glazer and B-Z. Shilo, unpublished results). Thus, genetic dissection of the role of a given RTK in Drosophila is without complications resulting from functional redundancy. The tyrosine kinase activity predicted from the structure of Drosophila RTKs was indeed confirmed for the EGF (12, 13) and insulin (11) receptor homologs, as well as for sevenless (14). This function is crucial, as RTK mutants in which the kinase is inactive show no biological function (12, 15). The concept of dimerization as an essential step in signal transduction was also illustrated genetically in Drosophila by the ability of specific combinations of two mutant alleles in the EGF receptor homolog (DER), each defective in a different region of the cytoplasmic domain, to complement each other (16). Two types of RTKs can be identified in Drosophila: receptors that have multiple roles and are expressed in a broad range of tissues, such as DER (17, 18), and receptors that are found at a single phase and in a single organ, such as sevenless (19, 20). Genetic dissection provides a more direct approach to studying the function of RTKs, by analysis of the phenotypes resulting from mutations in their loci. The review will be restricted to those RTKs in which the function has been studied genetically. They will be discussed in the order in which they participate in the life cycle of the fly, starting at oogenesis and continuing through embryogenesis and development of the compound eye.

MATERNAL POLARITY

DETERMINATION

OF

EMBRYONIC

The initial cues for the formation of the embryonic structures are provided by maternal information during oocyte development in the ovary (21). In the case of the anteriorposterior axis, the information is provided by maternal transcripts supplied to the developing oocyte by the nurse cells. These transcripts become localized to the anterior [bicoid (22)1 or posterior [nanos (23)] poles of the embryo. A different mechanism in used to provide information for the terminal embryonic structures and the dorsoventral axis. The developing egg chamber is composed of two tissues that have a different embryonic origin. The nurse cells and oocyte are germ line cells, derived from the pole cells of the embryo. They are surrounded by approximately 1000 follicle cells that come from a somatic, mesodermal origin (24). The maternal cues for the terminal and dorsoventral structures are generated by an intimate communication between the follicle cells and the oocyte. These interactions are mediated by two RTKs, torso and torpedo, respectively.

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torso The maternal pathway for generating the terminal structures was defined by a collection of female sterile mutations that give rise to embryos lacking the terminal structures (acron and telson) but retain all other structures (25-29). Experi-

ments with genetic mosaics and transplantation

A

of pole cells

whether the normal function of a given mutation in the somatic follicle cells or in the oocyte. Douexperiments established an epistasis relationship, and showed that the loci required in the follicle cells function in the pathway prior to the loci that are required in the oocyte (29). Thus, the directionality of the signal for the induction of the terminal structures was identified: it is initiated in the follicle cells and transmitted into the oocyte. The torso gene (which is required in the oocyte) was cloned and shown to encode an RTK with a split kinase domain similar to the PDGF receptor class (5). The transmembrane receptor structure of torso suggested that it represents the molecule that receives the cues from the follicle cells, torso transcripts are uniformly distributed in the syncytial blastoderm embryo (5, 30). The repertoire of torso mutants indetermined is required ble mutant

cludes both loss of function

alleles defective

torpedo There are many similarities between the establishment of the terminal pattern and the dorsoventral axis of the embryo. A group of maternal mutations was shown to give rise to yen-

KINASES IN DROSOPHILA

perivitelline space putative

ligand

torso

in tyrosine

kinase activity as well as dominant gain of function alleles which are likely to lead to constitutive receptor dimerization. The ability of the dominant torso alleles to induce development of terminal structures in the central portion of the embryo (31, 32) suggested that the postreceptor elements are not spatially restricted. Therefore, during normal development, torso may be specifically activated only in the terminal regions of the embryo due to a spatial restriction of the ligand that triggers it. Mosaic experiments suggest that the torso-like mutation required in the follicle cells may represent this spatially restricted ligand (29). It appears to be required and is presumably expressed only by the follicle cells at the two terminal regions. Analysis of the phenotypes resulting from injection of RNAs encoded by torso mutant alleles into different positions in the embryo, and into embryos of torso mutant backgrounds, has suggested the following model (21; F. Sprenger and C. N#{252}sslein-Volhard, personal communication): maternal torso RNA is translated in the embryo during the initial stages of embryogenesis, resulting in the accumulation of significant amounts of torso protein in the membrane. Until that stage, the ligand that has been secreted by the terminal follicle cells at the final stages of oogenesis remains tethered to the vitelline membrane (a rigid membrane that covers the plasma membrane of the oocyte). The ligand is then released from the vitelline membrane, and begins to diffuse in the perivitelline space. However, because the torso receptors are highly abundant, the ligand is trapped and internalized by the receptors found at the terminal region of the embryo, leading to activation of the torso kinase only in those regions. Such a mechanism may result in a graded activation of torso, as receptors found just beyond the terminal region are expected to encounter less ligand than the receptors at the termini. Indeed, it was shown that the type of terminal patterns formed depends on the level of activation of torso, where the highest level of activation gives rise to the development of the most terminal structures (30). The model is presented in Fig. 2A, Fig. 2B.

RECEPTOR TYROSINE

torso/ike

chorion vitellirie coat Figure 2. A model for the transmission of the information on the position embryonic terminal structures through torso. A) During oogenesis, a restricted population of follicle cells at the anterior and posterior termini of the oocyte are responsible for synthesis of the torso ligand (torso-like?), and its deposition in the vitelline membrane. In parallel, the maternal torso transcript is synthesized by the nurse cells, and transferred to the oocyte. B) At the syncytial blastoderm stage of embryogenesis, the torso protein has been synthesized from maternal torso mRNA and incorporated into the membrane. The putative ligand that was tethered to the vitelline membrane during oogenesis, at the time it was deposited by the follicle cells, is released. The free ligand will be trapped by the receptors at the terminal regions of the embryo before it can diffuse further in the perivitelline space. Activation of the tyrosine kinase activity of torso at the terminal regions will induce the formation of embryonic terminal structures.

tralized embryos in ventralized egg shells (33). This phenotype suggested that the polarities of the follicle cells and the embryo are intimately associated. Several ventralizing mutations (gurlosn and cornichon) were found to be required in the oocyte, whereas a single locus (torpedo) appeared to be required in the follicle cells (33). torpedo was shown to be encoded by the EGF receptor homolog (DER) (12, 34). The requirement for the function of torpedo by the follicle cells suggested that the signal for dorsoventral polarity is initiated in the oocyte and received by the dorsal follicle cells through the EGF receptor. DER is expressed by all follicle cells (R. Schweitzer, N. B. Zak and B. -Z. Shio, unpublished results). Downstream elements also appear to be ubiquitous, because in the proper genetic background all follicle cells can become dorsalized (35). Thus the basis for the restricted spatial activation of DER appears to lie in the localization of its yet unidentified ligand. Activation of DER at the dorsal region may trigger the follicle cells to become dorsalized (35). In the absence of DER activation, the follicle cells follow the default pathway and become ventralized. Figure 3 shows a scheme of the model. The ventral follicle cells subsequently transmit

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nurse cells

oocyte

DER (torpedo)

follicle cells (dorsal)

putative ligand follicle cells (ventral)

Figure 3. A model for the involvement of DER (torpedo) in the establishment of dorsoventral polarity in the follicle cells. DER is expressed by all follicle cells and displayed on their membranes. The putative ligand (gurken, cornichon?) is synthesized by the oocyte (or nurse cells), and becomes more concentrated at the dorsal side of the oocyte. Activation of DER (expressed on the surface of the dorsal follicle cells) by the ligand induces their dorsal fate. In contrast, the ventral follicle cells in which DER has not been activated follow the default pathway and become ventralized.

a signal back into the embryo (via the dorsal pathway), thus providing the cues to form the ventral and mesodermal embryonic structures. As the signal is transmitted from the oocyte to the follicle cells expressing DER, it may be refined. The oocyte is a single giant cell. The initial cues for dorsoventral polarity that are established in it [perhaps by the asymmetric localization of the nucleus (36)] must be very coarse. Transmission of this crude information to the layer of 1000 follicle cells may refine the signal, as each follicle cell may encounter a different level of DER activation, depending on its dorsoventral position. The level of DER activation could thus lead to a gradation of dorsal fates.

ZYGOTIC

EMBRYONIC

faint

ball

little

FUNCTIONS

The torpedo allele discussed results from a subtle reduction of the normal activity in the DER locus. More severe defects in the gene lead to embryonic lethality. The embryonic phenotype of null or severe alleles of the DER locus was termedfain#{128}little ball (fib), and for a good reason (12, 37, 38). The cuticle of mutant embryos has a rounded shape due to failure of the germ band to retract and the absence of head structures. The cuticle is also missing the ventral denticle belts. Other characteristics of the phenotype include severe collapse of the central nervous system (CNS), discontinuities of the longitudinal axon tracts, and fusion of commissures (12). The description of the fib phenotype raises a new set of questions. Both the head and CNS structures appear to develop normally and collapse only at a later stage of embryogenesis. Is the receptor involved in actual determination of cell fate in the affected tissues, or is it required later in em-

bryogenesis for survival and Dissection of the embryonic the protein is expressed in stages of embryogenesis

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maintenance of these tissues? role of DER is complicated, as multiple tissues and in many (18). The availability of a

temperature-sensitive allele has allowed dissection of the complex embryonic fib phenotype and determination of the temporal requirements for DER activity (39). These experiments show that although the disintegration of tissues in fib embryos occurs late in embryogenesis, the actual function of the receptor is required very early in embryonic development. For example, the collapse of the CNS can be prevented by providing the activity of DER early, when the neuroblasts are delaminating from the neuroectoderm. DER is expressed in the ectoderm but not in the neuroblasts or the neuronal cells. Thus, cell-cell interactions in the ectoderm that are mediated by DER are crucial for neuroblasts to achieve their final identity. The temperature-sensitivefib allele also allowed to identify the late embryonic functions of DER (39). At these stages, DER is specifically expressed in the CNS in three pairs of glial cells in each segment (midline glial, MG cells). In the absence of DER activity, these cells either fail to differentiate or die. Normally the MG cells migrate between the commissures of each segment and physically separate them (40). In the absence of the MG cells, the commissures remain fused. breathless In contrast to DER, which is expressed in multiple tissues and gives rise to a complex phenotype, the homolog of the FGF receptor is restricted in its embryonic pattern of expression and in its function. DFGF-Ri is expressed only in two embryonic tissues: the developing tracheal system and the ventral midline cells, which will form the glial and neural cells of the CNS midline (8). The tracheal system develops in two phases. First, the division of epithelial cells in the tracheal placodes generates 100 cells per tracheal pit on both sides of each segment. In the second phase, no cell division occurs, and the entire tracheal tree is generated from about 2000 cells that appear identical to each other and that follow an intricate pattern of cell migration and extension (41). The major extension of tracheal processes takes place rapidly, within several hours. This migration phase is in many respects analogous to axonal outgrowth, displaying a strikingly extensive and precise pattern of extension. It may be simpler to understand, however, because only a single cell type participates in these dramatic events. Mutations in the locus show no defects in the cell divisions that generate the tracheal pits. However, the process of tracheal migration and extension is completely blocked, and the cells remain in the tracheal pits (8; C. Kl#{228}mbt,L. Glazer and B-Z. Shio, unpublished results). The mutation was thus termed breathless. Based on the structure and phenotype of breathless, it is tempting to suggest that the guidance for migration of the tracheal cells is presented by neighboring cells or the extracellular matrix and transmitted into the tracheal cells through DFGF-R1. However, proof for this model depends on the ability to show that the yet unidentified ligand of DFGF-Ri is displayed on the surface of ectodermal or mesodermal cells in a prepattern that precedes the migration of tracheal cells.

DEVELOPMENT

OF

THE

COMPOUND

EYE

Morphogenesis of the compound eye in Drosophila from a uniform epithelium of cells in the eye imaginal disc of the third instar larva represents one of the most striking examples for the importance of cell-cell interactions in morphogenesis (42, 43). Because there are no lineage constraints on

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cells participating in this process, the different fates of the cells must be dictated exclusively by cell-cell interactions. Two RTKs were shown to play a role in eye development: Ellipse (DER) participates in the initial phase in which the number and spacing of photoreceptor clusters is determined, whereas sevenless is crucial for the differentiation of the last photoreceptor cell, termed R7.

Ellipse During the third instar larva, the eye disc is transformed from a nondifferentiated epithelium to the ordered array of photoreceptor clusters, each containing eight photoreceptor cells and the cone cells. The differentiation is initiated in the morphogenetic furrow, which progresses from the posterior end to the anterior end of the disc. At the furrow, photoreceptor clusters can first be identified. Very little is known about this initial phase of differentiation. The position of the preclusters does not appear to follow an existing prepattern. Expression of genes like scabarous demonstrates that the spacing between clusters is actually generated slightly anterior to the furrow (44). An appealing mechanism for the generation of an ordered spacing from the undifferentiated epithelium is that differentiation begins stochastically. The differentiated founder cells inhibit their neighbors from assuming a similar fate. Thus, an ordered spacing is achieved. The phenotype of the Ellipse mutation, which is dominant, suggested that it may be participate in generating the correct spacing between clusters. In homozygous Ellipse flies, the ommatidia in the eye are dramatically reduced to about 1/10th the normal number, but the ommatidia that do form appear normal. The Ellipse mutation was shown to represent a dominant allele in the locus of DER, which was discussed previously in the context of oocyte polarity and embryonic development (45). One possibility is that DER is responsible for transmitting such inhibitory signals to the nondifferentiated neighboring cells. The Ellipse protein, which represents a hyperactive version of DER, may transmit these signals without any stimulation, thus resulting in fewer differentiated ommatidia. However, in the absence of knowledge about the ligand of DER in the eye, it is hard to provide any support for the model. Moreover, as the nature and the basis for the hyperactivity of Ellipse are unknown, it is difficult to extrapolate the phenotype to the normal role of DER in eye development. A complementary approach may be to generate mosaic clones of cells in the eye disc that are homozygous for loss-of function alleles of DER, and follow the differentiation pattern within these clones. sevenless After the number of photoreceptor preclusters is established, cells join the cluster and assume the correct neuronal identity based on the differentiated cells they come in contact with. Differentiation of the last photoreceptor cell, R7, is intimately associated with another member of the RTK family, sevenless (9, 46, 47). In the absence of a functional sevenless protein, R7 fails to differentiate and assumes instead the identity of a cone cell. The transmembrane receptor structure of sevenless suggested that it is responsible for receiving the inductive signals for R7 differentiation. This notion was formally proved by genetic mosaic experiments showing that the function of sevenless is indeed cell autonomous, i.e., required in the same cell in which the phenotype is observed (46). Is the sevenless signal an essential prerequisite for R7 differentiation or is it sufficient to trigger the process? Generation of constitutive, ligand independent sevenless constructs

RECEPTOR TYROSINE


has demonstrated that these proteins can induce the differentiation of multiple R7 cells by recruiting a population of cells that is normally destined to produce the cone cells (48). This result ascertains that the signal transmitted by the sevenless kinase is sufficient to trigger R7 differentiation. The experiment suggested that normally the receptor is not triggered in the cells that will become the cone cells, thus allowing only a single R7 cell to be formed in each cluster. The spatial regulation of sevenless activity does not result from a restricted pattern of expression of the sevenless protein, as it was shown to be expressed in several cell types (19, 20). Furthermore, ectopic expression of the receptor in all cells did not give rise to an aberrant phenotype (49, 50). The specificity must therefore reside in a restricted presentation of the sevenless ligand to the nondifferentiated cells. Genetic and biochemical approaches have identified the boss protein as the sevenless ligand (51-53). This is the first ligand that was identified for RTKs in Drosophila. Some of its properties may provide a paradigm for the ligands of the other RTKs. boss is expressed only in R8 cells. The protein has seven transmembrane domains and a long NH2-terminal extracellular region, which is likely to associate with sevenless. The transmembrane structure of the ligand and its restricted pattern of expression demonstrate that it triggers sevenless by local cell-cell interactions. Table 1 summarizes the roles of RTKs in Drosophila development.

ELEMENTS PATHWAYS

IN THE OF RTKS

SIGNAL

TRANSDUCTION

Identification of mutant phenotypes for Drosophila RTKs provides an opportunity to use powerful genetic screens to identify the elements in their pathways of signal transduction. This may be achieved in several ways. Simplistically, if we regard the pathway of each RTK as a linear cascade of events, then we would expect mutations in other stages of the pathways to give rise to a similar phenotype. This methodology did not prove successful for isolation of mutations with a similar phenotype to fib. However, it identified the boss locus (51) as well as loci participating in different steps of the torso pathway (25-28). It was rewarding to find that (l)polehole, a downstream element of torso, represents the Drosophila homolog of the c-raf kinase (28, 54). The raf protein was shown to associate with the vertebrate PDGF receptor (3). A more sensitive screen for interacting elements proved to be highly informative when used in dissecting the pathways of RTKs (55). The basis for the screen was to use a genetic background of a temperature-sensitive sevenless mutation and

select for second-site suppressor mutations that would make the phenotype more severe. This was achieved by screening at a temperature in which the activity of sevenless is barely above the necessary threshold. The prediction was that by mutating one of the two alleles encoding an element in the signal transduction pathway, the level of the signal would be reduced below the threshold. Seven suppressor loci were identified by this screen. Further characterization of these mutations has demonstrated that the simplistic view of the signal transduction pathway as a linear cascade should be modified. In contrast to the sevenless phenotype, which is not lethal, the homozygous phenotype of the suppressors was lethal. Therefore, although the mutants were selected on the basis of their interaction with sevenless in the eye disc, they may have broader developmental roles. A second observation was that four of

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TABLE

1. The roles of receptor tyrosine kinases in Drosophila development

Drosophila RTK

Function

torso DER torpedo faint little ball

Ellipse DFGF-R sevenless

Induction of embryonic terminal structures Establishment of dorsoventral polarity in the follicle cells Differentiation of neuronal cells and midline glial cells in the embryo Germ band retraction of the embryo Differentiation of embryonic ventral ectodermal cells Attachment of somatic embryonic muscles Imaginal disc development Participates in establishing the number and spacing of photoreceptor clusters Essential for migration and extension of embryonic tracheal cells Induces differentiation of photoreceptor cell R7 in the eye imaginal disc

1 (breathless)

these loci modified not only the phenotype of sevenless, but also the eye phenotype of another RTK, Ellipse (55, 56). This finding has profound implications on the signal transduction mechanism of RTKs, as it indicates that different receptors may use common downstream elements. It is in accordance with biochemical experiments that identified complexes of different RTKs with common target molecules such as PLC’y, P1 kinase, and GAP. It also fits with the idea that the association between RTKs and their substrates is mediated by the SH2 domains of the substrates, which have a relatively broad specificity of binding. What is the nature of the downstream elements? Two com-

mon suppressors have been cloned, and their structure points to the ras pathway (55). One is the Drosophila ras 1 gene; the other, Sos, is homologous to the CDC 25 protein of Saccharomyces cerevisiae, which facilitates GTP/GDP exchange on ras proteins. The identification of ras as a downstream element of RTKs was also demonstrated by genetic experiments in Caenorhbolitis elegans (57). The question of whether RTKs activate ras by up-regulation of its activators such as Sos/CDC 25 or by down-regulation of its inhibitors such as GAP, is still open.

CONCLUDING

REMARKS

The genetic study of RTKs in Drosophila has provided a wealth of information regarding their developmental roles and signal transduction pathways. Four Drosophila RTKs have been genetically analyzed to date, and the accumulating information presents common themes. The major surprise was that none of these RTKs are required for regulation of cell proliferation. Rather, they appear to represent developmental switches dictating cell fate. These switches may be bin..’y ones, as with sevenless, or they may cover a wider gradation depending on the level of activation of the receptor, as may be the situation with torso. In cases where the same receptor triggers distinct decisions at different phases (such as DER), the context of the cell in which the pathway is activated may lead to the specific end result. The observation that different RTKs use common downstream elements also raises the question as to how the cell can distinguish between the signals transmitted by each of the pathways. One would have to assume that each pathway also uses unique elements not shared by other RTKs. Indeed, in the sevenless suppressor screen three of the loci did not appear to participate in the Ellipse pathway. Another common theme concerns the level of expression of RTKs, and the spatial and temporal regulation of RTK activity. Because RTKs represent the first step in the trans-

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in the eye disc

mission of information into the cell, their function is cell autonomous. Unless triggered by the proper ligand, the receptor is found in the membrane in an inactive form. Thus, the level of expression of the receptor does not have to be tightly regulated. For torso, it is even imperative that the receptor would be found in excess to trap the ligand once it is released. The spatial and temporal regulation of RTK expression does not appear to be critical as well. sevenless is expressed in the eye disc not only in the precursor of R7, but also in cells R 3,4 and in the cone cells. Ectopic expression of sevenless under the regulation of the heat shock promoter had no deleterious consequences (49, 50). Similar results were obtained with ectopic expression of DER in the embryo (R. Schweitzer and B-Z. Shio, unpublished results). The receptors have to be present in the right tissues at the time when the ligand is produced. Expression of RTKs in tissues in which the ligand is absent, however, does not appear to be harmful. Postreceptor elements are also not implicated in providing the temporal and spatial specificity, as several postreceptor elements appear to represent abundant components that may participate in the pathways of different RTKs. In addition, constitutive activation of torso (31, 32) or sevenless (48) and deregulated activation of DER in the ovary (35) could induce fate changes in cells that are not normally affected by these RTKs. The regulation of RTK activity is thus likely to be dictated at the level of ligand expression or presentation. Because processes controlled by RTKs in Drosophila determine cell fate, they require very stringent temporal and spatial regulation. A freely diffusible ligand may not be able to provide such an accurate control. It is tempting to speculate that the ligands would be anchored, at least until the time they are required to trigger the receptor, to the cell membrane, the vitelline membrane or the extracellular matrix. The only ligand identified to date is boss, the ligand of sevenless. Indeed, it fulfills these predictions. boss is restricted to the surface of the R8 cell, and may be presented to the neighboring precursor cells only at a time when cells R3 and R4 have already initiated their differentiation. The spatial restriction of boss is important, as ectopic expression of boss drives the cone cells into an R7 cell fate (58). The structure of boss shows that it has multiple transmembrane domains. Binding of boss to sevenless and internalization of the bound complex do not require proteolytic cleavage of boss. Isolation of the ligands for the other Drosophila RTKs will be crucial for further understanding of their regulation and function. In conclusion, RTKs serve a pivotal role in pathways determining cell fate in Drosophila. Their position in the membrane allows them to carry out the primary step in receiving the external information and transmitting it into the cell.

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The structure, mode of action, and some of the downstream elements are extremely conserved between Drosophila and vertebrates. This raises the question, is the normal role of RTKs in vertebrates also associated with the control cell fate? The mouse White spotting mutation in the c-kit locus (59), leads to defects in the development of hemopoietic cells, melanocytes, and sperm cells (60), but the basis for these abnormalities may be in cell survival rather than in cell determination (61, 62). The capacity of FGF as a potent angiogenic factor, promoting division and movement of endothelial cells to form blood vessels (63), may have features in common with the role of breathless (DFGF-R1) and its putative ligand in the migration of tracheal cells leading to the establishment of the tracheal tree. As the organismal phenotypes of mutations in additional vertebrate RTKs are analyzed in detail (64), the degree of similarity in the function of RTKs between the two phyla should be uncovered.

E!i I would like to thank all members of my lab for participating in the work that led to some topics discussed in this review, and for continuous stimulating and creative discussions that contributed to solidify the concepts that were presented. The work was supported by grants from the National Institutes of Health, Israel Academy of Science, and Minerva. REFERENCES 1. Yarden, Y., and Ullrich, A. (1988) Growth factor receptor tyrosine kinases. Annu. Rev. Biochem. 57, 443-478 2. Ullrich, A., and Schlessinger, J. (1990) Signal transduction by receptors with tyrosine kinase activity. Cell 61, 203-212 3. Cantley, L. C., Auger, K. R., Carpenter C., Duckworth, B., Graziani, A., Kapeller, R., and Soltoff, S. (1991) Oncegenes and signal transduction. Cell 64, 281-302 4. Matsuda, M., Mayer, B., Fukui, Y., and Hanafusa, H. (1990) Binding of transforming protein, P47”5, to a broad range of phosphotyrosine-containing proteins. Science 248, 1537-1539 5. Sprenger, F, Stevens, L. M., and N#{252}sslein-Volhard,C. (1989) The Drosophila gene torso encodes a putative receptor tyrosine kinase. Nature (London) 338, 478-483 6. Livneh, E., Glazer, L., Segal, D., Schlessinger, J., and Shio, B-Z. (1985) The Drosophila EGF receptor gene homolog: conservation of both hormone binding and kinase domains. Cell 40, 599-607 7. Schejter, E. D., Segal, D., Glazer, L., and Shio, B-Z. (1986) Alternative 5 exons and tissue-specific expression of the Drosophila EGF receptor homolog transcripts. Cell 46, 1091-1101 8. Glazer, L., and Shilo, B-Z. The Drosophila FGF receptor homolog is expressed in the embryonic tracheal system and appears to be required for directed tracheal cell extension. Genes & Devel. 5, 697-705 9. Hafen, E., Basler, K., Edstroem,J-E., and Rubin, G. M. (1987) sevenless, a cell-specific homeotic gene of Drosophila, encodes a putative transmembrane receptor with a tyrosine kinase domain. Science 236, 55-63 10. Bowtell, D. D. L., Simon, M. A., and Rubin, G. M. (1988) Nucleotide sequence and structure of the sevenless gene of Drosophila melanogaster Genes & DeveL 2, 620-634 11. Petruzzelli, L., Herrara, R., Arenas-Garcia, R., Fernandez, R., Birnbaum, M. J., and Rosen, 0. M. (1986) Isolation of a Drosophila genomic sequence homologous to the kinase domain of the human insulin receptor and detection of the phosphoiylated Drosophila receptor with anti-peptide antibody. Proc. Natl. Acad. Sd. USA 83, 4710-4714 12. Schejter, E. D., and Shio, B-Z. (1989) The Drosophila EGF receptor gene is allelic to faint little ball, a locus essential for embryonic development. Cell .56, 1093-1104 13. Wides, R. J., Zak, N. B., and Shio, B-Z. (1990) Enhancement of tyrosine kinase activity of the Drosophila EGF receptor homoRECEPTOR TYROSINE


log (DER) by alterations of the transmembrane domain. Eur. j Biochem. 189, 637-645 14. Simon, M. A., Bowtell, D. D. L., and Rubin, G. M. (1989) Structure and activity of the sevenless protein: a protein tyrosine kinase receptor required for photoreceptor development in Drosophila. Proc. Nail. Acad. Set USA 86, 8333-8337 15. Basler, K., and Hafen, E. (1988) Control of photoreceptor cell fate by the sevenless protein requires a functional tyrosine kinase domain. Cell 54, 299-311 16. Raz, E., Schejter, E. D., and Shio, B-Z. (1991) Inter-allelic cornplementation among DER/ftb alleles: implications on the mechanism of signal transduction by receptor-tyrosine kinases. Genetics 129, 191-201 17. Zak, N. B., Wides, R. J., and Shilo, B-Z. (1990) Localization of the DER/ftb protein in embryos: implications on the faint little ball phenotype. Development 109, 865-874 18. Shio, B-Z., and Raz, E. (1991) Developmental control by the Drosophila EGF receptor homolog DER. Trendc Genet. 7, 388-392 19. Tomlinson, A., Bowtell, D. D. L., Hafen, E., and Rubin, G. M. (1987) Localization of the sevenless protein, a putative receptor for positional information, in the eye imaginal disc of Drosophila. Cell 51, 143-150 20. Baneijee, U., Renfranz, P. J., Hinton, D. R., Rabin, B. A., and Benzer, S. (1987) The sevenless protein in expressed apically in cell membranes of developing Drosophila retina; it is not restricted to cell R7. Cell 51, 151-158 21. N#{252}sslein-Volhard,C. (1991) Determination of the embryonic axes of Drosophila. Development SuppL 1, 1-10 22. Driever, W., and N#{252}sslein-Volhard,C. (1988) A gradient of bicold protein in Drosophila embryos. Cell 54, 83-93 23. Wang, C., and Lehmann, R. (1991) Nanos is the localized posterior determinant in Drosophila. Cell 66, 637-647 24. King, R. C. (1970) Ovarian Development in Drosophila melanogaster, Academic, New York 25. Schupbach, T, and Wieschaus, E. (1986) Wilhelm Roux’s Arch. Dcv. BioL 195, 302-317 26. Degelmann, A., Hardy, P. A., Perrimon, N., and Mahawald, A. P. (1986) Developmental analysis of the torso-like phenotype in Drosophila produced by a maternal-effect locus. Dcv. Biol. 115, 479-489 27. Perrimon, N., Moheler, D., Engstrom, L., and Mahawald, A. P. (1986) X-linked female-sterile loci in Drosophila melanogoster. Genetics 113, 695-712 28. Perrimon, N., Engstrom, L., and Mahawald, A. P. (1985) A pupal lethal mutation with a paternally influenced maternal effect on embryonic development in Drosophila melanogaster. Dcv. Biol. 110, 480-491 29. Stevens, L. M., Frohnh#{246}fer, H. G., Klinger, M., and N#{252}ssleinVolhard, C. (1990) Localized requirement for torso-like expression in follicle cells for development of terminal anlagen of the Drosophila embryo. Nature (London) 346, 660-663 30. Casanova, J., and Struhl, G. (1989) Localized surface activity of torso, a receptor tyrosine kinase, specifies terminal body patterns in Drosophila. Genes & Dcv. 3, 2025-2038 31. Klinger, M., Erdelyi, M., Szabad, J., and N#{252}sslein-Volhard,C. (1988) Function of torso in determining the terminal anlagen of the Drosophila embryo. Nature (London) 335, 275-277 32. Strecker, T R., Halsell, S. R., Fisher, W. W., and Lipshitz, H. D. (1989) Reciprocal effects of the hyper- and hypoactivity mutations in the Drosophila pattern gene torso. &ience 243, 1062-1066 33. Schupbach, T (1987) Germ line and soma cooperate during oogenesis to establish dorsoventral pattern of egg shell and embryo in Drosophila melanogoster. Cell 49, 699-707 34. Price, J. V., Clifford, R. J., and Schupbach, T. (1989) The maternal ventralizing locus torpedo is allelic to faint little ball, an embryonic lethal, and encodes the Drosophila EGF receptor homolog. Cell 56, 1085-1092 35. Manseau, L. J., and Schupbach, T. (1989) cappucdino and spire: two unique maternal effect loci required for both anteroposterior and dorsoventral patterns of the Drosophila embryo. Genes &Dev. 3, 1437-1452 36. Monteil, D. J., Kashishian, H., and Spradling, A. C. (1991) Laser ablation studies of the role of the Drosophila oocyte nucleus in pattern formation. Science 254, 290-293 2921


37. N#{252}sslein-Volhard,C., Wieschaus, E., and Kluding, H. (1984) Mutations affecting the pattern of the larval cuticle in Drosophila melanogoster. I. Zygotic loci on the second chromosome. Wilhelm Roux’s Arch. Dcv. BioL 193, 267-282 38. Clifford, R. J., and Sch#{252}pbach,T (1990) Coordinately and differentially mutable activities of torpedo, the Drosophila melanogaster homolog of the vertebrate EGF receptor gene. Genetics 123, 771-787 39. Raz, E., and Shilo, B-Z. (1992) Dissection of the faint little ball (fib) phenotype: determination of the development of the Drosophila central nervous system by early interactions in the cctoderm. Development 114, 113-123 40. Kl#{227}rnbt, C., Jacobs, R., and Goodman, C. S. (1991) The midline of the Drosophila central nervous system: a model for the genetic analysis of cell fate, cell migration, and growth cone guidance. Cell 64, 801-815 41. Campos-Ortega, J. A., and Hartenstein, V. (1985) The Embryonic Development of Drosophila melanogaster. Springer-Verlag, Berlin 42. Banerjee, U., and Zipursky, S. L. (1990) The role of cell-cell interaction in the development of the Drosophila visual system. Neuron 4, 177-187 43. Hafen, E., and Basler, K. (1991) Specification of cell fate in the developing eye of Drosophila. Development SuppL 1, 123-130 44. Baker, N. E., Mlodzik, M., and Rubin, G. M. (1990) Spacing differentiation in the developing Drosophila eye: a fibrinogenrelated lateral inhibitor encoded by scabarous. Science 250, 13 70-13 7 7 45. Baker, N. E., and Rubin, G. M. (1989) Effect on eye development of dominant mutations in the Drosophila homologue of the EGF receptor. Nature (London) 340, 150-153 46. Tornlinson, A., and Ready, D. F (1987) Cell fate in the Drosophila ommatidium. Dcv. BioL 120, 264-275 47. Rubin, G. M. (1991) Signal transduction and the fate of the R7 photoreceptor in Drosophila. Trends Genet. 7, 372-377 48. Basler, K., Christen, B., and Hafen, E. (1991) Ligandindependent activation of the sevenless receptor tyrosine kinase changes the fate of cells in the developing Drosophila eye. Cell 64, 1069-1082 49. Basler, K., and Hafen, E. (1989) Ubiquitous expression of sevenless: position-dependent specification of cell fate. Science 243, 931-934 50. Bowtell, D. D. L., Simon, M. A., and Rubin, G. M. (1989) Ommatidia in the developing Drosophila eye require and can respond to sevenless for only a restricted period. Cell 56, 931-936 51. Reinke, R., and Zipursky, S. L. (1988) Cell-cell interaction in the Drosophila retina: the bride of sevenless gene is required in photoreceptor cell R8 for R7 cell development. Cell 55, 321-330

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52. Hart, A. C., Kramer, H., Van Vactor, D. L., Jr., Paidhungat, M., and Zipursky, S. L. (1990) Induction of cell fate in the Drosophila retina: the bride of sevenless protein is predicted to contain a large extracellular domain and seven transmembrane segments. Gene & Dcv. 4, 1835-1847 53. Kramer, H., Cagan, R. L., and Zipursky, S. L. (1991) Interaction of bride of sevenless membrane-bound ligand and the sevenless tyrosine kinase receptor. Nature (London) 352, 207-212 54. Nishida, Y., Hata, M., Ayakai, T., Ryo, H., Yamagata, M., Shimizu, K., and Nishizuka, Y. (1988) Proliferation of both somatic and germ cells is affected in the Drosophila mutants of the raf proto-oncogene. EMBO j 7, 7 75-781 55. Simon, M. A., Bowtell, D. D. L., Dodson, G. S., Laverty, T. R., and Rubin, G. M. (1991) Ras 1 and putative guanine nucleotide exchange factor perform crucial steps in signalling by the sevenless protein tyrosine kinase. Cell 67, 701-716 56. Rogge, R. D., Karlovitch, C. A., and Banerjee, U. (1991) Genetic dissection of a neurodevelopmental pathway: son of sevenless functions downstream of the sevenless and EGF receptor tyrosine kinases. Cell 64, 39-48 57. Aroian, R. V., Koga, M., Mendel, J. E., Ohshima, Y., and Sternberg, P. W. (1990) The let-23 gene necessary for Caenorhbditic elegans vulval induction encodes a tyrosine kinase of the EGF receptor subfamily. Nature (London) 348, 693-699 58. Van Vactor, D. L.,Jr., Cagan, R. L., Kramer, H., and Zipursky, S. L. (1992) Induction in the developing compound eye of Drosophila: multiple mechanisms restrict R7 induction to a single retinal precursor cell. Cell 67, 1145-1156 59. Chabot, B., Stephenson, D. A., Chapman, V. M., Besmer, P., and Bernstein, A. (1988) The proto-oncogene c-kit encoding a transmembrane tyrosine kinase receptor maps to the mouse W locus. Nature (London) 335, 88-89 60. Silvers, W. K. (1979) The Coat Colours of mice: A Model For Mammalian Gene Action and Interaction, pp. 243-267, Springer-Verlag, New York 61. Godin, I., Deed, R., Cooke, J., Zsebo, K., Dexter, M., and Wylie, C. C. (1991) Effects of the steel gene product on mouse premordial germ cells in culture. Nature (London) 352, 807-809 62. Dolci, S., Williams, D. E., Ernst, M. K., Rensnick, J. L., Brannan, C. I., Lock, L. F, Lyman, S. D., Boswell, H. S., and Donovan, P. J. (1991) Requirement for mast cell growth factor for primordial germ cell survival in culture. Nature (London) 352, 809-811 63. Folkman, J., and Klagsbrun, M. (1987) Angiogenic factors. Science 235, 442-447 64. Pawson, T, and Bernstein, A. (1990) Receptor tyrosine kinases: genetic evidence for their role in Drosophila and mouse development. Trends Genet. 6, 350-356

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